Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein

The MoFe protein of the complex metalloenzyme nitrogenase folds as a heterotetramer containing two copies each of the homologous alpha and beta subunits, encoded by the nifD and the nifK genes respectively. Recently, the functional expression of a fusion NifD-K protein of nitrogenase was demonstrated in Azotobacter vinelandii, strongly implying that the MoFe protein is flexible as it could accommodate major structural changes, yet remain functional [M.H. Suh, L. Pulakat, N. Gavini, J. Biol. Chem. 278 (2003) 5353-5360]. This finding led us to further explore the type of interaction between the fused MoFe protein units. We aimed to determine whether an interaction exists between the two fusion MoFe proteins to form a homodimer that is equivalent to native heterotetrameric MoFe protein. Using the Bacteriomatch Two-Hybrid System, translationally fused constructs of NifD-K (fusion) with the full-length lambdaCI of the pBT bait vector and also NifD-K (fusion) with the N-terminal alpha-RNAP of the pTRG target vector were made. To compare the extent of interaction between the fused NifD-K proteins to that of the beta-beta interactions in the native MoFe protein, we proceeded to generate translationally fused constructs of NifK with the alpha-RNAP of the pTRG vector and lambdaCI protein of the pBT vector. The strength of the interaction between the proteins in study was determined by measuring the beta-galactosidase activity and extent of ampicillin resistance of the colonies expressing these proteins. This analysis demonstrated that direct protein-protein interaction exists between NifD-K fusion proteins, suggesting that they exist as homodimers. As the interaction takes place at the beta-interfaces of the NifD-K fusion proteins, we propose that these homodimers of NifD-K fusion protein may function in a similar manner as that of the heterotetrameric native MoFe protein. The observation that the extent of protein-protein interaction between the beta-subunits of the native MoFe protein in BacterioMatch Two-Hybrid System is comparable to the extent of protein-protein interaction observed between the NifD-K fusion proteins in the same system further supports this idea.     

Mechanism of promoter activity of the beta-amyloid precursor protein gene in different cell lines: identification of a specific 30 bp fragment in the proximal promoter region

The amyloid beta-protein (Abeta) deposited in brains of Alzheimer's disease (AD) patients is proteolytically derived from a large Abeta precursor protein (APP). APP gene expression patterns in the AD brain region indicate that abnormalities of gene regulation may be important in AD pathology. To understand the contribution of different cell types to APP gene expression, we studied it at four levels: promoter activity (by reporter gene assay of transfected cells), DNA-nuclear protein interaction (by electrophoretic mobility shift assay), RNA message and protein (by northern and western blotting, respectively). APP mRNA and protein expression levels were greater in neuroblastoma and PC12 cells than in glial or cervix epithelial cells. Relative activity among 12 different promoter regions and within single regions varied according to cell type/cell line. An upstream regulatory region containing a GATA-1 site is necessary for activity in PC12 and glial cells but not in neuroblastoma cells. DNA-protein interactions were examined in three distal and one proximal promoter elements in nuclear extracts belonging to neuronal and non-neuronal cells. The proximal promoter region is important for cell line-specific APP gene expression. Characterization of the APP regulatory region's interaction with cell type-specific nuclear factor(s) is important to understand tissue-specific expression of APP seen in AD subjects.     

Linkage of recessive Robinow syndrome to a 4 cM interval on chromosome 9q22

Autosomal recessive Robinow syndrome is a form of mesomelic dwarfism with multiple rib and vertebral anomalies. Using autozygosity mapping we have identified a genetic locus (RBNW1) for this syndrome at chromosome 9q22 in seven consanguineous families from Oman. Our results indicate that the gene lies within a 4 cM region between markers D9S1836 and D9S1803 (maximum multipoint LOD score 12.3). In addition, we have analysed two non-Omani families with autosomal recessive Robinow and found no genetic heterogeneity.     

Effects of changes in central blood volume on carotid-vasomotor baroreflex sensitivity at rest and during exercise.

The purpose of this investigation was to examine whether the effect of changes in central blood volume on carotid-vasomotor baroreflex sensitivity at rest was the same during exercise. Eight men (means +/- SE: age 26 +/- 1 yr; height 180 +/- 3 cm; weight 86 +/- 6 kg) participated in the present study. Sixteen Torr of lower body negative pressure (LBNP) were applied to decrease central venous pressure (CVP) at rest and during steady-state leg cycling at 50% peak O2 uptake (104 +/- 20 W). Subsequently, infusions of 25% human serum albumin solution were administered to increase CVP at rest and during exercise. During all protocols, heart rate, arterial blood pressure, and CVP were recorded continuously. At each stage of LBNP or albumin infusion, the maximal gain (G(max)) of the carotid-vasomotor baroreflex function curve was measured using the neck pressure and neck suction technique. LBNP reduced CVP and increased the G(max) of the carotid-vasomotor baroreflex function curve at rest (+63 +/- 25%, P = 0.006) and during exercise (+69 +/- 19%, P = 0.002). In contrast to the LBNP, increases in CVP resulted in the G(max) of the carotid-vasomotor baroreflex function curve being decreased at rest -8 +/- 4% and during exercise -18 +/- 5% (P > 0.05). These findings indicate that the relationship between CVP and carotid-vasomotor baroreflex sensitivity was nonlinear at rest and during exercise and suggests a saturation load of the cardiopulmonary baroreceptors at which carotid-vasomotor baroreflex sensitivity remains unchanged.     

An improved protein structure evaluation using a semi-empirically derived structure property

Background

In the backdrop of challenge to obtain a protein structure under the known limitations of both experimental and theoretical techniques, the need of a fast as well as accurate protein structure evaluation method still exists to substantially reduce a huge gap between number of known sequences and structures. Among currently practiced theoretical techniques, homology modelling backed by molecular dynamics based optimization appears to be the most popular one. However it suffers from contradictory indications of different validation parameters generated from a set of protein models which are predicted against a particular target protein. For example, in one model Ramachandran Score may be quite high making it acceptable, whereas, its potential energy may not be very low making it unacceptable and vice versa. Towards resolving this problem, the main objective of this study was fixed as to utilize a simple experimentally derived output, Surface Roughness Index of concerned protein of unknown structure as an intervening agent that could be obtained using ordinary microscopic images of heat denatured aggregates of the same protein.

Result

It was intriguing to observe that direct experimental knowledge of the concerned protein, however simple it may be, might give insight on acceptability of its particular structural model out of a confusion set of models generated from database driven comparative technique for structure prediction. The result obtained from a widely varying structural class of proteins indicated that speed of protein structure evaluation can be further enhanced without compromising with accuracy by recruiting simple experimental output.

Conclusion

In this work, a semi-empirical methodological approach was provided for improving protein structure evaluation. It showed that, once structure models of a protein were obtained through homology technique, the problem of selection of a best model out of a confusion set of Pareto-optimal structures could be resolved by employing a structure agent directly obtainable through experiment with the same protein as experimental ingredient. Overall, in the backdrop of getting a reasonably accurate protein structure of pathogens causing epidemics or biological warfare, such approach could be of use as a plausible solution for fast drug design.

     

A putative <Emphasis Type="Italic">NEM1</Emphasis> homologue regulates lipid droplet biogenesis <Emphasis Type="Italic">via PAH1</Emphasis> in <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Nuclear envelope morphology protein 1 (NEM1) along with a phosphatidate phosphatase (PAH1) regulates lipid homeostasis and membrane biogenesis in yeast and mammals. We investigated four putative NEM1 homologues (TtNEM1A, TtNEM1B, TtNEM1C and TtNEM1D) in the Tetrahymena thermophila genome. Disruption of TtNEM1B, TtNEM1C or TtNEM1D did not compromise normal cell growth. In contrast, we were unable to generate knockout strain of TtNEM1A under the same conditions, indicating that TtNEM1A is essential for Tetrahymena growth. Interestingly, loss of TtNEM1B but not TtNEM1C or TtNEM1D caused a reduction in lipid droplet number. Similar to yeast and mammals, TtNem1B of Tetrahymena exerts its function via Pah1, since we found that PAH1 overexpression rescued loss of Nem1 function. However, unlike NEM1 in other organisms, TtNEM1B does not regulate ER/nuclear morphology. Similarly, neither TtNEM1C nor TtNEM1D is required to maintain normal ER morphology. While Tetrahymena PAH1 was shown to functionally replace yeast PAH1 earlier, we observed that Tetrahymena NEM1 homologues did not functionally replace yeast NEM1. Overall, our results suggest the presence of a conserved cascade for regulation of lipid homeostasis and membrane biogenesis in Tetrahymena. Our results also suggest a Nem1-independent function of Pah1 in the regulation of ER morphology in Tetrahymena.     

Kinetics of Avibactam Inhibition against Class A,C, and D β-Lactamases

Avibactam is a non-β-lactam β-lactamase inhibitor with a spectrum of activity that includes β-lactamase enzymes of classes A, C, and selected D examples. In this work acylation and deacylation rates were measured against the clinically important enzymes CTX-M-15, KPC-2, Enterobacter cloacae AmpC, Pseudomonas aeruginosa AmpC, OXA-10, and OXA-48. The efficiency of acylation (k₂/K_i) varied across the enzyme spectrum, from 1.1 × 10¹ m⁻¹s⁻¹ for OXA-10 to 1.0 × 10⁵ for CTX-M-15. Inhibition of OXA-10 was shown to follow the covalent reversible mechanism, and the acylated OXA-10 displayed the longest residence time for deacylation, with a half-life of greater than 5 days. Across multiple enzymes, acyl enzyme stability was assessed by mass spectrometry. These inhibited enzyme forms were stable to rearrangement or hydrolysis, with the exception of KPC-2. KPC-2 displayed a slow hydrolytic route that involved fragmentation of the acyl-avibactam complex. The identity of released degradation products was investigated, and a possible mechanism for the slow deacylation from KPC-2 is proposed.     

Comparative analysis of serum zinc,copper, magnesium,calcium and iron level in acute and chronic patients of visceral leishmaniasis

ProjectChronic visceral leishmaniasis (VL) is an increasingly common problem in disease endemic states of India. Identification of prognosis risk factor in patients with VL may lead to preventive actions, toward decreasing its mortality in chronic individuals. Though serum Zinc levels are decreased in patients of VL, limited information is available regarding trace elements status in acute and chronic VL patients. The present study was undertaken to compare serum trace elements concentrations in acute and chronic VL patients.ProcedureAcute (mean age = 28.64 years), chronic (mean age = 23.68 years) VL patients and healthy controls (mean age = 23.05 years) who agreed to provide blood specimens for laboratory investigations participated in this study. Serum zinc (Zn), copper (Cu), iron (Fe), magnesium (Mg) and calcium (Ca) were measured spectrophotometrically using chemistry analyzer.ResultsSerum Zn concentration was comparatively much decreased in chronic VL than to acute ones (p = 0.007) while serum Mg was higher in chronic VL than acute (p = 0.002) ones. There was no statistically significant difference between acute and chronic VL in serum concentrations of Cu, Fe and Ca.ConclusionsSerum Zn levels were much decreased and serum Mg were increased in chronic VL as compared to acute cases. The serum concentrations of Fe and Ca did not show any difference between two groups. The serum Cu was increased in both groups but more in chronic ones. Serum Zn and Mg could be a potential prognosis factor for chronic VL patients. We hypothesize zinc supplementation as a chemo preventive agent for chronic VL cases, particularly in endemic areas.