Vibrio damsela, a Marine Bacterium, Causes Skin Ulcers on the Damselfish Chromis punctipinnis.

A previously undescribed marine bacterium, Vibrio damsela, was isolated from naturally occurring skin ulcers on a species of temperate-water damselfish, the blacksmith (Chromis punctipinnis). Laboratory infection of the blacksmith with Vibrio damsela produced similar ulcers. Vibrio damsela was pathogenic for four other species of damselfish but not for members of other families of fish. The bacterium has also been isolated from water and from two human wounds and may be a cause of human disease.

Gene-expressed RNA as a therapeutic: issues to consider, using ribozymes and small hairpin RNA as specific examples.

In recent years there has been a greater appreciation of both the role of RNA in intracellular gene regulation and the potential to use RNA in therapeutic modalities. In the latter case, RNA can be used as a therapeutic target or a drug. The chapters in this volume cover the varied and potent actions of RNA as antisense, ribozymes, aptamers, microRNA and small hairpin RNA in gene regulation, as well as their use as potential therapeutics for metabolic and infectious diseases. Our group has been involved in the development of anti-HIV gene expression constructs to treat HIV. In this chapter, we address the relevant scientific and some of the commercial issues in the use of RNA as a therapeutic. Specifically, the chapter discusses delivery, expression, potency, toxicity and commercial development using, as examples, hammerhead ribozymes and small hairpin RNA.

Plasma levels of growth-related oncogene (CXCL1-3) associated with fibrosis and platelet counts in HCV-infected patients.

BACKGROUND: Fibrosis progression in hepatitis C virus (HCV)-infected patients varies greatly between individuals. Chemokines recruit immune cells to the infected liver and may thus play a role in the fibrosis process. AIM: To investigate plasma levels of a diverse chemokine panel in relation to liver fibrosis. METHODS: African-American and Caucasian HCV genotype 1 infected patients were treated with peginterferon (pegIFN) and ribavirin (RBV) for 48 weeks (VIRAHEP-C cohort). Plasma levels of 13 cytokines were studied at baseline (n = 386). Subsequently, GROα levels were assessed in a sub cohort (n = 99) at baseline, and at 4 and 12 weeks after start of pegIFN/RBV treatment. RESULTS: Increased severity of fibrosis (Ishak fibrosis score 0-2 vs. 3-6) was associated with increased plasma IP-10 (CXCL10) and IL-8 (CXCL8) levels, and decreased plasma levels of the chemokine growth-related oncogene (GRO, CXCL1-3). Plasma GRO levels were also positively correlated with platelet counts, and were higher in African-American as compared to Caucasian patients. In response to pegIFN/RBV treatment, GROα levels increased in Caucasian but not African-American patients from week 4 onwards. CONCLUSIONS: The association with severity of fibrosis and platelet count positions plasma GRO as a potential biomarker for liver fibrosis in HCV-infected patients. The secretion of GRO by platelets may explain the correlation between GRO plasma level and platelet count. The ethnic difference in GRO levels both pre-treatment and in response to pegIFN/RBV might be driven by a genetic polymorphism in GROα associated with higher plasma levels and more common in the African-American population.

RNA as a target for host defense and anti-HIV drugs.

Viruses have many strategies for negotiating entry into cells and harnessing the cellular machinery for their propagation. The diversity of strategies is however bound by the central process of translating protein from RNA. The co-evolution of cellular responses to signature viral RNA intermediates and the counter measures employed by viruses, highlight the vulnerability of this aspect of viral replication and the potential of viral RNA as a drug target. In this review we will discuss novel efforts to target the RNA intermediates of the HIV life cycle.

A survey on the knowledge and attitudes of anaesthesia providers in the United States of America, United Kingdom and Singapore on visual experiences during cataract surgery.

BACKGROUND AND OBJECTIVE: To assess the knowledge, beliefs and attitudes of anaesthesia providers on the patients' possible intraoperative visual experiences during cataract surgery under local anaesthesia. METHODS: Anaesthesia providers from the Ophthalmic Anaesthesia Society (USA); British Ophthalmic Anaesthesia Society (UK); Alexandra Hospital, National University Hospital, Tan Tock Seng Hospital, Singapore General Hospital and Changi General Hospital (Singapore) were surveyed using a structured questionnaire. RESULTS: A total of 146 anaesthesiologists (81.6%), 10 ophthalmologists (5.6%) and 23 nurse anaesthetists (12.8%) responded to the survey. Most respondents believed that patients would experience light perception and many also felt that patients might encounter other visual sensations such as movements, flashes, colours, surgical instruments, hands/fingers and the surgeon during the surgery. A significantly higher proportion of anaesthesia providers with previous experience of monitoring patients under topical anaesthesia believed that patients might experience the various visual sensations compared to those who have not previously monitored. For both topical and regional anaesthesia, anaesthesia providers who routinely counsel their patients are (1) more likely to believe that preoperative counselling helps or (2) were previously told by patients that they could see intraoperatively and/or that they were frightened by their visual sensations. These findings were statistically significant. CONCLUSIONS: The majority of anaesthesia providers in the USA, UK and Singapore are aware that patients may experience a variety of visual sensations during cataract surgery under regional or topical anaesthesia. Those who have previously managed patients undergoing cataract surgery under topical anaesthesia are more likely to believe this compared to those who have not.

Escherichia coli mutant with elevated nicotinamide adenine dinucleotide phosphate (reduced) oxidase activity.

A slow-growing mutant of Escherichia coli with greatly elevated nicotinamide adenine dinucleotide phosphate (reduced; NADPH) oxidase activity has been isolated. The oxidase activity of the wild-type organism, normally low at pH 7.5, was increased when the assay was performed at pH 6.0. Sucrose density gradients of sonic extracts of the mutant and wild-type strains revealed several peaks of NADPH oxidase activity at pH 6.0. The parent organism had a peak of activity of high molecular weight which was absent from the mutant. The mutant strain had an activator capable of increasing the activity of all wild-type density gradient peaks, especially the one of high molecular weight. The activator was either missing or masked in the wild type. Agar gel electrophoresis of the extracts uncovered a rapidly moving band from the wild type, missing from the mutant; the material in this band had weak NADPH-diaphorase activity and strongly inhibited the activity of the mutant NADPH oxidase. It was concluded that, in wild-type E. coli, NADPH oxidase activity is regulated by a proper balance of an activator and an inhibitor. The absence of the inhibitor, as in the mutant, or the inactivation of the inhibitor at acid pH levels, results in a high level of NADPH oxidase activity. The relation of high NADPH oxidase levels and subsequent decrease of the NADPH pool to the decrease in growth rate is considered.

Aeromonas jandaei (formerly genospecies DNA group 9 A. sobria), a new sucrose-negative species isolated from clinical specimens.

A large numerical taxonomy study conducted in 1988 of 165 mostly clinical Aeromonas strains from diverse geographic sources produced a cluster (S = 84%, SSM) of four sucrose-negative strains that included the DNA definition strain for DNA group 9 A. sobria (CDC 0787-80). These four strains, together with five additional strains received in 1989, were subjected to DNA-DNA hybridization (hydroxyapatite, 32P, 60 and 75 degrees C), and all eight strains were closely related to the ninth labeled DNA group 9 definition strain CDC 0787-80 (73 to 86% relatedness at 60 degrees C and 68 to 80% relatedness at 75 degrees C; percent divergence, 2.0 to 3.5). Type strains and DNA definition strains for all other established Aeromonas species were only 35 to 72% related (60 degrees C) to CDC 0787-80. We propose the name Aeromonas jandaei for this highly related group of nine strains, formerly known as DNA group 9 A. sobria. The type strain was designated ATCC 49568 (CDC 0787-80). The nine strains were examined at 36 degrees C and were found to be resistant to 0/129 (vibriostatic agent) and uniformly positive for oxidase, gas production from glucose, indole, lysine decarboxylase, arginine dihydrolase, o-nitrophenyl-beta-D-galactopyranoside, motility (25 degrees C), nitrate reduction, citrate utilization, hemolysis on sheep blood agar, and growth in Trypticase soy broth with no added NaCl. They all fermented D-glucose, D-mannitol, and mannose but did not ferment sucrose, cellobiose, L-arabinose, inositol, salicin, or D-sorbitol. They were uniformly negative for esculin and urea hydrolysis, elastase production, ornithine decarboxylation, and the string test. The antibiogram of A. jandaei resembled that of other aeromonads (resistance to ampicillin and cephalothin), but it differed from most other aeromonads because of resistance to single dilution of colistin and differed from clinical A. veronii biogroup sorbria (formerly A. sobria) by its nearly uniform resistance to cephalothin. The esculin-, sucrose-, and cellobiose-negative and colistin-resistant profile distinguished A. jandaei from other Aeromonas species. These A. jandaei strains were isolated from blood (two strains), wounds (two strains), diarrheal stools (four strains), and a prawn (one strain). The blood and wound isolates, in particular, suggest that there is a possible clinical significance for this species and justify identification of and further research on this group of motile aeromonads.

Isolation of Ewingella americana from mollusks.

Twenty-three of 2446 strains of Enterobacteriaceae isolated from mollusks were identified as Ewingella americana both biochemically and by DNA hybridization with strain S6/1111. The biochemical characteristics of the new strains showed few differences from previously reported strains obtained from human clinical specimens. These are the first strains of E. americana isolated from animals.

Comprehensive, serologically equivalent DNA typing for HLA-B by PCR using sequence-specific primers (PCR-SSP).

Polymorphic products of HLA class I genes from the human major histocompatibility complex (MHC) are traditionally assigned by serology with additional heterogeneity detectable using one-dimensional isoelectric focusing (1D-IEF). With the increased availability of HLA class I DNA sequence information it has become feasible to genotype for class I by polymerase chain reaction utilising sequence-specific primers (PCR-SSP). We describe here a comprehensive HLA-B PCR-SSP typing system based on available HLA nucleotide sequences which can detect all serologically defined antigens in most heterozygous combination in 48 one-step PCR reactions. In addition, four new unsequenced variants have been identified. DNA samples from 57 International Histocompatibility Workshop reference cell lines and 160 control individuals have been typed by the HLA-B PCR-SSP technique. 3/57 cell line types and 12/160 normal control individuals types were discrepant with the reported serological types. The SSP system has been designed to be higher resolution than serology but is not a complete allele-specific PCR although many single alleles can be identified. The system is entirely complementary to previous published PCR-SSP systems for HLA-Class II and HLA-Class I in that the same PCR conditions and controls are used which allows us to do one step PCR-SSP for all relevant HLA loci in under 3 hours in a system suitable for the typing of cadaver donors.

Origin of post-depolarization hyperpolarizations in a grease-gap recording preparation.

In grease-gap recording preparations, depolarizing responses evoked by agonists are often followed by a hyperpolarization (post-depolarization hyperpolarization). We have investigated the origin of these post-depolarization hyperpolarizations in the rat CA1-subiculum slice. They were evoked by L-glutamate, N-methyl-d-aspartate or alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate in approximately 80% of the slices tested. The post-depolarization hyperpolarizations evoked by perfusion of N-methyl-d-aspartate through the CA1 compartment of the chamber, persisted when N-methyl-d-aspartate receptors in the subicular compartment were blocked with D-2-amino-5-phosphonopentanoate. Carbachol only blocked the post-depolarization hyperpolarizations evoked by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate at concentrations which also blocked the depolarization. The post-depolarization hyperpolarization was selectively blocked by perfusion with Ca(2+)-free medium and by administration of ouabain, and showed a marked sensitivity to temperature. It is concluded that the post-depolarization hyperpolarizations observed in this preparation are not a consequence of diffusion of the agonist through the slice. The evidence is, however, consistent with them being generated by activation of the electrogenic Na(+)-K+ pump, although we cannot exclude an additional contribution from Ca(2+)- or voltage-dependent K+ currents.

Deoxyribonucleic acid relatedness among species of Erwinia and between Erwinia species and other enterobacteria.

Relatedness in species of Erwinia was assessed by determining the extent of reassociation in heterologous deoxyribonucleic acid preparations. Thermal elution chromatography on hydroxyapatite was used to separate reassociated nucleotide sequences from nonreassociated sequences and to determine the thermal stability of related nucleotide sequences. An apparent 15% core of relatedness is present between fire blight, soft-rot, and "atypical" Erwinia species. All Erwinia species showed low to moderate reaction with representative enteric bacteria.

Interleukin-10 gene promoter polymorphism in English and Polish healthy controls. Polymerase chain reaction haplotyping using 3' mismatches in forward and reverse primers.

A polymerase chain reaction with sequence-specific primers (PCR-SSP) system using primers with mismatches at the 3' ends was developed to determine polymorphisms in IL-10 promoter region. Three previously described biallelic polymorphisms in IL-10 were linked in a 12 reaction PCR-SSP system and the method used to provide genotype data on 233 UK and 166 Polish controls. There are eight possible polymorphic combinations in IL-10 promoter gene but only three were observed in both control groups. Population frequencies of IL-10 genotypes show, in contrast to HLA, that UK and Polish frequencies are remarkably similar.

Cytokine (TNF alpha, LT alpha and IL-10) polymorphisms in inflammatory bowel diseases and normal controls: differential effects on production and allele frequencies.

The influence of biallelic polymorphisms in the tumour necrosis factor-alpha (TNF alpha), lymphotoxin-alpha (LT alpha) and interleukin-10 (IL-10) genes on stimulated TNF alpha and IL-10 production was studied in ulcerative colitis (UC) patients, Crohn's disease (CD) patients and in healthy controls. A polymerase chain reaction sequence-specific primer (PCR-SSP) system was developed to type nine biallelic polymorphisms, three in each of the TNF alpha, LT alpha and IL-10 genes. Production of the TNF alpha and IL-10 was measured by ELISA in lipopolysaccharide (LPS) stimulated whole blood. Four haplotypes of the TNF alpha gene, three haplotypes of LT alpha and three haplotypes of IL-10 were identified. No significant differences in haplotype frequencies were found between patients and controls overall. On subgroup analysis however, haplotype TNF-2 was more frequent in women with extensive colitis compared to distal colitis (31% vs 12%; P = 0.028). This difference was even greater for the combined TNF-2-LT alpha-2 haplotype (56% vs 21%; P = 0.0007). The TNF-2 and LT alpha-2 haplotypes were associated with higher TNF alpha production in CD patients, and the TNF-4 haplotype was associated with lower TNF alpha production in UC patients. The A allele in the IL-10 promoter region at position -1082 was associated with decreased IL-10 production in CD patients and controls (P = 0.005, P = 0.015 respectively). These data provide evidence that the effect of TNF alpha, LT alpha and IL-10 gene polymorphisms on cytokine production differ in CD, UC patients and controls.

Polymerase chain reaction haplotyping using 3' mismatches in the forward and reverse primers: application to the biallelic polymorphisms of tumor necrosis factor and lymphotoxin alpha.

A polymerase chain reaction with sequence-specific primers (PCR-SSP) system that operates under identical conditions to HLA phototyping was devised for characterizing polymorphisms in tumor necrosis factor (TNF) and lymphotoxin alpha (LT-alpha). Mismatches at the 3' end were incorporated into the forward and reverse primers of each PCR so as to unequivocally establish the cis/trans status between the biallelic sites. Three previously described biallelic polymorphisms in TNF and three in LT-alpha were characterized in a 24-reaction PCR-SSP system. The method was used to genotype 20 cell lines and 201 HLA class I and II typed controls from the United Kingdom at the TNF and LT-alpha loci. Population frequencies of TNF haplotypes were determined as was linkage disequilibrium with HLA-A, B, Cw, DRB1 and DQB1 loci. In each gene there were 8 theoretical polymorphic combinations; 4 were observed in TNF and 4 in LT-alpha. A total of 11 TNF-LT-alpha haplotypes were determined from apparent homozygous controls and statistical analysis.

Emended description of Buttiauxella agrestis with recognition of six new species of Buttiauxella and two new species of Kluyvera: Buttiauxella ferragutiae sp. nov., Buttiauxella gaviniae sp. nov., Buttiauxella brennerae sp. nov., Buttiauxella izardii sp. nov., Buttiauxella noackiae sp. nov., Buttiauxella warmboldiae sp. nov., Kluyvera cochleae sp. nov., and Kluyvera georgiana sp. nov.

A total of 219 strains belonging to the genera Buttiauxella and Kluyvera were studied; 171 of these strains were isolated from mollusks, mainly snails and slugs, obtained from around the world. On the basis of DNA-DNA hybridization data, the strains were grouped into 11 genomospecies. A total of 44 phenotypic characters were used to differentiate the genera Buttiauxella and Kluyvera at the genus level and to identify genomospecies. There were significantly higher phenotypic probability distances between the genomospecies in the genus Battiauxella and the genomospecies in the genus Kluyvera than between the genomospecies in the same genus. Therefore, the existence of Buttiauxella and Kluyvera as different genera was confirmed. The existence of new species necessitated broadening the definitions of both genera. In two cases, Buttiauxella species could not be quantitatively differentiated biochemically, and several other pairs of species could be separated only by the results of one biochemical test. Nonetheless, combinations of several characteristics were used in differentiate all of the species with levels of certainly ranging from log 10.79 to log 57.77 (calculated as probability distances). The following new species are proposed: Buttiauxella ferragutiae (type strain, ATCC 51602 [DSM 9390]), Buttiauxella gaviniae (type strain, ATCC 51604 [DSM 9393]), Buttiauxella brennerae (type strain, ATCC 51605 [DSM 9396]), Buttiauxella izardii (type strain, ATCC 51606 [DSM 9397]), Buttiauxella noackiae (type strain, ATCC 51607 [DSM 9401]), Buttiauxella warmboldiae (type strain, ATCC 51608 [DSM 9404]), Kluyvera cochleae (type strain, ATCC 51609 [DSM 9406]), and Kluyvera georgiana (type strain, ATCC 51603 [DSM 9409]).

Mutation leading to increased sensitivity to chromium in Salmonella typhimurium.

Corwin, L. M. (Walter Reed Army Institute of Research, Washington, D.C.), G. R. Fanning, F. Feldman, and P. Margolin. Mutation leading to increased sensitivity to chromium in Salmonella typhimurium. J. Bacteriol. 91:1509-1515. 1966.-Certain deletion mutants including the tryptophan operon in Salmonella typhimurium are unable to utilize several sugars as carbon sources in solid media, although they are able to grow in liquid media with these sugars. The addition of citrate or washing the agar with ethylenediaminetetraacetic acid permits growth on solid media. Analysis of the agar revealed that Fe(3+) and Cr(3+) were present at concentrations of 22 and 75 mum, respectively. The addition of Fe(3+) to liquid media in 0.5 mm concentrations did not inhibit the wild type or the mutants. A similar concentration of Cr(3+) did not inhibit the wild type, but concentrations as low as 0.01 to 0.05 mm inhibited the deletion mutants. Other metals were inhibitory at various concentrations, but none showed any significant differential effects on the mutants and the wild type. The increased sensitivity of the mutants to chromium may be due either to an increased permeability to Cr(3+), resulting in higher effective intracellular concentrations and inhibition of one or more metabolic functions, or to a binding of Cr(3+) to an altered cell wall, resulting in decreased permeability of required substrates.