Information

Is NADH found in all type of cell in human body?

Is NADH found in all type of cell in human body?


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Is NADH found in all kinds of cell in the human body? I am curious about microglia specifically.


As NAD+/NADH is involved in both glycolysis and in the tricarboxylic acid cycle it is difficult to imagine any cell capable of generating energy not having some NADH. (Are there any cells that are not capable of this? Calcified cells such as bone?)

A quick internet search for 'microglia' and 'metabolism' indicates that microglia have energy metabolism and so I would expect them to have NADH. How much (compared to NAD+) would depend on their metabolic state.


Basic Types of Cells

Even though there are several hundred cell types in the body, all of them can be grouped into just four main categories, or tissues. This makes them easier to understand.

These four main tissues are formed from:

    . These cells are tightly attached to one another. They cover over the interior of hollow organs, like blood vessels or digestive organs, or else form the surface of things, like the skin. There are dozens of types of epithelial cells. Without epithelial cells, you would have no skin to protect your body from injury and would have no stomach to digest your food! . These cells are specialized for communication. They send signals from the brain to muscles and glands that control their functions. They also receive sensory information from the skin, the eyes, and the ears, and send this information to the brain. There are dozens of varieties of nerve cells in the body, each with their own shapes and functions. You would have no consciousness or control over your body without nerve cells. . These cells are specialized for contraction. Without muscle cells, you would not be able to move! There are three kinds of muscle cells. They pull and tug on bones and tendons to produce motion. They also form the thick outer walls of hollow organs, like blood vessels and digestive organs, and can contract to regulate the diameter of these hollow organs. . These cells provide structural strength to the body and also defend against foreign invaders like bacteria. Two types of cells—fibroblasts and fat cells—are native to connective tissue. Other cells migrate into connective tissue from the bloodstream to fight diseases. Special types of connective tissue—cartilage and bone—are designed to be stronger and more rigid than most connective tissues.

Related Topics

Choose one of the following categories to see related pages:

Share this Page

Written by John Young

John K. Young is a retired professor of Cell Biology. He worked in the Department of Anatomy at Howard University College of Medicine in Washington, DC for 35 years, teaching his students about cells. During his career, Dr. Young published scientific articles about a part of the brain called the hypothalamus and also wrote a number of books about cells and about the brain.


Is NADH found in all type of cell in human body? - Biology

Adult Stem Cells (ASCs):

ASCs are undifferentiated cells found living within specific differentiated tissues in our bodies that can renew themselves or generate new cells that can replenish dead or damaged tissue.  You may also see the term “somatic stem cell” used to refer to adult stem cells.  The term “somatic” refers to non-reproductive cells in the body (eggs or sperm).  ASCs are typically scarce in native tissues which have rendered them difficult to study and extract for research purposes.

Resident in most tissues of the human body, discrete populations of ASCs generate cells to replace those that are lost through normal repair, disease, or injury. ASCs are found throughout ones lifetime in tissues such as the umbilical cord, placenta, bone marrow, muscle, brain, fat tissue, skin, gut, etc.   The first ASCs were extracted and used for blood production in 1948.  This procedure was expanded in 1968 when the first adult bone marrow cells were used in clinical therapies for blood disease. 

Studies proving the specificity of developing ASCs are controversial some showing that ASCs can only generate the cell types of their resident tissue whereas others have shown that ASCs may be able to generate other tissue types than those they reside in.  More studies are necessary to confirm the dispute.

Types of Adult Stem Cells:

    • Hematopoietic Stem Cells (Blood Stem Cells)
    • Mesenchymal Stem Cells
    • Neural Stem Cells
    • Epithelial Stem Cells
    • Skin Stem Cells

    Embryonic Stem Cells (ESCs):

    During days 3-5 following fertilization and prior to implantation, the embryo (at this stage, called a blastocyst), contains an inner cell mass that is capable of generating all the specialized tissues that make up the human body.  ESCs are derived from the inner cell mass of an embryo that has been fertilized in vitro and donated for research purposes following informed consent.  ESCs are not derived from eggs fertilized in a woman’s body. 

    These pluripotent stem cells have the potential to become almost any cell type and are only found during the first stages of development.  Scientists hope to understand how these cells differentiate during development.  As we begin to understand these developmental processes we may be able to apply them to stem cells grown in vitro and potentially regrow cells such as nerve, skin, intestine, liver, etc for transplantation. 

    Induced Pluripotent Stem Cells (iPSCs)

    Induced pluripotent stem cells are stem cells that are created in the laboratory, a happy medium between adult stem cells and embryonic stem cells.  iPSCs are created through the introduction of embryonic genes into a somatic cell (a skin cell for example) that cause it to revert back to a “stem cell like” state.  These cells, like ESCs are considered pluripotent Discovered in 2007, this method of genetic reprogramming to create embryonic like cells, is novel and needs many more years of research before use in clinical therapies.  


    University of Nebraska Medical Center
    42nd and Emile, Omaha, NE 68198
    402-559-4000 | Contact Us


    Enzymes

    Reinhard Renneberg , . Vanya Loroch , in Biotechnology for Beginners (Second Edition) , 2017

    2.3 The Role of Cofactors in Complex Enzymes

    Not all enzymes consist exclusively of protein, as does lysozyme. Many include additional chemical components or cofactors which serve as tools. Such enzymes are known as qualified enzymes and have more complicated reaction mechanisms.

    Cofactors can consist of one or more inorganic ions (such as Fe 3+ , Mg 2+ , Mn 2+ , or Zn 2+ ) or more complex organic molecules, known as coenzymes. Some enzymes require both types of cofactors.

    Coenzymes are organic compounds that bind to the active site of enzymes or near it. They modify the structure of the substrate or move electrons, protons, and chemical groups back and forth between enzyme and substrate, negotiating considerable distances within the giant enzyme molecule. When used up, they separate from the molecule.

    Many coenzymes are derived from vitamin precursors, which explains why we require a constant low-level supply of certain vitamins. One of the most essential coenzymes, NAD + (nicotinamide adenine dinucleotide), is derived from niacin. Most water-soluble vitamins of the vitamin B group act as coenzyme precursors very much like niacin.

    Otto Heinrich Warburg (1883–1970, Fig. 2.3 ) discovered the respiratory enzyme cytochrome oxidase ( Fig. 1.14 ) and NAD. His discovery and subsequent structural analysis was one of the shining hours of modern biochemistry. In the absence of niacin in the diet, certain enzymes (e.g., dehydrogenases) cannot work effectively in the body. The affected human will develop pellagra, a disease caused by vitamin B (niacin) deficiency. Otto Warburg developed an optical test making it possible to quantify reduced NADH at a wavelength of 340 nm (the oxidized NAD + does not absorb light at this wavelength). It was now possible to measure essential enzyme reactions, such as the detection of glucose using glucose dehydrogenase (see Chapter: Analytical Biotechnology and the Human Genome ).

    Figure 2.3 . Otto Heinrich Warburg (1883–1970) discovered the cofactor nicotinamide adenine dinucleotide (NAD) and respiratory enzymes containing iron, such as cytochrome oxidase (see Fig. 1.14 ). He was awarded the Nobel Prize in 1941.

    Nowadays, vitamins like B2 (riboflavin), B12 (cyanocobalamin), and C (ascorbic acid) are produced by the ton using biotechnological methods (see Chapter: White Biotechnology: Cells as Synthetic Factories ).

    Cofactors that are covalently bonded to the enzyme are called prosthetic groups. Flavin adenine dinucleotide acts as a prosthetic group for GOD. Peroxidase and cytochrome P-450 contain a heme group, as found in myoglobin and hemoglobin. The heme group itself consists of a porphyrin ring incorporating an iron ion in its center.

    Coenzymes, by contrast, have only loose bonds, and, just like substrates, they undergo changes in the binding process and are used up. Unlike substrates, however, they bind to a whole host of enzymes (e.g., NADH and NADPH of nearly all dehydrogenases) and are regenerated and recycled inside the cells (see Section 2.13 ). Enzymes that bind to the same coenzyme usually resemble each other in their chemical mechanisms.

    While we referred to the cofactors as “tools,” the protein section of the enzyme is the “craftsman” using these tools, who is responsible for their effectiveness. As always, craftsmen and tools rely on each other to achieve the best possible result.


    How Free-Energy Currency Works

    Coupled reactions are frequently used in the body to drive important biochemical processes. Separate chemical reactions may be added together to form a net reaction. The free-energy change ( D G) for the net reaction is given by the sum of the free-energy changes for the individual reactions. For example, the phosphorylation of glycerol is a necessary step in forming the phospholipids that comprise cell membranes. (Recall from the experiment, "Membranes and Proteins: Dialysis, Detergents, and Proton Gradients," that the phospholipids that form cell membranes are formed from glycerol with a phosphate group and two fatty-acid chains attached.) This step actually consists of two reactions: (1) the phosphorylation of glycerol, and (2) the dephosphorylation of ATP (the free-energy-currency molecule). The reactions may be added as shown in Equations 2-4, below:

    ATP is the most important "free-energy-currency" molecule in living organisms (see Figure 2, below). Adenosine triphosphate (ATP) is a useful free-energy currency because the dephosphorylation reaction is very spontaneous i.e., it releases a large amount of free energy (30.5 kJ/mol). Thus, the dephosphorylation reaction of ATP to ADP and inorganic phosphate (Equation 3) is often coupled with nonspontaneous reactions (e.g., Equation 2) to drive them forward. The body's use of ATP as a free-energy currency is a very effective strategy to cause vital nonspontaneous reactions to occur.

    Figure 2

    This is the two-dimensional (ChemDraw) structure of ATP, adenosine triphosphate. The removal of one phosphate group (green) from ATP requires the breaking of a bond (blue) and results in a large release of free energy. Removal of this phosphate group (green) results in ADP, adenosine diphosphate.

    As these coupled reactions (e.g., Equations 2-4) occur, we use up ATP. In a typical cell, an ATP molecule is used within a minute of its formation. During strenuous exercise, the rate of utilization of ATP is even higher. Hence, the supply of ATP must be regenerated. We consume food to provide energy for the body, but the majority of the energy in food is not in the form of ATP. The body utilizes energy from other nutrients in the diet to produce ATP through oxidation-reduction reactions (Figure 3).

    Figure 3

    This flowchart shows that the energy used by the body for its many activities ultimately comes from the chemical energy in our food. The chemical energy in our food is converted to reducing agents (NADH and FADH2). These reducing agents are then used to make ATP. ATP stores chemical energy, so that it is available to the body in a readily accessible form.

    How is Food Used to Make the Reducing Agents Needed for the Production of ATP?

    To make ATP, energy must be absorbed. This energy is supplied by the food we eat, and then used to synthsize two reducing agents, NADH and FADH2 that are needed to produce ATP. One of the principal energy-yielding nutrients in our diet is glucose (see structure in Table 1 in the blue box below), a simple six-carbon sugar that can be broken down by the body. When the chemical bonds in glucose are broken, free energy is released. The complete breakdown of glucose into CO2 occurs in two processes: glycolysis and the citric-acid cycle. The reactions for these two processes are shown in the blue box below.

    Reactions for Glycolysis and the Citric-Acid Cycle

    The first process in the breakdown of glucose is glycolysis (Equation 5), in which glucose is broken down into two three-carbon molecules known as pyruvate. The pyruvate is then converted to acetyl CoA (acetyl coenzyme A) and carbon dioxide in an intermediate step (Equation 6). In the second process, known as the citric-acid cycle (Equation 7), the three-carbon molecules are further broken down into carbon dioxide. The energy released by the breakdown of glucose ( red ) can be used to phosphorylate (add a phosphate group to) ADP, forming ATP ( green ). The net reactions for glycolysis (Equation 5) and the citric-acid cycle (Equation 7) are shown below. (Note: In the equations below, glucose and the carbon compounds into which glucose is broken are shown in red energy-currency molecules are shown in green, and reducing agents used in the synthesis of ATP are shown in blue.)

    Glycolysis

    Hence the overall reaction for the oxidation of NADH paired with the reduction of O2 has a negative change in free energy ( D G = -220 kJ ) i.e., it is spontaneous. Thus, the higher the electrical potential of a reduction half reaction, the greater the tendency for the species to accept an electron.

    Just as in the box above, the electrical potential for the overall reaction (electron transfer) between two electron carriers is the sum of the potentials for the two half reactions. As long as the potential for the overall reaction is positive the reaction is spontaneous. Hence, from Table 2 below, we see that cytochrome c1 (part of the cytochrome reductase complex, #3 in Figure 9) can spontaneously transfer an electron to cytochrome c (#4 in Figure 9). The net reaction is given by Equation 16, below.

    reduced cytochrome c1 --> oxidized cytochrome c1 + e - e oxidation = - .220 V (14)
    oxidized cytochrome c + e - --> reduced cytochrome c e reduction = .250 V (15)
    NET: reduced cyt c1 + oxidized cyt c -->
    oxidized cyt c1 + reduced cyt c
    e rxn = 0.030 V (16) Spontaneous

    We can also see from Table 2 that cytochrome c1 cannot spontaneously transfer an electron to cytochrome b (Equation 19):

    reduced cyt c1 --> oxidized cyt c1 + e - e oxidation = - .220 V (17)
    oxidized cyt b + e - --> reduced cyt b e reduction = - 0.34 V (18)
    NET: reduced cyt c1 + oxidized cyt c -->
    oxidized cyt c1 + reduced cyt c
    e rxn = - 0.56 V (19) NOT Spontaneous

    Table 2 lists the reduction potentials for each of the cytochrome proteins (i.e., the last three steps in the electron-transport chain before the electrons are accepted by O2) involved in the electron-transport chain. Note that each electron transfer is to a cytochrome with a higher reduction potential than the previous cytochrome. As described in the box above and seen in Equations 14-19, an increase in potential leads to a decrease in D G (Equation 13), and thus the transfer of electrons through the chain is spontaneous.

    Complex Name

    Half Reaction

    Reduction Potential

    (also known as cytochrome b-c1 complex)

    (3 in Figure 9)

    (4 in Figure 9)

    (5 in Figure 9)

    Table 2

    To view the cytochrome molecules interactively using RASMOL, please click on the name of the complex to download the pdb file.

    Hence, the electron-transport chain (which works because of the difference in reduction potentials) leads to a large concentration gradient for H + . As we shall see below, this huge concentration gradient leads to the production of ATP.

    Questions on Electron Carriers: Steps in the Electron-Transport Chain Reduction Potentials and Relationship to Free Energy

    • Briefly, explain why electrons travel from NADH-Q reductase, to ubiquinone (Q), to cytochrome reductase, rather than in the opposite direction.
    • One result of the transfer of electrons from NADH-Q reductase down the electron transport chain is that the concentration of protons (H + ions) in the intermembrane space is increased. Could cells move protons (H + ions) from the matrix to the intermembrane space without transporting electrons? Why or why not?

    ATP Synthetase: Production of ATP

    We have seen that the electron-transport chain generates a large proton gradient across the inner mitochondrial membrane. But recall that the ultimate goal of oxidative phosphorylation is to generate ATP to supply readily-available free energy for the body. How does this occur? In addition to the electron-carrier proteins embedded in the inner mitochondrial membrane, a special protein called ATP synthetase (Figure 9, the red-colored protein) is also embedded in this membrane. ATP synthetase uses the proton gradient created by the electron-transport chain to drive the phosphorylation reaction that generates ATP (Figure 7c).

    ATP synthetase is a protein consisting of two important segments: a transmembrane proton channel, and a catalytic component located inside the matrix. The proton-channel segment allows H + ions to diffuse from the intermembrane space, where the concentration is high, to the matrix, where the concentration is low. Recall from the Kidney Dialysis tutorial that particles spontaneously diffuse from areas of high concentration to areas of low concentration. Thus, since the diffusion of protons through the channel component of ATP synthetase is spontaneous, this process is accompanied by a negative change in free energy (i.e., free energy is released). The catalytic component of ATP synthetase has a site where ADP can enter. Then, using the free energy released by the spontaneous diffusion of protons through the channel segment, a bond is formed between the ADP and a free phosphate group, creating an ATP molecule. The ATP is then released from the reaction site, and a new ADP molecule can enter in order to be phosphorylated.

    Questions on ATP Synthetase: Production of ATP


    Discussion

    There are a number of factors that support our underlying hypothesis that the fluorescence measured in this study originates from the coenzyme NADH. In breast epithelial cells (MCF10A pZIP), there are three principle fluorophores: NADH, flavin adenine dinucleotide (FAD), and tryptophan, the spectral characteristics of which have been characterized previously ( 28). At the equivalent single-photon excitation wavelength used in this study (370 nm), the fluorescence spectrum measured from the MCF10A pZIP cells has the same spectrum as NADH ( 28). Second, it is well known that the addition of KCN to cells will inhibit electron transport and thus the ratio of NADH and NAD + ( 29). This will cause an increase in the fluorescence intensity of NADH. In the KCN study reported in this article, the fluorescence intensity increased by a factor of ∼1.3 after addition of KCN, which is consistent with what is expected ( 25– 27). Finally, the average fluorescence lifetimes recorded for the two fluorescence emitting species in the MCF10A pZIP cells in this study are consistent with that reported for free and bound NADH in the literature ( 17, 24, 25). It is unlikely that tryptophan will contribute to the fluorescence at a two-photon excitation wavelength of 740 nm, because it has a single-photon absorption peak of ∼280 nm ( 30). Efficient two-photon excitation of tryptophan can be achieved at a wavelength of ∼560 nm ( 30), which is far from the 740-nm excitation used in our experiments. This, combined with the fact that compared with NADH and FAD, tryptophan has a particularly low two-photon absorption cross-section ( 31), would deem it an improbable source of the fluorescence measured in this study. It should also be pointed out that the measured fluorescence lifetimes in Results (Effect of cell confluence on the lifetimes and ratio of free and protein-bound NADH) are shorter in all cases by a factor of ∼2 compared with that reported for FAD ( 24).

    An increase in cell confluence increases cell crowding and thus likely decreases oxygen consumption. A decrease in oxygen consumption should in turn increase the NADH/NAD + ratio. It can be inferred from the results of the control study that a decrease in the fluorescence lifetime of both the free and protein-bound components of NADH and a decrease in the contribution of protein-bound NADH with increasing confluence is related to an increase in the NADH/NAD + ratio.

    KCN treatment inhibits electron transport ( 29). This should also result in an increase in the NADH/NAD + ratio. Thus, it can be inferred that the decrease in the fluorescence lifetime of the free and protein-bound components of NADH of KCN-treated cells at confluences of 25,000 and 100,000 and a decrease in the contribution of protein-bound NADH of KCN-treated cells at all three confluences is related to an increased NADH/NAD + ratio. This corroborates the experimental findings from the control study. However, the lack of statistically significant differences between the fluorescence lifetime of protein-bound and free NADH in the KCN-treated and control cells at a confluence of 1,000,000 is not clear at this time. It is speculated that KCN treatment does not further affect the NADH/NAD + ratio of the fully confluent cells.

    Serum is full of precursors for both glycolysis (such as glucose) and the citric acid cycle (such as pyruvate), which under normal conditions are essential for cellular metabolism ( 29). Therefore, removal of serum is expected to slow down cellular metabolism and shift the reduction-oxidation ratio towards increased NADH. Thus, it is not surprising that serum-starved and KCN-treated cells yield similar changes in the fluorescence lifetimes and relative contributions of free and protein-bound NADH relative to that of control cells.

    Previous studies of NADH in cell culture found that the effective lifetime of NADH (mean lifetime of free and protein-bound NADH) decreases with oxidative stress induced with the respiratory chain inhibitor rotenone ( 17). KCN, like rotenone, is a respiratory chain inhibitor and has a similar effect on the lifetime of free and protein-bound NADH in our study. In another study, multiphoton microscopy of lifetime and anisotropy in brain tissue slices revealed lifetime shortening with hypoxia and this was attributed to a redistribution of protein-bound NADH to enzyme binding sites with shorter lifetimes and due to a decrease in the lifetime of free NADH ( 32). KCN treatment, like hypoxia, caused a decrease in the lifetime of both protein-bound and free NADH in our study. Both KCN and hypoxia cause an increase in the NADH/NAD + ratio (KCN inhibits the transfer of electrons from NADH to oxygen and hypoxia reduces the supply of oxygen that can accept electrons from NADH). Thus, the lifetime shortening caused by both hypoxia and KCN is likely related to an increased ratio of NADH/NAD + .

    In addition to lifetime imaging, cell proliferation was determined using propidium iodide staining and flow cytometry in the serum starved and control cells at the three different confluences. Results averaged from a total of two independent experiments showed that there is negligible difference in the percentage of cells in S-G2-M phase for the control or serum starved cells at different confluences. However, a parallel imaging experiment done on cells plated on the same day and under the same conditions showed a decrease in the fluorescence lifetime of free and protein-bound NADH and a decrease in the contribution of protein-bound NADH with increased plating density and serum starvation (consistent with Figs. 4 and 5). This additional study indicates that the observed changes in the fluorescence lifetime variables are not due to changes in cell proliferation.

    In the current study, lifetime analyses were done on several pixels per cell (which were arbitrarily identified from the cytoplasmic region). To test the robustness of this approach, lifetime analyses were also done on all pixels from the cytoplasmic region of the cell. Both methods of analysis yielded very similar results (this was only evaluated on images collected from control cells at different confluences) thus indicating that the method of analysis does not affect the conclusions reported in this study.


    Difference Between Pluripotent and Totipotent

    The entire human body is made up of over 200 cell types. All these cell types basically arise from a single type of cell called ‘stem cells’. Stem cells are defined as the cells having the ability to self-renewal and to differentiate into one or all of the more than 200 cell types that make the entire body. There are four different stem cells found in the body those are unipotent, which gives rise to only a single cell type, multipotent, which produces a limited number of cell types, totipotent, which forms all types of cell at any stage of development, and pluripotent, which gives rise to all the cell types in the adult body. Out of these four types, pluripotent and totipotent have the ability to form any cell type in different stages of human development.

    Pluripotent

    Pluripotent cells are the stem cells that give rise to any kind of cells developing from all three embryonic germ layers including endoderm, ectoderm and mesoderm. That means pluripotent cells can form any type of cell in the adult body. Pluripotent cells have the same capabilities of totipotent cells with one exception they do not form trophoblast. Because of this exception, pluripotent cells cannot develop into full human being.

    Totipotent cell is defined as the cell having the ability to create all types of cells in an organism at any stage of development. Unlike the other stem cells, totipotent stem cells are extremely rare. In humans, only the first eight cells that form zygote are only totipotent as they have the ability to turn into any type of cell during the embryonic development. Therefore, unlike the other stem cells, totipotent cells have the ability to form an entire human being.

    What is the difference between Pluripotent and Totipotent?

    • Totipotent cells have the ability to form any cell type at any stage of development, whereas pluripotent cells have the ability to form any cell type after first few cleavages of embryo.

    • All the cells including pluripotent cells are derived from totipotent cells during the embryonic development.

    • Unlike pluripotent, totipotent is extremely rare.

    • Unlike the pluripotent cells, totipotent cells have the ability to form an entire human being.

    • Totipotent forms the trophoblast, whereas pluripotent does not.

    • Totipotent cells have potential of becoming an embryo, whereas pluripotent cells do not.


    Materials and Methods

    Media and Strains. Mtb H37Rv was a gift from C. Imperatrice (Clinical Infectious Diseases, Hospital of the University of Pennsylvania) and Mycobacterium smegmatis Mc 2 155 was obtained from V. Mizrahi (National Health Laboratory Service, Johannesburg). Bacteria were cultured in 7H9 broth supplemented with 10% oleic acid-albumin-dextrose catalase/0.5% glycerol/0.05% Tween 80. Solid agar (15 g/liter) was added to liquid media to create solid media as required.

    Spectroscopy. Aerobically grown, gamma-irradiated Mtb H37Rv whole cells were obtained from John Belisle (Colorado State University, Fort Collins) under National Institute of Allergy and Infectious Diseases Contract NO1-A1-75320. Cells (12 g of wet weight) were washed with 10 ml of phosphate–digitonin buffer (50 mM sodium phosphate, pH 7.4, containing 0.1% digitonin) and resuspended in the same buffer. The bacteria were lysed by passage through a French press at 14,000 kPa, and the suspension was centrifuged at 12,000 × g for 15 min to remove cellular debris. The supernatant was further clarified by centrifugation at 50,000 × g for 50 min. This supernatant was used as the membrane-containing fraction (or cell-free extract) for spectral studies. To isolate cell membranes, the 50,000 × g supernatant was centrifuged at 150,000 × g for 60 min. The resulting pellet was washed with phosphate–digitonin buffer, resuspended, and then centrifuged at 150,000 × g for 60 min a second time. Protein concentrations were determined by bicinchoninic assay (BCA, Pierce) by using BSA as the standard. UV-visible spectra were recorded at room temperature on a Cary 4E dual-beam spectrophotometer (Varian) by using a scan speed of 120 nm/min and a slit width of 1 nm. Reduction of 21.3 mg/ml membrane protein was accomplished by a 3-min incubation with 5 mM NADH. To illustrate the spectral perturbation due to CO binding of terminal oxidases, CO gas was gently bubbled into the sample for 5 min before NADH reduction. For the samples containing trifluoperazine (TPZ), drug was added to the sample and incubated for 5 min before the addition of NADH.

    Amperometric Assay. Oxygen consumption by respiration of Mtb cytoplasmic membranes was measured by a polarographic method. Mtb membranes (50 mg/ml) were suspended in 0.1 M potassium phosphate buffer (pH 7.4), and 10 mM NADH was added to initiate the respiration. Subsequently, 1 mM TPZ or vehicle and then 10 mM sodium ascorbate plus 1 mM 3,3,5,5-tetramethylphenylenediamine (TMPD) were added into the reaction mixture, as shown in Fig. 2B .

    The reduced and the CO-binding pigments of H37Rv cell extracts. (A) NADH-reduced minus air-oxidized difference spectrum. Reduction was accomplished 5 min after the addition of 10 mM NADH. (B) (NADH-reduced plus CO) minus (NADH-reduced) difference spectrum. To show the spectral perturbation due to CO binding, CO was bubbled through the sample before reduction.

    NADH-Quinone Oxidoreductase Activity Assay. For the NADH-quinone oxidoreductase activity assay of Mtb membrane, the membrane was dialyzed against 100 mM potassium phosphate buffer (pH 7.5) containing 1 mM EDTA at 4°C overnight to remove endogenous reducing substrates. The Mtb membrane (3 mg/ml) was suspended in the phosphate buffer containing 10 mM KCN and 100 μM menaquinone 1 (MK1) or Q1. Drug or vehicle was added as desired, and the reaction mixture was incubated for 5 min at 30°C. The reaction was initiated by adding 100 μM NADH, and the absorbance change at 340 nm was monitored by using a custom-built 1098 spectrophotometer (Hitachi, Tokyo). NADH oxidation did not occur in the absence of membrane protein or quinone. For the purified Ndh or NdhA, NADH-quinone oxidoreductase activity was measured in 50 mM sodium phosphate buffer (pH 7.0) containing 150 mM NaCl and 2% (wt/vol) potassium cholate. The reaction mixture contained 50 μMQ2/1 μg of purified enzyme, and it was initiated by adding 100 μM NADH and monitored by following the absorbance change at 340–400 nm with a PerkinElmer 557 double-beam dual-wavelength spectrophotometer at 37°C. For chlorpromazine (CPZ)-inhibition assays, ≈1 μg of purified enzyme and 50 μM Q2 were incubated in the reaction mixture with various amounts of CPZ for 2–3 min at room temperature before the addition of NADH.

    Expression and Purification of Mtb ndh and ndhA in Escherichia coli. The Mtb ndh and ndhA genes were amplified by PCR using the Mtb genomic DNA and template, and the products were cloned in pET16b expression plasmid, which contains an N-terminal His-6-tag. Mtb Ndh and NdhA were reproducibly expressed in E. coli strain BL21(DE3), and the products were localized to the cytoplasmic membrane. The E. coli cell suspension in buffer A (50 mM Hepes, pH 7.0/100 mM KCl/1 mM PMSF) was passed through a French press twice at 14,000 kPa. Next, the suspension was centrifuged at 12,000 × g for 15 min to remove unbroken cells and debris. The resulting supernatant was ultracentrifuged at 130,000 × g for 30 min. The membrane pellet was resuspended in buffer A and ultracentrifuged at 130,000 × g again for 15 min. The cytoplasmic membranes thus obtained were resuspended in buffer A, 2% (wt/vol) sodium cholate (pH 7.2) was added, and the mixture was solubilized by incubation at 4°C for 1 h. The mixture was next ultracentrifuged at 130,000 × g for 30 min, and solubilized proteins in the supernatant were collected. We added 1 ml of buffer A-equilibrated Talon (BD Biosciences) resin, and the mixture was incubated at 4°C for 1 h. The resin was transferred into a column was washed with 30 ml of buffer A with 1% cholate, followed by 50 ml of buffer A with 1% cholate and 10 mM imidazole. Last, the bound proteins were eluted with buffer A with 1% cholate and 100 mM imidazole. Purified recombinant Mtb Ndh and NdhA were used immediately for activity assays.

    Determination of the Minimum Inhibitory Concentration (MIC) by the Bactec MGIT 960. The MIC for each compound was determined by the Bactec MGIT (mycobacteria growth indicator tube) 960 system. Mtb H37Rv was grown in Middlebrook 7H9 broth until the growth index (GI) reached 75 (GI is a scale in the Bactec system (Becton Dickinson), which reflects the amount of growth) and was then diluted 2,500-fold and used as the inocula. The vials were dispensed with different dilutions of drug to reach final concentrations ranging 0.2–26 μg/ml. All of the drug-containing vials were inoculated with 0.5 ml of the bacterial suspensions prepared as described above. Six drug-free controls were included with each test three were inoculated with 0.5 ml of the suspension, and the remaining three were inoculated with 0.5 ml of a 1:100 dilution of the suspension. The vials were incubated at 37°C and read in a Bactec 960 reader every day until the GI in the control diluted 1:100 reached 75, with an increase in the GI of at least 10 for 3 consecutive days. The time to positive was 10 days for the undiluted control. MIC was defined as the lowest concentration of the drug that caused an increase in the GI equal to or less than the increase in the GI of the control diluted 1:100.

    Animal Studies: Test of Phenothiazine Efficacy in a BALB/c Mouse Model of Acute Mtb Infection. Each treatment group consisted of five female mice intranasally infected with 10 2 colony-forming units (cfu) of H37Rv Mtb on day 0. INH, RIF, or compound 1 was given orally on days 1–11. After 11 days of the indicated treatment, mice were killed, and the lungs and spleens were aseptically collected. Serial 10-fold dilutions were prepared of tissue homogenates in 7H9 media and were plated on 7H11 agar at 37°C for cfu enumeration after 4 weeks of incubation.


    Adult stem cell

    Adult stem cells are undifferentiated cells found throughout the body that divide to replenish dying cells and regenerate damaged tissues.

    Also known as somatic stem cells, they can be found in children, as well as adults.

    Research into adult stem cells has been fueled by their abilities to divide or self-renew indefinitely and generate all the cell types of the organ from which they originate &mdash potentially regenerating the entire organ from a few cells.

    Unlike embryonic stem cells, the use of adult stem cells in research and therapy is not controversial because the production of adult stem cells does not require the destruction of an embryo.

    Adult stem cells can be isolated from a tissue sample obtained from an adult.

    They have mainly been studied in humans and model organisms such as mice and rats.

    The rigorous definition of a stem cell requires that it possesses two properties: Self-renewal - the ability to go through numerous cycles of cell division while maintaining the undifferentiated state.

    Multipotency or multidifferentiative potential - the ability to generate progeny of several distinct cell types, for example both glial cells and neurons, opposed to unipotency - restriction to a single-cell type.

    Some researchers do not consider this property essential and believe that unipotent self-renewing stem cells can exist.

    Stem Cell Treatments Due to the ability of adult stem cells to be harvested from the patient, their therapeutic potential is the focus of much research.

    Adult stem cells, similar to embryonic stem cells, have the ability to differentiate into more than one cell type, but unlike embryonic stem cells they are often restricted to certain lineages.

    The ability of a stem cell of one lineage to become another lineage is called transdifferentiation.

    Different types of adult stem cells are capable of transdifferentiation more than others, and for many there is no evidence of its occurrence.

    Consequently, adult stem therapies require a stem cell source of the specific lineage needed and harvesting and or culturing them up to the numbers required is a challenge.

    Adult stem cell treatments have been used for many years to treat successfully leukemia and related bone/blood cancers through bone marrow transplants.


    Layers of the Skin

    The skin is composed of 3 layers namely:

    3 Layers of the Skin (Source: Wikimedia)

    1. Epidermis

    This is the outer most superficial layer which is made up of 5 inner layers. They are Stratum basale, Stratum spinosum, Stratum granulosum, Stratum lucidum, and Stratum corneum.

    In general, skin’s epidermal layer is subjected to constant wear & tear from external factors such as sunlight, chemicals such as soaps, and pollution.

    2. Dermis

    Dermis cover the significant portion of the skin’s layer. The dermis layer has connective tissues, blood vessels, oil and sweat glands, nerves, hair follicles, and other structures.

    The dermis is made up of two inner layers namely – a thin upper layer called the papillary dermis, and a thick lower layer called the reticular dermis.

    3. Subcutaneous Layer

    Subcutaneous layer is also known as hypodermis. The hypodermis is the innermost layer of the skin. This layer hosts fat and connective tissues that house larger blood vessels and various nerves.

    The primary function of the hypodermis is to act as an insulator for regulating the body temperature.

    Why are tattoos permanent though skin cells die and get replaced?

    The answer lies hidden in the second layer of the skin (Dermis). The permanent tattoo ink is injected till the dermis layer so that it stays permanent. If it is put on the outer layer, then it will be worn out as time progresses. That is why permanent tattooing is always a harrowing and painful experience.



Comments:

  1. Samulabar

    Just what you need. An interesting topic, I will participate.

  2. Goltirr

    It is time to become reasonable. It is time to come in itself.

  3. Virgilio

    You are mistaken. Write to me in PM.

  4. Tearlach

    You have hit the mark. Thought good, it agree with you.

  5. Abd Er Rahman

    have understood Quickly))))



Write a message