Burrowing behavior and ecology of the crab-eating Indian snake eel Pisoodonophis boro

Synopsis The snake eel Pisoodonophis boro burrows, causing leaks in the embankments and damaging the paddy fields and salt pans near estuaries. Field observations and laboratory experiments were made to study this behavior. P. boro was burrowing to eat the fiddler crab Uca annulipes in the mud flats. The eel showed a patchy distribution within the Uca zone. Salinity and the physical nature of the deposits controlled the distribution of the eel. Eel population density was low when the estuary was completely filled with neritic waters during the summer and fresh water during the monsoon period. The region of greatest abundance contained a good mixture of sand, silt and clay. Eels were not found where medium and fine sand formed the bulk of the substratum. The laboratory experiments showed that P. boro preferred loam soil although it could invariably burrow into hard substratum like sand for protection. The eel adapted itself to the experimental substrates ranging from sand to fine clay. However, their natural distribution was determined by Uca distribution. As U. annulipes is not found either in salt pans or in paddy fields P. boro rarely occurs in these habitats.     

Protease Inhibitors. Part 2. Weakly Basic Thrombin Inhibitors Incorporating Sulfonyl-Aminoguanidine Moieties as S1 Anchoring Groups: Synthesis and Structure-Activity Correlations

Abstract

Two series of derivatives have been prepared and assayed as inhibitors of two physiologically relevant serine proteases, human thrombin and human trypsin. The first series includes alkyl-/ aralkyl-/aryl- and hetarylsulfonyl-aminoguanidines. It was thus observed that sulfanilyl-aminoguanidine possesses moderate but intrinsically selective thrombin inhibitory properties, with K_i values around 90 and 1400 nM against thrombin and trypsin respectively. Further elaboration of this molecule afforded compounds that inhibited thrombin with K_i values in the range 10–50 nM, whereas affinity for trypsin remained relatively low. Such compounds were obtained either by attaching benzyloxycarbonyl- or 4-toluenesulfonylureido-protected amino acids (such as D-Phe, L-Pro) or dipeptides (such as Phe-Pro, Gly-His, β-Ala-His or Pro-Gly) to the N-4 atom of the lead molecule, sulfanilyl-aminoguanidine, or by attaching substituted-pyridinium-propylcarboxamido moieties to this lead. Thus, this study brings novel insights regarding a novel non-basic S1 anchoring moiety (i, e., SO₂NHNHC(=NH)NH₂), and new types of peptidomimetic scaffolds obtained by incorporating tosylureido-amino acids/pyridinium-substituted-GABA moieties in the hydrophobic binding site(s). Structure-activity correlations of the new serine protease inhibitors are also discussed based on a QSAR model described previously for a large series of structurally-related derivatives (Supuran et al. (1999) J. Med. Chem., in press).     

Binding of Biotinylated Thrombin Receptor Peptide to Cloned Human Thrombin Receptor Overexpressed in Baby Hamster Kidney Cells

Abstract

Baby hamster kidney (BHK) cells transfected with an expression vector for the human thrombin receptor, and then treated with basic fibroblast growth factor, were found to express specific and saturable binding sites for biotinylated thrombin receptor peptide (SFLLRNPNDKYEPF). Analysis of the binding to live BHK cells yielded an equilibrium dissociation constant (Kd) of 3.0±0.3 μmol/l and a maximal binding capacity (Bmax) of 31.0±0.5 nmol/mg of protein. In competitive binding experiments, the thrombin receptor agonist peptide (SFLLRN), which is a strong inducer of human platelet aggregation, was the most potent competitor. In contrast, position 1 to 2 inverted peptides such as FSLLRNPNDKYEPF and FSLLRNP, which fail to induce for the platelet aggregation, were less potent. This simple and convenient binding assay system using the biotinylated thrombin receptor peptide as a labeled ligand and the cloned thrombin receptor overexpressed in BHK cells may be useful for exploring specific antagonists of the receptor.     

Ramachandran analysis of conserved glycyl residues in homologous proteins of known structure

High conservation of glycyl residues in homologous proteins is fairly frequent. It is commonly understood that glycine tends to be highly conserved either because of its unique Ramachandran angles or to avoid steric clash that would arise with a larger side chain. Using a database of aligned 3D structures of homologous proteins we identified conserved Gly in 288 alignment positions from 85 families. Ninety‐six of these alignment positions correspond to conserved Gly residue with (φ, ψ) values allowed for non‐glycyl residues. Reasons for this observation were investigated by in‐silico mutation of these glycyl residues to Ala. We found in 94% of the cases a short contact exists between the C^β atom of the introduced Ala with the atoms which are often distant in the primary structure. This suggests the lack of space even for a short side chain thereby explaining high conservation of glycyl residues even when they adopt (φ, ψ) values allowed for Ala. In 189 alignment positions, the conserved glycyl residues adopt (φ, ψ) values which are disallowed for Ala. In‐silico mutation of these Gly residues to Ala almost always results in steric hindrance involving C^β atom of Ala as one would expect by comparing Ramachandran maps for Ala and Gly. Rare occurrence of the disallowed glycyl conformations even in ultrahigh resolution protein structures are accompanied by short contacts in the crystal structures and such disallowed conformations are not conserved in the homologues. These observations raise the doubt on the accuracy of such glycyl conformations in proteins.     

Length–weight relationships for four fish species from lower stretch of River Ganga,India

Length‐weight relationships (LWRs) for four fish species from the River Ganga (India) is presented. Sampling was conducted in the lower stretch of the river (Buxar: 25°33′43.90″N and 83°56′3.10″E to Freserganj: 21°35′40.58″N and 88°15′28.92″E) on tri‐monthly basis from September 2016 to December 2017. Specimens were caught in gill nets (mesh, 18–68 mm), cast nets (mesh, 12–14 mm), seine nets (mesh, 12–14 mm) and various traditional traps those were put over night and lifted in early morning. Total length and wet body weight of fish were measured to the nearest 0.1 cm and 0.01 g by a digital caliper and electronic balance respectively. From LWRs, the estimated b values were found to be 2.88 (Pisodonophis boro) to 3.17 (Gagata sexualis) whereas a value ranged from 0.001 (Pisodonophis boro) to 0.009 (Botia lohachata). As per FishBase, the species Gagata sexualis and Botia lohachata had new TLmax reported for LWR estimation.     

2,4,6-trinitrobenzenesulfonic acid modification of the carboxyl-terminal region (C-domain) of calreticulin

The role of the primary amino groups of lysine sidechains in Ca²⁺ binding to calreticulin was evaluated by chemical modification of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS binding to calreticulin could be described by two steps: (i) a fast reaction, with low affinity, and (ii) a slow reaction with a relatively high affinity. Inclusion of Ca²⁺ and/or Mg²⁺ decreased both the amount of TNBS bound to calreticulin and the apparent affinity constant of the slower reaction. In contrast, the properties of the faster reaction for TNBS binding were not sensitive to Ca²⁺ and/or Mg²⁺. Analysis of TNBS binding to the carboxyl-terminal (C-domain) and aminoterminal (N-domain) of calreticulin revealed that theC-domain andN-domain are responsible for the slow and fast component of the TNBS binding, respectively. In keeping with this, in the presence of Ca²⁺, TNBS binding to theC-domain was significantly reduced, whereas modification of theN-domain was unaffected. TNBS modification of calreticulin significantly decreased Ca²⁺ binding to the low affinity/high capacity Ca²⁺ binding site(s) which are localized to theC-domain but had no effect on the high affinity/low capacity Ca²⁺ binding localized to theN-domain.In theC-domain of calreticulin, which contains the low affinity/high capacity Ca²⁺ binding sites, acidic residues are interspersed at regular intervals with one or more positively charged lysine and arginine residues. Our results indicate that the aminogroups of the lysine sidechains in theC-domain of calreticulin have a role in the low affinity/high capacity Ca²⁺ binding that is characteristic of this region of the protein and which is proposed to contribute significantly to the capacity of the endoplasmic reticulum Ca²⁺ store. (Mol Cell Biochem130: 19–28, 1994)Abbreviations TNBS 2,4,6-Trinitrobenzenesulfonic Acid - GST Glutathione S-Transferase - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EDTA Ethylenediaminetetraacetic Acid - EGTA Ethylene Glycol bis(-aminoethylether)-N,N,N,N-tetraacetic Acid - MOPS 4-Morpholinepropanesulfonic Acid     

The role of individual lysine residues of horse cytochrome <Emphasis Type="Italic">c</Emphasis> in the formation of reactive complexes with components of the respiratory chain

A number of mutant forms of horse cytochrome c with single or double substitutions of lysine residues near the heme cavity involved in interaction of mitochondrial cytochrome c with ubiquinol:cytochrome c reductase (EC 1.10.2.2) (complex III) and cytochrome c oxidase (EC 1.9.3.1) (complex IV) were prepared.. The succinate:cytochrome c reductase and cytochrome c oxidase activities of mitoplasts of rat liver were measured in the presence of mutant forms of cytochrome c. The lysine residues in positions 8, 27, 72, 86, and 87 were shown to be the main contribution to the formation of a reactive complex with ubiquinol:cytochrome c reductase of the respiratory chain, whereas the lysine residues in positions 13, 79, 86, and 87 were predominantly responsible for the formation of a complex with cytochrome c oxidase.     

Glycine Activating Enzyme in the Posterior Silkgland of Silkworm

1. Aminoacyl tRNA synthetase was extracted from the silkgland of silkworm (Bombyxmori Linné) and fractionated on a DEAE-cellulose column. Activities were estimated by ATP-PP_i exchange reaction as well as glycyl tRNA formation.

2. Two peaks, A and B, having ATP-PP_i exchange activity were found in the separated fractions, respectively. There was also observed a marked difference between the both peaks with respect to the pH optimum and activity dependence on MgCl₂ concentration.

3. Peak A showed no activity of glycyl tRNA formation. Only a part of peak B coincided with the activity of glycyl tRNA formation. The activities of both the ATP-PP_i exchange reaction and glycyl tRNA formation were found to be dependent on MgCl₂ concentration, and the optimum concentration was different between two peaks.

4. It also seemed to exist two peaks of activities, a and b, in glycyl tRNA formation which could be separated with a DEAE-cellulose column.     

A thrombin inhibitor from the gut of <Emphasis Type="Italic">Boophilus microplus</Emphasis> ticks

A thrombin inhibitor was identified for the first time in the gut of the cattle tick Boophilus microplus. Here we present the partial purification and characterization of this new molecule, which was purified from the gut extract by three chromatographic steps: ion-exchange, gel filtration and affinity chromatography in a thrombin–Sepharose resin. In SDS-PAGE the inhibitor showed an apparent molecular mass of circa 26 kDa, which is different from the two thrombin inhibitors present in the saliva of this tick. The new inhibitor delays bovine plasma clotting time and inhibits both thrombin induced fibrinogen clotting and thrombin induced platelet aggregation. However, it does not interfere with thrombin amidolytic activity upon a small substrate (H-D-Phe-Pip-Arg-para-nitroanilide), which does not require binding to thrombin exosites. Therefore, the inhibitor does not block thrombin active site, although it must interfere with one of the thrombin exosites. B. microplus gut thrombin inhibitor (BmGTI) is also capable of enhancing activated protein C (APC) activity upon its specific substrate (H-D-Glu-Pro-Arg-para-nitroanilide), an activity never described before among B. microplus molecules.     

Induction of C-FOS Expression in Rat Vascular Smooth Muscle Reporter Cells by Selective Activation of the Thrombin Receptor

Abstract

The rat vascular smooth muscle cell (VSMC) line A10 (ATCC CRL 1476) was stably transfected with a human c-fos promoter-driven luciferase reporter gene to monitor thrombin receptor activation and subsequent induction of c-fos expression. Selective activation of the endogeneous thrombin receptor by the thrombin receptor activating peptide (TRAP_1-6), SFLLRN, is shown here to result in a significant transient increase of intracellular [Ca²⁺], dose-dependent induction of c-fos promoter-mediated luciferase activity, and stimulation of DNA synthesis. These data demonstrate that A10 cells and reporter line derivatives thereof possess a functional thrombin receptor very similar or identical to that previously described. Results obtained with various signal transduction modulating or inhibiting agents support previous notions showing that thrombin receptor activation by SFLLRN is coupled to events involving p21^ras activation, protein tyrosine kinase, and activation of PKC. The A10 reporter line described here proved to be a helpful and reliable tool to study α-thrombin and TRAP_1-6-mediated intracellular events, since it retained most of the spectrum of biological responses found in primary VSMC cultures.     

A comparative SAR study of thrombin receptor derived non peptide mimetics: Importance of phenyl/guanidino proximity for activity

Summary Thrombin, the most potent physiological platelet agonist interacts with cells through a specific G protein-coupled receptor which has been cloned and sequenced. Synthetic thrombin receptor peptides (TRAPS) comprising the first 5 amino acids (SFLLR and SFLLR-NH₂) of the new N-terminus tethered ligand of the thrombin receptor that is generated by thrombin's proteolytic activity were found to cause full platelet aggregation. During the screening of novel thrombin receptor derived non-peptide mimetics in the platelet aggregation assay we found that 1-phenylacetyl-4-(6-guanidohexanoyl)-piperazine (1) and 1-(6-guanidohexanoyl)-4-(phenylacetylamidomethyl)-piperidine (2) exertedin vitro antagonist activities (56% and 40% correspondingly) as it is depicted by the platelet aggregation assay. Using Molecular Modeling, the synthetic compounds were overlayed with SFFLR. All three superimposed low energy structures had Phe and Arg aminoacids in spatial close proximity. The superimposition results revealed that1 resembled more the stereoelectronic environment of SFLLR than2. This difference may be related to their different antagonist efficacy.     

Genotype and environment effects on rice (Oryza sativa L.) grain arsenic concentration in Bangladesh

Genetic analysis of 38 rice varieties released by the Bangladesh Rice Research Institute (BRRI) identified 34 as indica, 2 as admixed between indica and aus, and 4 as belonging to the aromatic/Group V subpopulation. Indica varieties developed for the two major rice-growing seasons, the wet monsoon (aman) and the dry winter (boro), were not genetically differentiated. The Additive Main Effect and Multiplicative Interaction (AMMI) model was used to assess the effect of genotype (G), environment (E) and genotype-environment interaction (GEI) on grain arsenic (As) concentration when these rice varieties were grown at ten BRRI research stations located across diverse agro-ecological zones in Bangladesh. G, E and GEI, significantly influenced grain As concentration in both seasons. Overall, E accounted for 69%–80%, G 9%–10% and GEI 10%–21% of the observed variability in grain As. One site, Satkhira had the highest mean grain As concentration and the largest interaction principle component analysis (IPCA) scores in both seasons, indicating maximum interaction with genotypes. Site effects were more pronounced in the boro than in the aman season. The soil level of poorly crystalline Fe-oxide (AOFe), the ratio of AOFe to associated As, soil phosphate extractable As and soil pH were important sub-components of E controlling rice grain As concentration. Irrespective of environment, the mean grain As concentration was significantly higher in the boro (0.290 mg As kg^?1) than in the aman (0.154 mg As kg^?1) season (p?<?0.0001), though the reasons for this are unclear. Based on mean grain As concentration and stability across environments, the variety BR3 is currently the best choice for the boro season, while BR 23 and BRRI dhan 38 are the best choices for the aman season. Popular varieties BR 11 (aman) and BRRI dhan 28 and 29 (boro) had grain As concentrations close to the mean value and were fairly stable across environments, while high-yielding, short-duration aman season varieties (BRRI dhan 32, 33 and 39) developed for intensified cropping had relatively high grain As concentrations. Results suggest that genetic approaches to reducing As in rice grain will require the introduction of novel genetic variation and must be accompanied by appropriate management strategies to reduce As availability and uptake by rice.     

The formation of dipeptides from amino acids and the 2′(3′)-glycyl ester of an adenylate

Summary The yields of dipeptide obtained from the reaction of 0.2M 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) and 0.2M amino acid at pH 8.2 ranged from 0.1% to 35.5% for a group of 15 amino acids. The yields of glyser (35.3%), gly-cys (11.8%) and gly-thr (5.4%) were considerably greater than dipeptide yields obtained from any of the other 12 amino acids ( 1.7%). Aminolysis of 0.05M 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by 0.4M serine ethyl ester yielded 53% glycylserine diketopiperazine, via N-(glycyl)-serine ethyl ester as a transient intermediate. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - serOEt serine ethyl ester - gly-serOEt N-(glycyl)-serine ethyl ester - Boc-gly N-tertbutyloxycarbonylglycine - cyclo-(gly-ser-) glycylserine diketo-piperazine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - gly-ser N-glycylserine     

Conformationally restricted analogs of the direct thrombin inhibitor FM 19

The serine protease thrombin plays several key roles in the clotting cascade within the hemostatic system, such as in fibrin formation and platelet activation. Thus, development of an inhibitor that binds to the enzyme’s active site (a direct thrombin inhibitor) offers an approach for the treatment of thrombus-associated diseases. Previous structure–activity relationship studies originally based on the bradykinin breakdown product Arg-Pro-Pro-Gly-Phe (RPPGF) led to the development of lead compound FM 19 (d-Arg-Oic-Pro-d-Ala-Phe(p-Me)-NH₂). The recently determined X-ray structure of FM 19 in the active site of thrombin has revealed sites of modification to potentially improve inhibition. In this study, we report the synthesis and biological characterization of nine peptides that replace only the d-Arg residue of the FM 19 sequence, investigating ways to add conformational restriction, modification of the basic moiety at the end of the side chain, and removal of the charge from the N-terminus. Two of these peptides, 6 and 7 (IC₅₀ values of 0.51 and 0.45 μM, respectively), show similar potency to the best compounds in the FM 19 series reported thus far.     

Alkylation of phosphorothioated thrombin binding aptamers improves the selectivity of inhibition of tumor cell proliferation upon anticoagulation

BackgroundRecently, aptamers have been extensively researched for therapy and diagnostic applications. Thrombin-binding aptamer is a 15 nt deoxyribonucleic acid screened by SELEX, it can specifically bind to thrombin and inhibit blood coagulation. Since it is also endowed with excellent antitumor activity, the intrinsic anticoagulation advantage converted to a main potential side effect for its further application in antiproliferative therapy.MethodsSite-specific alkylation was conducted through nucleophilic reaction of phosphorothioated TBAs using bromide reagents. Circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR) measurements were used to evaluate anticoagulation activity, and a CCK-8 assay was used to determine cell proliferation activity.ResultsThe CD spectra of the modified TBAs were weakened, and their affinity for thrombin was dramatically reduced, as reflected by the K_D values. On the other hand, their inhibition of A549 cells was retained.ConclusionsIncorporation of different alkyls apparently disrupted the binding of TBA to thrombin while maintaining the antitumor activity.General significanceA new modification strategy was established for the use of TBA as a more selective antitumor agent.     

Location of the structural gene for glycyl ribonucleic acid synthetase by means of a strain ofEscherichia coli possessing an unusual enzyme

Summary E. coli KB (Benzer) differs from other common laboratory strains in possessing a glycyl sRNA synthetase with a 50 to 100 times elevated K _m for glycine. The degree of charging of glycyl sRNA in this strain can be increased by supplementing the growth medium with glycine. The altered enzyme has been used as a marker by which to map its structural gene. Linkage analysis of recombinants from uninterrupted matings, and cotransduction (80%) of the synthetase withxyl, indicate that this gene is located betweenxyl andmalt, close toxyl, at min 69.5 on the map drawn byTaylor andThoman (1964).     

Heparin-like glycosaminoglycans influence growth and phenotype of human arterial smooth muscle cells in vitro. II. The platelet-derived growth factor A-chain contains a sequence that specifically binds heparin

Summary Synthetic oligopeptides were used to study the specificity of the interaction between heparin and platelet-derived growth factor (PDGF) in competition experiments. DNA synthesis in PDGF-dependent human arterial smooth muscle cell (hASMC) cultures was used as a biological tracer of PDGF activity. Oligo-108-124 (corresponding to amino acid residues 108-124 of the long PDGF A-chain isoform) had no effect on DNA synthesis in itself but competed at 10⁻¹⁰ M concentration effectively with PDGF for binding to heparin and released the block on thymidine incorporation induced by heparin. Poly-lysine-serine (lysine:serine ratio 3:1) was also effective but at a considerably higher concentration (10⁻⁶ M). Poly-arginine-serine did not compete with PDGF for heparin as deduced from the cell assay. This suggested that among basic amino acids, lysine was more important than arginine for heparin binding. Deletion of lysine residues 115 and 116 in Oligo-108-124 abolished its effect on the interaction between PDGF and heparin in the cell assay. Likewise, Oligo-69-84 (corresponding to the PDGF A-chain residues 69–84), with three lysine residues interrupted by a proline, was ineffective. In Oligo-108-124, the lysine residues are interrupted by an arginine. Our results suggested that the binding between PDGF and heparin is specific and that the amino acid sequence [-Lys¹¹⁵-Lys¹¹⁶-Arg¹¹⁷-Lys¹¹⁸-Arg¹¹⁹-] is of major importance. They do not however, exclude other domains of the PDGF A or B chains as additional binding sites for heparin nor do they exclude the possibility that heparin and the PDGF receptor share a common binding site.