Leading vs. Lagging strand replication

Leading strand 
 The leading strand is the template strand of the DNA double helix so that the replication fork moves along it in the 3' to 5' direction. This allows the newly synthesized strand complementary to the original strand to be synthesized 5' to 3' in the same direction as the movement of the replication fork. On the leading strand, a polymerase "reads" the DNA and adds nucleotides to it continuously. This polymerase is DNA polymerase III (DNA Pol III) in prokaryotes and presumably Pol ε in yeasts. In human cells the leading and lagging strands are synthesized by Pol α and Pol δ within the nucleus and Pol γ in the mitochondria. Pol ε can substitute for Pol δ in special circumstances.


Lagging strand The lagging strand is the strand of the template DNA double helix that is oriented so that the replication fork moves along it in a 5' to 3' manner. Because of its orientation, opposite to the working orientation of DNA polymerase III, which moves on a template in a 3' to 5' manner, replication of the lagging strand is more complicated than that of the leading strand. On the lagging strand, primase "reads" the DNA and adds RNA to it in short, separated segments. In eukaryotes, primase is intrinsic to Pol α. DNA polymerase III or Pol δ lengthens the primed segments, forming Okazaki fragments. Primer removal in eukaryotes is also performed by Pol δ.[18] In prokaryotes, DNA polymerase I "reads" the fragments, removes the RNA using its flap endonuclease domain (RNA primers are removed by 5'-3' exonuclease activity of polymerase I [weaver, 2005], and replaces the RNA nucleotides with DNA nucleotides (this is necessary because RNA and DNA use slightly different kinds of nucleotides). DNA ligase joins the fragments together.

Avery–MacLeod–McCarty experiment

The Avery–MacLeod–McCarty experiment was an experimental demonstration, reported in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty, that DNA is the substance that causes bacterial transformation. It was the culmination of research in the 1930s and early 1940s at the Rockefeller Institute for Medical Research to purify and characterize the "transforming principle" responsible for the transformation phenomenon first described in Griffith's experiment of 1928: killed Streptococcus pneumoniae of the virulent strain type III-S, when injected along with living but non-virulent type II-R pneumococci, resulted in a deadly infection of type III-S pneumococci. In their paper "Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types: Induction of Transformation by a Deoxyribonucleic Acid Fraction Isolated from Pneumococcus Type III", published in the February 1944 issue of the Journal of Experimental Medicine, Avery and his colleagues suggest that DNA, rather than protein as widely believed at the time, may be the hereditary material of bacteria, and could be analogous to genes and/or viruses in higher organisms.

Nanomedicine Animation



Nanomedicine is the medical application of nanotechnology. It covers areas such as nanoparticle drug delivery and possible future applications of molecular nanotechnology (MNT) and nanovaccinology. Current problems for nanomedicine involve understanding the issues related to toxicity and environmental impact of nanoscale materials

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Nanomedicine research is directly funded, with the US National Institutes of Health in 2005 funding a five-year plan to set up four nanomedicine centers. In April 2006, the journal Nature Materials estimated that 130 nanotech-based drugs and delivery systems were being developed worldwide

In the near future, advancement in nanomedicine will deliver a valuable set of research tools and clinically helpful devices. The National Nanotechnology Initiative expects new commercial applications in the pharmaceutical industry that will include advanced drug delivery systems, new therapies, and in vivo imaging. The most important innovations are taking place in drug delivery which involves developing nanoscale particles or molecules to improve bioavailability. Bioavailability refers to the presence of drug molecules where they are needed in the body and where they will do the most good. Drug delivery focuses on maximizing bioavailability both at specific places in the body and over a period of time. Over 65 billion dollars is wasted every year because of poor bioavailability. In vivo imaging is another area where tools and devices are being developed. Using nanoparticle contrast agents, images such as ultrasound and MRI have a favorable distribution and improved contrast. The new therapies and surgeries that are being developed might be effective in treating illnesses and diseases such as cancer. Finally, a shift from the possible to the potential will be made when nanorobots such as neuro-electronic interfaces and cell repair machines are discussed.


Drug delivery systems, lipid- or polymer-based nanoparticles, can be designed to improve the pharmacological and therapeutic properties of drugs. The strength of drug delivery systems is their ability to alter the pharmacokinetics and biodistribution of the drug. Nanoparticles have unusual properties that can be used to improve drug delivery. Where larger particles would have been cleared from the body, cells take up these nanoparticles because of their size. Complex drug delivery mechanisms are being developed, including the ability to get drugs through cell walls and into cells. Efficiency is important because many diseases depend upon processes within the cell and can only be impeded by drugs that make their way into the cell. Triggered response is one way for drug molecules to be used more efficiently. Drugs are placed in the body and only activate on encountering a particular signal. For example, a drug with poor solubility will be replaced by a drug delivery system where both hydrophilic and hydrophobic environments exist, improving the solubility. Also, a drug may cause tissue damage, but with drug delivery, regulated drug release can eliminate the problem. If a drug is cleared too quickly from the body, this could force a patient to use high doses, but with drug delivery systems clearance can be reduced by altering the pharmacokinetics of the drug. Poor biodistribution is a problem that can affect normal tissues through widespread distribution, but the particulates from drug delivery systems lower the volume of distribution and reduce the effect on non-target tissue. Potential nanodrugs will work by very specific and well-understood mechanisms, one of the major impacts of nanotechnology and nanoscience will be in leading development of completely new drugs with more useful behavior and less side effects.


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Nanorobots

The somewhat speculative claims about the possibility of using nanorobots in medicine, advocates say, would totally change the world of medicine once it is realized. Nanomedicine would make use of these nanorobots, introduced into the body, to repair or detect damages and infections. A typical blood borne medical nanorobot would be between 0.5-3 micrometres in size, because that is the maximum size possible due to capillary passage requirement. Carbon would be the primary element used to build these nanorobots due to the inherent strength and other characteristics of some forms of carbon (diamond/fullerene composites). Cancer can be treated very effectively, according to nanomedicine advocates. Nanorobots could counter the problem of identifying and isolating cancer cells as they could be introduced into the blood stream. These nanorobots would search out cancer affected cells using certain molecular markers. Medical nanorobots would then destroy these cells, and only these cells. Nanomedicines could be a very helpful and hopeful therapy for patients, since current treatments like radiation therapy and chemotherapy often end up destroying more healthy cells than cancerous ones. From this point of view, it provides a non-depressed therapy for cancer patients. Nanorobots could also be useful in treating vascular disease, physical trauma , and even biological

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Neuro-electronic Interfaces

Neuro-electronic interfaces are a visionary goal dealing with the construction of nanodevices that will permit computers to be joined and linked to the nervous system. This idea requires the building of a molecular structure that will permit control and detection of nerve impulses by an external computer. The computers will be able to interpret, register, and respond to signals the body gives off when it feels sensations. The demand for such structures is huge because many diseases involve the decay of the nervous system (ALS and multiple sclerosis). Also, many injuries and accidents may impair the nervous system resulting in dysfunctional systems and paraplegia. If computers could control the nervous system through neuro-electronic interface, problems that impair the system could be controlled so that effects of diseases and injuries could be overcome. Two considerations must be made when selecting the power source for such applications. They are refuelable and nonrefuelable strategies. A refuelable strategy implies energy is refilled continuously or periodically with external sonic, chemical, tethered, or electrical sources. A nonrefuelable strategy implies that all power is drawn from internal energy storage which would stop when all energy is drained.

One limitation to this innovation is the fact that electrical interference is a possibility. Electric fields, EMP pulses, and stray fields from other in vivo electrical devices can all cause interference. Also, thick insulators are required to prevent electron leakage, and if high conductivity of the in vivo medium occurs there is a risk of sudden power loss and “shorting out.” Finally, thick wires are also needed to conduct substantial power levels without overheating. Little practical progress has been made even though research is happening. The wiring of the structure is extremely difficult because they must be positioned precisely in the nervous system so that it is able to monitor and respond to nervous signals. The structures that will provide the interface must also be compatible with the body’s immune system so that they will remain unaffected in the body for a long time. In addition, the structures must also sense ionic currents and be able to cause currents to flow backward. While the potential for these structures is amazing, there is no timetable for when they will be available.

Cell repair machines

Using drugs and surgery, doctors can only encourage tissues to repair themselves. With molecular machines, there will be more direct repairs. Cell repair will utilize the same tasks that living systems already prove possible. Access to cells is possible because biologists can stick needles into cells without killing them. Thus, molecular machines are capable of entering the cell. Also, all specific biochemical interactions show that molecular systems can recognize other molecules by touch, build or rebuild every molecule in a cell, and can disassemble damaged molecules. Finally, cells that replicate prove that molecular systems can assemble every system found in a cell. Therefore, since nature has demonstrated the basic operations needed to perform molecular-level cell repair, in the future, nanomachine based systems will be built that are able to enter cells, sense differences from healthy ones and make modifications to the structure.


The possibilities of these cell repair machines are impressive. Comparable to the size of viruses or bacteria, their compact parts will allow them to be more complex. The early machines will be specialized. As they open and close cell membranes or travel through tissue and enter cells and viruses, machines will only be able to correct a single molecular disorder like DNA damage or enzyme deficiency. Later, cell repair machines will be programmed with more abilities with the help of advanced AI systems.

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Nanocomputers will be needed to guide these machines. These computers will direct machines to examine, take apart, and rebuild damaged molecular structures. Repair machines will be able to repair whole cells by working structure by structure. Then by working cell by cell and tissue by tissue, whole organs can be repaired. Finally, by working organ by organ, health is restored to the body. Cells damaged to the point of inactivity can be repaired because of the ability of molecular machines to build cells from scratch. Therefore, cell repair machines will free medicine from reliance on self repair.

A new wave of technology and medicine is being created and its impact on the world is going to be monumental. From the possible applications such as drug delivery and in vivo imaging to the potential machines of the future, advancements in nanomedicine are being made every day. It will not be long for the 10 billion dollar industry to explode into a 100 billion or 1 trillion dollar industry, and drug delivery, in vivo imaging and therapy is just the beginning.


Source:Wikepedia

Function of the Neuromuscular Junction

A neuromuscular junction (NMJ) is the synapse or junction of the axon terminal of a motoneuron with the motor end plate, the highly-excitable region of muscle fiber plasma membrane responsible for initiation of action potentials across the muscle's surface, ultimately causing the muscle to contract. In vertebrates, the signal passes through the neuromuscular junction via the neurotransmitter acetylcholine.
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Anatomy
When a motor neuron enters a muscle, it loses its myelin sheath and splits into many terminal branches. Motor neuron (efferent) axons originating in the spinal cord enter muscle fibers, where they split into many unmyelinated branches. These terminal fibers run along the myocytes to end at the neuromuscular junction, which occupies a depression in the sarcolemma. Each motor neuron can innervate from one to over 2000 muscle fibers, but each muscle fiber receives inputs from only one motor neuron.

In the terminal bouton of the motor nerve, structures known as presynaptic active zones accumulate synaptic vesicles filled with the neurotransmitter acetylcholine.

On the muscle side of the junction, the muscle fiber is folded into grooves called postjunctional folds that mirror the presynaptic active zones, the spaces between the folds contain acetylcholine receptors.


The muscle surface is covered by the synaptic basal lamina. Postjunctional folds are characteristic of skeletal muscle, particularly in fast muscle fibers.
Mechanism of action
Upon the arrival of an action potential at the axon terminal, voltage-dependent calcium channels open and Ca2+ ions flow from the extracellular fluid into the motor neuron's cytosol. This influx of Ca2+ triggers excitation-contraction coupling, a biochemical cascade that causes neurotransmitter-containing vesicles to fuse to the motor neuron's cell membrane and release acetylcholine into the synaptic cleft.

Acetylcholine diffuses across the synaptic cleft and binds to the nicotinic acetylcholine receptors that dot the motor end plate.

The receptors are ligand-gated ion channels, and when bound by acetylcholine, they open, allowing sodium and potassium ions to flow in and out of the muscle's cytosol, respectively.

Because of the differences in electrochemical gradients across the plasma membrane, more sodium moves in than potassium out, producing a local depolarization of the motor end plate known as an end-plate potential (EPP).

This depolarization spreads across the surface of the muscle fiber into transverse tubules, eliciting the release of calcium from the sarcoplasmic reticulum, thus initiating muscle contraction.

The action of acetylcholine is terminated when the enzyme acetylcholinesterase degrades the neurotransmitter and the unhydrolysed neurotransmitter diffuses away.

HIV Replication Animation

Entry to the cell
HIV enters macrophages and CD4+ T cells by the adsorption of glycoproteins on its surface to receptors on the target cell followed by fusion of the viral envelope with the cell membrane and the release of the HIV capsid into the cell.
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Entry to the cell begins through interaction of the trimeric envelope complex (gp160 spike, discussed above) and both CD4 and a chemokine receptor (generally either CCR5 or CXCR4, but others are known to interact) on the cell surface. The gp160 spike contains binding domains for both CD4 and chemokine receptors. The first step in fusion involves the high-affinity attachment of the CD4 binding domains of gp120 to CD4. Once gp120 is bound with the CD4 protein, the envelope complex undergoes a structural change, exposing the chemokine binding domains of gp120 and allowing them to interact with the target chemokine receptor. This allows for a more stable two-pronged attachment, which allows the N-terminal fusion peptide gp41 to penetrate the cell membrane.Repeat sequences in gp41, HR1 and HR2 then interact, causing the collapse of the extracellular portion of gp41 into a hairpin. This loop structure brings the virus and cell membranes close together, allowing fusion of the membranes and subsequent entry of the viral capsid.
Once HIV has bound to the target cell, the HIV RNA and various enzymes, including reverse transcriptase, integrase, ribonuclease and protease, are injected into the cell. During the microtubule based transport to the nucleus, the viral single strand RNA genome is transcribed into double strand DNA, which is then integrated into a host chromosome.
HIV can infect dendritic cells (DCs) by this CD4-CCR5 route, but another route using mannose-specific C-type lectin receptors such as DC-SIGN can also be used. DCs are one of the first cells encountered by the virus during sexual transmission. They are currently thought to play an important role by transmitting HIV to T cells once the virus has been captured in the mucosa by DCs.
Replication and transcription
Once the viral capsid enters the cell, an enzyme called reverse transcriptase liberates the single-stranded (+)RNA from the attached viral proteins and copies it into a complementary DNA.This process of reverse transcription is extremely error-prone and it is during this step that mutations may occur. Such mutations may cause drug resistance. The reverse transcriptase then makes a complementary DNA strand to form a double-stranded viral DNA intermediate (vDNA). This vDNA is then transported into the cell nucleus. The integration of the viral DNA into the host cell's genome is carried out by another viral enzyme called integrase.
This integrated viral DNA may then lie dormant, in the latent stage of HIV infection. To actively produce the virus, certain cellular transcription factors need to be present, the most important of which is NF-κB (NF kappa B), which is upregulated when T cells become activated. This means that those cells most likely to be killed by HIV are those currently fighting infection.
Rev-mediated HIV mRNA transport. Rev (red) binds the Rev response element (RRE, blue) to mediate export of unspliced and singly spliced mRNA from the nucleus to the cytoplasm.
In this replication process, the integrated provirus is copied to mRNA which is then spliced into smaller pieces. These small pieces produce the regulatory proteins Tat (which encourages new virus production) and Rev. As Rev accumulates it gradually starts to inhibit mRNA splicing. At this stage, the structural proteins Gag and Env are produced from the full-length mRNA. The full-length RNA is actually the virus genome; it binds to the Gag protein and is packaged into new virus particles.
HIV-1 and HIV-2 appear to package their RNA differently; HIV-1 will bind to any appropriate RNA whereas HIV-2 will preferentially bind to the mRNA which was used to create the Gag protein itself. This may mean that HIV-1 is better able to mutate (HIV-1 infection progresses to AIDS faster than HIV-2 infection and is responsible for the majority of global infections).
Assembly and release
The final step of the viral cycle, assembly of new HIV-1 virons, begins at the plasma membrane of the host cell. The Env polyprotein (gp160) goes through the endoplasmic reticulum and is transported to the Golgi complex where it is cleaved by protease and processed into the two HIV envelope glycoproteins gp41 and gp120. These are transported to the plasma membrane of the host cell where gp41 anchors the gp120 to the membrane of the infected cell. The Gag (p55) and Gag-Pol (p160) polyproteins also associate with the inner surface of the plasma membrane along with the HIV genomic RNA as the forming virion begins to bud from the host cell. Maturation either occurs in the forming bud or in the immature virion after it buds from the host cell. During maturation, HIV proteases cleave the polyproteins into individual functional HIV proteins and enzymes. The various structural components then assemble to produce a mature HIV virion. This cleavage step can be inhibited by protease inhibitors. The mature virus is then able to infect another cell.

Gleevecs Effects on tryosine Kinase

Imatinib is a drug used to treat certain types of cancer. It is currently marketed by Novartis as Gleevec (USA) or Glivec (Europe/Australia) as its mesylate salt, imatinib mesilate (INN). It was originally coded during development as CGP57148B or STI-571 (these terms are used in early preclinical publications). It is used in treating chronic myelogenous leukemia (CML), gastrointestinal stromal tumors (GISTs) and a number of other malignancies.
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The animation begins by introducing the Philadelphia Chromosome, the result of a reciprocal translocation between chromosomes 9 and 22. More specifically the breakpoint cluster region (BCR) of chromosome 22 is fused with part of the Abelson (ABL) gene on chromosome 9. The resulting BCR-ABL genetic domain now located within chromosome 22 and codes for a mutant tyrosine kinase also known as BCR-ABL. Under normal circumstances tyrosine kinase proteins respond to external cellular messaging proteins, and ultimately initiate a series of reactions that culminate in cellular replication.
Conversely, BCR-ABL is constitutively active, meaning it does not require activation by the aforementioned cellular messaging proteins in order to stimulate cellular replication. This results in acceleration of cell division, an inhibition of DNA repair, overall genomic instability, and the fatal blast crisis characteristic of chronic myelogenous leukemia. The animation progresses to introduce Gleevec (imatinib), the first in a class of drugs that specifically target and competively inhibit the ATP binding site on BCR-ABL tyrosine kinase. This prevents the ABL domain from phosphorylating the tyrosine residue, and as a result preventing the proliferation of hematopoietic cells that express BCR-ABL. Therapy with imatinib results in a dramatic reduction of tumor clone cells and the occurrence of blast crisis', through targeted drug treatment which leaves health cells unscathed.

Tissue Lysate Preparation (Denatured) Video

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