Uridine-diphosphate-glucose 4-epimerase from saccharomyces fragilis: inactivation by heat and reconstitution of the inactive enzyme

UDP-glucose 4-epimerase from Saccharamyces fragilis is rapidly inactivated by heating at 42 °C for 7 min and at 45 °C for 4 min. The effector site. specific for sugar phosphates, is destroyed still earlier. The enzyme is inactivated by the dissociation of NAD from it leaving the dimeric structure unaffected. It call be reactivated by mercaptoetlianol and NAD, both of which are essential for reactivation, and NAD becomes associated with the dimeric protein moiety

Understanding the bulk electronic structure of Ca1-xSrxVO3

We investigate the electronic structure of Ca1-xSrxVO3 using careful state-of-the-art experiments and calculations. Photoemission spectra using synchrotron radiation reveal a hitherto unnoticed polarization dependence of the photoemission matrix elements for the surface component leading to a substantial suppression of its intensity. Bulk spectra extracted with the help of experimentally determined electron escape depth and estimated suppression of surface contributions resolve outstanding puzzles concerning the electronic structure in Ca1-xSrxVO3.Comment: 4 pages including 3 figure

Comparative study of syndromic and etiological diagnosis of reproductive tract infections/sexually transmitted infections in women in Delhi

SummaryBackgroundThe adequacy of the World Health Organization's syndromic approach for the diagnosis and management of sexually transmitted diseases (STDs), especially at primary health centers (PHCs) and at other levels, is still debatable in different settings in India and requires validation.ObjectivesA cross-sectional study was carried out in women attending the peripheral government clinics of Delhi in order to (1) enumerate their self-reported reproductive tract infection (RTI)/sexually transmitted infection (STI) symptoms; (2) assess their clinical status; (3) determine the syndromic diagnosis of RTI/STI in symptomatic women and etiological diagnosis in both symptomatic and asymptomatic women; and (4) compare the level of agreement between self-reporting of morbidity and syndromic and etiological diagnosis.Materials and methodsThe study was conducted over 26 months in 4090 women attending peripheral government healthcare centers, both rural and urban, in four zones of Delhi. They were recruited into four different study groups: group I, non-pregnant, reporting with symptoms of RTI/STI; group II, with a bad obstetric history or infertility; group III, pregnant women in any trimester attending the antenatal clinic; and group IV, the control group. Gynecological examination, followed by the collection of genital specimens and blood, were performed after informed and written consent was obtained. Every symptomatic patient was managed on the basis of algorithms of the syndromic approach as recommended by the National AIDS Control Organisation (NACO), India. All specimens were transported to the STD Reference Laboratory, Safdarjung Hospital, New Delhi and processed by standard methods to diagnose the various STDs. Laboratory reports were sent to the clinicians and appropriate treatment was instituted. Data were analyzed by applying statistical methods.ResultsOverall, self-reporting of morbidity was 65.0%. However, the percentage of women with some STD-related syndrome was 71.4%. The rural women were observed to have significantly more STD syndromes than their urban counterparts. The etiological diagnosis could be established in only 32.2% of cases.ConclusionsThis study highlights the wide variation between self-reporting of morbidity and syndromic- and etiology-based diagnosis in women from both rural and urban settings. This has implications for the syndromic approach to STI case management. These observations call for a review of the diagnostic policy for RTIs/STIs by national authorities in order to avoid the overuse of antimicrobials. The study also highlights the need for the introduction and/or strengthening of facilities for simple diagnostic tests for RTIs/STIs, especially at the peripheral healthcare level

A microspectroscopic study of the electronic homogeneity of ordered and disordered Sr2FeMoO6

Besides a drastic reduction in saturation magnetization of disordered Sr2FeMoO6 compared to highly ordered samples, magnetizations as a function of the temperature for different disordered samples may also show qualitatively different behaviors. We investigate the origin of such diversity by performing spatially resolved photoemission spectroscopy on various disordered samples. Our results establish that extensive electronic inhomogeneity, arising most probably from an underlying chemical inhomogeneity in disordered samples is responsible for the observed magnetic inhomogeneity. It is further pointed out that these inhomogeneities are connected with composition fluctuations of the type Sr2Fe1+xMo1-xO6 with Fe-rich (x>0) and Mo-rich (x<0) regions.Comment: 14 pages, 4 figure

Molecular association of glucose-6- phosphate isomerase and pyruvate kinase M2 with glyceraldehyde-3-phosphate dehydrogenase in cancer cells

Background: For a long time cancer cells are known for increased uptake of glucose and its metabolization through glycolysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key regulatory enzyme of this pathway and can produce ATP through oxidative level of phosphorylation. Previously, we reported that GAPDH purified from a variety of malignant tissues, but not from normal tissues, was strongly inactivated by a normal metabolite, methylglyoxal (MG).Molecular mechanism behind MG mediated GAPDH inhibition in cancer cells is not well understood. Methods: GAPDH was purified from Ehrlich ascites carcinoma (EAC) cells based on its enzymatic activity. GAPDH associated proteins in EAC cells and 3-methylcholanthrene (3MC) induced mouse tumor tissue were detected by mass spectrometry analysis and immunoprecipitation (IP) experiment, respectively. Interacting domains of GAPDH and its associated proteins were assessed by in silico molecular docking analysis. Mechanism of MG mediated GAPDH inactivation in cancer cells was evaluated by measuring enzyme activity, Circular dichroism (CD) spectroscopy, IP and mass spectrometry analyses. Result: Here, we report that GAPDH is associated with glucose-6-phosphate isomerase (GPI) and pyruvate kinase M2 (PKM2) in Ehrlich ascites carcinoma (EAC) cells and also in 3-methylcholanthrene (3MC) induced mouse tumor tissue. Molecular docking analyses suggest C-terminal domain preference for the interaction between GAPDH and GPI. However, both C and N termini of PKM2 might be interacting with the C terminal domain of GAPDH. Expression of both PKM2 and GPI is increased in 3MC induced tumor compared with the normal tissue. In presence of 1 mM MG,association of GAPDH with PKM2 or GPI is not perturbed, but the enzymatic activity of GAPDH is reduced to 26.8 ± 5 % in 3MC induced tumor and 57.8 ± 2.3 % in EAC cells. Treatment of MG to purified GAPDH complex leads to glycation at R399 residue of PKM2 only, and changes the secondary structure of the protein complex. Conclusion: PKM2 may regulate the enzymatic activity of GAPDH. Increased enzymatic activity of GAPDH in tumor cells may be attributed to its association with PKM2 and GPI. Association of GAPDH with PKM2 and GPI could be a signature for cancer cells. Glycation at R399 of PKM2 and changes in the secondary structure of GAPDH complex could be one of the mechanisms by which GAPDH activity is inhibited in tumor cells by MG

Udpglucose-4 epimerase from Saccharomyces fragilis: interaction with sugar phosphates at an effector site

UDP glucose-4 epimerase from Saccharomyces fragilis was found to be activated at low substrate concentrations by some metabolically related sugar phosphates. The stimulation of the enzyme activity showed a sigmoidal response to the increasing concentration of glucose-6 phosphate at a fixed substrate concentration. The activated enzyme was allosterically inhibited by UMP which otherwise acted as a strictly competitive inhibitor for the enzyme. The interaction with sugar phosphates was not accompanied by any change in the aggregation state of the molelcule

Evidence for the presence of a critical histidine residue at the active site in glyceraldehyde-3-phosphate dehydrogenase of Ehrlich ascites carcinoma cells

7-13Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (GA3PD) (EC. 1.2.1.12) was completely inactivated by diethyl pyrocarbonate (DEPC), a fairly specific reagent for histidine residues in the pH range of 6.0-7.5. The rate of inactivation was dependent on pH and followed pseudo-first order reaction kinetics. The difference spectrum of the inactivated and native enzymes showed an increase in the absorption maximum at 242 nm, indicating the modification of histidine residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of one essential histidine residue to be responsible for loss of the catalytic activity of EAC cell GA3PD. DEPC inactivation was protected by substrates, D-glyceraldehyde-3-phosphate and NAD, indicating the presence of essential histidine residue at the substrate-binding region of the active site. Double inhibition studies also provide evidence for the presence of histidine residue at the active site

Udpglucose 4-epimerase from saccharomyces fragilis : desensitization with heat

The allosteric kinetics exhibited by UDP glucose 4-epimerase from Saccharomyces fragilis changes over to a normal hyperbolic kinetics when the enzyme is heated at 41° for 2 mins. The native enzyme is completely insensitive to inhibition by UMP in the allosteric region. The desensitized enzyme is however, strongly inhibited by UMP at this low concentrations. Apparently, desensitization by heat converts the enzyme to its ultimate catalytic form