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PubMed2002

Molecular immunogenetics in susceptibility to bovine dermatophilosis: a candidate gene approach and a concrete field application.

To identify molecular genetic markers of resistance or susceptibility to dermatophilosis in cattle, we used a functional candidate gene approach to analyze the DNA polymorphisms of targeted genes encoding molecules implicated in known mechanisms of both nonspecific and specific immune responses existing in the pathogen/host interface mechanisms. The most significant results were obtained within the Major Histocompatibility Complex (MHC) where the BoLA-DRB3 and DQB genes encode molecules involved in the antigen presentation to T cell receptors. A unique BoLA class II haplotype, made up of one DRB3 exon 2 allele and one DQB allele, highly correlates with the susceptibility character (P < 0.001). This haplotype marker of susceptibility was also found and validated in other bovine populations. A eugenic marker-assisted selection was developed in the field by eliminating only the animals having this haplotype. The disease prevalence was thereby reduced from 0.76 to 0.02 over 5 years. A crossbreeding plan is in progress to study the genetic transmission of the genotypic and phenotypic characters of susceptibility to dermatophilosis. In conclusion, we discuss several hypotheses at the molecular and cellular levels to better define the exact role of the MHC molecules in disease control and to answer the question: How is MHC diversity selectively maintained by natural selection imposed by pathogens?

Actinomycetales Infections↗
PubMed1980

Achievements of the seventies: community and school fluoridation.

Communal water fluoridation continues to be the cornerstone of an ideal caries prevention program. Its efficacy in reducing caries prevalence by about 50 to 60 percent was demonstrated throughout the seventies from Perth to Toronto. During the past decade, opponents of fluoridation have relentlessly attacked the safety of the procedure, but careful scientific analyses have not sustained their criticisms. The health and other scientific communities continue to endorse and recommend the practice of water fluoridation for improving dental health. During the seventies an additional 17 million Americans have benefitted from drinking fluoridated water, but this progress is too slow, particularly when compared with that of the fifties and sixties. Fluoridation campaigns in Seattle, Greater Boston, and California's Marin County and East Bay Municipal Utility district provide examples of successful tactics. An analysis of unsuccessful campaigns in Eugene, Los Angeles, and the State of Utah reveals mistakes which should not be repeated in future elections. For the eighties new approaches will be needed, particularly to reach the many smaller remaining communities. It is essential that the ADA and the USPHS maintain a stable office with sufficient staff and expertise on fluoridation. Training programs are needed to help prepare local community leaders and to assist them in obtaining fluoridation for their communities. Additional studies have documented the efficacy of school fluoridation programs for areas lacking a public water supply. Many school fluoridation programs were introduced during the seventies; nevertheless, they serve a very small number of children.

Attitude↗
PubMed2004

Selective knock-down of P2X7 ATP receptor function by dominant-negative subunits.

Among the family of P2X ATP-gated cation channels, the P2X7 receptor is a homomeric subtype highly expressed in immune cells of the monocyte-macrophage lineage. We report here that the WC167-168AA mutation in the ectodomain of P2X7 produced nonfunctional subunits with strong dominant-negative effect on wild-type P2X7 receptors (77% inhibition with cotransfection of wild-type and mutant DNA at a ratio of 3:1). The C168A single mutant was also very effective in suppressing P2X7 receptor function (72% reduction at a DNA ratio of 3:1), indicating the major role played by the C168A mutation in this inhibition. The dominant-negative effect is selective; the mutant subunit did not suppress the function of other receptor-channel subtypes. The reduced current responses in cells coexpressing wild-type and dominant-negative subunits display wild-type characteristics in both agonist affinity and ionic selectivity, strongly suggesting that the heteromeric channels are functionally impaired. The mutant subunits also suppressed the P2X7-dependent pore formation as assessed by uptake of the propidium dye YO-PRO-1 (Molecular Probes, Eugene, OR) in response to 2',3'-O-(4-benzoyl)-benzoyl-ATP (BzATP) in transfected human embryonic kidney 293 cells. Native responses to BzATP as well as ATP-induced ethidium dye uptake were significantly knocked down (31 +/- 9% and 25 +/- 7% of control, respectively) in mouse macrophage cell line RAW264.7 transfected with the mutant subunits. Therefore, these dominant-negative subunits provide selective genetic tools to investigate the functional roles of native P2X7 receptors.

Animals↗
PubMed1980

Hummingbirds see near ultraviolet light.

Three species of hummingbird (Archilochus alexandri, Lampornis clemenciae, and Eugenes fulgens) were trained to make visual discriminations between lights of different spectral content. On the basis of initial choices of feeders following a period of conditioning, birds of all three species were able to distinguish near ultraviolet (370 nanometers, 20-nanometer half bandwidth) from darkness (unilluminated viewing screen) or from the small amount of far red light that leaked through the ultraviolet-transmitting glass filter. A human observer was unable to make either discrimination. The birds were also able to distinguish white lights lacking wavelengths shorter than 400 nanometers from the full spectrum of the quartz-halogen bulbs. One can infer that the cone oil droplets, which have been lost from the retinas of most mammals, provide a potentially more flexible system for restricting the short wavelength end of the visible spectrum than does the filtering action of lens and macula that serves this function in the human eye.

Animals↗
PubMed2000

A simple, rapid method for demonstrating bacterial flagella.

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.

Bacteria↗
PubMed2002

Double-staining method for differentiation of morphological changes and membrane integrity of Campylobacter coli cells.

We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells.

Campylobacter coli↗
PubMed2004

Microscale and molecular assessment of impacts of nickel, nutrients, and oxygen level on structure and function of river biofilm communities.

Studies were carried out to assess the influence of nutrients, dissolved oxygen (DO) concentration, and nickel (Ni) on river biofilm development, structure, function, and community composition. Biofilms were cultivated in rotating annular reactors with river water at a DO concentration of 0.5 or 7.5 mg liter(-1), with or without a combination of carbon, nitrogen, and phosphorus (CNP) and with or without Ni at 0.5 mg liter(-1). The effects of Ni were apparent in the elimination of cyanobacterial populations and reduced photosynthetic biomass in the biofilm. Application of lectin-binding analyses indicated changes in exopolymer abundance and a shift in the glycoconjugate makeup of the biofilms, as well as in the response to all treatments. Application of the fluorescent live-dead staining (BacLight Live-Dead staining kit; Molecular Probes, Eugene, Oreg.) indicated an increase in the ratio of live to dead cells under low-oxygen conditions. Nickel treatments had 50 to 75% fewer 'live' cells than their corresponding controls. Nickel at 0.5 mg liter(-1) corresponding to the industrial release rate concentration for nickel resulted in reductions in carbon utilization spectra relative to control and CNP treatments without nickel. In these cases, the presence of nickel eliminated the positive influence of nutrients on the biofilm. Other culture-dependent analyses (plate counts and most probable number) revealed no significant treatment effect on the biofilm communities. In the presence of CNP and at both DO levels, Ni negatively affected denitrification but had no effect on hexadecane mineralization or sulfate reduction. Analysis of total community DNA indicated abundant eubacterial 16S ribosomal DNA (rDNA), whereas Archaea were not detected. Amplification of the alkB gene indicated a positive effect of CNP and a negative effect of Ni. The nirS gene was not detected in samples treated with Ni at 0.5 mg liter(-1), indicating a negative effect on specific populations of bacteria, such as denitrifiers, resulting in a reduction in diversity. Denaturing gradient gel electrophoresis revealed that CNP had a beneficial impact on biofilm bacterial diversity at high DO concentrations, but none at low DO concentrations, and that the negative effect of Ni on diversity was similar at both DO concentrations. Notably, Ni resulted in the appearance of unique bands in 16S rDNA from Ni, DO, and CNP treatments. Sequencing results confirmed that the bands belonged to bacteria originating from freshwater and marine environments or from agricultural soils and industrial effluents. The observations indicate that significant interactions occur between Ni, oxygen, and nutrients and that Ni at 0.5 mg liter(-1) may have significant impacts on river microbial community diversity and function.

Biofilms↗
PubMed2002

Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces.

Little is known about fungal biofilms, which may cause infection and antibiotic resistance. In this study, biofilm formation by different Candida species, particularly Candida albicans and C. parapsilosis, was evaluated by using a clinically relevant model of Candida biofilm on medical devices. Candida biofilms were allowed to form on silicone elastomer and were quantified by tetrazolium (XTT) and dry weight (DW) assays. Formed biofilm was visualized by using fluorescence microscopy and confocal scanning laser microscopy with Calcofluor White (Sigma Chemical Co., St. Louis, Mo.), concanavalin A-Alexafluor 488 (Molecular Probes, Eugene, Oreg.), and FUN-1 (Molecular Probes) dyes. Although minimal variations in biofilm production among invasive C. albicans isolates were seen, significant differences between invasive and noninvasive isolates (P < 0.001) were noted. C. albicans isolates produced more biofilm than C. parapsilosis, C. glabrata, and C. tropicalis isolates, as determined by DW assays (P was <0.001 for all comparisons) and microscopy. Interestingly, noninvasive isolates demonstrated a higher level of XTT activity than invasive isolates. On microscopy, C. albicans biofilms had a morphology different from that of other species, consisting of a basal blastospore layer with a dense overlying matrix composed of exopolysaccharides and hyphae. In contrast, C. parapsilosis biofilms had less volume than C. albicans biofilms and were comprised exclusively of clumped blastospores. Unlike planktonically grown cells, Candida biofilms rapidly (within 6 h) developed fluconazole resistance (MIC, >128 microg/ml). Importantly, XTT and FUN-1 activity showed biofilm cells to be metabolically active. In conclusion, our data show that C. albicans produces quantitatively larger and qualitatively more complex biofilms than other species, in particular, C. parapsilosis.

Antifungal Agents↗
PubMed1961

Role of one-carbon precursors in the biosynthesis of deoxyribonucleic acid in bacteriophage infected and growing cells of Escherichia coli.

Nester, Eugene W. (Western Reserve University, Cleveland, Ohio) and John Spizizen. Role of one-carbon precursors in the biosynthesis of deoxyribonucleic acid in bacteriophage-infected and growing cells of Escherichia coli. J. Bacteriol. 82:867-874. 1961.-The ability of growing and T2 bacteriophage-infected cells of Escherichia coli to incorporate serine-3-C(14), glycine-2-C(14), formate-C(14), and formaldehyde-C(14) into purine and pyrimidine moieties of deoxyribonucleic acid (DNA) was determined. All four one-carbon precursors are effective contributors to the DNA-purines, but only glycine-2-C(14) and serine-3-C(14) are incorporated into the side chains of the pyrimidines. In addition, formate-C(14) becomes incorporated only into position 8 of the purine ring, whereas isotope from serine-3-C(14) and glycine-2-C(14) is incorporated equally into the 2 and 8 positions. No qualitative differences were observed in the patterns of incorporation of any one-carbon units in growing or bacteriophage-infected cells. However, the 3-carbon of serine serves as a more effective precursor of the 2 and 8 positions of the DNA purine ring when the cells are infected. Formate-C(14) and to a slight extent glycine-2-C(14) are somewhat better precursors of these positions when the cells are infected under appropriate conditions.

Bacteriological Techniques↗
PubMed1963

ELECTRON MICROSCOPY OF PLASMOLYSIS IN ESCHERICHIA COLI.

Cota-Robles, Eugene H. (University of California, Riverside). Electron microscopy of plasmolysis in Escherichia coli. J. Bacteriol. 85:499-503. 1963.-Escherichia coli cells plasmolyzed in 0.35 m sucrose reveal plasmolysis at one tip of a cell or in the center of dividing cells in which protoplast partition has been complete. Central plasmolysis reveals that protoplast separation can be completed before the invagination of the cell wall is complete. These studies support the concept that these cells divide by constriction. The strength of the union between cell wall and cytoplasm is not uniform around the entire cell. It is strongest along the sides of these rod-shaped cells and weakest at one tip of the single cell. Thus, a single cell generally forms one cup-shaped vacuole in which the cytoplasm has collapsed away from one tip of the cell.

Cell Division↗
PubMed1963

INTRACELLULAR M PROTEIN OF GROUP A STREPTOCOCCUS.

Fox, Eugene N. (University of Chicago, Chicago, Ill.). Intracellular M protein of group A Streptococcus. J. Bacteriol. 85:536-540. 1963.-A heat-labile M protein antigen in protoplasts of a type 14 strain of group A Streptococcus has been demonstrated in a soluble form in the cytoplasm, and also bound to the protoplasmic membrane. When trypsinized whole cells (from which the M protein on the cell wall had been digested) or protoplasts were extracted with hot HCl, no M protein antigen was detected, although the antigen was routinely extracted from the cell walls of normal streptococci by the hot acid procedure. Various serological techniques, including the use of fluorescein-labeled type-specific antisera, were employed to demonstrate the M antigen in association with the membrane from osmotically lysed protoplasts.

Bacterial Proteins↗
PubMed1963

QUANTITATION OF VIRUSES BY THE PLAQUE TECNHIQUE.

Berg, Gerald (Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio), Eugene K. Harris, Shih L. Chang, and Kenneth A. Busch. Quantitation of viruses by the plaque technique. J. Bacteriol. 85:691-700. 1963.-This paper presents the results of a study on overcrowding as it occurred with several strains of enteroviruses on monkey kidney cell layers. Each cell sheet area was 4,500 mm(2). Dispersion analysis of all plaque counts in the study revealed that the percentage error, in the absence of overcrowding, fluctuated within a range of 7 to 36% about a mean value of about 17%, which is close to what is expected from purely statistical variation in sampling. Overcrowding with Mahoney virus, a mixture of particles producing either rapidly or slowly expanding plaques, resulted primarily from the obscuring effect produced as rapidly expanding plaques obliterated infected foci produced by virus particles responsible for slow-forming plaques. Overcrowding occurred at plaque levels in excess of 35 to 40 per cell sheet. Overcrowding with Mahoney LP virus, a strain derived from a rapidly expanding Mahoney plaque which produced uniformly expanding plaques, did not occur until counts in excess of 60 to 70 per cell sheet were reached. Plaque obscuring was again responsible. Apparent overcrowding with Coxsackie A9 virus, when plaques were not permanently marked from the first day of counting, resulted from coalescence at levels above about 40 to 50 plaques per cell sheet. When plaques were permanently marked, overcrowding resulted from plaque obscuring which did not occur until levels in excess of about 120 per cell sheet were reached.

Enterovirus↗
PubMed1964

Proteolytic activity and general characteristics of a marine bacterium, Aeromonas proteolytica sp. N.

Merkel, Joseph R. (Fort Johnson Marine Biological Laboratory, College of Charleston, Charleston, S.C.), Eugene D. Traganza, Barid B. Mukherjee, Travis B. Griffin, and J. M. Prescott. Proteolytic activity and general characteristics of a marine bacterium, Aeromonas proteolytica sp. n. J. Bacteriol. 87:1227-1233. 1964.-A highly proteolytic bacterial species was isolated from the alimentary canal of the marine borer, Limnoria. The morphological and biochemical characteristics of the organism indicated that it was a new Aeromonas species, for which the name A. proteolytica is proposed. When freshly isolated, the organism required seawater for growth; but, upon prolonged culture in the laboratory, it was able to grow in media of greatly reduced salt concentration, provided that relatively large amounts of peptone were supplied. Peptone or hydrolysates of casein were capable of supplying all organic nutrients required for growth and proteinase production. Certain individual amino acids were also able to furnish all energy, carbon, and nitrogen requirements. Inorganic nitrogen was utilized in the presence of citrate, but could not serve as the only source of nitrogen in the presence of glucose. The organism was facultatively anaerobic, but best growth and proteinase production occurred only with vigorous aeration. The amount of growth obtained in 24 hr increased rapidly as the incubation temperature was increased up to a maximum of 40 C, but no growth occurred at 42 C.

Aeromonas↗
PubMed1964

NEW TRANSPORT MEDIUM FOR SHIPMENT OF CLINICAL SPECIMENS. I. FECAL SPECIMENS.

Cary, Sylvia G. (Walter Reed Army Institute of Research, Washington, D.C.), and Eugene B Blair. New transport medium for shipment of clinical specimens. I. Fecal specimens. J. Bacteriol. 88:96-98. 1964.-A new transport medium for the collection and shipment of clinical specimens is described. Preliminary studies indicate that, with initial fecal specimens, Salmonella and Shigella can be recovered for as long as 49 days, Vibrio comma for 22 days, and Pasteurella pestis for at least 75 days.

Culture Media↗
PubMed1964

EPITHELIAL CELL PENETRATION AS AN ESSENTIAL STEP IN THE PATHOGENESIS OF BACILLARY DYSENTERY.

LaBrec, Eugene H., Herman Schneider, Thomas J. Magnani, and Samuel B. Formal. Epithelial cell penetration as an essential step in the pathogenesis of bacillary dysentery. J. Bacteriol. 88:1503-1518. 1964.-A parent strain of Shigella flexneri 2a and a colonial mutant derived from it were studied in three animal models. Both strains were equally virulent for mice when living cells suspended in hog gastric mucin were injected by the intraperitoneal route. Feeding the parent strain to starved guinea pigs, followed by the intraperitoneal injection of opium, resulted in the formation of ulcerative lesions in the intestinal tract and in the death of these animals. When the colonial variant was fed to similarly prepared animals, the animals survived and the intestinal tract remained normal. The parent produced diarrheal symptoms and intestinal lesions after its oral administration to rhesus monkeys; the variant caused neither symptoms nor pathology in this species. Studies were carried out to define the characteristics present in the parent strain and absent in the colonial mutant, which would enable the parent to produce ulcerative lesions of the bowel and death in the guinea pig model or intestinal lesions and diarrheal symptoms in the monkey. Neither serological studies nor growth studies conducted both in vitro and in vivo offered a clue to explain this difference. The virulent parent strain was shown to penetrate the bowel epithelium and enter the lamina propria; the avirulent mutant did not do this. Entrance to the lamina propria was by way of the epithelial cell of the mucosa. The avirulent mutant did not possess the capacity to penetrate this cell. This observation was extended to show that the virulent parent possesses the ability to infect and multiply within HeLa cells; furthermore, the organisms are able to penetrate epithelial cells of the guinea pig cornea, causing ulcerative lesions. The avirulent variant possesses neither of these capacities. It is suggested that epithelial cell penetration is a major factor in determining the pathogenicity of dysentery bacilli.

Journal Article↗
PubMed1965

EFFECT OF OXYGEN ON GROWTH AND THE SYNTHESIS OF BACTERIOCHLOROPHYLL IN RHODOSPIRILLUM MOLISCHIANUM.

Sistrom, W. R. (University of Oregon, Eugene). Effect of oxygen on growth and the synthesis of bacteriochlorophyll in Rhodospirillum molischianum. J. Bacteriol. 89:403-408. 1965.-The basis of the strict anaerobiosis of the nonsulfur purple bacterium Rhodospirillum molischianum was investigated. The bacterium is unable to grow aerobically in the dark. In the light, the only immediate effect of oxygen is to inhibit the biosynthesis of bacteriochlorophyll and carotenoid pigments. Growth and protein synthesis are not inhibited directly, nor are the cells killed by oxygen. The inhibition of bacteriochlorophyll synthesis ultimately leads to inhibition of growth. Oxygen inhibits photopigment synthesis by means of a metabolic control system which is apparently identical with the one already known in facultatively aerobic species such as R. rubrum. Whole cells of R. molischianum respire in the dark; illumination inhibits respiration by about 50%. Cell-free extracts display significant levels of dark reduced nicotinamide adenine dinucleotide oxidase activity. Both these properties are in accord with the physiological behavior of the organism.

Anaerobiosis↗
PubMed1965

OBSERVATIONS ON THE GROUP C STREPTOCOCCAL BACTERIOPHAGE AND LYTIC ENZYME SYSTEM.

Fox, Eugene N. (La Rabida-University of Chicago Institute, Chicago, Ill.), and Masako K. Wittner. Observations on the group C streptococcal bacteriophage and lytic enzyme system. J. Bacteriol. 89:496-502. 1965.-The phage-associated lytic enzyme of group C streptococci was assayed by measuring the solubilized portion of radioactive cell walls. By this sensitive assay system, the induced synthesis of the lytic enzyme was observed intracellularly during phage infection; at least half of the total enzyme was synthesized and remained intracellular during the eclipse period, and was then released with the liberation of mature phage. Lytic enzyme could be detected in only two of eight lysogenic strains during temperate-phage production after ultraviolet induction. Virulent phage purified by density-gradient centrifugation contained lytic enzyme presumably associated with the virus per se. No hyalyronidase was detected in association with the phage, nor was this enzyme induced during phage synthesis. Variant strains of group C streptococci, no longer serologically active, were isolated as phage-resistant mutants. These strains still adsorbed the phage, but without subsequent virus reproduction, indicating that the group polysaccharide was not the primary receptor for the virus.

Bacteriolysis↗
PubMed1965

OXIDATION OF SECONDARY ALCOHOLS BY EXTRACTS OF A CORYNEBACTERIUM.

Rosenberg, Eugene (University of California, Los Angeles), and Paul Holmes. Oxidation of secondary alcohols by extracts of a Corynebacterium. J. Bacteriol. 89:1212-1216. 1965.-A Corynebacterium was isolated from soil by use of the enrichment culture technique. This Corynebacterium oxidized and grew on the chemically synthesized secondary alcohol, 3-tetrahydrofuranol. Extracts of this organism contained at least two different nicotinamide adenine dinucleotide-requiring soluble secondary alcohol dehydrogenases. These enzymes were distinguished by the relative rates at which they oxidized thymidylic acid and 3-tetrahydrofuranol. In addition to their substrate specificity, the two enzymes differed in pH optima and thermal stability. Also, the 3-tetrahydrofuranol dehydrogenase was induced by 3-tetrahydrofuranol, whereas the thymidylic acid dehydrogenase was constitutive.

Alcohol Oxidoreductases↗