A Review and Meta-Analysis of the Prevalence and Health Impact of Polycystic Ovary Syndrome Among Medical and Dental Students.

Women currently comprise the majority of students graduating from medical school and face unique stressors not seen with their male counterparts. In particular, during their medical education, women with polycystic ovary syndrome (PCOS) experience symptoms of the disorder, which strongly impact both their academic and social lives. This in turn influences their academic and professional future. Although women as medical professionals, in general, are happy with their careers, awareness, and understanding on the part of medical educators will definitely be helpful to women medical students in their path to being successful medical professionals. The first objective of our current study is to find the prevalence of PCOS in medical and dental students. The second objective is to find the academic and health impacts of PCOS and what type of interventions are being adopted to relieve the symptoms. Using the keywords such as "PCOS," "medical students," and "dental students", the search engines PubMed, Embase, and Scopus were used to retrieve relevant articles published from 2020 to 2022. After removing the duplicates, 11 prospective cross-sectional studies were utilized for qualitative and quantitative analysis. The pooled prevalence of 2,206 female medical students diagnosed with PCOS was 24.7%. The students in the various studies were aware of their PCOS diagnosis and were taking therapeutic medications. The most common associated complications reported were BMI abnormalities, abnormal hair growth, and acne, along with other complications such as stress and poor academic and social life. The majority also had significant family histories of concurrent clinical conditions such as diabetes, hypertension, and other menstrual abnormalities. Considering the huge impact of PCOS, medical educators, policymakers, and all stakeholders should take proactive measures to accommodate students' needs and bridge the social gap. Special awareness of needed lifestyle changes should be a part of the medical education curriculum for an inclusive educational environment, as it will minimize the gender discrepancy in academic satisfaction and professional life.

Efficacy of Lanthanum Carbonate and Sevelamer Carbonate as Phosphate Binders in Chronic Kidney Disease-A Comparative Clinical Study.

(1) Background: Hyperphosphatemia is correlated with an increased rate of mortality and morbidity due to cardiovascular diseases in chronic kidney disease (CKD) patients. It can be improved by restricting dietary intake of phosphate and oral phosphate binders, such as lanthanum carbonate and sevelamer carbonate. (2) Objective: To evaluate the clinical efficacy of sevelamer carbonate in comparison to lanthanum carbonate as phosphate binders for the treatment of hyperphosphatemia in CKD patients. (3) Methods: A randomized control comparative clinical study was conducted for one year on 150 CKD patients associated with hyperphosphatemia, divided into two groups, i.e., Group 1 (n = 75) treated with sevelamer carbonate 800 mg thrice daily and Group 2 (n = 75) treated with lanthanum carbonate 500 mg thrice daily. The patients were assessed at the time of enrollment in the study, after three months and after six months from baseline for different parameters, i.e., complete blood count, liver function tests, renal function tests, electrolytes, and serum phosphate level. (4) Results: 150 CKD patients aged 51-60 participated in the study. The mean age of patients was 54 ± 4.6 years, and males (55.71%) were more common than females (44.29%). Hypertension was the common comorbidity in both groups with chronic kidney disease. After six months of treatment, the mean serum phosphate level was significantly decreased from 8.31 ± 0.09 mg/dL to 5.11 ± 0.18 (38%) in Group 1 and from 8.79 ± 0.28 mg/dl to 4.02 ± 0.12 (54%; p < 0.05) in Group 2, respectively. In both groups, no significant difference was found in other parameters such as parathyroid hormone, calcium, uric acid, LFT, RFT, CBC, etc. (5) Conclusion: Lanthanum carbonate is more efficacious in lowering serum phosphate concentrations and effectively managing hyperphosphatemia as compared to sevelamer carbonate.

Virtual Versus Light Microscopy Usage among Students: A Systematic Review and Meta-Analytic Evidence in Medical Education.

BACKGROUND: The usage of whole-slide images has recently been gaining a foothold in medical education, training, and diagnosis. OBJECTIVES: The first objective of the current study was to compare academic performance on virtual microscopy (VM) and light microscopy (LM) for learning pathology, anatomy, and histology in medical and dental students during the COVID-19 period. The second objective was to gather insight into various applications and usage of such technology for medical education. MATERIALS AND METHODS: Using the keywords "virtual microscopy" or "light microscopy" or "digital microscopy" and "medical" and "dental" students, databases (PubMed, Embase, Scopus, Cochrane, CINAHL, and Google Scholar) were searched. Hand searching and snowballing were also employed for article searching. After extracting the relevant data based on inclusion and execution criteria, the qualitative data were used for the systematic review and quantitative data were used for meta-analysis. The Newcastle Ottawa Scale (NOS) scale was used to assess the quality of the included studies. Additionally, we registered our systematic review protocol in the prospective register of systematic reviews (PROSPERO) with registration number CRD42020205583. RESULTS: A total of 39 studies met the criteria to be included in the systematic review. Overall, results indicated a preference for this technology and better academic scores. Qualitative analyses reported improved academic scores, ease of use, and enhanced collaboration amongst students as the top advantages, whereas technical issues were a disadvantage. The performance comparison of virtual versus light microscopy meta-analysis included 19 studies. Most (10/39) studies were from medical universities in the USA. VM was mainly used for teaching pathology courses (25/39) at medical schools (30/39). Dental schools (10/39) have also reported using VM for teaching microscopy. The COVID-19 pandemic was responsible for the transition to VM use in 17/39 studies. The pooled effect size of 19 studies significantly demonstrated higher exam performance (SMD: 1.36 [95% CI: 0.75, 1.96], p < 0.001) among the students who used VM for their learning. Students in the VM group demonstrated significantly higher exam performance than LM in pathology (SMD: 0.85 [95% CI: 0.26, 1.44], p < 0.01) and histopathology (SMD: 1.25 [95% CI: 0.71, 1.78], p < 0.001). For histology (SMD: 1.67 [95% CI: -0.05, 3.40], p = 0.06), the result was insignificant. The overall analysis of 15 studies assessing exam performance showed significantly higher performance for both medical (SMD: 1.42 [95% CI: 0.59, 2.25], p < 0.001) and dental students (SMD: 0.58 [95% CI: 0.58, 0.79], p < 0.001). CONCLUSIONS: The results of qualitative and quantitative analyses show that VM technology and digitization of glass slides enhance the teaching and learning of microscopic aspects of disease. Additionally, the COVID-19 global health crisis has produced many challenges to overcome from a macroscopic to microscopic scale, for which modern virtual technology is the solution. Therefore, medical educators worldwide should incorporate newer teaching technologies in the curriculum for the success of the coming generation of health-care professionals.

A novel SYNJ1 homozygous variant causing developmental and epileptic encephalopathy in an Afro-Caribbean individual.

BACKGROUND: SYNJ1 encodes Synaptojanin-1, a dual-function poly-phosphoinositide phosphatase that is expressed in the brain to regulate neuronal synaptic vesicle dynamics. Biallelic SYNJ1 variants cause a spectrum of clinical manifestations, from early onset parkinsonism to developmental and epileptic encephalopathy. METHODS: Proband-only exome sequencing was used to identify a homozygous SYNJ1 pathogenic variant in an individual with epileptic encephalopathy. Sanger sequencing was used to confirm the variant. RESULTS: We present an Afro-Caribbean female who developed uncontrollable seizures shortly after birth, accompanied by developmental delay and severe generalized dystonia. She had homozygosity for a novel c.242-2A > G variant in SYNJ1 with both parents being heterozygous carriers. An older sister was reported to have had a similar presentation but was not examined. Both siblings died at an approximate age of 16 years. CONCLUSIONS: We report a novel pathogenic variant in SYNJ1 present in homozygosity leading to developmental and epileptic encephalopathy. Currently, there are only 4 reports describing 10 individuals with SYNJ1-related developmental and epileptic encephalopathy. This case expands the clinical knowledge and the allelic heterogeneity associated with SYNJ1 variants.

Epithelial vectorial ion transport in cystic fibrosis: Dysfunction, measurement, and pharmacotherapy to target the primary deficit.

Cystic fibrosis patients display multi-organ system dysfunction (e.g. pancreas, gastrointestinal tract, and lung) with pathogenesis linked to a failure of Cl- secretion from the epithelial surfaces of these organs. If unmanaged, organ dysfunction starts early and patients experience chronic respiratory infection with reduced lung function and a failure to thrive due to gastrointestinal malabsorption. Early mortality is typically caused by respiratory failure. In the past 40 years of newborn screening and improved disease management have driven the median survival up from the mid-teens to 43-53, with most of that improvement coming from earlier and more aggressive management of the symptoms. In the last decade, promising pharmacotherapies have been developed for the correction of the underlying epithelial dysfunction, namely, Cl- secretion. A new generation of systemic drugs target the mutated Cl- channels in cystic fibrosis patients and allow trafficking of the immature mutated protein to the cell membrane (correctors), restore function to the channel once in situ (potentiators), or increase protein levels in the cells (amplifiers). Restoration of channel function prior to symptom development has the potential to significantly change the trajectory of disease progression and their evidence suggests that a modest restoration of Cl- secretion may delay disease progression by decades. In this article, we review epithelial vectorial ion and fluid transport, its quantification and measurement as a marker for cystic fibrosis ion transport dysfunction, and highlight some of the recent therapies targeted at the dysfunctional ion transport of cystic fibrosis.

Elective colon resection without curative intent in stage IV colon cancer.

BACKGROUND: Evidence suggests that elective primary colon resection (ePCR) in patients with asymptomatic colon tumors and unresectable metastases is not required and may expose patients to unnecessary operative risk. METHODS: Stage IV colon cancer patients with liver metastases from 2000 to 2011 were identified with SEER-Medicare data. Liver-based therapy or urgent/emergent colectomies were excluded. Chemotherapy alone was compared to ePCR ±â€¯chemotherapy. Univariate and multivariate analyses were used to identify predictors of ePCR. Multivariate Cox regression compared survival. RESULTS: 5139 patients were identified. The ePCR rate decreased over time; 84% underwent ePCR in 2000, compared to 52% in 2011 (p < 0.001). In multivariate analysis, older patients were more likely to undergo ePCR, as were patients from rural areas (OR 1.65, p < 0.001). The odds of PCR in high poverty areas (>10%) were almost 25% higher than those in low poverty areas (OR 1.23, p = 0.03). African-Americana were less likely to undergo PCR than Caucasians (OR 0.76, p = 0.01). In multivariate survival analysis, PCR was associated with a significant survival benefit (HR 0.59, p < 0.001). CONCLUSIONS: Although ePCR is not recommended with unresectable metastases and the rate has decreased significantly, over 50% of patients with untreated hepatic metastases underwent ePCR in 2011. Disparities exist in use of ePCR that are likely multifactorial and deserve further study.

Identification of a de novo case of COL5A1-related Ehlers-Danlos syndrome in an infant in the West Indies leading to improved targeted clinical care.

A 1-year-old girl from an underserved community presented with irritability, pain, and delayed motor skills. Our genetics outreach program facilitated the diagnosis of Ehlers-Danlos syndrome masquerading as developmental delay after noting hyperextensible skin. Diagnosis for this family allows for state-of-the-art cardiac monitoring and appropriate symptomatic treatment for this rare disease.

Orexin Receptor Multimerization versus Functional Interactions: Neuropharmacological Implications for Opioid and Cannabinoid Signalling and Pharmacogenetics.

Orexins/hypocretins are neuropeptides formed by proteolytic cleavage of a precursor peptide, which are produced by neurons found in the lateral hypothalamus. The G protein-coupled receptors (GPCRs) for these ligands, the OX1 and OX2 orexin receptors, are more widely expressed throughout the central nervous system. The orexin/hypocretin system has been implicated in many pathways, and its dysregulation is under investigation in a number of diseases. Disorders in which orexinergic mechanisms are being investigated include narcolepsy, idiopathic sleep disorders, cluster headache and migraine. Human narcolepsy has been associated with orexin deficiency; however, it has only rarely been attributed to mutations in the gene encoding the precursor peptide. While gene variations within the canine OX2 gene hcrtr2 have been directly linked with narcolepsy, the majority of human orexin receptor variants are weakly associated with diseases (the idiopathic sleep disorders, cluster headache and polydipsia-hyponatremia in schizophrenia) or are of potential pharmacogenetic significance. Evidence for functional interactions and/or heterodimerization between wild-type and variant orexin receptors and opioid and cannabinoid receptors is discussed in the context of its relevance to depression and epilepsy.

Cysteinyl Leukotrienes Pathway Genes, Atopic Asthma and Drug Response: From Population Isolates to Large Genome-Wide Association Studies.

Genetic variants associated with asthma pathogenesis and altered response to drug therapy are discussed. Many studies implicate polymorphisms in genes encoding the enzymes responsible for leukotriene synthesis and intracellular signaling through activation of seven transmembrane domain receptors, such as the cysteinyl leukotriene 1 (CYSLTR1) and 2 (CYSLTR2) receptors. The leukotrienes are polyunsaturated lipoxygenated eicosatetraenoic acids that exhibit a wide range of pharmacological and physiological actions. Of the three enzymes involved in the formation of the leukotrienes, arachidonate 5 lipoxygenase 5 (ALOX5), leukotriene C4 synthase (LTC4S), and leukotriene hydrolase (LTA4H) are all polymorphic. These polymorphisms often result in variable production of the CysLTs (LTC4, LTD4, and LTE4) and LTB4. Variable number tandem repeat sequences located in the Sp1-binding motif within the promotor region of the ALOX5 gene are associated with leukotriene burden and bronchoconstriction independent of asthma risk. A 444A > C SNP polymorphism in the LTC4S gene, encoding an enzyme required for the formation of a glutathione adduct at the C-6 position of the arachidonic acid backbone, is associated with severe asthma and altered response to the CYSLTR1 receptor antagonist zafirlukast. Genetic variability in the CysLT pathway may contribute additively or synergistically to altered drug responses. The 601 A > G variant of the CYSLTR2 gene, encoding the Met201Val CYSLTR2 receptor variant, is associated with atopic asthma in the general European population, where it is present at a frequency of ∼2.6%. The variant was originally found in the founder population of Tristan da Cunha, a remote island in the South Atlantic, in which the prevalence of atopy is approximately 45% and the prevalence of asthma is 36%. In vitro work showed that the atopy-associated Met201Val variant was inactivating with respect to ligand binding, Ca2+ flux and inositol phosphate generation. In addition, the CYSLTR1 gene, located at Xq13-21.1, has been associated with atopic asthma. The activating Gly300Ser CYSLTR1 variant is discussed. In addition to genetic loci, risk for asthma may be influenced by environmental factors such as smoking. The contribution of CysLT pathway gene sequence variants to atopic asthma is discussed in the context of other genes and environmental influences known to influence asthma.

Reduced levels of mitochondrial complex I subunit NDUFB8 and linked complex I + III oxidoreductase activity in the TgCRND8 mouse model of Alzheimer's disease.

Bioenergetic failure is a feature of Alzheimer's disease (AD). We examined mitochondrial function in the amyloid-ß protein precursor transgenic 'TgCRND8' mouse model of AD. Activities of NADH: cytochrome c reductase (complex I + III) and cytochrome oxidase (complex IV) of the electron transport chain, as well as those of α-ketoglutarate dehydrogenase (α-KGDH) and pyruvate dehydrogenase (PDH) were assessed in brains of 45 week-old mice. Complex I + III activity was reduced by almost 50%, whereas complex IV, α-KGDH, and PDH activities were unaffected. Reduced activity coincided with decreased expression of NDUFB8, a nuclear-DNA encoded subunit integral to the assembly of complex I. The composition and availability of cardiolipin, a major phospholipid in inner mitochondrial membranes, was not altered. To determine whether mitochondrial output is affected by the selective reduction in complex I + III activity, we examined tissue levels of high-energy phosphates. ATP was maintained whereas creatine increased in the cortex and hippocampus. These results suggest disruption of complex I function and the likely role of creatine in sustaining ATP at late stages of dysfunction in TgCRND8 mice.

Pain management and osteopathic manipulative medicine in the Army: new opportunities for the osteopathic medical profession.

Osteopathic manipulative medicine (OMM) is the most visible distinction between osteopathic physicians and allopathic physicians, and the osteopathic medical profession recognizes the proven clinical efficacy and value of OMM. The recently published report by the US Army Surgeon General's Pain Management Task Force provides a strong endorsement for the Army to increase the practice of OMM, to create OMM continuing medical education opportunities, and to improve research in OMM's role in pain management. This comprehensive restructuring of the Army's approach to pain management provides a unique opportunity for the civilian osteopathic medical profession-the true OMM experts-to collaborate with Army osteopathic physicians in OMM training, teaching, and research.

Identification of drug candidates which increase cytochrome c oxidase activity in deficient patient fibroblasts.

Cytochrome c oxidase (COX) activity reflects the expressed level of respiratory chain complexes, mtDNA levels, titer and mass of mitochondria. Activity is also indicative of the overall fitness of mt-transcription factors and the import, transcription and translation of mt-proteins. We have developed a high-throughput assay to measure COX activity using live cells to screen chemical libraries for compounds capable of increasing COX activity. These libraries have revealed four examples which elevated the activities of COX in NIH-3T3 fibroblasts and in fibroblasts from patients with COX defects independent of the peroxisome proliferator activated receptor family.

Oxidative stress alters the regulatory control of p66Shc and Akt in PINK1 deficient cells.

Mitochondrial dysfunction is involved in the underlying pathology of Parkinson's Disease (PD). PINK1 deficiency, which gives rise to familial early-onset PD, is associated with this dysfunction as well as increased oxidative stress. We have established primary fibroblast cell lines from two patients with PD who carry mutations in the PINK1 gene. The phosphorylation of Akt is abrogated in the presence of oxidative stressors in the complete absence of PINK1 suggesting enhanced apoptotic signalling. We have found an imbalance between the production of reactive oxygen species where the capacity of the cell to remove these toxins by anti-oxidative enzymes is greatly reduced. The expression levels of the anti-oxidant enzymes glutathione peroxidase-1, MnSOD, peroxiredoxin-3 and thioredoxin-2 were diminished. The p66(Shc) adaptor protein has recently been identified to become activated by oxidative stress by phosphorylation at residue Ser36 which then translocates to the mitochondrial inner membrane space. The phosphorylation of p66(Shc) at Ser36 is significantly increased in PINK1 deficient cell lines under normal tissue culture conditions, further still in the presence of compounds which elicit oxidative stress. The stable transfection of PINK1 in the fibroblasts which display the null phenotype ameliorates the hyper-phosphorylation of p66(Shc).

Disruption of a mitochondrial RNA-binding protein gene results in decreased cytochrome b expression and a marked reduction in ubiquinol-cytochrome c reductase activity in mouse heart mitochondria.

Mice homozygous for a defect in the PTCD2 (pentatricopeptide repeat domain protein 2) gene were generated in order to study the role of this protein in mitochondrial RNA metabolism. These mice displayed specific but variable reduction of ubiquinol-cytochrome c reductase complex activity in mitochondria of heart, liver and skeletal muscle due to a decrease in the expression of mitochondrial DNA-encoded cytochrome b, the catalytic core of the complex. This reduction in mitochondrial function has a profound effect on the myocardium, with replacement of ventricular cardiomyocytes by fibro-fatty tissue. Northern blotting showed a reduction in the mRNA for the mitochondrial DNA encoded proteins cytochrome b (cytb) and ND5 (NADH dehydrogenase subunit 5) and an elevation in a combined pre-processed ND5-CYTB transcript. This suggests that the PTCD2 protein is involved in processing RNA transcripts involving cytochrome b derived from mitochondrial DNA. This defines the site for PTCD2 action in mammalian mitochondria and suggests a possible role for dysfunction of this protein in the aetiology of heart failure.

Identification of a canine model of pyruvate dehydrogenase phosphatase 1 deficiency.

Exercise intolerance syndromes are well known to be associated with inborn errors of metabolism affecting glycolysis (phosphorylase and phosphofructokinase deficiency) and fatty acid oxidation (palmitoyl carnitine transferase deficiency). We have identified a canine model for profound exercise intolerance caused by a deficit in PDP1 (EC 3.1.3.43), the phosphatase enzyme that activates the pyruvate dehydrogenase complex (PDHc). The Clumber spaniel breed was originated in 1760 by the Duc de Noailles, as a hunting dog with a gentle temperament suitable for the 'elderly gentleman'. Here we report that 20% of the current Clumber and Sussex spaniel population are carriers for a null mutation in PDP1, and that homozygosity produces severe exercise intolerance. Human pyruvate dehydrogenase phosphatase deficiency was recently characterized at the molecular level. However, the nature of the human mutation (loss of a single amino acid altering PDP1 activity) made it impossible to discern the role of the second phosphatase isoform, PDP2, in the deficient phenotype. Here we show that the null mutation in dogs provides a valuable animal model with which to study the effects of dysregulation of the PDHc. Knowledge of the molecular defect has allowed for the institution of a rapid restriction enzyme test for the canine mutation that will allow for selective breeding and has led to a suggested dietary therapy for affected dogs that has proven to be beneficial. Pharmacological and genetic therapies for PDP1 deficiency can now be investigated and the role of PDP2 can be fully characterized.

Wild-type PINK1 prevents basal and induced neuronal apoptosis, a protective effect abrogated by Parkinson disease-related mutations.

Mutations in the PTEN-induced kinase 1 (PINK1) gene have recently been implicated in autosomal recessive early onset Parkinson Disease (1, 2). To investigate the role of PINK1 in neurodegeneration, we designed human and murine neuronal cell lines expressing either wild-type PINK1 or PINK1 bearing a mutation associated with Parkinson Disease. We show that under basal and staurosporine-induced conditions, the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive cells was lower in wild-type PINK1 expressing SH-SY5Y cells than in mock-transfected cells. This phenotype was due to a PINK1-mediated reduction in cytochrome c release from mitochondria, which prevents subsequent caspase-3 activation. We show that overexpression of wild-type PINK1 strongly reduced both basal and staurosporine-induced caspase 3 activity. Overexpression of wild-type PINK1 also reduced the levels of cleaved caspase-9, caspase-3, caspase-7, and activated poly(ADP-ribose) polymerase under both basal and staurosporine-induced conditions. In contrast, Parkinson disease-related mutations and a kinase-inactive mutation in PINK1 abrogated the protective effect of PINK1. Together, these results suggest that PINK1 reduces the basal neuronal pro-apoptotic activity and protects neurons from staurosporine-induced apoptosis. Loss of this protective function may therefore underlie the degeneration of nigral dopaminergic neurons in patients with PINK1 mutations.

Pyruvate dehydrogenase phosphatase deficiency: identification of the first mutation in two brothers and restoration of activity by protein complementation.

CONTEXT: Pyruvate dehydrogenase phosphatase (PDP) deficiency has been previously reported as an enzymopathy, but the genetic basis for such a defect has never been established. OBJECTIVE: The aim of this study was to identify the cause of the defect in two patients who presented with PDP deficiency. PATIENTS: We studied two brothers of consanguineous parents who presented with neonatal hypotonia, elevated lactate, and less than 25% native pyruvate dehydrogenase complex (PDHc) activity in skin fibroblasts compared with controls. The activity of the complex could be restored to normal values by preincubation of the cells with dichloroacetate or by treating cell extracts with calcium. RESULTS: These two individuals were found to be homozygous for a 3-bp deletion in the coding sequence of the PDP isoform 1 (PDP1), which removes the amino acid residue leucine from position 213 of the protein. A recombinant version of this protein was synthesized and found to have a very reduced (<5%) ability to activate purified PDHc. Reduced steady-state levels of PDP1 in the patient's fibroblasts coupled with the low catalytic activity of the mutant PDP1 resulted in native PDHc activity being reduced, but this could be corrected by the addition of recombinant PDP1 (wild type). CONCLUSION: We have identified mutations in PDP1 in two brothers with PDP deficiency and have proven that the mutation is disease-causing. This is the first demonstration of human disease due to a mutation in PDP1.

Phosphorylated derivatives that activate or inhibit mammalian adenosine kinase provide insights into the role of pentavalent ions in AK catalysis.

The enzyme adenosine kinase (AK) exhibits a nearly complete dependency on the presence of pentavalent ions (PVI) such as phosphate, arsenate, and vanadate. To understand its basis, the effect of a large number of phosphorylated compounds on AK activity was examined. Several compounds, such as phosphoribosyl pyrophosphate, phosphoenol pyruvate, creatine phosphate, phosphorous acid, phosphonoformic acid, and inorganic pyrophosphate, were found to substitute for PVI in stimulating AK activity. Similar to PVI, these compounds lowered the Km of AK for adenosine. In contrast, many other structurally related compounds (i.e., phosphonoacetic acid, 2-carboxyethyl phosphonic acid, N-phosphonomethyl glycine, N-phosphonomethyl iminodiacetic acid) inhibited AK activity. These compounds seemed to compete with the activators for binding to AK. Structural comparisons of different compounds indicate that all activating compounds contain a net positive charge on the pentavalent atom (e.g., phosphorous), which should enable it to act as an acceptor for a nucleophilic group. We suggest that a phosphate (or other activator) bound near the active site participates in AK catalysis by forming a transient pentavalent intermediate with a nonbridging oxygen of the beta-phosphate in ATP. This interaction likely facilitates the transfer of gamma-phosphate from ATP to adenosine, thus accounting for the stimulating role of PVI in AK catalysis. The insight provided by these studies concerning the structural features of activators and inhibitors should also prove helpful in the design of more potent inhibitors of AK.

Regulation of NADH/CoQ oxidoreductase: do phosphorylation events affect activity?

We had previously suggested that phosphorylation of proteins by mitochondrial kinases regulate the activity of NADH/CoQ oxidoreductase. Initial data showed that pyruvate dehydrogenase kinase (PDK) and cAMP-dependent protein kinase A (PKA) phosphorylate mitochondrial membrane proteins. Upon phosphorylation with crude PDK, mitochondria appeared to be deficient in NADH/cytochrome c reductase activity associated with increased superoxide production. Conversely, phosphorylation by PKA resulted in increased NADH/cytochrome c reductase activity and decreased superoxide formation. Current data confirms PKA involvement in regulating Complex I activity through phosphorylation of an 18 kDa subunit. Beef heart NADH/ cytochrome c reductase activity increases to 150% of control upon incubation with PKA and ATP-gamma-S. We have cloned the four human isoforms of PDK and purified beef heart Complex I. Incubation of mitochondria with PDK isoforms and ATP did not alter Complex I activity or superoxide production. Radiolabeling of mitochondria and purified Complex I with PDK failed to reveal phosphorylated proteins.

Pentavalent ions dependency is a conserved property of adenosine kinase from diverse sources: identification of a novel motif implicated in phosphate and magnesium ion binding and substrate inhibition.

The catalytic activity of adenosine kinase (AK) from mammalian sources has previously been shown to exhibit a marked dependency upon the presence of pentavalent ions (PVI), such as phosphate (PO4), arsenate, or vanadate. We now show that the activity of AK from diverse sources, including plant, yeast, and protist species, is also markedly enhanced in the presence of PVI. In all cases, PO4 or other PVI exerted their effects primarily by decreasing the Km for adenosine and alleviating the inhibition caused by high concentrations of substrates. These results provide evidence that PVI dependency is a conserved property of AK and perhaps of the PfkB family of carbohydrate kinases which includes AK. On the basis of sequence alignments, we have identified a conserved motif NXXE within the PfkB family. The N and E of this motif make close contacts with Mg2+ and PO4 ions in the crystal structures of AK and bacterial ribokinase (another PfkB member which shows PVI dependency), implicating these residues in their binding. Site-directed mutagenesis of these residues in Chinese hamster AK have resulted in active proteins with greatly altered phosphate stimulation and substrate inhibition characteristics. The N239Q mutation leads to the formation of an active protein whose activity was not stimulated by PO4 or inhibited by high concentrations of adenosine or ATP. The activity of the E242D mutant protein was also not significantly altered in the presence of phosphate. Although PO4 had no effect on the KmAdenosine for this mutant, the KmATP, K(i)Adenosine, and K(i)ATP were significantly decreased. In contrast to these mutations, N239L or E242L mutant proteins showed greatly decreased activity with an altered Mg2+ requirement. These observations support the view that N239 and E242 play an important role in the binding of PO4 and Mg2+ ions required for the catalytic activity of adenosine kinase.