A rat model for the energetic regulation of gonadotropin secretion: role of the glucose-sensing mechanism in the brain.

Energy availability has been considered to regulate gonadal activity by modulating the release of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) at various reproductive phases, such as lactation and puberty in domestic as well as wild animals. Experimental models with rats and sheep have demonstrated that fasting or glucoprivation suppresses pulsatile LH release. From those experiments, the information on energy deficiency is considered to be detected by specific central sensors and conveyed to the hypothalamus to regulate LH release as well as food intake. Noradrenergic neurons, originating in the medulla oblongata and projecting to the hypothalamic paraventricular nucleus (PVN), is reported to be one of the pathways mediating the response of LH release to energy deficiency. The other component is considered to be an energy-sensing mechanism in the brain. Glucose or other oxidizable fuels may function as a metabolic signal to regulate LH release. Previous studies suggest the presence of a glucose-sensing mechanism in the rat hindbrain. From our previous results in the rat, the ependymocytes lining the wall of the cerebroventricle could possibly serve as a glucose sensor to regulate GnRH/LH release. Greater understanding of the nature of the energy-sensing mechanism in the brain will contribute to the nutritional manipulation of reproductive performance in domestic animals in various conditions.

Partial characterization of an enzyme fraction with protease activity which converts the spore peptidoglycan hydrolase (SleC) precursor to an active enzyme during germination of Clostridium perfringens S40 spores and analysis of a gene cluster involved in the activity.

A spore cortex-lytic enzyme of Clostridium perfringens S40 which is encoded by sleC is synthesized at an early stage of sporulation as a precursor consisting of four domains. After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present study was undertaken to characterize GSP. In the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), a nondenaturing detergent which was needed for the stabilization of GSP, GSP activity was extracted from germinated spores. The enzyme fraction, which was purified to 668-fold by column chromatography, contained three protein components with molecular masses of 60, 57, and 52 kDa. The protease showed optimum activity at pH 5.8 to 8.5 in the presence of 0.1% CHAPS and retained activity after heat treatment at 55 degrees C for 40 min. GSP specifically cleaved the peptide bond between Val-149 and Val-150 of SleC to generate mature enzyme. Inactivation of GSP by phenylmethylsulfonyl fluoride and HgCl(2) indicated that the protease is a cysteine-dependent serine protease. Several pieces of evidence demonstrated that three protein components of the enzyme fraction are processed forms of products of cspA, cspB, and cspC, which are positioned in a tandem array just upstream of the 5' end of sleC. The amino acid sequences deduced from the nucleotide sequences of the csp genes showed significant similarity and showed a high degree of homology with those of the catalytic domain and the oxyanion binding region of subtilisin-like serine proteases. Immunochemical studies suggested that active GSP likely is localized with major cortex-lytic enzymes on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reactions.

[A case of intracerebral and subarachnoid hemorrhage of unknown origin with preceding headache].

We report a case of a 31-year-old female with multiple intracerebral hemorrhage and subarachnoid hemorrhage. She presented with headache one week before hemorrhage, and a CT scan performed at that time showed no abnormal findings. Neurological examination on admission revealed mild disturbance of consciousness, papilledema, and mild left hemiparesis. CT scans demonstrated intracerebral hemorrhage in the right caudate head and left frontal subcortex, and diffuse subarachnoid hemorrhage. Cerebral angiogram and laboratory examination revealed no abnormal findings. Erythrocyte sedimentation rate, C reactive protein and antiphospholipid antibody were within normal ranges. The patient underwent removal of hematoma by craniotomy. One week after the operation, a subcutaneous hematoma in the area of the craniotomy was found. Cerebral angiography demonstrated an aneurysm of the right superficial temporal artery, which was remote from the craniotomy. This aneurysm was surgically removed and examined. Histopathological examination revealed the presence of a pseudoaneurysm but no inflammation. Although primary angitis of the central nervous system was suspected to be the cause of this disease, a definite diagnosis could not be obtained.

The N-terminal prepeptide is required for the production of spore cortex-lytic enzyme from its inactive precursor during germination of Clostridium perfringens S40 spores.

A spore cortex-lytic enzyme of Clostridium perfringens S40 is synthesized during sporulation as a precursor consisting of four domains. After cleavage of an N-terminal preregion and a C-terminal proregion, inactive proenzyme (termed C35) is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present results demonstrated that the cleaved N-terminal prepeptide remained associated with C35. After the isolated complex was denatured and dissociated in 6 M urea solution, removal of urea regenerated a prepeptide-C35 complex which produces active enzyme when incubated with GSP. However, isolated C35 alone could not be activated by GSP. The prepeptide-C35 complex was more heat stable than active enzyme. Thus, non-covalent attachment of the prepeptide to C35 is required to assist correct folding of C35 and to stabilize its conformation, suggesting that the prepeptide functions as an intramolecular chaperone. Recombinant proteins, which have prepeptide covalently bonded to C35, were processed by GSP as well as the in vivo prepeptide-C35 complex, and the full length of the N-terminal presequence was needed to fulfil its role. Although the C-terminal prosequence is present as an independent domain which is not involved in the activation process of the enzyme, it appears that the N-terminal prosequence contributes to the regulation of enzyme activity as an inhibitor of the enzyme.

Intracerebroventricular administration of melanin-concentrating hormone suppresses pulsatile luteinizing hormone release in the female rat.

Melanin-concentrating hormone (MCH) has been reported to be involved in the regulation of feeding behaviour in rats and mice. Because many neuropeptides that influence ingestive behaviour also regulate reproductive function, the present study was designed to determine if central administration of MCH changes pulsatile secretion of luteinizing hormone (LH) in the rats. Wistar-Imamichi strain female rats were ovariectomized and implanted with oestradiol to produce a moderate inhibitory feedback effect on LH release. The effects of i. c.v. injections of MCH on LH release were examined in freely moving animals. Blood samples were collected every 6 min for 3 h through an indwelling cannula. After 1 h of sampling, MCH (0.1, 1 or 10 microg/animal) or vehicle (saline) was injected into the third cerebroventricle. Because MCH is also reported to affect the hypothalamo-pituitary-adrenal (HPA) axis, which in turn, can influence reproductive function, plasma corticosterone concentrations were determined in the same animals at 30-min intervals during the first and last hours and every 12 min during the second hour of the 3-h sampling period. When expressed as per cent changes, mean plasma LH concentrations after MCH administration were significantly lower in the animals injected with all doses of the peptide compared with vehicle-treated animals; LH pulse frequency was significantly lowered by 1 microg of MCH. Per cent changes in mean plasma corticosterone levels were not significantly affected by MCH administration. These results in oestradiol-treated ovariectomized rats indicate that central MCH is capable of inhibiting pulsatile LH secretion. We have previously shown that 48-h fasting suppresses pulsatile LH release in the presence of oestrogen. Take together, these results raise the possibility that MCH could play a role in mediating the suppression of LH secretion during periods of reduced nutrition.

Peripheral or central administration of motilin suppresses LH release in female rats: a novel role for motilin.

Motilin is secreted in a clear episodic pattern during fasting or during the interdigestive phase, but feeding promptly stops this secretory pattern, and plasma concentrations of motilin decrease. We have previously determined that fasting markedly suppresses pulsatile luteinizing hormone (LH) secretion in female rats in the presence of oestrogen. In the present study, we wished to learn if motilin may mediate the fasting-induced suppression of LH secretion by determining the effects of motilin administration on LH release and on food intake. Intravenous (i.v.) injection of motilin (37 nmol/rat) suppressed LH release and significantly decreased mean LH concentrations both in ovariectomized (OVX) and oestradiol-implanted ovariectomized (OVX+E2) rats. Food intake was significantly increased by i.v. motilin injection in OVX rats, but not in OVX+E2 rats. It is likely that motilin inhibits LH release via inhibition of the gonadotrophin-releasing hormone (GnRH)-releasing mechanism at the hypothalamic level, because motilin (3.7 nmol/rat) also suppressed LH secretion when centrally administered, and because LH release in i.v. motilin-treated rats increased in response to exogenous GnRH. These results suggest that motilin may be a peripheral signal for the suppression of LH secretion through central sensors.

Inhibitory effect of microfibril wheat bran on azoxymethane-induced colon carcinogenesis in CF1 mice.

Microfibril wheat bran (MFW), a processed dietary fiber prepared by milling of coarse wheat bran (WB), is softer and has a more pleasant taste than WB. In this study, we examined the inhibitory effect of MFW on azoxymethane (AOM)-induced colon carcinogenesis in female CF1 mice and compared its effect with that of WB and cellulose (CL). The mice were fed a modified AIN 76 A diet supplemented with either MFW, WB, or CL at a final concentration of 20% (w/w). Six weekly s.c. injections of AOM (10 mg/kg body weight) were administered per mouse commencing 1 week after the start of the feeding period. Control mice were injected with saline only. Thirty-three weeks after the initial injection, the mice were sacrificed, examined for tumors, and the cecal contents were analyzed to determine the moisture content and the concentrations of short-chain fatty acids (SCFA). The average number of total tumors per mouse in the MFW (2.9 +/- 0.6, P = 0.017) and WB (5.3 +/- 1.3, P = 0.373) diet groups was lower than that of the CL diet group (7.5 +/- 1.3), though there was no significant difference in tumor incidence (94.7%, 90.0% and 94.7%, respectively) between the groups. More than 90% of the tumors in each group were adenocarcinomas. The incidence of adenoma and that of carcinoma in situ in the MFW diet group (5.3% and 0%, respectively) were also lower than those in the CL diet group (26.3 and 26.3%, respectively; P = 0.180 and P = 0.046, respectively). Analysis of the cecal contents revealed a significantly higher moisture content and significantly higher concentrations of SCFA, butyrate in particular, in the MFW and WB diet groups. The results of this study indicate that the source and texture of dietary fiber can influence tumor development in CF1 mice, and more specifically that MFW is a promising and useful dietary supplement with properties serving to protect against the development of colon cancer.

Germination-specific cortex-lytic enzymes from Clostridium perfringens S40 spores: time of synthesis, precursor structure and regulation of enzymatic activity.

Germination-specific enzymes, an amidase and a muramidase, of Clostridium perfringens S40 were synthesized at the time of forespore formation during sporulation. The amidase had a unique precursor structure consisting of four domains: the N-terminal pre-sequence, the N-terminal pro-sequence, mature enzyme and the C-terminal pro-sequence. The N-terminal pre-sequence and the C-terminal pro-sequence were sequentially processed at the time of development of phase-bright spores, and the resulting inactive pro-enzyme was activated by cleavage of the N-terminal pro-sequence with a specific protease during germination. A possible mechanism for the regulation of activity of muramidase, which is produced as a mature form and does not need processing for activation, is presented.

Expression of a germination-specific amidase, SleB, of Bacilli in the forespore compartment of sporulating cells and its localization on the exterior side of the cortex in dormant spores.

A germination-specific amidase of bacilli is a major spore-lytic enzyme that is synthesized with a putative signal sequence and hydrolyses spore cortex in situ. The sleB gene encoding this amidase in Bacillus subtilis and Bacillus cereus was expressed in the forespore compartment of sporulating cells under the control of sigmaG, as shown by Northern blot and primer extension analyses. The forespore-specific expression of B. subtilis sleB was further indicated by the forespore-specific accumulation of a SleB-green fluorescent protein fusion protein from which a putative secretion signal of SleB was deleted. Immunoelectron microscopy with anti-SleB antiserum and a colloidal gold-immunoglobulin G complex showed that the enzymes from both Bacillus species are located just inside the spore coat layer in the dormant spore, and in the dormant spore, the amidases appear exist in a mature form lacking a signal sequence. These results indicate that SleB is translocated across the forespore's inner membrane by a secretion signal peptide and is deposited in cortex layer synthesized between the forespore inner and outer membranes. The peripheral location of the spore-lytic enzymes in the dormant spore suggests that spore germination is initiated at the exterior of the cortex.

Effect of band 3 subunit equilibrium on the kinetics and affinity of ankyrin binding to erythrocyte membrane vesicles.

The membrane-spanning protein, band 3, anchors the spectrin-based membrane skeleton to the lipid bilayer via the bridging protein, ankyrin. To understand how band 3 subunit stoichiometry influences this membrane-skeletal junction, we have induced changes in the band 3 association equilibrium and assayed the kinetics and equilibrium properties of ankyrin binding. We observe that band 3 oligomers convert slowly to dimers and ultimately monomers following removal of ankyrin. Addition of excess ankyrin back to these membranes enriched in dissociated band 3 then shifts band 3 almost entirely to tetramers, confirming that the tetrameric form of band 3 constitutes the preferred oligomeric state of ankyrin binding. 4, 4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) labeling of band 3, which is shown to shift most of the band 3 population to dimers, eliminates the majority of ankyrin-binding sites on the membrane and greatly reduces retention of band 3 in detergent-extracted membrane skeletons. Furthermore, DIDS- modified membranes lack all low affinity ankyrin-binding sites and roughly half of all high affinity sites. Since labeled membranes lack the rapid kinetic phase of ankyrin binding and exhibit only half of the normal amplitude of the slow kinetic phase, it can be concluded that the rapid phase of ankyrin association involves low affinity sites and the slow phase involves high affinity sites. A model accounting for these data and most previous data on ankyrin-band 3 interactions is provided.

A cysteine-dependent serine protease associated with the dormant spores of Bacillus cereus: purification of the protein and cloning of the corresponding gene.

Subtilisin-like serine protease, which is associated with the dormant spores of Bacillus cereus, was solubilized by washing the spores with 2 M KCl and purified to homogeneity by carbobenzoxy-D-phenylalanine-liganded affinity column chromatography and hydrophobic interaction column chromatography. Enzyme activity was completely inhibited by reagents for sulfhydryl groups such as HgCl2 as well as by conventional subtilisin inhibitors, suggesting the enzyme to be cysteine-dependent. The enzyme retained activity in 5 M urea at 4 degrees C for at least 2 months, and the specific activity was 50 times that of subtilisin BPN when measured for a common chromogenic substrate, carbobenzoxy-glycyl-glycyl-L-leucine p-nitroanilide. The gene encoding this protease was cloned in Escherichia coli, and its nucleotide sequence was analyzed. The deduced amino acid sequence suggested that the protease is produced as a precursor comprising three portions; a signal sequence (28 amino acid residues), a prosequence (80 amino acid residues) and a mature enzyme (289 amino acid residues). The mature region of the enzyme had high similarity with a thermitase from Thermoactinomyces vulgaris (72% identity) and a thermostable alkaline protease from Thermoactinomyces sp. E79 (66% identity), which have the N-terminal sequence showing scarcely noticeable similarity with corresponding stretches of subtilisins and mercuric ion-sensitive free cysteine in the equivalent position of the primary structure.

Localization of germination-specific spore-lytic enzymes in Clostridium perfringens S40 spores detected by immunoelectron microscopy.

The localization of germination-specific spore-lytic enzymes, an amidase and a muramidase, in Clostridium perfringens S40 spores was examined by immunoelectron microscopy with respective antisera raised against the enzymes and a colloidal gold-immunoglobulin G complex. For both antisera, immunogold particles were visualized on the outside of the cortex of dormant spores, and they were not detected in germinated spores and decoated spores.

Molecular characterization of a germination-specific muramidase from Clostridium perfringens S40 spores and nucleotide sequence of the corresponding gene.

The exudate of fully germinated spores of Clostridium perfringens S40 in 0.15 M KCI-50 mM potassium phosphate (pH 7.0) was found to contain another spore-lytic enzyme in addition to the germination-specific amidase previously characterized (S. Miyata, R. Moriyama, N. Miyahara, and S. Makino, Microbiology 141:2643-2650, 1995). The lytic enzyme was purified to homogeneity by anion-exchange chromatography and shown to be a muramidase which requires divalent cations (Ca2+, Mg2+, or Mn2+) for its activity. The enzyme was inactivated by sulfhydryl reagents, and sodium thioglycolate reversed the inactivation by Hg2+. The muramidase hydrolyzed isolated spore cortical fragments from a variety of wild-type organisms but had minimal activity on decoated spores and isolated cell walls. However, the enzyme was not capable of digesting isolated cortical fragments from spores of Bacillus subtilis ADD1, which lacks muramic acid delta-lactam in its cortical peptidoglycan. This indicates that the enzyme recognizes the delta-lactam residue peculiar to spore peptidoglycan, suggesting an involvement of the enzyme in spore germination. Immunochemical studies indicated that the muramidase in its mature form is localized on the exterior of the cortex layer in the dormant spore. A gene encoding the muramidase, sleM, was cloned into Escherichia coli, and the nucleotide sequence was determined. The gene encoded a protein of 321 amino acids with a deduced molecular weight of 36,358. The deduced amino acid sequence of the sleM gene indicated that the enzyme is produced in a mature form. It was suggested that the muramidase belongs to a separate group within the lysozyme family typified by the fungus Chalaropsis lysozyme. A possible mechanism for cortex degradation in C. perfringens S40 spores is discussed.

A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to L-alanine-mediated germination.

The Bacillus subtilis sleB gene, which codes for the enzyme homologous to the germination-specific amidase from Bacillus cereus, was cloned and its nucleotide sequence was determined. Sequence analysis showed that it had an open reading frame of 918 bp, coding for a polypeptide of 305 amino acids with a putative signal sequence of 29 residues. Enzyme activity was not found in germination exudate of B. subtilis spores, which differs from the case of B. cereus enzyme. A B. subtilis mutant with an insertionally inactivated sleB gene revealed normal behavior in growth and sporulation. However, the sleB mutant was unable to complete germination mediated by L-alanine.

A germination-specific spore cortex-lytic enzyme from Bacillus cereus spores: cloning and sequencing of the gene and molecular characterization of the enzyme.

A gene (sleB) encoding a 24-kDa germination-specific spore cortex-lytic enzyme, probably an N-acetylmuramyl-L-alanine amidase, was cloned from Bacillus cereus, and its nucleotide sequence was determined. It was indicated that the enzyme is produced as a 259-residue protein with a signal sequence of 32 residues and is present in dormant spores in its active form. Sulfhydryl reagents inactivated the enzyme, but mutation of a single cysteine of the protein, Cys-258, to Gly did not cause complete inactivation of the enzyme, suggesting that the residue does not function as the catalytic center of enzyme.

Two family G xylanase genes from Chaetomium gracile and their expression in Aspergillus nidulans.

With oligonucleotides based on the amino-terminal and internal amino-acid sequences of a xylanase, two xylanase genes, cgxA and cgxB, were isolated and sequenced from Chaetomium gracile wild and mutant strains. Each gene isolated from both strains was essentially the same as far as nucleotide sequences were compared. The mature CgXA and CgXB xylanases comprise 189 and 211 amino acids, respectively, and share 68.5% homology. The CgXA was found to be the major enzyme in the mutant strain. Comparison of these amino-acid sequences with xylanase sequences from other origins showed that they have a high degree of identity to the family G xylanases. The cgxA and cgxB genes were introduced into Aspergillus nidulans and found to be expressed with their own promoters.

A gene (sleC) encoding a spore-cortex-lytic enzyme from Clostridium perfringens S40 spores; cloning, sequence analysis and molecular characterization.

Antiserum was raised against a 31 kDa spore-cortex-lytic enzyme, which is released during germination of Clostridium perfringens S40 spores. Western blotting of dormant spore and vegetative cell fractions separated by SDS-PAGE indicated that the 31 kDa enzyme is spore-specific and that the enzyme in the dormant spore exists as a 36 kDa protein which has no cortex-lytic activity. A gene encoding the 31 kDa enzyme, sleC, was cloned into Escherichia coli using a synthetic oligonucleotide as a hybridization probe and the nucleotide sequence of the entire gene was determined. The N-terminal amino acid sequence of the 36 kDa protein was found in this reading frame, confirming that the 36 kDa protein is a pro-form of the 31 kDa enzyme. The deduced amino acid sequence indicated that the 31 kDa enzyme is produced as a precursor, comprising three portions; an N-terminal prepro-sequence (114 amino acid residues), a pro-sequence (35 amino acid residues) and a mature enzyme (289 amino acid residues). It is suggested that the 36 kDa pro-enzyme is non-covalently attached to the exterior of the cortex layer, and that the proform is processed to release the active enzyme during germination.

Partial characterization of the cytoplasmic domain of human kidney band 3.

The major anion exchanger in type A intercalated cells of the cortical and medullary collecting ducts of the human kidney is a truncated isoform of erythrocyte band 3 (AE1) that lacks the N-terminal 65 residues. Because this missing sequence has been implicated in the binding of ankyrin, protein 4.1, several glycolytic enzymes, hemoglobin, and hemichromes in erythrocytes, we have undertaken examination of the structure and peripheral protein interactions of this kidney isoform. The cytoplasmic domain of kidney band 3, kidney CDB3, was expressed in Escherichia coli and purified to homogeneity. The kidney isoform exhibited a circular dichroism spectrum and Stokes radius similar to its larger erythrocyte counterpart. Kidney CDB3 was also observed to engage in the same conformational equilibrium characteristic of erythrocyte CDB3. In contrast, the tryptophan and cysteine clusters of kidney CDB3 behaved very differently from erythrocyte CDB3 in response to pH changes and oxidizing conditions. Furthermore, kidney CDB3 did not bind ankyrin, protein 4.1, or aldolase, and expression of erythrocyte CDB3 was toxic to its bacterial host, whereas expression of kidney CDB3 was not. Taken together, these data suggest that the absence of the N-terminal 65 amino acids in kidney CDB3 eliminates the major function currently ascribed to CDB3 in erythrocytes, i.e. that of peripheral protein binding. The primary function of residues 66-379 found in kidney CDB3 thus remains to be elucidated.

Local structural difference between human and bovine band 3 in the anion transport inhibitor-binding region.

We have examined molecular properties of inhibitor-complexed human and bovine band 3, an anion transport protein of erythrocyte membrane, in order to demonstrate the structural characteristics of the inhibitor binding region. Band 3 modified with DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonate), a potent anion transport inhibitor, generated a positive circular dichroic band at a wavelength of 345 nm, corresponding to a DIDS chromophore. The dichroic spectra of human band 3-DIDS complex and its bovine counterpart differed markedly in their ellipticity. Under the conditions that H2DIDS (the dihydro-derivative of DIDS) cross-linked two chymotryptic fragments of human band 3, the reagent failed to cross-link the equivalent bovine fragments. The inhibitory effect of PLP (pyridoxal 5'-phosphate), a substrate and affinity label, on phosphate influx into red blood cells was more pronounced for human band 3 than for bovine band 3. The residue Lys-562 of human band 3 was found to be modified with PLP, while the corresponding residue of bovine band 3 was devoid of reactivity with PLP.