Differentiation between thermophilic Campylobacter species by species-specific antibodies

P.L. GRIFFITHS. G.S. MORENO AND R.W. A. PARK. 1992. The four species of thermophilic camplyobacters, Campylobacter jejuni, C. coli, C. upsaliensis and C. lari , are difficult to distinguish from each other because of their lack of reactivity in many conventional biochemical and physiological tests. Those tests which do discriminate sometimes give discordant results. Species-specific antibody preparations (APs), capable of discriminating between the thermophilic campylobacter species by dot-ELISA, were raised by inoculation of mice with partially purified membrane protein. The APs produced were absorbed with cells of cross-reactive species and tested by dot-ELISA against reference and natural strains, the identities of which were confirmed by DNA/DNA hybridization. The results showed that such APs could be useful as an alternative to DNA/DNA hybridization for rapid species identification, for example in epidemiological surveys. Western blotting experiments with the APs showed that the specificity of the antibodies was not due to a single antigen.     

Meiosis in NEUROSPORA CRASSA. I. the Isolation of Recessive Mutants Defective in the Production of Viable Ascospores

A scheme has been devised for efficient isolation of recessive meiotic mutants of Neurospora crassa. These mutants were detected by their reduced fertility or by the abortion of ascospores. Their isolation involved the selection and screening of the strains arising from ascospores disomic (n + 1) for linkage group I (LG I), which bears the mating-type locus. These strains are self-fertile heterokaryons that contain two types of haploid nuclei of opposite mating types (A + a). Selfings of these strains are homozygous for genes on all linkage groups except LGI and therefore allow the expression of recessive mutants with an altered sexual cycle. Using this selection procedure, three classes of mutants were detected. In one class, mutants had an early block in perithecial development (class I), and in another mutants had altered perithecia, but apparently unaltered fertility (class III). No recessive mutants were observed and all mutants tested (eight of class I and two of class III) were expressed only when used as the maternal parent. A third mutant class displayed normal production of perithecia, but defective formation of asci (class IIA), or black ascospores (class IIB). Four of 13 class IIA mutants were analyzed, and two of them [asc(DL131) and asc (DL400)] were definitely recessive analysis of 10 of 13 class IIB mutants disclosed six recessive, mutually complementing mutants: ase(DL95), asc(DL243), asc(DL711), asc(DL879), asc(DL917m) and asc(DL961). Mutants asc(DL95), asc(DL243) and the previously studied mei-1 mutant (Smith 1975) complemented one another in crosses, but did not recombine. These may be alleles of the same gene, or they may comprise a gene cluster.     

DNA synthesis in permeabilized WI38 and MRC5 cells

DNA synthesis was examined in cultures of growing WI38 and MRC5 cells made permeable to deoxyribonucleotides. Cells from late passage cultures showed a reduced rate of deoxythymidine triphosphate (dTTP) uptake as compared to cells from early- to mid-passage cultures. This reduction became evident earlier in WI38 cultures (passage 33) than in MRC5 cultures (passage 41). Although this reduced rate of incorporation appeared to be primarily due to a reduced percentage of replicating (S phase) cells in later passage cultures, some effect on the rate of DNA synthesis in replicating cells was also evident.     

Evidence for lysosomal reduction of cystine residues.

Previously reported evidence for the existence of a thiol: protein disulphide oxidoreductase in rat liver lysosomes has been re-examined and ambiguous results obtained. However, incubation of purified rat liver lysosomes with ¹²⁵I-labelled insulin at pH 5.5 shows that cathepsin D and a thiol-dependent enzyme other than cathepsin B or L are important in its digestion. The latter enzyme is most probably a thiol: protein disulphide oxidoreductase.     

Effect of hypertonic stress on mammalian cell lines and its relevance to freeze-thaw injury

The effect of subjecting the mammalian cell lines MRC-5 and CHO to hypertonic salt concentrations (0.16 to 2.4 m) and returning them to isotonic conditions was investigated. Parameters for measuring cell size, viability and release of radiochemical markers were used to determine the relative susceptibilities of the two cell lines to hypertonic stress and the relative effects of increasing and decreasing hypertonicity. The aim of this study was to determine how great a role hypertonic stress plays in freeze-thaw damage of mamalian cells. This type of study has been extensively used for erythrocytes but not for nucleated mamamlian cell lines.The findings were that considerable cell shrinkage occurred, with a minimum size at 0.6 m NaCl, but that this caused no cell injury or death. Injury, measured by cation leakage and release of membrane and cytoplasmic labels occurred whilst the cell was swelling after reaching its minimum volume. MRC-5 cells succumbed at relatively low salt concentrations and became denatured. CHO cells withstood far high salt concentrations but were then damaged during dilution back to isotonic conditions. Comparison of the data obtained from hypertonic stress experiments and freeze-thaw experiments showed many similarities for CHO cells and indicated that the cell membrane could withstand high salt concentrations both at constant and changing temperatures but were prone to injury on dilution back to isotonic conditions. MRC-5 cells were shown to be very prone to cold shock and the results indicated that they probably succumb to damage and death during the hypertonic phase of cooling rather than thawing thus explaining their much lower survival from freeze-thaw experiments than CHO cells. The influence of DMSO in delaying cell damage to higher salt concentrations and lessening disruptive swelling during dilution were also demonstrated.     

Alterations in tRNAs containing 2-methylthio-N6-(delta2-isopentenyl)-adenosine during growth of enteropathogenic Escherichia coli in the presence of iron-binding proteins.

Escherichia coli grown in chemically produced iron-deficient media have well characterized alterations in the chromatographic properties of tRNAs containing the modified nucleoside 2-methylthio-N6-(delta2-isopentenyl) adenosine. The present report shows that similar tRNA alterations occur in enteropathogenic E. coli inhibited by human milk and bovine colostrum, the inhibited bacteria containing 10% or less of the normal tRNA species. Adding sufficient iron to saturate the iron-binding capacity of the lactoferrin present in milk and colostrum reversed these changes which are probably due to a failure to methylthiolate the isopentenyladenosine. Although adding iron led to a rapid replacement of abnormal tRNA by the chromatographically normal species, and to a resumption of multiplication, the tRNA alterations are not directly related to the inhibition of growth. Strains of E. coli which grew normally in milk, colostrum and in defined media containing the iron-binding protein transferrin or ovotransferrin also contained about 90% of the abnormal species. Rapid conversion of abnormal tRNA to normal tRNA occurred on adding iron and in the absence of RNA synthesis. The tRNA changes are discussed in relation to their possible connection with both the adaptation of E. coli to growth under the iron-restricted conditions imposed by iron-binding proteins in tissue fluids and with bacterial pathogenicity.     

The fine structure of conidial development in the genus Torula. III. T. graminis Desm.

Torula graminis produced blastoconidia in acropetalous chains after the evagination of a characteristic conidiogenous cell. Conidia consisted of up to 15 cells and their cell wall was differentiated into an outer melanized zone and an inner hyaline zone. A consistent cytoplasmic feature of conidial cells was the presence of dictyosomal-like membranous stacks often closely associated with the nucleus. Vesicles that developed from the dictyosomal-like cisternae were probably involved in conidial wall synthesis.