Purification of rat intestinal cinnabarinate synthase and its possible role in hyperplasia of the small intestine in diabetic rats

Streptozotocin diabetic rats fed ad libitum exhibited hyperplasia of the small intestine. As compared to the control animals, the intestine of experimental animals grew in weight, length and total RNA and DNA contents. Intestinal cinnabarinate synthase activity in diabetic rats was however significantly lower. Developmental studies in albino rats indicated that, attainment of the terminal and highest activity of the enzyme tends to correspond with cessation of further increase in RNA and DNA contents of the intestine, thereby suggesting a possible relationship between cinnabarinate synthase and the hyperplastic changes observed. It was also observed that some properties of this enzyme, such as Km and Vmax are modified in diabetic condition. The enzyme was purified to apparent homogeneity and some of its kinetic and other properties were studied.     

Mechanisms of transmembrane cation transport studied by nuclear magnetic resonance spectroscopy

Several molecules like ionophores, vitamins, ion-binding cyclic peptides, acidic phospholipids, surfactants are known to expose the inner side of vesicles, to the externally added cations. Whereas ionophores and certain other systems bring about these changes by a selective transport (influx) of the cation by specialized mechanisms known as the carrier and channel mechanism, other systems cause lysis and vesicle fusion. These systems have been successfully studied using¹H,³¹ P and¹³C nuclear magnetic resonance spectroscopy after the demonstration, fifteen years ago, of the ability of paramagnetic lanthanide ions to distinguish the inside of the vesicle from the outside. The results of these ’nuclear magnetic resonance kinetics’ experiments are reviewed.     

Two preferentially expressed proteins protect vascular endothelial cells from an attack by peptide-specific CTL

Vascular endothelial cells (EC) are an exposed tissue with intimate contact with circulating Ag-specific CTL. Experimental in vitro and clinical data suggested that endothelial cells present a different repertoire of MHC class I-restricted peptides compared with syngeneic leukocytes or epithelial cells. This endothelial-specific peptide repertoire might protect EC from CTL-mediated cell death. The HLA-A*02-restricted peptide profile of human EC and syngeneic B lymphoblastoid cells was biochemically analyzed and compared. For EC selective peptides, source protein expression, peptide binding affinity, and peptide-HLA-A*02 turnover were measured. The significance of abundant peptide presentation for target cell recognition by immunodominant CTL was tested by small interfering RNA treatment of EC to knock down the source proteins. High amounts of two peptides, PTRF(56-64) and CD59(106-114), were consistently detected in EC. This predominance of two endothelial peptides was explained by cell type-specific source protein expression that compensated for poor HLA-A*02 binding affinity and short half-live of peptide/HLA-A*02 complexes. Knocking down the source proteins containing the abundant endothelial peptide motifs led to a nearly 100-fold increase of surface expression of SMCY(311-319), an immunodominant minor histocompatibility Ag, as detected by cytotoxicity assays using SMCY(311-319)-specific CTL. We conclude that EC express and present preferentially two distinct HLA-A*02-restricted peptides at extraordinary high levels. These abundant self-peptides may protect EC from CTL-mediated lysis by competing for HLA-A*02 binding sites with immunodominant scarcely expressed antigenic peptides.     

Biotic stress resistance in agriculture through antimicrobial peptides

Antimicrobial peptides (AMPs) are the hosts' defense molecules against microbial pathogens and gaining extensive research attention worldwide. These have been reported to play vital role of host innate immunity in response to microbial challenges. AMPs can be used as a natural antibiotic as an alternative of their chemical counterpart for protection of plants/animals against diseases. There are a number of sources of AMPs including prokaryotic and eukaryotic organisms and are present, both in vertebrates and invertebrates. AMPs can be classified as cationic or anionic, based on net charges. Large number of databases and tools are available in the public domain which can be used for development of new genetically modified disease resistant varieties/breeds for agricultural production. The results of the biotechnological research as well as genetic engineering related to AMPs have shown high potential for reduction of economic losses of agricultural produce due to pathogens. In this article, an attempt has been made to introduce the role of AMPs in relation to plants and animals. Their functional and structural characteristics have been described in terms of its role in agriculture. Different sources of AMPs and importance of these sources has been reviewed in terms of its availability. This article also reviews the bioinformatics resources including different database tools and algorithms available in public domain. References of promising biotechnology research in relation to AMPs, prospects of AMPs for further development of genetically modified varieties/breeds are highlighted. AMPs are valuable resource for students, researchers, educators and medical and industrial personnel.     

Lactobacillus protected bone damage and maintained the antioxidant status of liver and kidney homogenates in female wistar rats

The aim of the study was to evaluate protective property of Lactobacillus casei and Lactobacillus acidophilus in minimizing oxidative stress associated with arthritis from liver and kidney. Subsequently, protective property of Lactobacillus against the bone damage was also taken into consideration. Arthritis was induced by injecting freund's complete adjuvant (100 μl) into sub plantar surface of hind paw. Oral administration of culture, vehicle, and drug started after induction of arthritis (i.e. on day 9th). Indomethacin was used as a standard drug. Radiographic analysis of the hind paw knee joint was also done at the end of the 21st day. Oxidative stress parameters were studied from liver and kidney homogenate. Histopathology of liver and kidney was also performed. Lactobacillus treatment significantly rescued the enzymatic level of catalase, superoxide dismutase, reduced glutathione, and glutathione peroxidase in both liver and kidney homogenates, whereas it has decreased the malonaldehyde accumulation. Oral administration of Lactobacillus also significantly decreased the serum ceruloplasmin level. Radiographic analysis also corroborated these findings. Lactobacillus treatment maintained the normal histopathology of liver and kidney. Results of this study clearly suggest that L. casei and L. acidophilus, alone or in combination, decreased the bone damaged and effectively restored antioxidant status of liver and kidney. Lactobacillus has a significant antiarthritic and antioxidant activity against freund's complete adjuvant induced arthritis in rats.     

Sequence-based novel genomic microsatellite markers for robust genotyping purposes in foxtail millet [<Emphasis Type="Italic">Setaria italica</Emphasis> (L.) P. Beauv.]

The unavailability of microsatellite markers and saturated genetic linkage map has restricted the genetic improvement of foxtail millet [Setaria italica (L.) P. Beauv.], despite the fact that in recent times it has been documented as a new model species for biofuel grasses. With the objective to generate a good number of microsatellite markers in foxtail millet cultivar ‘Prasad’, 690 clones were sequenced which generated 112.95 kb high quality sequences obtained from three genomic libraries each enriched with different microsatellite repeat motifs. Microsatellites were identified in 512 (74.2%) of the 690 positive clones and 172 primer pairs (pp) were successfully designed from 249 (48.6%) unique SSR-containing clones. The efficacies of the microsatellite containing genomic sequences were established by superior primer designing ability (69%), PCR amplification efficiency (85.5%) and polymorphic potential (52%) in the parents of F₂ mapping population. Out of 172 pp, functional 147 markers showed high level of cross-species amplification (~74%) in six grass species. Higher polymorphism rate and broad range of genetic diversity (0.30–0.69 averaging 0.58) obtained in constructed phylogenetic tree using 52 microsatellite markers, demonstrated the utility of markers in germplasm characterizations. In silico comparative mapping of 147 foxtail millet microsatellite containing sequences against the mapping data of sorghum (~18%), maize (~16%) and rice (~5%) indicated the presence of orthologous sequences of the foxtail millet in the respective species. The result thus demonstrates the applicability of microsatellite markers in various genotyping applications, determining phylogenetic relationships and comparative mapping in several important grass species.     

Isolation and characterization of microcystin producing Microcystis from a Central Indian water bloom

Microcystis aeruginosa Kütz, a well-known microcystin (hepatotoxin) producing cyanobacterium was the dominant bloom-forming organism in a mesotrophic lake at Nagpur in Central India, which was isolated and characterized for morphospecies and microcystin content. Compact spherical colonies, formation of daughter colonies, and clathration of older colonies leading to release of solitary cells, were characteristics of laboratory grown M. aeruginosa. Its growth, monitored as increase in optical density (OD) measured at 678 nm (the wavelength selected using dilution curve technique), exhibited a maximum specific growth rate (μ_max) of 0.34 day⁻¹ which, was attained on the 5th day of the experiment with a doubling time of 3.25 days. Though the morphological characters of the M. aeruginosa under field conditions were not retained under laboratory conditions, the microcystin content and type of variants did match with bloom samples. Reverse phase high performance liquid chromatography (RP-HPLC) analyses revealed that the laboratory grown isolate of Microcystis produced microcystin-RR (732 μg g⁻¹ dry weight biomass) and demethylated microcystin-RR (165 μg g⁻¹ dry weight biomass) variants, which are reported to be less toxic when compared to microcystin-LR. LC/ESI/MS further confirmed the presence of these two variants. Geographical distribution of microcystin variants and their prevailing concentrations need to be considered during formulation of guideline values for drinking and recreational waters.     

The apolipoprotein A-I mimetic peptide 4F prevents defects in vascular function in endotoxemic rats

High density lipoprotein (HDL) and apolipoprotein A-I (apoA-I) reduce inflammatory responses to lipopolysaccharide (LPS). We tested the hypothesis that the apoA-I mimetic peptide 4F prevents LPS-induced defects in blood pressure and vascular reactivity. Systolic blood pressure (SBP) was measured in rats at baseline and 6 h after injection of LPS (10 mg/kg) or saline vehicle. Subgroups of LPS-treated rats also received 4F (10 mg/kg) or scrambled 4F (Sc-4F). LPS administration reduced SBP by 35% compared with baseline. 4F attenuated the reduction in SBP in LPS-treated rats (17% reduction), while Sc-4F was without effect. Ex vivo studies showed a reduced contractile response to phenylephrine (PE) in aortae of LPS-treated rats (ED₅₀ = 459 ± 83 nM) compared with controls (ED₅₀ = 57 ± 6 nM). This was associated with nitric oxide synthase 2 (NOS2) upregulation. 4F administration improved vascular contractility (ED₅₀ = 60 ± 9 nM), reduced aortic NOS2 protein, normalized plasma levels of NO metabolites, and reduced mortality in LPS-treated rats. These changes were associated with a reduction in plasma endotoxin activity. In vivo administration of ¹⁴C-4F and Bodipy-LPS resulted in their colocalization and retention in the HDL fraction. It is proposed that 4F promotes the localization of LPS to the HDL fraction, resulting in endotoxin neutralization. 4F may thus prevent LPS-induced hemodynamic changes associated with NOS2 induction.     

Structural consequences of replacement of an alpha-helical Pro residue in Escherichia coli thioredoxin

While it is well known that introduction of Pro residues into the interior of protein alpha-helices is destabilizing, there have been few studies that have examined the structural and thermodynamic effects of the replacement of a Pro residue in the interior of a protein alpha-helix. We have previously reported an increase in stability in the P40S mutant of Escherichia coli thioredoxin of 1-1.5 kcal/mol in the temperature range 280-330 K. This paper describes the structure of the P40S mutant at a resolution of 1.8 A. In wild-type thioredoxin, P40 is located in the interior of helix two, a long alpha-helix that extends from residues 32 to 49 with a kink at residue 40. Structural differences between the wild-type and P40S are largely localized to the above helix. In the P40S mutant, there is an expected additional hydrogen bond formed between the amide of S40 and the carbonyl of residue K36 and also additional hydrogen bonds between the side chain of S40 and the carbonyl of K36. The helix remains kinked. In the wild-type, main chain hydrogen bonds exist between the amide of 44 and carbonyl of 40 and between the amide of 43 and carbonyl of 39. However, these are absent in P40S. Instead, these main chain atoms are hydrogen bonded to water molecules. The increased stability of P40S is likely to be due to the net increase in the number of hydrogen bonds in helix two of E.coli thioredoxin.