Histomorphology of preorbital gland in territorial and non-territorial male blackbuck Antelope cervicapra, a critically endangered species

The preorbital gland is a specialized dermal gland of antelopes which plays an important role in territorial marking behavior and pheromonal communication. To our knowledge, there is little information available on the role of preorbital gland marks in Indian antelopes (blackbucks). Males are seen averting the gland during behavioural display and territorial marking but the functional aspect of this gland has not been examined. Hence, the aim of this study was to describe the histomorphology of the preorbital gland in territorial and non-territorial male blackbucks to determine its morphology and secretory function. The results showed that the preorbital gland is composed of modified sebaceous and apocrine glands. The apocrine gland is lined by simple cuboidal epithelial cells; the serous parts of the secretory products are often seen in the apical portions of the cells. The myoepithelial cells contain actin filaments lying on the basal membranes of the apocrine gland. There are some considerable histological changes in the presence of the sebaceous and apocrine glands in territorial males in comparison to non-territorial males. The following histological changes associated with occurrence of the sebaceous and apocrine glands have been observed in territorial and non-territorial male blackbucks: (1) increase of size of sebaceous and apocrine glands and (2) increase in density of sebaceous and apocrine glands in territorial males compared to non-territorial males. It is suggested that the higher development (i.e., size) and density of sebaceous and apocrine glands in territorial males could depend on hormone production (i.e., testosterone). Based on the histological observation and the role of sebaceous and apocrine glands in the preorbital gland supported by literature, it is possible to conclude that both territorial and non-territorial blackbuck males may produce pheromonal substances through preorbital gland (secretion) for olfactory communication.     

Identification of bacteria in culture negative and polymerase chain reaction (PCR) positive intraocular specimen from patients with infectious endopthalmitis

A novel Denaturing High-Performance Liquid Chromatography (dHPLC)-based technique allows rapid high-resolution analysis of PCR products. We show the application of this PCR/dHPLC approach for direct detection and identification of bacterium from the Eubacterial PCR amplified products of aqueous and vitreous aspirates from patients with endopthalmitis and to differentially identify the culture negative cases and initiate appropriate therapy. The aim of this study is to identify culture negative PCR positive cases by the application of PCR based DNA sequencing. A total of 116 intraocular specimens were subjected for the study. Sixty-nine different bacteria were identified using dHPLC based DNA sequencing of which predominant ones were Gram-positive bacteria and cannot be cultured by conventional methods. Forty eight different bacteria detected in this study is being reported for the first time in infectious endopthalmitis.     

Biochemical analysis of female mice urine with reference to endocrine function: a key tool for estrus detection

Species-specific chemical signals released through urine, sweat, saliva and feces are involved in communication between animals. Urinary biochemical constituents along with pheromones may contribute to variation across reproductive cycles and facilitate to estrus detection. Hence, the present study was designed to analyze such biochemical profiles, such as proteins, carbohydrates, lipids, fatty acids, in response with steroid hormones such as estradiol and progesterone. The experimental groups were normal, prepubertal, ovariectomized, and ovariectomized with estrogentreated female mice. In normal mice, the protein and lipid concentrations in urine were significantly higher in proestrus and estrus phases and the quantity of fatty acids was also comparatively higher in estrus. Furthermore, certain fatty acids, namely tridecanoic, palmitic and oleic acids, were present during proestrus and estrus phases, but were exclusively absent in ovariectomized mice. However, the carbohydrate level was equally maintained throughout the four phases of estrous cycle. For successful communication, higher concentrations of protein and specific fatty acids in estrus are directly involved. The significant increase in estradiol at estrus and progesterone at metestrus seems to be of greater importance in the expression pattern of biochemical constituents and may play a notable role in estrous cycle regulation. Thus, we conclude that the variations observed in the concentration of the biochemical constituents depend on the phase of the reproductive cycle as well as hormonal status of animals. The appearance of protein and specific fatty acids during estrus phase raises the possibility to use these as a urinary indicators for estrus detection.     

A peptidoglycan monomer with the glutamine to serine change and basic peptides bind in silico to TLR-2 (403–455)

Bacterial cell wall polysaccharides, such as PGN, bind and activate TLR-2, NOD2 and PGRP on monocytes/macrophages and activate inflammation. We found that the peptides containing basic amino acids (cations) at N -terminus and tyrosine at C-terminus interfered with activating ability of PGN. This finding is significant because the ECD of TLR-2 in vivo encounters a large number of proteins or peptides. Some should bind ECD and “pre-form” TLR-2 to respond or not to its activators, although they cannot activate TLR-2 alone. TLR-2 is receptor for a large number of ligands, including lipopeptides and bacterial cell wall glycoproteins. A binding site for lipopeptides has been identified; however, a binding site for soluble or multimeric PGN has not been proposed. To identify the candidate binding sites of peptides and PGN on TLR-2, we modeled docking of peptides and of the PGN monomer (PGN-S-monomer) to extracellular domain (ECD-TLR-2) of the unbound TLR-2. Quantification, in silico, of free energy of binding (DG) identified 2 close sites for peptides and PGN. The PGN-S-monomer binding site is between amino acids TLR-2, 404–430 or more closely TLR-2, 417–428. The peptide-binding site is between amino acids TLR-2, 434–455. Molecular models show PGN-S-monomer inserts its N -acetyl-glucosamine (NAG) deep in the TLR-2 coil, while its terminal lysine interacts with inside (Glu⁴⁰³) and outside pocket (Tyr³⁷⁸). Peptides insert their two N -terminal arginines or their C-terminal tyrosines in the TLR-2 coil. PGN did not bind the lipopeptide-binding site in the TLR-2. It can bind the C-terminus, 572–586 (DG = 0.026 kcal), of “lipopeptide-bound” TLR-2. An additional, low-affinity PGN-binding site is TLR-2 (227–237). MTP, MDP, and lysine-less PGN bind to TLR-2, 87–113. This is the first report identifying candidate binding sites of monomer PGN and peptides on TLR-2. Experimental verification of our findings is needed to create synthetic adjuvant for vaccines. Such synthetic PGN can direct both adjuvant and cancer antigen to TLR-2.     

Synthesis and characterization of chimeric proteins based on cellulase and xylanase from an insect gut bacterium

Insects living on wood and plants harbor a large variety of bacterial flora in their guts for degrading biomass. We isolated a Paenibacillus strain, designated ICGEB2008, from the gut of a cotton bollworm on the basis of its ability to secrete a variety of plant-hydrolyzing enzymes. In this study, we cloned, expressed, and characterized two enzymes, β-1,4-endoglucanase (Endo5A) and β-1,4-endoxylanase (Xyl11D), from the ICGEB2008 strain and synthesized recombinant bifunctional enzymes based on Endo5A and Xyl11D. The gene encoding Endo5A was obtained from the genome of the ICGEB2008 strain by shotgun cloning. The gene encoding Xyl11D was obtained using primers for conserved xylanase sequences, which were identified by aligning xylanase sequences in other species of Paenibacillus. Endo5A and Xyl11D were overexpressed in Escherichia coli, and their optimal activities were characterized. Both Endo5A and Xyl11D exhibited maximum specific activity at 50°C and pH 6 to 7. To take advantage of this feature, we constructed four bifunctional chimeric models of Endo5A and Xyl11D by fusing the encoding genes either end to end or through a glycine-serine (GS) linker. We predicted three-dimensional structures of the four models using the I-TASSER server and analyzed their secondary structures using circular dichroism (CD) spectroscopy. The chimeric model Endo5A-GS-Xyl11D, in which a linker separated the two enzymes, yielded the highest C-score on the I-TASSER server, exhibited secondary structure properties closest to the native enzymes, and demonstrated 1.6-fold and 2.3-fold higher enzyme activity than Endo5A and Xyl11D, respectively. This bifunctional enzyme could be effective for hydrolyzing plant biomass owing to its broad substrate range.     

Enzymatic bioremediation of organophosphorus insecticides by recombinant organophosphorous hydrolase

The gene for organophosphorous hydrolase (OPH) (GenBank accession no. M20392) was chemically synthesised with a codon bias toward E. coli, followed by its cloning and heterologous over-expression in E. coli under induction with 0.1 M isopropyl-d-thiogalactopyranoside (IPTG). The protein was localised in the membrane fraction and no amount of the expressed protein was soluble, thus hindering its purification and further downstream utility. The expressed enzyme was solubilised by mild treatment with ionic detergent [sodium dodecyl sulphate (SDS 1.0% (w/v))]. An innovative step of incubation at 4 °C was used to precipitate SDS, resulting in catalytically active OPH in the supernatant and detergent at the bottom. This solubilised, SDS-free, recombinant OPH was able to detoxify parathion and methylparathion ranging between 10-80% and 3.6-45% in enzyme reaction cycles after immobilization on Ca-alginate and agar-agar, respectively.     

A role for F-actin in hexokinase-mediated glucose signaling

HEXOKINASE1 (HXK1) from Arabidopsis (Arabidopsis thaliana) has dual roles in glucose (Glc) signaling and in Glc phosphorylation. The cellular context, though, for HXK1 function in either process is not well understood. Here we have shown that within normal experimental detection limits, AtHXK1 is localized continuously to mitochondria. Two mitochondrial porin proteins were identified as capable of binding to overexpressed HXK1 protein, both in vivo and in vitro. We also found that AtHXK1 can be associated with its structural homolog, F-actin, based on their coimmunoprecipitation from transgenic plants that overexpress HXK1-FLAG or from transient expression assays, and based on their localization in leaf cells after cryofixation. This association might be functionally important because Glc signaling in protoplast transient expression assays is compromised by disruption of F-actin. We also demonstrate that Glc treatment of Arabidopsis seedlings rapidly and reversibly disrupts fine mesh actin filaments. The possible roles of actin in HXK-dependent Glc signaling are discussed.     

Development of fructosyl valine binding polymers by covalent imprinting

Molecularly imprinted polymers (MIPs) against fructosyl valine (Fru-Val), the N-terminal constituent of hemoglobin A1c β-chains, were prepared by cross-linking of β-d-Fru-Val-O-bis(4-vinylphenylboronate) with an excess of ethylene glycol dimethacrylate (EDMA) or trimethylolpropane trimethacrylate (TRIM). Control MIPs were prepared in analogy by cross-linking the corresponding vinylphenylboronate esters of fructose and pinacol. After template extraction batch rebinding studies were performed using different pH values and buffer compositions. The Fru-Val imprinted TRIM cross-linked polymer binds about 1.4 times more Fru-Val than the fructose imprinted polymer and 2.7 times more Fru-Val than pinacol imprinted polymer. The highest imprinting effect was obtained in 100 mM sodium carbonate/10% methanol (pH 11.4). The TRIM cross-linked Fru-Val imprinted polymer showed a better specificity than the EDMA cross-linked polymer. The binding of valine was very low. Thermo gravimetric analysis indicated that the generated Fru-Val imprinted polymer has high thermo stability. No change in binding was observed after incubation of the polymers in buffer at 80 °C for 36 h. Since the functional group of the polymers (phenyl boronic acid) targets the sugar part of Fru-Val the imprint technique used should also be applicable for the development of MIPs against other glycated amino acids and peptides.     

Redox signalling from NADPH oxidase targets metabolic enzymes and developmental proteins in Fusarium graminearum

NADPH oxidase (NOX) is one of the sources of reactive oxygen species (ROS) that modulates the activity of proteins through modifications of their cysteine residues. In a previous study, we demonstrated the importance of NOX in both the development and pathogenicity of the phytopathogen Fusarium graminearum. In this article, comparative proteomics between the wild-type and a Nox mutant of F. graminearum was used to identify active cysteine residues on candidate redox-sensing proteins. A two-dimensional gel approach based on labelling with monobromobimane (mBBR) identified 19 candidate proteins, and was complemented with a gel-free shotgun approach based on a biotin switch method, which yielded 99 candidates. The results indicated that, in addition to temporal regulation, a large number of primary metabolic enzymes are potentially targeted by NoxAB-generated ROS. Targeted disruption of these metabolic genes showed that, although some are dispensable, others are essential. In addition to metabolic enzymes, developmental proteins, such as the Woronin body major protein (FGSG_08737) and a glycosylphosphatidylinositol (GPI)-anchored protein (FGSG_10089), were also identified. Deletion of either of these genes reduced the virulence of F. graminearum. Furthermore, changing the redox-modified cysteine (Cys³²⁵) residue in FGSG_10089 to either serine or phenylalanine resulted in a similar phenotype to the FGSG_10089 knockout strain, which displayed reduced virulence and altered cell wall morphology; this underscores the importance of Cys³²⁵ to the function of the protein. Our results indicate that NOX-generated ROS act as intracellular signals in F. graminearum and modulate the activity of proteins affecting development and virulence in planta.