Correlations of geographic distribution and temperature of embryonic development with the nuclear DNA content in the Salamandridae (Urodela, Amphibia).

We used flow cytometry to measure the nuclear DNA content in erythrocytes of 27 salamandrid species. Across these species, diploid genome size varied more than 2 fold (51.3-104.4 pg). According to genome size and geographic distribution, 3 groups of newt species were recognized: West Palearctics with smaller amounts of nuclear DNA; Nearctic, with intermediate values; and East Asiatic, with higher genome sizes. Viviparous West Palearctic salamanders differed from most of the oviparous West Palearctic newts in possessing larger genome sizes. The nuclear DNA content strongly correlates with species range limits. At the same temperature, embryos of salamandrid species with larger genome sizes have a markedly longer developmental time than those with smaller genomes. We present an analysis of the relationships between the amount of nuclear DNA and water temperature at the breeding sites.     

Use of cultivated plants and non-plant remedies for human and animal home-medication in Liubań district,Belarus

Background

To use any domestic remedy, specific knowledge and skills are required. Simple logic dictates that the use of wild plants in the context of limited interaction with nature requires prior identification, while in the case of non-plant remedies and cultivated plants this step can be omitted. This paper aims to document the current and past uses of non-plant remedies and cultivated plants in the study region for human/animal medication; to analyze the human medicinal and veterinary use areas in the context of the remedy groups; to qualitatively compare the results with relevant historical publications; and to compare the intensity and purpose of use between the remedy groups.

Methods

During field studies 134 semi-structured interviews were conducted with locals from 11 villages in the Liubań district of Belarus. Currently used home-remedies as well as those used in the past were documented by employing the folk history method. The subject was approached through health-related uses, not by way of remedies. Interview records were digitalized and structured in Detailed Use Records in order to ascertain local perceptions. An Informant Consensus Factor (FIC) was calculated for remedy groups as well as for different use categories.

Results

In the human medication area the use of nearby remedies was neither very diverse nor numerous: 266 DUR for 45 taxa belonging to 27 families were recorded for cultivated plants along with 188 DUR for 58 different non-plant remedies. The FIC values for both remedy groups were lower than for wild plants. In the ethnoveterinary medicine use area there were 48 DUR referring to the use of 14 cultivated plant taxa from 12 families and 72 DUR referring to the use of 31 non-plant remedies. The FIC value for the whole veterinary use area of cultivated plants was relatively low, yet similar to the FIC of wild plants.

Conclusions

Differences between remedy groups were pronounced, indicating that in domestic human medicine cultivated plants and non-plant remedies are either remarkably less important than wild ones or not considered worth talking about. In ethnoveterinary medicine non-plant remedies are almost equally important as wild plants, while cultivated plants are the least used. People in study area seem to still more often rely on, or are more willing to talk to strangers about, wild plants, as promoted by both official medicine and popular literature.

     

Improvement of plasmid encoded lactococcal bacteriophage resistance by mutator strain Epicurian coli XL1-Red

A random mutation strategy using mutator strain, Epicurian coli XL1-Red, was applied to a plasmid, pND018, constructed by inserting a Lactococcus lacis bacteriophage resistance gene (abiI) into a L. lactis/E. coli shuttle vector (pDL278), to introduce random mutations throughout the plasmid. Following transformation of the mutated plasmid library to a plasmid free and phage sensitive strain of L. lactis (LM0230), mutated plasmids were screened by cross-streaking and efficiency of plaquing (EOP) assays. Two strains with enhanced resistance were obtained, as well as several phage sensitive strains. Repeated transformation of the mutated plasmids to LM0230 confirmed that the observed phenotypes were caused by mutations located on the plasmids. The EOP values and plaque morphology of two enhanced phage resistance mutants were characterized at 30°C and 37°C. These results indicate that this simple procedure can be applied to generate modified plasmids with improved phage resistance, which may be of commercial value.     

Phylogeographic patterns of genetic diversity in the common spadefoot toad, Pelobates fuscus (Anura: Pelobatidae), reveals evolutionary history, postglacial range expansion and secondary contact

Based on allozyme variation of 410 newly collected individuals from 52 localities, we reconstructed range-wide phylogeography of the widespread Western Palearctic anuran, Pelobates fuscus. To study genetic diversity, evolutionary history, postglacial range expansion and secondary contact zones, we used a multidisciplinary approach combining information from various genetic analyses and ecological niche modeling. We confirmed the presence of two main groups in P. fuscus, initially revealed by genome size variation. Pelobates f. vespertinus presents a monomorphic group, but two main groups can be identified in P. f. fuscus: an East European and a West European group. We suggest the existence of at least four different Last Glacial refugia for P. fuscus: (1) the area between the Caspian and Azov Seas as the origin for the expansion of P. f. vespertinus; (2) the northwestern part of the Black Sea area for the East European P. f. fuscus; (3) the southwestern part of the Pannonian Plain and (4) the Po Plain for the West European P. f. fuscus. The routes of postglacial range expansions from the refugia are discussed. We newly identified a hybrid zone between P. f. fuscus and P. f. vespertinus. The width of this zone is about 12.5 km. In light of these findings, the two subspecies of P. fuscus constitute distinct evolutionary lineages and merit recognition as separate species. Our data do not provide support for the validity of P.f. insubricus. We therefore propose to synonymize this subspecies with P. f. fuscus.     

Role of depolarization and calcium in contractions of canine trachealis from endogenous or exogenous acetylcholine

The relationships of the electrical to the mechanical responses of the canine trachealis muscle during stimulation of its cholinergic nerves or exposure to exogenous acetylcholine were recorded in the single or the double sucrose gap. At 27 degrees C, the responses to a train of stimuli consisted of a transient depolarization excitatory junction potential of 10-30 mV followed by fading oscillations and contractions. When stimulus parameters were varied in the single sucrose gap, contractions were more closely associated with the occurrence of and varied in duration with the oscillations rather than with the amplitude of the EJP. Acetylcholine superfused at a concentration of 10(-6) M for 30 s caused a prolonged depolarization of 10-20 mV, but a much larger contraction than could be elicited by nerve stimulation. None of the responses to acetylcholine was significantly affected by the Ca channel antagonists, nifedipine, nitrendipine, or verapamil in Ca channel blocking concentrations. When tissues were exposed to a Ca-free medium, the excitatory junction potentials and oscillations rapidly disappeared, but the electrical and mechanical responses to acetylcholine persisted and only gradually disappeared with repetitive exposures. Furthermore, in a medium with normal Ca2+ in the double sucrose gap, depolarization by 10-15 mV with an applied current caused no contraction, and repolarization to the normal membrane potential during acetylcholine-induced contraction caused no relaxation. Tetraethylammonium ion (20 mM) depolarized the membrane, increased membrane resistance, and enhanced the secondary oscillations and contractions after field stimulation. No other K(+)-channel blocker tested (Ba2+, apamin, 4-aminopyridine, glibenclamide, charybdotoxin) had the effect of prolonging secondary oscillations.(ABSTRACT TRUNCATED AT 250 WORDS)     

New role for Agrin in T cells and its potential importance in immune system regulation

Agrin plays a crucial role in the maintenance of the neuromuscular junction. However, it is expressed in other tissues as well, including T lymphocytes, where cell activation induces its expression. Agrin from activated T cells has the capacity to induce aggregation of key receptors and to regulate signalling. Interestingly, T cells isolated from patients with systemic lupus erythematosus over-express Agrin and its co-stimulation with the T cell receptor enhances production of pathogenic cytokines. These early studies point to an important function for Agrin in T cell biology and make the case for a more thorough and systematic investigation into its role in the immune system.     

A nuclear gene for higher level phylogenetics: phosphoenolpyruvate carboxykinase tracks mesozoic-age divergences within Lepidoptera (Insecta)

The sequence of phosphoenolpyruvate carboxykinase (PEPCK) has been previously identified as a promising candidate for reconstructing Mesozoic-age divergences (Friedlander, Regier, and Mitter 1992, 1994). To test this hypothesis more rigorously, 597 nucleotides of aligned PEPCK coding sequence (approximately 30% of the coding region) were generated from 18 species representing Mesozoic-age lineages of moths (Insecta: Lepidoptera) and outgroup taxa. Relationships among basal Lepidoptera are well established by morphological analysis, providing a strong test for the utility of a gene which has not previously been used in systematics. Parsimony and other phylogenetic analyses were conducted on nucleotides by codon positions (nt1, nt2, nt3) separately and in combination, and on amino acids, for comparison to the test phylogeny. The highest concordance was achieved with nt1 + nt2, for which one of two most-parsimonious trees was identical to the test phylogeny, and with all nucleotides when nt3 was down-weighted sevenfold or higher, for which a single most-parsimonious tree identical to the test phylogeny resulted. Substitutions in nt3 approached saturation in many, but not all, pairwise comparisons and their exclusion or severe downweighting greatly increased the degree of concordance with the test phylogeny. Neighbor-joining analysis confirms this finding. The utility of PEPCK for phylogenetics is demonstrated over a time span for which few other suitable genes are currently available.      

Molecular relationships in Genista L. Sect. Spartocarpus Spach (Fabaceae)

 A molecular investigation on the species of Genista belonging to sect. Spartocarpus was undertaken with the aim of inferring phylogenetic relationships within the section. The combined cladistic analysis of ITS 1 and 2 and of the chloroplast DNA trnL^(UAA) intron yielded a single most parsimonious cladogram with G. aetnensis as sister group to the rest of the section, which is in turn divided into two major clades, one of which includes G. nissana, G. aucheri, G. sessilifolia, G. spartioides and G. haenseleri, and the other the remaining species of the section. This latter group has G. holopetala and G. radiata forming a clade, which is a sister group to a not entirely resolved monophylum including the G. ephedroides species complex. The latter shows a basal collapse of G. cilentina, G. dorycnifolia and G. numidica, and then a clade including G. ephedroides and G. valsecchiae, as well as a collapsed monophylum with G. demarcoi, G. gasparrini and G. thyrrena. The clade including the G. ephedroides species group has the lowest within-group molecular variation and appears to be recent in origin, as compared to the rest of the section. The pattern of cladogeny in the section seems to be related to geographical isolation, and is broadly congruent with palynological and biogeographical evidence. Received March 3, 2001 Accepted October 8, 2001     

Primary human CD4+ T cells have diverse levels of membrane lipid order that correlate with their function

Membrane lipid microdomains (lipid rafts) play an important role in T cell function by forming areas of high lipid order that facilitate activation. However, their role in regulating T cell differentiation and function remains controversial. In this study, by applying a new approach involving microscopy and flow cytometry, we characterize membrane lipid order in ex vivo primary human CD4(+) T cells. We reveal that differential membrane lipid order dictates the response to TCR stimulation. T cells with high membrane order formed stable immune synapses and proliferated robustly, intermediate order cells had reduced proliferative ability accompanied by unstable immune synapse formation, whereas low order T cells were profoundly unresponsive to TCR activation. We also observed that T cells from patients with autoimmune rheumatic disease had expanded intermediate order populations compared with healthy volunteers. This may be important in dictating the nature of the immune response since most IFN-γ(+)CD4(+) T cells were confined within intermediate membrane order populations, whereas IL-4(+)CD4(+) T cells were contained within the high order populations. Importantly, we were able to alter T cell function by pharmacologically manipulating membrane order. Thus, the results presented from this study identify that ex vivo CD4(+) T cells sustain a gradient of plasma membrane lipid order that influences their function in terms of proliferation and cytokine production. This could represent a new mechanism to control T cell functional plasticity, raising the possibility that therapeutic targeting of membrane lipid order could direct altered immune cell activation in pathology.     

Novel Strategies for Enhanced Removal of Persistent Bacillus anthracis Surrogates and Clostridium difficile Spores from Skin

Background

Removing spores of Clostridium difficile and Bacillus anthracis from skin is challenging because they are resistant to commonly used antimicrobials and soap and water washing provides only modest efficacy. We hypothesized that hygiene interventions incorporating a sporicidal electrochemically generated hypochlorous acid solution (Vashe^®) would reduce the burden of spores on skin.

Methods

Hands of volunteers were inoculated with non-toxigenic C. difficile spores or B. anthracis spore surrogates to assess the effectiveness of Vashe solution for reducing spores on skin. Reduction in spores was compared for Vashe hygiene interventions versus soap and water (control). To determine the effectiveness of Vashe solution for removal of C. difficile spores from the skin of patients with C. difficile infection (CDI), reductions in levels of spores on skin were compared for soap and water versus Vashe bed baths.

Results

Spore removal from hands was enhanced with Vashe soak (>2.5 log₁₀ reduction) versus soap and water wash or soak (~2.0 log₁₀ reduction; P <0.05) and Vashe wipes versus alcohol wipes (P <0.01). A combined approach of soap and water wash followed by soaking in Vashe removed >3.5 log₁₀ spores from hands (P <0.01 compared to washing or soaking alone). Bed baths using soap and water (N =26 patients) did not reduce the percentage of positive skin cultures for CDI patients (64% before versus 57% after bathing; P =0.5), whereas bathing with Vashe solution (N =21 patients) significantly reduced skin contamination (54% before versus 8% after bathing; P =0.0001). Vashe was well-tolerated with no evidence of adverse effects on skin.

Conclusions

Vashe was safe and effective for reducing the burden of B. anthracis surrogates and C. difficile spores on hands. Bed baths with Vashe were effective for reducing C. difficile on skin. These findings suggest a novel strategy to reduce the burden of spores on skin.