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In seasonally breeding vertebrates, extrinsic factors like photoperiod and temperature are major determinants, controlling the annual reproductive cycle. In teleosts, kisspeptin, which occurs in two molecular forms: kisspeptin1 (Kiss1) and kisspetin2 (Kiss2), has been reported to alter gonadotropin (Lh and Fsh) secretion but its effect on gonadotropin-releasing hormone (Gnrh) secretion is not unequivocally proved. In the catfish Heteropneustes fossilis, we isolated and characterized kiss2 and gnrh2 cDNAs and the present work reports effects of altered photo-thermal conditions and melatonin (MT, a pineal hormone) on their expressions in the brain. The exposure of the catfish to long photoperiod (LP, 16 h light) at normal temperature (NT) or high temperature (HT, 28 °C) at normal photoperiod (NP) for 14 or 28 days stimulated both kiss2 and gnrh2 expression in both gonad resting and preparatory phases with the combination of LP + HT eliciting maximal effects. Short photoperiod (SP, 8 h light) under NT or HT altered the gene expression according to the reproductive phase and temperature. MT that mediates photo-thermal signals to the brain inhibited brain kiss2 and gnrh2 gene expression in the NP + HT, LP + NT, and SP + NT groups. The altered photo-thermal conditions elicited changes in steroidogenic pathway as evident from changes in plasma E2, progesterone, and testosterone levels. The results show that brain kiss2-gnrh2 signaling is involved in photo-thermal-mediated mechanisms controlling reproduction.

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Kisspeptin (Kiss) is considered an upstream regulator of gonadotropin-releasing hormone in mammals but its role in non-mammalian vertebrates is not unequivocally established. In the catfish Heteropneustes fossilis, a 605 bp long cDNA was identified from the brain by cloning as well as by retrieving from the catfish transcriptome database. The open reading frame (ORF, 93-405 bp) codes for a 113 amino acids long precursor protein. Homology and phylogenetic analyses showed that the predicted protein belongs to the vertebrate Kiss2 type with a high degree of conservation in the Kiss2-10 region (FNFNPFGLRF). The kiss2 transcripts were expressed highly in the brain and gonads in a dimorphic manner with a female bias. In the brain, kiss2 transcripts showed regional differences with higher expression in the medulla oblongata and forebrain regions. The kiss2 transcripts showed significant seasonal variations with the highest expression in the brain in spawning phase and in the gonads in prespawning phase. The kiss2 transcripts were localized in the brain (nucleus preopticus, habenular nucleus, nucleus recessus posterioris, nucleus recessus lateralis) and stratum periventriculare (radial glial cells) of optic tectum, pituitary and ovary (follicular layer and germinal vesicle). Ovariectomy (1, 2, 3 and 4 weeks) decreased brain kiss2 mRNA levels and a single injection of estradiol-17ß (E2; 0.5 µg/g body weight) in 3- week ovariectomized (OVX) and sham operated fish resulted in an increase in the transcript levels after 24 h. The E2 receptor antagonist Tamoxifen (TMX) produced biphasic effects on the kiss2 expression in the dose- response study. TMX inhibited the expression in the OVX fish, but elicited a stimulatory effect in the OVX + E2-treated fish. Testosterone (T) decreased, and progesterone (P4) inhibited (resting phase) or stimulated (prespawning phase) the transcript level in 3-week OVX fish. In the 3-week sham groups, E2 increased, and TMX, T and P4 inhibited the kiss2 transcript levels. The results suggest that Kiss2 is an important regulator of the brain- pituitary- gonadal- endocrine axis, and in habenular and optic tectum functions.

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Gonadotropin-releasing hormone 2 (Gnrh2) is one of the three classes of Gnrh distributed in vertebrates and is highly conserved. In the present study, the cDNA encoding Gnrh2 was isolated and characterized in the ostariophysan catfish Heteropneustes fossilis (hf). The cDNA is 611 bp long with an open reading frame (ORF) of 261 bp that encodes a highly conserved protein of 86 amino acids. The deduced Gnrh2 precursor protein clustered with the vertebrate Gnrh2 type. The sequence identity of hfgnrh2 is 94% with African catfish (Clarias gariepinus) gnrh2 mRNA (accession no. X78047). The hfgnrh2 transcripts were expressed only in the brain and gonads with a higher expression in the female brain and ovary in both resting and prespawning phases. The expression was higher in the prespawning phase than the resting phase. The gnrh2 expression in the brain and ovary showed significant seasonal variations but with opposite patterns. In the brain, the expression was the highest in the preparatory phase, decreased progressively to low levels in the postspawning and resting phases. In the ovary, the transcript level was low in the resting and preparatory phases, increased sharply in the prespawning phase reaching the peak level in the spawning phase and declined sharply in the postspawning phase. The gnrh2 mRNA showed the highest expression in the hind brain-medulla oblongata and moderate to low expression in forebrain regions and pituitary. Ovariectomy resulted in a duration-dependent inhibition of hfgnrh2 mRNA levels in the resting and prespawning phases. Steroid (E2, testosterone and progesterone) replacement treatments (0.5 µg/g body weight) in the 3- week ovariectomized fish restored the inhibition due to ovariectomy, elevated the expression over and above the sham level in the resting phase (E2 group), and raised the levels almost to that of the sham group (testosterone and progesterone groups) in the prespawning phase. In the sham control groups, the steroid replacement resulted in a significant reduction in the mRNA levels. The expression of the gnrh2 mRNA in the brain-pituitary-gonadal axis and its regulation by gonadal steroids suggest that Gnrh2 may have a reproductive role in the catfish.

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AIM: To isolate and characterize bacteriocin, BL8, from the bacteria identified as Bacillus licheniformis from marine environment. METHODS AND RESULTS: One-hundred and twelve bacterial isolates from sediment and water samples collected off the coast of Cochin, India, were screened for antibacterial activity. Strain BTHT8, identified as Bacillus licheniformis, inhibited the growth of Gram-positive test organisms. The active component labelled as bacteriocin BL8 was partially purified by ammonium sulphate fractionation and was subjected to glycine SDS-PAGE. The band exhibiting antimicrobial activity was electroeluted and analysed using MALDI-TOF mass spectrometry, and the molecular mass was determined as 1.4 kDa. N-terminal amino acid sequencing of BL8 gave a 13 amino acid sequence stretch. Bacteriocin BL8 was stable even after boiling at 100 °C for 30 min and over a wide pH range of 1-12. CONCLUSION: A novel, pH-tolerant and thermostable bacteriocin BL8, active against the tested Gram-positive bacteria, was isolated from Bacillus licheniformis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a stable, low molecular weight bacteriocin from Bacillus licheniformis. This bacteriocin can be used to address two important applications: as a therapeutic agent and as a biopreservative in food processing industry.

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N-Lauroyl sarcosine (LS), a cationic, non-toxic and biodegradable detergent readily permeabilized whole cells of baker's yeast (Saccharomyces cerevisiae). Permeabilization was carried out to increase assayable cellular catalase activity, an enzyme of great physiological and industrial importance, and to release 5'-nucleotides which find food/nutritional applications. The event of permeabilization was concentration, time and temperature dependent. Maximum permeabilization of yeast cells were observed when 1 g wet weight (0.2 g dry wt) of cells were permeabilized with 1.0 ml of 2% LS at 45 degrees C for 15 min. LS-permeabilized cells showed 350-fold increase in catalase activity and the supernatant obtained after permeabilization was rich in 5'-nucleotides. LS-permeabilized baker's yeast cells can be used as a source of biocatalyst and to isolate valuable by-products.

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Mango sap (latex) from four Indian varieties was studied for its composition. Sap was separated into non-aqueous and aqueous phases. Earlier, we reported that the non-aqueous phase contained mainly mono-terpenes having raw mango aroma (Phytochemistry 52 (1999) 891). In the present study biochemical composition of the aqueous phase was studied. Aqueous phase contained little amount of protein (2.0-3.5 mg/ml) but showed high polyphenol oxidase (147-214 U/mg protein) and peroxidase (401-561 U/mg protein) activities. It contained low amounts of polyphenols and protease activities. On native PAGE, all the major protein bands exhibited both polyphenol oxidase and peroxidase activities. Both polyphenol oxidase and peroxidase activities were found to be stable in the aqueous phase of sap at 4 degrees C. Sap contained large amount of non-dialyzable and non-starchy carbohydrate (260-343 mg/ml sap) which may be responsible for maintaining a considerable pressure of fluid in the ducts. Thus, the mango sap could be a valuable by-product in the mango industry as it contains some of the valuable enzymes and aroma components.

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The cellular D-amino acid oxidase (DAAO) and catalase activities of Rhodotorula gracilis were greatly increased upon the treatment of the cells with cetyltrimethylammonium bromide (CTAB). However, these enzymes, slowly leaks out from the permeabilized cells. The released DAAO was rapidly inactivated in the absence of ethylenediaminotetraacetic acid (EDTA), beta-mercaptoethanol, and glycerol. DAAO within the permeabilized cells did not require these stabilizing agents. Treating the CTAB-permeabilized cells with 0.2% glutaraldehyde (GA) at 4 degrees C for 10 min prevented the leakage of both DAAO and catalase. Alternately, stabilized whole cell DAAO and catalase was prepared by treating the whole yeast cells with 1% GA at 4 degrees C for 60 min, followed by permeabilization with CTAB, a method which was equally efficient but easy to scale up. CTAB-permeabilized cells converted D-phenylalanine to 97% phenylpyruvate and 3% phenylacetate, and these cells were reused up to 3 cycles in a batchwise reaction. On the other hand, GA-treated CTAB-permeabilized cells produced more than 99% phenylpyruvate and the cells could be reused up to 20 cycles.

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Reactive oxygen species contribute to male infertility by reducing sperm function. Our laboratory has recently demonstrated that reactive oxygen species stimulate the expression of adenosine A(1) receptor which confers cytoprotection in a variety of tissues. Since the adenosine A(1) receptor is highly expressed in the testis, the goal of this study was to determine whether this testicular adenosine A(1) receptor could also be regulated in vivo by reactive oxygen species. Cisplatin, a chemotherapeutic agent shown to alter testicular function, was used to generate reactive oxygen species in vivo. Testes obtained from Sprague-Dawley rats treated with cisplatin (8 mg kg(-1)) demonstrate increased lipid peroxidation and induction of heat shock protein by day 3. In addition, radioligand binding and Western blotting studies indicate an increase in testicular adenosine A(1) receptor in these rats. Scatchard analysis of [3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX) binding data indicates a significant increase in adenosine A(1) receptor number (B(max)) from 309+/-77 to 540+/-69 fmol mg(-1) protein in the cisplatin-treated group. The respective equilibrium dissociation constants (K(d)s) were 3.2+/-1.5 and 3.0+/-0.7 nM for the control and cisplatin-treated groups, respectively. Northern blotting analysis of rat testicular poly (A)(+) RNA indicates two adenosine A(1) receptor transcripts migrating at 3.4 and 5.6 kb, whose combined levels were increased by 49.3+/-9.3% following cisplatin treatment. These results indicate that cisplatin enhances adenosine A(1) receptor expression in the rat testis, possibly through promotion of oxidative stress.

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The rat testis expresses high levels of A1 adenosine receptors (A1 AR) that couple to the inhibition of adenylyl cyclase activity. However, the physiological role of these receptors in the testis is not clear. Previous studies have documented a number of changes in the testis associated with the aging process. The goal of this study was to assess whether alteration in the expression and function of the testicular A1 AR occurs in aging, using the Fischer 344 rats as an aging model. Quantitation of A1 AR expression by radioligand binding of [3H]1,3-dipropyl-8-cyclopentylxanthine, an antagonist radioligand, indicates reductions in receptor number by 35 +/- 13.3 and 53 +/- 18.2% in 18- and 25-mo-old rats, respectively, compared with 3-mo-old rats. Similar reductions in A1 AR expression were determined using Western blotting and receptor autoradiography. Quantitation of the Gi proteins using selective antibodies indicate age-dependent reductions in the levels of alpha i-1,2-, alpha i-3- and beta-subunits. Furthermore, the modulatory influences of guanosine 5'-O-(3-thiotriphosphate) on the binding of agonist and antagonist radioligands to the A1 AR were substantially reduced. Northern blotting analysis of rat testicular poly(A)+ RNA indicates both a 3.4-kb transcript and a 5.6-kb transcript that hybridized to the canine A1 AR cDNA probe. The levels of the 5.6-kb transcript were decreased by 24 +/- 18 and 52 +/- 3% in the 18- and 25-mo-old rats, respectively, compared with the 3-mo-old rats. These results indicate age-dependent deficits in the A1 AR signal transduction pathway in the testes and predict concomitant reductions in the action of adenosine.

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The feasibility of using permeabilized whole cells as a source of intracellular enzymes instead of isolated expensive enzymes for the estimation of biomolecules has been studied. Alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6PDH), hexokinase (HK), and beta-galactosidase (beta-GAL) activities of cetyltrimethylammonium bromide (CTAB)-permeabilized whole yeast cells were employed to estimate ethyl alcohol, glucose, and lactose. The method using permeabilized cells was comparable to that of isolated enzymes and was applicable for the estimation of these analytes in complex samples such as blood, milk, and fermented samples. The usefulness of permeabilized cells as a single source of more than one enzyme required for coupled enzyme assays was demonstrated.

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Alcohol dehydrogenase (ADH) and glucose-6-phosphate dehydrogenase (G6PDH) activities of cetyltrimethylammonium bromide permeabilized baker's yeast whole cells were employed to prepare reduced nicotinamide nucleotides NADH and NADPH from their corresponding oxidised forms. Both NADH and NADPH were found to be stable in the presence of permeabilized cells under the conditions of preparation. No dephosphorylation of NADP+ to NAD+ or of NADPH to NADH was found. Reduction is complete and the prepared NADH and NADPH are chromatographically pure. Since readily available Baker's yeast cells were used instead of expensive isolated enzyme the method described here is simple, economical, and easy to scale up.

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The yeast, Kluyveromyces fragilis was permeabilized to a number of low-molecular-weight substrates using digitonin. The activities of intracellular yeast enzymes, viz., alcohol dehydrogenase (ADH), beta-galactosidase, glucose-6-phosphate dehydrogenase, aspartase, and hexokinase were found to be much higher in the permeabilized cells than the untreated cells. The optimum conditions for permeabilization with reference to ADH were 0.1% digitonin at 37 degrees C for 15 min. The ADH activity in permeabilized cells was several-fold higher than that in cell free extracts prepared by either physical or chemical methods.

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The results of a prospective study, aimed at having a fresh look at the clinical features of secondary syphilis in 89 patients, are presented. Eighty-one (91.0%) had syphilides, and of these, 24 (29.6%) had atypical morphology. Two or more groups of lymph nodes were enlarged in 60, and hepatosplenomegaly was seen in 20 (22.5%) patients. Condylomata data in atypical sites occurred in six patients. A total of 10 patients had alopecia on the scalp, and anterior uveitis was seen in 7 (7.9%). The clear CSF showed minimal elevation of lymphocytes in one of the 21 patients on whom lumbar puncture was performed and may, therefore, be considered unnecessary as a routine procedure. An awareness of the varied clinical presentations would assist in early diagnosis of the disease and help reduce its complications.

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The role of oleic acid in the regulation of the hydrolysis of cholesteryl oleate in lipid films at the air--buffer interface was investigated by using initial rate techniques. A small quantity of enzyme is rapidly adsorbed to substrate-containing films; however, a much greater, although slower, adsorption occurs if oleic acid is present. The rate constant for the slow adsorption is independent of the phase distribution of cholesteryl oleate but is markedly dependent upon both the concentration of oleic acid head groups and the acyl chain packing density in the film. Adsorption is controlled by two ionizable groups, one of which may be the carboxyl group of oleic acid. In contrast to adsorption, catalysis by the surface excess of enzyme is pH independent between 5.5 and 7.5 and is relatively specific for substrate in the monolayer phase. The second-order rate constants for the hydrolysis of cholesteryl oleate in the monolayer phase and the interfacial layer of the double-layer phase are 27 and 2 cm2 s-1 fmol-1. These results indicate that adsorption and catalysis occur at functionally. if not physically, distinct sites on the protein. The adsorption of enzyme to a hydrolysis product, oleic acid, constitutes a form of product activation which presumably helps keep it at the interface during intraluminal fat digestion. The catalytic properties of the adsorbed enzyme suggest that substrate specificities determined for cholesterol esterase in complex reaction systems may largely reflect the availability of substrate in the appropriate physical state at the lipid-water interface.

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The synthesis/hydrolysis of cholesteryl oleate as catalyzed by porcine pancreatic cholesterol esterase has been studied in lipid films at the air-buffer interface. With only reactants and products initially present at the interface, equilibrium is rapidly attained at subphase enzyme concentrations of 4 x 10(-8) M or less. The equilibrium constant for the reaction, 1.4 x 10(-8) mol/cm2, is independent of pH, initial composition, and surface pressure. Lecithin, if present in molar excess relative to the sum of free and esterified cholesterol, is inhibitory. Inhibition is associated with division of the substrate into reactive and unreactive pools which are not exchangeable. Bile salts and other surfactants reverse the inhibition at concentrations one-tenth their critical micelle concentrations. Presumably this occurs through formation of a surfactant surface excess at the lipid-water interface which disrupts the unreactive lecithin-substrate complex. The adsorption of cholesterol esterase to oleic acid monolayers is first order with respect to enzyme and is saturable. At saturation, the enzyme forms a close packed monolayer at the lipid-water interface with a molecular area of 4510 A2. Adsorption of cholesterol esterase to lecithin monolayers is less than one-tenth that to oleic acid monolayers and is proportional to subphase enzyme concentration. With either lipid monolayer, enzyme denaturation at the interface was negligible. In the presence of substrate, differences in enzyme absorption can only partially account for the observed inhibition of catalysis by lecithin, indicating that the reactivity or availability of substrate to the adsorbed enzyme is also affected.

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Mutants in the gene flu-2 of the free-living nematode Caenorhabditis elegans are characterised by an altered autofluorescence of the intestine cells, from the light blue of wild-type to a dull green colour. The properties of flu-2 mutants have been investigated. L-kynureninase activity has been detected in wild-type C. elegans. The flu-2 mutants have markedly reduced kynureninase activity, as predicted earlier from chromatographic analysis of tryptophan catabolites of wild-type and mutant worms. Associated with this enzymatic block, all flu-2 mutants have enhanced sensitivity to ethyl methane sulfonate (EMS) and gamma-rays.

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The microsomes of rabbit kidney medulla converted arachidonic acid into prostaglandin E2 in the presence of hemoglobin, tryptophan and glutathione as activators. When themicrosomal suspension was treated with 1% Tween 20, a solubilized enzyme was obtained which catalyzed the conversion of arachidonic acid to prostaglandins G2 and H2. The solubilized enzyme was adsorbed to and then eluted from an omega-aminooctyl Sepharose 4B column, resulting in about 10-fold purification over the microsomes. The partially purified enzyme produced predominantly prostaglandin G2 in the presence of hemoglobin, while prostaglandin H2 was produced in the presence of both hemoglobin and tryptophan. The stimulation of prostaglandin endoperoxide formation was also observed with other heme and aromatic compounds. Prostaglandin H2 synthesis was inhibited by a variety of compounds including non-steroidal anti-inflammatory drugs, thiol compounds and prostaglandin analogues with a thiol group(s).

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