Overexpression and characterization of dimeric and tetrameric forms of recombinant serine hydroxymethyltransferase from Bacillus stearothermophilus

Serine hydroxymethyltransferase (SHMT), a pyridoxal-5′-phosphate (PLP) dependent enzyme catalyzes the interconversion of L-Ser and Gly using tetrahydrofolate as a substrate. The gene encoding for SHMT was amplified by PCR from genomic DNA of Bacillus stearothermophilus and the PCR product was cloned and overexpressed in Escherichia coli. The purified recombinant enzyme was isolated as a mixture of dimer (90%) and tetramer (10%). This is the first report demonstrating the existence of SHMT as a dimer and tetramer in the same organism. The specific activities at 37°C of the dimeric and tetrameric forms were 6.7 U/mg and 4.1 U/mg, respectively. The purified dimer was extremely thermostable with a Tm of 85°C in the presence of PLP and L-Ser. The temperature optimum of the dimer was 80°C with a specific activity of 32×4 U/mg at this temperature. The enzyme catalyzed tetrahydrofolate-independent reactions at a slower rate compared to the tetrahydrofolate-dependent retro-aldol cleavage of L-Ser. The interaction with substrates and their analogues indicated that the orientation of PLP ring of B. stearothermophilus SHMT was probably different from sheep liver cytosolic recombinant SHMT (scSHMT)

Differential Effects of Leptin on the Invasive Potential of Androgen-Dependent and -Independent Prostate Carcinoma Cells

Obesity has been linked with an increased risk of prostate cancer. The formation of toxic free oxygen radicals has been implicated in obesity mediated disease processes. Leptin is one of the major cytokines produced by adipocytes and controls body weight homeostasis through food intake and energy expenditure. The rationale of the study was to determine the impact of leptin on the metastatic potential of androgen-sensitive (LNCaP) cells as well as androgen-insensitive (PC-3 and DU-145) cells. At a concentration of 200 nm, LNCaP cells showed a significant increase (20% above control; P < .0001) in cellular proliferation without any effect on androgen-insensitive cells. Furthermore, exposure to leptin caused a significant (P < .01 to P < .0001) dose-dependent decrease in migration and invasion of PC3 and Du-145 prostate carcinoma cell lines. At the molecular level, exposure of androgen-independent prostate cancer cells to leptin stimulates the phosphorylation of MAPK at early time point as well as the transcription factor STAT3, suggesting the activation of the intracellular signaling cascade upon leptin binding to its cognate receptor. Taken together, these results suggest that leptin mediates the invasive potential of prostate carcinoma cells, and that this effect is dependent on their androgen sensitivity

Importance of tyrosine residues of Bacillus stearothermophilus serine hydroxymethyltransferase in cofactor binding and L-allo-Thr cleavage

Serine hydroxymethyltransferase (SHMT) from Bacillus stearothermophilus (bsSHMT) is a pyridoxal 5&#8242;-phosphate-dependent enzyme that catalyses the conversion of L-serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. In addition, the enzyme catalyses the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids and transamination. In this article, we have examined the mechanism of the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids by SHMT. The three-dimensional structure and biochemical properties of Y51F and Y61A bsSHMTs and their complexes with substrates, especially L-allo-Thr, show that the cleavage of 3-hydroxy amino acids could proceed via C&#945; proton abstraction rather than hydroxyl proton removal. Both mutations result in a complete loss of tetrahydrofolate-dependent and tetrahydrofolate-independent activities. The mutation of Y51 to F strongly affects the binding of pyridoxal 5&#8242;-phosphate, possibly as a consequence of a change in the orientation of the phenyl ring in Y51F bsSHMT. The mutant enzyme could be completely reconstituted with pyridoxal 5&#8242;-phosphate. However, there was an alteration in the &#955;max value of the internal aldimine (396 nm), a decrease in the rate of reduction with NaCNBH3 and a loss of the intermediate in the interaction with methoxyamine (MA). The mutation of Y61 to A results in the loss of interaction with C&#945; and C&#946; of the substrates. X-Ray structure and visible CD studies show that the mutant is capable of forming an external aldimine. However, the formation of the quinonoid intermediate is hindered. It is suggested that Y61 is involved in the abstraction of the C&#945; proton from 3-hydroxy amino acids. A new mechanism for the cleavage of 3-hydroxy amino acids via C&#945; proton abstraction by SHMT is proposed

Timing Offset Calibration of CZTI instrument aboard ASTROSAT

The radio as well as the high energy emission mechanism in pulsars is yet not understood properly. A multi-wavelength study is likely to help in better understanding of such processes. The first Indian space-based observatory, ASTROSAT, has five instruments aboard, which cover the electromagnetic spectrum from infra-red (1300 $\AA$) to hard X-ray (380 KeV). Cadmium Zinc Telluride Imager (CZTI), one of the five instruments is a hard X-ray telescope functional over an energy range of 20-380 KeV. We aim to estimate the timing offset introduced in the data acquisition pipeline of the instrument, which will help in time alignment of high energy time series with those from two other ground-based observatories, viz. the Giant Meterwave Radio Telescope (GMRT) and the Ooty Radio Telescope (ORT). PSR B0531+21 is a well-studied pulsar with nearly aligned radio and hard X-ray pulse profiles. We use simultaneous observations of this pulsar with the ASTROSAT, the ORT and the GMRT. The pulsar was especially observed using the ORT with almost daily cadence to obtain good timing solutions. We also supplement the ORT data with archival FERMI data for estimation of timing noise. The timing offset of ASTROSAT instruments was estimated from fits to arrival time data at the ASTROSAT and the radio observatories. We estimate the offset between the GMRT and the ASTROSAT-CZTI to be -4716 $\pm$ 50 $\mu s$. The corresponding offset with the ORT was -29639 $\pm$ 50 $\mu s$. The offsets between the GMRT and Fermi-LAT -5368 $\pm$ 56 $\mu s$. (Abridged)Comment: 6 pages, 5 figures, 2 tables, Revised and Updated, accepted for publication in A&

Seasonal intercomparison of observational rainfall datasets over India during the southwest monsoon season

The Indian monsoon is an important component of Earth's climate system, accurate forecasting of its mean rainfall being essential for regional food and water security. Accurate measurement of the rainfall is essential for various water-related applications, the evaluation of numerical models and detection and attribution of trends, but a variety of different gridded rainfall datasets are available for these purposes. In this study, six gridded rainfall datasets are compared against the India Meteorological Department (IMD) gridded rainfall dataset, chosen as the most representative of the observed system due to its high gauge density. The datasets comprise those based solely on rain gauge observations and those merging rain gauge data with satellite-derived products. Various skill scores and subjective comparisons are carried out for the Indian region during the south-west monsoon season (June to September). Relative biases and skill metrics are documented at all-India and sub-regional scales. In the gauge-based (land-only) category, Asian Precipitation-Highly-Resolved Observational Data Integration Towards Evaluation of water resources (APHRODITE) and Global Precipitation Climatology Center (GPCC) datasets perform better relative to the others in terms of a variety of skill metrics. In the merged category, the Global Precipitation Climatology Project (GPCP) dataset is shown to perform better than the Climate Prediction Center Merged Analysis of Precipitation (CMAP) for the Indian monsoon in terms of various metrics, when compared with the IMD gridded data. Most of the datasets have difficulty in representing rainfall over orographic regions including the Western Ghats mountains, in north-east India and the Himalayan foothills. The wide range of skill scores seen among the datasets and even the change of sign of bias found in some years are causes of concern. This uncertainty between datasets is largest in north-east India. These results will help those studying the Indian monsoon region to select an appropriate dataset depending on their application and focus of research

Purine nucleoside phosphorylase: A new marker for free oxygen radical injury to the endothelial cell

The effect of ischemia and reperfusion on purine nucleoside phosphorylase was studied in an isolated perfused rat liver model. This enzyme is localized primarily in the cytoplasm of the endothelial and Kupffer cells; some activity is associated with the parenchymal cells. Levels of this enzyme accurately predicted the extent of ischemia and reperfusion damage to the microvascular endothelial cell of the liver. Livers from Lewis rats were subjected to 30, 45 and 60 min of warm (37° C) no flow ischemia that was followed by a standard reperfusion period lasting 45 min. Purine nucleoside phosphorylase was measured at the end of the no flow ischemia and reperfusion periods as was superoxide generation (O2‐). Bile production was monitored throughout the no flow ischemia and reperfusion periods. Control perfusions were carried out for 120 min. A significant rise in purine nucleoside phosphorylase levels as compared with controls was observed at the end of ischemia in all the three groups. The highest level, 203.5 ± 29.2 mU/ml, was observed after 60 min of ischemia. After the reperfusion period, levels of purine nucleoside phosphorylase decreased in the 30‐ and 45‐min groups 58.17 ± 9.66 mU/ml and 67.5 ± 17.1 mU/ml, respectively. These levels were equal to control perfusions. In contrast, after 60 min of ischemia, levels of purine nucleoside phosphorylase decreased early in the reperfusion period and then rose to 127.8 ± 14.8 mU/ml by the end of reperfusion (p < 0.0001). Superoxide generation at the beginning of reperfusion was higher than in controls with similar values observed at the end of 30, 45 and 60 min of ischemia. During reperfusion, production of superoxide continued. Bile production was significantly lower at the end of 30 min (0.044 ± 0.026 μl/min/gm), 45 min (0.029 ± 0.0022 μ/min/gm) and 60 min of ischemia (0.022 ± 0.008 μ/min/gm) when compared with bile production by control livers during the corresponding time (0.680 ± 0.195, 0.562 ± 0.133 and 0.480 ± 0.100 μ/min/gm respectively; p < 0.001). During reperfusion, rates of bile production were normal after 30 and 45 min of ischemia. In contrast, significantly lower rates of bile production, 0.046 ± 0.36 μ/min/gm (p < 0.001) occurred during reperfusion after 60 min of ischemia. Control livers during the same period produced 0.330 ± 0.056 μl/min/gm of bile. The results indicate that purine nucleoside phosphorylase levels may be a good index of oxidative injury to the liver in ischemia reperfusion and reliably predict the functional state of the organ after reperfusion. Copyright © 1990 American Association for the Study of Liver Disease