Structural studies of the capsular polysaccharide of Klebsiella type 37

The structure of the Klebsiella type 37 capsular polysaccharide has been investigated. Methylation analysis, various specific degradations, and n.m.r. spectroscopy were the principal methods used. It is concluded that the polysaccharide is composed of tetrasaccharide repeating-units having the structure 4-O-Lac-d-GlcA  4-O-[(S)-1-carboxyethyl]-d-glucuronic acid:

     

FRET-Based Trilateration of Probes Bound within Functional Ryanodine Receptors

To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca²⁺ channels, we developed an approach that combines fluorescence resonance energy transfer (FRET), simulated-annealing, cryo-electron microscopy, and crystallographic data. DPc10 is identical to the 2460–2495 segment within the cardiac muscle RyR isoform (RyR2) central domain. DPc10 binding to RyR2 results in a pathologically elevated Ca²⁺ leak by destabilizing key interactions between the RyR2 N-terminal and central domains (unzipping). To localize the DPc10 binding site within RyR2, we measured FRET between five single-cysteine variants of the FK506-binding protein (FKBP) labeled with a donor probe, and DPc10 labeled with an acceptor probe (A-DPc10). Effective donor positions were calculated from simulated-annealing constrained by both the RyR cryo-EM map and the FKBP atomic structure docked to the RyR. FRET to A-DPc10 was measured in permeabilized cardiomyocytes via confocal microscopy, converted to distances, and used to trilaterate the acceptor locus within RyR. Additional FRET measurements between donor-labeled calmodulin and A-DPc10 were used to constrain the trilaterations. Results locate the DPc10 probe within RyR domain 3, ∼35 Å from the previously docked N-terminal domain crystal structure. This multiscale approach may be useful in mapping other RyR sites of mechanistic interest within FRET range of FKBP.     

Studies on the differentiation capacity of neural epithelium cells in chick embryos

Cell and Tissue Research -     

Nitric oxide synthase-containing, peptide-containing, and acetylcholinesterase-positive nerves in the cat lower oesophagus

Summary The innervation of the cat lower oesophagus, including the lower oesophageal sphincter, was studied by enzyme histochemistry, immunohistochemistry, and confocal microscopy. In the lower oesophageal sphincter, and at a level 2 cm above it, no apparent differences were seen in the nerve distribution pattern. Among the nerve populations studied, acetylcholinesterase (AChE)-positive nerves were the most abundant in both these regions. The density of AChE-positive nerves was particularly marked in the circular muscle layer. A rich supply of nitric oxide synthase (NOS)-containing nerves was identified by using an antiserum against neuronal NOS, or by enzyme histochemical staining for NADPH diaphorase activity. Vasoactive intestinal peptide (VIP)-immunoreactive nerves had a similar distribution pattern as NOS-immunoreactive nerves, and nerves displaying immunoreactivity for NOS and VIP often showed profiles coinciding with AChE-positive nerves. As judged by confocal microscopy, immunoreactivities for helospectin, pituitary adenylate cyclase-activating peptide (PACAP) and VIP, to a large extent were found in the same nerves. At a level 7 cm above the lower oesophageal sphincter, the total nerve supply was less than in the sphincter itself and 2 cm above it. Immunoreactivity towards VIP, PACAP and helospectin was also found to co-exist with NOS and neuropeptide Y within the same nerve structures. It is concluded that there is an intricate innervation pattern in the feline lower oesophagus reflecting the complexity in the regulation of its motility.     

Effects of Variations in Glutamic Acid Decarboxylase Activity on Acute Oxygen Poisoning

Abstract— A study was made to test the influence of rapid variations in glutamic acid decarboxylase (GAD) activity on the susceptibility of rats to hyperbaric oxygen (HBO). GAD was inhibited by the convulsant drug unsymmetrical dimethylhydrazine (UDMH) and reactivated by pyridoxine (PYR) after onset of convulsive activity. There was a relatively long induction period after UDMH injection until the onset of convulsions and the predictable interictal periods between successive periodic convulsions made it possible to study the impact of variations in GAD activity on survival rates, suspectibility to HBO and brain glycogen levels in a time sequence after UDMH administration. The experiments showed that UDMH interferes with aerobic metabolism in brain in such a way that profound alterations in resistance to acute oxygen poisoning resulted. An accumulation of substrate proximal to the enzyme block is assumed to develop during UDMH poisoning. The protective effect against HBO toxicity that was achieved after reactivation of GAD by PYR injection, as well as the rapid re-establishment of glycogen levels, is believed to speak in favour of this hypothesis.     

Heterozygosity-fitness correlations within inbreeding classes: local or genome-wide effects?

Marker-based studies of inbreeding may lead to an enhanced understanding of inbreeding depression in natural populations, which is a major concern in conservation genetics. Correlations between marker heterozygosity and variation in fitness-associated traits—‘heterozygosity-fitness correlations’ (HFCs)—are of particular importance and have been widely applied in natural populations. In partially inbred populations, HFCs can be driven by selection against inbred individuals and thus reflect inbreeding depression. However, other explanations for HFCs also exist, such as functional effects of the markers per se or that the markers reveal selection on linked fitness genes due to extended linkage disequilibrium (LD) in the population. Accordingly, HFCs do not only arise in partially inbred populations, they may also occur within inbreeding classes such as families, i.e. in situations when there is no variation in the inbreeding coefficient. In this study we focus on the importance of LD for within-family HFCs, thereby aiming at enhancing our general understanding of HFCs. For non-coding markers, within-family HFCs have been proposed to be caused in two ways: either by ‘local effects’ at linked fitness genes in LD with the markers, or by ‘general effects’ due to a correlation between proportion of heterozygous markers (H _M ) and heterozygosity at genome-wide distributed fitness genes (H _GW ). To evaluate these contrasting hypotheses for within-family HFCs, we analysed simulated data sets of sexually reproducing populations with varying levels of LD. The results confirmed that segregation induces variation in both H _M and H _GW at a fixed level of inbreeding; as expected, the variation in H _M declined with increasing number of markers, whereas the variation in H _GW declined with decreasing LD. However, less intuitively, there was no positive correlation between the variation in H _M and H _GW within inbreeding classes when the local component of H _GW was accounted for (i.e. when the part of the chromosome in LD with the markers was excluded). This strongly suggests that within-family HFCs are not caused by general effects. Instead, our results support the idea that HFCs at a known level of inbreeding can be driven by local effects in populations with high to moderate LD. Note however that we define the local component of H _GW as the part of the chromosomes in LD with the markers. This implies that when LD is high, the local component will consist of a substantial part of the genome and thus provides a rather ‘genome-wide’ view. We caution against routinely interpreting positive HFCs as evidence of inbreeding depression and non-significant HFCs as lack thereof, especially when few markers are used.     

Occludin is overexpressed in Alzheimer's disease and vascular dementia

The tight junctions (TJs) are key players in the control of blood-brain barrier (BBB) properties, the most complex TJs in the vascular system being found in the endothelial cells of brain capillaries. One of the main TJs proteins is occludin, which anchors plasma membranes of neighbour cells and is present in large amounts in the brain endothelia. Previous studies demonstrated that disruption of BBB in various pathological situations associates with changes in occludin expression, and this change could be responsible for malfunction of BBB. Therefore in this study, applying an immunohistochemical approach, we decided to explore the occludin expression in frontal cortex (FC) and basal ganglia in ageing control, Alzheimer's disease (AD), and vascular dementia (VD) brains, as far as all these pathologies associate microangiopathy and disruption of BBB. Strikingly, we found selected neurons, astrocytes and oligodendrocytes expressing occludin, in all cases studied. To estimate the number of occludin-expressing neurons, we applied a stereological approach with random systematic sampling and the unbiased optical fractionator method. We report here a significant increase in ratio of occludin-expressing neurons in FC and basal ganglia regions in both AD and VD as compared to ageing controls. Within the cerebral cortex, occludin was selectively expressed by pyramidal neurons, which are the ones responsible for cognitive processes and affected by AD pathology. Our findings could be important in unravelling new pathogenic pathways in dementia disorders and new functions of occludin and TJs.     

Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy

Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO₂ NMs with different surface chemistry – hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO – uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO₂ NMs produced by two different manufacturing techniques – precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO₂>TiO₂). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO₂ and SiO₂, while the embryonic stem cell test (EST) classified the TiO₂ NMs as potentially ‘weak-embryotoxic’ and ZnO and SiO₂ NMs as ‘non-embryotoxic’. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO₂ NM-203 > SiO₂ NM-200 > TiO₂ NM-104 > TiO₂ NM-103). This ranking was different in the case of embryonic tissues, for which TiO₂ displayed higher toxicity compared with ZnO and SiO₂. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.