UV Raman spectroscopy of hydrocarbons.

In this paper, the UV Raman spectra of a large number of saturated and alkyl-substituted monocyclic, bicyclic and polycyclic aromatic hydrocarbons are obtained at 220 and 233 nm excitation wavelengths. Also included are nitrogen- and sulphur-containing hydrocarbons. The spectra obtained are fluorescence free, even for such highly fluorescent compounds as perylene, consistent with earlier reports of UV Raman spectra of hydrocarbons. The hydrocarbon UV Raman spectra exhibit greatly improved signal-to-noise ratio when in the neat liquid or solution state compared with the neat solid state, suggesting that some surface degradation occurs under the conditions used here. Assignments are given for most of the bands and clear marker bands for the different classes of hydrocarbons are readily observable, although their relative intensities vary greatly. These results are discussed in the context of structure and symmetry to develop a consistent, molecular-based model of vibrational group frequencies.

Fourier transform Raman spectroscopy of Syncrude Sweet Blend distillation fractions: the 200-1800 cm-1 region.

The fingerprint (200-1800 cm(-1)) region in FT-Raman spectra of Syncrude Sweet Blend (SSB) and its three constituent distillation fractions (naphtha, light gas oil and heavy gas oil) was analyzed in detail in this study. Approximately 50 bands were observed and assigned to functional groups in saturated (alkanes) and unsaturated (aromatics) species. Characteristic bands for mono-, bi-, and tricyclic aromatics were identified and used to quantify these groups in SSB and the distillation fractions. Total aromatics content was determined using the carbon-carbon stretching bands in the 1600-cm(-1) region, and shown to agree with earlier results obtained from the C-H stretching region. The bands due to mono- and bicyclic aromatics permitted calculation of the relative abundances of these species with an accuracy equivalent to that obtained using NMR spectroscopy, a traditional method for measuring this quantity.

Evaluation of a patient-held record for patients with cancer.

The objective of this study was to evaluate a patient-held record (PHR) for patients with cancer. A randomized controlled trial (RCT) was conducted of a PHR to be used by patients newly diagnosed with lung or colorectal cancer (hospital), patients with cancer at any stage (community) and professionals involved in their care, together with surveys of health professionals to gauge views on PHR. Main outcome measures were patient satisfaction with information and communication, and patient and healthcare professionals' views of PHR. The only significant difference was 86% of control compared with 58% of intervention patients were very satisfied with information received at the end of treatment (odds ratio 4.4, 95% confidence interval 1.2-15.6, P < 0.05). Fifty-three per cent of intervention respondents found the PHR helpful (63% hospital vs. 38% community patients), and 69% felt that it would be useful to them in the future. Primary healthcare (PHC) professionals found the PHR of more benefit than those working in hospitals (P < 0.05). The PHR did not improve measures of patient satisfaction with information or communication. Despite its limited use by many health professionals, the PHR was well received by recently diagnosed patients, and those who did not receive negative responses to it from staff involved in their care. It was also positively valued by staff in PHC. An evaluation of a customized record provided at the time of diagnosis is warranted.

Identification of Escherichia coli O157:H7 and other enterohemorrhagic serotypes by EHEC- hlyA targeting, strand displacement amplification, and fluorescence polarization.

Human disease caused by enterohemorrhagic E. coli O157:H7 and other serotypes (EHEC) has been associated with bovine fecal contamination of food and the environment. The range of serotypes, low infectious dose, and numerous transmission vehicles for EHEC render development of detection methods for this pathogen complex. In this study, the hemolysin gene (EHEC- hly A) was targeted with oligonucleotides, and probe-target hybrids were amplified using strand displacement amplification (SDA). Amplicons were resolved in the complete reaction mix through changes in the fluorescence polarization (FP) of a fluorescein-labeled detector probe hybridized to the amplicons during amplification. Results combining EHEC- hly A, SDA, and FP were obtained within 35 min of reaction initiation. The test specificity was determined on EHEC strains representing 13 serotypes (49 isolates); and control uropathogenic, commensal, and other organisms (10 isolates). Statistical analysis of results indicated a sensitivity in the reaction vessel to 4.3 bacteria (95% confidence interval), and a specificity for EHEC (n=59) at 100% (P=5.11E-17; i.e. P<<0.05). Detection based on combining EHEC- hly A, SDA, and FP was compatible with water sources directly associated with human infection (drinking and recreational supplies), and bovine drinking trough water representing an environmental matrix linked to the maintenance of an EHEC animal reservoir.

Fourier transform Raman spectroscopy of Syncrude sweet blend distillation fractions derived from Athabasca bitumen.

The C-H stretching region in FT-Raman spectra of Syncrude sweet blend (SSB) and three distillation fractions (naphtha, light gas oil and heavy gas oil) was analyzed in detail in this investigation. The frequencies and intensities of the 11 aliphatic and three aromatic C-H bands used to fit the spectrum of SSB were equal to the averages (weighted sums) of the corresponding quantities in the spectra of the fractions. The additivity of the spectra, thought to be a consequence of the large number of discrete compounds contained in each fraction, makes it possible to estimate the composition of other SSB samples using the spectra of the fractions reported in this work. In the aromatic C-H region, total intensities can be used to calculate the distribution of aromatics among the distillation fractions; these data also permit calculation of the fractional aromaticity (per cent aromatic carbon) for SSB and each fraction, with accuracies comparable to those obtained using NMR spectroscopy.

Purification and characterization of a cytotonic protein expressed In vitro by the live cholera vaccine candidate CVD 103-HgR.

Cholera vaccines developed by the deletion of CTX genes from Vibrio cholerae induce a residual reactogenicity in up to 10% of vaccinees. A novel cytotonic agent named secreted CHO cell elongating protein (S-CEP) was purified from culture supernatants of CVD 103-HgR (Levine et al., Lancet ii:467-470, 1988). Five fractionation steps yielded electrophoretically pure S-CEP with an M(r) of 79,000. A partially purified preparation caused fluid accumulation in the sealed infant mouse model. The amino terminus bore a unique sequence with strong homology to a cytotonic toxin of El Tor V. cholerae.

Identification of a CHO cell-elongating factor produced by Vibrio cholerae O1.

Vibrio cholerae strains with all known toxin genes deleted or inactivated still cause diarrhoea in some volunteers, suggesting the presence of an unknown virulence factor or factors. Lysozyme-EDTA treated cells of JBK70, a genetically manipulated cholera toxin negative strain of Vibrio cholerae O1, biotype El Tor, release a factor that causes elongation of Chinese hamster ovary (CHO) cells. CHO cell-elongating toxin (Cef) was purified by FPLC chromatography (anion exchange; Q Sepharose High Performance) followed by 2D electrophoresis (isoelectric focusing gel, IEF; pH 3-9 and SDS-PAGE, 8-25% gradient gel). Partly purified toxin (anion exchange or IEF-eluted concentrate) caused fluid accumulation in sealed infant mice suggesting that Cef shows some properties of an enterotoxin. On SDS-PAGE (8-25%) and IEF (pH 2.5-5.0) gels, CHO cell activity was associated with a single band at 85 kDa and a pI of 3.8, respectively. A unique amino terminal sequence, XGDETNSSGASTEVVYESYIQQ, was determined by automated Edman degradation of gel-purified protein. The unique molecular mass, N-terminal sequence and activity on CHO cells indicate that this factor is not zonula occludens toxin (Zot) or accessory cholera enterotoxin (Ace) or the Hly A haemolysin. Partly purified Cef did not increase cyclic AMP or prostaglandin E(2)levels in CHO cells which suggests that its mechanism of action differs from that of cholera toxin.

Characterization of a 20-kDa pilus protein expressed by a diarrheogenic strain of non-O1/non-O139 Vibrio cholerae.

A diarrheogenic strain of non-O1/non-O139 Vibrio cholerae (10,325) belonging to serogroup O34 was earlier shown to express a new type of pilus composed of a 20-kDa subunit protein. Amino-terminal sequence data (determined up to 20 amino acid residues) of this protein showed it to be different from the subunit proteins of other known types of pili of V. cholerae. On the other hand, it showed complete homology with the corresponding sequence of a 22-kDa outer membrane protein (OmpW) of V. cholerae. Expression of 10,325 pili was favored in AKI rather than in NB medium and at 30 degrees C rather than at 37 degrees C. Further, cultural conditions favoring pilus expression also enhanced autoagglutination and adherence properties of strain 10,325. An antiserum to the 20-kDa protein induced passive protection against challenge with the parent organism 10,325, but not against V. cholerae O1 strains. Such protection was shown to be mediated by inhibition of intestinal colonization in vivo.

Developing a funding model for an after-hours primary medical care service in a rural town.

The study described in this paper aimed to determine a funding model for an after-hours primary medical care service in the rural town of Moe, a socioeconomically disadvantaged area of Victoria suffering the rigours of industry restructuring and privatisation. It has 12.5 equivalent full-time general practitioners servicing 21,966 persons. A break-even analysis of the financial viability compared the expected costs of providing the service with the anticipated income. A mixed funding model is recommended. This would incorporate a general practitioner incentive scheme and State Government underwriting of infrastructure and basic non-medical staffing costs during the business development phase to supplement the income from the Health Insurance Commission.

Biochemical and physiological characteristics of HlyA, a pore-forming cytolysin of Vibrio cholerae serogroup O1.

Among the various toxins produced by the bacterial species Vibrio cholerae is HlyA, a cytolytic protein commonly called the E1 Tor hemolysin. HlyA is synthesized and processed in a complex manner involving various processed or degraded forms, that may co-purify and complicate the interpretation of biochemical and physiological experiments. In this study a single form of HlyA was purified by gel filtration and chromatofocusing using fast protein liquid chromatography in the presence of protease inhibitors. A 45-fold purification was obtained, with a final recovery of 17% of pure 60,000 mol. wt HlyA. A significant improvement in specific activity to 8.5 x 10(6) Chinese hamster ovary tissue culture units per mg protein was obtained. Physiological activity studies indicated that cytolysis of erythrocytes (hemolysis) was inhibited by oxygen: storage of HlyA under oil, and experimentation in N2-flushed buffers maintained activity. HlyA-mediated lysis of human erythrocytes was characterized by a significant lag phase, followed by a rapid induction of hemolysis. Hemolysis was inhibited by sucrose, an osmotic protectant, suggesting that the initial action of HlyA on erythrocytes is to raise the basal cation permeability of the cell membrane. The most likely cytolytic mechanism is thus the formation of transmembrane lesions such as homopolymer pores in target cells, as has been found for toxins from numerous other bacterial pathogens.

A search for cholera toxin (CT), toxin coregulated pilus (TCP), the regulatory element ToxR and other virulence factors in non-01/non-0139 Vibrio cholerae.

Twenty-four selected non-O1/non-O139 Vibrio cholerae strains were examined for the presence of virulence associated genes like ctxA, tcpA, toxR and the repetitive sequence (RS element). Seventeen of these were isolated from diarrhoeal stool samples while the remaining seven were of local environmental origin. Nine and four respectively of these strains were positive for ctxA and tcpA by Multiplex PCR analysis. The majority (16 out of 18 tested) of the strains (including the four tcpA + strains) contained toxR sequences as determined by another PCR assay. The presence of RS element was demonstrable in ctxA+ strains only. Interestingly, three of these non-O1/non-O139 strains were shown to contain all the three virulence associated genes (ctxA, tcpA and toxR) as well as the RS element. Two of these belonged to serogroups 037 (V2) and 064 (CG15) while the third one (V315-1) was untypable. These three strains also produced cholera toxin, expressed toxin coregulated pilus (TCP) and/or TcpA related antigens when grown under appropriate culture conditions. Southern hybridization analysis of their chromosomal DNA fragments using DNA probes representing ctxA, zot, ace and RS element revealed that the strains V2 and CG15 contained, at least, two complete copies of the CTX genetic element, while the strain V315-1 had three or more copies of the same. Presence of the RS element in these strains led to tandem duplication of the CTX genetic element in the chromosome of V2 and V315-1, but not in CG15 where the copies were likely to be present at different loci. These results also indicate the presence of additional copies of incomplete "core region' with zot and ace genes, but not ctxA, in strains V2 and CG15. The significance of these results in terms of the pathogenic and epidemic potential of V. cholerae strains is discussed.

Membrane attack induced by HlyA, a pore-forming toxin of Vibrio cholerae.

Determining the activity of purified toxins has generally provided the basis for establishing their role in the host-pathogen relationship. The bacterial genus Vibrio produces a number of exotoxins in addition to cholera toxin, including haemolysin A (HlyA; Vibrio cholerae) and thermostable direct haemolysin (TDH; Vibrio parahaemolyticus), both of which possess membrane-targeting cytolytic activity. The action of HlyA has been analyzed using protocols previously applied to TDH: lysis and flux experiments on human erythrocytes showed that HlyA similarly causes lysis after cell swelling (by colloid osmosis) due to an elevation of cation permeability. However, kinetic measurements of flux, haemolysis and cation selectivity showed that HlyA and TDH form pores with distinct and characteristic features.

SB 203347, an inhibitor of 14 kDa phospholipase A2, alters human neutrophil arachidonic acid release and metabolism and prolongs survival in murine endotoxin shock.

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 fatty acyl group [predominantly arachidonic acid (AA)] of membrane phospholipids, the products of which are further metabolized, forming a variety of eicosanoids and/or platelet-activating factor. PLA2 activity is significantly enhanced during inflammation and therefore offers an intriguing target in designing anti-inflammatory drugs. SB 203347 (2-[2-[3,5-bis (trifluoromethyl) sulfonamido]-4- trifluoromethylphenoxy] benzoic acid) potently inhibits rh type II 14 kDa PLA2 (IC50 = 0.5 microM) but exhibits a 40-fold weaker inhibition of 85 kDa PLA2 (IC50 = 20 microM) using [3H]-AA E. coli as substrate. A specific interaction with rh type II 14 kDa PLA2 was confirmed both by observing the pH dependence of its IC50 and by demonstrating linear inhibition in a "scooting" kinetic model using radiolabeled phospholipid reporter substrate in a 1,2-dimyristoyl phosphatidylmethanol vesicle. Before evaluating the effect of SB 203347 on AA metabolism in intact human neutrophil, we showed that it fully inhibits PLA2 activity in acid extracted-intact human neutrophil homogenate (IC50 = 4.7 microM). SB 203347 inhibited A23187-induced intact human neutrophil AA mass release in a concentration-dependent manner (IC50 = 1 microM), which coincided with reductions in the biosynthesis of platelet-activating factor (IC50 = 1.5 microM) and leukotriene B4 (IC50 = 2.3 microM). Finally, SB 203347 prolonged survival in a mouse model of endotoxin shock delivered i.p. Taken together, the data support a role of cellular 14 kDa PLA2 in the formation of AA-derived proinflammatory lipid mediator.(ABSTRACT TRUNCATED AT 250 WORDS)

Vibrio cholerae non-O1 serogroup associated with cholera gravis genetically and physiologically resembles O1 E1 Tor cholera strains.

Until recently, only Vibrio cholerae strains of the O1 serogroup have been associated with epidemic cholera. In December 1992, an outbreak of cholera gravis in Vellore, India, was attributed to a new serogroup of V. cholerae recently designated O139. Serogroup O139 cholera has since spread to 13 countries and has reached pandemic proportions. Serogroup O139 cholera evades immunity to O1 cholera and is not detected by the standard O1 antigen test. Understanding the origins of O139 cholera and determining the relatedness of O139 to O1 cholera are necessary to device strategies for detecting, reporting, and controlling this new pandemic. In order to determine the origins of this novel cholera serogroup, O139 was analyzed for virulence genes, for virulence proteins and their regulation, and for its genomic background. We found that O139 and O1 V. cholera strains of the E1 Tor biotype possess highly homologous virulence genes encoding cholera toxin and toxin-coregulated pili and that the regulation of virulence protein expression likewise was indistinguishable between O139 and O1. Pulsed-field gel electrophoresis (PFGE) revealed the restriction digest pattern of O139 strains to be closely related to that of O1 serogroup E1 Tor biotype cholera strains from the Indian subcontinent. However, PFGE showed minor differences among individual O139 cholera isolates, suggesting that O139 V. cholerae is evolving.

Cation flux studies of the lesion induced in human erythrocyte membranes by the thermostable direct hemolysin of Vibrio parahaemolyticus.

Vibrio parahaemolyticus, an important agent of seafood-borne gastroenteritis, expresses several putative virulence factors that could account for the disease symptoms of infected humans, namely, diarrhea, nausea, and abdominal cramps. The pathogenicity of V. parahaemolyticus correlates well with the Kanagawa phenomenon (the hemolytic ability of strains grown on Wagatsuma blood agar), implicating the thermostable direct hemolysin (TDH) as the predominant toxin responsible for pathogenicity. TDH-induced hemolysis could be inhibited by the addition of the osmolyte sorbitol to the extracellular solution, supporting the hypothesis that hemolysis occurs through colloid osmosis secondary to an increase in the cation permeability of the membrane. The effect of TDH on cation permeability was investigated by measuring K+ (congener, 86Rb+) influx into human erythrocytes in which the endogenous cation transporters had been blocked (by use of ouabain, bumetanide, and nitrendipine). TDH increased K+ influx into these cells; this increase was rapid in onset and constant in magnitude, suggesting a direct action by TDH on the membrane. The kinetics of leak generation were examined; the relationship between counts accumulated and hematocrit indicated that the TDH-induced lesion is multihit in nature. TDH-induced K+ influx was sensitive to Zn2+. Time courses of hemolysis in isosmotic solutions of monovalent cation chlorides were used to obtain the selectivity series for the TDH-induced leak: Cs+ > Li+ > K+ > Rb+ > Na+. Both the Zn2+ sensitivity and this selectivity series were obtained for crude culture supernatants, suggesting that TDH is the predominant leak-inducing agent. Thus, we have identified several features of the TDH-induced leak likely to be important in the diarrhetic action of V. parahaemolyticus in the human intestine.

Substrate specificities and properties of human phospholipases A2 in a mixed vesicle model.

Studies of the specificity of phospholipases A2 (PLA2s) for different substrates have usually been carried out in vesicles or mixed micelles, where differences in shape, size, or charge of vesicles formed with different phospholipids may give misleading results. Another factor is binding of the enzyme to the phospholipid surface, which has recently been addressed using vesicles of an anionic phospholipid, dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) to which some extracellular PLA2s were shown to bind with a very high affinity (Jain, M. K., and Berg, O. G. (1989) Biochem. Biophys. Acta 1002, 127-156). In the present report we have used a similar system to study the substrate preferences of two human PLA2s that are thought to be physiologically relevant in the metabolism of arachidonic acid: a recombinant form of the human synovial fluid (14 kDa) PLA2 and the cytosolic (85 kDa) PLA2 found in monocytic cells. It is shown that both human enzymes bind tightly to DMPM vesicles and follow the basic characteristics of processive hydrolysis in this model using analysis of progress curves and substrate competition experiments. Mixed vesicles containing DMPM with small amounts (3-5 mol%) of other phospholipids have been used to study the substrate selectivity of the two human isoenzymes. The synovial fluid PLA2 shows a clear preference (approximately 7-fold) for sn-glycero-3-phosphoethanolamine over sn-glycero-3-phosphocholine. Within glycerophosphocholines, this enzyme displays little preference for the sn-2 fatty acyl group, and a slight preference for phospholipids with sn-1-acyl versus sn-1-alkyl substituents. In contrast, the cytosolic PLA2 shows a marked selectivity for arachidonoyl in the sn-2 position and only minor differences in selectivity for the polar head group in the sn-3 position. This enzyme does not distinguish between sn-1-acyl and sn-1-alkyl subclasses of glycerophosphocholines.

Further consideration of the clonal nature of Salmonella typhi: evaluation of molecular and clinical characteristics of strains from Indonesia and Peru.

We examined envelope protein profiles, chromosomal restriction endonuclease digest patterns, and immune responses to envelope proteins for collections of Salmonella typhi strains isolated in Peru and Indonesia. Only minor differences in envelope protein patterns were apparent among strains. Strains from 7 of 20 Indonesian patients had a distinct chromosomal digest pattern compared with patterns of Peruvian and other Indonesian strains. Strains with this pattern carried the gene for the j flagellar antigen (H1-j); differences in response to envelope proteins of j and d strains were noted on immunoblot analysis. Our data suggest that there are genotypic and phenotypic differences among S. typhi strains. The clinical importance of these differences remains to be fully evaluated; however, in this study it was not possible to show a clear correlation between strain characteristics and disease severity.