Responses of Listeria monocytogenes to acid stress and glucose availability revealed by a novel combination of fluorescence microscopy and microelectrode ion-selective techniques

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H(+) at either side of the bacterial membrane in real time and therefore to evaluate the rate of H(+) flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH(o)) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH(i)) and net H(+) fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH(i) remained above 7.0 within a pH(o) range of 4 to 6 and decreased below pH(o) 4. Above pH(o) 4, H(+) extrusion increased correspondingly, with the maximum value at pH(o) 5.5, and below pH(o) 4, a net H(+) influx was observed. Without glucose in the medium, the pH(i) decreased, and a net H(+) influx was observed below pH(o) 5.5. A high correlation (R = 0.75 to 0.92) between the pH(i) and net H(+) flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH(i) homeostasis in L. monocytogenes subjected to acid stress.     

Preliminary genetic linkage map of Miscanthus sinensis with RAPD markers

We have used an "offspring cross" mapping strategy in combination with the random amplified polymorphic DNA (RAPD) assay to construct the first genetic map of the species Miscanthus sinensis (2 n = 2 x = 38). This map is based on an outbred population of 89 individuals resulting from the cross between two genotypes from a previously designed cross. Consequently, both parents are fullsibs. The same proportion of bi-parental markers (heterozygotic in both parents) and pseudo-testcross markers (heterozygotic in one parent and null in the other), mono-parental markers, have been obtained. A total of 383 RAPD markers were analysed within the 89 F1 plants. Out of these markers, 257 were mapped into 28 linkage groups which spanned a total map length of around 1,074.5 cM with an average density of 4.2 cM per marker. Out of 257 mapped markers, 62 were inherited from F1.1 (P1), 63 from F1.7 (P7) and 132 were bi-parental markers. The contribution to the map was equal from both parents. This map provides a useful tool for genetic analyses of agronomically interesting characters in M. sinensis such as flowering, yield, plant height, stem diameter and mineral constitution. The offspring cross mapping strategy is proposed to obtain a higher efficiency in developing integrated maps including both parents.     

Presence of Pleurotus ostreatus in Patagonia,Argentina

Specimens belonging to the genus Pleurotus were collected growing on fallen trunks of Araucaria araucana, a native tree with a poorly known mycoflora, which grows in Patagonia, Argentina. Fruitbodies were produced in culture on sawdust from an isolated strain. Interspecific pairing tests performed between mating types of Pleurotus from Patagonia and tester strains of P. pulmonarius and P. ostreatus showed the Patagonia strain to be 100% compatible with P. ostreatus and incompatible with P. pulmonarius. Dikaryons obtained on sawdust were fertile, since they were able to produce fruitbodies and viable spores. This is the first documented record of P. ostreatus from Argentina and the first gilled fungus found growing on Araucaria araucana.     

Diversity of European Lyme Disease Spirochetes at the Southern Margin of Their Range

We determined whether the genospecies diversity of Lyme disease spirochetes in vector ticks questing on a subtropical island is as broad as that in Central Europe. Although spirochetes infected <1% of the ticks sampled on Madeira Island, these infections included all three genospecies implicated in human disease. Therefore, spirochetal diversity is as great at the southern margin as it is in the center of this pathogen’s range.     

Localization of epitopes for monoclonal antibodies to urokinase-type plasminogen activator: relationship between epitope localization and effects of antibodies on molecular interactions of the enzyme.

We localized the epitopes for several murine mAbs to human urokinase-type plasminogen activator (uPA) by Ala scanning mutagenesis and related the localization to the effects of the mAbs on the molecular interactions of uPA. Several antibodies against the serine proteinase domain (SPD) were found to have overlapping epitopes composed of variable combinations of Arg178, Arg179, His180, Arg181, Tyr209, Lys211, and Asp214 in the so-called 37-loop and 60-loop, located near the active site and taking part in the binding of uPA to plasminogen activator inhibitor-1 (PAI-1). Besides inhibiting uPA-catalysed plasminogen activation, all antibodies to SPD strongly delayed the binding of uPA to PAI-1, decreasing the second-order rate constant 15- to 6500-fold. There was no correlation between the relative effects of the 37-loop and 60-loop substitutions on the second-order rate constant and on the binding of the antibodies, indicating that the antibodies did not delay complex formation by blocking residues of specific importance for the uPA-PAI-1 reaction, but rather by steric hindrance of the access of PAI-1 to the active site. The affinity of the SPD antibodies for the uPA-PAI-1 complex was only slightly lower than that for free uPA, indicating that the 37-loop and 60-loop are exposed in the complex. The epitopes for two antibodies to the kringle included Arg108, Arg109, and Arg110. The ability of these antibodies to block the binding of uPA to polyanions correlated with a reduced uPA-polyanion affinity after substitution of the three Arg residues.     

Comparison of barley malt α-amylase isozymes 1 and 2: construction of cDNA hybrids by in vivo recombination and their expression in yeast

Germinating barley produces two α-amylase isozymes, AMY1 and AMY2, having 80% amino acid (aa) sequence identity and differing with respect to a number of functional properties. Recombinant AMY1 (re-AMYI) and AMY2 (re-AMY2) are produced in yeast, but whereas all re-AMYI is secreted, re-AMY2 accumulates within the cell and only traces are secreted. Expression of AMY1::AMY2 hybrid cDNAs may provide a means of understanding the difference in secretion efficiency between the two isozymes. Here, the efficient homologous recombination system of the yeast, Saccharomyces cerevisiae, was used to generate hybrids of barley AMY with the N-terminal portion derived from AMY1, including the signal peptide (SP), and the C-terminal portion from AMY2. Hybrid cDNAs were thus generated that encode either the SP alone, or the SP followed by the N-terminal 21, 26, 53, 67 or 90 aa from AMY1 and the complementary C-terminal sequences from AMY2. Larger amounts of re-AMY are secreted by hybrids containing, in addition to the SP, 53 or more aa of AMY1. In contrast, only traces of re-AMY are secreted for hybrids having 26 or fewer aa of AMY1. In this case, re-AMY hybrid accumulates intracellularly. Transformants secreting hybrid enzymes also accumulated some re-AMY within the cell. The AMY1 SP, therefore, does not ensure re-AMY2 secretion and a certain portion of the N-terminal sequence of AMY1 is required for secretion of a re-AMYI::AMY2 hybrid.     

Reduced childhood mortality after standard measles vaccination at 4-8 months compared with 9-11 months of age.

OBJECTIVE--To evaluate the impact on mortality of standard Schwarz measles immunisation before 9 months of age. DESIGN--Children vaccinated in 1980-3 at 4-5, 6-8, and 9-11 months of age were followed to migration, death, or the age of 5 years. SETTING--One urban district and nine villages in two rural areas of Guinea-Bissau. SUBJECT--307 children vaccinated at 4-8 months and 256 at 9-11 months. MAIN OUTCOME MEASURES--Mortality from 9 months to 5 years of age for children immunised at 4-5, 6-8, and 9-11 months. RESULTS--Mortality was significantly lower in children vaccinated at 6-8 months than at 9-11 months (mortality ratio = 0.63, (95% confidence interval 0.41 to 0.97), p = 0.047). As vaccination was provided in semiannual or annual campaigns it is unlikely that age at vaccination reflected a selection bias. The trend was the same in all three study areas. Improved survival after early immunisation was not related to better protection against measles infection. With a Cox multivariate regression model to adjust for age, sex, season at risk, season at birth, measles infection, and region, children vaccinated at 4-8 months had a mortality ratio of 0.61 (0.40 to 0.92, p = 0.020) compared with children vaccinated at 9-11 months. Reimmunised children tended to have lower mortality than children who received only one vaccine (0.59 (0.28 to 1.27, p = 0.176)). CONCLUSION--Standard measles vaccination before 9 months is not associated with higher childhood mortality than is the currently recommended strategy of immunising from 9 months, and it may reduce mortality. This has implications for measles immunisation strategy in developing countries.     

Equilibrium sorption of Cr6+ by a consortia of denitrifying bacteria

Summary The accumulation of metal ions on to biological materials is a promising method for removing these toxic materials from waste or ground-waters. A fractional factorial statistical experimental design was used to ascertain which experimental parameters affect the sorption of Cr⁶⁺ by a consortium of denitrifying bacteria using a minimum number of experiments. Analysis of this set of experiments found that the steady state sorption behavior could be described by Langmuir isotherms. Observed variations in the isotherm parameters were due to the imposed changes in the experimental variables. In particular, solution pH and the state of the biomass were found to have the greatest influence upon the amount of chromium that was taken up, while other factors had less significance, but were still important.