Synapsin I-associated phosphatidylinositol 3-kinase mediates synaptic vesicle delivery to the readily releasable pool

Maintaining synaptic transmission requires replenishment of docked synaptic vesicles within the readily releasable pool (RRP) from synaptic vesicle clusters in the synapsin-bound reserve pool. We show that synapsin forms a complex with phosphatidylinositol 3-kinase (PI 3-kinase) in intact nerve terminals and that synapsin-associated kinase activity increases on depolarization. Disruption of either PI 3-kinase activity or its interaction with synapsin inhibited replenishment of the RRP, but did not affect exocytosis from the RRP. Thus we conclude that a synapsin-associated PI 3-kinase activity plays a role in synaptic vesicle delivery to the RRP. This also suggests that PI 3-kinase contributes to the maintenance of synaptic transmission during periods of high activity, indicating a possible role in synaptic plasticity.     

Computational analysis of multivalency in lectins: structures of garlic lectin-oligosaccharide complexes and their aggregates

Multivalency in lectins is a phenomenon that has been discussed at considerable length. The structural basis for the role of multivalency in garlic lectin has been investigated here through computational studies. Biochemical studies have shown that the binding affinity of garlic lectin for high mannose oligosaccharides is orders of magnitude greater than that for mannose. Modeling and energy calculations clearly indicate that such increase in affinity cannot be accounted for by binding of these oligosaccharides at any of the six sites of a garlic lectin dimer. These studies also indicate that a given oligosaccharide cannot bind simultaneously to more than one binding site on a lectin dimer. The possibility of a given oligosaccharide simultaneously binding to and hence linking two or more lectin molecules was therefore explored. This study showed that trimannosides and higher oligomers can cross-link lectin dimers, amplifying the protein-oligosaccharide interactions severalfold, thus explaining the role of multivalency in enhancing affinity. A comprehensive exploration of all possible cross-links posed a formidable computational problem. Even a partial exploration involving a carefully chosen region of the conformational space clearly showed that a given dimer pair can be cross-linked not only by a single oligosaccharide molecule but also simultaneously by two oligosaccharides. The number of such possible double cross-links, including those forming interesting tetrameric structures, generally increases with the size of the oligosaccharide, correlating with the biochemical data. In addition to their immediate relevance to garlic lectin, these studies are of general interest in relation to lectin-oligosaccharide interactions.     

Chromium accumulation and toxicity in aquatic vascular plants

Chromium poisoning among leather tanners has long been known. The workers have been found to suffer from ulcers, allergic dermatitis, lung cancer, and liver necrosis due to prolonged contact with chromium salts. One of the highly catastrophic incidences of lung cancer as a result of inhaling dust containing Cr (VI) was reported in 1960 from the Kiryama factory of the Nippon-Denko concern on the island of Hokkaido, Japan. Pollution of water resources, both surface and underground, by indiscriminate discharge of spent wastes of chromium-based industries has become a serious global concern, for it has created an acute scarcity of safe drinking water in many countries. In August 1975 it was observed that underground drinking water in Tokyo near the chromium (VI))-containing spoil heaps contained more than 2000 times the permissible limit of chromium. In Ludhiana and Chennai, India, chromium levels in underground water have been recorded at more than 12 mg/L and 550–1500 ppm/L, respectively. Chromium is widely distributed in nature, occupying 21st position in the index of most commonly occurring elements in the earth’s crust. Chromium occurs in nature in the form of a compound (chromium + oxygen + iron) known as “chromite.” The geographical distribution of chromite mines is uneven. Over 95% of economically viable chromite ores are situated in the southern part of Africa. Its annual global production is ca. 9 million tons, mainly mined in the former Soviet Union, Albania, and Africa. In India, over 90% of chromite deposits are located in Sukinda Valley of Orissa. Chromium occurs in several oxidation states, ranging from Cr²⁺ to Cr⁶⁺, with trivalent and hexavalent states being the most stable and common in the terrestrial environment. Chromium (III) is used for leather tanning because it forms stable complexes with amino groups in organic material. In the presence of excessive oxygen, chromium (III) oxidizes into Cr (VI), which is highly toxic and more soluble in water than are other forms. Chromium (VI) can easily cross the cell membrane, whereas the phosphate-sulphate carrier also transports the chromite anions. On the other hand, Cr (III) does not utilize any specific membrane carrier and hence enters into the cell through simple diffusion. The diffusion is possible only after the formation of appropriate lipophilic ligands. Use of chromium as industrial material was discovered only 100 years ago. It was used for the first time in the production of corrosion-resistant steel (stainless steel) and coatings. Subsequently, chromium was widely deployed in various industries; namely, electroplating, dyes and pigments, textiles, photography, and wood processing. The tanning industry is one of the major users of chromium (III) salts. During leather processing the conversion of putrefactive proteinaceous matter, skin, into non-putricible is carried out by the treatment of chromium sulphate solution. According to an estimate, ca. 32 tons of chromium sulphate salts are used annually in Indian tanneries. As a result of unplanned disposal of spent tannery wastes, ca. 2000–3200 tons of chromium as element escapes into the environment. This has raised severe ecological concern and reduced the forest cover considerably. Aquatic vascular plants play an important role in the uptake, storage, and recycling of metals. The uptake of metals depends on the chemical form present in the system and on the life form of the macrophytes (floating, free floating, well rooted, or rootless). The free-floating species (Eichhornia, Lemna, Pistia) absorb elements through the roots/leaves, whereas the rootless speciesCeratophyllum demersum absorbs mainly through the finally divided leaves. Submerged species showed higher chromium accumulation than do floating and emergent ones. The order is:Elodea canadensis > Lagarosiphon major > Potamogeton crispes > Trapa natans > Phragmitis communis. Roots of water hyacinth (Eichhornia crassipes) showed an accumulation of 18.92 μmol (g dry tissue wt^-1) Cr. AlthoughCeratophyllum demersum andHydrodictyon reticulatum showed lower levels of chromium accumulation, their bioconcentration factor values were very high. Floating-species duckweeds (Lemna, Spirodela) are potential accumulators of heavy metals. They have bioconcentrated Fe and Cu, as high as 78 times their concentration in wastewater. Duckweeds have also shown the ability to accumulate chromium substantially. Although duckweeds attain higher concentrations of chromium in their tissues than do other macrophytes, their bioconcentration factor (BCF) values were much lower than those reported in other aquatic species. A moderate accumulation of chromium has been found in emergent species. Plants ofScirpus validatus andCyperus esculentus accumulated 0.55 kg and 0.73 kg^-1 Cr, respectively. InBacopa monnieri andScirpus lacustris accumulations of 1600 and 739 μg g^-1 dw Cr, respectively, have been reported when exposed to 5 mg L^-1 Cr for 168 hours in solution culture. The accumulation of Cr was greater in the root than the shoot. Higher accumulations of chromium in roots and least in shoots of emergent species have also been recorded. Phytotoxicity of chromium in aquatic environment has not been studied in detail. The mechanism of injury in terms of ultrastructural organization, biochemical changes, and metabolic regulations has not been elucidated. It has been pointed out that while considering the toxicity of heavy metals, a distinction should be made between elements essential to plants and metals that have no proven beneficial biochemical effects. For example, an increased level of chromium may actually stimulate growth without being essential for any metabolic process. In aquatic species—namely,Myriophyllum spicatum— the maximum increase in shoot length was found at 50 μgl^-1 Cr. Higher concentrations up to 1000 μ gl^-1 caused an almost linear reduction both in shoot weight and length. Duckweeds showed relatively greater tolerance to chromium. However, an inhibition of growth inSpirodela andLemna was found at 0.02 mM and 0.00002 mM Cr concentrations, respectively. Mortality ofL. aequinoctialis was found at 0.005 mM Cr and higher concentrations. The effective chromium concentrations (EC-50) for some aquatic species have been reported as follows:Lemna minor, 5.0 mg L^-1, 14 days EC;L. Paucicostata, 1.0 mg L-¹, 20 days EC;Myriophyllum spicatum, 1.9 mg L^-1, 32 days EC; andSpirodela polyrrhiza, 50 mg L^-1, 14 days EC. Chromium toxicity on biochemical parameters showed a reduction in photosynthetic rate at 50 μgl^-1 Cr inMyriophyllum spicatum. Decrease in chlorophyll and protein contents were also recorded inNajas indica, Vallisneria spiralis, andAlternanthera sessilis with an increase in chromium concentration. InLimnanthemum cristatum, a slight reduction in chlorophyll and almost no change in control were found due to chromium toxicity. Submerged species (Ceratophyllum demersum, Vallisneria spiralis) and an emergent one (Alternanthera sessilis) showed decreases in chlorophyll as well as in protein contents when treated with chromium. Chromium-induced morphological and ultrastructural changes have been reported in several aquatic vascular plants: InLemna minor andCeratophyllum demersum, chromium-induced changes in chloroplast fine structure disorganized thylakoids with loss of grain and caused formation of many vesicles in the chloroplast. Chromium (VI) has caused stunting and browning of roots produced from the chromium-treated excised leaves ofLimnanthemum cristatum. At 226 μg/g Cr dry wt leaf tissue concentration, development of brown coloration in the hydathodes of juvenile leaves ofLimnanthemum cristatum is a characteristic chromiuminduced alteration. Aquatic vascular plants and algae may serve as effective bioindicators in respect to metals in aquatic environments. Chromium-induced morphological and ultrastructural changes inLimnanthemum cristatum have significant indicator values and could be used for assessing the level of chromium in ambient water.Wolffia globosa, a rootless duckweed, showed substantial chromium accumulation and high concentration factor (BCF) value at very low ambient chromium concentrations, suggesting its feasibility in detecting chromium pollution in water resources. Methylene blue-stained cells ofScenedesmus acutus become uniformly dark blue during chromium (VI) treatment. This may serve as an indicator of chromium pollution.     

In vitro micropropagation and ex vitro acclimation of Bupleurum kaoi-an endangered medicinal plant native to Taiwan

Summary This study reports an improved protocol for in vitro-shoot multiplication and ex vitro acclimation of Bupleurum kaoi, an endangered medicinal herb. Nodal segments were cultured in half-strength Murashige and Skoog (MS) basal medium supplemented with different concentrations of benzyladenine (BA) and kinetin. The presence of 0.25 mg l⁻¹ BA induced the highest number of shoots per explant after 8 wk of culture. Although BA was more effective than kinetin on shool multiplication, it induced hyperhydric shoots at all concentrations tested. The use of dispense paper (DP) instead of aluminum foil (AF) for container closure was found to reduce hyperhydricity and improve ex vitro acclimation. The best survival rate (61%) was obtained when plantlets were grown in MS basal medium containing 0.5 mg l⁻¹ indole-3-butyric acid and 0.1–0.2 mg l⁻¹ α-naphthaleneacetic acid using DP as container closure. Leaves of the plant treated with AF6 (two layers of AF as container closure and 6 wk of incubation) lacked epicuticular wax and possessed larger stomata, higher stomata density, and fewer functional stomata compared to those of plants treated with AF2+DP4 (two layers of AF for 2 wk, then replaced AF by three layers of DP for 4wk) and ex vitro-acclimated plantlets.     

Biodegradation of the major color containing compounds in distillery wastewater by an aerobic bacterial culture and characterization of their metabolites

This study deals the biodegradation of the major color containing compounds extracted from distillery wastewater (DWW) by an aerobic bacterial consortium comprising Bacillus licheniformis (DQ79010), Bacillus sp. (DQ779011) and Alcaligenes sp. (DQ779012) and characterization of metabolic products. The degradation of color containing compounds by bacteria was studied by using the different carbon and nitrogen sources at different environmental conditions. Results revealed that the bacterial consortium was efficient for 70% color removal in presence of glucose (1.0%) and peptone (0.1%) at pH 7.0 and temperature 37°C. The HPLC analysis of control and bacterial degraded samples has shown the reduction in peak area as well as shifting of peaks compared to control indicating the bacterial degradation as well as transformation of color containing compounds from DWW. The comparative LC–MS–MS and other spectrophotometric analysis has shown the presence of dihydroxyconiferyl alcohol, 2, 2′-bifuran-5-carboxylic acid, 2-nitroacetophenone, p-chloroanisol, 2, 3-dimethyl-pyrazine, 2-methylhexane, methylbenzene, 2, 3-dihydro-5-methylfuran, 3-pyrroline, and acetic acid in control samples that were biodegraded and biotransformed into 2-nitroacetophenone, p-chloroanisol, 2, 2′-bifuran, indole, 2-methylhexane, and 2, 3-dihydro-5-methylfuran by bacterial consortium. In this study, it was observed that most of the compounds detected in control samples were diminished from the bacterial degraded samples and compounds 2, 2′-bifuran and indole with molecular weight 134 and 117 were produced as new metabolites during the bacterial degradation of color containing compounds from DWW.     

Open source tool for prediction of genome wide protein-protein interaction network based on ortholog information

Background  

Protein-protein interactions are crucially important for cellular processes. Knowledge of these interactions improves the understanding of cell cycle, metabolism, signaling, transport, and secretion. Information about interactions can hint at molecular causes of diseases, and can provide clues for new therapeutic approaches. Several (usually expensive and time consuming) experimental methods can probe protein - protein interactions. Data sets, derived from such experiments make the development of prediction methods feasible, and make the creation of protein-protein interaction network predicting tools possible.     

Effects of peristaltic and longitudinal wave motion of the channel wall on movement of micro-organisms: application to spermatozoa transport

A mathematical model is presented to study the motion of the spermatozoa in the cervical canal by considering the transverse waves along its tail and the transverse and longitudinal motions of the cervical wall. In an attempt to control fertility by reducing the speed of sperm, the transverse waves have been considered in the direction opposite to the motion of the spermatozoa. It has been shown that by having appropriate transverse wave motion and longitudinal velocity, the sperm may not be able to move towards the oviduct even if it could continue to have its own propelling velocity. A particular case of the motion of a thin plane sheet in a channel under peristaltic motion of its walls has also been obtained and studied.     

Role of the N-terminal domain of the calcitonin receptor-like receptor in ligand binding

Calcitonin receptor-like receptor (CRLR) is a seven-transmembrane (7-TM) domain class B G protein-coupled receptor (GPCR) which requires coexpression of different receptor activity modifying proteins (RAMP) to become a functional calcitonin gene-related peptide (CGRP) receptor or an adrenomedullin (AM) receptor. The N-terminal (Nt) extracellular region of class B GPCRs in ligand binding has been reported for receptors such as glucagon and parathyroid hormone. We hypothesize that the Nt-domain of CRLR (Nt-CRLR) is an autonomously folded unit possessing a well-defined structure and is involved in ligand binding and specificity. To obtain structural and functional information on the Nt-CRLR, we cloned and expressed the Nt-CRLR as a fusion protein in Escherichia coli. Overexpressed protein formed an inclusion body, which was refolded and purified, resulting in a soluble monomeric protein. Far-UV CD and fluorescence spectra of Nt-CRLR showed characteristics of a folded protein. The ability of Nt-CRLR to bind CGRP and AM independent of RAMPs was determined by studying inhibition of (125)I-CGRP and (125)I-AM binding to pregnant rat uterine membrane in the presence of Nt-CRLR protein. We observe that Nt-CRLR inhibits (125)I-CGRP and (125)I-AM binding to rat uterus in a dose-dependent fashion (IC(50) = 0.25 and 0.29 muM, respectively). Taken together, our data provide evidence that Nt-CRLR is structured and further that a significant part of the binding affinity comes from binding to the Nt-domain.