Taurine transport rates between plasma and tissues in adult and 7-day-old mice.

Transport rates for taurine from plasma to liver, kidney, heart, spleen and femoral muscle were evaluated in adult and 7-day-old mice in vivo. The mice were injected with [35S]taurine and the specific radioactivity of taurine was determined in the above tissues at varying intervals from 10 min up to 48 hr after the injection. A multicompartment model was fitted to the data and the transport rates with their confidence limits were estimated using a digital computer. The tissue-plasma exchange rate was generally faster in adult mice than in 7-day-old mice. The transport rates between the plasma and the brain or muscle were low, while taurine penetrated into the liver and kidneys very rapidly. There was no distinct correlation between the calculated transport rates and the tissue taurine concentrations. The metabolic breakdown of taurine in the tissues was slow, since only negligible amounts of radioactivity were recovered in the metabolites of taurine, isethionic acid and inorganic sulphate. It seems unlikely that either the magnitudes of the transport rates between the plasma and the tissues or taurine breakdown rates in situ act as the primary factor determining the taurine levels in tissues.     

Structural Basis for Complement Evasion by Lyme Disease Pathogen Borrelia burgdorferi

Borrelia burgdorferi spirochetes that cause Lyme borreliosis survive for a long time in human serum because they successfully evade the complement system, an important arm of innate immunity. The outer surface protein E (OspE) of B. burgdorferi is needed for this because it recruits complement regulator factor H (FH) onto the bacterial surface to evade complement-mediated cell lysis. To understand this process at the molecular level, we used a structural approach. First, we solved the solution structure of OspE by NMR, revealing a fold that has not been seen before in proteins involved in complement regulation. Next, we solved the x-ray structure of the complex between OspE and the FH C-terminal domains 19 and 20 (FH19-20) at 2.83 Å resolution. The structure shows that OspE binds FH19-20 in a way similar to, but not identical with, that used by endothelial cells to bind FH via glycosaminoglycans. The observed interaction of OspE with FH19-20 allows the full function of FH in down-regulation of complement activation on the bacteria. This reveals the molecular basis for how B. burgdorferi evades innate immunity and suggests how OspE could be used as a potential vaccine antigen.     

The Salivary Scavenger and Agglutinin (SALSA) in Healthy and Complicated Pregnancy

Pre-eclampsia is a leading cause of maternal and perinatal morbidity and mortality worldwide. The etiology is not clear, but an immune attack towards components of placenta or fetus has been indicated. This involves activation of the complement system in the placenta. We have previously described the presence of the complement-regulating protein salivary scavenger and agglutinin (SALSA) in amniotic fluid. In this study we investigated the potential role of SALSA in pregnancy by analyzing its presence in amniotic fluid and placental tissue during healthy and complicated pregnancies. SALSA levels in amniotic fluid increased during pregnancy. Before 20 weeks of gestation the levels were slightly higher in patients who later developed pre-eclampsia than in gestation age-matched controls. In the placenta of pre-eclamptic patients syncytial damage is often followed by the formation of fibrinoid structures. SALSA was found clustered into these fibrinoid structures in partial co-localization with complement C1q and fibronectin. In vitro analysis showed direct protein binding of SALSA to fibronectin. SALSA binds also to fibrin/fibrinogen but did not interfere with the blood clotting process in vitro. Thus, in addition to antimicrobial defense and epithelial differentiation, the data presented here suggest that SALSA, together with fibronectin and C1q, may be involved in the containment of injured placental structures into fibrinoids.     

KCC2-Mediated Cl− Extrusion Modulates Spontaneous Hippocampal Network Events in Perinatal Rats and Mice

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Mitochondrial genes reveal cryptic diversity in plant-breeding frogs from Madagascar (Anura, Mantellidae, Guibemantis)

One group of mantellid frogs from Madagascar (subgenus Pandanusicola of Guibemantis) includes species that complete larval development in the water-filled leaf axils of rainforest plants. This group consists of six described species: G. albolineatus, G. bicalcaratus, G. flavobrunneus, G. liber, G. pulcher, and G. punctatus. We sequenced the 12S and 16S mitochondrial rRNA genes ( approximately 1.8 kb) from multiple specimens (35 total) of all six species to assess phylogenetic relationships within this group. All reconstructions strongly supported G. liber as part of the Pandanusicola clade, even though this species does not breed in plant leaf axils. This result confirms a striking reversal of reproductive specialization. However, all analyses also indicated that specimens assigned to G. liber include genetically distinct allopatric forms that do not form a monophyletic group. Most other taxa that were adequately sampled (G. bicalcaratus, G. flavobrunneus, and G. pulcher) likewise consist of several genetically distinct lineages that do not form monophyletic groups. These results suggest that many of the recognized species in this group are complexes of cryptic species.     

Substituted salicylaldehydes as potential antimicrobial drugs: minimal inhibitory and microbicidal concentrations

Substituted salicylaldehydes are potent antibacterial and antifungal agents and may have chemotherapeutic potential. In the clinical setting, the minimal inhibitory concentration (MIC) as well as the minimal bactericidal and fungicidal concentrations (MBC and MFC, respectively) are of fundamental interest. Therefore, we have now, using a panel of five microbial species (Bacillus cereus, Candida albicans, Escherichia coli, Saccharomyces cerevisiae, and Staphylococcus aureus), determined the MIC and MBC/MFC values of a total of 22 aromatic aldehydes, including 19 substituted salicylaldehydes and the unsubstituted parent compounds benzaldehyde and salicylaldehyde (2-hydroxybenzaldehyde). The results clearly indicate that both of the yeasts studied are remarkably sensitive to various salicylaldehydes and, especially, to halogenated ones. Some congeners clearly merit consideration as potential therapeutic agents for Candida infections. The MIC values of the most potent congeners are of roughly the same magnitude as that of amphotericin B, and the results of the MFC measurements indicate that the compounds are fungicidal. All of the bacteria studied are also sensitive to at least some of the compounds tested but, clearly, this class of antimicrobials has superior activity against yeasts. Structure-activity relationships are discussed for each microbial species and compared with each other. The comparison of the results of MIC and MBC/MFC measurements with those of agar diffusion tests revealed aspects that are of interest concerning the methodology of antimicrobial activity screening. Unexpectedly, it was found that some compounds that are completely devoid of activity in agar diffusion tests had potent activity in MIC tests, indicating that if only agar diffusion methodology is used in drug discovery, some highly active compounds may be missed.     

Illuminating the evolutionary history of liverworts (Marchantiophyta)—towards a natural classification

The phylogenetic relationships of liverworts were reconstructed using the sequence data of four genome regions including rbcL, rps4 and trnL‐F of the chloroplast and 26S large subunit ribosomal rRNA gene of the nucleus, and 90 characters of morphological, ultrastructural and developmental aspects. The taxa sampled consisted of 159 species including 135 liverworts (108 genera, 54 families and 29 suborders), 13 mosses, two hornworts, seven vascular plants and two charophyte algae. Analyses based on maximum parsimony using both direct optimization (POY) and static alignment (NONA), as well as Bayesian inference (MrBayes) were done. All the data sets were analyzed simultaneously. Our study confirms that liverworts compose a monophyletic group which consists of three classes. The class Treubiopsida including both Treubia and Haplomitrium is resolved as the earliest diverging liverwort lineage. Blasia and the complex thalloids are assigned to the Marchantiopsida, under which Blasiidae and Marchantiidae are divided. Marchantiidae include Sphaerocarpales and Marchantiales. The simple thalloid and leafy liverworts form the Jungermanniopsida, which is further divided to subclasses Pelliidae subclassis nov., Metzgeriidae and Jungermanniidae. Metzgeriidae here is defined to include only Metzgeriaceae, Aneuraceae and Vandiemeniaceae, and is the sister group to the leafy liverworts. The leafy liverworts Jungermanniidae include the orders Pleuroziales, Porellales and Jungermanniales. It is assumed that the Porellales and the Jungermanniales have split early, at least in the Jurassic period. In the Porellales, the diversification rate may have remained relatively constant for long periods of time but speeding up only recently within some of the families, associated with an explosive radiation of angiosperms. The Jungermanniales are most probably a recently diversified group which has attained the greatest profusion of structure and the most remarkable diversity of leaf development and protective devices for maturing sporophytes. A detailed classification scheme for liverworts is presented. © The Willi Hennig Society 2006.     

Green fluorescent protein-propidium iodide (GFP-PI) based assay for flow cytometric measurement of bacterial viability.

BACKGROUND: Several staining protocols have been developed for flow cytometric analysis of bacterial viability. One promising method is dual staining with the LIVE/DEAD BacLight bacterial viability kit. In this procedure, cells are treated with two different DNA-binding dyes (SYTO9 and PI), and viability is estimated according to the proportion of bound stain. SYTO9 diffuses through the intact cell membrane and binds cellular DNA, while PI binds DNA of damaged cells only. This dual-staining method allows effective separation between viable and dead cells, which is far more difficult to achieve with single staining. Although SYTO9-PI dual staining is practical for various bacterial viability analyses, the method has a number of disadvantages. Specifically, the passage of SYTO9 through the cell membrane is a slow process, which is significantly accelerated when the integrity of the cell membrane is disrupted. As a result, SYTO9 binding to DNA is considerably enhanced. PI competes for binding sites with SYTO9 and may displace the bound dye. These properties diminish the reliability of the LIVE/DEAD viability kit. In this study, we investigate an alternative method for measuring bacterial viability using a combination of green fluorescent protein (GFP) and PI, with a view to improving data reliability. METHODS: Recombinant Escherichia coli cells with a plasmid containing the gene for jellyfish GFP were stained with PI, and green and red fluorescence were measured by FCM. For comparison, cells containing the plasmid from which gfp was removed were stained with SYTO9 and PI, and analyzed by FCM. Viability was estimated according to the proportion of green and red fluorescence. In addition, bioluminescence and plate counting (other methods to assess viability) were used as reference procedures. RESULTS: SYTO9-PI dual staining of bacterial cells revealed three different cell populations: living, compromised, and dead cells. These cell populations were more distinct when the GFP-PI combination was used instead of dual staining. No differences in sensitivity were observed between the two methods. However, substitution of SYTO9 with GFP accelerated the procedure. Bioluminescence and plate counting results were in agreement with flow cytometric viability data. CONCLUSIONS: In bacterial viability analyses, the GFP-PI combination provided better distinction between current viability stages of E. coli cells than SYTO9-PI dual staining. Additionally, the overall procedure was more rapid. No marked differences in sensitivity were observed.