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Sickle Cell Anemia, Symptoms, Genetics, Disease, Diagnosis and Treatment
Posted by Harshil Sardhara
Sickle cell disease also called sickle cell anemia is a genetic disease, where red blood cells can take the shape of a crescent or sickle, and that change allows them to more easily be destroyed causing anemia among other things. Sickle cell disease is caused by defective hemoglobin, which is the oxygen carrying protein in red blood cells. Hemoglobin is actually made up of four peptide chains, each bond to a heme group.
Different hemoglobins have diffrent combination of these chains. Hemoglobin-A or Hb-A made up of two α-globin and two
β-globin peptide chains. This is the primary hemoglobin affected in the sickle cell. Specifically, the
β-globin chains end up misshapen. This is because of a mutation in the beta globin gene or Hb-B gene.
Sickle cell is an autosomal recessive disease so a mutation in both copies of the beta globin gene is needed to get the disease; if the person has just one copy of the mutation and one normal Hb-B gene, then they are a sickle cell carrier, also called sickle trait. Having sickle trait doesn't cause health problems unless the person is exposed to extreme conditions like high altitude or dehydration.
Where some sickle cell disease like symptoms can crop up what it does do is decrease the severity of infection by plasmodium falsiparum malaria, so in part of the world with a high malaria burden, like Africa and pockets of southern Asia, those with sickle trait actually have no evolutionary advantage. This phenomenon is called heterozygote advantage, and it's unfortunate consequence is a high rate of sickle cell disease in people from these part of the world.
Almost always sickle cell mutation is a non-conservative missense mutation that happens into the 6th amino acid of beta globin being a valine instead of glutamic acid. A non conservative substitution means that the new amino acid valine, which is hydrophobic has different properties than the one it replaced glutamic acid, which is hydrophilic.
A hemoglobin tetramer with two alpha-globin and two mutated beta-globin proteins is called sickle hemoglobin or Hb-S. Hb-S carries oxygen perfectly well, but when deoxygenated Hb-S change it's shape, which allows it to aggregate with other Hb-S proteins and form long polymers that distort the red blood cell into a crescent shape, a process called sickling. Conditions favorable for sickling include acidosis, which decreases hemoglobins affinity for oxygen and small low flow vessels where red blood cell's hemoglobin molecules have plenty of time to dump lots of oxygen molecules. Repeated sickling of red blood cell damages their cell membranes and promotes premature destruction, since this happens within the vasculature, its called intra vascular hemolysis.
Symptoms and sign
This destruction of red blood cells not only leads to anemia, which is a deficiency in red blood cells or loss of the normal levels of hemoglobin, but also means a lot of hemoglobin spilling out. Free hemoglobin in the plasma is bound by a molecule called haptoglobin and gets recycled; which is why a low haptoglobin levels is a sign of intro vascular hemolysis recycling of that heme group yields unconjugated bilirubin, which at high concentration can cause scleral icterus, jaundice and bilirubin gallstones.
To counter the anemia of sickle cell disease the bone marrow makes increased numbers of reticulocytes, which are immature red blood cells. This ends up causing new bone formation and the modulatory cavities of the skull can expand out ward, which causes enlarged cheeks and a 'hair-on-end' appearance on skull x-ray. Extra medullary hematopoiesis, which is red blood cell production cut side of the bone marrow can also happen, most often in the liver which can causes hepatomegaly.
In sickled from red blood cells tend to get stuck in capillaries, called vasco-occlusion. starting in infancy, they can clog up blood flow in bones of the hands and the feet, causing dactylitis, or swelling and pain of the digits. Later, they get stuck in other bone, causing a sickle cell pain crisis or a vascular necrosis of the bone. Red blood cells can also clog up the spleen, which can lead to an infarct of the spleen as well as an enormous back up of blood in the spleen called splenic sequestration, a life threatening complications.
Over time, splenic infarcts might can lead to an auto-splenectomy, where the spleen scars down and fibrosis to basically nothing. Having an absent or non functional spleen makes a person susceptible to encapsulated bacteria like streptococcus pneumonia, haemophilus influenza, Neisseria meningitis, and salmonella species, since encapsulated bacteria are normally opsonized and phagocytosed by macrophages in the spleen. It also leaves a person with Howell jolly bodies. Basophilic nuclear remnants in red blood cells, which can be seen on a peripheral blood cells, which can be seen on a peripheral blood smear. Sickled red blood cells can get stuck in the cerebral vasculature, causing vessel damage resulting in strokes.
Extensive sickle cell damage to the brain vessels may resulting Moya-Moya disease, which is named for the "puff-of-smoke". Collateral vessels that by pass blocked arteries they can also get stuck in the blood vessels of the lungs, leading to acute chest syndrome. This is particularly dangerous because it sets up a vicious cycle of preventing other red blood cells from getting oxygen, and this is made worse by the lung's natural tendency for hypoxia vasoconstriction, which is blood vessel.
Constriction in ares of the lungs that are lower in oxygen. In addition, clogging in the renal papillae can cause necrosis which can manifest as hematuria and proteinuria-blood and protein spilling out into take urine and in men, clogging the of the vasculature of the penis can cause priapism, a painful and prolonged erection.
Given all of these symptoms, it's important to diagnose sickle cell anemia. So it's included in the new born blood spot screen in some countries and can also be identified with a blood smear looking for sickle cells or by identifying Hb-S using protein electrophoresis.
Treatments of sickle cell anemia
The factor that cause red blood cell to sickle, which are hypoxia, dehydration and/or acidosis, can be improved with oxygen and fluids, which are mainstays of treatment. In addition, opioids are usually used to manage pain and antibiotics are used to treat any underlying bacterial infections causing acute chest syndrome. Occasionally blood transfusion are also used built the risk of multiple transfusions include iron overload and risk of developing new antibodies against future blood transfusion.
Finally, children with sickle cell typically get prophylaxis with penicillin and an additional polysaccharide vaccine against streptococcus pneumonia, to help prevent sepsis and meningitis with encapsulated bacteria, which works by increasing the amount of gamma globin, which results in more fetal hemoglobin (Hb-F). Fetal hemoglobin is made up of two alpha and two gamma globin chains, so it doesn't include the mutated beta globins.
Since it can't polymerase, it gets in the way of Hb-S polymers being made and prevents sickling. Hb-F is the primary hemoglobin at birth, which explains why sickle cell symptoms don't happen until a few months of life when adult hemoglobin starts to predominate, which contains the mutated beta globin.more rarely bone marrow transplants have been used in some patients and given that sickle cell involves a single point mutation Gene Therapy is another option being researched.
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AIDS : HIV virus invasion & Future treatment of HIV
A divers array of viruses occur among animal. A good way to gain a general idea of what they are like is to look at one animal virus in detail. Here we look at the virus responsible for a comparatively new and total viral disease. Acquired immuno-deficiency syndrome (AIDS). AIDS was first reported in the United States in 1981. It was not long before the infections agent, human immunodeficiency virus (HIV), was identified by laboratories in France and United States. Study of HIV revealed it to be closely related to a chimpanzee virus, suggesting that it might have been introduced to humans in central Africa from chimpanzees. Infected individuals have little resistance to infection, and nearly all of them eventually die of diseases that non-infected individuals easily ward off. Few who contract AIDS survive more than a few years untreated.
The risk of HIV transmission from an infected individual to a healthy one in the cause of day to day contact is essentially non existent. However, the transfer of body fluids, such as blood, semen, or vaginal fluid, or the use of nonsterile needles, between infected and healthy individuals poses a severe risk.
The incidence of AIDS is growing very rapidly in the United States. Over one million people were estimated to have been infected by HIV by the mid 1990s. Many perhaps all of them will eventually come down with AIDS. AIDS incidence is already very high in many African countries.
How HIV Compromises the Immune System
In normal individuals, an army of specialised cells patrots the blood stream, attacking and destroying any invading bacteria or viruses. In AIDS patients, this army of defenders is vanquished. One special kind of white blood cell, called a CD4+ T cell is required to cause the defending cells to action. In AIDS patients the virus homes in on CD4+ T cells, infecting and killing them until none are left. Without these crucial immune system cells, the body cannot mount a defense against invading bacteria or viruses. AIDS patients die of infections that a healthy person could fight off.
Clinical symptoms typically do not begin to develop until after a long latency period generally, 8 to 10 years after the initial inflation with HIV. During this long interval, carriers of HIV have no clinical symptoms but are apparently fully infectious, which makes the spread of HIV very difficult to control. The reason for the long latency period puzled researchers for some times. In some viral infections, such as Herpes, the DNA inserts itself into the host cells. Chromosome as a provirus, much as a bacteriophage does, where it remains inactive until some future event causes it to be removed from the chromosome and resume activity. This does not appear to be the case with HIV. Evidence indicates that the HIV infection cycle continues throughout the latent period, the immune system suppressing the ongoing infection. Eventually, however, a random mutational event in the virus allows it to quickly overcome the immune defense.
The HIV Infection Cycle
The way HIV infects humans provides a good example of how animal viruses replicate. Most other viral infections follow a similar course, although the details of entry and replication differ in individual cases.
When HIV is introduced into the human blood stream, the virus particle circulates throughout the entire body but will only infect CD4+ T-cells. Most other animal viruses are similarly narrow in their requirements. Polio goes only to certain spinal nerves, hepatities to the liver, and rabies to the brain. How does a virus such as HIV recognize a specific kind of Larget cell? Every kind of cell in the human body has specific array of cell surface glycoprotein markers at serve to identify them to other similar cells. Each HIV particle possesses a glycoprotein at precisely fits a cell surface marker protein called on its surface are infected CD4 on the surface of immune system cells called macrophages and T-cells. Macrophages are infected cells.
Entry into Macrophages
After docking onto the CD4 receptor of a macrophage, HIV requires a second macrophage receptor, called CCR5, to pull itself across the cell membrane. After gp 120 binds to CD4, it goes through conformational change that allows it to bind to CCRS. Investigators speculate that after the conformational change, the second receptor passes the gp 120-CD4 complex through the cell membrane, triggering passage of the contents of the HIV virus into the cell by endocytosis, with the cell membrane bolding inward to form a deep cavity around the virus.
Once inside the macrophage, the HIV particle sheds its protective coat. This leaves virus RNA floating in the cytoplasm, along with a virus enzyme that was also within the virus shell. This enzyme, called reverse transcriptase, synthesizes a double strand of DNA complementary to the virus RNA, often making mistakes and so creating new mutations. This double- stranded DNA directs the host cell machinery to produce many copies of the virus. HIV does not rupture and kill the macrophage cells it infects. Instead the new viruses are released from the cell by exocytosis. HIV synthesizes large numbers of viruses in this way, challenging the immune system over a period of years.
Entry into T Cells
During this time, HIV is constantly replicating and mutating. Eventually, by chance, HIV alters the gene for gp 120 in a way that causes the gp120 protein to change it's second receptor allegiance. This new form of gp 120 protein prefers to bind instead to a different second receptor, CXCR4, a receptor that occurs on the surface of T- lymphocyte CD4+ cells. Soon the body's T-lymphocytes become infected with HIV. This has deadly consequences, as new viruses exist the cell by rupturing the cell membrane, effectively killing the infected T-cell. Thus, the shift to the CXCR4 second receptor is followed swiftly by a steep drop in the number of T-cells. This destruction of the body's T-cell blocks the immune response and leads directly to the onset of AIDS, with cancers and opportunistic infections free to invade the defenseless body.
The Future of HIV Treatment
New discoveries of how HIV works continue to fuel research on devising ways to counter HIV. For example, scientists are testing drugs and vaccines that act on HIV receptors, researching the possibility of blocking CCR5, and looking for defects in the structures of HIV receptors in individuals that are infected with HIV but have not developed AIDS.
Combination Drug Therapy
A variety of drugs inhibit HIV in the test tube. These include AZT and its analogs (which inhibit virus nucleic acid replication) and protease inhibitors (which inhibit the cleavage of the large polyproteins encoded by gag, poll, and env genes into functional capsid, enzyme, and envelope segments).
When combinations of these drugs were administered to people with HIV in controlled studies, their condi- tion improved. A combination of a protease inhibitor and two AZT analog drugs entirely eliminated the HIV virus from many of the patients' bloodstreams. Importantly, all of these patients began to receive the drug therapy within three months of contracting the virus, before their bodies had an opportunity to develop tolerance to any one of them.Widespread use of this combination therapy has cut the US AIDS death rate by three-fourths since its introduction in the mid-1990s, from 43,000 AIDS deaths in 1995 to 31,000 in 1996, and just over 10,000 in 1999.
Unfortunately, this sort of combination therapy does not appear to actually succeed in eliminating HIV from the body. While the virus disappears from the blood- stream, traces of it can still be detected in lymph tissue ol the patients. When combination therapy is discontinued, virus levels in the bloodstream once again rise. Because of demanding therapy schedules and many side efects, long-term combination therapy does not seem a proms ing approach.
Using a defective HIV Gene to Develop Vaccines and Drug therapy
Recently, five people in Australia who are HIV- positive but have not developed AIDS in 14 years were found to have all received a blood transfusion from the samc HIV-positive person, who also has not developed AIDS. This led scientists to believe that the strain of virus transmitted to these people has some sort of genetic defect that prevents it from effectively disabling the human immune system. In subsequent research, a defect was found in one of the nine genes present in the AIDS virus.
This Gene is called nef, named for "negative factor", and the defective version of nef in the six Australians seems to be missing some pieces. Viruses with the defective gene may not be able to reproduce as much, allowing the immune system to keep the virus in check.
This finding has exciting implications for developing a vaccine against AIDS. Before this
Scientists have been unsuccessful in trying to produce a harmless strain of AIDS that can elicit effective immune response. The Australian strain with the defective nef gene has the potential to be used in a vaccine that would use the immune system against this and other strains of HIV.
Another potential application of this discovery is its use in developing drugs that inhibit HIV proteins that speed virus replication. It seems that the protein produced from the nef gene is one of these critical HIV proteins, Because viruses with defective forms of nef donot reproduce as scen in the cases of the six Australians. Research is currently underway to develop a drug that targets the nef Protein.
CHEMOKINES AND CAF
In the laboratory, chemicals called chemokines appear to inhibit HIV infection by binding to and blocking the CCRS and CXCR4 coreceptors. As one might expect, people long infected with the HIV virus who have not developed the disease prove to have high levels of chemokines in their blood.
The search for HIV inhibiting chemokies is intense. Not all results are promising. Rescarchers report that in tests, the levels of chemokines were not different between patients in which the discase was not progressing and those in which it was rapidly progressing. More promising, levels of another factor called CAF (CD8+ cell antiviral factor) are different between these two groups.
Researchers have not yet succeeded in isolating CAF, which seems not to block receptors that HIV uses to gain entry to cell, but, instead, to prevent replication of the virus once it has infected the cells. Research continues on the use of chemokines in treatments for HIV infection, either increasing the amount of chemokines or disabling the CCR5 receptor. However, promising research on CAF suggests that it may be an even better target for treatment and prevention of AIDS.
One problem with using chemokines as drugs is that they are also involved in the inflammatory response of the immune system. The function of chemokines is to attract white blood cells to arcas of infection. Chemokines work beautifully in small amounts and in local areas, but chemokines in mass numbers can cause an inflammatory response that is worse than the original infection. Injections of chemokines may hinder the immune system's ability to respond to local chemokines, or they may even trigger an out of control inflammatory response. Thus, scientists caution that injection of chemokines could make patients more susceptible to infections and they continue to research other methods of using chemokines to treat AIDS.
DISABLING
Scientists have found that a mutation in the gene that codes for the second receptor CCR5, consisting of a 32-base-pair deletion appears to block or inhibit HIV infection. Individuals homozygons for the mutated gene allele who have been exposed to HIV or are at high risk of HIV infection have not developed AIDS. Furthermore, individuals heterozygous for the mutated allele may have some protection and may develop the disease more slowly. In one study of 1955 people, scientist found no individuals who were infected and homozygous for the mutated allele. The allele seems to be more common in Caucasian populations (10% to 11%) than in African-American populations (2%) and absent in African and Asian populations.
Treatment for AIDS involving disruption of CCR5 looks promising, as research indicates that people live perfectly well without CCR5. Attempts to block or disable CCR5 are being sought in numerous laboratories.
A cure for AIDS is not yet in hand, but many new approaches look promising.
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Bacterial Spores - Property, Structure, Endospore formation and Germination
Spores are the dormant structures of prokaryotes. Endospores are the differentiated cells that are especially resistant to various environmental stresses and remain dormant. The bacterial spores develop within bacterial cell, so they are called endospores.
Bacterial genera that form endospores
Endospore is formed characteristically in gram-positive bacteria only. The genera that possess capability to form spores include-
Bacillus
Sporolactobacillus
Sporosarcina
Desulfatomaculum
Paeniabacdllus (Earlier this was member of genus Bacillus)
Thermoactinomyces (This actinomyces are endo-spore formers)
Properties of endospores :
Bacterial spores are the dormant structures, which can perform up to 100000 years. They do not perform any metabolic activity. Such a state of dormancy is known as Cryptobiosis.
Bacterial spores are extraordinarily resistant to environmental stresses like heat, radiations (ultraviolet rays. gamma rays), various chemical disinfectants, and desiccation.
Spores are highly resistant to staining process. This is mainly due to the impermeability of spore wall.
They are the refractile bodies, which cannot be stained easily by usual methods of staining.
Shapes and location of spore :
Bacterial spores are formed with in the cell. Hence, they are called endospores. Their shape and position within the cell is characteristic of the species by which It is formed
They can be central, sub terminal or terminal in their position within the cell.
Usually their size is same as that of cell size. But some bacteria produce spores having diameter greater than that of cell.
They may be round or oval in shape. having smooth or wrinkled surface.
Structure of endospore :
Detailed ultra structure of the endospore can be studied by transmission electron microscopy. The endospore has a very complex structure, consisting of -
Exosporium
Spore coat
Spore protoplast
Exosporium is a thin and delicate covering of spore. It is supposed to be originated from cell membrane during sporulation.
A layer located below the exosporium is called spore coat. It is thick and consists of several layers of proteins.
It is impermeable to toxic chemicals and thus account for resistance to chemicals.
It is believed to contain enzymes for germination.
Cortex is a structure below the spore coat. It forms more than half of the spore volume.
It contains relatively less cross-linked peptidoglycans.
It also possesses a high content of calcium dipicolonate (Ca-DPA).
They constitute about 15% of the cellular dry weight.
It is the core region of spore inside the cortex. It consists of core wall (spore wall), the cytoplasmic membrane, cytoplasm, ribosomes and nucleoid.
It is metabolically Inert.
The water content in spore protoplast exists as bound water. water complexed with protein.
Factors associated with resistance in endospore
Heat resistance of bacterial spores is attributed to various factors. They Include
Thick layer of spore coat and cortex.
High content of Ca-DPA present in cortex of spore
Presence of very little amount of free water in spore protoplast.
Endospore formation- Sporulation or sporogenesis
Sporogenesis is a complex process of differentiation that in several stages.
Sporulation results differentiation of vegetative bacterial cell in to altogether a different morphological structure, recognised as spore.
The process of sporogenesis is a genetically defined character and it involves about 200 genes. The proteins produced by these genes catalyse series of events concerned with sporogenesis.
The process of sporulation takes about ten hours.
Stages of endospore formation
Stage 1.
It Involves coalescence of replicated nucleoids. The a compact and elongate nuclear material forms structure which gets arranged as axial filament.
Now, the cell membrane invaginates or folds inward to form a septum. It divides the cell unequally, Smaller cell is called forespore, which contains cytoplasm and DNA.
It is the engulfment of forespore. In this, the extension of membrane takes place to cover the future endospore. It develops a second membrane.
It is stage of accumulation of cortex in the space between two membranes.
It is concerned with formation of protein coat around the cortex.
It is concerned with maturation of endospore. During this, spore coat is formed completely and there is an increase in refractivity.
It is the last stage of sporulation. In this stage, the endospore is released from the cell. The release is caused by action of several autolytic enzymes, which digest cell wall of sporulating bacterium.
Germination of Spore :
Transformation of dormant spore into vegetative cells called germination.
A group of scientists claimed in then report about the revival of 25 - 40 million years old spore from the gut of an extinct bee.
It suggests that spore well-designed structure that can remain dormant for hundreds of thousands of years.
Dormancy may be long, but return to vegetative stage is very rapid. It occurs in three stage.
Out growth
It is conditioning of the spores to germinate when nutrients are available.
It can be achieved by:
Heat treatment, at sub lethal temperatures in which spores are kept at 60 to 70°C for several minutes.
Storage of spore suspension at 4°C or at room temperature.
It is concerned with the breakdown of spore dormancy. Changes taking place during germination stage include .....
Swelling. rupture or absorption of spore coat.
Loss of calcium dipicolinate and cortex components.
Increase in stainability.
Loss of refractivity.
Loss of resistance to heat and other stresses.
Release of spore components.
Increase in metabolic activity.
Loss of calctum dipicolinate.
It can be triggered by metabolites or nutrients like sugars and amino acids.
It is concerned with emergence of cell from the spore.
It is associated with visible swelling due to water uptake. Spore protoplast makes new components like RNA. protein and DNA. It turns Into an active bacterium.
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What is gene therapy ?
When we introduced some concepts in biotechnology we briefly mentioned the notion of gene therapy and alluded to it's enormous potential for treating genetic disorders. This is a fascinating and promising area of study.
Image source: Wikipedia.Org
There are number of disorders that can be traced to a single defective gene. Some mutation has a risen, which alters the products of gene expression and the resulting protein does not perform it's function as intended. Which creates problems for that cell, and by extension the organism. But what if we could fix this gene? What is a normal allele could take the place of this defective one? That would necessarily solve the problem be done for every cell that possesses the mutation. It would solve the problem for the organism definitively curing the disease that is precisely.
What gene therapy seeks to do?
Take for example a type of severe combined immunodeficiency that causes bone marrow cells to be unable to produce a vital enzyme an issue which stems from single gene. Because bone marrow cells include stem cell that give rise to all the cells in the blood and immune system. This can be a huge problem, a solution to this is as follows. we can synthesize an RNA version of the normal allele for the gene of interest and insert it into a retrovirus.
Recall from our study of viruses that a retrovirus has the ability to generate a DNA transcript of it's RNA genome, which it then inserts into a host cell for replication. We then allow this retrovirus containing our cloned gene, to infect bone marrow cells that have been removed from the patient the virus is taken into these cells and Viral DNA containing the normal version of the gene of interest is inserted into the genome. These recombinant cells are then injected back into the bone marrow of the patient, and as these continually divide over an extended period of time, as bone marrow cells do more and more cells have the capacity to produce the vital enzyme, and the disorder is alleviated.
Gene therapy can involve inserting a normal allele into a genome to compensate for the activity of a mutated gene. It can also involve introducing a completely novel gene into an organism. It can even involved inactivation or knocking out a mutated gene, so that it will not be expressed. In addition. The novel DNA is not always delivered by a virus. There are techniques that involved the introduction of foreign genes into cells by electroporation. This is where an electric field is used to increase the permeability of the cell membrane, so DNA can pass through tiny temporary holes. DNA can even be injected into cells with incredibly thin needles.
There have been some complications with gene therapy, largely due to the uncertainty associated with where the insertion of the retroviral vactor will occur on the genome. it is also difficult to control the manner in which this new gene is expressed. However there is still cause for caution optimism, as a number of very serious genetic disease have been treated with significant success, and this is an area of ongoing study.
There are those that cite ethical concerns with this kind of practice. In addition to the obvious technical challenges is it appropriate to modify the genome of a living human? Well it is worth noting that this has already been done through blood donation and organ transplantation. These both introduce living cells with foreign DNA into someone's body.
Is gene therapy really different?
Of course one could argue that it is a slippery stape. Will this technology be used to genetically engineer humans and if so, according to what guidance? This type of thought could lead to the practice of eugenics, where by efforts are made to control the genomes of a population. This has been disastrous in the past, and under the wrong political influence, could be disastrous in the future.
Organization of Genes - Fine structure & Types
A gene can be described as a polynucleotide chain, which is a segment of DNA. It is a functional unit controlling a particular trait such as hair colour or eye colour.
According to Lodish and others, gene is defined as the entire nucleic acid sequence that is necessary for the synthesis of a functional gene product, which be polypeptide or any type of RNA. In addition to structural genes i.e. coding genes it also includes all the control sequences and non coding introns.
Most prokaryotic genes transcribe polycistronie mRNA and most eukaryotic genes transcribe monocistronic mRNA. Total number of genes on a single chromosome is different in different organisms.
Bacteriophage virus R17 consists of only three genes, SV40 consists 1 mm long chromosome, of 5-10 genes.
E. coli bacteria have more than 3000 genes on single
Fine Structure of a Gene :
A gene is present only in one strand of DNA, which is a double stranded helix. A gene consists of several different regions. The main region of gene is the coding sequence which carries information regarding amino acid sequence of polypeptide chains.
The region lies on the left side of coding sequence i.e. upstream or minus region and on the right side i.e. downstream or plus region consists of fairly fixed regulatory sequences.
Regulatory sequences of gene made up of promoters which are different in prokaryotes and eukaryotes. There are some genes which are different from normal genes either in terms of their nucleotide sequences or their functions. Split gene, jumping gene, overlapping gene and pseudo gene are some examples of such genes.
(1) Split Genes:
Usually a gene has a continuous sequence of nucleotides. In other words, there is no interruption in the nucleotide sequence of a gene. Such nucleotide sequence codes for a particular single polypeptide chain.
Nevertheless, it was observed that the sequence of nucleotides was not continuous in case of some genes; the sequences of nucleotides were interrupted by intervening sequences. Such genes which carry nucleotides with interrupted sequence are referred to as split genes or interrupted genes. Thus, split genes have two types of sequences, viz., normal sequences and interrupted sequences.
Normal sequence:
Normal sequence represents the sequence of nucleotides which are included in the mRNA which is translated from DNA of split gene. These sequences code for a particular polypeptide chain and are known as exons.
Interrupted sequence:
The intervening or interrupted sequences of split gene are known as introns. These interrupted sequences do not code for any peptide chain, moreover, interrupted sequences are not included into mRNA which is transcribed from DNA of split genes.
An interrupted gene showing introns (light) and exon (dark) portions
( 2 ) Jumping Genes :
In general, a gene occupies a specific position on the chromosome called locus. However, in some cases within the chromosome and also between the chromosomes of the same genome, a gene keeps on changing its position. Such genes are known as jumping genes or transposons or transposable elements.
( 3 ) Overlapping Genes :
The genes which code for more than one protein are known as overlapping genes. In case of overlapping genes, the complete nucleotide sequence codes for one protein and a part of such nucleotide sequence can code for another protein.
( 4 ) Pseudogenes :
Especially in eukaryotes, there are some DNA sequences present which are non - functional or defective copies of normal genes. These sequences do not have any function, such DNA sequences or genes are known as pseudogenes. These pseudogenes have been reported in humans, mouse and drosophila.
Genome :
The basic set of chromosomes is refer as genome. In a genome, each type of chromosome is represented only once. The overall organization of plant nuclear genome revealed that coding capacity is relatively constant among plants as seen in comparison of genome of Arabidopsis and Maize.
Comparing genomic nature of these two plants also reveals genomic codes for same numbers of genes but differ in their genome size. Similarly, maize and sorghum plant contains 10 chromosomes but the maize genome is three times larger than the size as that of sorghum.
Several striking similarities were observed on the arrangement of genes on chromosome of sorghum and maize. The extra DNA that accounts for differences in maize and sorghum genome size is mainly non coding repetitive sequence between genes. This clearly indicated that in most of the organisms only 1 % of the DNA is utilized for protein production and rest may have a significant role in structure and organization of the genome.
4 Phases of Bacterial Growth Curve
When bacteria are inoculated in to a fresh medium and are allowed to grow, they show a characteristic pattern of growth. This is known as a normal growth curve. This characteristic pattern is observed in batch or closed culture system.
Batch culture is the one, where bacteria once inoculated in the medium, are allowed to complete their growth without further adding any nutrients in the medium. This culture is also known as closed culture because once organisms are inoculated in the culture medium they are allowed to complete their growth in the same medium. The normal growth curve of bacteria appears sigmoidal in nature, when growth is plotted graphically.
Typical bacterial growth curve.
A lag phase; B log(logarithmic) or exponential phase, C stationary phase; D death or decline phase
The curve can be divided in to four phases as under :
Lag phase or initial phase
Log phase or logarithmic phase or exponential phase.
Maximum stationery phase.
Death phase.
The occurrence of different growth phase is due to constantly changing environment in the medium due to bacterial activity.
Lag phase
When bacteria are inoculated into a fresh medium, initially for some time, there is no visible increase in cell number and cell mass. This is referred to as lag phase of growth This is attributed to various reasons.
If the inoculum is obtained from old culture or culture from death phase , it may carry with it toxic metabolites which may continue to Inhibit the growth.
Such cells may also be metabolically weak , having very few numbers of ribosome, RNA and proteins.
Much often, the nutrient environment in fresh medium may be quite different than the one that might be in the old / previous culture. Hence organisms may require sometime to adapt to the new environment.
If the inoculum is obtained from spore forming bacteria and consists mainly of spores, they must germinate and get converted to vegetative cells before starting the growth. This time for spore germination may also account for the lag phase of the growth.
Changes in cells during lag phase
Though there is no visible increase in cell number during lag phase. metabolic and physiological changes do occur in the bacteria, to prepare them to undergo rapid growth Hence, we cannot consider lag phase as idle phase. During this phase,
The number of ribosome, amount of RNA,DNA and proteins increase in cell. This shows physiological activation of the weak cells.
The overall rate of metabolic activity and respiration Increases.
The cell size also increases.
Factors affecting the length of lag phase
The length of lag phase is influenced by a number of factors They may include :
1). The type of Inoculum
If the inoculum is obtained from previous culture occurring in log phase. It will show a shorter lag phase.
This is because cells from log phase are metabolically more active than those occurring in death phase.
Similarly, Inoculum containing large number of spores will show longer lag phase.
2). Medium composition
If the composition of fresh medium is similar to that of previous one or if it is rich, a smaller lag phase in obtained.
3). Size of Inoculum
If the inoculum is large the culture shows shorter lag phase and vice versa.
Exponential or logarithmic phase of growth (log phase)
By the end of lag phase, most of organisms in the culture are active and large enough to divide. Hence they start dividing causing logarithmic increase in cell number.
Since the environmental and nutrient conditions are most favorable, these bacteria will show maximum rate of growth and minimum generation time.
Usually, the medium contains nutrients in excess. Therefore even though nutrients are being continuously depleted from the medium due to their utilisation during the growth Initially, the change in their concentration has very little effect on growth. Hence the bacteria continue to grow logarithmically for many generations.
However, during the growth the chemical environment of the medium undergoes a constant change and gradually becomes unfavorable for growth.
This results in to gradual decline in growth rate. Hence, the increase in growth slows down, allowing it to other stationery phase.
The development of unfavorable chemical and physical environment of medium occurs due to following reasons:
During growth, bacteria consume nutrients as well as dissolved oxygen. Therefore, gradually, their supply becomes limiting and unfavorable for growth
Further, as growth increases, the need for nutrients Increases more and more. Hence this depletion becomes more significant during later part of log phase.
Increase in cell mass due to growth causes increase in Viscosity of medium significantly. This also provides unfavorable condition to the organisms for uptake of nutrients as well as O2 by the organisms.
Even enough space is also not available for the organisms to have free movement in the medium dut a to increased cell density.
Due to respiratory and metabolic activities of the organisms during growth, a drop in pH of medium is normally observed. This also contributes to the development of unfavorable condition for the organisms.
The metabolic activities of organisms results in to excretion of various waste metabolites by the organisms. These metabolites may have toxic effect on the microorganisms and they have unfavorable effect on growth and tend to retard growth.
Stationary phase
When the bacteria are allowed to continue their growth in the same medium for prolonged time , condition in medium becomes more and more unfavorable for the growth. This is due to
Depletion of nutrients from the medium, due to their utilisation in log phase.
More and more accumulation of toxic waste metabolites.
Increased cell density can cause limiting space available for the organisms and increase in viscosity.
Greater changes in pH to unfavorable value etc.
As a result, growth of the bacteria becomes Inhibited and their growth rate decreases and finally organisms stop dividing. This results in to development of stationary phase.
Perhaps, the rate of new cell being formed may also become equal to the death of cell. Thus the equivalent growth rate and death rate helps in establishing stationery growth phase.
Death or Decline phase of growth
Following stationary phase, the bacterial growth curve enters death phase, if they are still continued to remain in the same medium. Due to the development of still more vigorous unfavorable conditions in the medium, bacteria stop growing. Their death rate exceeds growth rate and hence number of living cells from the culture decrease.
The decrease in number of bacteria may even become logarithmic. Hence, this phase of growth may also be called as negative logarithmic or negative exponential growth phase.
The factors that contribute death of bacteria include :
Sharp decrease in essential nutrients.
Excessive accumulation of toxic waste metabolites.
However, death rate will not be same for organisms of all species. It may differ with the type of organisms. Usually gram-negative bacteria decline sharply and hence very few bacteria may survive at the end. While gram - positive bacteria die slowly and viable cells may persist for prolonged periods in the culture.
Electron Transport Chain (ETC) and ETC Inhibitors
Electron Transport Chain (ETC)
The goal of the electron transport chain is to couple energy stored in electron acceptors to a proton gradient that derives ATP synthesis. ETC is takes place in inner mitochondrial membrane.
In inner mitochondrial membrane has a series of complexes.
There are five complex proteins, which helps in ATP synthesis. Additionally the presence of two other molecules that act not as complex but they are embedded in this area of the electron transport chain. Those are Coenzyme-Q and Cytochrome-C, now technically these are broken into two distinct parts of oxidative phosphorylation.
The complex portion from 1 to 4 including coenzyme-Q and cytochrome-C is known as the electron transport chain, because this is where electrons get shuffled from one complex to the other. The ATP synthase which pumps the proton gradient to generation ATP is technically known as chemiosmosis. The combination of the electron transport chain plus chemiosmosis that is togather known as oxidative phosphorylation.
How ETC works ?
Protons that are present in the mitochondrial metrix. TCA cycle goal is to produce NADH and FADH2 for use in the electron transport chain. so along comes NADH and NADH2 approaches complex-1, NADH can give up it's proton and give up it's electrons and become NAD+.
In the process it donates it's electron to complex-1. When the electron enter complex-1 complex-1 becomes supercharged. It has the energy to pump the proton from the mitochondrial matrix into the inter membrane space as it does this it pumps more and more protons from the mitochondrial matrix into inter membrane space and you get the accumulation of protons on the other site of the membrane. This pumping is only made possible by the electron given up from NADH and supercharge complex-1.
After while the electron will sit in complex-1 and the proton gradient is beginning to from on the top in the inter membrane space you have much more protons than exists.
Now at this point the gradient is being to form and complex will pass it's electrons to Coenzyme-Q. The electrons will go to Co-Q and sit there awaiting further instruction.
Now at this point FADH2 comes along and approaches complex -2 Just like NADH, FADH2 was produced in the TCA cycle. it migrates here in ETC and begins it's electrons and turn into FAD in this process it donates it's electron to complex-2.
Complex -2 however can not become supercharged and can not pump protons from the mitochondrial metrix into the inter membrane space.
So the electron sits in complex-2 and awaits further instruction and ultimately gets passed to Co-Q. NADH only works at complex-1, FADH2 only works at complex-2.
So the electrons given up from NADH go from one to Co-Q and the electrons given up by FADH2, from complex-2 to Co-Q.
Now it's important to understand something that's very high yield Co-Q is common electron acceptor from both complex-1 and complex-2. It's also incredibly important and very high yield to remember that NADH only gives up it's electrons at complex-1 and FADH2 only gives up it's electrons at complex-2 at this point the electrons are sitting in Co-Q and they are passed to complex-3.
It's supercharges complex-3 which creates enough energy potential to pump the proton from the mitochondrial Matrix through complex-3 into the inter membrane space. Complex-3 is being super charged by the shuffling of electrons both from complex-1 and complex-2 to Co-Q to complex-3 supercharging it and helping to created this proton gradient in the inter membrane space.
You are getting the accumulation of protons there's a much greater positive charge on the inter membrane space. than there is in the mitochondrial Matrix so we are containing to form a very big proton gradient at this point complex-3 will pass it's electrons on to cytochrome-C.
The electrons arrive and then get post to complex-4. At complex-4 the electron enter it and supercharge. It just like we have seen in charged complex-3 and in complex-1,once supercharged complex-4 has enough energy to pump protons from the mitochondrial Matrix into the inter membrane space.
Again the proton gradient continues to form the inter membrane space is laced with tons of positively charged protons. So there's a proton gradient compared to the mitochondrial matrix which has fewer protons at this point complex-4.
Electrons sitting inside of it and it needs to pass to the final electron acceptor the final electron acceptors is oxygen. The electrons are passed to oxygen.
The electrons are passed to oxygen. Which splits into two oxygen ions and protons are added creating two water molecul. Now at this point we have formed a massive proton gradient there are so many protons in the inter membrane space and so fewer protons in the mitochondrial Matrix.
Now at this point that ATP synthase comes into play. ATP synthase is going to make use of this proton gradient to generate massive amounts of ATP. So along comes the molecule ATP and ADP. Wants to turn into ATP which is a higher energy throughout the body but in order to catalyze the conversion.
We have to put energy source into this reaction because you can't just go from a lower energy source ADP to a higher energy molecule ATP. Without some type of energy input it's at this point that ATP synthase takes advantage of the proton gradient which was being formed by complexes 1,3 & 4.
The protons will always want to flow down it's gradient that is to say molecules in general like to flow from high energy states to low energy states to achieve equilibrium.
Protons will flow from the inter membrane space down through ATP synthase Bach to the mitochondrial matrix and when they do this it is an energy input that catalyses the conversion of ADP to ATP. That is how the energy is formed and massive amounts of ATP are formed during that step because there's such a large proton gradient that can continuously flow down to hill.
Now as those protons come across ATP synthase they build back up on the mitochondrial matrix. So they are sitting in front of complexes 1,3 & 4 ready to be pumped back up into the inter membrane space.
When complex 1,3 & 4 gets super charged as you can see the cycle continuous and the electron transport chain can continue to turn out ATP, so long as NADH2 and FADH2 are being shuffled from the TCA cycle to the electron transport chain to continue the flow of protons.
Inhibitors Electron Transport Chain
• Rotenone inhibits complex-1
• Antimycin inhibits complex-3
• Cyanide and carbon monoxide
inhibits complex-4 and
cytochrome-C
• Oligomycin inhibits ATP
synthase and uncoupling
such as 2 for DNP,uncouples
the proton gradient and
inhibits
the proton gradients ability to pump
protons down through ATP
synthase.
these are five electron transport chain inhibitors.
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Krebs (TCA) Cycle and it's Products
Krebs cycle also known as tricarboxylic acid cycle, it is a biochemical pathway that is used to generate energy to the oxidation of acetyl-coA. It is also used for the synthesis of NADH and for the production of the amino acid. Krebs cycle takes place in mitochondria of the eukaryotes and cytosol of the prokaryotes.
The first steps involved is actually a preparatory step which begins with pyruvate. Pyruvate is derived through the glycolysis of glucose which is 6 carbon compound. The next step includes the oxidation of pyruvate into acetyl-coA by the enzyme pyruvate dehydrogenase complex and in this reaction a molecule of carbon dioxide and a molecule of NADH is generated. The acetyl-coA is a 2 carbon compound, in the next step the acetyl-coA combines with oxygen as it eight.which is a four carbon compound to form citrate and hence the resulting molecule is six carbon compound this reaction is catalyzed by the enzyme citratesynthase. In the next step the citrate is isomerized in to isocitrate by the enzyme aconitase.
Next the isocitrate is oxidized into alpha-ketoglutarate by the enzyme isocitrate dehydrogenase and in this reaction a molecule of NAD is reduced to NADH and one molecule of carbon dioxide is generated. Hence the alpha-ketoglutarate is a 5 carbon compound since one molecule of carbon is lost into carbon dioxide. Next the alpha-ketoglutarate is converted into succinyle-coA by the enzyme alpha ketoglutarate dehydrogenase and in this reaction also a molecule of NAD is reduced to NADH and a molecule of carbon dioxide is released, hence the succinyl-coA is four carbon compound. In the next step this succinyl-coA is converted into succinate by the enzyme succinyl-coA synthase, in this reaction a molecule of GTP is generated. Next succinate is converted into fumarate by the enzyme succinate dehydrogenase in this reaction a molecule of QH2 is generated which is used for the production of FADH2. Fumarate is then converted into malate by the enzyme fumese. In the last step the malate is converted into oxaloacetate by the enzyme malate dehydrogenase, in this reaction also NAD is reduced to NADH.
Let's look at the result of the kreb's cycle through each cycle of the Krebs cycle following products are generated
3 molecular of NADH, 1 molecule of FADH2, 1molecule of GTP and 2 molecule of carbon dioxide. Now since glucose is split into two pyruvate compounds, for each molecule of glucose this cycle runs twice, hence the products formed are 6 NADH, 2 FADH2, 2 GTP and 4 CO2.
All the NADH and FADH2 are next fed to the electron transport chain for the generation of ATP.
What are the Causes of Cancer ?
What are the Causes of Cancer?
The process of conversion of a normal cell to malignancy is called carcinogenesis and the agents which cause this are called carcinogens.
Carcinogenesis is a complete process involving interaction of many factors, some of which favour development and others which appear to provide some protection against it.
Cancer can be viewed as a normal process mitosis that in mistimed or misplaced. Proteins such as growth factors, kinases and cyclins partially control the pace of mitosis, and because these, proteins are constructed using genetic information, genes play a role in causing cancer.
Two major classes of genes contribute to causing cancer. ONCOGENES must be activated to cause cancer. TUMOUR SUPPRESSORS, which normally hold mitosis in check, must be inactivated or removed to eleminate control of the cell cycle and initiate cancer.
ONCOGENES
Oncogenes are genes that normally activate cell activates cell division in the wrong time or place, cell, they are normal, essential genes that have undergone a mutation. In its normal nonmutated cancer may result. Oncogenes are not alien to the state, it is a proto-oncogene, a gene that can be transformed into on oncogene.
In 1911. Francis Peyton Rous reported that a virus, later named Rous Sarcoma Virus (RSV), is capable of causing sarcoma, a type of cancer in chickens, RSV is an RNA virus. After the virus infects a cell, it inserts a DNA transcript of its RNA genome, which includes a gene known as sre (for sarcoma gene), into host chromosome, The sre gene is now known to be an oncogene. It is possible that the sre gene is under the control of a viral promotor that has turned it on.
The oncogenes most frequently involved in human cancers belong to the ras gene family. An alteration of only a single nucleotide pair is sufficient to convert a normally functioning ras proto-oncogene to an oncogene. The ras K oncogene is Yound in about 25% of lung cancers, 50% of colon cancer and 90% of pancreatic cancer. The ras N oncogene is associated 'with leukaemias and lymphomas and botch ras oncogenes are frequenty found in thyroid cancers.
Activation of protooncogenes to oncogenes is achieved by at least five mechanism ( pramoter and enhancer insertion, chromosomal translocation, gene amplification, point mutation).
TUMOR SUPPRESSOR GENE
Tumor suppressor gene is so called because it prevents cancer from occurring. Researchers have identified about a half dozen tumour suppressor genes. When these gene malfunction, a tumour results. Tumour suppressor genes are row recognized transe players in the genesis of cancer. The effect on the oncogenes on cell growth has been compared to putting one's foot on the accelerator of an automobile, whereas the action of tumour suppressor prote resembles taking one's foot off the brakes. hard a Important tumour suppressor genes include RBI and P 53, both of which are nuclear phosphoproteins and probably affect the_transcription of genes involved in regulating events in the cell cycle.
A genetic model for colorectal cancer invokes the interplay of tumour suppressor genes and the K-ras oncogene. Mutations in mismatch repair genes have been found to be associated with hereditary and loss of nonpolyposis colon cancer, responsiveness to the growth inhibitory effects of TGF important in development of this type of tumour. A number of cancer susceptibility genes have been isolated. They include RB 1,P 53, BRCA 1, BRCA 2. Tumour progression reflects an instability of the tumour genome, probably due to at least in part to defects In DNA repair systems, activation to additional oncogenes, and inactivation to additional tumour suppressor genes.
The RB tumour suppressor gene was discovered by Alfred Knudson. RB tumour- suppressor gene has now been found to malfunction in cancers of breast, prostate, bladder. Loss of RB tumour suppressor gene through chromosomal deletion is particularly frequent in a type of lung carcinoma.
It is now known that signaling proteins often regulate the transcription of genes whose products are needed for cell division, and this appears to be true of the RB protein. When a particular factor attaches to a receptor, the RB protein is activated. An active RB protein turns off the expression of a proto-oncogene, whose product initiates cell division. Translocations of genes may occur as result of mistakes during mitosis, or they may be caused by carcinogens or viral infection.
Another major tumour-suppressor gene is called P 53, a gene that is more frequently mutated in human cancers than any other known gene. It has been found that the P 53 protein acts an a transcription factor and as such is involved in turning on the expression of a gene called WAP I or Clp 1 The product of this gene is a cell cycle inhibitor. A number of cell proteins combine with the p 53 protein. When a protein combines with p 53, it is hard to tell whether it is activating p 53 or is being activated by p 53. This intricate situation is now being unraveled.
Discovering the normal function of the p 53 gene is a top priority in biomedical research so far, the normal form of gene is implicated in cell cycle control, is cell's expression of other genes, and in several metabolic processes. Understanding p 53 will open the door to understanding many types of of cancer.
While oncogenes and tumour-suppressors play a part in causing cancer, other pOssible causes include certain chemicals, nutrient deficiencies, and radiations.
Direct carcinogens cause cancer when they are applied to fibroblast cells growing in culture. Benzene is an example for this.
Procarcinogens are safe outside the body, but inside they are metabolized to produce intermediate compounds that cause cancer. They include certain organic dyes, cigarettes, nitrites & nitrates used to preserve processed meats.
Promoters are chemicals that make other carcinogens more powerful. They include alcohol, certain hormones and various chemicals in cigrette smoke.
Polycyclic hydrocarbons produce cancer at the site of local application. Aromatic amines like 2- naphthylamine and benzidine cause cancer of urinary bladder. A20-dyes cause liver cancer and amino-fluorenes also cause cancer of liver and urinary bladder. Alkylating agents like mustard gas, methylnitrosourea and nitrosamine and various inorganic compounds like asbestos fibres, arsenic, beryllium, nickel, cadmium, and chromates are all carcinogenic.
X-rays, a,ß and Y rays and ultraviolet light all induce tumours in man and animals. While passing through the tissue, these release energy which alters various macromolecules of the cell including nucleic acids. This causes increased of mutations in the irradiated cells.
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