The Central Dogma of Life

All cells within a complex multicellular organism such as a human being contain DNA. All the DNA together make up that organisms genome. There are many different types of cells within a complex organism. What, then makes a cardiomyocyte different than a hepatocyte? The answer lies within how each cell controls its genome. DNA consists of genes, which are short sequences of nucleic acids that code for particular molecular structures or protein that carries out a specialized function. Each cell can control its unique set of genes. Some are expressed, and others are repressed. This dictates cellular morphology and function. That is how a myocyte differs from a hepatocyte. Its not the DNA itself. All cells contain the same DNA. Rather, it’s how each cell is individualized, and controls how it uses the set of DNA. This expression and repression is highly regulated by cues both within and extrinsic to the cell. This article will serve to cover the first part of what DNA actually is and how it codes for specific polypeptides that carry out downstream functions. This is the central dogma of life.

Nucleic Acids

Nucleic acids are what transfers genetic material as well as participate in cell signaling and other metabolic processes. Often considered the building blocks of cells. There are two main categories of nucleic acids; Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Both DNA and RNA are polymers of nucleotides. Both RNA and DNA structures are similar in that they consist of a sugar (either Ribose, or deoxyribose) with a nitrogen containing base (either a pyrimidine or purine) bonded to a phosphate group, except that DNA does not have a hydroxide group at Carbon-2, where as RNA does.

Bases

Nitrogenous bases are categorized as either a pyrimidine or a purine. A pyrimidine is a heterocyclic aromatic ring structure, whereas a purine is a two ringed heterocyclic aromatic ring. The pyrimidines are Uracil, which only exists in RNA, thymine, which only exists in DNA, and cytosine which coexists. The purines are adenine and guanine, which coexist in both DNA and RNA. Base-pairing is what creates the characteristic double-helix feature of DNA, and the single-stranded RNA structure. Each base-pair consists of one pyrimidine and one purine which are called base complements. In DNA, thymine always binds adenine (T-A), and guanine always binds to cytosine (G-C) through double or triple hydrogen bonds.

DNA molecule

DNA being a double helix is proven to be advantageous in multiple ways; the nitrogenous base which contains the nucleic information is locked within the complex, facing each other in the centre of the molecule, as opposed to in RNA, where the nucleic acid base is exposed to the cellular environment which provides more opportunity for it to be mutated. DNA is more chemically stable than RNA and less susceptible to degradation. Having two complementary strands allows for greater proof-reading mechanisms. Thymine is much more stable than uracil (RNA). Due to deamination cytosine is often changed to uracil, which in DNA is quickly corrected because uracil is not supposed to be there. In RNA, it’s impossible for the cell to know if it’s truly supposed to be there or not.

DNA replication

DNA replication is the biological process of producing two identical copies of DNA from a single original DNA molecule. This process occurs in all living organisms. It beings at specific locations within the genome called origins of replication. These origins of replication are areas of high T-A base pairing. This is important because there are less hydrogen bonds between the A-T bases so the strand is easier to break at those points. This is known as initiation and at this point the DNA strand is unwound by proteins and enzymes known as helicases, which expose the two strands and result in replication forks that are bi-directional from the origin. Topoisomerases are enzymes used to temporarily break the strands of DNA to relieve tension. These two strands then serve as a template for the leading and lagging strands which will be created as a DNA polymerase will match complementary nucleotides to the templates. DNA is always synthesized in the 5′ to 3′ direction and because at the replication fork the template strands are oriented in opposite directions the leading strand is the strand of nascent DNA which is being synthesized in the same direction as the growing replication fork and replication is continuous in the 5′ to 3′ direction. Within the lagging strand the nascent DNA being synthesized is in the opposite direction, and because of this replication lags and is fragile. Primase is an enzyme that synthesizes a short RNA primer with a free 3′ OH group which is then elongated by a DNA polymerase. The leading strand only receives one RNA primer, while the lagging strand receives several. The leading strand is continuously extended by a DNA polymerase, while the lagging strand is extended discontinuously from the multiple primers forming Okazaki fragments. DNA clamp proteins form a sliding clamp around the DNA, this allows the DNA polymerase to maintain constant contact with the template strands, and enhancing processivity. Once DNA polymerase reaches the end of the template strands and runs into double-strand DNA the sliding clamp protein complex undergoes a conformational change and releases the DNA polymerase. RNase then removes the RNA primers and is replaced by DNA ligase which joins together the multiple parts of the DNA.

DNA replication occurs at multiple points within the chromosome of an organism and because of the linear nature of chromosomes these replication forks are unable to reach the terminal ends and as a consequence a small amount of DNA is lost each replication cycle. Telomeres are regions of repetitive DNA close to the ends of the chromosomes that help mitigate the loss of genomic material during replication. These telomeres are finite and thus each cell has a terminal number of replications. This is otherwise known as the Hayflick limit. When DNA is passed down the germ cell line telomerase is an enzyme that extends the repetitive sequences of the telomere region. Telomerase can become mistakenly up-regulated in somatic cells, which can sometimes lead to cancer.


This is the first release of a 3 part series of articles dedicated to the Central Dogma of life. This article covered what RNA, DNA and DNA replication are.

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Enzyme-Linked Immunosorbent Assays (ELISA)

The first step in any ELISA assay is the immobilization of the antigen within the sample to the wall of the wells within a microtiter plate. These microtiter plates are usually 96-wells. This is by direct adsorption to the plates surface or by using a capture antibody. The capture antibody has to be specific to the  target antigen. After immobilization, another antibody is added called the detection antibody. This detection antibody binds to the adsorbed antigen which forms an antigen:antibody complex. This detection antibody is either directly conjugated to an enzyme, such as horseradish peroxidase (HRP), or provides an antibody-binding site for a secondary labeled antibody. There are four different types of ELISAs which will all be discussed below. ELISAs take advantage of an enzymatic label to produce a signal that can be quantified and correlated to the binding of an antibody to an antigen. The final assay signal is measured using spectophotometry.

Direct ELISA

In the direct ELISA, the detection antibody is conjugated with either alkaline phosphatase (AP) or horseradish peroxidase (HRP). These substrates produce a colorimetric output that is then measured. The advantages of a direct ELISA is that it is a short protocol which saves time and reagent, and money. There is no cross-reactivity from a secondary antibody that can cause interference. The disadvantages are that there is no signal amplification, so the primary antibody must be conjugated for it to work.

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Indirect ELISA

In the indirect ELISA, antibodies can be conjugated to biotin, which is then followed by a streptavidin-conjugated enzyme step. This is becoming more common place within the clinical laboratory. Alternatively, the detection antibody is typically a human IgG antibody that binds to the antigen within the wells. This primary antibody has multiple antibody-binding sites on it. A secondary rabbit anti-human IgG antibody conjugated with an enzymatic substrate is added. This secondary antibody binds to the first antibody and gives off a colorimetric signal which can be quantified by spectrophotometry. There are advantages over the direct ELISA, mainly that there is signal amplification by using several antibodies, allowing for high flexibility. This also creates a longer protocol, and increases the chances for cross-reactivity, which can be deemed disadvantages.

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Sandwich ELISA

The sandwich ELISA is less common, but is highly efficient in antigen detection. It quantifies antigens using multiple polyclonal or monoclonal antibodies. Monoclonal antibodies recognize a single epitope, while a polyclonal antibody recognizes multiple antigen epitopes. The antigen that is to be measured must contain at least two antigenic epitopes capable of binding to an antibody for this reason. The first step is to coat the microtiter plate wells with the capture antibody within a carbonate/bicarbonate buffer (pH 9.6). Proceed to incubate the plate overnight at 4 degrees Celsius. Wash the plate twice using PBS. Incubate the plate again for at least 2 hours at room temperature. Wash the plate again using PBS. The next step is to add diluted unknown samples to each well. Its important to run unknown samples against those of a standard curve by running standards in duplicates or triplicates. Incubate for 90 minutes at 37 degrees Celsius. then remove the sample and wash with PBS again. Next, add diluted detection antibody to each well. Its important to make sure that the detection antibody recognizes a different epitope on the target antigen than the capture antibody. The prevents interference with antibody binding. To maximize specificity and efficiency, use a tested matched pair. Once the detection antibody has been added, incubate for 2 hours at room temperature. Wash once again with PBS. After washing, add conjugated secondary antibody to each well. Incubate once again at room temperature, then proceed to wash. Once again, horseradish peroxidase and alkaline phosphatase are used as enzymes conjugated to the secondary antibody. The substrates for HRP are called HRP chromogens. Cleavage of hydrogen peroxide is coupled to an oxidation reaction which changes color. Another common substrate used is ABTS. The end product is green.

Sandwich-ELISA

The sandwich ELISA employs high specificity, even when using complex samples. Within the sandwich ELISA, both direct and indirect methods can be used. It can be challenging to find two different antibodies against the same target the recognize different epitopes.

Competitive ELISA

The competitive ELISA is exactly what its name suggests; it is a competitive binding process which is produced by the sample antigen, and an add-in known concentration of antigen. A primary unlabeled antibody is incubated with the unknown sample antigen. This creates antigen:antibody complexes, which are then conjugated to a microtiter plate which is pre-coated with the same antigen. Any free antibody binds to the same antigen on the well. Unbound antibody is removed by washing the microtiter plate. The more antigen within the unknown sample means that less antibody will be able to bind to the antigens within the wells, hence the assay gets its name. Its a competition. A secondary conjugated antibody that is specific for the primary antibody bound to the antigen on the pre-coated on the wells is added. When a substrate is added, the reaction elicits a chromogenic or fluorescent signal. The higher the sample antigen concentration, the weaker the eventual signal.

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References

https://www.bio-rad-antibodies.com/elisa-procedure.html

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-elisa.html

Cardiac Markers

Approximately every 42 seconds an American will suffer from a myocardial infarction. A MI occurs in a hypoxic state and sections of the heart are unable to get the oxygen it needs. According to the CDC about 610,000 die of heart disease in the United States every year, an occurrence of about 1 in every 4 deaths. Heart disease is the leading cause of death in both men and women.

Also about 47% of sudden cardiac deaths occur outside a hospital environment suggesting that many people with acute heart disease either don’t recognize the early signs or they don’t act on them.

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There are cardiac markers that can give a physician a better picture of what is going on. These are routinely measured in a clinical laboratory and are almost all of the time STAT tests. The quick turn-around-time of these tests is imperative, because the sooner a patient with a heart condition gets treated, the better their prognosis.

The Troponin test is the most sensitive and specific test for myocardial damage. Troponin is released during and MI from the cytosolic pool of the myocytes. Its release is prolonged by the degradation of the actin and myosin filaments. Isoforms of protein, T, and I are specific markers for myocardium. After myocardial injury, troponin is released in 2-4 hours and peaks after 12 hours. It persists for up to 7 days after MI.

The Creatine Kinase-MB test is relatively specific when skeletal muscle damage is not present. Creatine kinase is an enzyme that is present in various tissues and cell types. It catalyzes the conversion of creatine to phosphocreatine utilizing adenosine triphosphate (ATP). The phosphocreatine serves as an energy reservoir in tissues that consumes ATP, especially the skeletal muscle and the brain. When mitochondrial creatine kinase is involved in the formation of phosphocreatine from mitochondrial ATP, cytosolic CK regenerates ATP from ADP and phosphocreatine kinase. In this instance, CK acts as an ATP regenerator. Clinically creatine kinase levels are assayed as a marker for damage of the CK-rich tissues in pathological states of myocardial infarction, rhabdomyolysis, muscle dystrophy, autoimmune diseases, and acute kidney injuries. There are two subunits of the cytosolic CK enzymes; Brain type (B) or Muscle type (M). The two subunits create three different isoenzymes CK-MM, CK-BB, and CK-MB. The different isoenzymes are present in different levels in various tissues. In skeletal tissue CK-MM is predominantly expressed, and in myocardial tissue, CK-MM and CK-MB is measured. Therefore measuring CK-MB levels is a good diagnostic test for heart damage from myocardial infarctions. CK-MB peaks about 10-24 hours after the attack and normalizes within 2-3 days.

Lactate dehydrogenase (LDH) was talked about in one of my previous posts and it can aid in the diagnosis of MI. Although it has been most recently replaced by the more specific and sensitive troponin test. LDH catalyzes the conversion of pyruvate to lactate. LDH-1 is found in the myocardium and LDH-2 is found in the serum. Normally LDH-2 is the predominate isoenzyme, but in cases of MI, LDH-1 is the predominate isoenzyme assayed and found. LDH takes about 72 hours to peak and normalizes within 10-14 days.

Myoglobin is an iron and oxygen binding molecule found in the muscle tissue. Myoglobin is a cytoplasmic protein that only harbors one heme group, although in contrast it has a much higher affinity for oxygen than does hemoglobin because its primary role is to store oxygen, where hemoglobins function is to transport oxygen. It contains a porphyrin ring with a proximal histidine group attached to the iron in its center. Myoglobin is only found in the bloodstream after muscle injury such as rhabdomyolysis. Myoglobin is a sensitive marker for muscle injury, making it a potential marker for MIs. However it lacks specificity and should be taken into account with other clinical findings to make a diagnosis. Myoglobin peaks the earliest of all other cardiac markers, that is within two hours, but it also falls quickly, usually before troponin or CK-MB.

Pro-brain natriuretic peptide is used as marker for acute congestive heart failure. The BNP is a hormone secreted by the cardiomyocytes in the ventricles of the heart in response to stretching caused by increased ventricular blood volume. The actions of BNP cause a decrease in systemic vascular resistance and venous pressure which causes a drop in blood pressure and causes after load. This causes a decrease in cardiac output.

Cardiac markers should be used to add to a clinical diagnosis, they should not be solely used to diagnose a patient with MI or CHF.

-Caleb

Lactate Dehydrogenase

Lactate dehydrogenase (LD, LDH) is an enzyme that is found in all cells in all tissues of the body. It catalyzes the reversible conversion of lactate to pyruvic acid in glycolysis and gluconeogenesis. It is released from various anatomical sites of the body in response to cellular injury and damage. It is used as a common marker for tissue damage and disease such as heart failure. Its down-fall is that it isn’t very specific to which tissue is damaged, but there are subtle hints that can clue a physician in particular directions.

Lactate dehydrogenase is structurally composed of four subunits, but the two common subunits are LDHA known as LDH-M, and LDHB, known as LDH-H. The only difference between the two subunits is that their is an amino acid substitution of alanine with glutamine within the H subunit. This amino acid change slightly changes the biochemical properties of the two subunits slightly in that the H subunit can bind faster, but the catalytic activity of the M subunit does not deteriorate at the same rate as the H subunit, it holds well.

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The two subunit of LDH can form five isomers which are found in various sites within the body;

LDH-1 (4H)- Found in the heart, RBCs, and the brain.

LDH-2 (3H1M)- Found in the RES.

LDH-3 (2H2M)- Found in the lungs.

LDH-4 (1H3M)- Found in the kidneys, placenta, and the pancreas.

LDH-5 (4M)- Found in the liver and striated muscle.

LDH is a protein that is found in small amounts normally in the body and there are various conditions that can cause an elevation. Cancer can raise the LDH levels within the body. Cancer cells rely on increased glycolysis due to their high energy demand. LDH elevation in cancer is often times referred to the Warburg effect which allows malignant cells to convert glucose stores into lactate even in the presence of aerobic respiration. This shifts glucose metabolism from simple energy production to accelerate cell growth and proliferation.

Hemolysis can be measured as LDH is abundant in RBCs and can be measured. Although measures should be taken to correctly receive the sample as incorrect procedures an cause hemolysis and a false-positive elevation in LDH levels among other substrates and electrolytes.

It can also be used as a marker for myocardial infarction. Normally LDH-2 is at a higher level than LDH-1. When someone experiences a myocardial infarction, levels of LDH-1 will be significantly elevated to a level higher than LDH-2. This is known as the LDH flip and is diagnostic in patients who have experienced myocardial infarction. Elevation of LDH peaks 3-4 days after MI, and can remain elevated for up to 10 days. LDH has since been replaced by the troponin test, which is a much more specific and sensitive test in diagnosing MI.

High levels of LDH in the cerebrospinal fluid (CSF) can indicate bacterial meningitis. Elevated LDH levels in viral meningitis is indicative of a poor prognosis.

LDH is an important tool that physicians don’t always utilize to its lack of specificity, but it can still be helpful in a diagnosis. Its important not to ignore any test and any result as it still contributes to the whole picture.

-Caleb

 

Case Study Mini-Series; Diagnosis

The patient was diagnosed with subclinical DIC because complications from Acute Promyelocytic Leukemia (APL)

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The characteristic chromosomal translocation of Acute Promyelocytic Leukemia is the break and fusion of the PML gene located on chromosome 15 and the RARA gene located on chromosome 17. This results in a t(15;17) which is detectable in more than 90% of cases. The PML gene has a physiological role in apoptotic pathways and in genomic stability. The t(15;17) breakpoint in PML can occur in three different sites; bcr1 within intron 6, bcr2 within exon 6, and bcr3 within intron 3 . The RARA receptor is active in different variations within each tissue and is important for granulopoiesis. The PML-RARA fusion transcripts impair signaling which is mediated by RARA and interact with proteins that leads to the delocalization of normal PML from its nuclear structures known as NBs. It is in this way that the PML-RARA oncoprotein negatively acts on the normal physiology of the native PML protein. APL is a subtype of AML that has distinctive morphological, biological and clinical characteristics. It is classified as AML-M3 in the French-American-British (FAB) classification system. The cure rate for APL is ~80-90% for patients who survive induction therapy with ATRA. Before ATRA, the 10-day survival rate with treatment was 9.4%. A high blast count was significantly associated with hemorrhagic events and fatality within the first 10 days. A high blast count and thrombocytopenia was associated with death within 24 hours upon admission and treatment. APL predominantly affects a wide spectrum of individuals between the ages 20 and 59 with no gender discrimination. 10-15% of all AML diagnosed in adults is APL, although it can be seen in distinct populations in a higher percentage. 28.2% of all AML diagnosed in Brazil is APL, and 20% of all AML in Venezuela is APL.

APL presents as a bleeding diathesis and coagulopathy. The more common hypergranular variant of APL presents with leukopenia while the less common microgranular variant tends to be more aggressive and presents with leukocytosis. The malignant promyelocytes have specific properties that interact with the host cells. Maligant APL cells express tumor associated procoagulants; Tissue factor (TF) and cancer procoagulant (CP). Tissue factor is an activator of coagulation and the relative expression is elevated significantly in patients with APL.

APL is characterized as a hyperfibrinolysis state. Fibrinolysis is normally activated by thrombin as the fibrin clot develops and coagulation comes to an end. Malignant promyelocytes highly express annexin-II. Annexin-II is a protein receptor that has a strong affinity to plasminogen and tissue-type plasminogen activator (tPA) which results in strong yield of plasmin which initiates fibrinolysis. Annexin-II is highly expressed in the cerebral microvascular endothelial cells explaining the high prevalence of intracerebral hemorrhage in patients with APL. Cytokine release of IL-1B and TNFa by malignant promyelocytes upregulate apoptosis and upregulate the expression of tissue factor on endothelial cells. It is also common for the cytokines to cause loss of the anti-coagulant cofactor thrombomodulin. These various factors lead to APL-associated coagulopathy commonly seen.

Patients with APL present low fibrinogen levels, low platelet count, and an elevated PT-INR, aPTT, and D-dimer. In DIC secondary to APL, fibrinogen survival is markedly decreased due to rapid consumption and the liver can’t produce the product fast enough. Sometimes more specialized tests are needed to diagnose the coagulopathy in APL. Levels of thrombin-antithrombin complex (TAT), prothrombin fragment 1 and 2, and fibrinopeptide A are all increased and all indicate coagulation activation. Decreased levels of plasminogen, and a-2-antiplasmin further support the hyperfibrinolysis state. Sometimes it is helpful to further evaluate the coagulation process and its components. Protein C and antithrombin III are synthesized in the liver and are relatively normal in APL associated coagulopathy unless the maligancy is accompanied by hepatic dysfunction.

The next installment of the mini-series will focus on the key points of what lead to the diagnosis, what I look for as a medical laboratory professional in aiding the doctor in the diagnosis, and how to treat appropriately.

 

-Caleb