Histology and ultrastructure of male mullerian gland of Uraeotyphlus narayani (Amphibia: Gymnophiona)

This study reports the anatomy, histology, and ultrastructure of the male Mullerian gland of the caecilian Uraeotyphlus narayani, based on dissections, light microscopic histological and histochemical preparations, and transmission electron microscopic observations. The posterior end of the Mullerian duct and the urinogenital duct of this caecilian join to form a common duct before opening into the cloaca. The boundary of the entire gland has a pleuroperitoneum, followed by smooth muscle fibers and connective tissue. The Mullerian gland is composed of numerous individual tubular glands separated from each other by connective tissue. Each gland has a duct, which joins the central Mullerian duct. The ducts of the tubular glands are also surrounded by abundant connective tissue. The tubular glands differ between the column and the base in regard to the outer boundary and the epithelial organization. The basement membrane of the column is so thick that amoeboid cells may not penetrate it, whereas that around the base of the gland is thin and appears to allow migration of amoeboid cells into and out of the basal aspect of the gland. The epithelium of the column has nonciliated secretory cells with basal nuclei and ciliated nonsecretory cells with apical nuclei. In the epithelium of the base there are secretory cells, ciliated cells, and amoeboid cells. The epithelium of ducts of the tubular glands is formed of ciliated dark cells and microvillated light cells. The epithelium of the central duct is formed of ciliated dark cells also possessing microvilli, ciliated light cells also possessing microvilli, and microvillated light cells that lack cilia. It is regressed during March to June when the testis lobes are in a state of quiescence. The Mullerian gland is active in secretion during July to February when the testis is active in spermatogenesis.     

High-throughput fluorescent tagging of full-length Arabidopsis gene products in planta

We developed a high-throughput methodology, termed fluorescent tagging of full-length proteins (FTFLP), to analyze expression patterns and subcellular localization of Arabidopsis gene products in planta. Determination of these parameters is a logical first step in functional characterization of the approximately one-third of all known Arabidopsis genes that encode novel proteins of unknown function. Our FTFLP-based approach offers two significant advantages: first, it produces internally-tagged full-length proteins that are likely to exhibit native intracellular localization, and second, it yields information about the tissue specificity of gene expression by the use of native promoters. To demonstrate how FTFLP may be used for characterization of the Arabidopsis proteome, we tagged a series of known proteins with diverse subcellular targeting patterns as well as several proteins with unknown function and unassigned subcellular localization.     

Humoral defence factors in Indian river prawn, Macrobrachium malcolmsonii

A preliminary study on humoral defence factors of Indian river prawn, Macrobrachium malcolmsonii was carried out. The serum of animals collected from intermoult stage had an average total protein content of 9.65 g dl(-1) and lysozyme-like activity of 0.04 unit ml(-1). The phenoloxidase activity in haemocyte lysate supernatant was triggered significantly (P < 0.05) by chitosan, Gram-positive and Gram-negative bacterial cells in comparison to other elicitors viz., levamisole, trypsin and glucan. The serum contained an agglutinin against Gram-negative bacteria and a haemagglutinin against a wide range of vertebrate erythrocytes. The haemagglutinin was stable over a wide range of pH (3.0-7.0) and temperatures (-30 degrees C to 60 degrees C). A 413 kDa N-acetyl galactosamine-specific haemagglutinin was purified by single step affinity chromatography and the protein was found to be calcium ion-dependent in nature and made up of five different subunits of varied molecular weights on denaturing SDS-PAGE.     

Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases

Modular polyketide synthases (PKSs) are large multi-enzymatic, multi-domain megasynthases, which are involved in the biosynthesis of a class of pharmaceutically important natural products, namely polyketides. These enzymes harbor a set of repetitive active sites termed modules and the domains present in each module dictate the chemical moiety that would add to a growing polyketide chain. This modular logic of biosynthesis has been exploited with reasonable success to produce several novel compounds by genetic manipulation. However, for harnessing their vast potential of combinatorial biosynthesis, it is essential to develop knowledge based in silico approaches for correlating the sequence and domain organization of PKSs to their polyketide products. In this work, we have carried out extensive sequence analysis of experimentally characterized PKS clusters to develop an automated computational protocol for unambiguous identification of various PKS domains in a polypeptide sequence. A structure based approach has been used to identify the putative active site residues of acyltransferase (AT) domains, which control the specificities for various starter and extender units during polyketide biosynthesis. On the basis of the analysis of the active site residues and molecular modelling of substrates in the active site of representative AT domains, we have identified a crucial residue that is likely to play a major role in discriminating between malonate and methylmalonate during selection of extender groups by this domain. Structural modelling has also explained the experimentally observed chiral preference of AT domain in substrate selection. This computational protocol has been used to predict the domain organization and substrate specificity for PKS clusters from various microbial genomes. The results of our analysis as well as the computational tools for prediction of domain organization and substrate specificity have been organized in the form of a searchable computerized database (PKSDB). PKSDB would serve as a valuable tool for identification of polyketide products biosynthesized by uncharacterized PKS clusters. This database can also provide guidelines for rational design of experiments to engineer novel polyketides.     

Enzymatic E-colicins bind to their target receptor BtuB by presentation of a small binding epitope on a coiled-coil scaffold

Toxins and viruses often initiate their attacks by binding to specific proteins on the surfaces of target cells. Bacterial toxins (e.g. bacteriocins) and viruses (bacteriophages) targeting Gram-negative bacteria typically bind to outer membrane proteins. Bacterial E-colicins target Escherichia coli by binding to the outer membrane cobalamin transporter BtuB. Colicins are tripartite molecules possessing receptor-binding, translocation, and toxin domains connected by long coiled-coil alpha-helices. Surprisingly, the crystal structure of colicin E3 does not possess a recognizable globular fold in its receptor-binding domain. We hypothesized that the binding epitope of enzymatic E-colicins is a short loop connecting the two alpha-helices that comprise the coiled-coil region and that this flanking coiled-coil region serves to present the loop in a binding-capable conformation. To test this hypothesis, we designed and synthesized a 34-residue peptide (E-peptide-1) corresponding to residues Ala366-Arg399 of the helix-loop-helix region of colicin E3. Cysteines placed near the ends of the peptide (I372C and A393C) enabled crosslinking for reduction of conformational entropy and formation of a peptide structure that would present the loop epitope. A fluorescent analog was also made for characterization of binding by measurement of fluorescence polarization. Our analysis shows the following. (i). E-peptide-1 is predominantly random coil in aqueous solution, but disulfide bond formation increases its alpha-helical content in both aqueous buffer and solvents that promote helix formation. (ii). Fluorescein-labeled E-peptide-1 binds to purified BtuB in a calcium-dependent manner with a Kd of 43.6 +/- 4.9 nm or 2370 +/- 670 nm in the presence or absence of calcium, respectively. (iii). In the presence of calcium, cyanocobalamin (CN-Cbl) displaces E-peptide-1 with a nanomolar inhibition constant (Ki = 78.9 +/- 5.6 nm). We conclude that the BtuB binding sites for cobalamins and enzymatic E-colicins are overlapping but inequivalent and that the distal loop and (possibly) the short alpha-helical flanking regions are sufficient for high affinity binding.     

Paternal reciprocal translocation t(11;16)(p13;q24.3) in a Silver-Russel syndrome patient

We describe a 7-month-old male child with Silver-Russel syndrome (SRS) phenotype, presented with two major clinical features: low birth weight, short stature, and minor features, such as macrocephaly, clinodactyly, essential for the diagnosis of SRS. Routine cytogenetic studies with GTG-banding showed 46,XY,t(11;16)(p13;q24.3). Fluorescence in situ hybridisation (FISH) with single copy probes BAC (11p13) and PAC (16q24.3), showed a reciprocal translocation. Chromosomal analysis of the mother was normal and the phenotypically normal father had apparently identical translocation t(11;16)(p13;q24.3). The disruption of growth factor genes at 11p and 16q breakpoint regions due to reciprocal translocation in the father might have caused SRS phenotype in the child.     

Enzymatic digestion--a safe and rapid technique for individual separation of Macrobrachium rosenbergii embryos for cryopreservation studies

A new, safe, and rapid technique for the individual separation of the embryos of giant freshwater prawn Macrobrachium rosenbergii de Man is described. Two protease enzymes, e.g., trypsin and collagenase were used. Embryos in the advanced stage of development (gray embryos with eyespot and heart beat) were selected for the study. Treatment with collagenase and trypsin at respective concentrations of 0.05 and 0.25% for 30 min resulted in 100% separation of 35-40 mg of embryonic mass (approximately 180 embryos). A chelating agent, EDTA (ethylenediaminetetraacetic acid disodium salt: dihydrate) at 400 mg l(-1) enhanced the activity of trypsin. Trypsin and collagenase, when used together, were found to act synergistically. The separated embryos revealed no morphological injury when observed under the microscope. Further, in vitro hatching of the separated embryos was successful indicating that the present technique is safe and effective in achieving individual separation of prawn embryos.     

Effect of proline on the production of singlet oxygen

Molecular oxygen in electronic singlet state is a very powerful oxidant. Its damaging action in a variety of biological processes has been well recognized. Here we report the singlet oxygen quenching action of proline. Singlet oxygen (1O2) was produced photochemically by irradiating a solution of sensitiser and detected by following the formation of stable nitroxide radical yielded in the reaction of 1O2 with the sterically hindered amine (2,2,6,6-tetramethylpiperidine, TEMP). Illumination of a sensitiser, toluidine blue led to a time dependent increase in singlet oxygen production as detected by the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) by EPR spectrometry. Interestingly, the production of TEMPO was completely abolished by the presence of proline at concentration as low as 20mM. These results show that proline is a very effective singlet oxygen quencher. Other singlet oxygen generating photosensitizer like hematopophyrin and fluorescein also produced identical results with proline. Since proline is one of the important solutes which accumulate in many organisms when they are exposed to environmental stresses, it is likely that proline accumulation is related to the protection of these organisms against singlet oxygen production during stress conditions. A possible mechanism of singlet oxygen quenching by proline is discussed.     

Stretch-induced Ca(2+) release via an IP(3)-insensitive Ca(2+) channel

Various mechanicalstimuli increase the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). A part of the increase in [Ca²⁺]_i isdue to the release of Ca²⁺ from intracellular stores. Wehave investigated the effect of mechanical stimulation produced bycyclical stretch on the release of Ca²⁺ from theintracellular stores. Permeabilized VSMC loaded with ⁴⁵Ca²⁺ were subjected to 7.5% average (15%maximal) cyclical stretch. This resulted in an increase in⁴⁵Ca²⁺ rate constant by 0.126 ± 0.0035. Inhibition of inositol 1,4,5-trisphosphate (IP₃),ryanodine, and nicotinic acid adenine dinucleotide phosphate channels(NAADP) with 50 µg/ml heparin, 50 µM ruthenium red, and 25 µMthio-NADP, respectively, did not block the increase in⁴⁵Ca²⁺ efflux in response to cyclical stretch.However, 10 µM lanthanum, 10 µM gadolinium, and 10 µMcytochalasin D but not 10 µM nocodazole inhibited the increase in⁴⁵Ca²⁺ efflux. This supports the existence of anovel stretch-sensitive intracellular Ca²⁺ store in VSMCthat is distinct from the IP₃-, ryanodine-, and NAADP-sensitive stores.