Evidence for the direct involvement ofβ-hemolysin inAeromonas hydrophila enteropathogenicity

Culture filtrates of 19 of 21 (90%) -hemolytic isolates ofAeromonas hydrophila caused fluid accumulation in permanently ligated rabbit ileal loops, whereas no fluid was accumulated with filtrates of eight non--hemolytic isolates. Antiserum to purified -hemolysin neutralized the ileal loop activity of culture filtrates from four of four -hemolytic isolates, and treatment at 56°C for 10 min eliminated the loop activity of six additional isolates. These results support the conclusion that -hemolysin alone causes significant changes in intestinal permeability and that it is a more common pathogenic mechanism than the heat-stable cytotonic enterotoxin. Electrophoretic and serological assays showed evidence for production of only one species of -hemolysin byA. hydrophila.     

Genetic mapping of the component chains of Ia antigens

The genes encoding the two polypeptide chains ( and) that comprise the murine Ia antigens were localized within distinct regions of the major histocompatibility complex (MHC). This was accomplished by correlating allelic forms of the and chains with the MHC congenic strains of mice from which they were isolated. Allelic forms of and chains were distinguished by their unique structural markers, such as isoelectric points, amino acid sequences or peptide maps. The results indicate that the structural genes for both the and chains of I-A subregion antigens are located within the K to I-A genetic interval. In contrast, the gene encoding the chain of I-E subregion antigens is located outside of theI-E subregion and within the K to I-B genetic interval. These findings may have important implications for analysis of observations that complementation by twoI-region genes is sometimes required for development of immune responses.     

Isozyme polymorphism ofβ-glucosidase inAspergillus nidulans

Electrophoretic analysis of the distribution of various electromorphs at different -glucosidase zones was carried out in natural populations ofA. nidulans, theA. nidulans group, and various species belonging to the genusAspergillus from diverse geographical areas of India. The data show the existence of three segregating zones for -glucosidase, designated -GluI, -GluII and -GluIII. All three zones are present in wild isolates ofA. nidulans, and only two, i.e., -GluI and -GluIII, in theA. nidulans group and -GluII and -GluIII in different species ofAspergillus exceptA. terreus, A. flavus, andA. brevipes, where only -GluIII is present. Overall nine electromorphs are observed at -GluI, three at -GluII, and six at -GluIII zones, respectively, It can be concluded that there may be three structural genes for -glucosidase coding the three polymorphic zones inA. nidulans.This research work was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi.     

Lactose utilization byVibrio vulnificus

The ability of wild-type strains ofVibrio vulnificus to utilize lactose as a sole source of carbon and energy and produce acid in lactose-containing media is associated with the appearance of spontaneous lactose-utilizing mutants. These contain increased activities of an enzyme able to hydrolyzeo-nitrophenyl--d-galactoside as well as lactose. This activity is constitutive in some mutants and inducible by both lactose and isopropyl--d-thiogalactoside in others. A limited survey of otherVibrio species indicates thatV. pelagius also can acquire, by mutation, the ability to grow on and make acid from lactose. No immunological cross-reaction was detected between the enzymes fromVibrio and the -galactosidases ofEscherichia coli andKlebsiella.     

Chemical activity of simple basic peptides

Alternating all-L poly(leucyl-lysyl) increases markedly the rate of hydrolysis of oligoribonucleotides. Pure D poly (leucyl-lysyl) is as active as the all-L polymer. The homochiral polypeptides adopt a-sheet structure when complexed to the oligonucleotides. Alternating poly(D,L-Leu-D,L-Lys) made of racemic amino acids is much less efficient and is unable to adopt a-sheet structure. A set of alternating poly (leucyl-lysyl) ranging from the racemic to the homochiral all-L polymer has been checked. Their conformations can be described as a mixture of random coil and-sheet conformations, the amount of-sheet increasing with the optical purity of the polymer. The hydrolytic activity follows the proportion of-sheets, suggesting that the chemical activity is related to the geometry of the chain. Short peptides were prepared in order to evaluate the critical chain length required for the hydrolytic activity. A decapeptide is long enough to present 90% of the activity of the corresponding polypeptide.     

Cloning and expression of β-galactosidase gene from Streptococcus thermophilus in Saccharomyces cerevisiae

Summary The -galactosidase gene ofStreptococcus thermophilus was cloned into plasmid vector, pVT100-U, and used to transform a strain ofEscherichia coli andSaccharomyces cerevisiae. Transformants which expressed -galactosidase activity were obtained in bothE. coli andSaccharomyces cerevisiae, the highest activity found in a yeast recombinant. The expression and thermostability of the cloned -galactosidase genes from different plasmid constructions were compared with the streptococcal -galactosidase. The recombinant protein was equivalent to the specific activity and thermostability ofS. thermophilus.     

Modification of β-adrenoceptor signal transduction pathway by genetic manipulation and heart failure

The -adrenoceptor (-AR) mediated signal transduction pathway in cardiomyocytes is known to involve ₁- and ₂-ARs, stimulatory (G_s) and inhibitory (G_i) guanine nucleotide binding proteins, adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA). The activation of ₁- and ₂-ARs has been shown to increase heart function by increasing Ca²⁺-movements across the sarcolemmal membrane and sarcoplasmic reticulum through the stimulation of G_s-proteins, activation of AC and PKA enzymes and phosphorylation of the target sites. The activation of PKA has also been reported to increase phosphorylation of some myofibrillar proteins (for promoting cardiac relaxation) and nuclear proteins (for cardiac hypertrophy). The activation of ₂-AR has also been shown to affect G_i-proteins, stimulate mitogen activated protein kinase and increase protein synthesis by enhancing gene expression. ₁- and ₂-ARs as well as AC are considered to be regulated by PKA- and protein kinase C (PKC)-mediated phosphorylations directly; both PKA and PKC also regulate -AR indirectly through the involvement of -AR kinase (ARK), -arrestins and G-protein subunits. Genetic manipulation of different components and regulators of -AR signal transduction pathway by employing transgenic and knockout mouse models has provided insight into their functional and regulatory characteristics in cardiomyocytes. The genetic studies have also helped in understanding the pathophysiological role of ARK in heart dysfunction and therapeutic role of ARK inhibitors in the treatment of heart failure. Varying degrees of defects in the -AR signal transduction system have been identified in different types of heart failure to explain the attenuated response of the failing heart to sympathetic stimulation or catecholamine infusion. A decrease in ₁-AR density, an increase in the level of G_i-proteins and overexpression of ARK are usually associated with heart failure; however, these attenuations have been shown to be dependent upon the type and stage of heart failure as well as region of the heart. Both local and circulating renin-angiotensin systems, sympathetic nervous system and endothelial cell function appears to regulate the status of -AR signal transduction pathway in the failing heart. Thus different components and regulators of the -AR signal transduction pathway appears to represent important targets for the development of therapeutic interventions for the treatment of heart failure.     

Localization of melanotropin-like peptides in the central nervous system of two insect species,the migratory locust,Locusta migratoria,and the fleshfly,Sarcophaga bullata

Summary By use of well characterized antisera in the peroxidase-antiperoxidase method, we were able to demonstrateMSH andMSH immunoreactive cells and nerve fibres within the nervous system of adults and larvae ofLocusta migratoria and 3-, 5- and 8-day-old adultSarcophaga bullata. In neither of these insect species, any immunoreaction was obtained with a ₃MSH-antiserum. Double immuno-histochemical stainings revealed thatMSH-like andMSH-like substances are located in different cells. These cells show no immunoreactivity to a number of antisera against other POMC-derivatives (anti-lipotropin, anti-endorphin, anti-ACTH_1–24); thus they appear to containMSH- orMSH-like material in a specific way. The function of the immunologically detected peptides remains to be demonstrated. The distribution of the immunoreactive material suggests that, like in amphibians and other lower vertebrates, the synthesis or release of melanotropins might be under the influence of external stimuli. The present observations support the recently developed concept that even some of the smallest neuropeptides, the melanotropins, have been highly conserved during a long period of evolution.     

Fluorescence studies of normal and sickle beta apohemoglobin self-association

The acrylamide quenching of the intrinsic tryptophanyl fluorescence of normal and sickle apohemoglobins has been studied in 0.05 M potassium phosphate buffer,pH 7.5, at 5°C over a protein concentration range from 1 to 50M. Analysis of quenching dynamics revealed a strong dependence on acrylamide concentration for the intrinsic fluorescence of both normal and sickle apohemoglobins, suggesting that one tryptophanyl residue [presumably that at position 37(C3)], was more accessible to collisional quencher than the other tryptophanyl residue [15(A12)]. Additional studies, which altered viscosity and subunit assembly experimental parameters, supported the assignment of residue 37 as the more dynamically accessible residue. Finally, the quenching data were also found to be dependent on protein concentration, implying that this difference in the mobility between the two residues is a sensitive probe of self-aggregation. Extrapolated dynamic quenching constants at low concentration of acrylamide were used to estimate the dimer-monomer equilibrium dissociation constants of normal and sickle apohemoglobins, and were found to be 5.6 and 2.4M, respectively, thus demonstrating distinct self-association properties of _A and _S apohemoglobins.     

Characterization of carotene accumulation inUstilago violacea using high-performance liquid chromatography

Quantitative analysis of carotene accumulation in white, pink, pumpkin, orange, and yellow haploid strains ofUstilago violacea by high-performance liquid chromatography indicated that specific patterns of carotene accumulation are primarily responsible for the white, pumpkin, orange, and yellow phenotypes. The yellow strains accumulated primarily -zeacarotene and -carotene. The white strains accumulated primarily the colorless carotene, phytoene, or did not accumulate any carotene at all. Carotene accumulation in pink haploid strains followed the same patterns as for the white, pumpkin, orange, or yellow strains. Pink diploid and disomic strains ofU. violacea with various parental combinations of the color mutations accumulated either cis--zeacarotene and -carotene or only -carotene. The pattern of carotene accumulation in conjunction with the available genetic information for the carotene loci inU. violacea was used as a basis for the construction of a new genetic model for carotene biosynthesis inU. violacea. The model employs three dehydrogenases and one cyclase for the synthesis of -carotene from phytoene, and accounts for the carotene accumulation patterns of either cis--zeacarotene and -carotene or lycopene, -carotene, and -carotene.     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

The β-tubulin gene family evolution in theDrosophila montium subgroup of themelanogaster species group

The 1-, 2-, and 3-tubulin genes have been mapped by in situ hybridization on the polytene chromosomes of 11 selected species (15 strains) belonging to theDrosophila montium subgroup. Although the hybridization pattern among the strains of the same species does not differ, this pattern is significantly different among the species. The -tubulin genes in themontium subgroup seem to be organized in a cluster, or in a semi-cluster, or are completely dispersed. The clustered arrangement is found in the North-Oriental sibling speciesD. auraria, D. triauraria, andD. quadraria. The semi-clustered arrangement, wherein the 1 and 2 genes are located at the same locus while 3 is at a different one, appears in the South-Oriental speciesD. bicomuta, D. serrata, andD. birchii, as well as in the Afrotropical speciesD. diplacantha andD. seguyi. The complete separation of the genes is observed in the Indian speciesD. kikkawai andD. jambulina and in the Afrotropical speciesD. vulcana. Based on the above results, a possible mode of evolution of the -tubulin genes in the montium subgroup is attempted. In addition, phylogenetic relationships among themontium species are discussed. Correspondence to: Z.G. Scouras     

Steroid dehydrogenases in the adrenal gland of four species of birds: a histochemical study

Synopsis The presence of⁵-3-hydroxysteroid dehydrogenase, II-hydroxysteroid dehydrogenase, 17-hydroxysteroid dehydrogenase and glucose-6-phosphate dehydrogenase has been demonstrated histochemically in the adrenal gland of the rain quailCoturnix coromendalica, barn owlTyto alba, brown crakeAmaurornis akool and painted partidgeFrancholinus pictus. All these enzymes occurred in the inter-renal cells. No activity was observed in the chromaffin cells. It is suggested that the inter-renal cells of these four species of birds are capable of synthesizing both corticosteroids and sex steroids.     

Compartmentation of the early steps of cholesterol biosynthesis in mammalian liver

Summary Cholesterol synthesis was studied in the isolated perfused rat liver and with cell-free preparations by incorporation measurements of³H from³HOH and of carbon label from [1-¹⁴C]-acetate. Using specific inhibitors such as (-)-hydroxycitrate, kynurenate, and avidin the following conclusions were reached:Fatty acid and cholesterol biosynthesis share a common substrate pool of cytoplasmic acetyl-CoA. The substrate of mevalonate synthesis is furnished by an extramitochondrial pathway of-hydroxy--methylgluraryl-CoA synthesis, which does not include malonyl-CoA. This favors the assumption of a sequence including cytoplasmic thiolase and-hydroxy--methylglutaryl-CoA synthase.Besides its inhibitory action on ATP citrate lyase (-)-hydroxycitrate was found to stimulate acetyl-CoA carboxylase.Acetyl-CoA synthetase activity of liver is localized predominantly in the cytoplasm. The regulatory behavior of the cytoplasmic enzyme points to a lipogenetic function.The control of cholesterol biosynthesis and the role of cytoplasmic acetyl-CoA synthetase in the maintenance of the extramitochondrial acetyl-CoA pool are considered in light of the reported findings.     

Delineation of sodium-stimulated amino acid transport pathways in rabbit kidney brush border vesicles

Summary We have confirmed previous demonstrations of sodium gradient-stimulated transport ofl-alanine, phenylalanine, proline, and -alanine, and in addition demonstrated transport of N-methylamino-isobutyric acid (MeAIB) and lysine in isolated rabbit kidney brush border vesicles. In order to probe the multiplicity of transport pathways available to each of these¹⁴C-amino acids, we measured the ability of test amino acids to inhibit tracer uptake. To obtain a rough estimate of nonspecific effects, e.g., dissipation of the transmembrane sodium electrochemical potential gradient, we measured the ability ofd-glucose to inhibit tracer uptake.l-alanine and phenylalanine were completely mutually inhibitory. Roughly 75% of the¹⁴C-l-alanine uptake could be inhibited by proline and -alanine, while lysine and MeAIB were no more effective thand-glucose. Roughly 50% of the¹⁴C-phenylalanine uptake could be inhibited by proline and -alanine; lysine was as effective as proline and -alanine, and the effects of pairs of these amino acids at 50mm each were not cumulative. MeAIB was no more effective thand-glucose. We conclude that three pathways mediate the uptake of neutral,l, -amino acids. One system is inaccessible to lysine, proline, and -alanine. The second system carries a major fraction of thel-alanine flux; it is sensitive to proline and -alanine, but not to lysine. The third system carries half the¹⁴C-phenylalanine flux, and it is sensitive to proline, lysine, and -alanine. Since the neutral,l, -amino acid fluxes are insensitive to MeAIB, we conclude that they are not mediated by the classicalA system, and since all of thel-alanine flux is inhibited by phenylalanine, we conclude that it is not mediated by the classicalASC system.l-alanine and phenylalanine completely inhibit uptake of lysine. MeAIB is no more effective thand-glucose in inhibiting lysine uptake, while proline and -alanine appear to inhibit a component of the lysine flux. We conclude that the¹⁴C-lysine fluxes are mediated by two systems, one, shared with phenylalanine, which is inhibited by proline, -alanine, andl-alanine, and one which is inhibited byl-alanine and phenylalanine but inaccessible to proline, -alanine, and MeAIB. Fluxes of¹⁴C-proline and¹⁴C-MeAIB are completely inhibited byl-alanine, phenylalanine, proline, and MeAIB, but they are insensitive to lysine. Proline and MeAIB, as well as alanine and phenylalanine, but not lysine, inhibit¹⁴C--alanine uptake. However, -alanine inhibits only 38% of the¹⁴C-proline uptake and 57% of the MeAIB uptake. We conclude that two systems mediate uptake of proline and MeAIB, and that one of these systems also transports -alanine.     

Cellulase and beta-glucosidase production by mexied culture of trichoderma reesei rut C30 and aspergillus phoenicis

Summary When lignocellulosic materials are hydrolysed using cellulase fromTrichoderma reesei, cellobiose accumulates due to a deficiency in -glucosidase activity of the enzyme complex. Cellobiose decreases the rate and extent of hydrolysis through feedback inhibition of exo -1,4-glucanase. Cellulase produced through mixed culture ofTrichoderma andAspergillus showed increased -glucosidase activity and greatly improved hydrolytic potential.     

Isolation and characterization of β-glucosidases from Aspergillus nidulans mutant USDB 1183

Two extracellular -glucosidases (cellobiase, EC 3.2.1.21), I and II, from Aspergillus nidulans USDB 1183 were purified to homogeneity with molecular weights of 240,000 and 78,000, respectively. Both hydrolysed laminaribiose, -gentiobiose, cellobiose, p-nitrophenyl--L-glucoside, phenyl--L-glucoside, o-nitrophenyl--L-glucoside, salicin and methyl--L-glucoside but not -linked disaccharides. Both were competitively inhibited by glucose and non-competitively (mixed) inhibited by glucono-1,5-lactone. -Glucosidase I was more susceptible to inhibition by Ag⁺ and less inhibited by Fe²⁺ and Fe³⁺ than -glucosidase II.     

Comparison of different strains ofCellulomonas for production of cellulolytic and xylanolytic enzymes from biomass produced on saline lands

A comparison of the rate of carboxymethyl cellulase (CMCase), avicelase, xylanase, -glucosidase and -xylosidase production and rates of growth by 4 different strains ofCellulomonas revealed a wide range of behaviour; with some strains producing more CMCase, avicelase, xylanase, -glucosidase and -xylosidase from complex lignocellulosic (LC) biomass (from saline land) and CMC while some others producing small amounts of these enzymes. One strain,C. biazotea, was better with respect to enzyme production potential and growth behaviour than most of the other strains and has been chosen as a starting strain for genetic improvement for producing enzymes of the cellulase complex.