Thursday, July 29, 2010

Drug addiction












What is Drug Addiction?
Drug addiction is a complex brain disease. It is characterized by compulsive, at times uncontrollable, drug craving, seeking, and use that persist even in the face of extremely negative consequences.Drug seeking becomes compulsive, in large part as a result of the effects of prolonged drug use on brain functioning and, thus, on behavior. For many people, drug addiction becomes chronic, with relapses possible even after long periods of abstinence.

Symptoms and Diagnosis of Alcoholism, Addiction
Many times, determining whether someone is an actual alcoholic or not is really not all that important. If someone's drinking and drugging has advanced to the point to where it has become a problem for them and those around them, then it is a problem, period. If the person has tried to quit -- swore to themselves and others "never again!" -- and simply cannot stop, then chances are they have become dependent or addicted.
What Is Alcoholism?
Alcohol problems occur at different levels of severity, from mild and annoying to life-threatening. Although alcohol dependence (alcoholism) is the most severe problem, less severe drinking problems can also be dangerous. The term "alcoholism" refers to a disease known as alcohol dependence syndrome, the most severe stage of a range of drinking problems.
Symptoms of Alcoholism
Alcoholism is called a progressive disease, meaning that over time the symptoms and effects of drinking alcohol become more intense and severe. The symptoms in the early stages differ from those during later stages as the disease progresses from binge drinking to alcohol abuse to alcohol dependence.
Diagnosing Alcoholism

Because one of the most common symptoms of alcoholism is denial, diagnosing alcoholism can be difficult -- the diagnosis depends on the individual's willingness to answer questions about their drinking honestly. Usually, the friends and family members closest to the drinker see the problem long before it is diagnosed in a medical setting.

Is Alcoholism Inherited?

Alcoholism does tend to run in families, and scientific studies indicate that genetics play a role in a person's risk of developing alcohol problems. But research also shows that a individual's environment and peer influences also affect the risk of becoming alcoholic.

Abuse Signs, Symptoms, and Help for Drug Problems and Substance

Are you struggling with a drug problem that’s spiraled out of control? If so, you may feel isolated, helpless, or ashamed. Or perhaps you’re worried about a friend or family member’s drug use. In either case, you’re not alone. Addiction is a problem that many people face.
The good news is that you or your loved one can get better. There is hope—no matter how bad the substance abuse problem and no matter how powerless you feel. Learning about the nature of addiction—how it develops, what it looks like, and why it has such a powerful hold—will give you a better understanding of the problem and how to deal with it.


How addiction developsThe path to drug addiction starts with experimentation. You or your loved one may have tried drugs out of curiosity, because friends were doing it, or in an effort to erase another problem. At first, the substance seems to solve the problem or make life better, so you use the drug more and more.
But as the addiction progresses, getting and using the drug becomes more and more important and your ability to stop using is compromised. What begins as a voluntary choice turns into a physical and psychological need. The good news is that drug addiction is treatable. With treatment and support, you can counteract the disruptive effects of addiction and regain control of your life.
5 Myths about Drug Addiction and Substance Abuse
MYTH 1: Overcoming addiction is a simply a matter of willpower. You can stop using drugs if you really want to. Prolonged exposure to drugs alters the brain in ways that result in powerful cravings and a compulsion to use. These brain changes make it extremely difficult to quit by sheer force of will.
MYTH 2: Addiction is a disease; there’s nothing you can do about it. Most experts agree that addiction is a brain disease, but that doesn’t mean you’re a helpless victim. The brain changes associated with addiction can be treated and reversed through therapy, medication, exercise, and other treatments.
MYTH 3: Addicts have to hit rock bottom before they can get better. Recovery can begin at any point in the addiction process—and the earlier, the better. The longer drug abuse continues, the stronger the addiction becomes and the harder it is to treat. Don’t wait to intervene until the addict has lost it all.
MYTH 4: You can’t force someone into treatment; they have to want help. Treatment doesn’t have to be voluntary to be successful. People who are pressured into treatment by their family, employer, or the legal system are just as likely to benefit as those who choose to enter treatment on their own. As they sober up and their thinking clears, many formerly resistant addicts decide they want to change.
MYTH 5: Treatment didn’t work before, so there’s no point trying again; some cases are hopeless. Recovery from drug addiction is a long process that often involves setbacks. Relapse doesn’t mean that treatment has failed or that you’re a lost cause. Rather, it’s a signal to get back on track, either by going back to treatment or adjusting the treatment approach.


The far-reaching effects of drug abuse and drug addictionWhile each drug of abuse produces different physical effects, all abused substances share one thing in common. They hijack the brain’s normal “reward” pathways and alter the areas of the brain responsible for self-control, judgment, emotional regulation, motivation, memory, and learning.
Whether you’re addicted to nicotine, alcohol, heroin, Xanax, speed, or Vicodin, the effect on the brain is the same: an uncontrollable craving to use that is more important than anything else, including family, friends, career, and even your own health and happiness.
Using drugs as an escape: A short-term fix with long-term consequences
Many people use drugs in order to escape physical and emotional discomfort. Maybe you started drinking to numb feelings of depression, smoking pot to deal with stress at home or school, relying on cocaine to boost your energy and confidence, using sleeping pills to cope with panic attacks, or taking prescription painkillers to relieve chronic back pain.
But while drugs might make you feel better in the short-term, attempts to self-medicate ultimately backfire. Instead of treating the underlying problem, drug use simply masks the symptoms. Take the drug away and the problem is still there, whether it be low self-esteem, anxiety, loneliness, or an unhappy family life. Furthermore, prolonged drug use eventually brings its own host of problems, including major disruptions to normal, daily functioning. Unfortunately, the psychological, physical, and social consequences of drug abuse and addiction become worse than the original problem you were trying to cope with or avoid.


Signs and symptoms of drug abuse and drug addiction
Although different drugs have different physical effects, the symptoms of addiction are the same no matter the substance. The more drugs begin to affect and control your life, the more likely it is that you’ve crossed the line from drug use to abuse and drug addiction. Unfortunately, when you’re in the middle of it, you may be in denial about the magnitude of the problem or the negative impact it's had on your life. See if you recognize yourself in the following signs and symptoms of substance abuse and addiction. If so, consider talking to someone about your drug use. You’re on a dangerous road, and the sooner you get help, the better.
Common signs and symptoms of drug abuse
You’re neglecting your responsibilities at school, work, or home (e.g. flunking classes, skipping work, neglecting your children) because of your drug use.
You’re using drugs under dangerous conditions or taking risks while high, such as driving while on drugs, using dirty needles, or having unprotected sex.
Your drug use is getting you into legal trouble, such as arrests for disorderly conduct, driving under the influence, or stealing to support a drug habit.
Your drug use is causing problems in your relationships, such as fights with your partner or family members, an unhappy boss, or the loss of old friends.
Common signs and symptoms of drug addiction
You’ve built up a drug tolerance. You need to use more of the drug to experience the same effects you used to with smaller amounts.
You take drugs to avoid or relieve withdrawal symptoms. If you go too long without drugs, you experience symptoms such as nausea, restlessness, insomnia, depression, sweating, shaking, and anxiety.
You’ve lost control over your drug use. You often do drugs or use more than you planned, even though you told yourself you wouldn’t. You may want to stop using, but you feel powerless.
Your life revolves around drug use. You spend a lot of time using and thinking about drugs, figuring out how to get them, and recovering from the drug’s effects.
You’ve abandoned activities you used to enjoy, such as hobbies, sports, and socializing, because of your drug use.
You continue to use drugs, despite knowing it’s hurting you. It’s causing major problems in your life—blackouts, infections, mood swings, depression, paranoia—but you use anyway.
What drugs are most commonly abused and what are the signs and symptoms?
Almost all drugs have the potential for addiction and abuse, from caffeine to prescription medication. However, the majority of non-alcohol related addictions are due to a short list of drugs including sleeping pills, painkillers, cocaine, marijuana, methamphetamine, and heroin.
Click here for a PDF factsheet on the symptoms and effects of commonly abused drugs.
Warning signs that a friend or family member is abusing drugs
Drug abusers often try to conceal their symptoms and downplay their problem. If you’re worried that a friend or family member might be abusing drugs, look for the following warning signs:
Physical warning signs of drug abuse
Bloodshot eyes or pupils that are larger or smaller than usual.
Changes in appetite or sleep patterns. Sudden weight loss or weight gain.
Deterioration of physical appearance and personal grooming habits.
Unusual smells on breath, body, or clothing.
Tremors, slurred speech, or impaired coordination.
Behavioral signs of drug abuse
Drop in attendance and performance at work or school.
Unexplained need for money or financial problems. May borrow or steal to get it.
Engaging in secretive or suspicious behaviors.
Sudden change in friends, favorite hangouts, and hobbies.
Frequently getting into trouble (fights, accidents, illegal activities).
Psychological warning signs of drug abuse
Unexplained change in personality or attitude.
Sudden mood swings, irritability, or angry outbursts.
Periods of unusual hyperactivity, agitation, or giddiness.
Lack of motivation; appears lethargic or “spaced out.”
Appears fearful, anxious, or paranoid, with no reason.
Warning Signs of Teen Drug Use
There are many warning signs of drug use and abuse in teenagers. The challenge for parents is to distinguish between the normal, sometimes volatile, ups and downs of the teen years and the red flags of substance abuse.
Being secretive about friends, possessions, and activities.
New interest in clothing, music, and other items that highlight drug use.
Demanding more privacy; locking doors; avoiding eye contact; sneaking around.
Skipping class; declining grades; suddenly getting into trouble at school.
Missing money, valuables, or prescriptions.
Acting uncharacteristically isolated, withdrawn, or depressed.
Using incense, perfume, or air freshener to hide the smell of smoke or drugs.
Using eyedrops to mask bloodshot eyes or dilated pupils.

Drug addiction and denial
One of the most dangerous effects of drug abuse and addiction is denial. The urge to use is so strong that the mind finds many ways to rationalize the addiction. You may drastically underestimate the quantity of drugs you’re taking, how much it impacts your life, and the level of control you have over your drug use.
Denial is an unconscious defense mechanism. Minimizing and rationalizing the addiction is less scary than admitting that your drug use is dangerously out of control. But the cost of denial can be extremely high—including the loss of important relationships, your job, financial security, and your physical and mental health.

Do you have a substance abuse problem?
Do you feel like you can’t stop, even if you wanted to?
Do you ever feel bad or guilty about your drug use?
Do you need to use drugs to relax or feel better?
Do your friends or family members complain or worry about your drug use?
Do you hide or lie about your drug use?
Have you ever done anything illegal in order to obtain drugs?
Do you spend money on drugs that you really can’t afford?
Do you ever use more than one recreational drug at a time?
If you answered “yes” to one or more of the questions, you may have a drug problem.

Getting help for drug abuse and drug addiction

If you’re ready to admit you have a drug problem, congratulations! Recognizing that you have a problem is the first step on the road to recovery, one that takes tremendous courage and strength.
Facing your addiction without minimizing the problem or making excuses can feel frightening and overwhelming, but recovery is within reach. If you’re ready to make a change and willing to seek help, you can overcome your addiction and build a satisfying, drug-free life for yourself.
Support is essential to addiction recovery
Don’t try to go it alone; it’s all too easy to get discouraged and rationalize “just one more” hit or pill. Whether you choose to go to rehab, rely on self-help programs, get therapy, or take a self-directed treatment approach, support is essential. Recovering from drug addiction is much easier when you have people you can lean on for encouragement, comfort, and guidance

LUCIFERASE GENE AND ITS APPLICATIONS








Background
Bioluminescence is a form of light produced by a chemical reaction in living
organisms. The function of bioluminescence may vary from one organism to the other,
such as for defense against predators, for predation or for communication with their
mates . It is exhibited by a diverse group of organisms
although their number is very less compared to the total number of known species. It has
been estimated that luminous organisms may have come from about 30 different
evolutionarily distinct origins .
The enzyme luciferase and its substrate luciferin are discovered to be the agents
responsible for Bioluminescence. The biochemical and physiological mechanisms
responsible for the bioluminescence are different among the luminous organisms
. Hastings and Morin in 1991 have reported that the genes and
proteins responsible for bioluminescence are not evolutionarily conserved. These genes
and proteins are evolved independently and are not similar to each other.
Shimomura et al., first discovered luciferase in 1962. They extracted the light
emitting protein from jellyfish “Aequorea aequorea” and found the compound to be
emitting blue color instead of expected green color. This protein was later named as
“aequorin”. The expected green color was emitted by another protein, which absorbed the
blue color emitted by aequorin and re-emitted the energy at a higher wavelength. This
protein was later named “green fluorescence protein” (GFP).
There have been many studies on luciferase enzyme systems in different groups
of luminescent organisms. There are also many applications of this enzyme and the
associated GFP in molecular biology fields. In the following review the different aspects
of the enzyme luciferase and its applications will be addressed.
Chemistry
The chemistry behind bioluminescence has two major components. One is the
substrate which is generically called luciferin and the other is the enzyme that catalyses
the substrate to emit a photon called as luciferase.




The basic reaction involves luciferase catalyzing the oxidation of luciferin
resulting in light and an inactive "oxyluciferin" In most cases, fresh luciferin must be
brought into the system, either through the diet or by internal synthesis.
Luciferin + O2 Oxyluciferin + light
Luciferase
Sometimes the luciferin and luciferase (as well as a co-factor such as oxygen) are
bound together in a single unit called a "photoprotein". This molecule can be triggered to
produce light when a particular type of ion is added to the system (frequently
calcium).




Types of Luciferin
Luminous organisms are intensively studied for their difference in the luminous
systems. The luminous systems differ in the structures of the luciferins and luciferases,
although they have some properties in common. All known luciferases are oxygenases
and they oxidize the luciferin using molecular oxygen to intermediate enzyme bound
peroxide, which dissociates to produce an intermediate or product in its excited state to
emit photon. Luminous systems of luminous organisms are classified into four types
based on the different type of luciferin they possess. They are 1) Coelenterate luciferin
(Coelenterazine), 2) Bacterial luciferin, 3) Dinoflagellate luciferin, and 4) Firefly
luciferin.
Coelenterazine is the most popular of the marine luciferins, found in a variety of
phyla. This luciferin is biochemically similar to other cnidarian luminescent systems. It
possesses an imidazopyrazine skeleton and is widely distributed among the cnidarian
group.
Coelenterazine
Bacterial luciferin is a reduced riboflavin phosphate, which is oxidized in
association with a long-chain aldehyde, oxygen, and a luciferase. Luminous bacteria are
observed ubiquitously in seawater samples. All bacterial luciferases are heterodimeric
containing two polypeptides alpha and beta. Fisher et al. in 1995 reported the X-ray
structure of luciferases.
Bacterial Luciferin




Dinoflagellate luciferin is a novel tetrapyrrole related to chlorophyll. In the genus
Gonyaulax, at pH 8 the molecule is "protected" from the luciferase by a "luciferinbinding
protein", but when the pH lowers to around 6, the free luciferin reacts and light is
produced. A modified form of this luciferin is also found in herbivorous euphausiid
shrimp, perhaps indicating a dietary link for the acquisition of luciferin.




Firefly luciferin is a unique benzothiazolethat requires ATP to form luciferyl
adenylate intermediate. The adenylate then reacts with with oxygen to form a cyclic
luciferyl perooxy speces, which breaks down to yield CO2 and an excited state of the
carbonyl product. Wood in 1995 reported that the long chain acyl-CoA synthase is
homologous with the firefly luciferase indicating the evolutionary origin of the gene




Luciferase Gene organisation
Bacterial luciferase gene

Luciferase genes are cloned and sequenced from luminescent bacteria of Vibrio,
Xenorhabdus and Photobacterium genera (Szittner, 1990). Two genes code for the two á
and â subunits of bacterial luciferase , named luxA and luxB respectively. The
luciferase genes occur as two major operons, also containing other proteins associated
with the luciferase activity that is involved in the synthesis of myristic aldehyde
(Meighen, 1991) . The operon also contains genes that control the development
and expression of luminescence by a mechanism of “autoinduction” (Nealson et al.,
1970). The luciferase operon contains about 10 genes and they are transcribed only by the
presence of an autoinducer which is a homoserine lactone produced by the gene present
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in the operon itself named luxI gene. The ecological importance of this mechanism was
discovered in 1994 by Fuqua et al., who dubbed this phenomenon to be Quarum sensing.
It is also discovered that the autoinducer is responsible for the cell-cell signaling in
luminous bacteria.
Plate 2 Bacterial luciferase




Dinoflagellate luciferase gene
The Gonyaulax polyedra luciferin binding protein (LBP) gene and the luciferase
gene are the first dinoflagellate luciferase gene cloned and sequenced by Lee et al.,
(1993) and Bae and Hastings (1994) respectively. They sequenced the cDNA of the LBP
gene and found that the open reading frame of the gene is 2004 nucleotides in length and
has no introns. It is also found that it has no polyadenylation sites. The open reading
frame codes for about 688 aminoacids (~ 75kDa). The interesting circadian phenomenon
is seen in the dinoflagellate LBP gene, which causes the gene to be transcribed only once
a day and the protein destroyed every day (Hastings et al., 1993).
The luciferase gene was found to be 4.1kb and the luciferase enzyme is 130kDa.
This gene also did not have any introns. Cloning of the cDNA of the gene in Escherichia
coli showed the expression of the active luciferase protein (Bae and Hastings, 1994). It
was found that the LBP is responsible for the pH dependent binding of luciferin and the
luciferase is responsible for the oxidation of the luciferin to produce light. Li et al.,
(1997) reported a unique case of a 137kDa protein gene responsible of catalyzing the
light-emitting oxidation of the dinoflagellate luciferin. They also found that the gene had
three tandem repeat sequences of (~377, 377 and 379 bp) with no intervals, capable of
producing individually active luciferase.




Okamoto et al., in 2001 reported three new classes of the luciferase gene in new
members of dinoflagellate, Pyrocystis lunula and Lingulodinium polyedrum. The three
genes are named lcfA, lcfB and lcfC, which are unique and not related to the luciferase
genes of other dinoflagellates. The lcfA and lcfB genes are connected by a 2.2kb
intergenic region. In P.lunula, the lcfC gene has an intron (Figure 5). They also found the
presence of an intron in lcfC of P. lunula luciferase genes; different sizes of untranslated
regions in P. lunula and L. polyedrum luciferase genes, sharing no significant sequence
similarities; and notable differences in the frequency of synonymous substitutions in the
center of the repeat luciferase domains between the two species




Firefly luciferase gene
The Firefly luciferase gene was first cloned and sequenced by Wet et al., in 1985
from Photuris pennsylvanica, belonging to the Photurinae subfamily of the Lampyridae.
The gene sequence was about 1.8kb in length, coding for 545 aminoacid . The 5'
noncoding region was 61 bp and the 3' noncoding region is 135 bp in length. The
poly(A)tail was 24-nucleotide in length (Figure 6). Wood in 1995 studied the homology
of the luciferase enzyme aminoacid sequences with other known enzyme sequences. He
showed the enzyme’s similarity with adenylate kinase, the putative AMP-binding
domain, luciferin 4-monooxygenase, 4-coumarate CoA ligase, long-chain fatty acid CoA
ligase, 2-acylglycerophosphoethanolamine acyltransferase, the microbody-directing
sequence, peptide-synthesizing complexes, and acyladenylate-synthesizing enzymes.
Choi et al., in 2003 studied the luciferase gene organization in the Hotaria-group of
fireflies. They also studied the phylogenetic diversity of the luciferase gene in fireflies of
the Hotaria group.
Plate




Applications of Luciferase
Initial applications of luciferase were dependent on the type substrate that were
used for emitting light. The apoaequorin, which functions like the luciferase in
coelenterate system, was used to monitor intracellular calcium levels (Tanahashi et al.,
1990). Saran et al., (1994) used cnidarian luciferases to monitor intracellular calcium
changes in response to cyclic adenosinemonophosphate (cAMP) stimulation. Johnson et
al., (1995) used coelenterate luciferased to investigate the circadian changes in free
cytosolic calcium level in the chloroplasts of tobacco and Arabidopsis. Similarly bacterial
lux genes have been used as reporters for visualizing gene expression in Streptomyces
(Schauer et al., 1988) and to study the circadian transcriptional regulation in
cyanobacteria (Kondo et al., 1994). The firefly luciferase was widely used to measure
ATP (Hastings et al., 1997). Recently a combined use of two different luminous systems,
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such as firefly and Renilla luciferase enzymes are used to assay two different luciferins
has been reported by Sherf et al., 1997.
Luciferases are nowadays successfully used as reporter genes for gene expression
studies in living cells. Their application is growing in all aspects of the biological field
such as Animal research, environmental and plant research.
Luciferase in Animal Research
Molecular imaging of live cells
Luciferase is widely used in molecular imaging of cells in vivo. Luciferases are
used to track the in vivo behaviour of any tissue. Bhaumik and Gambhir (2002) validated
that the bioluminescence from Renilla luciferase (rluc) and the firefly luciferase enzyme
both can be imaged using different substrates for each and their kinetics can be studied in
vivo in the tissue. They carried out the experiment in mice by transfecting the mice with
C6 cells containing Firefly luciferase (fluc) and (rluc) (Plate 4).
Plate 4 Kinetics of light production from mice carrying s.c. C6-Fluc and C6-Rluc cells after simultaneous tail-vein injection of both Dluciferin
and coelenterazine. A mouse was injected s.c. with C6-Fluc (A), C6-Rluc (B), and C6 control cells (C) on right forearm, left
forearm, and right thigh regions, respectively. Simultaneous injection of both coelenterazine and D-luciferin mixture via tail-vein
shows bioluminescence from both the sites simultaneously but with distinct kinetics. A series of image at 2-min intervals is shown
from the same mouse. Each image represents a scan time of 1 min. The signal from C6-Rluc cells (B) peaks early and is near
extinguished within 10 min. Bioluminescence from C6-Fluc cells (A) shows a relatively strong signal beyond 10 min. The region of
control cells does not show any significant bioluminescence. R and L represent the right and left side of the mouse resting in supine
position.
Applications in Gene therapies
Molecular imaging of reporter therapeutic genes could play a critical role in gene
therapy to achieve controlled and effective delivery of genes to target cells avoiding
ectopic expression. Luciferases play a major role in reporting the level, the location and
the duration of the gene expression. A study by Weng et al., (2000) used somatic cell
gene transfer of the fluc linked heme oxygenase-1 (HO-1) transgene to the lung alveoli of
neonatal mice through transpulmonary injection. They monitored the gene expression in
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real time and were able to detect targeted expression of the gene in the cells of the
alveolar epithelium rather than other cells of the lung




Application in transgenic animal research
A study carried out by Voojis et al., (2002) involved the use of a conditional
mouse model for retinoblastoma suppressor gene-dependent pituitary cancer development
with co-expression of fluc genes enabling long term bioluminescence imaging,
quantification of tumour and assessment of chemotherapeutic response(Plate 6) .
Plate 6 Pseudocolor images of longitudinal tumor growth in a POMCcre-POMCluc;RbF19/F19 animal.
Quantification of theemitted photons shows an approximately exponential tumor growth between 12 and 16 weeks.
Another study carried out by Carlsen et al., (2002) developed transgenic mice that
express the fluc gene under the control of the nuclear factor 6B (NF-6B) promoter. This
enabled then for real-time imaging of the NF-6B promoter activity and its modulation in
living mice (Plate 7




Luciferases in Environmental research
Applications as biosensors

Luciferase reporter systems are developed into luminescent bacterial biosensors to
detect a wide range of pollutants and also to assess the bioavailability in the
environmental samples. Hollis et al., in 1999 transformed a Saccharomyces cerevisiae
with firefly luciferase gene from Photinus pyralis to be used as a biosensor. They studied
the cell health upon exposure to toxins directly correlating with the endogenous supply of
energy by ATP. A bioluminescent plasmid reporter that shows a sensitive response
towards 2-4-Dichlorophenoxyacetic acid and 2, 4-Dichlorophenol in soil was constructed
and introduced into the chromosome of Ralstonia eutropha. The transformed bacterium is
capable of detecting the concentrations of 1.2mM 2,4-D and 1.1X102 μM of 2,4-
dichlorophenol (Hay et al., 2000). Pellinen et al., (2002) developed Escherichia coli K-12
strain for specific detection of the tetracycline family of antimicrobial agents. It was
optimized to work with fish samples. The biosensing strain contains a plasmid
incorporating the bacterial luciferase operon of Photorhabdus luminescens under the
control of the tetracycline responsive element from transposon Tn10. The lowest levels of
detection of tetracycline and oxytetracycline from spiked fish tissue were 20 and 50
μg/kg, respectively, in a 2-h assay.



Wednesday, July 28, 2010

Transgenic Animals
















A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using recombinant DNA methodology. In addition to a structural gene, the DNA usually includes other sequences to enable it
to be incorporated into the DNA of the host and
to be expressed correctly by the cells of the host.
Transgenic sheep and goats have been produced that express foreign proteins in their milk.
Transgenic chickens are now able to synthesize human proteins in the "white" of the eggs. These animals should eventually prove to be valuable sources of proteins for human therapy.
In July 2000, researchers from the team that produced Dolly reported success in producing transgenic lambs in which the transgene had been inserted at a specific site in the genome and functioned well. [More]
Transgenic mice have provided the tools for exploring many biological questions.
An example:Normal mice cannot be infected with polio virus. They lack the cell-surface molecule that, in humans, serves as the receptor for the virus. So normal mice cannot serve as an inexpensive, easily-manipulated model for studying the disease. However, transgenic mice expressing the human gene for the polio virus receptor
can be infected by polio virus and even
develop paralysis and other pathological changes characteristic of the disease in humans.
Two methods of producing transgenic mice are widely used:
transforming embryonic stem cells (ES cells) growing in tissue culture with the desired DNA;
injecting the desired gene into the pronucleus of a fertilized mouse egg.
The Embryonic Stem Cell Method (Method "1")Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of mouse blastocysts. They can be grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes.





. Make your DNAUsing recombinant DNA methods, build molecules of DNA containing
the structural gene you desire (e.g., the insulin gene)
vector DNA to enable the molecules to be inserted into host DNA molecules
promoter and enhancer sequences to enable the gene to be expressed by host cells
2. Transform ES cells in cultureExpose the cultured cells to the DNA so that some will incorporate it.
3. Select for successfully transformed cells. [Method]
4. Inject these cells into the inner cell mass (ICM) of mouse blastocysts.
5. Embryo transfer
Prepare a pseudopregnant mouse (by mating a female mouse with a vasectomized male). The stimulus of mating elicits the hormonal changes needed to make her uterus receptive.
Transfer the embryos into her uterus.
Hope that they implant successfully and develop into healthy pups (no more than one-third will).
6. Test her offspring
Remove a small piece of tissue from the tail and examine its DNA for the desired gene. No more than 10–20% will have it, and they will be heterozygous for the gene.
7. Establish a transgenic strain
Mate two heterozygous mice and screen their offspring for the 1:4 that will be homozygous for the transgene.
Mating these will found the transgenic strain.





The Pronucleus Method (Method "2")
1. Prepare your DNA as in Method 1
2. Transform fertilized eggs
Harvest freshly fertilized eggs before the sperm head has become a pronucleus.
Inject the male pronucleus with your DNA.
When the pronuclei have fused to form the diploid zygote nucleus, allow the zygote to divide by mitosis to form a 2-cell embryo.
3. Implant the embryos in a pseudopregnant foster mother and proceed as in Method 1.
An Example This image (courtesy of R. L. Brinster and R. E. Hammer) shows a transgenic mouse (right) with a normal littermate (left). The giant mouse developed from a fertilized egg transformed with a recombinant DNA molecule containing:
the structural gene for
human growth hormone
a strong mouse gene promoterThe levels of growth hormone in the serum of some of the transgenic mice were several hundred times higher than in control mice.

Random vs. Targeted Gene InsertionThe early vectors used for gene insertion could, and did, place the gene (from one to 200 copies of it) anywhere in the genome. However, if you know some of the DNA sequence flanking a particular gene, it is possible to design vectors that replace that gene. The replacement gene can be one that
restores function in a mutant animal or
knocks out the function of a particular locus.In
either case, targeted gene insertion requires
the desired gene
neor, a gene that encodes an enzyme that inactivates the
antibiotic neomycin and its relatives, like the drug G418, which is lethal to mammalian cells;
tk, a gene that encodes thymidine kinase, an enzyme that phosphorylates the nucleoside analog gancyclovir. DNA polymerase fails to discriminate against the resulting nucleotide and inserts this nonfunctional nucleotide into freshly-replicating DNA. So ganciclovir kills cells that contain the tk gene.
Step 1Treat culture of ES cells with preparation of vector DNA.
Results:
Most cells fail to take up the vector; these cells will be killed if exposed to G418.
In a few cells: the vector is inserted randomly in the genome. In random insertion, the entire vector, including the tk gene, is inserted into host DNA. These cells are resistant to G418 but killed by gancyclovir.
In still fewer cells:
homologous recombination occurs. Stretches of DNA sequence in the vector find the homologous sequences in the host genome and the region between these homologous sequences replaces the equivalent region in the host DNA.
Step 2Culture the mixture of cells in medium containing both G418 and ganciclovir.
The cells (the majority) that failed to take up the vector are killed by G418.
The cells in which the vector was inserted randomly are killed by gancyclovir (because they contain the tk gene).
This leaves a population of cells transformed by homologous recombination (enriched several thousand fold).
Step 3Inject these into the inner cell mass of mouse blastocysts.
Knockout Mice: What do they teach us?
If the replacement gene (A* in the diagram) is nonfunctional (a "null" allele), mating of the heterozygous transgenic mice will produce a strain of "knockout mice" homozygous for the nonfunctional gene (both copies of the gene at that locus have been "knocked out"). Knockout mice are valuable tools for discovering the function(s) of genes for which mutant strains were not previously available. Two generalizations have emerged from examining knockout mice:
Knockout mice are often surprisingly unaffected by their deficiency. Many genes turn out not to be indispensable. The mouse genome appears to have sufficient redundancy to compensate for a single missing pair of alleles.
Most genes are pleiotropic. They are expressed in different tissues in different ways and at different times in development.
Tissue-Specific Knockout Mice





While "housekeeping" genes are expressed in all types of cells at all stages of development, other genes are normally expressed in only certain types of cells when turned on by the appropriate signals (e.g. the arrival of a hormone).
Link to a discussion of cell-specific gene expression.
To study such genes, one might expect that the methods described above would work. However, it turns out that genes that are only expressed in certain adult tissues may nonetheless be vital during embryonic development. In such cases, the animals do not survive long enough for their knockout gene to be studied. Fortunately, there are now techniques with which transgenic mice can be made where a particular gene gets knocked out in only one type of cell.
The Cre/loxP System
One of the bacteriophages that infects E. coli, called P1, produces an enzyme — designated Cre — that cuts its DNA into lengths suitable for packaging into fresh virus particles. Cre cuts the viral DNA wherever it encounters a pair of sequences designated loxP. All the DNA between the two loxP sites is removed and the remaining DNA ligated together again (so the enzyme is a recombinase). Using "Method 1" (above), mice can be made transgenic for
the gene encoding Cre attached to a promoter that will be activated only when it is bound by the same transcription factors that turn on the other genes required for the unique function(s) of that type of cell;
a "target" gene, the one whose function is to be studied, flanked by loxP sequences. In the adult animal,
those cells that
receive signals (e.g., the arrival of a hormone or cytokine)
to turn on production of the transcription factors needed
to activate the promoters of the genes whose products are needed by that particular kind of cellwill also turn on transcription of the Cre gene. Its protein will then remove the "target" gene under study.
All other cells will lack the transcription factors needed to bind to the Cre promoter (and/or any enhancers) so the target gene remains intact.
The result: a mouse with a particular gene knocked out in only certain cells.
Knock-in Mice
The Cre/loxP system can also be used to
remove DNA sequences that block gene transcription. The "target" gene can then be turned on in certain cells or at certain times as the experimenter wishes.
replace one of the mouse's own genes with a new gene that the investigator wishes to study.
Such transgenic mice are called "knock-in" mice.
Transgenic Sheep and Goats
Until recently, the transgenes introduced into sheep inserted randomly in the genome and often worked poorly. However, in July 2000, success at inserting a transgene into a specific gene locus was reported. The gene was the human gene for alpha1-antitrypsin, and two of the animals expressed large quantities of the human protein in their milk.
This is how it was done.
Sheep fibroblasts (connective tissue cells) growing in tissue culture were treated with a vector that contained these segments of DNA:
2 regions homologous to the sheep COL1A1 gene. This gene encodes Type 1 collagen. (Its absence in humans causes the inherited disease osteogenesis imperfecta.)
This locus was chosen because fibroblasts secrete large amounts of collagen and thus one would expect the gene to be easily accessible in the chromatin.
A neomycin-resistance gene to aid in isolating those cells that successfully incorporated the vector. [Link to technique]
The human gene encoding alpha1-antitrypsin.
Some people inherit two non- or poorly-functioning genes for this protein. Its resulting low level or absence produces the disease Alpha1-Antitrypsin Deficiency (A1AD or Alpha1). The main symptoms are damage to the lungs (and sometimes to the liver).
Promoter sites from the beta-lactoglobulin gene. These promote hormone-driven gene expression in milk-producing cells.
Binding sites for ribosomes for efficient translation of the mRNAs. Successfully-transformed cells were then
fused with enucleated sheep eggs [Link to description of the method] and
implanted in the uterus of a ewe (female sheep).
Several embryos survived until their birth, and two young lambs have now lived over a year.
When treated with hormones, these two lambs secreted milk containing large amounts of alpha1-antitrypsin (650 µg/ml; 50 times higher than previous results using random insertion of the transgene).
On June 18, 2003, the company doing this work abandoned it because of the great expense of building a facility for purifying the protein from sheep's milk. Purification is important because even when 99.9% pure, human patients can develop antibodies against the tiny amounts of sheep proteins that remain. However, another company, GTC Biotherapeutics, has persevered and in June of 2006 won preliminary approval to market a human protein, antithrombin, in Europe. Their protein — the first made in a transgenic animal to receive regulatory approval for human therapy — was secreted in the milk of transgenic goats.
Transgenic ChickensChickens
grow faster than sheep and goats and large numbers can be grown in close quarters;
synthesize several grams of protein in the "white" of their eggs. Two methods have succeeded in producing chickens carrying and expressing foreign genes.
Infecting embryos with a viral vector carrying
the human gene for a therapeutic protein
promoter sequences that will respond to the signals for making proteins (e.g. lysozyme) in egg white.
Transforming rooster sperm with a human gene and the appropriate promoters and checking for any transgenic offspring.
Preliminary results from both methods indicate that it may be possible for chickens to produce as much as 0.1 g of human protein in each egg that they lay.
Not only should this cost less than producing therapeutic proteins in culture vessels, but chickens will probably add the correct sugars to glycosylated proteins — something that E. coli cannot do.
Transgenic Pigs
Transgenic pigs have also been produced by fertilizing normal eggs with sperm cells that have incorporated foreign DNA. This procedure, called sperm-mediated gene transfer (SMGT) may someday be able to produce transgenic pigs that can serve as a source of transplanted organs for humans. [More]
Transgenic Primates
In the 28 May 2009 issue of Nature, Japanese scientists report success in creating transgenic marmosets. Marmosets are primates and thus our closest relatives (so far) to be genetically engineered. In some cases, the transgene (for green fluorescent protein) was incorporated into the germline and passed on to the animal's offspring. The hope is that these transgenic animals will provide the best model yet for studying human disease and possible therapies





Transgenic plants








Transgenic plants are plants possessing a single or multiple genes, transferred from a different species. Though DNA from another species can be integrated into a plants' genome via natural processes, the term "transgenic plants" refers to plants created in a laboratory using recombinant DNA technology.

The aim of creating transgenic plants is to design plants with specific characteristics through artificial insertion of genes from other species (or taxonomically up to different kingdoms).

Varieties containing genes of two distinct plant species are frequently created by classical breeders who deliberately force hybridization between distinct plant species when carrying out interspecific or intergeneric wide crosses with the intention of developing disease resistant crop varieties.

Classical plant breeders use a number of in vitro techniques such as protoplast fusion, embryo rescue or mutagenesis to generate diversity and produce plants that would not ordinarily exist in nature (see also Plant breeding, Heterosis, New Rice for Africa).

Such traditional techniques (used from ca 1930) have never been controversial[opinion], or been given wide publicity except among professional biologists, and have allowed crop breeders to develop varieties of basic food crop. Hope is one such wheat variety bred by E. S. McFadden with a gene from a wild grass to have rust resistance. Hope saved American wheat growers from devastating stem rust outbreaks in the 1930s[citation needed].

Methods used in traditional breeding that generate plants with DNA from two species by non-recombinant methods are widely familiar to professional plant scientists, and serve important roles in securing a sustainable future for agriculture by protecting crops from pests and helping land and water to be used more efficiently.[citation needed] (see also Food security, International Fund for Agricultural Development, International development)

Natural gene flow between species




Natural flow of genes between bacterial species, often called horizontal gene transfer or lateral gene transfer, can occur because of gene transfer mediated by natural processes.
This natural gene movement between bacteria has been widely detected during genetic investigation of various natural mobile genetic elements, such as transposons, and retrotransposons that naturally translocate to new sites in a genome, and often move to new species over an evolutionary time scale.
There are many types of natural mobile DNAs, and they have been detected abundantly in food crops such as rice .
These various mobile genes play a major role in dynamic changes to chromosomes during evolution and have often been given whimsical names, such as Mariner, Hobo, Trans-Siberian Express (Transib), Osmar, Helitron, Sleeping Princess, MITE and MULE, to emphasize their mobile and transient behavior.
Genetically mobile DNA constitutes a major fraction of the DNA of many plants, and the natural dynamic changes to crop plant chromosomes caused by this natural transgenic DNA mimics many of the features of plant genetic engineering currently pursued in the laboratory, such as using transposons as a genetic tool, and molecular cloning. See also transposon, retrotransposon, integron, provirus, endogenous retrovirus, heterosis, Gene duplication and exon shuffling by helitron-like transposons generate intraspecies diversity in maize.
There is new scientific literature about natural transgenic events in plants, through movement of natural mobile DNAs called MULEs between rice and Setaria millet.
It is becoming clear that natural rearrangements of DNA and horizontal gene transfer play a pervasive role in natural evolution. Importantly many, if not most, flowering plants evolved by transgenesis[citation needed] - that is, the creation of natural interspecies hybrids in which chromosome sets from different plant species were added together. There is also the long and rich history of interspecies cross-breeding with traditional methods.




Deliberate creation of transgenic plants
Production of transgenic plants in wide-crosses by plant breeders has been a vital aspect of conventional plant breeding for about a century[citation needed]. Without it, security of our food supply against losses caused by crop pests such as rusts and mildews would be severely compromised[citation needed]. The first historically recorded interspecies transgenic cereal hybrid was actually between wheat and rye .
In the 20th century, the introduction of alien germplasm into common foods was repeatedly achieved by traditional crop breeders by artificially overcoming fertility barriers. Novel genetic rearrangements of plant chromosomes, such as insertion of large blocks of rye (Secale) genes into wheat chromosomes ('translocations'), has also been exploited widely for many decades [5].
By the late 1930s with the introduction of colchicine, perennial grasses were being hybridized with wheat with the aim of transferring disease resistance and perenniality into annual crops, and large-scale practical use of hybrids was well established, leading on to development of Triticosecale and other new transgenic cereal crops. In 1985 Plant Genetic Systems (Ghent, Belgium), founded by Marc Van Montagu and Jeff Schell, was the first company to develop genetically engineered (tobacco) plants with insect tolerance by expressing genes encoding for insecticidal proteins from Bacillus thuringiensis (Bt).




Transgenic resistance traits in bread wheat varieties

The lists in this article may contain items that are not notable, encyclopedic, or helpful. Please help out by removing such elements and incorporating appropriate items into the main body of the article. (January 2010)
Important transgenic pathogen and parasite resistance traits in current bread wheat varieties (gene, eg "Lr9" followed by the source species) are:
Disease resistance
Leaf rust
Lr9 (from Aegilops umbellulata)
Lr18 Triticum timopheevi
Lr19 Thinopyrum
Lr23 T. turgidum
Lr24 Ag. elongatum
Lr25 Secale cereale
Lr29 Ag. elongatum
Lr32 T. tauschii
Stem rust
Sr2 T. turgidum ("Hope" ) McFadden, E. S. (1930) J. Am. Soc. Agron. 22, 1020-1031 .
Sr22 Triticum monococcum
Sr36 Triticum timopheevii
Stripe rust
Yr15 Triticum dicoccoides
Powdery mildew
Pm12 Aegilops speltoides
Pm21 Haynaldia villosa
Pm25 T. monococcum
Wheat streak mosaic virus
Wsm1 Ag. elongatum
Pest resistance
Hessian fly
H21 S. cereale H23,
H24 T. tauschii
H27 Aegilops ventricosa
Cereal cyst nematode
Cre3 (Ccn-D1) T. tauschii
Lepidoptera
Bt Bacillus thuringiensis




Genetically engineered plants

Plums that have been genetically engineered to be resistant to the plum pox virus
The intentional creation of transgenic plants by laboratory based recombinant DNA methods is more recent (from the mid-70s on[citation needed]) and has been a controversial development in the field of biotechnology opposed vigorously by many NGOs, and several governments, particularly in Europe.
These transgenic recombinant plants are transforming agriculture in those regions that have allowed farmers to adopt them, and the area sown to these crops has continued to grow globally in every years since their first introduction in 1996.
As of 2006 there were around 250 million acres of genetically engineered crops being grown commercially in 22 countries[citation needed]. The U.S. has adopted the technology most widely whereas Europe has almost no genetically engineered crops. The EU had a formal ban on GM crops, until it was overturned in 2006; in a controversial move GM crops are now regulated by the EU.Transgenic recombinant plants are generated in a laboratory by adding one or more genes to a plant's genome,and the techniques frequently called transformation. Transformation is usually achieved using gold particle bombardment or through the process of Horizontal gene transfer using a soil bacterium, Agrobacterium tumefaciens, carrying an engineered plasmid vector, or carrier of selected extra genes.
Transgenic recombinant plants are identified as a class of genetically modified organism(GMO); usually only transgenic plants created by direct DNA manipulation are given much attention in public discussions[opinion].
Transgenic plants have been deliberately developed for a variety of reasons: longer shelf life, disease resistance, herbicide resistance, pest resistance, non-biological stress resistances, (e.g. drought or nitrogen starvation), and nutritional improvement (see Golden rice) and frost tolerance (see Fish tomato).
The first modern recombinant crop approved for sale in the U.S., in 1994, was the FlavrSavr tomato, which had a longer shelf life. The first conventional transgenic cereal created by scientific breeders was actually a hybrid between wheat and rye (triticale) in 1876 (Wilson, 1876). The first transgenic cereal may have been wheat, which itself is a natural transgenic plant derived from at least three parental species.
Genetically modified organisms came before commercially viable crops as the FlavrSavr tomato, only strictly grown indoors (in laboratories). However, after the introduction of the Flavr Savr tomato, certain GMO-crops (e.g. GMO-soy, GMO-corn, etc.) in the US were being grown outdoors on large scales.
Commercial factors, including high regulatory and research costs, have so far restricted modern transgenic crop varieties to major traded commodity crops[citation needed]. Recently, R&D has targeted enhancement of crops that are locally important in developing countries, such as insect-resistant cow-pea for Africa and insect-resistant Brinjal eggplant for India.
Transgenic plants have been used for bioremediation of contaminated soils. Mercury, selenium and organic pollutants such as polychlorinated biphenyls (PCBs) have been removed from soils by transgenic plants containing genes for bacterial enzymes.




Wednesday, July 21, 2010

Golden rice



Golden rice burst on a world ready and eager for a new beginning.
Announced with a flourish in January 2000, it promised to save millions of people from blindness and disease.
It can certainly help to improve nutrition and health in many developing countries.
But, as the publicists' dust settled, it became clear that golden rice was never going to be a silver bullet.
It is a genetically modified (GM) strain of rice that has been engineered to produce beta-carotene.
That not only gives it its eponymous golden colour, but enables people eating it to produce vitamin A.
The World Health Organisation estimates about 250 million people globally are deficient in vitamin A, increasing their risk of blindness, immune problems and other serious conditions.
Improving on Nature
So golden rice sounded like a real answer to a genuine problem, especially since the biotech company responsible, Zeneca, said it would offer the seeds freely to farmers in poor countries.
The reality, though, appears a little more prosaic. For a start, the genes for beta-carotene are already present in conventional rice.
It is just that they do not work as well in the "natural" varieties as in the novel version.
Beyond that though, poorly-fed people are unlikely to be able to absorb beta-carotene even when they eat golden rice. To use it, they need a diverse diet, including green leafy vegetables.
But the sorts of vegetables people used to be able to find have declined in number as the green revolution of the 60s and 70s emphasised monocultures of new varieties.
Household consumption of vegetables in India has fallen by 12% in two decades.
The prospects for golden rice receded a little further in 2002, when scientists published the draft sequences of the rice genome.
Short cut
That promised quicker results from conventional plant breeding, partly because it established where the beta-carotene "pathway" sat in the rice code.
A scientist from the biotech company Syngenta, which now includes Zeneca, said: "All the genes are present in rice. One could make a non-GM vitamin-A rice simply by studying those genes in a more focused way."
Golden rice may prove part of the answer to vitamin A deficiency, though not the comprehensive solution it seemed to be.
But it would be an answer that came with a hefty price-tag: the persistent concerns about the safety of GM technology to human health and to wild species.
Golden rice looks like being a special case, anyway, because the biotech industry is unlikely to give poor farmers free access to all its inventions.
People who campaign against GM crops are sometimes accused of wanting to deny the wretched of the Earth the chance to escape poverty and disease, all in the name of their own ideological obsession. But some impressive figures echo their concerns.
False dawn
Dr Richard Horton, editor of the British medical journal The Lancet, said: "Seeking a technological food fix for world hunger may be... the most commercially malevolent wild goose chase of the new century."
And from the biotech industry itself, Steve Smith, who worked for Syngenta Seeds before his death in June 2003, said: "If anyone tells you that GM is going to feed the world, tell them that it is not... To feed the world takes political and financial will - it's not about production and distribution."
Every day 800 million people go to bed with empty stomachs. Every day more than 30,000 under-fives die, from easily prevented diseases or from hunger.
The world is out of joint, and it will stay that way until those of us who are well-fed care enough to wage a war on hunger as ferocious as that against terrorism.
Science, perhaps including GM technology, can provide the weapons for that war - but that won't ever be a silver bullet.
** NOTICE: In accordance with Title 17 U.S.C. Section 107, this material is distributed for research and educational purposes only

The Biopharmaceuticals


FDA-Approved Biopharmaceutical Drugs and Vaccines

The following biopharmaceutical agents have been approved by the Food and Drug Administration for marketing in the United States. The trademarked name is followed by the generic name of the drug in parentheses.
Acctimmune (gamma interferon)
Genentech, Inc.Approved for use in the management of chronic granulomatous disease (December, 1990).
Activase (recombinant alteplase)
Genentech, Inc.Approved for use in the treatment of myocardial infarction (November, 1987) and acute pulmonary embolism (June, 1990).
Adagen (adenosine deaminase)
Enzon, Inc.Approved for use in the treatment of infants and children with severe immunodeficiency (March, 1990).
Alferon N
Interferon Sciences, Inc.Approved for use in treating genital warts (October, 1989).
Betaseron (recombinant interferon beta 1-B)
Berlex Laboratories/Chiron Corp.Approved for use in the management of relapsing, remitting multiple sclerosis (August, 1993).
Ceredase (alglucerase)
Genzyme Corp.Approved for use in treating Type 1 Gaucher's disease (April, 1991).
Cerezyme (imiglucerase)
Genzyme Corp.Approved for use in treating Type 1 Gaucher's disease (June, 1994).
Engerix-B
SmithKline BeechamApproved for use as a hepatitis B vaccine (September, 1989).
EPOGEN (epoetin alfa)
Amgen Ltd.Approved for use in treating anemia associated with chronic renal failure and anemia in Retrovir-treated, HIV-infected patients (June, 1989).
Humatrope (somatropin)
Eli Lilly & Co.Approved for treating human growth hormone deficiency in children (March, 1987).
Humulin (recombinant human insulin)
Eli Lilly & Co.Approved for the treatment of diabetes (October, 1982).
Intron A (alpha-interferon)
Schering-Plough Corp.Approved for the treatment of hairy cell leukemia (June, 1986), genital warts (June, 1988), AIDS-related Kaposi's sarcoma (November, 1988), non-A, non-B hepatitis (February, 1991), and hepatitis B (July, 1992).
KoGENate (antihemophiliac factor)
Miles, Inc.Approved for use in the treatment of hemophilia A (February, 1993).
Leukine (yeast-derived GM-CSF)
Immunex Corp.Approved for use in autologous bone marrow transplantation (March, 1991).
Neupogen
Amgen Ltd.Approved for use in treating cheomtherapy-induced neutropenia (February, 1991) and bone marrow transplant-associated neutropenia (June, 1994).
Oncaspar (pegaspargase)
Enzone/Rhone-Poulenc RorerApproved for use in treating acute lymphoblastic leukemia (February, 1994).
Orthoclone OKT 3
Ortho BiotechApproved for use in reversal of acute kidney transplant rejection (June, 1986).
Procrit (epoetin alfa)
Ortho BiotechApproved for use in the treatment of anemia associated with chronic renal failure (December, 1990) and anemia in Retrovir-treated, HIV-infected patients and chemotherapy-associated anemia (April, 1993).
Proleukin, IL-2
Chiron Corp.Approved for the treatment of kidney (renal) carcinoma (May, 1992).
Protropin (somatrem)
Genentech, Inc.Approved for treating human growth hormone deficiency in children (May, 1985).
Pulmozyme (DNase)
Genentech, Inc.Approved for use in the management of cystic fibrosis (December, 1993).
Recombinate rAHF (recombinant antihemophiliac factor)
Baxter HealthcareRecombinate rAHF is the recombinant version of blood clotting factor VIII, and it has been approved for the treatment of hemophilia A (December, 1992).
Recombivax HB
Merck & Co.Approved as a hepatitis B prevention vaccine (July, 1986).
Roferon-A (recombinant alfa-interferon)
Hoffman-La RocheApproved for the treatment of hairy cell leukemia (June, 1986) and AIDS-related Kaposi's sarcoma (November, 1988

drug metabolism & pharmacokinetics



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In Vitro ADMET
In vitro ADMET parameters are a set of factors that describe how a drug behaves in the human body and can be a major cause of drug failure. By focusing on in vitro ADMET data you can predict at an early stage which compounds not only possess good binding affinity for a specific target, but also pass the test for good bioavailability and safety. Our wide range of automated in vitro assays yields information in the areas of metabolism, toxicity, and physicochemical characteristics.
In vitro screening assays:
Aqueous solubility
Cytochrome P450 enzyme inhibition
Cytochrome P450 induction
Cytochrome P450 reaction phenotyping
Cell permeability and efflux assays (CaCo-2)
Cell proliferation and cytotoxicity
Drug-drug interaction
Enzyme assays
hERG inhibition
Melanin binding
Metabolic plasma and buffer stability
Metabolite profiling
Metabolite assessment and identification
Plasma protein binding

In Vivo Pharmacokinetic/ADME Screening
Whether you have one compound or a library of compounds requiring drug metabolism assessment, the skilled scientists at Charles River can design, conduct, and interpret the in vivo
pharmacokinetic/ADME components of your program as part of our streamlined non-GLP screening services. Quick study initiation, multiple species, resident animal colonies with chronic surgical models included, and various dose regimens and administration routes plus bioanalytical support provide the necessary tools you need to rapidly identify and optimize potential drug candidates.
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Applications of biotechnology in the modern world


The scientific knowledge which is applied to practical ways in industry for the benefit of human beings is called as technology. The biological sciences have recently passed through more advanced technologies in different spheres of life and activities. One of those advanced technologies is the Bio- technology.The applications of advances made in the techniques and instrumentations in research in biological sciences are called as Bio- technology. Some people define bio- technology as – the manipulation of organisms to make products that benefit human beings.

The characteristics of a living organism are determined by some physical units called as genes that are arranged on chromosomes found inside the nucleus of a cell. A gene is made of molecules of deoxyribonucleic acid (DNA), which is called as the Genetic Blue Print of each living cell. The characteristics and structure of DNA was studied by Rosalind Franklin, Francis Crick, James Watson and Maurice Wilkins (1953), for which Crick, Watson and Wilkins were awarded Nobel Prize for the year 1962, though the contribution of Rosalind Franklin remained un-noticed and she died four years earlier to the award of the Nobel Prize to these workers. Later, Dr. H.G. Khorana, R.H. Holley and M.W. Nirenberg were awarded Nobel Prize for the year 1968, for their pioneering works related to the synthesis of gene. These studies of gene and DNA formed the basis if development of bio- technological studies in India and abroad. The presentation of double helical model of DNA and discoveries pertaining to gene, mark the beginning of the modern era of Bio- technology.

Rosalind Elsie Franklin (1920- 1958)The Pioneer of the discovery of DNA

Helical Structure of DNA Molecule

Double Helix of DNA
Biotechnology: its origin and development
The pioneering achievement in the field of Bio- technology was the production of greater genetic varieties of plants and animals through the techniques of Hybridization in 5000BC. It was through hybridization that present day High Yielding Varieties of plants and animals were evolved. This technique is being continuously used in improved forms in our programmes concerned with food production. Norman E. Borlaug, the American Agriculturist, received the Nobel Prize for the year 1970 for his important contribution of increasing the world food supply through different techniques like hybridization, Selective Breeding and Gene Transfer. Hybridization is the technology or process of crossing individuals of opposite sex of different species so as to produce off springs of combined character. The Indian Scientist Dr. M.S.Swaminathan (1967) initiated and developed technologies for increasing food production in India. Dr.Swaminathan is now being called as the Father of Economic Ecology.
In 1960, Werner Arber discovered specific enzymes called as Restriction Enzyme in bacteria. These enzymes can cut the DNA strand of an organism at a precise point. Further, in 1973, Stanley Cohen and Herbert Boyer became successful in removing a specific gene from a bacterium and inserting it into another bacterium by the application of the restriction enzyme. This discovery led to the development of the Recombinant DNA Technology which is commonly called as Genetic Engineering.
Another major achievement in the field of bio- technological development was the manipulation of bacteria to produce Human Protein (1977). Afterwards, the American Molecular Biologist Lydia Villa- Komaroff developed basic bio- technology for the production of Rat Insulin with the help of genetically engineered bacteria. By using genetically engineered bacteria, scientists became successful in producing Human Insulin in 1978 and within a period of 5 years the Human Insulin became the first biopharmaceutical product in the market to control Diabetes Mellitus.
Scope of Bio- Technology
Bio- technology has scope in the fields of Agriculture and Animal Husbandry, Molecular Medicine, Forensic Science, Microbial Genetics, Environmental Sciences etc.
Agriculture and Animal Husbandry
The scope of bio- technological applications in Agriculture and Animal Husbandry is concerned with- the development of disease and pest resistant varieties of plants and animals; the production of food containing high nutrient value; the production of vaccines and the development of drought resistant crop varieties.
Molecular Medicine
This field of Bio- technology incorporates- disease diagnosis through cultural and different biotechnological methods and through Gene Therapy (cure of diseases through improvement and modification of genes.
Forensic Science
This science relates to the identification of criminals and researches pertaining to crime. This field of bio- technology incorporates- the DNA- matching tests; the establishment of genetic relationships; DNA-mapping; DNA- Fingerprinting and Genetic Analysis etc.
Microbial Genetics
This field of bio- technology incorporates- Study of microorganisms and their genetic make up; the study of genetic behavior and possibilities of genetic alterations; recombination etc.The microbial Genetics is also concerned with the study of bio- weapons; bio-wars and bio- fuels.
Environmental Science
This field of bio- technology incorporates- studies pertaining to the detection of microorganisms causing health hazards; environmental pollutions and the monitoring of environmental problems concerned with the living world.
Applications of biotechnology
The application of bio- technology can be categorized as Specific Applications and General Applications.
(a) Genetic Engineering: The experiments of Stanley Cohen and Herbert Boyer (1973) pertaining to the removal of specific gene from a bacterium and the insertion of the same gene into another bacterium, marks the beginning of the Recombinant DNA- technology which is also called as the Genetic Engineering. In their experiments, the “restriction enzymes” were used for cutting the segments of DNA. The Enzymes are specific proteins which can speed up biochemical processes without being altered in the chemical reactions.
The science of changing the pattern of development and behavior of an organism by making alterations in its genes is called as genetic engineering. The organisms produced by altering their genetic make up through DNA recombinant technology or through genetic engineering are called as Genetically Modified Organisms or GMOs. In agriculture, GM-crops are also being produced through genetic engineering. The food produced from genetically modified crops is called as GM- food.
The genetically engineered organism in which a foreign gene has been introduced to obtain desired results is called as a Transgenic Organism. Such an organism may be a plant, an animal, bacteria or fungi. These organisms are developed by injecting a foreign gene into the embryo or the fertilized egg of an organism.
Importance of genetic engineering
. Through genetic engineering the alterations in the genetic make up of an organism can be done successfully,
. Genetic engineering can be employed in increasing food production from plants and animals,
. It is applied in the diagnosis of diseases and improvements in the medical treatment.
(b) Plant Tissue Culture: The technique of growing plant tissues on artificial nutrient medium under laboratory conditions, so as to produce new plants, is called as plant tissue culture. These may be the Meristem Culture, Embryo Culture and Anther Culture and so on.
(c) Monoclonal Antibody: The specialized types of protein molecules produced in the laboratory are called as monoclonal antibodies. These are produced naturally in our bodies when any bacteria or virus invades it. Monoclonal antibodies are produced in our blood and protect us from different types pf diseases. These bodies are used in identifying different types of cells. These are also employed in many diagnostic tests for bacteria and viruses. The experiments on using monoclonal antibodies for fighting against cancer are also going on across the world.
The genetic engineers have engineered the bacterial species E. coli to synthesize a specific protein called as Interferon. These proteins are produced naturally by body cells to oppose viral infections. The biologically synthesized interferon has been tested and found successful.
(d) Synthesis of Enzymes: The Proteinaceous chemical substances of biological origin that accelerate biochemical reactions without undergoing any change are called as enzymes. The term “enzyme” was coined by William Kuhne in 1867 on the basis of his studies on yeast.
Specific enzymes can be synthesized through the application of bio- technology. These enzymes are used in various processes like removal of stains, softening of fabrics, preparation of digestible foods, processing of meat and, even the treatment of cancer.
(e). Synthesis of Biodegradable Plastics: Bio- technology is currently employed in the synthesis of plastic which is biodegradable i.e. unlike other plastics; this plastic can be broken down into simpler substances by microorganisms.
The biodegradable plastic is made from lactic acid which is produced at the time of bacterial fermentation of plant materials like discarded stalks of corn. In the process, molecules of Lactic Acid are chemically grouped to form the biodegradable plastic. In fact, the biodegradable plastic is a material which has most of the properties of plastic except the property of being non- biodegradable.
(f) Blood Substitute: Today the number of people needing blood transfusion is increasing due to frequent accidents and diseases. In view of these facts, biotechnologists are trying to synthesize artificial blood through bio- technology. The biotechnologists like Mary L. Nucci and Abraham Abuchowski (1998) are likely to get success in their experiments in this regard.
(g). Bio- technology and Mining: Different types of bacteria are being currently employed in the extraction of different metals like copper, zinc, lead and other metals. These bacteria act on the metallic compounds available inside the earth and help in the isolation of respective metals.
(h).Bio- technology and Blood Clotting Protein: Bio- technology has many types of dramatic applications in the field of medicine. It has produced Factor- VIII (1986), which is a blood clotting protein and which is not produced in haemophilic persons. Under these conditions the haemophilic people are always at the risk of bleeding to death. In the process of synthesis of Factor VIII, the human genes having codes of production of blood clotting protein are transplanted into the haemophilic person. The factor VIII is being produced commercially since 1992.
(i). Cloning: The bio- technology of production of cells or organisms that are originally derived from a single original cell or organism by asexual method under laboratory conditions is called as cloning. The copies of organisms produced during cloning have identical genetic make up and are known as clones. We can define a clone as an individual cell or organism which has been grown by a single body cell and which is genetically identical to its parent cell.
During 1950s, scientists cloned frogs and by 1980s they cloned mice. During 1996, Ian Wilmot and his team of researchers achieved success for the first time in cloning an adult sheep. They named the clone as Dolly. Scientists at Texas A & M University in College Station produced the first cloned cat on Dec. 22, 2001 through the Nuclear Transfer Technique. They named the clone – cc or the carbon copy. It is hoped that scientists may one day become successful in cloning extinct animals also.
(j).Applications in food and beverage industry: A number of food products are produced on industrial scale through the fermentation technology. Some of those products are wine, idlis, yoghurt, cheese, mycoproteins, quorn, bread etc. Besides this bio- technology helps in the production of different vitamins, amino acids and vinegar etc.
(K). Application of Bio- technology in Pharmaceuticals: Modification of microorganisms, animals and plants so as to get maximum yield of medicinally useful substances is called as pharmaceutical bio- technology. Some important examples are being given below-
(I) Production of human insulin from non- human sources.
(ii) Production of hormones like Interferon, Cytokinins, Steroids and human growth hormones.
(iii)Gene-therapy for prevention and control of diseases.
(iv) Development of vaccines and antibodies.
(l)Application of bio- technology in Agriculture: Tissue culture, cloning and hybridization are important bio- technologies that are being developed in agriculture today. Tissue culture is important for the propagation of high yielding varieties of plants for agriculture and floriculture. Hybridization is the technique of combining properties of two plants or animals to produce one better hybrid plant or animal. Though this process frequently occurs in nature, the adoption of this technology has supported the agriculture to produce more food to feed growing population in many countries. Similarly, hybridization technology has supported animal husbandry to produce more milk and meat.
The application of bio- technology in agriculture can make it more sustainable. The introduction of bio-fertilizers in soil can improve it’s composition besides making it fertile in a natural way. Similarly, the introduction of bio-pesticides can control pests through natural ways without contaminating the natural environment. The development of disease resistant and pest resistant crop varieties through bio- technological methods has further supported the agriculture.
Scientists are continuously experimenting for the improvement of crop plants like potato, tomato, cabbage and other vegetables and fruits as well as other crop plants like sugarcane, wheat, maize etc. to obtain better yield. Some new crop varieties like Triticale (a man made cereal) have also been developed to add nutrients in our food and increase food production.
(m) Application of bio- technology in Pollution Control: The natural tendency of microorganisms can be exploited through bio- technology for solving the problem of wastes in the environment. The crop residues and animal waste are used in making manure by the activity of aerobic and anaerobic bacteria. The bacterial species Pseudomonas aeruginosa has been developed to eat away the oil spilled on the sea surface. Solid wastes like crop residues can be put inside a biogas digester to produce biogas, a most suitable source of rural energy.
In nature, green plants control atmospheric carbon dioxide by utilizing it in photosynthesis. Hence, plantation of more and more fast growing trees may be an important bio- technological method of controlling carbon dioxide level of atmosphere. Plants fix carbon dioxide by the help of specific enzyme Ribulose- bi- phosphate Carboxilase. The function of this enzyme is controlled genetically. Hence, scientists are trying to manipulate this enzyme to increase the rate of photosynthesis for greater reduction of atmospheric carbon dioxide level.
(n) Application of bio- technology in Waste Water Treatment: The treatment of waste water comprises three major steps- the Primary Treatment, the Secondary Treatment and the Tertiary Treatment.
The primary treatment comprises many sub- steps like sedimentation, chemical coagulation and precipitation. These sub- steps remove most of the physical impurities or pollutants. The secondary treatment comprises biological process involving bio- technology of employing bacteria, fungi, algae etc. for the breaking down of complex pollutants. In this process, the effluent is passed through a microbial slime layer. The microbes present in this layer break down the organic and nitrogenous waste liberating carbon dioxide and nitrogen dioxide.
(o) Application of bio- technology in the degradation of pesticides: Different species of bacteria and fungi tend to degrade pesticides. These microorganisms can be genetically manipulated to degrade more and more of them. Some species of bacteria like Pseudomonas sp., Flavobacterium, Azotobacter, E. coli and Acromobacter tend to degrade different pesticides. It has been reported that a mixture of Phenerocheate (a fungus) and enzyme peroxidase in suitable proportion can degrade DDT.
(p) Bioremediation: The application of biotic agents like microorganisms in the correction and recovery of environmental damage is called as bioremediation. The removal of oil spilled on sea water by the help of bacteria is one example of bioremediation.
(q) Industrial Applications of bio- technology: Bio- technology is currently being applied in many areas of industry like the production of stain remover, detergents, bread, biotech-polyester, vitamins; stone washed jeans, bleached paper etc. Here are some examples-
(i) Detergents containing protease enzyme can remove stains of proteinaceous nature both on fabrics and lenses,
(ii) Detergents containing lipase enzyme can remove stains of oil and grease, and those containing amylase can remove starch grains stuck with fabrics.Now a day, polyesters are being synthesized from corn starch feed stock through the application of bio- technology. The enzyme cellulose is used for fading of jeans.
Key Words : Recombinant DNA Technology,deoxyribonucleic acid,Bio- technology,Dr. H.G. Khorana, R.H. Holley and M.W. Nirenberg ,Hybridization ,Ribulose- bi- phosphate Carboxilase,Nucci and Abraham Abuchowski,agriculture,Molecular Medicine, Forensic Science, Microbial Genetics, Environmental Sciences ,Detergents, pesticides, Human Insulin