Habitat occupancy of sloth bear Melursus ursinus in Chitwan National Park,Nepal

Mammals have experienced a massive decline in their populations and geographic ranges worldwide. The sloth bear, Melursus ursinus (Shaw, 1791), is one of many species facing conservation threats. Despite being endangered in Nepal, decades of inattention to the situation have hindered their conservation and management. We assessed the distribution and patterns of habitat use by sloth bears in Chitwan National Park (CNP), Nepal. We conducted sign surveys from March to June, 2020, in 4 × 4 km grids (n = 45). We collected detection/non‐detection data along a 4‐km trail that was divided into 20 continuous segments of 200 m each. We obtained environmental, ecological, and anthropogenic covariates to understand determinants of sloth bear habitat occupancy. The data were analyzed using the single‐species single‐season occupancy method, with a spatially correlated detection. Using repeated observations, these models accounted for the imperfect detectability of the species to provide robust estimates of habitat occupancy. The model‐averaged occupancy estimate for the sloth bear was 69% and the detection probability was 0.25. The probability of habitat occupancy by sloth bears increased with the presence of termites and fruits and in rugged, dry, open, undisturbed habitats. Our results indicate that the sloth bear is elusive, functionally unique, and widespread in CNP. Future conservation interventions and action plans aimed at sloth bear management must adequately consider their habitat requirements.     

Ultrastructural studies on nodules induced by pyrimidine auxotrophs of Sinorhizobium meliloti

Twenty three pyrimidine auxotrophs of Sinorhizobium meliloti Rmd201 were generated by random mutagenesis with transposon Tn5. On the basis of biochemical characters these auxotrophic mutants were classified into car, pyrC and pyrE/pyrF categories. All auxotrophs induced white nodules which were ineffective in nitrogen fixation. Light and electron microscopic studies revealed that the nodules induced by pyrC mutants were more developed than the nodules of car mutants. Similarly the nodules induced by pyrE/pyrF mutants had more advanced structural features than the nodules of pyrC mutants. The nodule development in case of pyrE/pyrF mutants was not to the extent observed in the parental strain. These results indicated that some of the intermediates and/or enzymes of pyrimidine biosynthetic pathway of S. meliloti play a key role in bacteroidal transformation and nodule development.     

Photoaffinity labeling of mouse fibroblast enzymes by a base excision repair intermediate. Evidence for the role of poly(ADP-ribose) polymerase-1 in DNA repair

To examine the interaction of mammalian base excision repair (BER) enzymes with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast cellular extracts. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by apurinic/apyrimidinic endonuclease creating a nick with 3'-hydroxyl and 5'-reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was added to the 3'-hydroxyl group. With near-UV light (312 nm) exposure of the extract/probe mixture, six proteins were strongly labeled. Four of these include poly(ADP-ribose) polymerase-1 (PARP-1) and the BER participants flap endonuclease-1, DNA polymerase beta, and apurinic/apyrimidinic endonuclease. The amount of the probe cross-linked to PARP-1 was greater than that cross-linked to the other proteins. The specificity of PARP-1 labeling was examined using various competitor oligonucleotides and DNA probes with alternate structures. PARP-1 labeling was stronger with a DNA representing a BER intermediate than with a nick in double-stranded DNA. These results indicate that proteins interacting preferentially with a photoreactive BER intermediate can be selected from the crude cellular extract.     

Molecular analysis of a carbohydrate antigen involved in the structure and function of zona pellucida glycoproteins

A lactosaminoglycan-associated antigen is associated with a carbohydrate moiety of all three zona pellucida (ZP) glycoproteins of pig and rabbit but is absent in the mouse and rat. A monoclonal antibody (PS1) recognizing this determinant was obtained by immunizing mice with a porcine ZP glycoprotein isoform purified by two-dimensional polyacrylamide gel electrophoresis. Conditions known to remove O-linked or sialic acid carbohydrate moieties (alkaline reduction; O-glycanase or neuraminidase enzymatic cleavage) did not remove the carbohydrate epitope. However, treatment with endo-beta-glycosidase, endoglycosidase F, or combinations of neuraminidase plus beta-galactosidase, totally removed the determinant, indicating that it is associated with a poly-N-acetyllactosaminoglycan structure present on an N-linked oligosaccharide. Molecular morphology studies using immunofluorescence and confocal microscopy techniques demonstrate that the PS1 antigen is localized at the surface of the ZP. Confirmation of this localization was obtained through studies that show that this antibody will inhibit homologous sperm binding to the pig ZP. Additional analyses using modular contrast microscopy and immunocytochemistry demonstrate that this carbohydrate-associated antigen is localized in discrete layers throughout the ZP matrix. These studies are the first to demonstrate the presence of a lactosaminoglycan type carbohydrate moiety in all three ZP proteins using a monoclonal antibody that appears to be involved in sperm recognition and structural organization.     

The mechanism of aconitase: 1.8 A resolution crystal structure of the S642a:citrate complex

The crystal structure of the S642A mutant of mitochondrial aconitase (mAc) with citrate bound has been determined at 1.8 A resolution and 100 K to capture this binding mode of substrates to the native enzyme. The 2.0 A resolution, 100 K crystal structure of the S642A mutant with isocitrate binding provides a control, showing that the Ser --> Ala replacement does not alter the binding of substrates in the active site. The aconitase mechanism requires that the intermediate product, cis-aconitate, flip over by 180 degrees about the C alpha-C beta double bond. Only one of these two alternative modes of binding, that of the isocitrate mode, has been previously visualized. Now, however, the structure revealing the citrate mode of binding provides direct support for the proposed enzyme mechanism.     

Effect of cisplatin on Allium cepa root meristem cells

Allium cepa root growth was retarded by cisplatin treatment in a dose-dependent manner. A decrease in the mitotic index (MI) and an increase in the number of interphase cells was seen in cisplatin treated root tips. An increase in the frequency of abnormal mitoses and chromosomal aberrations was also observed in cisplatin treated groups which indicates its genotoxic effect on plant cells. The endogenous glutathione (GSH) level in the root tips decreased significantly after cisplatin treatment which may favour its increased interaction with cellular DNA thereby developing enhanced chromosomal aberrations and affecting cell divisions and root growth. It is suggested that the decrease in endogenous GSH may be related to the development of cisplatin-mediated genotoxic effects in plants.     

Effect of cigarette smoke on lipid peroxidation and antioxidant enzymes in albino rat

Effect of cigarette smoke on lipid peroxidation (LPX) and antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione-S-transferase (GST) in various organs like brain, heart, lung, liver and kidney of the albino rats exposed to cigarette smoke for 30 min/day for a period of 30 days were assayed. It was observed that the lipid peroxide levels in liver, lung and kidney were enhanced in case of animals exposed to cigarette smoke, whereas brain and heart did not show any change as compared to control animals. The activity of the antioxidant enzymes was also elevated in liver, lung and kidney of the test animals whereas, brain and heart did not show any change in the activities of all of these antioxidant enzymes except glutathione-s-transferase which was increased in brain also. The level of reduced glutathione (GSH) was lowered in liver, lung and kidney of the tested animals when compared with the control animals but there was no significant change in brain and heart. The results of our study suggest that cigarette smoke induces lipid peroxidation in liver, lung and kidney, and the antioxidant enzymes levels were enhanced in order to protect these tissues against the deleterious effect of the oxygen derived free radicals. The depletion of reduced glutathione in these organs could be due to it's utilization by the tissues to mop off the free radicals.     

Activation of Glut1 glucose transporter in response to inhibition of oxidative phosphorylation.

We have previously shown that exposure of Clone 9 cells to hypoxia, cyanide, or azide results in an acute stimulation of glucose transport that is largely mediated by "activation" of glucose transporter (Glut1) sites preexisting in the plasma membrane. However, it is not known whether inhibition of oxidative phosphorylation only at its terminal step, or at any of its steps, leads to the glucose transport response. Hence, the effect of azide (5 mM), rotenone (1 microM), rotenone (1 microM) plus thenoyltrifluoroacetone (TTFA) (5 microM), antimycin A (0.3 microM), dinitrophenol (0.25 mM), carbonyl cyanide m-chlorophenylhydrazone (CCCP) (2.5 microM), and oligomycin B (0.15 microM) on glucose transport was determined. All of the above agents elicited a similar approximately 4-fold stimulation of cytochalasin B (CB)-inhibitable 3-O-methyl glucose (3-OMG) uptake in Clone 9 cells. The stimulatory effect of azide on 3-OMG uptake was not inhibited by antioxidants 2-mercaptopropionyl glycine (1.2 mM) and 1,10-phenanthroline (40 microM), while, in contrast, the antioxidants attenuated the stimulation of glucose transport in response to 250 microM H(2)O(2) by approximately 50%. To differentiate between an increase in the number of functional Glut1 sites in the plasma membrane (in the absence of "translocation") versus an increase in the "intrinsic activity" of Glut1, the effect of azide on the energy of activation (E(a)) of glucose transport was measured. The E(a) was determined by measuring the rate of CB-inhibitable 3-OMG uptake at 24.0, 28.0, 35. 0, and 40 degrees C. The E(a) of control Clone 9 cells and of cells exposed to 10 mM azide for 2 h was 32,530 +/- 1830 and 31,220 +/- 600 J/mol, respectively (P > 0.1), while the rate of CB-inhibitable 3-OMG uptake was 9.3 +/- 0.7-fold higher in azide-treated cells. It is concluded that (i) inhibition of oxidative phosphorylation, at any of its steps, leads to a stimulation of glucose transport, and (ii) the mechanism of stimulation of glucose transport in response to azide appears to be predominately mediated by an apparent increase in the number of functional Glut1 sites in the plasma membrane (instead of an increase in their "intrinsic activity"), suggesting an "unmasking" mechanism.     

Human LAT1, a subunit of system L amino acid transporter: molecular cloning and transport function

We report here on the cloning and functional characterization of human LAT1, a subunit of the amino acid transport system L. The hLAT1 cDNA, obtained from a human placental cDNA library, codes for a protein of 507 amino acids. When functionally expressed in mammalian cells together with the heavy chain of the rat 4F2 antigen (r4F2hc), hLAT1 induces the transport of neutral amino acids. When expressed independently, neither hLAT1 nor r4F2hc was capable of amino acid transport to any significant extent. Thus, the hLAT1-r4F2hc heterodimeric complex is responsible for the observed amino acid transport. The transport process induced by the heterodimer is Na+ independent and is not influenced by pH. It recognizes exclusively neutral amino acids with high affinity. LAT1-specific mRNA is expressed in most human tissues with the notable exception of the intestine.     

Thyrotropin releasing hormone stimulation of GABA-gated but not basal chloride ion influx in rat cerebellum

Potential interaction between gamma-amino butyric acid (GABA) and thyrotropin-releasing hormone (TRH) in eliciting a variety of central nervous system (CNS)-related biologic activities is well known; however, the mechanism underlying this interaction is not clearly defined. To gain further insight into this interaction, we examined the effects of TRH and two of its central nervous system selective analogs, DN 1417 and TA 0910, on basal and GABA-mediated chloride ion influx into rat cerebellar neurosynaptosomes. The results of these studies show that TRH may facilitate GABA action by augmenting chloride ion influx and hyperpolarization of cerebellar neurons.