Biosynthesis of myo-inositol and its role as a precursor of cell-wall polysaccharides in suspension cultures of wild-carrot cells

The biosynthesis of myo-inositol (MI) and its role as a precursor of cell-wall polysaccharides was studied in supension cultures of wild carrot (Daucus carota L.) cells. Suspension cultures, grown in the presence or absence of 2,4-dichlorophenoxyacetic acid for 7 and 14d were incubated with [U-¹⁴C]glucose and [2-³H]MI in the presence of different concentrations of unlabeled MI. Synthesis of [¹⁴C]MI from [U-¹⁴C]glucose occurred under all conditions. The amount of MI synthesized from glucose was sharply reduced when 10 mM MI was provided in the medium. Substantial quantities of ³H were incorporated in arabinose, xylose and galacturonic acid isolated and purified from the cell-wall polysaccharides of the cell cultures in various stages of growth or embryogenesis. No ³H was present in the glucose or galactose units of cell-wall polysaccharides. At the four stages of growth and states of development of the carrot cultures used, the MI oxidation pathway contributed to the synthesis of pentosyl and galacturonosyl units of the cell wall. However, the data indicate that the contribution of the MI oxidation pathway to pentosyl and galacturonosyl units is small.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MI myo-inositol     

The purification and properties of peroxidase in Mycobacterium tuberculosis H37Rv and its possible role in the mechanism of action of isonicotinic acid hydrazide.

Peroxidase from Mycobacterium tuberculosis H37Rv was purified to homogeneity. The homogeneous protein exhibits catalase and Y (Youatt's)-enzyme activities in addition to peroxidase activity. Further confirmation that the three activities are due to a single enzyme was accomplished by other criteria, such as differential thermal inactivation, sensitivity to different inhibitors, and co-purification. The Y enzyme (peroxidase) was separated from NADase (NAD+ glycohydrolase) inhibitor by gel filtration on Sephadex G-200. The molecular weights of peroxidase and NADase inhibitor, as determined by gel filtration, are 240000 and 98000 respectively. The Y enzyme shows two Km values for both isoniazid (isonicotinic acid hydrazide) and NAD at low and high concentrations. Analysis of the data by Hill plots revealed that the enzyme has one binding site at lower substrate concentrations and more than one at higher substrate concentration. The enzyme contains 6g-atoms of iron/mol. Highly purified preparations of peroxidases from different sources catalyse the Y-enzyme reaction, suggesting that the nature of the reaction may be a peroxidatic oxidation of isoniazid. Moreover, the Y-enzyme reaction is enhanced by O2. Isoniazid-resistant mutants do not exhibit Y-enzyme, peroxidase or catalase activities, and do not take up isoniazid. The Y-enzyme reaction is therefore implicated in the uptake of the drug.     

Neck region of gerbil spermatozoa

In the course of the reorganization and degeneration of the proximal centriole in the mature acentriolate spermatozoon of the Mongolian gerbil, both the proximal and distal centrioles appear in the early cap phase of spermatid development. During the acrosome phase, both distal and proximal centrioles become highly active in the formation of a segmented column. The proximal centriole becomes actively involved in the formation of the capitulum, while the distal centriole forms the axonemal complex and dense fibers. During the maturation phase of spermatid development, the “pinwheel” arrangement of the proximal centriole becomes an “S”-shaped structure, turned 90° on its vertical axis. The few “doublet” microtubules that can be detected later in that stage completely disappear during spermiation. The distal centriolar area develops a single central pair of microtubules and membranous elements. Another prominent feature in the neck region of the gerbil spermatozoa is the presence of two dense rudimentary columns in association with the mitochondria. Although their density is similar to that of the other columns, these two columns have no connection with the dense fibers; in fact, they are closely associated with the mitochondria.     

Radiation protection of human lymphocyte chromosomes in vitro by orientin and vicenin.

Orientin (Ot) and Vicenin (Vc), two water-soluble flavonoids isolated from the leaves of Indian holy basil Ocimum sanctum have shown significant protection against radiation lethality and chromosomal aberrations in vivo. In the present study the protective effect of Ot and Vc against radiation induced chromosome damage in cultured human peripheral lymphocytes was determined by micronucleus test. In order to select the most effective drug concentration, fresh whole blood was exposed to 4Gy of cobalt-60 gamma-radiation with or without a 30 min pre-treatment with 6.25, 12.5, 15.0, 17.5 or 20 microM of Ot/Vc. Micronucleus (MN) assay was done by cytochalasin induced cytokinesis block method. Radiation significantly increased the MN frequency (16 times normal). Pre-treatment with either Ot or Vc at all concentrations significantly (P<0.05-0.001) reduced the MN count in a concentration dependent manner, with the optimum effect at 17.5 microM. Therefore, fresh blood samples were incubated with/without 17.5 microM Ot/Vc for 30 min and then exposed to 0.5-4Gy of gamma-radiation. Radiation increased the MN frequency linearly (r(2)=0.99) with dose. Pre-treatment with Ot or Vc significantly (P<0.01-0.001) reduced the MN counts to 51-67% of RT alone values, giving DMFs of 2.62 (Ot) and 2.48 (Vc). Both the compounds showed significant antioxidant activity in vitro at the above concentrations, which was significantly higher than that of DMSO at equimolar concentrations. Thus, the results demonstrate that both the flavonoids give significant protection to the human lymphocytes against the clastogenic effect of radiation at low, non-toxic concentrations. The radioprotection seems to be associated with their antioxidant activity. The clinical potential of these protectors in cancer therapy needs to be investigated.     

Photorespiration in C3–C4 intermediate species of Alternanthera and Parthenium: Reduced ammonia production and increased capacity of CO2 refixation in the light

The pattern of photorespiratory ammonia (PR–NH₃) formation and its modulation by exogenous bicarbonate or glycine were investigated in C₃–C₄ intermediates of Alternanthera (A. ficoides and A. tenella) and Parthenium hysterophorus in comparison to those of C₃ or C₄ species. The average rates of PR–NH₃ accumulation in leaves of the intermediates were slightly less than (about 25% reduced) those in C₃ species, and were further low in C₄ plants (40% of that in C₃). The levels of PR–NH₃ in leaf discs decreased markedly when exogenous bicarbonate was present in the incubation medium. The inhibitory effect of bicarbonate on PR–NH₃ accumulation was pronounced in C₃ plants, very low in C₄ species and was moderate in the C₃–C₄ intermediates. Glycine, an intermediate of photorespiratory metabolism, raised the levels of PR–NH₃ in leaves of not only C₄ but also C₃–C₄ intermediates, bringing the rates close to those of C₃ species. The rate of mitochondrial glycine decarboxylation in darkness in C₃–C₄ intermediates was partially reduced (about 80% of that in C₃ species), corresponding to the activity-levels of glycine decarboxylase and serine hydroxymethyltransferase in leaves. The intermediates had a remarkable capacity of reassimilating photorespiratory CO₂ in vivo, as indicated by the apparent refixation of about 85% of the CO₂ released from exogenous glycine in the light. We suggest that the reduced photorespiration in the C₃–C₄ intermediate species of Alternanthera and Parthenium is due to both a limitation in the extent of glycine production/decarboxylation and an efficient refixation/recycling of internal CO₂.Abbreviations GDC glycine decarboxylase - GS glutamine synthetase - GOGAT glutamate-oxoglutarate aminotransferase - -HPMS -hydroxy-2-pyridinemethanesulfonic acid - INH isonicotinyl hydrazide - MSO L-methionine sulfoximine - PR–NH₃ photorespiratory-ammonia - SHMT serine hydroxymethyltransferase     

Synthesis,Biological Evaluation and in Silico Study of N-(2- and 3-Pyridinyl)benzamide Derivatives as Quorum Sensing Inhibitors against Pseudomonas aeruginosa

The effectiveness of treating bacterial infections is seriously threatened by the emergence of bacterial resistance to chemical treatment. Growth of microbes in biofilm is one of the main causes of resistance to antimicrobial drugs. Quorum sensing (QS) inhibition, which targets the QS signalling system by obstructing cell-cell communication, was developed as an alternative treatment by creating innovative anti-biofilm drugs. Therefore, the goal of this study is to develop novel antimicrobial drugs that are effective against Pseudomonas aeruginosa by inhibiting QS and acting as anti-biofilm agents. In this study, N-(2- and 3-pyridinyl)benzamide derivatives were selected to design and syntheses. Antibiofilm activity was revealed by all the synthesized compounds and the biofilm was visibly impaired, and the OD_595nm readings of solubilized biofilm cells presented a momentous difference between the treated and untreated biofilms. The best anti-QS zone was observed for compound 5d and found to be 4.96 mm. Through in silico research, the physicochemical characteristics and binding manner of these produced compounds were examined. For the purpose of understanding the stability of the protein and ligand complex, molecular dynamic simulation was also carried out. The overall findings showed that N-(2- and 3-pyridinyl)benzamide derivatives could be the key to creating effective newer anti-quorum sensing drugs that are effective against different bacteria.     

Use ofN,N′-polymethylenebis(iodoacetamide) derivatives as probes for the detection of conformational differences in tubulin isotypes

Mammalian brain tubulin is anαΒ heterodimer; bothα andΒ exist in 6–7 isotypic forms which differ in their amino acid sequences. By the use of isotype-specific monoclonal antibodies, we have previously shown that we can purify theαΒ _II,αΒ _III, andaΒ _IV tubulin dimers from bovine brain. We have also observed that these isotypes differ in their distributionin vivo and their polymerization and drug-binding propertiesin vitro. We have now explored the question of whether the isotypically purified dimers differ in their overall conformation using as probes compounds of theN,N′-polymethylenebis (iodoacetamide) series which are known to form discrete intrachain cross-links inΒ-tubulin. These compounds have the structure ICH₂CONH(CH₂) _n NHCOCH₂I. One of these cross-links, designatedΒ ^s, is between cys¹² and either cys²⁰¹ or cys²¹¹. The other, designatedΒ ^*, is between cys²³⁹ and cys³⁵⁴. TheΒ ^* cross-link forms inαΒ _II andαΒ _IV but not inαΒ _III; this is not surprising in view of the fact thatαΒ _III has serine at position 239 instead of cysteine. However,αΒ _III is also unable to form theΒ ^s cross-link, although it appears to have all three cysteines which may be involved in the cross-link. This suggests that at least one of the sulfhydryls involved in the cross-link may be inaccessible inαΒ _III. Although bothαΒ _II andαΒ _IV can form theΒ ^s cross-link, the dependence on cross-linker chain length is different.αΒ _II formsΒ ^s with derivatives in whichn=2, 4, 5, 6, and 7 but not with those in whichn=3 or 10. In contrast,αΒ _IV formsΒ ^s with derivatives in whichn=2, 3, 4, 5, 6, 7, and 10. These results imply that theΒ ^s sulfhydryls are slightly more accessible inαΒ _IV and are therefore less dependent on the conformation of the cross-linker to react with it. It appears, therefore, that theαΒ _II,αΒ _III, andαΒ _IV dimers each have unique conformations. This may help to explain the different assembly and drug-binding properties of these dimers.     

Purification and characterization ofFus sI3596, a 65 kd allergen ofFusarium solani

A component ofFusarium solani (F. solani), identified as the major allergen,Fus sI₃₅₉₆ ^* was purified to homogeneity from culture filtrate (CF) by means of anion-exchange column chromatography, gel filtration and FPLC. The homogeneity ofFus sI₃₅₉₆ ^* was assessed by IEF, PAGE, SDS-PAGE (non-reducing), immunoblot and HPLC.Fus sI₃₅₉₆ ^* was isolated as a glycoprotein of MW 65 kd and pI 3.6. The IgE ELISA-inhibition assay after periodate treatment of the fraction showed a lower IgE binding capacity suggesting involvement of carbohydrate moiety in IgE binding reactions of the allergen. Peptide fragments ofFus sI₃₅₉₆ ^* obtained after CNBr and trypsin treatment were analysed by immunoblotting for their allergenicity. This study indicated that there could be at least 3 allergenic determinants in the major allergen,Fus sI₃₅₉₆ ^* ofF. solani CF.Abbreviations DEAE diethyl aminoethyl - HPLC high performance liquid chromatography - SDS sodium dodecyl sulphate - PBS-Tween phosphate buffer saline containing 0.1% tween - DTT dithiothreitol - TFA triflouroacetate - FPLC fast protein liquid chromatography     

Larval stages of Neodiaptomus lindbergi Brehm, 1953 (Copepoda,Calanoida)

All the postembryonic developmental instars of Neodiaptomus lindbergi Brehm, 1953, reared in the laboratory from eggs, are described and illustrated. Further, the larval affinities of this species vis-a-vis certain other Indian diaptomid species are mentioned.