Cardiovascular actions of central neuromedin U in conscious rats

Neuromedin U (NMU) is a brain-gut peptide, which peripherally stimulates smooth muscle, increases of blood pressure, alters ion transport in the gut, controls local blood flow, and regulates adrenocortical function. Although intracerebroventricular (i.c.v.) administration of NMU is known to decrease food intake and body weight, little is known about its effect on other physiological functions. We examined the effects of i.c.v. administration of NMU on mean arterial pressure (MAP), heart rate (HR), and plasma norepinephrine in conscious rats. Neuromedin U (0.05 and 0.5 nmol) provoked an increase in MAP (93.8 +/- 0.5 to 123.5 +/- 1.7 and 94.7 +/- 0.8 to 132.7 +/- 3.0 mm Hg, respectively) and HR (334.9 +/- 6.0 to 494.1 +/- 6.9 and 346.3 +/- 3.3 to 475.1 +/- 8.9 beats/min, respectively). In contrast, plasma norepinephrine increased only with a high dose of neuromedin U. Intravenously administered NMU (0.5 nmol) elicited a small and short lasting increase in MAP, compared to that by i.c.v. NMU. These results indicate that central neuromedin U regulates sympathetic nervous system activity and affects cardiovascular function.     

Structure of the bc1 complex from Seculamonas ecuadoriensis,a jakobid flagellate with an ancestral mitochondrial genome

In eubacteria, the respiratory bc(1) complex (complex III) consists of three or four different subunits, whereas that of mitochondria, which have descended from an alpha-proteobacterial endosymbiont, contains about seven additional subunits. To understand better how mitochondrial protein complexes evolved from their simpler bacterial predecessors, we purified complex III of Seculamonas ecuadoriensis, a member of the jakobid protists, which possess the most bacteria-like mitochondrial genomes known. The S. ecuadoriensis complex III has an apparent molecular mass of 460 kDa and exhibits antimycin-sensitive quinol:cytochrome c oxidoreductase activity. It is composed of at least eight subunits between 6 and 46 kDa in size, including two large "core" subunits and the three "respiratory" subunits. The molecular mass of the S. ecuadoriensis bc(1) complex is slightly lower than that reported for other eukaryotes, but about 2x as large as complex III in bacteria. This indicates that the departure from the small bacteria-like complex III took place at an early stage in mitochondrial evolution, prior to the divergence of jakobids. We posit that the recruitment of additional subunits in mitochondrial respiratory complexes is a consequence of the migration of originally alpha-proteobacterial genes to the nucleus.     

Rapamycin treatment results in GATA factor-independent hyperphosphorylation of the proline utilization pathway activator in Saccharomyces cerevisiae

Treatment of Saccharomyces cerevisiae cells with the immunosuppressive drug rapamycin results in a variety of cellular changes in response to perceived nutrient deprivation. Among other effects, rapamycin treatment results in the nuclear localization of the global nitrogen activators Gln3p and Nil1p/Gat1p, which leads to expression of nitrogen assimilation genes. The proline utilization (Put) pathway genes were shown to be among the genes induced by rapamycin. Having previously shown that the Put pathway activator Put3p is differentially phosphorylated in response to the quality of the nitrogen source, we examined the phosphorylation status of Put3p after rapamycin treatment. Treatment with rapamycin resulted in the hyperphosphorylation of Put3p, which was independent of Gln3p, Nil1p, and Ure2p. The relative contributions of global nitrogen (Gln3p and Nil1p) and pathway-specific (Put3p) activators to rapamycin-induced expression of the target gene PUT1 were also examined. We found that Nil1p and Put3p, but not Gln3p, play major roles in rapamycin-induced PUT1 expression. Our findings show that perceived nitrogen deprivation triggered by rapamycin treatment and steady-state growth in nitrogen-derepressing conditions are associated with hyperphosphorylation of Put3p and increased PUT1 expression. Rapamycin treatment and nitrogen derepression may share some, but not all, regulatory elements, since Gln3p and Nil1p do not participate identically in both processes and are not required for hyperphosphorylation. A complex relationship exists among the global and pathway-specific regulators, depending on the nature and quality of the nitrogen source.     

Mechanisms of TCDD-induced abnormalities and embryo lethality in white leghorn chickens

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds in birds has been well-established in laboratory and field studies. Observed effects of TCDD and related chemicals in birds include developmental deformities, reproductive failure, liver damage, wasting syndrome and death. The mechanism of action of TCDD at the cellular level is primarily mediated through the aryl hydrocarbon receptor (AhR). However, the mechanism of toxic action at the organism level is poorly understood. In this study, the role of radical oxygen species and mixed function oxidize (MFO; cytochrome P4501A) in the mechanism of TCDD-induced abnormalities and lethality were examined by co-injecting radical scavengers and an MFO inhibitor (piperonyl butoxide). Egg injection studies were conducted to determine if in ovo TCDD exposure can cause oxidative stress in white leghorn chicken eggs. Test agents were injected into the yolk prior to incubation. Treatments included TCDD (150 ng/kg), triolein (vehicle control), and various co-treatments including MnTBAP (a mimetic of superoxide dismutase), piperonyl butoxide, piroxicam, vitamin A acetate, and vitamin E succinate. Phenytoin, which is known to cause teratogenesis through oxidative stress was used as a positive control. Eggs were incubated until hatch and then the following parameters were assessed: mortality, hatching success, abnormalities, weights for whole body, liver, heart and brain, and biochemical endpoints for oxidative stress. As a measure of exposure, concentrations of TCDD and ethoxyresorufin-O-deethylase (EROD) activities were measured in tissues of hatchlings. While greater mortality and abnormalities were observed in the TCDD treatment groups, the number of the replicates were not great enough to detect statistically significant differences in abnormality rates for the co-treatments. Some of the observed developmental abnormalities included edema, liver necrosis and bill, eye and limb deformities with TCDD treatments, bill and brain deformities with phenytoin treatments, eye abnormalities with Vitamin E treatments, and abnormal feather pigmentation with piperonyl butoxide treatments.     

Aromatic clusters: a determinant of thermal stability of thermophilic proteins

A number of factors have been elucidated as responsible for the thermal stability of thermophilic proteins. However, the contribution of aromatic interactions to thermal stability has not been systematically studied. In the present investigation we used a graph spectral method to identify aromatic clusters in a dataset of 24 protein families for which the crystal structures of both the thermophilic and their mesophilic homologues are known. Our analysis shows a presence of additional aromatic clusters or enlarged aromatic networks in 17 different thermophilic protein families, which are absent in the corresponding mesophilic homologue. The additional aromatic clusters identified in the thermophiles are smaller in size and are largely found on the protein surface. The aromatic clusters are found to be relatively rigid regions of the surface and often the additional aromatic cluster is located close to the active site of the thermophilic enzyme. The residues in the additional aromatic clusters are preferably mutated to Leu, Ser or Ile in the mesophilic homologue. An analysis of the packing geometry of the pairwise aromatic interaction in the additional aromatic clusters shows a preference for a T-shaped orthogonal packing geometry. The present study also provides new insights for protein engineers to design thermostable and thermophilic proteins.     

Evidence for the induction of apoptosis by endosulfan in a human T-cell leukemic line

Several organochlorinated pesticides including DDT, PCBs and dieldrin have been reported to cause immune suppression and increase susceptibility to infection in animals. Often this manifestation is accompanied by atrophy of major lymphoid organs. It has been suggested that increased apoptotic cell death leading to altered T-B cell ratios, and loss of regulatory cells in critical numbers leads to perturbations in immune function. The major objective of our study was to define the mechanism by which endosulfan, an organochlorinated pesticide, induces human T-cell death using Jurkat, a human T-cell leukemic cell line, as an in vitro model. We exposed Jurkat cells to varying concentrations of endosulfan for 0-48 h and analyzed biochemical and molecular features characteristic of T-cell apoptosis. Endosulfan lowered cell viability and inhibited cell growth in a dose- and time-dependent manner. DAPI staining was used to enumerate apoptotic cells and we observed that endosulfan at 10-200 M induced a significant percentage of cells to undergo apoptotic cell death. At 48 h, more than 90% cells were apoptotic with 50 M of endosulfan. We confirmed these observations using both DNA fragmentation and annexin-V binding assays. It is now widely being accepted that mitochondria undergo major changes early during the apoptotic process. We examined mitochondrial transmembrane potential (_m) in endosulfan treated cells to understand the role of the mitochondria in T-cell apoptosis. Within 30 min of chemical exposure, a significant percentage of cells exhibited a decreased incorporation of DiOC₆(3), a cationic lipophilic dye into mitochondria indicating the disruption of _m. This drop in _m was both dose- and time-dependent and correlated well with other parameters of apoptosis. We also examined whether this occurred by the down regulation of bcl-2 protein expression that is likely to increase the susceptibility of Jurkat cells to endosulfan toxicity. Paradoxically, the intracellular expression of bcl-2 protein was elevated in a dose dependent manner suggesting endosulfan-induced apoptosis occurred by a non-bcl-2 pathway. Based on these data, as well as those reported elsewhere, we propose the following sequence of events to account for T-cell apoptosis induced by endosulfan: uncoupling of oxidative phosphorylation excess ROS production GSH depletion oxidative stress disruption of _m release of cytochrome C and other apoptosis related proteins to cytosol apoptosis. This study reports for the first time that endosulfan can induce apoptosis in a human T-cell leukemic cell line which may have direct relevance to loss of T cells and thymocytes in vivo. Furthermore, our data strongly support a role of mitochondrial dysfunction and oxidative stress in endosulfan toxicity.     

Human carbonic anhydrase I: Effect of specific site mutations on its function

Carbonic anhydrase I (CAI) is one out of ten CA isoenzymes that have been identified in humans. X-ray crystallographic and inhibitor complex studies of human carbonic anhydrase I (HCAI) and related studies in other CA isoenzymes identified several residues, in particular Thr199, GlulO6, Tyr7, Glull7, His l07, with likely involvement in the catalytic activity of HCAI. To further study the role of these residues, we undertook, site-directed mutagenesis of HCAI. Using a polymerase chain reaction based strategy and altered oligonucleotide primers, we modified a cloned wild type hCAI gene so as to produce mutant genes encoding proteins with single amino acid substitutions. Thrl99Val, Thrl99Cys, Thr199Ser, GlulO6Ile, Glul06Gln, Tyr7Trp, Glu.117Gln, and His 107Val mutations were thus generated and the activity of each measured by ester hydrolysis. Overproduction of the Glu117Gln and HisI07Val mutant proteins inEscherichia coli resulted in a large proportion of the enzyme forming aggregates probably due to folding defect. The mutations Thr199Val, GlulO6Ile and GlulO6Gln gave soluble protein with drastically reduced enzyme activity, while the Tyr7Trp mutation had only marginal effect on the activity, thus s.uggesting important roles for Thr199 and Glu lO6 but not for Tyr7 in the catalytic function of HCAI.     

Rac1 Regulates Endometrial Secretory Function to Control Placental Development

During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal-endothelial and stromal-trophoblast crosstalk critical for placenta development and establishment of pregnancy.     

Rolling circle amplification-based detection of human topoisomerase I activity on magnetic beads

A high-sensitivity assay has been developed for the detection of human topoisomerase I with single molecule resolution. The method uses magnetic sepharose beads to concentrate rolling circle products, produced by the amplification of DNA molecules circularized by topoisomerase I and detectable with a confocal microscope as single and discrete dots, once reacted with fluorescent probes. Each dot, corresponding to a single cleavage–religation event mediated by the enzyme, can be counted due to its high signal/noise ratio, allowing detection of 0.3 pM enzyme and representing a valid method to detect the enzyme activity in highly diluted samples.     

Enhanced decolourization of Direct Red-80 dye by the white rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> employing sequential design of experiments

Decolourization of Direct Red 80 (DR-80) by the white rot fungus Phanerochaete chrysosporium MTCC 787 was investigated employing sequential design of experiments. Media components for growing the white rot fungus were first screened using Plackett-Burman design and then optimized using response surface methodology (RSM), which resulted in enhancement in the efficiency of dye removal by the fungus. For determining the effect of media constituents on the dye removal, both percent dye decolourization and specific dye removal due to maximum enzyme activity were chosen as the responses from the experiments, and the media constituents glucose, veratryl alcohol, KH₂PO₄, CaCl₂ and MgSO₄ were screened to be the most effective with P values less than 0.05. Central composite design (CCD) followed by RSM in the optimization study revealed the following optimum combinations of the screened media constituents: glucose, 11.9 g l⁻¹; veratryl alcohol, 12.03 mM; KH₂PO₄, 23.08 g l⁻¹; CaCl₂, 2.4 g l⁻¹; MgSO₄, 10.47 g l⁻¹. At the optimum settings of the media constituents, complete dye decolourization (100% removal efficiency) and a maximum specific dye removal due to lignin peroxidase enzyme of 0.24 mg U⁻¹ by the white rot fungus were observed.