Effect of Antimicrobial Peptides Divergicin M35 and Nisin A on <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> LSD530 Potassium Channels

The aim of this work was to study the effect of antimicrobial peptides: divergicin M35 and nisin A on Listeria monocytogenes LSD 530 potassium (K⁺) channels: ATP-sensitive (K_ATP), calcium-activated (BK_Ca), and depolarization-activated (K_v) types. Increase on K⁺ efflux and inhibition of cellular growth were observed after adding K⁺ channel activators pinacidil, NS1619, and cromakalim to divergicin M35. Increase in K⁺ efflux from log-phase cells was about 18 ± 1.1, 11 ± 0.63, and nmol mg⁻¹ of cell dry weight (CDW) for pinacidil and NS1619, respectively, over the efflux obtained with divergicin M35 alone. Increases in K⁺ efflux obtained by adding the same K⁺ channel activators to nisin A fit a completely different profile. Divergicin M35 activates K⁺ channels, particularly of the K_v and BK_Ca types and to a lesser extent the K_ATP type, causing K⁺ efflux and consequently cell death.     

Characterization and Optimization of Orodispersible Mosapride Film Formulations

Orodispersible film (ODF) technology offers new possibilities for drug delivery by providing the advantages of oral delivery coupled with the enhanced onset of action and convenience to special patient categories such as pediatrics and geriatrics. In this study, mosapride (MOS) was formulated in an ODF preparation that can be used for treatment of patients who suffer from gastrointestinal disorders, especially difficulty in swallowing due to gastroesophageal reflux disease. Poloxamer 188 was used to solubilize MOS to allow its incorporation into the film matrix. The films were prepared by solvent-casting method using different polymer ratios of maltodextrin and hydroxypropyl methylcellulose and plasticizer levels of glycerol and propylene glycol. A D-optimal design was utilized to study the effect of polymer ratio, plasticizer type, and level on film mechanical properties, disintegration time, and dissolution rate. Statistical analysis of the experimental design showed that the increase of maltodextrin fraction and plasticizer level conferred optimum attributes to the prepared films in terms of film elasticity, film disintegration time, and MOS release rate. The ODF formulations were further tested for moisture sorption capacity, with formulations containing a higher ratio of maltodextrin and percent plasticizer showing more moisture uptake. The optimum film composition was also tested in vivo for film palatability and disintegration time. An optimized mosapride orodispersible film formulation was achieved that could be of benefit to patients suffering from gastrointestinal disorders.     

Cellulose Buccoadhesive Film Bearing Glimepiride: Physicomechanical Characterization and Biophysics of Buccoadhesion

The present study aimed to develop buccoadhesive film of glimepiride with unique combination of polymers and to investigate its effect(s) on physicomechanical parameters, drug-release, and permeation of films. Drug-polymer interaction was examined by FTIR and DSC analysis. Films were prepared by solvent casting technique and characterized for film strength (320?±?8.5 g, 28.98?±?2.00 mJ), buccoadhesive strength (28.8?±?1.37 g, 3.04?±?0.32 mJ), and tensile strength (260?±?6.88 g, 18.00?±?0.44 mJ) by new instrumental techniques. Increase in polymer concentration augmented zeta potential of polymeric matrix-mucin mixture and exhibited strong buccoadhesion (electrical theory). Buccoadhesion was also influenced by particle size (adsorption theory) and swelling (wetting theory). Erosion behavior of films was observed in swelling and SEM studies. Film GM4 exhibited 98?±?2% in vitro drug release and 85?±?8% ex vivo drug permeation in 12 h with controlled diffusion mechanism. Films were compatible with oral probiotic microorganisms. Stability studies revealed no significant (P?<?0.05) variation in physicomechanical characteristics.     

Characterization and enhanced production of enterocin HJ35 byEnterococcus faecium HJ35 isolated from human skin

A strain named as HJ35 was isolated from the skin of sixty-five men and fourteen women for acne therapy, in order to find an effective antimicrobial agent againstPropionibacterium acnes. Isolate HJ35 was identified asEnterococcus faecium based on 16 rDNA sequence and produced enterocin HJ35 having antimicrobial activities against most lactic acid bacteria,Enterococcus spp.,Staphylococcus aureus, S. epidermidis, Clostridium perfringens, some bacilli,Micrococcus flavus, Listeria monocytogenes, L. ivanovii, Escherichia coli, Pseudomonas fluorescens andPropionibacterium acnes, in the modified well diffusion method. Especially, enterocin HJ35 showed a bactericidal activity againstPropionibacterium acnes P1. The antimicrobial activity of enterocin HJ35 was disappeared completely with the use of protease XIV. But enterocin HJ35 activity is very stable at high temperature (up to 100°C for 30 min), in wide range of pH 3.0∼9.0), and by treatment with organic solvents. The apparent molecular mass of enterocin HJ35 was estimated to be approximately 4∼4.5 kDa on detection of its bactericidal activity after SDS-PAGE. In batch fermentation ofE. faecium HJ35, enterocin HJ35 was produced at the midlog growth phase, and its maximum production was obtained up to 2,300 AU/mL at the late stationary phase. By employing fed-batch fermentation, the enhanced production of enterocin HJ35 was achieved up to 12,800 AU/mL by feeding with 10 g/L glucose or 6 g/L lactate.     

Partial Characterization of Artichoke Virus M

A virus with filamentous particles 697 nm in length was isolated from artichoke plants in Southern Italy and identified as a new possible member of Carlavirus group, for which the name artichoke virus M (AVM) is suggested. AVM could not be transmitted by sap inoculation to herbaceous hosts and was always present in artichoke in mixed infections with other viruses. Virus particles had a buoyant density in CsCl of 1.31 g × cm^?3 and contained a single species of nucleic acid with an apparent size of 7.5 Kb and a single coat protein species with a mol. wt of 31,000. The virus was distantly related serologically to carnation latent and poplar mosaic carlaviruses but not to other members of the group including the recently described artichoke latent S carlavirus. Cytological alterations consisted of complex cytoplasmic inclusions composed of deranged organelles, lipid droplets and accumulations of membranes.     

Physical Characterization of a New Skin Bioadhesive Film

Physical properties (roughness, gloss, mechanical, surface topography and adhesive) of a bioadhesive film for the transdermal delivery of drugs and its interactions with a skin model surface were studied. Roughness is a measurement of the small-scale variations in the height of a physical surface. No significant differences in Ra between the “x” and “y” dimensions for both the skin model and patch were detected, due to uniformity in their production. Scanning electron microscope pictures showed small particles projected from the film. Those particles resulted in increasing roughness and surface area. For the patch, gloss values measured at 20° were 6.0 ± 0.9 and at 60°, 32.2 ± 2.2 gloss units, respectively, indicating a semi-gloss material. Concerning the mechanical properties, the tensile strength of the film resulted four- to sevenfold greater than the peel force from the model skin used, indicating the suitability of the film for skin application. The adhesion to skin model depended on the amount of water used for film application and on the elapsed time between film application and removal. Finally, the model skin that was invented by Charkoudian can be used as an alternative to costly and highly variable human skin substrates since it possesses human topography.     

TcLr35小麦中病程相关蛋白1基因的克隆及分析

根据已发表的植物病程相关蛋白1基因设计引物,利用RT-PCR技术,从被小麦叶锈菌诱导的小麦抗叶锈病基因近等基因系材料TcLr35中获得一个病程相关蛋白1基因cDNA片段,长度为489bp,3′端包含21个poly(A),暂命名为PR12。利用5′RACE技术获得了818bp的PR12全长,该基因包含495bp的开放读码框,147bp的5′非翻译区(non translated region,NTR),155bp的3′非翻译区和21bp的多聚腺苷酸尾。编码164个通读的蛋白质氨基酸序列,基因产物具有植物防御体系中病程相关蛋白SCP保守结构域,与GenBank中多个植物病程相关蛋白1基因具有较高的同源性。Southern杂交显示,该基因在小麦基因组中为单拷贝。     

Purification and Characterization of the Pasteurella Haemolytica 35 Kilodalton Periplasmic Iron-Regulated Protein

ABSTRACT

Pasteurella haemolytica serovar A1 is the causative agent of acute fibrinohemorrahgic pneumonia also known as shipping fever. Many pathogens, including P. haemolytica, survive in their respective hosts through the up-regulation of an iron acquisition system. In this study we identified, purified and characterized a 35-kDa periplasmic iron-regulated protein. The N-terminal sequence of the iron-regulated protein ANEVNVYSYRQP YLIEPMLK was identical to the deduced amino acid sequence of the ferric binding protein, FbpA, of P. haemolytica. Growth of P. haemolytica in a synthetic medium (RPMI-1640), without iron and supplemented with 50 μM 2,2' dipyridyl, facilitated the expression, isolation and purification of the native P. haemolytica FbpA.     

Cloning and Characterization of a 35-kDa Mouse Mitochondrial Outer Membrane Protein MOM35 with High Homology to Tom40

We have cloned a 35-kDa protein from a mouse cDNA library with a 25% overall amino acididentity to yTom40 and 27% identity to nTom40. This homolog to Tom40 was named MOM35.It contains two possible start codons 36 amino acids apart from each other. Both the long andthe short version of MOM35 can be imported in vitro into mouse mitochondria. The identifiedprotein is imported into the outer mitochondrial membrane and comprises a trypsin-resistancepattern similar to that of nTom40. Tom40 of N. crassa, S. cerevisiae, and the protein identifiedherein contains a highly conserved region with possible physiological importance. Subsequentinvestigation has revealed that this region interacts specifically in vitro with preproteinsproposed to be imported by a Tom40-dependent pathway.     

A Novel Itraconazole Bioadhesive Film for Vaginal Delivery: Design, Optimization, and Physicodynamic Characterization

The purpose of this work was to design and optimize a novel vaginal drug delivery system for more effective treatment against vaginal candidiasis. Itraconazole was formulated in bioadhesive film formulations that could be retained in the vagina for prolonged intervals. The polymeric films were prepared by solvent evaporation and optimized for various physicodynamic and aesthetic properties. In addition, percentage drug retained on vaginal mucosa was evaluated using a simulated dynamic vaginal system as function of time. A polymeric film containing 100 mg itraconazole per unit (2.5 cm × 2.5 cm) have been developed using generally regarded as safe listed excipients. The pH of vaginal film was found to be slightly acidic (4.90 ± 0.04) in simulated vaginal fluid and alkaline (7.04 ± 0.07) in water. The little moisture content (7.66 ± 0.51% w/w) was present in the film, which helps them to remain stable and kept them from being completely dry and brittle. The mechanical properties, tensile strength, and percentage elongation at break (9.64 N/mm² and 67.56% for ITRF₆₅) reveal that the formulations were found to be soft and tough. The films (ITRF₆₅) contained solid dispersion of itraconazole (2.5)/hydroxypropyl cellulose (1)/polyethylene glycol 400 (0.5), which was found to be the optimal composition for a novel bioadhesive vaginal formulation, as they showed good peelability, relatively good swelling index, and moderate tensile strength and retained vaginal mucosa up to 8 h. Also, the film did not markedly affect normal vaginal flora (lactobacillus) and was noncytotoxic as indicated by the negligible decrease in cell viability.     

Characterization and enzymatic degradation of Sup35NM, a yeast prion-like protein

Transmissible spongiform encephalopathies (TSEs) are believed to be caused by an unconventional infectious agent, the prion protein. The pathogenic and infectious form of prion protein, PrPSc, is able to aggregate and form amyloid fibrils, very stable and resistant to most disinfecting processes and common proteases. Under specific conditions, PrPSc in bovine spongiform encephalopathy (BSE) brain tissue was found degradable by a bacterial keratinase and some other proteases. Since this disease-causing prion is infectious and dangerous to work with, a model or surrogate protein that is safe is needed for the in vitro degradation study. Here a nonpathogenic yeast prion-like protein, Sup35NM, cloned and overexpressed in E. coli, was purified and characterized for this purpose. Aggregation and deaggregation of Sup35NM were examined by electron microscopy, gel electrophoresis, Congo red binding, fluorescence, and Western blotting. The degradation of Sup35NM aggregates by keratinase and proteinase K under various conditions was studied and compared. These results will be of value in understanding the mechanism and optimization of the degradation process.     

Structural and Functional Characterization of Reston Ebola Virus VP35 Interferon Inhibitory Domain

Ebolaviruses are causative agents of lethal hemorrhagic fever in humans and nonhuman primates. Among the filoviruses characterized thus far, Reston Ebola virus (REBOV) is the only Ebola virus that is nonpathogenic to humans despite the fact that REBOV can cause lethal disease in nonhuman primates. Previous studies also suggest that REBOV is less effective at inhibiting host innate immune responses than Zaire Ebola virus (ZEBOV) or Marburg virus. Virally encoded VP35 protein is critical for immune suppression, but an understanding of the relative contributions of VP35 proteins from REBOV and other filoviruses is currently lacking. In order to address this question, we characterized the REBOV VP35 interferon inhibitory domain (IID) using structural, biochemical, and virological studies. These studies reveal differences in double-stranded RNA binding and interferon inhibition between the two species. These observed differences are likely due to increased stability and loss of flexibility in REBOV VP35 IID, as demonstrated by thermal shift stability assays. Consistent with this finding, the 1.71-Å crystal structure of REBOV VP35 IID reveals that it is highly similar to that of ZEBOV VP35 IID, with an overall backbone r.m.s.d. of 0.64 Å, but contains an additional helical element at the linker between the two subdomains of VP35 IID. Mutations near the linker, including swapping sequences between REBOV and ZEBOV, reveal that the linker sequence has limited tolerance for variability. Together with the previously solved ligand-free and double-stranded-RNA-bound forms of ZEBOV VP35 IID structures, our current studies on REBOV VP35 IID reinforce the importance of VP35 in immune suppression. Functional differences observed between REBOV and ZEBOV VP35 proteins may contribute to observed differences in pathogenicity, but these are unlikely to be the major determinant. However, the high level of similarity in structure and the low tolerance for sequence variability, coupled with the multiple critical roles played by Ebola virus VP35 proteins, highlight the viability of VP35 as a potential target for therapeutic development.     

Antisecretory effects of galanin and its putative antagonists M15, M35 and C7 in the rat stomach.

The neuropeptide galanin has been reported to have a wide range of biological actions both in the central nervous system and in the gastrointestinal tract. Recent works led to the discovery of selective galanin receptor antagonists including M15 (galanin(1-12)-Pro-substanceP(5-11)-amide), M35 (galanin(1-12)-Pro-bradykinin(2-9)-amide) and C7 (galanin(1-12)-Pro-spantide-amide). These antagonists were shown to competitively inhibit actions of galanin in the central nervous system. The present study was designed to investigate the effect of galanin, M15, M35 and C7 on gastric acid secretion and gastric emptying. Pentagastrin-stimulated gastric acid secretion was inhibited by galanin (0.1-9 nmol x kg(-1) x h(-1), i.v.) in a dose-dependent manner (ID50 = 1.8 +/- 0.3 nmol x kg(-1) x h(-1)). When 9 nmol x kg(-1) x h(-1) galanin infusion was given, inhibition became almost complete. M15, M35 and C7 (1-9 nmol x kg(-1) x h(-1)) did not modify responses of the stomach to galanin, but acted as agonists of galanin on acid secretion. Neither galanin nor its putative antagonists affected the emptying of non-caloric liquids from the stomach. In conclusion, galanin may play an antisecretory role in the regulation of gastric acid secretion but not in the control of gastric emptying of liquids in rats. Its antisecretory action on the stomach is mediated by galanin receptors that are distinct from those in the central nervous system.     

Cloning and Characterization of the Human Lectin P35 Gene and Its Related Gene

We previously cloned a novel human lectin, designated P35, with both collagen-like and fibrinogen-like domains. P35 recognizes GlcNAc residues and is opsonic toward microorganisms. The overall structure of P35 closely resembles those of two pig ficolins that are putative TGF-β1-binding proteins. In this study, we analyzed the exon–intron structure and chromosomal location of the P35 gene as well as its structural relationship to splicing variants. In addition, we isolated another distinct genomic clone corresponding to the upstream region of a P35-related gene that has an exon organization closely resembling that of the P35 gene. The sequences of exons in the P35-related gene were identical to the cDNA sequence reported for “human ficolin.” Northern blotting revealed that the P35 gene is expressed mainly in liver, whereas the P35-related gene is expressed in lung and peripheral blood leukocytes, demonstrating tissue-specific expression of these two genes. Both genes were assigned to a closely related region of chromosome 9 at 9q34.     

Characterization of alpha-fetoprotein secreted from cultured reuber H-35 hepatoma cells

Summary Reuber H-35 hepatoma cells were examined for their ability to synthesize protein in vitro, especially to produce alpha-fetoprotein (AFP). The presence of AFP in the culture supernatant solution was determined immunologically by the micro-Ouchterlony method. Charge heterogeneity of AFP was examined electrophoretically in continuous gradient polyacrylamide microgels. With regard to the duration of culture, there was no remarkable change in the ratio of two peaks of AFP, and which came out as a major combined peak and a similar peak by PAS staining on the condition of added SDS. These findings indicated that Reuber H-35 hepatoma cells had potential to produce two charge variants of AFP in vitro. A part of this study was performed in the Division of Biofunction Research, Biomedical Research Laboratories, The Jikei University School of Medicine, with support by Grant-in-Aid for Scientific Research for 1981 of the Ministry of Education, Science and Culture of Japan.     

Characterization of three new snRNAs from Saccharomyces cerevisiae: snR34, snR35 and snR36.

Genes for three novel snRNAs of Saccharomyces cerevisiae have been isolated, sequenced and tested for essentiality. The RNAs encoded by these genes are designated snR34, snR35 and snR36 respectively and contain 203, 204 and 182 nucleotides. Each RNA is derived from a single copy gene and all three RNAs are believed to be nucleolar, i.e. snoRNAs, based on extraction properties and association with fibrillarin. SnR34 and snR35 contain a trimethylguanosine cap, but this feature is absent from snR36. The novel RNAs lack elements conserved among several other snoRNAs, including box C, box D and long sequence complementarities with rRNA. Genetic disruption analyses showed each of the RNAs to be dispensable and a haploid strain lacking all three RNAs and a previously characterized fourth snoRNA (snR33) is also viable. No differences in the levels of precursors or mature rRNAs were apparent in the four gene knock-out strain. Possible roles for the new RNAs in ribosome biogenesis are discussed.