Molecular, genetic and biochemical characterization of lactate dehydrogenase-A enzyme activity mutations in Mus musculus

Four independent heterozygous lactate dehydrogenase (LDH) mutations with approximately 60% of wild-type enzyme activity in whole blood have been recovered. The mutant line Ldh1 ^a2Neu proved to be homozygous lethal, whereas for the three lines Ldh1 ^a7Neu, Ldh1 ^a11Neu, and Ldh1 ^a12Neu homozygous mutants with about 20% residual activity occurred in the progeny of heterozygous inter se matings. However, the number of homozygous mutants was less than expected, suggesting an increased lethality of these animals. Various physicochemical and kinetic properties of LDH are altered. Exons of the Ldh1 gene were PCR amplified and sequenced to determine the molecular lesion in the mutant alleles. Ldh1 ^a2Neu carried an A/T → G/C transition in codon 112 (in exon 3), resulting in an Asn → Asp substitution; Asn112 is part of the helix αD, which is involved in the coenzyme-binding domain. Ldh1 ^a7Neu contained an A/T → C/G transversion within the codon for residue 194 in exon 4, causing an Asp → Ala substitution, which may affect the arrangement of the substrate-binding site. Three base substituions were discovered for the mutation Ldh1 ^a11Neu in exon 7: the transition C/G → T/A, a silent mutation, and two transversions C/G → A/T and C/G → G/C, both missense mutations, which led to the amino acid replacements Ala319 → Glu and Thr321 → Ser, respectively, located in the αH helix structure of the COOH tail of LDHA. We suggest that the mutation is the result of a gene conversion event between Ldh1 ^a wild-type gene and the pseudogene Ldh1-ps. The alteration Ile → Thr of codon 241 in exon 6 caused by the base pair change T/A → C/G was identified in the mutation Ldh1 ^a12Neu; IIe241 is included in the helix α2G, a structure that is indirectly involved in coenzyme binding. Each of the sequence alterations has a potential impact on the structure of the LDHA protein, which is consistent with the decreased LDH activity and biochemical and physiological alterations. Received: 3 July 1997 / Accepted: 30 September 1997     

Ergosterol levels in two l-methionine-enriched mutants of the methylotrophic yeast Candida boidinii ICCF26

Two L-methionine-enriched mutants, SN-78 and SE-57, were isolated in a sulphur-deficient medium from the methylotrophic yeast Candida boidinii ICCF26. No significant differences were detected between the L-cysteine pools of the mutants and the wild-type. In mutant strain SE-57, S-adenosylmethionine and ergosterol levels were higher than in the wild-type strain, while in the other mutant, SN-78, the levels were lower. The evidence presented would suggest that, in both the mutants and the wild-type used in this study, S-adenosylmethionine was of key importance for the accumulation of L-methionine and ergosterol.     

Selective Interaction Model and Photoreceptor Cell Membrane

A mathematical model is proposed in order to describe, from the thermodynamic point of view, the changes in the photoreceptorcell membrane induced by light stimuli. The phenomenologicalbackground is the increase of the fly microvillar membrane ionicconductivity as a consequence of Ca^++-Na⁺ affinity modification under light action. On the basis of the analogywith the model of protein interaction in mixed solvents, themodel is focused on the selective interaction between ionchannels gates and two ionic ligands. Three possible theoretical cases are examined.     

Girdling Affects Ectomycorrhizal Fungal (EMF) Diversity and Reveals Functional Differences in EMF Community Composition in a Beech Forest

The relationships between plant carbon resources, soil carbon and nitrogen content, and ectomycorrhizal fungal (EMF) diversity in a monospecific, old-growth beech (Fagus sylvatica) forest were investigated by manipulating carbon flux by girdling. We hypothesized that disruption of the carbon supply would not affect diversity and EMF species numbers if EM fungi can be supplied by plant internal carbohydrate resources or would result in selective disappearance of EMF taxa because of differences in carbon demand of different fungi. Tree carbohydrate status, root demography, EMF colonization, and EMF taxon abundance were measured repeatedly during 1 year after girdling. Girdling did not affect root colonization but decreased EMF species richness of an estimated 79 to 90 taxa to about 40 taxa. Cenococcum geophilum, Lactarius blennius, and Tomentella lapida were dominant, colonizing about 70% of the root tips, and remained unaffected by girdling. Mainly cryptic EMF species disappeared. Therefore, the Shannon-Wiener index (H′) decreased but evenness was unaffected. H′ was positively correlated with glucose, fructose, and starch concentrations of fine roots and also with the ratio of dissolved organic carbon to dissolved organic nitrogen (DOC/DON), suggesting that both H′ and DOC/DON were governed by changes in belowground carbon allocation. Our results suggest that beech maintains numerous rare EMF species by recent photosynthate. These EM fungi may constitute biological insurance for adaptation to changing environmental conditions. The preservation of taxa previously not known to colonize beech may, thus, form an important reservoir for future forest development.In temperate and boreal forest ecosystems, most tree species form ectomycorrhizal fungal (EMF) associations. EM fungi ensheathe the root tip, forming characteristic mantlelike structures (1). The presence and lengths of hyphae emanating from the mantle are characteristic of different EMF species and establish different soil exploration types (2). It has been assumed that EMF communities are adapted specifically to mobilize sparse soil nutrient resources in boreal and temperate forests (11, 50). Current estimates indicate that about 80% of all nitrogen and phosphorus present in plants has been taken up via mycorrhizas (30, 41, 63).Unlike free-living soil microbes, EM fungi have direct access to reduced carbon from their host plants. More than 50 years ago, Melin and Nilsson (46) showed that ¹⁴C applied to leaves was recovered within one day in EM fungi, suggesting a strong dependence of fungal metabolism on host photosynthesis. Subsequent isotopic studies corroborated tight connections between current photosynthate and EM fungi (28, 42). EMF hyphae constitute the main path of plant-derived carbon into the soil (24, 29). Furthermore, EMF hyphae contribute substantially to soil respiration (25% from hyphae and 15% from roots) (27). As hyphal respiration decreases strongly in response to girdling of trees, a tight metabolic link between extramatrical mycelia and host photosynthetic activity must exist (5, 9, 32). In addition, fruiting body formation of EMF species was strongly dependent on host photosynthetic capacity (32, 40). In contrast, the significance of the current assimilate supply for EMF colonization of root tips and for community composition is not yet well understood. Since trees contain substantial stores of carbohydrates in the roots and stem (7), it may be expected that EM fungi can be maintained if this carbon resource is available. For example, defoliation experiments with conifers, which restricted but did not eliminate current photosynthate transfer to roots, showed no effects on root EMF colonization. Massive defoliation that negatively affected aboveground biomass production suppressed morphotypes with thick mantles compared to those with thin mantles, suggesting a shift to less-carbon-demanding EMF species (14, 40, 44, 54, 56). Earlier studies also reported decreased EMF colonization of root tips (21, 52).In a common garden experiment with young beech trees, strong shading over several years, which severely limited plant growth, suppressed EMF colonization and resulted in low EMF diversity (20). EMF community composition was affected strongly by shading and slightly by short-term girdling, suggesting that EMF taxa are sensitive to changes in plant internal carbohydrate resources (20). However, the overall EMF diversity was low, probably because the young trees were grown in nutrient-rich compost soil (20). The significance of photoassimilates for EMF abundance, diversity, and community composition, therefore, remains to be shown for adult forest trees, which usually have high EMF diversity and low nitrogen availability (10, 26, 53, 61).The aim of this work was to test the hypothesis that EMF abundance and diversity are independent of the current photoassimilate supply and can be maintained by internal resources. To investigate this concept, old-growth beech trees (Fagus sylvatica L.) were girdled to suppress carbon allocation to roots. Since disruption of the current assimilate flux affects the carbohydrate source strength, we hypothesized that changes in EMF taxon composition would occur if EMF species had different carbon demands. Tree carbohydrate status, root demography, EMF colonization, and EMF taxon abundance were measured repeatedly during 1 year after girdling. Since girdling also affects carbon release into and probably nutrient uptake from soil, the influence of possible feedback by changes in the ratio of dissolved organic carbon to dissolved organic nitrogen (DOC/DON) in the soil on EMF diversity was also assessed.     

Comparison of genomic profiles of Escherichia coli isolates from urinary tract infections

Formally included in the larger category of extraintestinal pathogenic Escherichia coli (ExPEC), the uropathogenic E. coli remains the most frequent cause of urinary tract infection (UTI), an important endemic health problem. The genomic DNA of E. coli urinary isolates from adults diagnosed with urinary tract infections and of E. coli fecal isolates from healthy subjects was analysed by PCR for the presence of virulence factor encoding genes pap, sfa/foc, afa, hly and cnf and by field inversion gel electrophoresis (FIGE) fingerprinting of XbaI DNA macrorestriction fragments. The aim was to obtain more detailed microbiological data regarding the community circulating strains in respect of their virulence potential and genetic relatedness. Almost 70% of the urinary strains carried at least one of the target virulence genes, and only 35.5% of the fecal E. coli strains were positive in the PCR screening. Taking into account the virulence genotypes exhibited, a part of the strains isolated from the urinary tract could be defined as belonging to the ExPEC pathotype. A unique FIGE profile was obtained for each of the selected isolates and the dendrogram generated by Taxotron software package analysis suggested a polyclonal population of potential uropathogenic strains clustered into 14 groups of only 60% similarity. For better understanding the epidemiology of UTIs, diseases commonly caused by such a heterogeneous species like E. coli, molecular analysis methods could be essential due to their increased power of identification and fingerprinting.     

Probiotic Strains Influence on Infant Microbiota in the In Vitro Colonic Fermentation Model GIS1

The main goal of our study was to evaluate the effect of the individual administration of five lyophilized lactic acid bacteria strains (Lactobacillus fermentum 428ST, Lactobacillus rhamnosus E4.2, Lactobacillus plantarum FCA3, Lactobacillus sp. 34.1, Weissella paramesenteroides FT1a) against the in vitro simulated microbiota of the human colon using the GIS1 system. The influence on the metabolic activity was also assessed by quantitative determination of proteins and polysaccharides at each segment of human colon. The obtained results indicated that the lactic acid bacteria L. rhamnosus E4.2 and W. paramesenteroides FTa1 had better efficiency in synthesising exopolysaccharides and also a better probiotic potential and therefore could be recommended for use in probiotics products or food industry.     

Collagen regulates the ability of endothelial progenitor cells to protect hypoxic myocardium through a mechanism involving miR‐377/VE‐PTP axis

The possibility to employ stem/progenitor cells in the cardiovascular remodelling after myocardial infarction is one of the main queries of regenerative medicine. To investigate whether endothelial progenitor cells (EPCs) participate in the restoration of hypoxia‐affected myocardium, we used a co‐culture model that allowed the intimate interaction between EPCs and myocardial slices, mimicking stem cell transplantation into the ischaemic heart. On this model, we showed that EPCs engrafted to some extent and only transiently survived into the host tissue, yet produced visible protective effects, in terms of angiogenesis and protection against apoptosis and identified miR‐377‐VE‐PTP axis as being involved in the protective effects of EPCs in hypoxic myocardium. We also showed that collagen, the main component of the myocardial scar, was important for these protective effects by preserving VE‐PTP levels, which were otherwise diminished by miR‐377. By this, a good face of the scar is revealed, which was so far perceived as having only detrimental impact on the exogenously delivered stem/progenitor cells by affecting not only the engraftment, but also the general protective effects of stem cells.     

School absenteeism as an adjunct surveillance indicator: experience during the second wave of the 2009 H1N1 pandemic in Quebec, Canada

Background

A school absenteeism surveillance system was implemented in the province of Quebec, Canada during the second wave of the 2009 H1N1pandemic. This paper compares this surveillance approach with other available indicators.

Method

All (3432) elementary and high schools from Quebec were included. Each school was required to report through a web-based system any day where the proportion of students absent for influenza-like illness (ILI) exceeded 10% of current school enrolment.

Results

Between October 18 and December 12 2009, 35.6% of all schools met the 10% absenteeism threshold. This proportion was greater in elementary compared to high schools (40% vs 19%) and in smaller compared to larger schools (44% vs 22%). The maximum absenteeism rate was reached the first day of reporting or within the next two days in 55% and 31% of schools respectively. The first reports and subsequent peak in school absenteeism provincially preceded the peak in paediatric hospitalization by two and one weeks, respectively. Trends in school surveillance otherwise mirrored other indicators.

Conclusion

During a pandemic, school outbreak surveillance based on a 10% threshold appears insufficient to trigger timely intervention within a given affected school. However, school surveillance appears well-correlated and slightly anticipatory compared to other population indicators. As such, school absenteeism warrants further evaluation as an adjunct surveillance indicator whose overall utility will depend upon specified objectives, and other existing capacity for monitoring and response.