Indian J Med Res, 1997 Jun, 105, 254 - 7 Isolation of Vibrio cholerae 0139 phages to develop a phage typing scheme; Chakrabarti AK et al.; Five V . cholerae 0139 phages isolated from different parts of India have been used for phage typing study . A strain isolated from Nagpur city (NPR-4) was used as the host for phage propagation . All but one of the 260 strains of V . cholerae 0139 were found to be typeable and could be clustered into 8 distinct phage types as revealed by lytic patterns . Phage type 1 was the predominant type (61.15%) followed by type 2 (18.46%) . The strains isolated from Madras exhibited 7 out of 8 phage types . These newly isolated phages could be adopted for phage typing of V . cholerae 0139 strains as an epidemiological tool. Glycoconj J, 1997 Jun, 14(4), 433 - 8 Synthesis of the dodecasaccharide fragment representing the O-polysaccharide of Vibrio cholerae O:1, serotype Ogawa, bearing an aglycon offering flexibility for chemical linking to proteins; Ogawa Y et al.; Two azidohexasaccharide building blocks, of which the glycosyl acceptor was the 5-(methoxycarbonyl)pentyl glycoside, were coupled using the trichloroacetimidate technology . The 12 azido functions present in the dodecasaccharide thus formed were then converted to amino groups using hydrogen sulfide as a reducing reagent . Subsequent N-acylation with 4-O-benzyl-L-glycero-tetronic acid, followed by catalytic debenzylation yielded the desired spacer-equipped, title dodecasaccharide. Immunobiology, 1997 Jun, 197(1), 82 - 96 Leukosialin (CD43) is proteolytically cleaved from stimulated HMC-1 cells; Weber S et al.; Leukosialin (CD43), the major sialoprotein on circulating leukocytes, has been previously described to be down-regulated on neutrophils following activation with phorbol myristate acetate (PMA) . The other single cells previously examined, blood lymphocytes, do not down-regulate CD43 when stimulated by PMA . Recently, we have characterized leukosialin on the human mast cell line HMC-1 and observed that leukosialin is down-regulated after stimulation with PMA . In the present study, we have investigated the mechanism of PMA-mediated down-regulation of CD43 on HMC-1 cells (subclone 5C6) . PMA caused the release of soluble leukosialin (123 kD) during HMC-1 cell activation . The molecular weight of soluble leukosialin was nearly identical to that of the cell-membrane bound molecule, suggesting a cleavage proximal from the cell membrane . Inhibitors of serine proteases, like phenylmethylsulphonyl fluoride (PMSF), benzamidine and 3, 4-dichloroisocoumarin, blocked the PMA-mediated cleavage of CD43 . In all experiments, the inhibition of CD43-down-regulation was dependent on the concentration of protease inhibitors . Treatment of HMC-1 cells with various proteases (trypsin, alpha-chymotrypsin, elastase, papain, nagarse) substantially decreased anti-CD43 binding capacity and caused the release of soluble leukosialin (116 kD) or its fragments into the supernatant . Pretreatment of HMC-1 cells with neuraminidases from Vibrio cholerae or Arthrobacter ureafaciens resulted in an increased sensitivity of CD43 against proteases, whereas the effects of PMA were not influenced . In conclusion, proteolytic cleavage of CD43 is described for the first time in a cell other than neutrophils, namely HMC-1 cells . Our results suggest that serine proteases are involved in the PMA-mediated down-regulation of leukosialin on HMC-1 cells. Vet Immunol Immunopathol, 1997 Jun, 57(1-2), 147 - 52 Immunostimulating effect of growth hormone: in-vivo administration of growth hormone in rainbow trout enhances resistance to Vibrio anguillarum infection; Sakai M et al.; In-vivo administration of purified (nGH) and recombinant (rGH) chum salmon, Oncorhynchus keta, growth hormone enhanced the survival of rainbow trout, O . mykiss, against virulent Vibrio anguillarum . Macrophages obtained from rainbow trout that were treated in vivo with either GH preparation showed enhanced stimulation of the chemiluminescent responses induced by opsonized V . anguillarum cells and increased phagocytic activities . However, the serum bactericidal activity against V . anguillarum was not enhanced in fish injected with nGH or rGH . These findings show that in-vivo administration of either GH preparation can effectively prime macrophages and increase the resistance to V . anguillarum in rainbow trout. Comp Biochem Physiol B Biochem Mol Biol, 1997 Jun, 117(2), 273 - 86 A heterogeneous sialic acid-binding lectin with affinity for bacterial LPS from horse mussel (Modiolus modiolus) hemolymph; Tunkijjanukij S et al.; A sialic acid-binding lectin that agglutinates a variety of erythrocytes and bacteria and react with sialoconjugates and purified lipopolysaccharides from marine vibrios has been affinity purified from hemolymph of the horse mussel Modiolus modiolus using Bovine submaxillary mucin conjugated to CNBr-activated Sepharose 4B . The lectin demonstrated heterogeneous activity, and at least two main entities were partially characterized, and are referred to as modiolin H and modiolin E activities for the agglutination of human and horse (equine) erythrocytes, respectively . Only modiolin E activity required calcium ions for hemagglutination . The M . modiolus lectin was mainly specific for NeuAc, although the lectin demonstrated a broader range of specificity, similarly to the Limulus polyphemus lectin . The purified lectin was a glycoprotein, and in the native state existed as aggregates with M(r) in the range of 100-1,300 kDa as observed by gradient-gel electrophoresis and gel filtration on Biogel and Superose . SDS-PAGE under reducing conditions revealed three subunits of M(r) 14, 17.5 and 20 kDa . Various marine bacteria adsorbed the hemagglutinating activities of the M . modiolus lectin . Purified LPS preparations from various pathogenic marine vibrios were also effective inhibitors, in particular for modiolin E activity . These results indicate that the lectin play a role in recognition of bacteria. Mol Microbiol, 1997 Jun, 24(5), 917 - 26 Regulation, replication, and integration functions of the Vibrio cholerae CTXphi are encoded by region RS2; Waldor MK et al.; CTXphi is a filamentous phage that encodes cholera toxin, one of the principal virulence factors of Vibrio cholerae . CTXphi is unusual among filamentous phages because it can either replicate as a plasmid or integrate into the V . cholerae chromosome at a specific site . The CTXphi genome has two regions, the 'core' and RS2 . Integrated CTXphi is frequently flanked by an element known as RS1 which is related to RS2 . The nucleotide sequences of RS2 and RS1 were determined . These related elements contain three nearly identical open reading frames (ORFs), which in RS2 were designated rstR, rstA2 and rstB2 . RS1 contains an additional ORF designated rstC . Functional analyses indicate that rstA2 is required for CTXphi replication and rstB2 is required for CTXphi integration . The amino terminus of RstR is similar to the amino termini of other phage-encoded repressors, and RstR represses the expression of rstA2 . Although genes with related functions are clustered in the genome of CTXphi in a way similar to those for other filamentous phages, the CTXphi RS2-encoded gene products mediating replication, integration and repression appear to be novel. Epidemiol Infect, 1997 Jun, 118(3), 221 - 5 Vibrio vulnificus infection reporting on death certificates: the invisible impact of an often fatal infection; Banatvala N et al.; This study assessed accuracy of (a) recording Vibrio vulnificus infection on death certificates and (b) International Classification of Disease (ICD)-9 codes for V . vulnificus . Patients with microbiologically confirmed V . vulnificus infection were identified as part of co-ordinated surveillance in four USA Gulf Coast states between 1989 and 1993 . Of 60 deaths, 51 death certificates were reviewed and V . vulnificus was recorded as the immediate cause of death on 11 (22%) . There was no ICD-9 code for V . vulnificus infection, thus no patients had an ICD-9 code indicating V . vulnificus infection . Of 23 certificates where V . vulnificus was recorded on the death certificate, only 5 (22%) were coded for Gram-negative, septicaemia . This study highlights the importance of teaching physicians how to provide epidemiologically meaningful data on death certificates and the need for accurate ICD mortality codes. Epidemiol Infect, 1997 Jun, 118(3), 207 - 14 Epidemic cholera among refugees in Malawi, Africa: treatment and transmission; Swerdlow DL et al.; Between 23 August and 15 December 1990 an epidemic of cholera affected Mozambican refugees in Malawi causing 1931 cases (attack rate = 2.4%); 86% of patients had arrived in Malawi < 3 months before illness onset . There were 68 deaths (case-fatality rate = 3.5%); most deaths (63%) occurred within 24 h of hospital admission which may have indicated delayed presentation to health facilities and inadequate early rehydration . Mortality was higher in children < 4 years old and febrile deaths may have been associated with prolonged i.v . use . Significant risk factors for illness (P < 0.05) in two case-control studies included drinking river water (odds ratio {OR} = 3.0); placing hands into stored household drinking water (OR = 6.0); and among those without adequate firewood to reheat food, eating leftover cooked peas (OR = 8.0) . Toxigenic V . cholerae O1, serotype Inaba, was isolated from patients and stored household water . The rapidity with which newly arrived refugees became infected precluded effective use of a cholera vaccine to prevent cases unless vaccination had occurred immediately upon camp arrival . Improved access to treatment and care of paediatric patients, and increased use of oral rehydration therapy, could decrease mortality . Preventing future cholera outbreaks in Africa will depend on interrupting both waterborne and foodborne transmission of this pathogenPIP: An epidemiologic investigation of a 1990 cholera outbreak among Mozambican refugees in the Nyamithuthu camp in Malawi highlighted the challenges of providing adequate treatment and prevention in this setting . Between August 23 and December 15, 1990, 1931 cholera patients were admitted to the camp's intravenous (IV) treatment tent (attack rate, 2.4%); 28% were under 6 years of age . There were 68 deaths among these patients, for a case-fatality rate of 3.5% . 84% of patients for whom data were available had come to Malawi less than 3 months before the onset of illness and 52% were admitted for treatment within 16 days of camp arrival . 60% of the 40 cholera deaths investigated in detail involved children under 4 years of age (17% of total cases) . Acute dehydration was the most common cause of death among the 63% who died within 24 hours of IV tent admission, suggesting delayed presentation and inadequate early rehydration . The remaining patients died from complications (e.g., infections with fever caused by prolonged IV use) . In 2 case-control studies, cholera was significantly associated with placing hands into the storage container holding household drinking water (odds ratio, 6.0), obtaining drinking water from the river (odds ratio, 3.0), and eating leftover unheated cooked peas (odds ratio, 8.0) . Toxigenic Vibrio cholerae O1, serotype Inaba, was isolated from patients and stored household water . Increased water rations and running water during cholera outbreaks are recommended to reduce contamination of stored drinking water during washing . More rapid referral to IV tents, administration of oral rehydration solution in addition to IV, quick removal or replacement of IV lines to prevent infection, and more attention to child cases also would reduce cholera mortality . Microbiology, 1997 Jun, 143 ( Pt 6), 1805 - 13 VlpA of Vibrio cholerae O1: the first bacterial member of the alpha 2-microglobulin lipocalin superfamily; Barker A et al.; We have identified a gene, vlpA, which is closely linked to the mfrA,B locus associated with mannose-fucose-resistant haemagglutination . VlpA is an outer-membrane protein which can be labelled with {3H}palmitate and whose processing is globomycin-sensitive, suggesting that it is a lipoprotein . Homology searches revealed that VlpA belongs to the group of lipocalins of the alpha 2-microglobulin superfamily which function as small hydrophobic molecule transporters, and is the first identified bacterial member of this group . Multiple copies of this gene are present in Vibrio cholerae O1 and O139 and Southern hybridization reveals a biotype-specific pattern of fragment sizes . Construction of strains capable of hyperproducing VlpA suggested that it is able to bind haemin with low affinity but this may be due to a simple hydrophobic interaction . Attempts to construct specific mutants in vlpA have been unsuccessful, presumably because of the multiple copies of vlpA genes and their linkage to the VCR element. J Bacteriol, 1997 Jun, 179(12), 4043 - 5 Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi; Bassler BL et al.; Different species of bacteria were tested for production of extracellular autoinducer-like activities that could stimulate the expression of the luminescence genes in Vibrio harveyi . Several species of bacteria, including the pathogens Vibrio cholerae and Vibrio parahaemolyticus, were found to produce such activities . Possible physiological roles for the two V . harveyi detection-response systems and their joint regulation are discussed. Appl Environ Microbiol, 1997 Jun, 63(6), 2464 - 7 Distribution of Vibrio vulnificus phage in oyster tissues and other estuarine habitats; DePaola A et al.; Phages lytic to Vibrio vulnificus were found in estuarine waters, sediments, plankton, crustacea, molluscan shellfish, and the intestines of finfish of the U.S . Gulf Coast, but no apparent relationship between densities of V . vulnificus and its phages was observed . Phage diversity and abundance in molluscan shellfish were much greater than in other habitats . V . vulnificus phages isolated from oysters did not lyse other mesophilic bacteria also isolated from oysters . Both V . vulnificus and its phages were found in a variety of oyster tissues and fluids with lowest densities in the hemolymph and mantle fluid . These findings suggest a close ecological relationship between V . vulnificus phages and molluscan shellfish. Appl Environ Microbiol, 1997 Jun, 63(6), 2200 - 5 Photoreactivation compensates for UV damage and restores infectivity to natural marine virus communities; Weinbauer MG et al.; We investigated the potential for photoreactivation to restore infectivity to sunlight-damaged natural viral communities in offshore (chlorophyll a, < 0.1 microgram liter-1), coastal (chlorophyll a, ca . 0.2 microgram liter-1), and estuarine (chlorophyll a, ca . 1 to 5 micrograms liter-1) waters of the Gulf of Mexico . In 67% of samples, the light-dependent repair mechanisms of the bacterium Vibrio natriegens restored infectivity to natural viral communities which could not be repaired by light-independent mechanisms . Similarly, exposure of sunlight-damaged natural viral communities to > 312-nm-wavelength sunlight in the presence of the natural bacterial communities restored infectivity to 21 to 26% of sunlight-damaged viruses in oceanic waters and 41 to 52% of the damaged viruses in coastal and estuarine waters . Wavelengths between 370 and 550 nm were responsible for restoring infectivity to the damaged viruses . These results indicate that light-dependent repair, probably photoreactivation, compensated for a large fraction of sunlight-induced DNA damage in natural viral communities and is potentially essential for the maintenance of high concentrations of viruses in surface waters. J Bacteriol, 1997 Jun, 179(11), 3808 - 12 Identification of a second endogenous Porphyromonas gingivalis insertion element; Wang CY et al.; In this study a second endogenous Porphyromonas gingivalis insertion element (IS element) that is capable of transposition within P . gingivalis was identified . Nucleotide sequence analysis of the Tn4351 insertion site in a P . gingivalis Tn4351-generated transconjugant showed that a complete copy of the previously unidentified IS element, designated PGIS2, had inserted into IS4351R in Tn4351 . PGIS2 is 1,207 bp in length with 19-bp imperfect terminal inverted repeats, and insertion resulted in a duplicated 10-bp target sequence . Results of Southern hybridization of chromosomal DNA isolated from several P . gingivalis strains with a PGIS2-specific probe demonstrated that the number of copies of PGIS2 per genome varies among different P . gingivalis strains . Computer analysis of the putative polypeptide encoded by PGIS2 revealed strong homologies to the products encoded by IS1358 from Vibrio cholerae, ISAS1 from Aeromonas salmonicida, and H-rpt in Escherichia coli K-12. J Bacteriol, 1997 Jun, 179(11), 3594 - 603 The plasmid R64 thin pilus identified as a type IV pilus; Kim SR et al.; The entire nucleotide sequence of the pil region of the IncI1 plasmid R64 was determined . Analysis of the sequence indicated that 14 genes, designated pilI through pilV, are involved in the formation of the R64 thin pilus . Protein products of eight pil genes were identified by the maxicell procedure . The pilN product was shown to be a lipoprotein by an experiment using globomycin . A computer search revealed that several R64 pil genes have amino acid sequence homology with proteins involved in type IV pilus biogenesis, protein secretion, and transformation competence . The pilS and pilV products were suggested to be prepilins for the R64 thin pilus, and the pilU product appears to be a prepilin peptidase . These results suggest that the R64 thin pilus belongs to the type IV family, specifically group IVB, of pili . The requirement of the pilR and pilU genes for R64 liquid mating was demonstrated by constructing their frameshift mutations . Comparison of three type IVB pilus biogenesis systems, the pil system of R64, the toxin-coregulated pilus (tcp) system of Vibrio cholerae, and the bundle-forming pilus (bfp) system of enteropathogenic Escherichia coli, suggests that they have evolved from a common ancestral gene system. Infect Immun, 1997 Jun, 65(6), 2475 - 9 The lipopolysaccharide O side chain of Vibrio vulnificus serogroup E is a virulence determinant for eels; Amaro C et al.; Vibrio vulnificus is a gram-negative bacterium capable of producing septicemic infections in eels and immunocompromised humans . Two biotypes are classically recognized, with the virulence for eels being specific to strains belonging to biotype 2, which constitutes a homogeneous lipopolysaccharide (LPS)-based O serogroup (which we have designated serogroup E) . In the present study we demonstrated that the O side chain of this LPS determines the selective virulence of biotype 2 for eels: (i) biotype 1 strains (which do not belong to serogroup E) are destroyed by the bactericidal action of nonimmune eel serum (NIS) through activation of the alternative pathway of complement, (ii) biotype 2 strains (of serogroup E) are resistant to NIS, and (iii) rough mutants of biotype 2 lacking the O polysaccharide side chain are sensitive to NIS and avirulent for eels. Infect Immun, 1997 Jun, 65(6), 2127 - 35 Coexpression of the B subunit of Shiga toxin 1 and EaeA from enterohemorrhagic Escherichia coli in Vibrio cholerae vaccine strains; Butterton JR et al.; A promoterless gene for the Shiga toxin 1 B subunit (stxB1) has been placed under transcriptional control of the Vibrio cholerae heat shock gene htpG . A chromosomal enterohemorrhagic Escherichia coli fragment containing eaeA and 400 bp of upstream DNA was added to the construct, downstream of stxB1; no transcription terminators were located between the two genes . The plasmid construct was confirmed by DNA sequencing; in vitro transcription-translation studies demonstrated expression of EaeA from the plasmid . The htpGp-->stxB1, eaeA construct was inserted into lacZ on the chromosome of Peru2, an El Tor V . cholerae strain with both attRS1 sequences and the entire cholera toxin genetic element deleted, and into lacZ in JRB10, a Peru2 derivative that has a second copy of htpGp-->stxB1 also inserted in the V . cholerae virulence gene irgA . Two plasmid constructs, one containing stxB1 under the control of the tac promoter and another containing htpGp-->stxB1,eaeA, were transformed into Peru2 . Expression of StxB1 by these constructs was quantified by enzyme-linked immunosorbent assay and was highest in the plasmid construct with stxB1 under the control of the tac promoter . Localization of EaeA to the outer membrane of the vector strains was demonstrated both by Western blotting and by immunofluorescence with an anti-EaeA antibody . A rabbit model for colonization by V . cholerae was used to compare the immune responses to the two heterologous antigens, StxB1 and EaeA, expressed by these strains . Rabbits immunized with Peru2 transformed with a plasmid carrying tac-->stxB1 developed neutralizing serum anti-StxB1 immunoglobulin G antibody responses . One of two rabbits immunized with a strain carrying a chromosomal copy of eaeA developed a marked immune response against EaeA . The plasmid construct containing htpGp-->stxB1,eaeA was unstable, producing low levels of StxB1 in vitro and not evoking anti-EaeA antibody responses in vivo following oral immunization . Chromosomal insertion of eaeA may be preferred for future expression of this antigen in V . cholerae vaccine constructs. Infect Immun, 1997 Jun, 65(6), 2107 - 11 Comparison of alternative buffers for use with a new live oral cholera vaccine, Peru-15, in outpatient volunteers; Sack DA et al.; During development of Peru-15, a new live oral vaccine for cholera, the role of buffer needed to be evaluated . Generally, oral bacterial vaccines are acid labile and need to be administered by using a formulation which protects them from gastric acid . We compared three different buffers for use with Peru-15, including a standard bicarbonate-ascorbic acid buffer, Alka-Seltzer, and a new electrolyte-rice buffer, CeraVacx . Saline served as the control . Thirty-nine healthy adult volunteers received Peru-15 (10(8) CFU) with one of the three buffers or saline in a double-masked study . The volunteers were monitored for symptoms for 7 days after the dose, serum was tested for antibody responses by vibriocidal antibody and immunoglobulin G antitoxin enzyme-linked immunosorbent assays, and stool samples were tested for excretion of the vaccine strain . Side effects were minimal in all groups . All 30 volunteers who took Peru-15 with a buffer showed significant rises in vibriocidal antibody titer . The magnitude of the rises was higher in the CeraVacx group than in the other two buffer groups . Four of nine volunteers who took the vaccine with saline also showed increased titers, but they were lower than those in any of the three buffer groups . Excretion of the vaccine strain was similar in the buffer groups, but excretion was not associated with the magnitude of the vibriocidal responses . Excretion of Peru-15 was not detected in the saline group . We conclude that buffer does amplify the serological response to Peru-15 and that CeraVacx may provide benefits not provided by other buffers. J Clin Microbiol, 1997 Jun, 35(6), 1633 - 5 Rapid detection of Vibrio cholerae O139 Bengal from stool specimens by PCR; Albert MJ et al.; In a previous study using pure bacterial cultures in a PCR assay, a primer pair corresponding to a unique chromosomal region of Vibrio cholerae O139 Bengal generated an amplicon from only V . cholerae O139 Bengal . PCR with the same primer pair was used to screen 180 diarrheal stool specimens . All the 67 V . cholerae O139 culture-positive stool specimens were positive by PCR, and the remaining specimens, which contained either other recognized enteric pathogens or no pathogens, were all negative by PCR. Biochim Biophys Acta, 1997 May 24, 1360(3), 277 - 82 A mutant cell line resistant to Vibrio parahaemolyticus thermostable direct hemolysin (TDH): its potential in identification of putative receptor for TDH; Tang G et al.; Thermostable direct hemolysin (TDH), a pore-forming toxin produced by Vibrio parahaemolyticus, is cytotoxic to Rat-1, a fibroblast cell line derived from rat embryo . Through mutagenesis of Rat-1 with nitrosoguanidine, we established a mutant cell line, MR-T1 . MR-T1 was over 200 times more resistant to the cytotoxic activity of TDH than Rat-1 . TDH increased membrane permeability of Rat-1 but not of MR-T1 . Binding analysis showed that, while being able to bind to Rat-1 . TDH failed to bind to MR-T1, indicating that MR-T1 is deficient in the putative receptor for TDH . Somatic hybrid cells between Rat-1 and MR-T1 were similarly sensitive to TDH as Rat-1 . Moreover, TDH could bind to the hybrid cells as well as to Rat-1 cells . These results indicate that MR-T1 is promising for complementation cloning of a gene related to the putative receptor for TDH. Biochim Biophys Acta, 1997 May 23, 1339(2), 247 - 52 Structure of membrane glutamate carboxypeptidase; Rawlings ND et al.; Membrane glutamate carboxypeptidase (mGCP) hydrolyses pteroylpoly-gamma-glutamates, methotrexate tri-gamma-glutamate and N-acetyl-aspartyl-alpha-glutamate . The enzyme is thought to be required for intestinal uptake of folate, for the resistance of some tumours to methotrexate, and for the metabolism of N-acetyl-aspartyl-glutamate, an abundant neuropeptide . It has recently been reported that mGCP is a protein also known as prostate-specific membrane antigen, homologous with transferrin receptor . This allows us to predict the domain structure of mGCP . Moreover, we have been able to assign the catalytic domain of mGCP to peptidase family M28, which contains cocatalytic zinc metallopeptidases . On the basis of the known structure of an aminopeptidase in family M28, we predict that Asp377, Asp387, Glu425, Asp453 and His553 are ligands of two atoms of zinc bound in the catalytic site of mGCP, and suggest that the aminopeptidases of Vibrio and Streptomyces can serve as valuable models in the design of inhibitors for this medically important enzyme. Carbohydr Res, 1997 May 19, 300(4), 329 - 39 Synthesis of methyl alpha-glycosides of some higher oligosaccharide fragments of the O-antigen of Vibrio cholerae O1, serotype Inaba and Ogawa; Zhang J et al.; The title oligosaccharides, the tri- through the hexasaccharide in the Inaba series and the penta- and the hexasaccharide in the Ogawa series, have been synthesized using 1-thioglycosides of precursors to 3-O-benzyl-perosamine (4-amino-4,6-dideoxy-D-mannose) as building blocks and N-iodosuccinimide/silver triflate as a promoter . The azido groups in the assembled oligosaccharides were reduced to amino groups, which were then acylated using 2,4-O-benzylidene-3-deoxy-L-glycero-tetronic acid as the derivatizing reagent . Catalytic hydrogenolysis, simultaneously of the benzyl and benzylidene groups, gave the desired products that were characterized by 1H and 13C NMR spectroscopy. FEMS Microbiol Lett, 1997 May 15, 150(2), 289 - 96 Analysis of functional domains of Vibrio parahaemolyticus thermostable direct hemolysin using monoclonal antibodies; Tang G et al.; Neutralizing monoclonal antibodies (mAbs) against Vibrio parahaemolyticus thermostable direct hemolysin (TDH) were used in probing the functional domains of this toxin . While pre-incubation of TDH with mAb 2A-13C inhibited further binding of TDH to erythrocytes, pre-incubation with another mAb 1-24 did not, indicating that mAb 1-24 epitope resides in a domain which is not involved in binding of TDH to erythrocytes . On the other hand after binding to erythrocytes, TDH could react with mAb 1-24 but poorly with mAb 2A-13C, indicating that the mAb 2A-13C epitope is masked, possibly by erythrocyte surface . As both antibodies are TDH-specific and do not react with TRH (TDH-related hemolysin), we used TDH/ TRH chimeric proteins to identify location of the epitopes for mAbs by inhibition ELISA as well as Western blotting . The results showed that the mAb 1-24 epitope resides on a region near the C-terminal of TDH (residues 99-139), while the mAb 2A-13C epitope resides on the N-terminal (residues 1-31) . All these results suggested that, in TDH, the N-terminal region may be involved in binding process while the region near C-terminal may be involved in postbinding process. FEMS Microbiol Lett, 1997 May 15, 150(2), 249 - 54 El Tor hemolysin of Vibrio cholerae O1 forms channels in planar lipid bilayer membranes; Ikigai H et al.; We investigated the channel formation by El Tor hemolysin (molecular mass, 65 kDa) of Vibrio cholerae O1 biotype El Tor in planar lipid bilayers . The El Tor hemolysin channel exhibited asymmetric and hyperbolic membrane current with increasing membrane potential, meaning that the channel is voltage dependent . The zero-current membrane potential measured in KCI solution showed that permeability ratio PK+/PCl- was 0.16, indicating that the channel is 6-fold more anion selective over cation . The hemolysin channel frequently flickered in the presence of divalent cations, suggesting that the channel spontaneously opens and closes . These data imply that the El Tor hemolysin damages target cells by the formation of transmembrane channels and, consequently, is the cause of osmotic cytolysis. Kansenshogaku Zasshi, 1997 May, 71(5), 417 - 20 {Incidence of Kanagawa phenomenon-positive and -negative Vibrio parahaemolyticus strains isolated from traveller's diarrhea and their relation to tdh and trh genes}; Suzuki N et al.; A total of 1,319 strains of Vibrio parahaemolyticus isolated from traveller's diarrhea were analysed for Kanagawa phenomenon (KP) with the Wagatsuma blood agar test and the results were also compared with those of analyses of tdh and trh genes which encode thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh) . The majority of the strains (1,152 strains) counting 87.3% had positive KP, among which 1,049 and 103 strains were only tdh and both tdh and trh-positive ones, respectively . However, 167 strains counting 12.7%, which is quite high compared to the previous report, were found to have negative KP, among which 94 and 24 strains were only trh and both tdh and trh-positive ones, respectively. Am J Trop Med Hyg, 1997 May, 56(5), 533 - 7 Volunteer studies investigating the safety and efficacy of live oral El Tor Vibrio cholerae O1 vaccine strain CVD 111; Tacket CO et al.; A live oral cholera vaccine should ideally protect against both classical and El Tor biotypes of Vibrio cholerae O1 . An El Tor biotype vaccine strain, therefore, would complement classical cholera vaccine strain CVD 103-HgR, a strain already in use in some countries . In this study, 25 healthy adult volunteers received a single dose of 10s colony-forming units of El Tor vaccine strain CVD 111, a derivative of El Tor Ogawa strain N16117 deleted in the virulence cassette . Three (12%) volunteers developed mild diarrhea (mean stool volume = 813 ml) but no systemic symptoms; 23 (92%) of the 25 volunteers developed serum vibriocidal antibodies (geometric mean titer = 1:2,291) . Five weeks after vaccination, 18 vaccines and eight uninimunized control volunteers underwent wild-type challenge with El Tor Ogawa strain 3008 . Three (16.7%) of 18 vaccinees and seven (87.5%) of eight controls developed diarrhea (P = 0.001) (vaccine efficacy = 80.9%) . Further studies are underway to determine a dosage of CVD 111 that will be more clinically acceptable but equally immunogenic and protective. Eur J Cell Biol, 1997 May, 73(1), 1 - 9 Effects of cell surface ganglioside sialidase inhibition on growth control and differentiation of human neuroblastoma cells; Kopitz J et al.; Gangliosides on the external side of the plasma membrane are important modulators of cellular functions . In previous work we had found that in cultured human SK-N-MC neuroblastoma cells a cell surface sialidase activity specifically cleaved terminal sialic acids from gangliosides, leading to a shift from higher sialylated species to GM1 and a decrease of GM3 . To further elucidate the function of the enzyme, we have now examined the consequences of ganglioside sialidase inhibition . When present in the culture medium, the ganglioside sialidase inhibitors 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en), heparin, and heparan sulfate caused dramatic changes in cell behavior . Thus, the inhibitors uniformly led to a complete release from contact inhibition of growth, and to the loss of the differentiation markers neuron-specific enolase and neurofilaments, and a decrease of cyclic AMP . In presence of NeuAc2en, cells that normally were spread out evenly and were firmly attached, appeared smaller, rounded, and only loosely adherent to the culture vessel . Exogenous addition of vibrio cholerae sialidase mimicked the action of the plasma membrane ganglioside sialidase by retarding cell proliferation and increasing intracellular acetylcholinesterase . That the ganglioside sialidase inhibitors in the culture medium indeed affected solely the cell surface enzyme and not also a lysosomal sialidase, was demonstrated in an experiment where the desialylation of exogenously added radioactive gangliosides was determined in absence and presence of NeuAc2en and NH4Cl, an inhibitor of lysosomal function . Taken together, our results suggest that the ganglioside sialidase on the surface of SK-N-MC cells is responsible for growth control and differentiation in this neuronal cell line. South Med J, 1997 May, 90(5), 544 - 5 Vibrio fluvialis: an underrecognized enteric pathogen in infants? Kolb EA, Eppes SC, Klein JD. We report a case of Vibrio fluvialis gastroenteritis in an infants 3 1/2 weeks old . The case was unusual because no likely epidemiologic risk factors were involved . Since several other such cases in young infants have been reported, V fluvialis should be considered in the differential diagnosis of infantile gastroenteritis. J Med Microbiol, 1997 May, 46(5), 398 - 402 Detection of Vibrio cholerae and V . mimicus heat-stable toxin gene sequence by PCR; Vicente AC et al.; Previously the heat-stable enterotoxin in Vibrio cholerae and V . mimicus has been detected by suckling mouse assay, a non-specific approach, and by DNA probes, a time-consuming method . This report describes a polymerase chain reaction (PCR) procedure for the detection of the stn (NAG-ST) and sto (O1-ST) gene sequences that is rapid and specific, allowing toxin gene molecular characterisation . A total of 34 V . cholerae and V . mimicus isolates was examined for ST and CT genes . The NAG-ST gene sequence was amplified in 13 of 22 non-O1/non-O139 V . cholerae and three of five V . mimicus strains . A new enterotoxin gene sequence pattern was found with MseI and TaqI restriction endonuclease PCR fragment digestion of two V . cholerae isolates, in addition to the pattern anticipated from the Genbank sequence, and found with the other ST+ . These results show that ST-PCR detection is useful for the characterisation of V . cholerae and V . mimicus. J Bacteriol, 1997 May, 179(10), 3116 - 21 The disulfide bond in the Aeromonas hydrophila lipase/acyltransferase stabilizes the structure but is not required for secretion or activity; Brumlik MJ et al.; Vibrio and Aeromonas spp . secrete an unusual 35-kDa lipase that shares several properties with mammalian lecithin-cholesterol acyltransferase . The Aeromonas hydrophila lipase contains two cysteine residues that form an intramolecular disulfide bridge . Here we show that changing either of the cysteines to serine does not reduce enzymatic activity, indicating that the disulfide bond is not required for correct folding . However, when either of the cysteines is replaced, the enzyme is more readily denatured by urea and more sensitive to degradation by trypsin than is the wild-type enzyme, evidence that the bridge has an important role in stabilizing the protein's structure . The two mutant proteins with serine-for-cysteine replacements were secreted by Aeromonas salmonicida containing the cloned genes, although the levels of both in the culture supernatants were lower than the level of the wild-type enzyme . When the general secretory pathway was blocked with carbonyl cyanide chlorophenylhydrazone, the cell-associated pools of the mutant enzymes appeared to be degraded, whereas the wild-type pool remained stable . We conclude that reduced extracellular levels of the mutant proteins are the result of their increased sensitivities to proteases encountered inside the cell during export. Appl Environ Microbiol, 1997 May, 63(5), 1674 - 8 Comparison of ribotyping and randomly amplified polymorphic DNA PCR for characterization of Vibrio vulnificus; Hoi L et al.; A total of 85 isolates of Vibrio vulnificus were characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by randomly amplified polymorphic DNA-PCR (RAPD-PCR) with a 10-mer oligonucleotide primer . The RAPD-PCR results were scanned, and the images were analyzed with a computer program . Ribotype membranes were evaluated visually . Both the ribotyping and the RAPD-PCR results showed that the collection of strains was genetically very heterogeneous . Ribotyping enabled us to differentiate U.S . and Danish strains and V . vulnificus biotypes 1 and 2, while the RAPD-PCR technique was not able to correlate isolates with sources or to differentiate the two biotypes, suggesting that ribotyping is useful for typing V . vulnificus strains whereas RAPD-PCR profiles may subdivide ribotypes . Two Danish clinical biotype 2 strains isolated from fishermen who contracted the infection cleaning eels belonged to the same ribotype as three eel strains (biotype 2), providing further evidence that V . vulnificus biotype 2 is an opportunistic pathogen for humans . One isolate (biotype 2) from Danish coastal waters also showed the same ribotype as the eel strains . This is, to our knowledge, the first time the isolation of V . vulnificus biotype 2 from coastal waters has been described. J Bacteriol, 1997 May, 179(9), 3004 - 12 Quorum sensing in Vibrio anguillarum: characterization of the vanI/vanR locus and identification of the autoinducer N-(3-oxodecanoyl)-L-homoserine lactone; Milton DL et al.; Certain gram-negative pathogens are known to control virulence gene expression through cell-cell communication via small diffusible signal molecules termed autoinducers . This intercellular signal transduction mechanism termed quorum sensing depends on the interaction of an N-acylhomoserine lactone (AHL) auto-inducer molecule with a receptor protein belonging to the LuxR family of positive transcriptional activators . Vibrio anguillarum is a gram-negative pathogen capable of causing a terminal hemorrhagic septicemia known as vibriosis in fish such as rainbow trout . In this study, we sought to determine whether V . anguillarum employs AHLs to regulate virulence gene expression . Spent V . anguillarum culture supernatants stimulated bioluminescence in a recombinant lux-based Escherichia coli AHL biosensor strain, whereas they both stimulated and inhibited AHL-mediated violacein pigment production in Chromobacterium violaceum . This finding suggested that V . anguillarum may produce multiple AHL signal molecules . Using high-performance liquid chromatography and high-resolution tandem mass spectrometry, we identified the major V . anguillarum AHL as N-(3-oxodecanoyl)-L-homoserine lactone (ODHL), a structure which was unequivocally confirmed by chemical synthesis . The gene (vanI) responsible for ODHL synthesis was cloned and sequenced and shown to belong to the LuxI family of putative AHL synthases . Further sequencing downstream of vanI revealed a second gene (vanR) related to the LuxR family of transcriptional activators . Although deletion of vanI abolished ODHL synthesis, no reduction of either metalloprotease production or virulence in a fish infection model was observed . However, the vanI mutant remained capable of weakly activating both bioluminescence and violacein in the E . coli and C . violaceum biosensors, respectively, indicating the existence of additional layers of AHL-mediated regulatory complexity. J Infect Dis, 1997 May, 175(5), 1134 - 41 Molecular characterization of Vibrio cholerae O1 biotype El Tor strains isolated between 1992 and 1995 in Calcutta, India: evidence for the emergence of a new clone of the El Tor biotype; Sharma C et al.; Sixty-one clinical strains of Vibrio cholerae O1 El Tor isolated in Calcutta before, during, and after the V . cholerae O139 Bengal outbreak were examined to see if the O1 strains of the post-O139 period were different from those in existence before . Comparison of the restriction fragment length polymorphism of the rRNA genes (ribotyping) and the CTX genetic element revealed that all "before" strains except 1 belonged to a single known ribotype, whereas all "after" strains except 2 belonged to a hitherto undescribed ribotype . Also, 23 of 25 "before" strains harbored two or more copies of CTX in tandem and also a "free" RS1 element away from CTX, whereas 19 of 21 "after" strains had a single copy of CTX and no free RS1 element . CTX occupied different chromosomal locations in "before" and "after" strains . These studies clearly showed that El Tor O1 strains, which displaced V . cholerae O139 in Calcutta, belonged to a new clone and suggested that there is a continuous genetic reassortment among El Tor strains of V . cholerae O1. Infect Immun, 1997 May, 65(5), 1830 - 5 Purification and characterization of a hemolysin produced by Vibrio mimicus; Miyoshi S et al.; Vibrio mimicus is a causative agent of human gastroenteritis . This pathogen secretes a pore-forming toxin, V . mimicus hemolysin (VMH), which causes hemolysis by three sequential steps: binding to an erythrocyte membrane, formation of a transmembrane pore, and disruption of the cell membrane . VMH with a molecular mass of 63 kDa was purified by ammonium sulfate precipitation and column chromatography with phenyl Sepharose HP and Superose 6 HR . The hemolytic reaction induced by VMH continued up to disruption of all erythrocytes in the assay system . Moreover, VMH that bound preliminarily to erythrocyte ghosts showed a sufficient ability to attack intact erythrocytes . These results suggest reversible binding of the toxin molecule to the membrane . The final cell-disrupting stage was effectively inhibited by various divalent cations . Additionally, some cations, such as Zn2+ and Cu2+, blocked the pore-forming stage at high concentrations . Although VMH could disrupt all kinds of mammalian erythrocytes tested, those from horses were most sensitive to the hemolysin . Horse erythrocytes were found to have the most toxin-binding sites and to be hemolyzed by the least amount of membrane-bound toxin molecules, suggesting that toxin binding to and pore formation on erythrocytes are more effective in horses than in other mammals . Purified VMH induced fluid accumulation in a ligated rabbit ileal loop in a dose-dependent manner . In addition, the antibody against the hemolysin obviously reduced enteropathogenicity of living V . mimicus cells . These findings clearly demonstrate that VMH is probably involved in the virulence of this human pathogen. J Clin Microbiol, 1997 May, 35(5), 1260 - 2 Food-borne disease outbreaks due to bacteria in Taiwan, 1986 to 1995; Pan TM et al.; Between 1986 and 1995, 852 outbreaks of food-borne disease involving 26,173 cases and 20 deaths were reported in Taiwan . About 80% of the outbreaks occurred in the warmer months, i.e., between April and October . Of the 852 reported outbreaks, 555 (65%) were caused by bacterial pathogens . The three most common bacteria involved were Vibrio parahaemolyticus (35%, 197 of 555 outbreaks), Staphylococcus aureus (30%, 169 of 555 outbreaks), and Bacillus cereus (18%, 104 of 555 outbreaks). J Clin Microbiol, 1997 May, 35(5), 1151 - 6 Molecular evolution of Vibrio cholerae O1 strains isolated in Lima, Peru, from 1991 to 1995; Dalsgaard A et al.; Following the emergence of cholera in Lima, Peru, in 1991, isolates of Vibrio cholerae O1 biotype El Tor recovered from patients in various parts of Lima were selected and characterized . Ribotyping and pulsed-field gel electrophoresis (PFGE) revealed four BglI ribotypes and eight NotI PFGE types among 50 V . cholerae O1 strains recovered from patients with cholera in Lima from 1991 to 1995, with certain genotypes appearing to cluster geographically . While differences in ribotype and PFGE type patterns suggest that genetic changes are occurring in the strain responsible for the Latin American cholera epidemic, more frequently than previously reported, 40 (80%) O1 strains showed an identical ribotype pattern and 41 (82%) strains showed closely related PFGE types, types 1, 2, or 3, that differed by less than three restriction fragments . All strains were susceptible to nine antibacterial agents studied . In 1991, more than 95% of the clinical V . cholerae O1 strains were serotype Inaba, whereas from 1992, serotype Ogawa began to predominate, with more than 90% of the isolates being of the Ogawa serotype in 1995 . The small differences in genotypes of V . cholerae O1 is remarkable because cholera is highly seasonal in coastal areas of Peru and support the hypothesis that the epidemic strain reemerges from an environmental source . However, the relative high rate of genetic changes within V . cholerae O1 as shown by ribotyping and PFGE should be taken into consideration when typing patterns of V . cholerae O1 associated with cholera in Latin America are evaluated. Curr Microbiol, 1997 May, 34(5), 314 - 7 Urea hydrolysis and suppressed production of thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus associated with presence of TDH-related hemolysin genes; Okitsu T et al.; A total of 18 strains of V . parahaemolyticus isolated from patients of past food poisoning cases occurring in Kanagawa Prefecture, Japan, were assayed for presence of the thermostable direct hemolysin (TDH) gene and the TDH-related hemolysin (TRH) genes (trh 1 and trh 2) with specific reference to their ability to hydrolyze urea and TDH production . A polymerase chain reaction assay revealed that all urea-hydrolyzing strains (9 strains) carried either trh 1 gene or trh 2 gene . The strains carrying the trh genes as well as the tdh gene produced TDH less by a factor of 4 to 16 than those carrying only the tdh gene, suggesting the expression of the tdh gene was suppressed by the presence of trh gene through a mechanism yet to be defined. J Mol Biol, 1997 Apr 25, 268(1), 137 - 46 Structure of TcpG, the DsbA protein folding catalyst from Vibrio cholerae; Hu SH et al.; The efficient and correct folding of bacterial disulfide bonded proteins in vivo is dependent upon a class of periplasmic oxidoreductase proteins called DsbA, after the Escherichia coli enzyme . In the pathogenic bacterium Vibrio cholerae, the DsbA homolog (TcpG) is responsible for the folding, maturation and secretion of virulence factors . Mutants in which the tcpg gene has been inactivated are avirulent; they no longer produce functional colonisation pili and they no longer secrete cholera toxin . TcpG is thus a suitable target for inhibitors that could counteract the virulence of this organism, thereby preventing the symptoms of cholera . The crystal structure of oxidized TcpG (refined at a resolution of 2.1 A) serves as a starting point for the rational design of such inhibitors . As expected, TcpG has the same fold as E . coli DsbA, with which it shares approximately 40% sequence identity . In addition, the characteristic surface features of DsbA are present in TcpG, supporting the notion that these features play a functional role . While the overall architecture of TcpG and DsbA is similar and the surface features are retained in TcpG, there are significant differences . For example, the kinked active site helix results from a three-residue loop in DsbA, but is caused by a proline in TcpG (making TcpG more similar to thioredoxin in this respect) . Furthermore, the proposed peptide binding groove of TcpG is substantially shortened compared with that of DsbA due to a six-residue deletion . Also, the hydrophobic pocket of TcpG is more shallow and the acidic patch is much less extensive than that of E . coli DsbA . The identification of the structural and surface features that are retained or are divergent in TcpG provides a useful assessment of their functional importance in these protein folding catalysts and is an important prerequisite for the design of TcpG inhibitors. Gene, 1997 Apr 21, 189(2), 203 - 7 The rpoH gene encoding sigma 32 homolog of Vibrio cholerae; Sahu GK et al.; The Vibrio cholerae rpoH gene coding for the heat-shock sigma factor, sigma 32, has been cloned and shown to functionally complement Escherichia coli rpoH mutants . The nt sequence of the gene has been determined and the deduced aa sequence is more than 80% homologous to the E . coli rpoH gene product . Downstream of the V . cholerae rpoH gene, an unidentified dehydrogenase gene (udhA) is present on the opposite strand facing rpoH . The predicted secondary structure of the 5'-proximal region of V . cholerae rpoH mRNA is apparently different from the conserved secondary structures of the rpoH mRNA reported for several bacterial species . The 'RpoH box', a stretch of 9 aa (QRKLFFNLR) unique to sigma 32 factors, and the 'downstream box' sequence complementary to a part of the 16S rRNA, have been detected. Gene, 1997 Apr 21, 189(2), 163 - 8 Cloning and characterization of the gene (empV) encoding extracellular metalloprotease from Vibrio vulnificus; Chuang YC et al.; A gene (empV) encoding the extracellular metalloprotease of Vibrio vulnificus CKM-1 has been cloned and sequenced . When the empV gene was expressed in minicells, a unique peptide of approx . 46 kDa was identified . Protease activity staining experiments also indicated a similar M(r) for the protease . The empV gene product (EmpV) is secreted into the periplasm of Escherichia coli, but not out of it . The crude enzyme prepared from the periplasmic fraction of recombinant E . coli was inhibited by a metalloprotease inhibitor and Zn2+ is essential for its protease activity . Nucleotide sequence analysis predicted a single open reading frame (ORF) of 1818 bp encoding a 606 amino acid (aa) polypeptide, with a potential 24 aa signal peptide followed by a long 'pro' sequence consisting of 172 aa . The N-terminal 20 aa sequence for the elastolytic protease (EepV), purified from the culture supernatant of V . vulnificus ATCC 29307, completely identified the beginning of the predicted mature protein within the deduced aa sequence except for 1 aa residue difference . The estimated pI and molecular weight of the predicted mature protein were 5.86 and 44.3 kDa, respectively, which are nearly identical to those of V . vulnificus L-180 extracellular neutral metalloprotease (EnmV) and of strain ATCC 29307 EepV . The estimated molecular weight also closely matches that determined by SDS-PAGE analysis of the minicells and by protease activity staining . The deduced aa sequence of EmpV showed high homology to V . anguillarum metalloprotease (EmpA), V . cholerae HA/protease (HprC), and V proteolyticus neutral protease (NprP), particularly with respect to active-site residues, zinc-binding residues, and cysteine residues. Biochim Biophys Acta, 1997 Apr 12, 1360(2), 102 - 4 Nucleotide sequence of the vmhA gene encoding hemolysin from Vibrio mimicus; Kim GT et al.; The structural gene (vmhA) of hemolysin from Vibrio mimicus (ATCC33653) was cloned and sequenced . The vmhA gene contains an open reading frame consisting of 2232 nucleotides which can code for a protein of 744 amino acids with a predicted molecular mass of 83,059 . The similarity of amino acid sequence shows 81.6% identity with Vibrio cholerae El Tor hemolysin. Lancet, 1997 Apr 5, 349(9057), 981 - 5 Epidemic cholera in Burundi: patterns of transmission in the Great Rift Valley Lake region; Birmingham ME et al.; BACKGROUND: After a 14-year hiatus, epidemic cholera swept through Burundi between January and May, 1992 . The pattern of transmission was similar to that in 1978, when the seventh pandemic first reached this region . Communities affected were limited to those near Lake Tanganyika and the Rusizi River . The river connects Lake Tanganyika with Lake Kivu to the north in Zaire and Rwanda . METHODS: To identify sources of infection and risk factors for illness, an epidemiological study was carried out in Rumonge, a lake-shore town where 318 people were admitted to hospital with cholera between April 9 and May 31, 1992 . The investigation included a case-control study of 56 case-patients and 112 matched controls . FINDINGS: Attack rates according to street increased with the street's proximity to Lake Tanganyika (chi 2 test for linear trend, p < 0.01) which suggests that exposure to the lake was a risk factor for illness . Comparison of the 56 case-patients with matched controls showed that bathing in the lake (odds ratio 1.6, attributable risk percentage 37%) and drinking its water (2.78, 14%) were independently and significantly (p < 0.05) linked with illness . No food-borne risk factors were identified . Vibrio cholera 01 was isolated from Lake Tanganyika during, but not after, the outbreak in Rumonge . Isolates from the lake and from patients with acute watery diarrhoea had the same serotype, biotype, and antimicrobial susceptibility profiles . The number of cases rapidly declined when access to the lake was blocked . INTERPRETATION: This study identifies bathing in contaminated surface water as a major risk factor for cholera in sub-Saharan Africa, and suggests that improving the quality of drinking water alone will have only limited impact on the transmission of the disease in the Great Rift Valley Lake region . The similarity in the patterns of transmission during the 1978 and 1992 epidemics suggests that extensive use of the Great Lakes and connecting rivers for transportation and domestic purposes may be the reason for the explosive cholera outbreaks that occur sporadically in this region. Rev Environ Health, 1997 Apr-Jun, 12(2), 63 - 80 Pathogenic vibrios in the natural aquatic environment; Chakraborty S et al.; In recent years, members belonging to the genus Vibrio of the family Vibrionaceae have acquired increasing importance because of the association of several of its members with human disease . The most feared of the Vibrio species is Vibrio cholerae, the causative agent of cholera, a devastating disease of global significance . Other important vibrios of medical importance are V . parahemolyticus, V . vulnificus, V . mimicus, and to a lesser extent V . fluvialis, V . furnissii, V . hollisae, and V . damsela . Recent studies have also implicated V . alginolyticus and V . metschnikovii in human disease, although their complete significance has not yet been established . The virulence of all medically important vibrios is aided by a variety of traits that help breach human defenses . In this review, we provide an overview of the environmental distribution of the pathogenic vibrios and the important virulence traits that enable them to cause disease. Genes Genet Syst, 1997 Apr, 72(2), 115 - 8 Molecular cloning and functional analysis of an rpoS homologue gene from Vibrio cholerae N86; Hiratsu K et al.; A homologue of the rpoS gene of Escherichia coli was cloned from Vibrio cholerae N86 by complementation of the phenotypes of the E . coli rpoS mutant strain . We determined the DNA sequence of this gene . Sequence alignments have indicated that the rpoS gene of V . cholerae N86 encoding a 39-kDa protein is very similar to that of E . coli . In addition, the nlpD-like gene was found in the upstream region of the rpoS gene in the same order as in E . coli . These results suggest that the organization of these genes is highly conserved between E . coli and V . cholerae. J Clin Microbiol, 1997 Apr, 35(4), 951 - 3 Rapid screening method for identification of cholera toxin-producing Vibrio cholerae O1 and O139; Osawa R et al.; A novel method of identifying cholera enterotoxin (CT)-producing Vibrio cholerae serogroups O1 and O139 was developed . The method uses degradation of NAD as a specific biochemical marker for the CT-producing strains . The substrate NAD at a concentration of 100 mumol/liter was markedly degraded when it was incubated at 37 degrees C for 2 h with the CT-producing stains at a final cell density equivalent to that of a twofold dilution of a McFarland no . 1 standard . NAD degradation was monitored by an enzyme-amplified color development assay . Subsequent tests conducted with a total of 119 strains of V . cholerae, including both clinical and environmental isolates, confirmed a significant correlation between NAD degradation and CT production for all V . cholerae strains belonging to serogroups O1 and O139 . Since 2 of 11 non-O1, non-O139 V . cholerae strains not carrying the CT gene degraded NAD, serotyping of the strains prior to the test is recommended. J Vet Med Sci, 1997 Apr, 59(4), 277 - 9 Selective survival of a thermostable direct hemolysin-producing Vibrio parahaemolyticus in the alimentary tract of a juvenile estuarine gastropod (Clithon retropictus); Kumazawa NH et al.; Juvenile estuarine gastropods (Clithon retropictus), maintained in ultraviolet ray-irradiated recirculating artificial seawater with a salinity of 20/1000 at 28 degrees C, preserved thermostable direct hemolysin (TDH)-producing strain D-3 of Vibrio parahaemolyticus at a level of 10(4)-10(5) colony forming units per gram (cfu/g) and TDH-non-producing strains N-18 and R-13 at a level of 10(1)-10(2) cfu/g in the alimentary tract for at least 21 days after ingestion . In adults, the numbers of the three strains decreased to a level of 10(0) cfu/g within 21 days under the same conditions . This evidence supports our recent observations that TDH-producing strains increased to a high level in the summer months in the presence of high levels of TDH-non-producing strains in the alimentary tract of juvenile C . retropictus at estuaries in Japan. Indian J Med Res, 1997 Apr, 105, 153 - 4 Resistance of Vibrio cholerae 01 to nalidixic acid; Jesudason MV et al.; Until 1987 all isolates of V . cholerae 01 at a tertiary care hospital in south India were susceptible to drugs commonly used to treat gastroenteritis including cholera . Since July 1987 strains resistant to co-trimoxazole have been encountered and since October 1995 strains resistant to nalidixic acid are being isolated . In this study the latter strains were examined by determining minimum inhibitory concentration levels of nalidixic acid as well as norfloxacin, the fluoroquinolone extensively used to treat diarrhoea . No cross resistance to norfloxacin was found in any of the nalidixic acid resistant V . cholerae 01 strains. Hybridoma, 1997 Apr, 16(2), 195 - 9 Detection of two isotypically different antibodies produced by a murine hybridoma; Chen D et al.; A hybridoma, F31P46B, secreting monoclonal antibodies (mAbs) comprised of mu and gamma heavy chains in association with a single kappa light chain, has been characterized . This hybridoma was prepared by fusing splenocytes, derived from a BALB/c mouse immunized with Vibrio vulnificus and SP2/O-Ag-14 mouse myeloma cells . The specificity of this hybridoma was determined by ELISA screening on a large number of bacterial strains . Hybridoma cells of F31P46B were cloned by limiting dilution to an average cell density of 0.1 cells/well and repeated 3 times to ensure monoclonality . Isotyping of 7 subclones was then performed by Ouchterlony gel double diffusion, as well as a Bio-Rad isotyping kit, and both methods showed that both IgM and IgG2b were secreted . PAGE and immunoblotting showed the presence of mu, gamma, and kappa chains with respective molecular weights of 80, 50, and 25kDa . A series of fractions, collected from F31P46B ascites during Superose 12 gel chromatography, were tested by the two isotyping methods and each confirmed the presence of two immunoglobulin products . These data indicated that the hybridoma secreted two separate immunoglobulins, IgM/kappa and IgG2b/kappa. Zentralbl Bakteriol, 1997 Apr, 285(4), 486 - 90 Arsenate resistance as a possible marker in the differentiation of environmental and clinical isolates of Vibrio parahaemolyticus; Chauhan BS et al.; Strains of Vibrio parahaemolyticus were isolated from clinical, marine and freshwater fish of Calcutta, West Bengal, India . Drug and metal resistance characteristics were compared for differentiation of clinical and environmental strains . Eighteen out of the twenty environmental isolates were resistant to arsenate, unlike the clinical isolates which were all susceptible . All the thirty-five isolates of V . parahaemolyticus were resistant to ampicillin and streptomycin. Trends Microbiol, 1997 Apr, 5(4), 161 - 5 The evolution of epidemic Vibrio cholerae strains; Mooi FR et al.; The emergence of the novel Vibrio cholerae strain, O139 Bengal, which caused a large epidemic in Southeast Asia, underlines the adaptability of pathogenic microorganisms . Recent studies reveal that horizontal transfer of cell-wall polysaccharide genes played a central role in the emergence of this strain and that its genesis may not be as unique as initially believed. Microb Pathog, 1997 Apr, 22(4), 199 - 208 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; Ghosh C et al.; 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. Toxicon, 1997 Apr, 35(4), 515 - 27 Biochemical and physiological characteristics of HlyA, a pore-forming cytolysin of Vibrio cholerae serogroup O1; Sathyamoorthy V et al.; 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. Infect Immun, 1997 Apr, 65(4), 1561 - 5 Promoter activities in Vibrio cholerae ctx phi prophage; Fando R et al.; Comparison of cholera toxin (CT) production directed by different gene constructs and S1 nuclease mapping revealed the presence of a ctxB-specific promoter within the ctxA coding sequence . Initiation of transcription in this region occurred in wild-type El Tor and classical biotype choleragenic vibrios . We propose that transcription from the ctxB-specific promoter and a stronger ribosomal binding site on the ctxB mRNA synergistically contribute to achieve the correct (5B:1A) subunit stoichiometry . Plasmid pB, a CT promoterless vector expressing only CTB, was used to detect promoter activity by restoration of A-subunit synthesis . Promoter activity expressed in vitro and in vivo was detected upstream of the zonula occludens toxin gene, suggesting that this factor could be produced in vivo to contribute to fluid accumulation . No promoter activity was detected in vitro and in vivo upstream from the accessory cholera enterotoxin gene. Infect Immun, 1997 Apr, 65(4), 1387 - 94 Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women; Kozlowski PA et al.; To determine which mucosal immunization routes may be optimal for induction of antibodies in the rectum and female genital tract, groups of women were immunized a total of three times either orally, rectally, or vaginally with a cholera vaccine containing killed Vibrio cholerae cells and the recombinant cholera toxin B (CTB) subunit . Systemic and mucosal antibody responses were assessed at 2-week intervals by quantitation of CTB-specific antibodies in serum and in secretions collected directly from mucosal surfaces of the oral cavity, rectum, cervix, and vagina with absorbent wicks . The three immunization routes increased levels of specific immunoglobulin G (IgG) in serum and specific IgA in saliva to similar extents . Rectal immunization was superior to other routes for inducing high levels of specific IgA and IgG in rectal secretions but was least effective for generating antibodies in female genital tract secretions . Only vaginal immunization significantly increased both specific IgA and specific IgG in both the cervix and the vagina . In addition, local production of CTB-specific IgG in the genital tract could be demonstrated only in vaginally immunized women . Vaginal immunization did not generate antibodies in the rectum, however . Thus, generation of optimal immune responses to sexually transmitted organisms in both the rectal and the genital mucosae of women may require local immunization at both of these sites. Infect Immun, 1997 Apr, 65(4), 1293 - 8 Potent membrane-permeabilizing and cytocidal action of Vibrio cholerae cytolysin on human intestinal cells; Zitzer A et al.; Many strains of Vibrio cholerae non-O1 and O1 El Tor that cause diarrhea do not harbor genes for a known secretogenic toxin . However, these strains usually elaborate a pore-forming toxin, hitherto characterized as a hemolysin and here designated V . cholerae cytolysin, whose action on intestinal cells has not yet been described . We report that V . cholerae cytolysin binds as a monomer to Intestine 407 cells and then assembles into detergent-stable oligomers that probably represent tetra- or pentamers . Oligomer formation is accompanied by generation of small transmembrane pores that allow rapid flux of K+ but not influx of Ca2+ or propidium iodide . Pore formation is followed by irreversible ATP depletion and cell death . Binding of fewer than 10(4) toxin molecules per cell in vitro is lethal . The possibility is raised that production of this toxin by bacteria that are in close contact with intestinal cells is rapidly cytocidal in vivo, and death of intestinal cells may be a cause of diarrhea. Clin Pharmacokinet, 1997 Apr, 32(4), 313 - 23 Oral delivery of antibodies . Future pharmacokinetic trends; Reilly RM et al.; Antibodies have been investigated as specific targeting agents for cancer diagnosis and therapy, to inactivate toxic substances including drugs and also as passive immunotherapy for neoplastic or infectious diseases . In most cases the antibodies were administered systemically by the intravenous route . More recently, however, there has been increasing interest in the oral administration of antibodies for localised treatment of infections or other conditions in the gastrointestinal tract . The normal physiological handling of ingested proteins is degradation by proteases in the stomach and intestine into small peptides or amino acids which are subsequently absorbed . Proteolytic enzymes involved in the degradation of orally administered immunoglobulins include pepsin, trypsin, chymotrypsin, carboxypeptidase and elastase . These enzymes initially degrade the antibodies to F(ab')2 . Fab and Fc fragments . The F(ab')2 and Fab fragments, however, retain some of their neutralising activity locally in the gastrointestinal tract . Various approaches are possible to increase the stability of orally administered antibodies against proteolysis, including formulation in liposomes, coating with polymers and genetic engineering of resistant forms . The clinical application of orally administered antibodies includes the treatment and prevention of gastrointestinal infections caused by enteric pathogens such as rotavirus, Escherichia coli or Vibrio cholerae in susceptible individuals including those with immunodeficiency diseases and patients with bone marrow transplants . There is also a suggestion that such agents may be useful in preventing chemotherapy-induced gastrointestinal mucositis . Future opportunities for research include the design of oral dosage forms of antibodies which resist proteolysis and can deliver a greater fraction of immunoreactive antibody locally in the gastrointestinal tract for the treatment of infections or perhaps even to allow the absorption of antibodies for the treatment or prevention of systemic conditions. J Biomed Mater Res, 1997 Apr, 35(1), 101 - 5 Evaluation of toxicity of medical devices using Spirotox and Microtox tests: I . Toxicity of selected toxicants in various diluents; Nalecz-Jawecki G et al.; Significant effort has been directed toward developing in vitro alternatives, which can be the first step of toxicity analysis . Tissue culture assays are currently the most popular in vitro tests for evaluating acute toxicity . The possibility of applying two bioassays using microorganisms in assessing the toxicity of extracts of medical devices was investigated . The Microtox test system--a luminescent bacteria toxicity test--assesses changes in light output from a luminescent bacteria, Vibrio fischeri . Spirotox used a large ciliate protozoan: Spirostomum ambiguum . The most widely used extraction solvent, 0.9% NaCl, must be concentrated up to 2% for Microtox and diluted nine times for the Spirotox test . The organic solvents ethanol, DMSO, and polyethylene glycol 400 were not toxic in either test in concentrations of 1-2% . The toxicity of reference compounds Hg, Cd, Zn, Pb, and SDS was examined in various diluents . The sequence of toxicity of the tested compounds in Spirotox and Microtox was: Hg > Cd > Zn > Pb > SDS, and Hg > Pb = Zn > SDS > Cd, respectively . Addition of organic solvents changed the toxicity of compounds tested in 60% of Spirotox tests and only in 25% of Microtox tests . Changes were low, not exceeding 100% in almost all cases . No correlation was observed between diluent and toxicant in either bioassay. Appl Environ Microbiol, 1997 Apr, 63(4), 1460 - 6 Phenotypic and genotypic characterization of Vibrio vulnificus: proposal for the substitution of the subspecific taxon biotype for serovar; Biosca EG et al.; The classification of Vibrio vulnificus strains into two biotypes has been maintained on the basis of phenotypic properties and eel virulence . Biotype 2 is virulent for eels, negative for the indole reaction, and serologically homogeneous (serogroup E), whereas strains of biotype 1 are avirulent, indole positive, and serologically heterogeneous . In the present study, we phenotypically and genotypically characterized 21 V . vulnificus isolates, recovered mainly from northern Europe, by comparing them with reference strains of both biotypes to look for new isolates of biotype 2 . The results of this work revealed that the majority of isolates virulent for eels presented phenotypic traits previously considered characteristics of biotype 2 and specific ribotypes with HindIII . However, among the new isolates we found (i) a serogroup E strain virulent for eels but indole positive and (ii) one isolate not belonging to serogroup E but pathogenic for eels . Since no biochemical test for specific serogroup can with certainty be associated with eel virulence, we propose to classify V . vulnificus strains into serovars instead of biotypes . Thus, we suggest serovar E as the denomination of those strains previously classified as biotype 2 . Finally, the occurrence of serogroup E in eels cultured in Norway and Sweden, as well as from human infections and shrimp, has been demonstrated. Appl Environ Microbiol, 1997 Apr, 63(4), 1449 - 52 Intraspecific characterization of Vibrio tapetis strains by use of pulsed-field gel electrophoresis, ribotyping, and plasmid profiling; Castro D et al.; A total of twenty-two strains of Vibrio tapetis, the causative agent of brown ring disease affecting cultured clams, were compared and evaluated in an investigation of strain heterogeneity using pulsed-field gel electrophoresis (PFGE), ribotyping, and plasmid profile analysis . A total of 90.9% of V . tapetis strains tested by using NotI showed the same PFGE pattern, consisting of 15 bands . In contrast, the V . tapetis strains showed a low degree of similarity with six reference Vibrio species tested . All V . tapetis strains harbored a large plasmid of 74.5 kb . This plasmid was not detected in any of the other Vibrio species . In addition, endonuclease restriction analysis of the plasmid content of the strains using EcoRI and HindIII clearly showed that all the strains of V . tapetis possessed the same cleavage pattern . The three enzymes used for ribotyping, PvuII, SmaI, and SalI, yielded patterns with 8 to 12 bands ranging in size from 2 to 23 kb . The application of the SalI and SmaI endonuclease rendered the separation of the strains tested in two ribotypes, while all the V . tapetis strains belonged to the same ribotype when the enzyme PvuII was used. Curr Microbiol, 1997 Apr, 34(4), 258 - 63 Molecular cloning and nucleotide sequence of the porphobilinogen deaminase gene, hemC, from Chlorobium vibrioforme; Majumdar D et al.; We previously reported the DNA sequence and expression of the Chlorobium vibrioforme glutamyl-tRNA reductase (hemA) gene (Majumdar et al., Arch Microbiol 156:281, 1991) . The sequence downstream of the hemA gene indicated homology to Escherichia coli and Bacillus subtilis porphobilinogen deaminase (hemC) gene . The Chlorobium gene was confirmed to be the porphobilinogen deaminase gene, and complete sequence of the structural gene was obtained . A 2.8-kb DNA fragment containing the 1.3-kb hemA gene of Chlorobium was cloned into a hemC auxotroph (Sz16) of Bacillus subtilis, and complementation of the auxotroph to prototrophy was achieved . DNA sequence data showed a single open reading frame of 840 bp coding a protein of 279 amino acid residues . The deduced amino acid sequence of the Chlorobium porphobilinogen deaminase revealed 39% to 46% homology with the corresponding prokaryotic and eukaryotic sequences. J Commun Dis, 1997 Mar, 29(1), 15 - 22 Changing patterns of Vibrio cholerae isolation over three consecutive cholera seasons (1992-1994) in east Delhi; Rudra S et al.; The emergence of new strains of Vibrio Cholerae has added a new dimension to the variability in pathogenicity and potential virulence of the organisms precipitating diarrhoeal diseases . Considering the shifting patterns of V . cholerae 01 there is a continuous need to monitor the strain characteristics . In this study total 541 stool specimens of acute secretory diarrhoea were investigated between May 1992 and November 1994 for strains of Vibrio Cholerae and anti-microbial susceptibility testing of all the confirmed V . Cholerae strains . In 1992, 50 of the 125 strains (40%) were positive for V . cholerae 01 predominantly biotype El Tor serotype ogawa, and 10 (80%) of non 01 type, with most strains susceptible to tetracycline (100%), chloramphenicol (98%) and Cotrimoxazole (98%), but all resistant to polymyxin B and furazolidine . In 1993, 44 (43.6%) of the 010 strains were positive for V . cholerae 0139 and the rest V . cholerae 01 . In 1994, another sero group of V . cholerae 010 emerged, with 42 (13.3%) being positive . Isolates did not agglutinate with any of these antisera and have been labelled as 'other than non-01 vibrio cholerae'. Zh Mikrobiol Epidemiol Immunobiol, 1997 Mar-Apr, (2), 43 - 6 {The characteristics of the protectiveness of a preparation of Vibrio cholerae outer membrane based on the data from a study using the ligated intestinal loop}; Urbanovich LIa et al.; The study aimed at finding out the antiadhesive capacity of antigenic preparation, earlier obtained from V . cholerae outer membrane and highly effective with respect to cholera infection, was undertaken . The study was made on previously immunized adult rabbits who had been subjected to laparotomy under anesthesia and the ligation of intestinal loops, subsequently inoculated with the broth culture of V . cholerae eltor (P-3122, serovar Ogawa) . The intestinal loops were studied histologically and bacteriologically with the calculation of the number of vibrios, the deduction of the adhesion index and the coefficient of the efficacy of immunization . The data thus obtained indicated that the specific immunization of rabbits with their subsequent inoculation with V . cholerae virulent strain suppressed the adhesive activity of the infective agent which was more pronounced in rabbits immunized with the preparation of V . cholerae outer membrane. J Med Microbiol, 1997 Mar, 46(3), 233 - 8 Isolation of a contact-dependent haemolysin from Mycobacterium tuberculosis; Deshpande RG et al.; Contact-dependent haemolytic activity was observed with cells of Mycobacterium tuberculosis H(37)Rv and M . tuberculosis H(37)Ra, but not with those of M . bovis, M . bovis BCG and M . africanum . Culture filtrates of all these strains did not exhibit any haemolytic activity . M . tuberculosis H(37)Rv was subsequently used for the isolation of haemolysin . Haemolytic activity was retained in the cell debris even after sonication of the cells and treatment with Tween 80 and lysozyme . Solubilisation of haemolysin was possible only after the cell debris was washed with ethanol 70% and then treated with Tween 80 0.1% . The haemolysin thus obtained showed a micellar M(r) of >200000 by gel-filtration on Sephadex G-200 and a subunit M(r) of 66000 by SDS-PAGE . It was sensitive to trypsin but stable when heated at 60 degrees C for 10 min . Polyclonal serum raised in rabbits against the haemolysin neutralised the haemolytic activity . The N-terminal amino-acid sequence of the 66-kDa subunit of haemolysin showed identity with TB66, the 66-kDa secretory protein of M . tuberculosis, and 30% homology with the haemolysin A precursor of Vibrio cholerae . Phosphatidylglycerol inhibited lysis of sheep erythrocytes by the haemolysin and is probably the receptor for the haemolysin . Haemolysin not only lysed erythrocytes, but was also cytotoxic to human lung cells . It appears that, among the members of the M . tuberculosis complex, the cell-bound contact-dependent haemolysin/cytolysin is restricted to M . tuberculosis and it may be associated with the pathogenesis of M . tuberculosis. Ecotoxicol Environ Saf, 1997 Mar, 36(2), 189 - 95 Mixture toxicity of nitrobenzene and trinitrobenzene using the marine bacterium Vibrio harveyi as the test organism; Thomulka KW et al.; Vibrio harveyi, a bioluminescent marine bacterium, was used to evaluate combined or mixture toxicity of two organic compounds, nitrobenzene and trinitrobenzene . An estimated median effective concentration (EC50) and confidence interval were determined for each chemical . These chemicals at their EC50 were evaluated in combination and an additive index method was used to determine a numerical toxicology value . Combinations at 20% intervals of the EC50 were performed using isopleths . The isopleths employed were the isobole plot and the isobologram . Bioluminescent change was also determined and graphed for evaluation of toxicity . Statistical evaluation of isopleths and the additive index method were employed by incorporating confidence intervals . Bioluminescent change and isopleths suggest that mixtures of nitrobenzene and trinitrobenzene are additive, while the additive index method is suggestive of synergism . Statistical evaluation between mixtures and single values, using the z test, was in some cases different at the 5% level . These data suggest that interaction of combinations should be evaluated and described by multiple methodologies . Evaluation of these data suggests, in part, that one mixture is statistically different for antagonism . This study supports the use of bioluminescent microbial toxicity tests with various evaluative methodologies for the determination of mixture interactions. Ecotoxicol Environ Saf, 1997 Mar, 36(2), 180 - 2 Effect of river and wetland sediments on toxicity of metolachlor; Karuppiah M et al.; Metolachlor is a preplant, preemergent herbicide applied to corn and soybean fields . Agricultural runoff after application can cause the herbicide to enter into natural waters . The objective of this study was to determine the effect of river and wetland sediments on the toxicity of metolachlor . Sediments from a river and a wetland (separately) were mixed with Ottawa sand (0, 25, 50, 75, and 100%) . Metolachlor (in water) and sediment were mixed in an orbital shaker for 8 hr; the mixture was centrifuged, and the supernatant liquid was tested for toxicity (EC50%) using the marine luminescent bacteria Vibrio fischeri (Microtox) . The toxicity (EC50%) of metolachlor with the river sediment was 64.61 . Metolachlor after interaction with the wetland sediment demonstrated no toxicity possibly due to increased adsorption on the higher amount of organic matter (10 times) and clay (3.5 times) present in the wetland sediment than the river sediment. Eur J Biochem, 1997 Mar 1, 244(2), 454 - 61 Redox titration of two {4Fe-4S} clusters in the photosynthetic reaction center from the anaerobic green sulfur bacterium Chlorobium vibrioforme; Scott MP et al.; Anaerobic green sulfur bacteria contain photosynthetic reaction centers analogous to photosystem I (PS I) of plants and cyanobacteria . These reaction centers, termed type I, are characterized by the presence of bound iron-sulfur clusters as the terminal electron acceptors . In this work, the iron-sulfur clusters in Chlorobium vibrioforme were studied using selective light-induced reduction protocols, spin quantifications, and chemical redox titrations coupled with EPR detection . Illumination of a dark-frozen sample at 12 K results in the appearance of a spectrum termed signal I . Chemical reduction in darkness at solution potentials between -414 mV and -492 mV results in the appearance of a different spectrum termed signal II . Illumination of these chemically poised samples at 12 K results in the appearance of signal I such that the sum of the intensity of signal I + signal II is nearly constant for every ratio of signal I/signal II . As the solution potential is lowered to -545 mV, the spectrum shifts to yet a third set of resonances, termed signal III . Concomitant with this shift is a loss of low temperature light-induced reduction of signal I . Photoaccumulation of a sample poised at a solution potential of -50 mV results also in the appearance of signal III at nearly the same spin concentration as the chemically reduced sample . Spin quantifications imply that signals I and II are both derived from the reduction of one iron-sulfur cluster, termed center I; signal III is derived from simultaneous reduction of two iron-sulfur clusters, centers I and II . By measuring the EPR signal intensities over a range of solution potentials, centers I and II were shown to have Em (pH 10.0) values of -446 mV and -501 mV, respectively . The observations are consistent with a structural and functional analogy of centers I and II with F(A) and F(B) of PS I. J Med Entomol, 1997 Mar, 34(2), 226 - 33 Parasites of the Asian tiger mosquito and other container-inhabiting mosquitoes (Diptera:Culicidae) in northcentral Florida; Fukuda T et al.; Seven microorganisms including 4 protozoans, 2 fungi, and a bacterium infected Aedes albopictus (Skuse) larvae collected from 12 counties in northecentral Florida . Ae albopictus and 14 other species of mosquitoes were collected from tires, flower-holding vases in cemeteries, other types of artificial containers, and treeholes . Ascogregarina taiwanensis (Lien & Levine) was the most common parasite of Ae . albopictus throughout the year . The microsporidium Vavraia culicis (Weiser) infected Aedes aegypti (L.), Ae . albopictus, Aedes triseriatus (Say), and Orthopodomyia sinifera (Coquillett) . A vibrio bacterium and 2 fungi (Leptolegnia sp . and Smittium culisetae Lichtwardt), infected Ae . albopictus larvae but were observed infrequently . A . taiwanensis, S . culisetae, and the vibrio bacterium previously have been reported from Ae . albopictus . This is the 1st report of the other 4 microorganisms parasitizing Ae . albopictus larvae. J Bacteriol, 1997 Mar, 179(6), 2038 - 46 Outer membrane translocation arrest of the TcpA pilin subunit in rfb mutants of Vibrio cholerae O1 strain 569B; Iredell JR et al.; The toxin-coregulated pilus (TCP) of Vibrio cholerae is a type 4-related fimbrial adhesin and a useful model for the study of type 4 pilus biogenesis and related bacterial macromolecular transport pathways . Transposon mutagenesis of the putative perosamine biosynthesis genes in the rfb operon of V . cholerae 569B eliminates lipopolysaccharide (LPS) O-antigen biosynthesis but also leads to a specific defect in TCP export . Localization of TcpA is made difficult by the hydrophobic nature of this bundle-forming pilin, which floats anomalously in sucrose density gradients, but the processed form of TcpA can be found in membrane and periplasmic fractions prepared from these strains . While TcpA cannot be detected by surface immunogold labelling in transmission electron microscope preparations, EDTA pretreatment facilitates immunofluorescent antibody labelling of whole cells, and ultrathin cryosectioning techniques confirm membrane and periplasmic accumulation of TcpA . Salt and detergent extraction, protease accessibility, and chemical cross-linking experiments suggest that although TcpA has not been assembled on the cell surface, subunit interactions are otherwise identical to those within TCP . In addition, TcpA-mediated fucose-resistant hemagglutination of murine erythrocytes is preserved in whole-cell lysates, suggesting that TcpA has obtained its mature conformation . These data localize a stage of type 4 pilin translocation to the outer membrane, at which stage export failure leads to the accumulation of pilin subunits in a configuration similar to that within the mature fiber . Possible candidates for the outer membrane defect are discussed. FEMS Microbiol Lett, 1997 Mar 1, 148(1), 101 - 6 Characterization of a mutant of Vibrio vulnificus for heme utilization; Miyoshi S et al.; Vibrio vulnificus, an opportunistic human pathogen, can obtain iron from a variety of heme proteins . This process involves the digestion of heme proteins by an exoprotease to liberate protoheme (iron-protoporphyrin IX) . In the present study, we isolated and characterized a mutant for protoheme utilization . One mutant isolated by treatment with a chemical mutagen was shown to be unable to use either protoheme or heme proteins, but multiplied in a medium supplemented with an iron siderophore, such as iron-vulnibactin . Like a wild-type strain, the mutant sensed iron depletion, so that the 74- and 79-kDa outer membrane proteins were expressed under iron-regulated conditions . Both the parent and mutant strains secreted hemolysin independent of the iron concentration of the medium . Whole cells of either of the strains were equally capable of binding of hematin . Taken together, the data suggest that the mutant may have a mutation in a gene encoding an inner membrane or a periplasmic protein which transports protoheme or iron dissociated from protoporphyrin IX into the cell. J Invertebr Pathol, 1997 Mar, 69(2), 177 - 82 In Vitro Activity of the Limulus Antimicrobial Peptide Tachyplesin I on Marine Bivalve Pathogens Morvan A, Iwanaga S, Comps M, Bachere E. Tachyplesin 1 is an antimicrobial peptide extracted from hemocytes of the Japanese horseshoe crab Tachypleus tridentatus . We studied the in vitro activity of tachyplesin I against bivalve pathogens: the oyster parasites Bonamia ostreae, the intrahemocytic parasite of the flat oyster Ostrea edulis and Perkinsus marinus, the histozoic parasite of the Eastern oyster Crassostrea virginica, and the bacterium Vibrio P1, pathogenic for the clam Tapes philippinarum . Viability of the protozoans was assessed microscopically by the uptake of the vital dyes acridine orange and ethidium bromide . Following exposure to tachyplesin I, B . ostreae and P . marinus viabilities were reduced in a dose-dependent manner, up to, respectively, 94 and 62% within a 500 mug/ml peptide concentration . The fine structure of P . marinus was highly altered by the peptide . Tachyplesin I also displayed a potent activity against marine vibrios, with a MIC of 0.4-0.8 mug/ml against Vibrio P1 . We examined the morphology of oyster hemocytes treated by tachyplesin I, together with the cell functional capabilities to produce chemiluminescence . No effect of the peptide was found on bivalve host cells . As transgenic technology is currently being applied to marine invertebrates, these results indicate that tachyplesin I may provide effective gene sequences to be manipulated in order to produce disease-resistant bivalves. Arch Biochem Biophys, 1997 Mar 1, 339(1), 47 - 54 Purification and characterization of isobutylamine N-hydroxylase from the valanimycin producer Streptomyces viridifaciens MG456-hF10; Parry RJ et al.; Streptomyces viridifaciens MG456-hF10 produces the antitumor agent valanimycin, which is a member of a family of antibiotics containing the azoxy group . An enzyme involved in the biosynthesis of valanimycin has been purified 360-fold from S . viridifaciens . This enzyme, isobutylamine N-hydroxylase, catalyzes the oxidation of isobutylamine to isobutylhydroxylamine in the presence of oxygen and a reduced flavin cofactor . Unlike other known N-hydroxylases, isobutylamine N-hydroxylase cannot carry out the reduction of the flavin cofactor . Rather, the reduced flavin is supplied by a separate flavin reductase that is present in extracts of S . viridifaciens . The reduced flavin cofactor could also be supplied by the flavin mononucleotide reductase of Vibrio fischeri . The requirement for molecular oxygen and a reduced flavin indicates that the N-hydroxylase is a flavin monooxygenase and that the mechanism for the hydroxylation is likely to proceed via the formation of a flavin 4a-hydroperoxide . Isobutylamine N-hydroxylase exhibited a subunit molecular mass of 40 kDa and existed in dimeric or trimeric form depending upon buffer conditions . The pI of the protein was found to be ca . 5.1 and the enzyme exhibited a sensitivity to thiol-directed reagents. J Bacteriol, 1997 Mar, 179(5), 1805 - 8 Primary structure and properties of the Na+/glucose symporter (Sg1S) of Vibrio parahaemolyticus; Sarker RI et al.; Previously, we cloned and sequenced a DNA fragment from Vibrio parahaemolyticus and found four open reading frames (ORFs) . Here, we clearly demonstrate that one of the ORFs, ORF1, is the gene (sglS) encoding a Na+/glucose symporter (SglS) . We characterize the Na+/glucose symporter produced in Escherichia coli mutant (JM1100) cells which lack original glucose transport activity and galactose transport activity . We also show that phlorizin, a potent inhibitor of the SGLT1 Na+/glucose symporter of animal cells, inhibited glucose transport, but not galactose transport, via the SglS system. J Bacteriol, 1997 Mar, 179(5), 1591 - 7 The light organ symbiont Vibrio fischeri possesses two distinct secreted ADP-ribosyltransferases; Reich KA et al.; We have previously described the purification, cloning, and initial characterization of a secreted ADP-ribosyltransferase, halovibrin (gene designation hvn), from the luminescent light organ symbiont Vibrio fischeri . This report describes a strategy for overexpression of halovibrin, the production and refinement of antihalo-vibrin antisera, and the molecular biological construction of a V . fischeri halovibrin null strain . Biochemical analysis of this mutant revealed that V . fischeri hvn null still possessed ADP-ribosyltransferase activity and that this activity is immunologically, genetically, and structurally distinct from the previously described enzyme . This unusual finding, of two ADP-ribosyltransferase enzymes produced by a microorganism, is complemented by the details of the purification to apparent homogeneity and in vitro regulation of this new protein, halovibrin-beta. J Clin Microbiol, 1997 Mar, 35(3), 624 - 30 Emergence of a new clone of toxigenic Vibrio cholerae O1 biotype El Tor displacing V . cholerae O139 Bengal in Bangladesh; Faruque SM et al.; The emergence of Vibrio cholerae O139 Bengal in 1993, its rapid spread in an epidemic form, in which it replaced existing strains of V . cholerae O1 during 1992 and 1993, and the subsequent reemergence of V . cholerae O1 of the El Tor biotype in Bangladesh since 1994 have raised questions regarding the origin of the reemerged El Tor vibrios . We studied 50 El Tor vibrio strains isolated in Bangladesh and four other countries in Asia and Africa before the emergence of V . cholerae O139 and 32 strains isolated in Bangladesh during and after the epidemic caused by V . cholerae O139 and 32 strains isolated in Bangladesh during and after the epidemic caused by V . cholerae O139 to determine whether the reemerged El Tor vibrios were genetically different from the El Tor vibrios which existed before the emergence of V . cholerae O139 . Analysis of restriction fragment length polymorphisms in genes for conserved rRNA, cholera toxin (ctxA), and zonula occludens toxin (zot) or in DNA sequences flanking the genes showed that the El Tor strains isolated before the emergence of V . cholerae O139 belonged to four different ribotypes and four different ctx genotypes . Of 32 El Tor strains isolated after the emergence of O139 vibrios, 30 strains (93.7%) including all the clinical isolates belonged to a single new ribotype and a distinctly different ctx genotype . These results provide evidence that the reemerged El Tor strains represent a new clone of El Tor vibrios distinctly different from the earlier clones of El Tor vibrios which were replaced by the O139 vibrios . Further analysis showed that all the strains carried the structural and regulatory genes for toxin-coregulated pilus (tcpA, tcpI, and toxR) . All strains of the new clone produced cholera toxin (CT) in vitro, as assayed by the GM1-dependent enzyme-linked immunosorbent assay, and the level of CT production was comparable to that of previous epidemic isolates of El Tor vibrios . Further studies are required to assess the epidemic potential of the newly emerged clone of V . cholerae O1 and to understand the mechanism of emergence of new clones of toxigenic V . cholerae. Gastroenterology, 1997 Mar, 112(3), 839 - 46 The enterotoxic effect of zonula occludens toxin on rabbit small intestine involves the paracellular pathway; Fasano A et al.; BACKGROUND & AIMS: Zonula occludens toxin is a novel toxin elaborated by Vibrio cholerae that modulates intestinal tight junctions . The aim of this study was to establish whether the permeabilizing effect of the toxin leads to intestinal secretion . METHODS: Rabbit intestine was mounted in Ussing chambers and exposed to increasing concentrations of purified toxin . The tissues were also fixed, exposed to zonula occludens toxin, and processed for fluorescence microscopy to determine the distribution of the toxin receptor within the intestine . Then purified toxin was simultaneously perfused in three distinct rabbit intestinal segments in vivo, and water and electrolyte absorption were measured . RESULTS: Zonula occludens toxin induced a time- and dose-dependent decrease of tissue resistance starting at a toxin concentration of 1.1 x 10(-13) mol/L . When tested in vivo, the toxin induced a secretion of water and chloride and the passage of polyethylene glycol 4000 in the bloodstream . Both the in vitro and in vivo effects of the toxin were observed only in the small intestine but not in the colon and paralleled the distribution of the toxin receptor within the intestine . CONCLUSIONS: The intestinal secretion induced by zonula occludens toxin follows the opening of tight junctions caused by the toxin, possibly representing a novel mechanism of intestinal secretion. Infect Immun, 1997 Mar, 65(3), 1131 - 4 Bile affects production of virulence factors and motility of Vibrio cholerae; Gupta S et al.; The effect of bile on the expression of cholera toxin (CT) and the major subunit of the toxin-coregulated pilus (TcpA) and on motility was examined in the Vibrio cholerae O1 classical-biotype strains 0395 and 569B . Although the motility of the cells increased significantly in the presence of bile, transcription of the ctxAB genes, encoding CT, and of the tcpA gene was drastically reduced . In toxR mutant strains, motility is higher than in the wild-type strain and was further increased, by about 150%, in the presence of bile . Bile represses CT production in strain 569B-55, a toxR mutant of strain 569B, which normally produces more than 80% of the amount of CT synthesized in the wild-type cells . These results suggest that bile may target some factor other than ToxR that is involved in the regulation of CT production and motility . Bile has no effect on the relative amounts of the two outer membrane porins, OmpU and OmpT, which are under ToxR control. Biochemistry (Mosc), 1997 Feb, 62(2), 225 - 30 The role of the sodium cycle of energy coupling in the emergence and persistence of natural foci of modern cholera; Brown II et al.; A hypothesis on the appearance and persistence of natural foci of cholera based on ecological and bioenergetic features of the process has been developed . The main causes of persistence and propagation of modern cholera are: 1) inability of various bacteria, including the genus Vibrio and many cyanobacterial species, to perform energy coupling, depending on external conditions, by means of two cycles (the proton and sodium cycles); induction of the sodium cycle of energy coupling increases the resistance of bacteria to various environmental factors, such as high concentrations of sodium, alkaline pH, and a high proton conductance of coupling membranes {1}, and probably the virulence of the vibrios; 2) development of cyanobacteria in an aquatic environment enriched with Na+ accelerates alkalization of the medium and stimulates the development of the community of cyanobacteria with Vibrio cholerae, an autochthonous inhabitant of saline water bodies and marine shallow waters; 3) salinization of water bodies accelerates their blooming and enriches them with soluble organic matter, a substrate for vibrios inhabiting the biotope; 4) further propagation of cholera infection is related to eating heat-untreated hydrobionts from blooming water bodies {2}. Hum Exp Toxicol, 1997 Feb, 16(2), 101 - 5 Membrane attack induced by HlyA, a pore-forming toxin of Vibrio cholerae; Huntley JS et al.; 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. FEMS Microbiol Lett, 1997 Feb 1, 147(1), 115 - 20 Purification and characterisation of a hemolysin with phospholipase C activity from Vibrio cholerae O139; Pal S et al.; A hemolysin was purified from a Vibrio cholerae O139 strain which moved as a single protein band of 67 kDa in SDS-PAGE . The hemolysin showed high level of phospholipase C activity . The purified phospholipase C-hemolysin demonstrated enterotoxic activity in rabbit ileal loop, suckling mice and enhanced permeability of rabbit skin . The pI of the purified hemolysin was 6.4 . Erythrocytes from rabbit, chicken, guinea pig, sheep and horse were sensitive to the purified hemolysin in decreasing order of intensity . Erythrocytes from human and cow were unaffected by purified hemolysin. Appl Environ Microbiol, 1997 Feb, 63(2), 537 - 42 An enzyme-linked immunosorbent assay for detection of Vibrio vulnificus biotype 2: development and field studies; Biosca EG et al.; Vibrio vulnificus biotype 2 is a primary eel pathogen which constitutes a lipopolysaccharide (LPS)-based homogeneous O serogroup within the species . In the present work, we have developed an enzyme-linked immunosorbent assay (ELISA) based on the specificity of LPS for the detection of this pathogen . The ELISA specificity was confirmed after testing 36 biotype 2 strains from laboratory cultures and environmental samples, 31 clinical and environmental biotype 1 isolates, and several strains of Vibrio, Aeromonas, and Yersinia species, including the fish pathogens V . anguillarum, V . furnissii, A . hydrophila, and Y . ruckerii . The detection limits for biotype 2 cells were around 10(4) to 10(5) cells/well, and the immunoassay was also able to detect cells in the nonculturable state . Artificially infected eels and environmental samples were analyzed, and the immunodetection was confirmed by cultural methods (isolation on selective and nonselective media before and after broth enrichment) . With this methodology, V . vulnificus biotype 2 was successfully detected in infected eels and asymptomatic carriers, which suggests that eels can act as a reservoir for this pathogen. Curr Microbiol, 1997 Feb, 34(2), 110 - 7 Alkaline serine protease is an exotoxin of Vibrio alginolyticus in kuruma prawn, Penaeus japonicus; Lee KK et al.; An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn(Penaeus japonicus) was partially purified by Fast Protein Liquid Chromatography with hydrophobic interaction (Phenyl Sepharose Hig hPerformance) chromatography and gel filtration columns . The toxin is an alkaline serine protease, inhibited by phenyl-methylsulfonyl fluoride (PMSF),and showed maximal activity at pH 10, having a molecular weight of about 33kDa estimated by SDS-PAGE and gel filtration chromatography . In addition, the toxin was also completely inhibited by FeCl2 but partially inhibited by CaCl2, CuCl2, CoCl2,MnCl2, and ZnCl2, and not inhibited by ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-amino-ethyl ether)N,N,N',N'-tetraacetic acid (EGTA), iodoacetamide, pepstatin A, sodium dodecylsulfate (SDS), and N-tosyl-l-phenyl-alanine chloromethyl ketone (TPCK) . Both the crude extracellular products (ECP) and the partially purified toxin are lethal for kuruma prawn at LD50 values of 0.30 and 0.27 microg protein/g body weight, respectively . The addition of PMSF completely inhibited the lethal toxicity of both the ECP and the partially purified toxin, indicating that this serine protease is a lethal factor produced by the bacterium . The 33-kDa protease is, therefore, suggested to be a new toxic protease produced by V . alginolyticus strain Swy. J Neurochem, 1997 Feb, 68(2), 878 - 81 Expression of sulfated gangliosides in the central nervous system; Farrer RG et al.; Several sulfated lipids were detected in the ganglioside fraction isolated from a cell line of oligodendrocyte progenitors that had been metabolically labeled with {35S} sulfate . Separation of the ganglioside fraction by two-dimensional TLC showed that, except for galactosylceramide-sulfate, none of the sulfate-labeled lipids comigrated with those glycosphingolipids visualized by orcinol staining, indicating that these sulfolipids were quantitatively minor components . At least eight sulfate-labeled lipid bands were susceptible to desialylation by Arthrobacter ureafaciens neuraminidase, which resulted in the formation of three new bands that retained the labeled sulfate . Six of the sulfate-labeled lipid bands containing sialic acid were also susceptible to Vibrio cholerae neuraminidase, which generated two labeled bands that appeared identical to the two major products formed after treatment with A . ureafaciens neuraminidase . In vivo labeling of lipids from 14-day-old rat brain with {35S}-sulfate demonstrated that the synthesis of sulfated lipids containing sialic acid also occurred in intact brain tissue . These results show that sulfated gangliosides are synthesized in the CNS and that oligodendrocytes are one cell type that contributes to this synthesis. Gene, 1997 Jan 31, 185(1), 43 - 7 The rfaD locus: a region of rearrangement in Vibrio cholerae O139; Vimont S et al.; We analyzed the rfaD locus of the novel epidemic Vibrio cholerae strain O139, a putative region of rearrangement . This region includes 4 orfs in the same orientation . Two orfs, rfaD(O139) and orf2(O139) were almost identical to those described in V . cholerae O1 . In contrast, the two other orfs upstream from rfaD(O139), designated orfA(O139) and orfB(O139), were absent from V . cholerae O1, but present in environmental strains of V . cholerae O22, O141 and O155 . These results suggest that a chromosomal rearrangement might have occurred in the vicinity of rfaD in V . cholerae O1, resulting in the emergence of V . cholerae O139 . The putative source of exogenous DNA might have been V . cholerae O22, O141 and O155. Biochemistry, 1997 Jan 28, 36(4), 665 - 72 Structure of bacterial luciferase beta 2 homodimer: implications for flavin binding; Tanner JJ et al.; The crystal structure of the beta 2 homodimer of Vibrio harveyi luciferase has been determined to 2.5 A resolution by molecular replacement . Crystals were grown serendipitously using the alpha beta form of the enzyme . The subunits of the homodimer share considerable structural homology to the beta subunit of the alpha beta luciferase heterodimer . The four C-terminal residues that are disordered in the alpha beta structure are fully resolved in our structure . Four peptide bonds have been flipped relative to their orientations in the beta subunit of the alpha beta structure . The dimer interface of the homodimer is smaller than the interface of the heterodimer in terms of buried surface area and number of hydrogen bonds and salt links . Inspection of the subunits of our structure suggests that FMNH2 cannot bind to the beta 2 enzyme at the site that has been proposed for the alpha beta enzyme . However, we do uncover a potential FMNH2 binding pocket in the dimer interface, and we model FMN into this site . This proposed flavin binding motif is consistent with several lines of biochemical and structural evidence and leads to several conclusions . First, only one FMNH2 binds per homodimer . Second, we predict that reduced FAD and riboflavin should be poor substrates for beta 2 . Third, the reduced activity of beta 2 compared to alpha beta is due to solvent exposure of the isoalloxazine ring in the beta 2 active site . Finally, we raise the question of whether our proposed flavin binding site could also be the binding site for flavin in the alpha beta enzyme. Lancet, 1997 Jan 25, 349(9047), 231 - 5 Field trial of a locally produced, killed, oral cholera vaccine in Vietnam; Trach DD et al.; BACKGROUND: Several studies have shown that orally administered killed cholera vaccines are safe and protective in populations at risk of cholera in developing countries . However, these vaccines have not been adopted for use in developing countries because of their expense and limited efficacy in young children . We have tested an inexpensive, killed whole-cell cholera vaccine developed and produced in Vietnam . METHODS: The efficacy of the vaccine was assessed in a large-scale, open field trial in people at least 1 year old residing in 22,653 households in the central coastal city of Hue . Alternate households were assigned vaccine (67,395 people; two doses per person) or no vaccine