Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans.

Isogenic mutant strains of V. cholerae O1 lacking elements of a genetic regulon controlled by toxR and implicated in virulence were tested in volunteers. A deletion mutation in ctxA, the gene encoding the A subunit of cholera toxin, markedly attenuated disease symptoms without affecting intestinal colonization. Deletion of toxR, the gene encoding the cholera toxin-positive regulatory protein resulted in a diminution in colonizing capacity. A deletion mutation in tcpA, encoding the major subunit of the toxin coregulated pilus (regulated by toxR), abolished the colonizing capacity of this strain. These results show for the first time the role of a specific pilus structure in colonization of the human intestine by V. cholerae O1 and exemplify the significance of a genetic regulon in pathogenesis.

Sulfur-containing nucleoside from yeast transfer ribonucleic acid: 2-thio-5(or 6)-uridine acetic acid methyl ester.

A nucleoside, isolated from yeast transfer RNA, has been assigned the structure 2-thio-5-uridine acetic acid methyl ester on the basis of high-esolution mass spectrometry, chemical properties, and ultraviolet spectra. The alternate 6-substituted isomeric structure cannot yet be completely ruled out.

Cytokinins in the soluble RNA of plant tissues.

The cytokinin, N(6)-(Delta(2)-isopentenyl) adenosine occurs in the soluble RNA of yeast and mammalian tissue and has now been detected in plant soluble RNA. A hydroxylated derivative of this cytokinin 6-(cis-4-hydroxy-3-methylbut-2-enylamino)-9-,beta-D-ribofuranosylpurine has also been identified as a constituent of plant soluble RNA.

Cytokinin activity of ureidopurine derivatives related to a modified nucleoside found in transfer RNA.

By use of a radioactive labeling technique N-(nebularin-6-ylcarbamoyl)threonine has been detected in plant transfer RNA. Derivatives of this nucleoside promote cell division of a cytokinin-requiring soya bean tissue.

A high molecular weight RNA fraction in chromatin.

Utilizing a new chromatin isolation and fractionation technique we have obtained a high molecular weight RNA fraction from L-929 cell chromatin. The synthesis of this RNA is not greatly inhibited by concentrations of 0.04 mug/ml actinomycin D in the medium. Its synthesis appears to be strongly inhibited by 2 mug/ml of alpha-amanitin. The RNA appears to be quickly degraded (or removed from the chromatin) and does not contain a poly(A) sequence at its 3'-OH terminal end. Our working hypothesis is that this RNA is "nascent" heterogenous nuclear RNA partially transcribed from regions of the chromatin.

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

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

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

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

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

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

Measles virus antibodies in a laboratory colony of owl monkeys (Aotus trivirgatus).

Serologic testing revealed that 17/84 (20.2%) of bought-in Aotus and 1/31 (3.2%) of colony-born Aotus had haemagglutination-inhibition antibody. Clinically-inapparent measles infections were detected in 3 monkeys by increased antibody titres. Following the detection of a recent infection, antibody titre persisted at a high level for at least 240 days. Although 84% of the monkeys were sero-susceptible, no further serological evidence of measles infection occurred.

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

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

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

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

A new threat to public health: organochlorines and food.

Ambient levels of persistent toxic chemicals, chemicals that persist for decades in the environment, have reached levels high enough to affect the health of children. The organochlorines (PCBs, DDT and the dioxin family) accumulate in human adipose tissue. Pregnant women pass the contamination to their fetuses. The developing nervous system is the most vulnerable. Neurobehavioral deficits, including short-term memory loss, are detected in children born to mothers at the high end of the distribution curve of organochlorines. Humans are not alone in their susceptibility to these subtle effects. Wildlife exposed to the same spectrum of organochlorines as humans suffer a variety of behavioral changes. Rats and Rhesus monkeys fed diets containing the organochlorines under laboratory conditions exhibit behavioral changes that persist into adulthood. For humans, food provides 80 percent of organochlorine contamination. Meat, fish, dairy and commercial fruit are the main sources. A vegetarian diet including unsprayed fruit minimizes contamination. The ultimate solution to this public health problem is elimination of the organochlorines from the environment.

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

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