The goal of this project was to identify deoxypyrimidine salvage pathways used to maintain dNTP pools in brain mitochondria, with a view to understanding the mechanisms by which the central nervous system displays a relative resistance to AZT in both treatment and toxicity when compared to other organ systems. These metabolic pathways are increasingly relevant not only to the treatment of HIV/AIDS, but also to targeting the role of mitochondrial dysfunction in developing new treatment options for neurological degenerative diseases and primary neoplasms of the CNS.
Expression, purification and spectroscopic characterization of the cytochrome...John Clarkson
K.J. McLean, M.R. Cheesman, S.L. Rivers, A. Richmond, D. Leys, S.K. Chapman, G.A. Reid, N.C. Price, S.M. Kelly, J. Clarkson, W.E Smith & A.W. Munro, “Expression, Purification and Spectroscopic Characterization of the Cytochrome P450 CYP121 from Mycobacterium Tuberculosis”, J. Inorganic Biochemistry, 91, 527-541, 2002.
— Eukaryotic cells contain two organelles originally derived from endosymbiotic bacteria: mitochondria and plastids (only plants). In eukaryotes, (owner mitochondria and chloroplast) ATP synthase complex is located in the inner membrane of mitochondria, and thylakoids membrane of chloroplast. ATP synthesis utilization and provision of both ADP and Pi need to be fine – tuned for optimal ATP synthase activity. Mitochondria and chloroplast have their DNA. The vast majority of mitochondrial and plastid proteins are encoded in the nucleus, synthesized by cytosolic ribosomes and subsequently imported into the organelles via active protein transport systems.
Expression, purification and spectroscopic characterization of the cytochrome...John Clarkson
K.J. McLean, M.R. Cheesman, S.L. Rivers, A. Richmond, D. Leys, S.K. Chapman, G.A. Reid, N.C. Price, S.M. Kelly, J. Clarkson, W.E Smith & A.W. Munro, “Expression, Purification and Spectroscopic Characterization of the Cytochrome P450 CYP121 from Mycobacterium Tuberculosis”, J. Inorganic Biochemistry, 91, 527-541, 2002.
— Eukaryotic cells contain two organelles originally derived from endosymbiotic bacteria: mitochondria and plastids (only plants). In eukaryotes, (owner mitochondria and chloroplast) ATP synthase complex is located in the inner membrane of mitochondria, and thylakoids membrane of chloroplast. ATP synthesis utilization and provision of both ADP and Pi need to be fine – tuned for optimal ATP synthase activity. Mitochondria and chloroplast have their DNA. The vast majority of mitochondrial and plastid proteins are encoded in the nucleus, synthesized by cytosolic ribosomes and subsequently imported into the organelles via active protein transport systems.
A quick revision of Carbohydrate metabolism with case- based discussions and ...Namrata Chhabra
Absorption of glucose, pathways of glucose utilization, TCA Cycle, Cori cycle, glycogen metabolism, metabolism of fructose and galactose, and disorders of carbohydrate metabolism
Alcohol induced metabolic alterations - A Case based discussionNamrata Chhabra
I shall proceed through a case based discussion and highlight a few of the metabolic alterations that have been found in the patient under study and of course these are the commonest metabolic alterations that change the whole scenario.
The objective of my discussion is to provide you with a solid foundation of alcohol induced metabolic alterations. The knowledge thus acquired will help you to make spontaneous diagnosis and plan the relevant treatment in the clinical settings.
The case details are with you. There are 3 questions related to the problems the patient is having in this case, and there are 4 options for each of the questions. Using your prior knowledge, try to select the most appropriate answer, you have only one minute to solve the answer.
Mitochondrial dysfunction has been identified as an important factor in many diseases and conditions beyond primary mitochondrial disease, including autism, ALS, Parkinson's, Alzheimer's, and diabetes. Exposure to toxins via medication, lifestyle, and the environment may lead to mitochondrial dysfunction, cell damage and organ dysfunction. Join us this month with Dr. Kendall Wallace, Ph.D., DABT, ATS to learn more about mitochondrial disorders which may be acquired by or aggravated by toxins. Dr. Wallace’ primary area of research is the mechanisms of involvement of mitochondria in the origination of metabolic diseases which may be caused by toxicity. - See more at: http://www.mitoaction.org/blog/mitochondrial-disease-and-toxins#sthash.PyxCUqlF.dpuf
A quick revision of Carbohydrate metabolism with case- based discussions and ...Namrata Chhabra
Absorption of glucose, pathways of glucose utilization, TCA Cycle, Cori cycle, glycogen metabolism, metabolism of fructose and galactose, and disorders of carbohydrate metabolism
Alcohol induced metabolic alterations - A Case based discussionNamrata Chhabra
I shall proceed through a case based discussion and highlight a few of the metabolic alterations that have been found in the patient under study and of course these are the commonest metabolic alterations that change the whole scenario.
The objective of my discussion is to provide you with a solid foundation of alcohol induced metabolic alterations. The knowledge thus acquired will help you to make spontaneous diagnosis and plan the relevant treatment in the clinical settings.
The case details are with you. There are 3 questions related to the problems the patient is having in this case, and there are 4 options for each of the questions. Using your prior knowledge, try to select the most appropriate answer, you have only one minute to solve the answer.
Mitochondrial dysfunction has been identified as an important factor in many diseases and conditions beyond primary mitochondrial disease, including autism, ALS, Parkinson's, Alzheimer's, and diabetes. Exposure to toxins via medication, lifestyle, and the environment may lead to mitochondrial dysfunction, cell damage and organ dysfunction. Join us this month with Dr. Kendall Wallace, Ph.D., DABT, ATS to learn more about mitochondrial disorders which may be acquired by or aggravated by toxins. Dr. Wallace’ primary area of research is the mechanisms of involvement of mitochondria in the origination of metabolic diseases which may be caused by toxicity. - See more at: http://www.mitoaction.org/blog/mitochondrial-disease-and-toxins#sthash.PyxCUqlF.dpuf
Describes the Classification of standards, standard-setting models and the Hofsee method of scaling, as employed in Medical University of Americas, as per USMLE guidelines.
What 33 Successful Entrepreneurs Learned From FailureReferralCandy
Entrepreneurs encounter failure often. Successful entrepreneurs overcome failure and emerge wiser. We've taken 33 lessons about failure from Brian Honigman's article "33 Entrepreneurs Share Their Biggest Lessons Learned from Failure", illustrated them with statistics and a little story about entrepreneurship... in space!
Objective: To investigate the changes in the retina due to deltamethrin toxicity and the process in cell inflammation and apoptosis.
Study Design: Sixteen Wistar albino rats were randomly divided into two groups as control (n=8) and deltamethrin (n=8) groups. Saline was given to the control group, and 0.5 mL of 5 mg/kg deltamethrin was given to the deltamethrin group for 14 days each. Blood was collected for biochemical analysis. Retinal tissue was processed for histological examination.
Results: Compared to the control group, MDA levels were high while GSH and CAT levels were low in the deltamethrin group. Histopathological analysis showed spaces between the pigment epithelium, irregularity in the delimiting membrane, degenerated ganglion, cone and bacillus cell, pyknotic nuclei, thinned inner limitation membrane, and thickened vascular wall. The control group showed FAS expression in the pigment layer limiting membranes, in the nuclei of many cone and bacillus cells, and ganglion cells in the control group sections. In the deltamethrin group, FAS expression was observed in the inner and outer limiting membranes of the pigment epithelium, cone and bacillus cells, and ganglion cell nuclei. In the control group, negative NOS expression in the pigment epithelium and outer limiting membranes, internal limitation membrane, and ganglion cells in the cone and bacillus cell nuclei were observed. In the deltamethrin group, NOS expression was positive in the pigment epithelium, cone and bacillus, and ganglion cell nuclei.
Conclusion: We suggest that deltamethrin toxicity induced apoptotic process due to increased inflammation in the retina and may cause visual impairment as a result of neural damage.
Keywords: deltamethrin, FAS, insecticides, NOS, nitric oxide synthase, retina
Metabolism of 5-FU, deoxyuridine, and dUTP in Brain Mitochondria:kathleenmccann
5-FU (5-flourouracil) is currently in clinical trials for treatment of gliomas.1 In neoplastic cells, this drug is metabolized by two pathways. 5-FU is converted to
5-dUMP by thymidine phosphorylase and thymidine kinase. 5-FdUMP then competitively inhibits the conversion of dUMP to TMP by thymidylate synthase. This increases dUTP and depletes the TTP needed for DNA synthesis and repair, leading to apoptosis. 5-FdUMP is further phosphorylated to 5-FdUTP and is incorporated into DNA. In the second pathway, 5-FU is phosphorylated by actions of uridine phosphorylase and uridine kinase to 5-FUMP and then further phosphorylated to 5-FUTP and incorporated into RNA. However, the metabolism of 5-FU is as yet undefined in brain. 5-FU neurotoxicity may present as leukoencephalopathy, cerebellar signs, or acute demyelination. The use of thymidine infusions to reverse symptoms strongly suggested that the neurotoxicity is caused by dNTP pool imbalance. It is necessary to understand the underlying mechanisms to minimize neurotoxicity and judiciously use therapeutic thymidine.
Deoxyuridine metabolism in the brain mitochondria is not fully understood. While a small amount is needed for RNA synthesis, incorporation of dUTP into mitochondrial DNA (mtDNA) can lead to mutagenesis and apoptosis, especially as base excision repair is known to be inefficient in brain mtDNA.
These investigations aim to establish the activity of the enzymes necessary for regulation of dUTP pools in brain mitochondria: dUTPase, thymidine phosphorylase, and thymidine synthase. We also investigate the metabolism of 5-flourouricil (5-FU), the metabolite of which, 5-FdUMP, functions as a dUMP analogue.
Objective: To study the effects of resveratrol in neuronal structures in traumatic brain injury (TBI).
Study Design: Thirty rats were categorized as (1) control group (n=10), saline solution administered i.p. for 14 days, (2) TBI group (n=10), trauma induced by weight-drop model on brain, and (3) TBI+Resveratrol group (n=10), 15 minutes after injury the rats were given resveratrol (10 μmoL/kg/i.p.) for 14 days. At the end of the experiment the cerebellum was excised for routine paraffin tissue protocol. Blood samples were tested for serum biochemical markers (MDA, SOD, CAT, and GSH-x).
Results: SOD, GPx, and CAT values were lowest in the TBI group. MDA and histological scores of dilations in vessels, inflammation, degeneration in neurons, apoptosis in microglia, ADAMTS8, and GFAP expressions were highest in the TBI group. Sections of the control group showed normal cerebellar histology. The trauma group showed degenerated ganglion layer, pyknotic and apoptotic Purkinje cell nuclei. Vascular thrombus was seen in the substantia alba and substantia grisea. In the Trauma+Resveratrol group, most pa- thologies observed in the TBI group were improved. In the control group, GFAP protein was expressed in granular cells, axons, dendrites, Purkinje cells, and microglia cells. In the trauma group, increased GFAP expression was observed in glial processes, neurons, and Purkinje cells. In the Trauma+Resveratrol group, GFAP was expressed in molecular layer and glial processes. In the control group, ADAMTS-4 activity was observed in granulosa layer, glial cells, and Purkinje cells. In the trauma group, ADAMTS-4 expression was positive in Purkinje cells and glial cells. In the Trauma+ Resveratrol group, ADAMTS-4 was expressed in Purkinje cells, granular cells, and glial cells.
Conclusion: GFAP and ADAMTS-4 proteins may be involved in regeneration of damaged astroglial cells and other glial cells, Purkinje cells, and synaptic extensions. We suggest that antioxidative drugs such as resveratrol may be alternative target agents in neurological disease.
Keywords: ADAMTS-4, brain, cerebellum, GFAP, rat, resveratrol, traumatic brain injury
Hypothesis: Whether mitochondrial function are involved in irisin-induced cardioprotective effects.
Purpose of study : To determine the effect of irisin on myocardial I /R injury in the Langendorff perfused
heart and cultured myocytes.
Methods : Adult C 5 7 /BL 6 mice were treated with irisin ( 1 0 0 mg /kg ) or vehicle for 3 0 min to elicit
preconditioning. The isolated hearts were subjected to 30 min ischemia followed by 30 min reperfusion. Left
ventricular function was measured and infarction size were determined using by tetrazolium staining .
Western blot was employed to determine myocardial SOD - 1 , active -caspase 3 , annexin V, p 3 8 , and
phospho -p 3 8 . H 9c 2 cardiomyoblasts were exposed to hypoxia and reoxygenation for assessment of the
effects of irisin on mitochondrial respiration and mitochondrial permeability transition pore (mPTP).
Result: Irisin treatment improved ventricular function and marked reduction of infarct size. Notably, irisin
treatment increased SOD-1 and p38 phosphorylation, by suppressing levels of active-caspase 3, cleaved
PARP, and annexin V. Furthermore , irisin treatments suppressed the opening of mPTP, mitochondrial
swelling, and protected mitochondria function.
Conclusion: This result indicate that, irisin-induced cardioprotective effects is linked with the improvement of
mitochondrial function.
First aid for usmle step 1 with uworld and nbme notes sampleusmlematerialsnet
First Aid For USMLE Step 1 with Uworld and NBME Notes
Download Full book from > usmlematerials.net
Pages: 798
Series: First Aid for the USMLE Step 1
NBMEs and Uworld Notes are added to this file.
La hipotermia moderada se ha estado estudiando y se ha demostrado que ayuda a prevenir el daño neuronal. El siguiente seminario se realiza en unos experimentos con unas neuronas y unas proteínas proapoptoticas (PARP1) y genes de apoptosis (AIF). Se demostró que la hipotermia tiene la capacidad de reducir el daño provocado por estas proteínas y estos genes, dando así una mayor posibilidad de estudio para en un futuro lograr hacer terapias y medicinas para la conservación cerebral.
D cell-hypothesis-schizophrenia-importance-trace-amine-associated-receptor-ty...dynajolly
Mesolimbic dopamine (DA) hyperactivity is a well-known pathophysiological hypothesis of schizophrenia. The author shows a hypothesis to clarify the molecular basis of mesolimbic DA hyperactivity of schizophrenia. An immunohistochemical method was used to show D-neuron (trace amine (TA) neuron) decrease in the nucleus accumbens (Acc) of postmortem brains with schizophrenia. The striatal D-neuron decrease in schizophrenia and consequent (TAAR1) stimulation decrease onto terminals of midbrain ventral tegmental area (VTA) DA neurons induces mesolimbic DA hyperactivity of schizophrenia. Dysfunction of sub ventricular neural stem cells (NSC), located partially overlapping Acc is the cause of D-neuron decrease in Acc. DA hyperactivity, which inhibits NSC proliferation, causes disease progression of schizophrenia. The highlight is the rational that the “D-cell hypothesis (TA hypothesis) of schizophrenia” is a pivotal theory to link NSC dysfunction hypothesis to DA hypothesis. From a therapeutic direction, TAAR1 agonists, DA D2 antagonists, and neurotropic substances have potential to normalize mesolimbic DA hyperactivity. To develop novel therapeutic strategies, metabolisms of TAAR1 ligands, and NSC- and D-neuron-pathophysiology of neuropsychiatric illnesses should further be explored.
![METABOLISM OF DEOXYPYRIMIDINES AND DEOXYPYRIMIDINE ANTIVIRAL ANALOGS IN ISOLATED BRAIN MITOCHONDRIA Kathleen McCann 1 , David Williams 2 , and Edward McKee 1,2 1 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, South Bend, IN 46617 USA 2 Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 USA The goal of this project was to identify deoxypyrimidine salvage pathways used to maintain dNTP pools in brain mitochondria, with a view to understanding the mechanisms by which the central nervous system displays a relative resistance to AZT in both treatment and toxicity when compared to other organ systems. These metabolic pathways are increasingly relevant not only to the treatment of HIV/AIDS, but also to targeting the role of mitochondrial dysfunction in developing new treatment options for neurological degenerative diseases and primary neoplasms of the CNS. Methodology: Mitochondria were isolated from freshly removed brains from adult Harlan Sprague Dawley rats. The protein content was measured by the method of Lowry and the intactness of the brain mitochondrial preparation was determined by measuring the respiratory control ratio (RCR). Mitochondria with RCR values over 5 were incubated at a final concentration of 4 mg protein /ml in media with labeled and unlabeled deoxynucleosides and deoxynucleoside analogs. Samples of the mitochondrial incubation were removed at specific time points and combined with an equal volume of 10% trichloroacetic acid to lyse mitochondria and precipitate the protein and nucleic acids. This mixture was placed on ice, centrifuged, and the supernatant extract neutralized by addition of AG-11A8 resin. Labeled deoxynucleosides and phosphorylated products in the filtered extracts were analyzed and quantitated by HPLC on an Alltech nucleoside-nucleotide reverse phase column coupled to an inline UV monitor and liquid scintillation counter as previously described. 1 Peaks were identified by comparison to standards. Figure 1: HPLC results of samples taken from mitochondrial incubations at 180 minutes. Isolated rat brain mitochondria were able to transport thymidine (A ) and dC (B) across the inner membrane into the matrix, and phosphorylate both to their mono-, di-, and tri-phosphates, demonstrating that they possess all enzymes necessary in this pathway, including TK-2 and TMPK . Deoxyuridine (dU) (C) was phosphorylated much more slowly than thymidine and only to dUMP. (D) AZT was phosphorylated to AZT-MP as readily as thymidine was phosphorylated to TMP. There was no evidence of AZT-TP. Figure 2: Time course for phosphorylation of H 3 -thymidine, H 3 -deoxycytidine, H 3 -deoxyuridine, H 3 -AZT. Figure 3: Rates of phosphorylation of AZT (A) and thymidine (B) in isolated brain mitochondria demonstrated a salvage pathway 10-20 times more active in brain mitochondria than mitochondria of heart or liver. 1,2 A B Figure 4: Michaelis-Menton graphs were constructed to calculate Vmax and Km of thymidine (panel A) and dC (panel D) phosphorylation. This same data was then plotted to construct Eadie-Hofstee graphs in panels B and E. These data demonstrate negative cooperativity. This is further supported by the Hill plots in panels C and F, both of which have slopes of less than 1. Incubation of Brain Mitochondria with H 3 -thymidine, H 3 -deoxycytidine, H 3 -deoxyuridine, and H 3 -AZT. Figure 5: Similar Michaelis-Menton graphs were constructed to calculate Vmax and Km of AZT phosphorylation (panel A). The Eadie-Hofstee and Hill plots in panels B and C did not support negative cooperativity for AZT phosphorylation. How do the deoxypyrimidines compete with each other for phosphorylation? Isolated brain mitochondria were incubated with (A) H 3 -thymidine and increasing concentrations (0 µM – 500 µM) of unlabeled deoxycytidine (dC) or (B) H 3 -dC and increasing concentrations (0 µM – 200 µM) of unlabeled thymidine Figure 5: While dC (A) inhibited thymidine phosphorylation at an IC 50 = 8.8 +/- 3.9 µM, thymidine (B) did not inhibit dC phosphorylation until concentrations far exceeded physiological levels (IC 50 = >400 µM). Effect of AZT (0 µM – 200 µM) on phosphorylation of H 3 -thymidine, H 3 -deoxycytidine, and H 3 -deoxyuridine in brain mitochodnria. Figure 6: AZT was shown to inhibit thymidine phosphorylation by 50% (IC 50 = 5.5 ± 1.7 μM) as well as phosphorylation of dU (IC 50 = 1.0±-0.1 μM), but AZT was not observed to inhibit dC phosphorylation except at levels > 100 µM. ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],A B brain liver heart liver heart Discussion: As previously noted in both human and murine models with TK-2 deficiency, in tissues with mostly non-replicating cells, the enzyme thymidine kinase 2 (TK-2) is crucial in maintaining dNTP pool balance, particularly that of TTP. 3,4 These investigations demonstrate that TK-2 in brain mitochondria recognizes thymidine, deoxycytidine, deoxyuridine, and AZT as substrates, and phosphorylates them to the monophosphate form. Unlike thymidine and dC, AZT and dU were not phosphorylated beyond the monophosphate. Toxicity of AZT, if present, cannot be mediated by AZT-TP inhibition of the mitochondrial DNA polymerase. Rather, we demonstrate that AZT inhibits thymidine phosphorylation, which may ultimately limit the amount of TTP made, leading to potential disruption of mtDNA replication. ,[object Object],[object Object],[object Object],[object Object],[object Object]](https://image.slidesharecdn.com/postertokyorev2-1305918787373-phpapp02-110520141651-phpapp02/85/Metabolism-of-Deoxypyrimidines-and-Deoxypyrimidine-Antiviral-Analogs-in-Isolated-Brain-Mitochondria-1-320.jpg)
