Chemical modification studies on Abrus agglutinin. Involvement of tryptophan residues in sugar binding.

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydrate-binding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.     

Subcellular distribution of various proteases in Escherichia coli.

It has been reported recently that Escherichia coli cells contain eight distinct soluble enzymes capable of degrading proteins to acid-soluble material. Two are metalloproteases that degrade [125I]insulin but not larger proteins: protease Pi, which is identical to protease III, is restricted to the periplasm, and protease Ci is restriction to the cytoplasm. The six others (named Do, Re, Mi, Fa, So, and La, which is the ATP-dependent protease) are serine proteases that degrade [14C]globin and [3H]casein, but not insulin. One of these (Mi) is localized to the periplasm, and one (Re) is distributed equally between the two cellular fractions. The others are present only in the cytoplasm.     

In vitro micropropagation of elite rosewood (Dalbergia latifolia Roxb.)

Induction of single and multiple shoots was obtained from nodal expiants of 60–80 year-old elite trees of rosewood on Murashige and Skoog's basal medium supplemented with 6-benzylaminopurine (1.0 mg 1^-1) and -Naphthalene acetic acid (0.05 mg 1^-1) or indole acetic acid (0.5 mg 1^-1). Multiplication of shoots was obtained on MS (reduced major elements) or Woody Plant Medium supplemented with 6-benzylaminopurine (1.0 mg 1^-1) and kinetin (0.5–1.0 mg 1^-1). Excised shoots were rooted on half-strength MS with IBA (2.0 mg 1^-1) to obtain complete plantlets. The regenerated plantlets have been acclimatized and successfully transferred to the soil.Abbreviations MS Murashige and Skoog's (1962) medium - B5 Gamborg (1968) medium - WPM Woody plant medium, Lloyd and McCown (1981) medium - NAA -naphthalene acetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzyl aminopurine - KIN kinetin - PVP polyvinyl pyrrolidone - CH casein hydrolysate - ADS adenine sulphate - L-Gl L-glutamine - L-Arg L-arginine - L-Asp L-asparagine - PG phloroglucinol     

Glycation of lens MIP26 affects the permeability in reconstituted liposomes.

We studied the role of glycation of lens putative gap junctional protein, MIP26, on the permeability as well as on calmodulin mediated gating activity in reconstituted liposomes. Calf lens membranes were incubated with 0-100 mM glucose for 3 days and MIP26 was isolated. There was a glucose concentration dependent increase in the glycation of MIP26 which reached to 2.48 moles/mole of protein with 100 mM glucose. Gel electrophoresis showed that there was no degradation of MIP26 to MIP22 during incubation. Channel permeability was determined by reconstituting MIP26 into asolectin liposomes. There was a MIP26 glycation dependent decrease in the permeability to sucrose. Furthermore, proteoliposomes containing nonglycated MIP26 showed complete uncoupling of the channels with calmodulin whereas the channels containing glycated MIP26 were only partially uncoupled. These results suggest that glycation of MIP26 does interfere with the gating activity in reconstituted liposomes.     

Isolation of Clostridium botulinum from tropical fish

Electroporation was used to facilitate transformation of Listeria species with plasmid DNA. Optimal conditions for transformation of L. monocytogenes were a field strength of 8.5 kV/cm, 200 Ohms resistance, 25 microF capacitor with a time constant of 5 ms. With these conditions, 3.9 x 10(6) transformants/micrograms DNA were obtained. Under the same conditions, L. innocua and L. ivanovii exhibited a frequency of transformation similar to that of L. monocytogenes but a somewhat lower level was obtained with L. seeligeri.     

Structure of spinach nitrite reductase: implications for multi-electron reactions by the iron-sulfur:siroheme cofactor

The structure of nitrite reductase, a key enzyme in the process of nitrogen assimilation, has been determined using X-ray diffraction to a resolution limit of 2.8 A. The protein has a globular fold consisting of 3 alpha/beta domains with the siroheme-iron sulfur cofactor at the interface of the three domains. The Fe(4)S(4) cluster is coordinated by cysteines 441, 447, 482, and 486. The siroheme is located at a distance of 4.2 A from the cluster, and the central iron atom is coordinated to Cys 486. The siroheme is surrounded by several ionizable amino acid residues that facilitate the binding and subsequent reduction of nitrite. A model for the ferredoxin:nitrite reductase complex is proposed in which the binding of ferredoxin to a positively charged region of nitrite reductase results in elimination of exposure of the cofactors to the solvent. The structure of nitrite reductase shows a broad similarity to the hemoprotein subunit of sulfite reductase but has many significant differences in the backbone positions that could reflect sequence differences or could arise from alterations of the sulfite reductase structure that arise from the isolation of this subunit from the native complex. The implications of the nitrite reductase structure for understanding multi-electron processes are discussed in terms of differences in the protein environments of the cofactors.     

Triacontanol negatively modulates the jasmonic acid-stimulated proteinase inhibitors in tomato (Lycopersicon esculentum)

Triacontanol (TRIA), a long chain aliphatic alcohol (C30H61OH) reverses the effect of jasmonic acid (JA) in inducing proteinase inhibitors (PIs) in tomato leaves. Porcine pancreas trypsin and Spodoptera litura gut proteinases were inhibited in the presence of leaf proteins treated with JA, and TRIA partially reverses this effect. Spodoptera litura larvae fed with tomato leaves treated with JA were reduced in body weight and TRIA is able to partially reverse this JA-induced effect. These results reflect the partial reversal effect of TRIA in down regulating the JA-induced production of proteinase inhibitors.     

Discovery and pharmacological characterization of a novel rodent-active CCR2 antagonist, INCB3344

This report describes the characterization of INCB3344, a novel, potent and selective small molecule antagonist of the mouse CCR2 receptor. The lack of rodent cross-reactivity inherent in the small molecule CCR2 antagonists discovered to date has precluded pharmacological studies of antagonists of this receptor and its therapeutic relevance. In vitro, INCB3344 inhibits the binding of CCL2 to mouse monocytes with nanomolar potency (IC(50) = 10 nM) and displays dose-dependent inhibition of CCL2-mediated functional responses such as ERK phosphorylation and chemotaxis with similar potency. Against a panel of G protein-coupled receptors that includes other CC chemokine receptors, INCB3344 is at least 100-fold selective for CCR2. INCB3344 possesses good oral bioavailability and systemic exposure in rodents that allows in vivo pharmacological studies. INCB3344 treatment results in a dose-dependent inhibition of macrophage influx in a mouse model of delayed-type hypersensitivity. The histopathological analysis of tissues from the delayed-type hypersensitivity model demonstrates that inhibition of CCR2 leads to a substantial reduction in tissue inflammation, suggesting that macrophages play an orchestrating role in immune-based inflammatory reactions. These results led to the investigation of INCB3344 in inflammatory disease models. We demonstrate that therapeutic dosing of INCB3344 significantly reduces disease in mice subjected to experimental autoimmune encephalomyelitis, a model of multiple sclerosis, as well as a rat model of inflammatory arthritis. In summary, we present the first report on the pharmacological characterization of a selective, potent and rodent-active small molecule CCR2 antagonist. These data support targeting this receptor for the treatment of chronic inflammatory diseases.     

Synthesis and characterization of novel 6-fluoro-4-piperidinyl-1,2-benzisoxazole amides and 6-fluoro-chroman-2-carboxamides: antimicrobial studies

Novel derivatives of 6-fluoro-4-piperidinyl-1,2-benzisoxazole amides 4(I-VI) were obtained by the condensation of different acid chlorides with 6-fluoro-3-piperidin-4yl-benzo[d]isoxazole. Also, 6-fluoro-chroman-2-carboxamides 6(I-III) were synthesized by using nebulic acid chloride with different amines in presence of triethylamine as acid scavenger and dichloroethane as solvent. The synthesized compounds were characterized by IR, 1H NMR, and CHN analysis. These molecules were evaluated for their efficacy as antimicrobials in vitro by disc diffusion and microdilution method against pathogenic strains such as Bacillus substilis, Escherichia coli, Pseudomonas fluorescens, Xanthomonas campestris pvs, X. oryzae, Aspergillus niger, A. flavus, Fusarium oxysporum, Trichoderma species, F. monaliforme, and Penicillum species. Compounds 4I, 4IV, 4V, 6I, 6II and 6III showed better inhibitory activity than compared to standard drugs. Among these compounds, 4IV and 6III showed potent inhibitory activity against all the strains and found to be nonstrain dependent. The title compounds represent a novel class of potent antimicrobial agents.     

Purification and characterization of thermostable β-amylase and pullulanase from high-yielding Clostridium thermosulfurogenes SV2

Thermostable β-amylase and pullulanase, secreted by the thermophilic anaerobic bacterium Clostridium thermosulfurogenes strain SV2, were purified by salting out with ammonium sulphate, DEAE-cellulose column chromatography, and gel filtration using Sephadex G-200. Maltose was identified as a major hydrolysis product of starch by β-amylase, and maltotriose was identified as a major hydrolysis product of pullulan by pullulanase. The molecular masses of native β-amylase and pullulanase were determined to be 180 and 100 kDa by gel filtration, and 210 and 80 kDa by SDS–PAGE, respectively. The temperature optima of purified β-amylase and pullulanase were 70 and 75°C, respectively, and both enzymes were completely stable at 70°C for 2h. The presence of starch further increased the stability of both the enzymes to 80°C and both displayed a pH activity optimum of 6.0. The starch hydrolysis products formed by β-amylase action had β-anomeric form.