Human TTRV30M localization within podocytes in a transgenic mouse model of transthyretin related amyloidosis: does the environment play a role?

Transthyretin related amyloidosis is a nosological entity that leads to disability, diminished quality of life, all stages of chronic kidney disease and eventually death. Podocytes are polarized, highly differentiated epithelial cells important for proper nephron function. In the present study we investigated whether deposited TTRVal30Met (TTRV30M) molecules could be localized within podocytes in situ under the effect of different housing conditions (i.e. specific pathogen free [SPF] vs. non-SPF). Murine renal glomeruli from human TTRV30M (hTTRV30M) transgenic mice were examined via direct and indirect immunofluorescence techniques for the presence of hTTRV30M, murine serum amyloid P, activated caspase-3 and NPHS1. Association strength and amount of colocalization for NPHS1?ChTTRV30M, NPHS1-activated caspase-3, hTTRV30M-murine serum amyloid P were estimated. Localization of hTTRV30M in podocytes was demonstrated by immuno-electron microscopy. Renal hTTRV30M gene and NPHS1 gene expression levels were estimated. Non-SPF transgenic mice showed increased glomerular hTTRV30M deposition compared to their SPF counterparts. Furthermore increased podocytic localization of hTTRV30M was noticed in non-SPF mice. Glomerular caspase-3 activation was increased only in the non SPF housing conditions. Podocytic caspase-3 activation was increased in SPF and in non-SPF transgenic mice when compared to non transgenic controls. Environmental conditions influence glomerular deposition and podocytic localization of hTTRV30M. In this context increased caspase-3 activation occurred.     

Development of a Selective Enterococcus Medium Based on Manganese Ion Deficiency, Sodium Azide, and Alkaline pH

Rogosa broth, without its salt supplement and dissolved in deionized water, was adapted for the selective isolation and enumeration of enterococci. This medium supported good growth of enterococci, but it suppressed growth of other lactic acid bacteria. The sensitivity and specificity of the medium were tested after addition of various increasing concentrations of NaN(3) against known strains of enterococci and other bacteria. Many strains of Streptococcus faecium showed low azide tolerance; optimal growth was obtained at a concentration of 0.01% NaN(3), which totally or partially inhibited unrelated species of lactic acid bacteria. The selectivity of the medium was further increased by pH adjustment to 9.6. Carbonate and Tween 80 were added to overcome partial inhibition of enterococcal growth by the new combination of selective conditions. The final medium was evaluated in agar form in isolations from human and animal feces, polluted water, meat, and dairy products. Counts were obtained after 16 to 17 h of incubation at 37 C. The isolates satisfactorily conformed to the group characteristics of enterococci.     

Insight into the protein and solvent contributions to the reduction potentials of [4Fe–4S]2+/+ clusters: crystal structures of the Allochromatium vinosum ferredoxin variants C57A and V13G and the homologous Escherichia coli ferredoxin

The crystal structures of the C57A and V13G molecular variants of Allochromatium vinosum 2[4Fe–4S] ferredoxin (AlvinFd) and that of the homologous ferredoxin from Escherichia coli (EcFd) have been determined at 1.05-, 1.48-, and 1.65-Å resolution, respectively. The present structures combined with cyclic voltammetry studies establish clear effects of the degree of exposure of the cluster with the lowest reduction potential (cluster I) towards less negative reduction potentials (E°). This is better illustrated by V13G AlvinFd (high exposure, E° = ?594 mV) and EcFd (low exposure, E° = ?675 mV). In C57A AlvinFd, the movement of the protein backbone, as a result of replacing the noncoordinating Cys57 by Ala, leads to a +50-mV upshift of the potential of the nearby cluster I, by removal of polar interactions involving the thiolate group and adjustment of the hydrogen-bond network involving the cluster atoms. In addition, the present structures and other previously reported accurate structures of this family of ferredoxins indicate that polar interactions of side chains and water molecules with cluster II sulfur atoms, which are absent in the environment of cluster I, are correlated to the approximately 180–250 mV difference between the reduction potentials of clusters I and II. These findings provide insight into the significant effects of subtle structural differences of the protein and solvent environment around the clusters of [4Fe–4S] ferredoxins on their electrochemical properties.     

Batch culture of bacteria under controlled gaseous conditions

Summary A flask, designed for direct gassing of batch cultures of bacteria, was evaluated for its use in studying oxygen absorption rates (OAR) and suitability for physiological studies under various controlled atmospheres. Such flasks, aerated directly without shaking, yielded an OAR (up to 1.2 mmol O₂/l/min) that was comparable to or higher than those obtained in conventional flasks aerated by shaking. Direct aeration in combination with shaking resulted in OAR values that were elevated and most favorable for growth of oxygen demanding bacteria (5 mmol O₂/l/min). In comparison with controls, the direct method of aeration in combination with shaking proved most efficient and least dependent on the surface to volume ratio of the aerated solution. In experiments with the facultative anaerobe Streptococcus faecalis 10Cl, grown in controlled aerobic, anaerobic, and mixed gas (CO₂-free air, air-plus-CO₂, N₂-plus-CO₂) environments, a specific anaerobic requirement for CO₂ could be established. The wide range of gaseous environments possible renders the newly tested flask useful for comparative biochemical studies, especially when the gaseous condition of culture is a factor of critical importance.     

Difference Between Manganese Ion Requirements of Pediococci and Enterococci

For maximal turbidity and dissimilatory activity, three strains of pediococci required the addition of Mn(2+) to Rogosa basal medium. Twenty-two strains of enterococci proved indifferent to this supplement.     

Protective Effects of Non-Anticoagulant Activated Protein C Variant (D36A/L38D/A39V) in a Murine Model of Ischaemic Stroke

Ischaemic stroke is caused by occlusive thrombi in the cerebral vasculature. Although tissue-plasminogen activator (tPA) can be administered as thrombolytic therapy, it has major limitations, which include disruption of the blood-brain barrier and an increased risk of bleeding. Treatments that prevent or limit such deleterious effects could be of major clinical importance. Activated protein C (APC) is a natural anticoagulant that regulates thrombin generation, but also confers endothelial cytoprotective effects and improved endothelial barrier function mediated through its cell signalling properties. In murine models of stroke, although APC can limit the deleterious effects of tPA due to its cell signalling function, its anticoagulant actions can further elevate the risk of bleeding. Thus, APC variants such as APC(5A), APC(Ca-ins) and APC(36-39) with reduced anticoagulant, but normal signalling function may have therapeutic benefit. Human and murine protein C (5A), (Ca-ins) and (36-39) variants were expressed and characterised. All protein C variants were secreted normally, but 5-20% of the protein C (Ca-ins) variants were secreted as disulphide-linked dimers. Thrombin generation assays suggested reductions in anticoagulant function of 50- to 57-fold for APC(36-39), 22- to 27-fold for APC(Ca-ins) and 14- to 17-fold for APC(5A). Interestingly, whereas human wt APC, APC(36-39) and APC(Ca-ins) were inhibited similarly by protein C inhibitor (t_½ - 33 to 39 mins), APC(5A) was inactivated ~9-fold faster (t_½ - 4 mins). Using the murine middle cerebral artery occlusion ischaemia/repurfusion injury model, in combination with tPA, APC(36-39), which cannot be enhanced by its cofactor protein S, significantly improved neurological scores, reduced cerebral infarct area by ~50% and reduced oedema ratio. APC(36-39) also significantly reduced bleeding in the brain induced by administration of tPA, whereas wt APC did not. If our data can be extrapolated to clinical settings, then APC(36-39) could represent a feasible adjunctive therapy for ischaemic stroke.     

A bacteria-specific 2[4Fe-4S] ferredoxin is essential in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Background  

Ferredoxins are small iron-sulfur proteins belonging to all domains of life. A sub-group binds two [4Fe-4S] clusters with unequal and extremely low values of the reduction potentials. These unusual properties are associated with two specific fragments of sequence. The functional importance of the very low potential ferredoxins is unknown.     

Productivity of verb-forming suffixes in Modern Greek: a corpus-based study

The paper studies the productivity of eight Greek verb-forming suffixes (-áro, -éno, -évo, -íno, -ízo, -(i)ázo, -jázo and -óno) in a corpus of 4,143,583 words. The corpus used comes from the Corpus of Greek Texts and includes all text types found in it in the same proportion. Our study follows similar research on other languages, investigating frequency and morphological productivity with a corpus-based methodology. The productivity of the suffixes was studied in the corpus as a whole, across its two subcorpora of written and spoken registers and across and within the text types included in the corpus. Three main sets of suffixes were identified: (a) the very productive -ízo and -óno, (b) the moderately productive -évo and -áro and (c) the least productive or unproductive -iázo, -jázo, -éno and -íno. The results from the study of text types suggest that the degree of productivity of each suffix is influenced by the text type in which it occurs. Quantitative evidence drawn from the measurement of productivity, by means of vocabulary growth curves and related methods, was combined with qualitative evidence on the semantic and other properties of verb-forming suffixes. On the basis of our results it cannot be argued that there is a default suffix for verb derivation in Modern Greek; it is rather suggested that the two most productive suffixes, -ízo and -óno, are used in Greek in a more or less complementary way, since they show a different preference for meanings and text types.