Preparation of poly(-lysine) adsorbents and application to selective removal of lipopolysaccharides

To remove endotoxins (lipopolysaccharides; LPS) from cell products used as drugs, water-insoluble poly(-lysine) (PL) particles were prepared by cross-linking with PL originating from Streptomyces albulus and chloromethyloxirane (CMO). The apparent pK_a (pK_a,app) and the anion-exchange capacity of the particles were easily adjusted by changing the PL ratio and the CMO ratio. The higher the pK_a,app, the greater the LPS-adsorption capacity of the particles. On the other hand, when the PL ratio (in the particles) increased to 75 unit-mol% or higher, the adsorption of bovine serum albumin by the particles also increased, but decreased with increasing ionic strength of the buffer to μ=0.2 or higher. The adsorption of γ-globulin increased with decreasing PL ratio to 65 unit-mol% or lower. As a result, when the PL ratio was 70 unit-mol% and the pK_a,app was 6.7, the PL/CMO particles selectively removed LPS from various protein solutions that were naturally contaminated with LPS, at pH 6.0 and μ=0.05.     

Localization of immunoreactive lamprey gonadotropin-releasing hormone in the rat brain

A highly specific antiserum against lamprey gonadotropin-releasing hormone (GnRH) was used to localize 1-GnRH in areas of the rat brain associated with reproductive function. Immunoreactive 1-GnRH-like neurons were observed in the ventromedial preoptic area (POA), the region of the diagonal band of Broca and the organum vasculosum lamina terminalis, with fiber projections to the rostral wall of the third ventricle and the organum vasculosum lamina terminalis. Another population of 1-GnRH-like neurons was localized in the dorsomedial and lateral POA, with nerve fibers projecting caudally and ventrally to terminate in the external layer of the median eminence. Other fibers apparently projected caudally and circumventrically to terminate around the cerebral aqueduct in the mid-brain central gray. By using a highly specific antiserum directed against mammalian luteinizing hormone-releasing hormone (m-LHRH), the localization of the LHRH neuronal system was compared to that of the 1-GnRH system. There were no LHRH neurons in the dorsomedial or the lateral region of the POA that contained the 1-GnRH neurons. As expected, there was a large population of LHRH neurons in the ventromedial POA associated with the diagonal band of Broca and organum vasculosum lamina terminalis. In both of these regions, there were many more LHRH neurons than 1-GnRH neurons and the LHRH neurons extended more dorsally and laterally than the 1-GnRH neurons. The LHRH neurons seemed to project to the median eminence in the same areas as those that were innervated by the 1-GnRH neurons. Absorption studies indicated that 1-GnRH cell bodies were eliminated by adding 1 microg of either 1-GnRH-I or 1-GnRH-III, but not m-LHRH to the antiserum before use. Fibers were largely eliminated by the addition of 1 microg 1-GnRH-III to the antiserum. No chicken GnRH-II neurons or nerve fibers could be visualized by immunostaining. Because the antiserum recognized GnRH-I and GnRH-III equally, we have visualized an 1-GnRH system in rat brain. The results are consistent with the presence of either one or both of these peptides within the rat hypothalamus. Because 1-GnRH-I has only weak nonselective gonadotropin-releasing activity, whereas 1-GnRH-III is a highly selective releaser of follicle-stimulating hormone, and because 1-GnRH neurons are located in areas known to control follicle-stimulating hormone release selectively, our results support the hypothesis that 1-GnRH-III, or a closely related peptide, may be mammalian follicle-stimulating hormone-releasing factor.     

Reduction of pentylenetetrazol-induced convulsions by the octadecaneuropeptide ODN

Intracerebroventricular injection of the octadecaneuropeptide ODN in mouse, at doses of 12.5-1000 ng, reduced the percentage of convulsing animals and increased the latency of convulsions elicited by pentylenetetrazol (50 mg/kg, intraperitoneal [i.p.]). ODN also reduced the percentage of mortality induced by pentylenetetrazol (100 mg/kg, i.p.). The COOH-terminal octapeptide fragment of ODN was approximately equally effective but acted more rapidly than ODN to reverse the convulsant effect of pentylenetetrazol. ODN (100 ng, intracerebroventricular [i.c.v.]) increased the convulsion latency and reduced the percentage of animals that convulsed after the administration of the inverse agonist of benzodiazepine receptors DMCM (13 mg/kg, i.p.), whereas the benzodiazepine receptor antagonist flumazenil (1 mg/kg, subcutaneously) abrogated the protective effect of ODN (100 ng, i.c.v.) on pentylenetetrazol-induced convulsions. ODN (100 ng, i.c.v.) also reduced the percentage of DBA/2J mice displaying audiogenic convulsions. In contrast, ODN did not reduce the percentage of mice displaying tonic or clonic convulsions when electrical interauricular stimulations were applied. It is concluded that ODN, or more likely a proteolytic fragment derived from ODN, reduces pentylenetetrazol-induced convulsions through activation of central-type benzodiazepine receptors.     

-hydroxy- and -ketoester functionalized thrombin inhibitors

-Hydroxy- and -ketoester functionalized D-Phe-Pro-Lys tripeptides were found to be potent thrombin active site inhibitors. The ketoester derivatives were characterized by slow binding kinetics. The most potent of the series was 9 (BMS 181, 412) with an overall inhibition constant K_i^* of 0.0017 μM.     

Preparation of clear noodles with mixtures of tapioca and high-amylose starches

Ways to simulate the making of clear noodles from mung bran starch were investigated by studying the molecular structures of mung bean and tapioca starches. Scanning electron micrographs showed that tapioca starch granules were smaller than those of mung bean starch. X-ray diffraction patterns of mung bean and tapioca starch were A- and C_A-patterns, respectively. Iodine affinity studies indicated that mung bean starch contained 37% of apparent amylose and tapioca starch contained 24%. Gel permeation chromatograms showed that mung bean amylopectin had longer peak chain-length of long-branch chains (DP 40) than that of tapioca starch (DP 35) but shorter peak chain-length of short-branch chains (DP 16) than that of tapioca starch (DP 21). P-31 n.m.r. spectroscopy showed that both starches contained phosphate monoesters, but only mung bean starch contained phospholipids. Physical properties, including pasting viscosity, gel strength, and thermal properties (gelatinization), were determined. The results of the molecular structure study and physical properties were used to develop acceptable products using mixtures of cross-linked tapioca and high-amylose maize starches. Tapioca starch was cross-linked by sodium trimetaphosphate (STMP) with various reaction times, pH values, and temperatures. The correlation between those parameters and the pasting viscosity were studied using a visco/amylograph. Starches, cross-linked with 0.1% STMP, pH 11.0, 3.5 h reaction time at 25, 35, and 45°C (reaction temperature), were used for making noodles. High-amylose maize starch (70% amylose) was mixed at varying ratios (9, 13, 17, 28, 37, and 44%) with the cross-linked tapioca starches. Analysis of the noodles included: tensile strength, water absorption, and soluble loss. Noodle sensory properties were evaluated using trained panelists. Noodles made from a mixture of cross-linked tapioca starch and 17% of a high-amylose starch were comparable to the clear noodles made from mung bean starch.     

Melanin-concentrating hormone (MCH) modifies memory retention in rats

The purpose of the present study was to evaluate the possible effect of melanin-concentrating hormone (MCH) on learning and memory by using the one-trial step-down inhibitory avoidance test in rats. The peptide was infused into hippocampus, amygdala, and entorhinal cortex. MCH caused retrograde facilitation when given at 0 or 4 h post-training into hippocampus, but only at 0 h into amygdala. From these results, it seems that MCH modulates memory early after training by acting on both the amygdala and hippocampus and, 4 h after training, on the hippocampus.     

Conformation and vasoreactivity of VIP in phospholipids: effects of calmodulin

The purpose of this study was to determine the conformation and vasorelaxant effects of vasoactive intestinal peptide (VIP) self-associated with sterically stabilized phospholipid micelles (SSM) and whether calmodulin modulates both of these processes. Circular dichroism spectroscopy revealed that VIP is unordered in aqueous solution at room temperature but assumes appreciable α helix conformation in SSM. This conformational transition was amplified at 37°C and by a low concentration of calmodulin (0.1 nM). Suffusion of VIP in SSM elicited significant time- and concentration-dependent potentiation of vasodilation relative to that elicited by aqueous VIP in the in situ hamster cheek pouch (P < 0.05). This response was significantly potentiated by calmodulin (0.1 nM). Collectively, these data indicate that exogenous calmodulin interacts with VIP in SSM to elicit conformational transition of VIP molecule from a predominantly random coil in aqueous environment to α helix in SSM. This process is associated with potentiation and prolongation of VIP-induced vasodilation in the in situ peripheral microcirculation.     

Mouse pancreatic polypeptide modulates food intake, while not influencing anxiety in mice

This study was designed to investigate the effects of synthetic mouse pancreatic polypeptide (mPP) on feeding and anxiety in mice. The intracerebroventricular (ICV) injection of mPP (0.003–3 nmol) dose-dependently increased food intake. A significant increase was observed 20 min after ICV injection and continued for 4 h. The intraperitoneal (IP) injection of mPP (0.03–30 nmol) dose-dependently decreased food intake. A significant decrease was observed 20 min after IP injection and continued for 4 h. In the elevated plus maze test, the ICV injection of mPP (0.003–3 nmol) did not affect anxiety behavior. These results suggest that mPP modulates food intake and the Y4 receptor in the brain may contribute to the regulation of feeding, whereas appearing not to influence anxiety in mice.     

Differential distribution of orexin-A and orexin-B immunoreactivity in the rat brain and spinal cord

The orexins are recently identified appetite-stimulating hypothalamic peptides. We used immunohistochemistry to map orexin-A and orexin-B immunoreactivity in rat brain, spinal cord, and some peripheral tissues. Orexin-A- and orexin-B-immunoreactive cell bodies were confined to the lateral hypothalamic area and perifornical nuclei. Orexin-A-immunoreactive fibers were densely distributed in the hypothalamus, septum, thalamus, locus coeruleus, spinal cord, and near the ventricles, but absent from peripheral sites investigated. In contrast, orexin-B-immunoreactive fibers were distributed sparsely in the hypothalamus. Orexin cells are strategically sited to contribute to feeding regulation, but their widespread projections suggest that orexins have other physiological roles.     

Polymorphism,haplotype composition,and selection in the <Emphasis Type="Italic">Mhc-DRB</Emphasis> of wild baboons

General patterns of organization in the major histocompatibility complex (MHC) have been successfully explained by the model of birth-and-death evolution, but understanding why certain MHC genes are maintained together into specific haplotypes remains challenging. The haplotype configurations of the functionally important class II DR region have been described in few primates and display important interspecific variability with respect to the extent of allelic variation, the number of loci and/or combinations of loci present. Understanding the evolutionary mechanisms driving such variation is conditional upon characterizing haplotypes in new species and identifying the selective pressures acting on haplotypes. This study explores the variability of haplotype configurations in the Mhc-DRB region (exon 2) for the first time in wild non-human primates, chacma baboons (Papio ursinus). Paur-DRB haplotypes were characterized through segregation studies and linkage disequilibrium. 23 Paur-DRB sequences and 15 haplotype configurations were identified in 199 animals. The Paur-DRB exon 2 is shown to be subjected to intense positive selection and frequent recombination. An approach recently developed for human vaccine studies was used to classify Paur-DRB sequences into supertypes, based on the physico-chemical properties of amino acids that are positively selected, thus most probably involved in antigen recognition. Sequences grouped into the same supertype (thus presumably sharing antigen-binding affinities) are non-randomly distributed within haplotypes, leading to an increased individual diversity of supertypes. Our results suggest that selection favoring haplotypes with complementary sets of DRB supertypes shapes functionally tuned haplotypes in this natural baboon population.     

Diet composition of wolves<Emphasis Type="Italic">Canis lupus</Emphasis> in the northeastern Qinghai-Tibet Plateau,China

From August 1998 to August 2001, 119 wolf scats were collected from the desert in a pastoral region in northeastern Qinghai-Tibet Plateau, China. The study area is the last habitat for the critically endangered Przewalski’s gazelleProcapra przewalskii. Wolf predation was hypothesized as a cause of the endangerment of the Przewalski’s gazelle. The diet of wolf during the plant green period (June-September) and the plant withering period (October-May) were determined using three scat-analysis methods: frequency of occurrence, mass in scats and the ingested biomass obtained with the linear regression models of Weaver (1993). Limited to mammalian prey, total agreement was found between the dry weight and biomass methods, but less so between the frequency of occurrence data and other methods. Hare, yak, and small rodents were the important prey species of the wolves during the plant green period, each accounting for 33, 27, and 20%. Yak, sheep and hare were the important prey species during the plant withering period, each accounting for 53, 25, and 17%. Other food types were badger, Przewalski’s gazelle, birds, reptiles, invertebrates and plants. The presence of the livestock remains in the wolf’s scats may imply scavenging behavior by the wolves, because few cases of missing livestock were reported during the study. No notable seasonal difference in the diet composition of the wolves was found. The food niche of wolf during the plant green period (0.55 ± 0.02) was similar to that during the plant withering period (0.50 ± 0.02).     

Purification and some properties of -poly-l-lysine-degrading enzyme from Kitasatospora sp. CCTCC M205012

An -poly-l-lysine-degrading enzyme (PLD) from Kitasatospora sp. CCTCC M205012 has been purified to homogeneity by three steps of anion-exchange chromatography including DEAE-Sepharose, Source 15Q and Mono Q, with a 500-fold increase in specific activity and 40.9% yield. The PLD has a molecular mass of approximately 87.0 kDa and consists of two identical subunits with a molecular mass of 43.6 kDa. Electrophoretic shows that the PLD isoelectric point was about 7.2. The optimum temperature and pH for the PLD was 30 °C and 7.0, respectively. The PLD was deactivated by EDTA, which was indicated that the enzyme was a metallo enzyme. The activity of PLD was stimulated by Co²⁺ and inhibited by Ca²⁺ remarkably. The apparent K_m with l-lysyl-p-nitroanilide as substrate was 0.216 mM and the V_max was 0.112 mmol/min mg. The PLD was an exo-type enzyme and monomers of l-lysine were detected during the enzymatic degradation of -PL.     

-cyano-substituted analogoues of decarboxylated S-adenosylmethionine as enzyme activated, irreversible inhibitors of S-adenosylmethionine decarboxylase

A pair of -cyano analogues of decarboxylated S-adenosylmethionine (2a and 2b) were synthesized as potential enzyme activated, irreversible inhibitors of the[pyruvoyl enzyme S-adenosylmethionine decarboxylase (AdoMet-DC). Each of these analogues acts as an irreversible inactivator for ADoMet-DC from Escherichia coli (IC₅₀ values of 9 and 50 μM, respectively). These analogues also inactivate human AdoMet-DC, with K_I values of 246.6 and 7.2 μM, and k_inact values of 0.29 and 0.03 min⁻¹, respectively.     

Preparation and characterization of well defined d-erythro sphingomyelins

A simple semisynthetic procedure for the preparation of various d-erythro sphingomyelins (SPMs), differing in their acyl chains, is described. They were prepared by one-step condensation of the desired free fatty acid with sphingosyl phosphorylcholine (SPC) using dicyclohexylcarbodiimide. The d-erythro SPMs were obtained in high purity, high yields and resemble bovine brain SPM in their chromatographic behavior, infrared, circular dichroism (CD) and proton NMR (PMR) spectra as well as in their rate of hydrolysis by Staphylococcus aureus sphingomyelinase. Multilamellar vesicles can be prepared from the semisynthetic SPMs. Their thermotropic behavior is dependent mainly on the acyl chain though it is also affected by the heterogeneity of the sphingosine base composition. Intact sealed small unilamellar vesicles (SUV) cannot be prepared from a single semisynthetic saturated SPM but can be prepared from their mixtures. This acylation procedure can also be applied for preparing simple neutral glycosphingolipids. The sphingolipids prepared by this method can be used to study metabolism, enzymology and physicochemical properties of d-erythro well defined simple sphingolipids.     

Xylanase of the micromycete <Emphasis Type="Italic">Rhizopus</Emphasis> var. <Emphasis Type="Italic">microsporus</Emphasis> 595: Preparation,structural and functional characteristics,and application

Procedures for the production of endo-1,4-β-xylanase have been developed. An active producer—Rhizopus var. microsporus BKMF-595—has been chosen, and the conditions of surface and submerged cultivation, as well as the composition of the culture medium for this strain, have been optimized to ensure maximum yield of the target enzyme. Activity of xylomicrosporin Px equaled 123 U/g, while the activity of xylomicrosporin Gx equaled 25 U/cm³. Homogeneous enzyme preparations, purified 59.44-fold and 72.6-fold, have been obtained. The dependence of endo-1,4-β-xylanase catalytic activity on temperature and pH of the reaction medium has been studied. The enzyme has been shown to be most stable in the pH range 5.0–6.0 and to be thermostable. Amino acid composition and subunit structure of the enzyme were determined; the molecular masses of the subunits equaled 50 and 56 kDa. Carboxyl groups of glutamic and aspartic acid residues of the active center were experimentally shown to play an important role in catalysis. The potential of this enzyme for beer production has been demonstrated.     

Stroke: development, prevention and treatment with peptidase inhibitors

Stroke is the leading cause of long-term disability in the United States and affects more people than any other neurologic disorder. Hypertension is a major risk factor associated with stroke. Several anti-hypertensive agents have been used to treat chronic hypertension to reduce the morbidity and mortality of stroke. Previous experimental studies have shown reduced stroke mortality with angiotensin-converting enzyme (ACE) inhibitors. This review discusses the development of stroke and potential use of ACE inhibitors in prevention and treatment of this disease. Furthermore, this review focuses on current investigations aimed at cellular mechanisms involved in stroke-induced microvascular alterations.     

Adult Brugia malayi 34 kDa (BMT-5) antigen offers Th1 mediated significant protection against infective larval challenge in Mastomys coucha

We earlier reported a sizeable protection conferred by ‘mitochondria rich’ (MT) fraction of adult B. malayi and the present study was planned to locate the candidate protective molecule/s in the active fraction. The MT fraction was subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the antigen bands showing strong immune-reactivity with the resistant mastomys sera were assayed for their lymphoproliferative response using splenocytes of protected animals. Of the eight such protein bands, one sub fraction with a molecular weight of  34 kDa (BMT-5) produced utmost cellular proliferation and was therefore exploited for vaccination study. BMT-5 emulsified in Freund's adjuvant produced discernible protection causing 69 and 67% reductions in microfilaraemia and adult worm burden respectively along with sterilization of 68% of the recovered female parasites. Significant levels of filaria-specific and non-specific lymphoproliferation along with enhanced release of Th1 cytokines (TNF-α, IFN-γ and IL-2) by splenocytes were observed in the vaccinated mastomys in addition to elevated levels of antigen-specific IgG, IgG2a, IgG2b and IgA. The peritoneal macrophages of immunized animals also revealed enhanced nitric oxide production in the presence of BMT-5. The findings suggest that  34 kDa (BMT-5) molecules present in the MT fraction of adult B. malayi provided sizeable protection against infective larval challenge by generating a Th1 biased milieu in the host.     

Determination of

Four of the six electrophoretically distinguishable isoenzymes of the l-lactate dehydrogenase (EC 1.1.1.27) from potato tubers were purified from crude extracts. The isoenzymes are tetrameric and exhibit MWs around 145000. They are composed of mixtures of different subunits. Two of the isoenzymes together contain at least three, the other two together contain six different subunits indicating that the actual number of isoenzymes may be even greater than the number of electrophoretically detectable isoenzymes. Since the isoenzymes agree largely with respect to their enzymatic properties and to their primary structure as suggested from fingerprinting and amino acid analysis, it is suggested that the variation of the subunits is caused by proteolytic processing in vivo rather than by different genetic coding. The amino acid sequence of the substrate-binding region (Arg₆ peptide) shows a high homology to that of the l-lactate dehydrogenases of animals and bacteria indicating a common origin of plant, animal and bacterial enzymes.     

Subunit composition and substrate binding region of potato l-lactate dehydrogenase

Four of the six electrophoretically distinguishable isoenzymes of the l-lactate dehydrogenase (EC 1.1.1.27) from potato tubers were purified from crude extracts. The isoenzymes are tetrameric and exhibit MWs around 145000. They are composed of mixtures of different subunits. Two of the isoenzymes together contain at least three, the other two together contain six different subunits indicating that the actual number of isoenzymes may be even greater than the number of electrophoretically detectable isoenzymes. Since the isoenzymes agree largely with respect to their enzymatic properties and to their primary structure as suggested from fingerprinting and amino acid analysis, it is suggested that the variation of the subunits is caused by proteolytic processing in vivo rather than by different genetic coding. The amino acid sequence of the substrate-binding region (Arg₆ peptide) shows a high homology to that of the l-lactate dehydrogenases of animals and bacteria indicating a common origin of plant, animal and bacterial enzymes.