Differential Expression of Micro-Heterogeneous LewisX-Type Glycans in the Stem Cell Compartment of the Developing Mouse Spinal Cord

Complex glycan structures and their respective carrier molecules are often expressed in a cell type specific manner. Thus, glycans can be used for the enrichment of specific cell types such as neural precursor cells (NPCs). We have recently shown that the monoclonal antibodies 487^LeX and 5750^LeX differentially detect the LewisX (LeX) glycan on NPCs in the developing mouse forebrain. Here, we analysed the staining pattern of both antibodies during late embryonic mouse spinal cord development. At E13.5 both antibodies strongly label the central canal region. Along these lines they detect the LeX glycan primarily on Nestin-positive NPCs at that age. Moreover, we show that spinal cord NPCs cultured as free floating neurospheres display a high immunoreactivity to both antibodies. In that context, we also demonstrate that the 487^LeX antibody can be used to deplete a subpopulation of neurosphere forming NPCs from a mixed E13.5 spinal cord cell suspension. Towards the end of embryogenesis the overall immunoreactivity to both antibodies increases and the staining appears very diffuse. However, the 5750^LeX antibody still labels the central canal region. The increase in immunoreactivity correlates with an expression increase of the extracellular matrix molecules Tenascin C and Receptor Protein Tyrosine Phosphatase β/ζ, two potential LeX carrier proteins. In line with this, immunoprecipitation analyses confirmed Tenascin C as a LeX carrier protein in the embryonic mouse spinal cord. However, the immunoreactivity to both antibodies appears only to be marginally affected in the absence of Tenascin C, arguing against Tenascin C being the major LeX carrier. In conclusion our study gives some novel insights into the complex expression of LeX glycans and potential carrier proteins during the development of the mouse spinal cord.     

Endocytosis by liver cells during stimulation of the mononuclear phagocyte system by yeast polysaccharides]

Endocytosis (phagocytosis, fluid-phase- and receptor-mediated endocytosis) by liver cells, lysosomal enzyme activities have been studied during macrophages stimulation by yeast polysaccharides. It was shown that like macrophages stimulator zymosan, yeast polysaccharides cryelan and rhodexman increased the carbon particles phagocytosis. The most effective was intravenous administration of yeast polysaccharides. Compared to rhodexman, the effect of cryelan was more prominent. Macrophages stimulation was followed by suppression of fluid-phase endocytosis by liver cells. Increased activity of cathepsin B was discovered on day 5 after macrophages stimulation (proteinase, most typical for macrophages enzymes).     

Noncompetitive Immunochemical Determination of Ribonuclease Using Transition Metal Ions and the Effect of Catalytic Hydrogen Release

A noncompetitive variant of immunochemical ribonuclease (RNase) determination has been developed, involving the use of Co(II) as a label. A variety of approaches to labeling the immunological reagent with the metal have been assessed. In the variant proposed, catalytic hydrogen release is used as a means of detecting the label, the amount of which is proportional to the RNase concentration. Conditions making it possible to record catalytic hydrogen release currents have been determined. In the presence of RNase, the electrocatalytic effect is maximum at a concentration of Co(II) in ammoniac buffer equal to 2 × 10^–4 M (pH 10.0). The dependence is linear in the RNase concentration range 2000–4 ng/ml (threshold concentration, 2 ng/ml).     

Characterization of Novel Chitin-degrading laceyella spp. Strains from New Athos Cave (Abkhazia) Producing Thermostable Chitinases

Microbiology - Nine chitin-degrading isolates of moderately thermophilic bacteria with temperature optimum for growth and chitin destruction at 50–55°C were isolated from soils of the...     

The characteristics of the humoral antibacterial immunity of young children with respiratory organ diseases]

The state of antibacterial humoral immunity in young children with acute bronchitis, acute obstructive bronchitis and bronchial asthma at the period of exacerbation has been shown with the use of the enzyme immunoassay. The concentration of antibodies to endotoxin positively correlates with the severity of clinical manifestations of the endogenic intoxication of the body. As the inflammatory process in the bronchial tree increases, the spectrum bacterial agents to which elevated concentrations of specific antibodies can be detected becomes wider, and this finds its maximum reflection in bronchial asthma.     

Factors affecting vigilance in springbok: importance of vegetative cover, location in herd, and herd size

Vigilance in vertebrates is often inversely related to group size. We present evidence that distance to bushes and location within the herd are also critical factors in vigilance in springbok (Antidorcas marsupialis) in Etosha National Park, Namibia, where they are the preferred prey of cheetahs (Acinonyx jubatus). Most springbok feed in heterospecific herds, both by grazing on grass and browsing on bushes. We studied 1245 animals; variations in vigilance (time alert) were explained by location within the herd, distance to bushes and roads, number of springbok in each herd, and gender and age. Vigilance time decreased with increasing herd size, with increasing distance to bushes and roads, and with density. Springbok on the edge of herds devoted significantly more time to vigilance than did those in other locations, and vigilance in edge animals decreased with group size. Adults were more vigilant than young, and males were more vigilant than females. Position within the herd, and distance from bushes, were the most important variables influencing vigilance. Location in the herd and gender/age affected both browsing and grazing springbok, although other factors accounted for the differences in vigilance between browsing and grazing springbok: 1) group size was not significant for browsers, but it was for grazers, and 2) distances to bushes and road were not significant for browsers, but they were for grazers. These data relate to the risk from predators and the benefits from other group members. Springbok in bushes cannot see all members of the herd, cannot derive early warning from many group members, and are more at risk from predators because the latter can hide in the bushes. Received: 17 May 1999 / Received in revised form: 30 August 1999 / Accepted: 29 November 1999     

A novel epileptic encephalopathy mutation in KCNB1 disrupts Kv2.1 ion selectivity,expression, and localization

The epileptic encephalopathies are a group of highly heterogeneous genetic disorders. The majority of disease-causing mutations alter genes encoding voltage-gated ion channels, neurotransmitter receptors, or synaptic proteins. We have identified a novel de novo pathogenic K⁺ channel variant in an idiopathic epileptic encephalopathy family. Here, we report the effects of this mutation on channel function and heterologous expression in cell lines. We present a case report of infantile epileptic encephalopathy in a young girl, and trio-exome sequencing to determine the genetic etiology of her disorder. The patient was heterozygous for a de novo missense variant in the coding region of the KCNB1 gene, c.1133T>C. The variant encodes a V378A mutation in the α subunit of the Kv2.1 voltage-gated K⁺ channel, which is expressed at high levels in central neurons and is an important regulator of neuronal excitability. We found that expression of the V378A variant results in voltage-activated currents that are sensitive to the selective Kv2 channel blocker guangxitoxin-1E. These voltage-activated Kv2.1 V378A currents were nonselective among monovalent cations. Striking cell background–dependent differences in expression and subcellular localization of the V378A mutation were observed in heterologous cells. Further, coexpression of V378A subunits and wild-type Kv2.1 subunits reciprocally affects their respective trafficking characteristics. A recent study reported epileptic encephalopathy-linked missense variants that render Kv2.1 a tonically activated, nonselective cation channel that is not voltage activated. Our findings strengthen the correlation between mutations that result in loss of Kv2.1 ion selectivity and development of epileptic encephalopathy. However, the strong voltage sensitivity of currents from the V378A mutant indicates that the loss of voltage-sensitive gating seen in all other reported disease mutants is not required for an epileptic encephalopathy phenotype. In addition to electrophysiological differences, we suggest that defects in expression and subcellular localization of Kv2.1 V378A channels could contribute to the pathophysiology of this KCNB1 variant.     

Cytotoxic effect of co-expression of human hepatitis A virus 3C protease and bifunctional suicide protein FCU1 genes in a bicistronic vector

Recent reports on various cancer models demonstrate a great potential of cytosine deaminase/5-fluorocytosine suicide system in cancer therapy. However, this approach has limited success and its application to patients has not reached the desirable clinical significance. Accordingly, the improvement of this suicide system is an actively developing trend in gene therapy. The purpose of this study was to explore the cytotoxic effect observed after co-expression of hepatitis A virus 3C protease (3C) and yeast cytosine deaminase/uracil phosphoribosyltransferase fusion protein (FCU1) in a bicistronic vector. A set of mono- and bicistronic plasmid constructs was generated to provide individual or combined expression of 3C and FCU1. The constructs were introduced into HEK293 and HeLa cells, and target protein synthesis as well as the effect of 5-fluorocytosine on cell death and the time course of the cytotoxic effect was studied. The obtained vectors provide for the synthesis of target proteins in human cells. The expression of the genes in a bicistronic construct provide for the cytotoxic effect comparable to that observed after the expression of genes in monocistronic constructs. At the same time, co-expression of FCU1 and 3C recapitulated their cytotoxic effects. The combined effect of the killer and suicide genes was studied for the first time on human cells in vitro. The integration of different gene therapy systems inducing cell death (FCU1 and 3C genes) in a bicistronic construct allowed us to demonstrate that it does not interfere with the cytotoxic effect of each of them. A combination of cytotoxic genes in multicistronic vectors can be used to develop pluripotent gene therapy agents.