Separation of bacterial capsular and lipopolysaccharides by preparative electrophoresis

Preparative gel electrophoresis was used to separate and purify extracellular, capsular and lipopolysaccharides (EPSs, CPSs, and LPSs, respectively) from crude bacterial extracts. The procedure effectively separates CPS from LPSs. In addition discreet size ranges of these various polysaccharides can be isolated. The 'rough' (R-type), 'smooth' (S-type), and 'semi-smooth' LPSs were separated from one another. In addition different size classes of 'semi-smooth', or S-type LPS, can be separated. This procedure was demonstrated for diverse bacterial species, including the soil bacteria Rhizobium fredii, and the enteric bacterial species, Salmonella enteritidis and Proteus mirabilis. In the latter case, it was also possible to separate capsular polysaccharide from its lipid-bound form.     

Quantitative relations in the retinal ganglion cell layer of the rat: neurons, glia and capillaries before and after optic nerve section

The severity of pulmonary fibrosis is the main prognostic factor for survival of patients with interstitial lung diseases (ILD). Unfortunately, lung biopsy, which is the best method to assess fibrosis quantitatively, is done only once during the evolution of the disease. In this study we analyzed the relationship between the degree of fibrosis and the exponential constant k, derived from the lung pressure-volume curve (LPVC) in 33 patients with chronic ILD, 19 with pigeon breeder's disease (PBD), and 14 with idiopathic pulmonary fibrosis (IPF). Pulmonary function tests, including the LPVC, were obtained before biopsy. A semiquantitative histologic assessment of the severity of fibrosis was performed on lung tissues. All patients showed a decrease of total lung capacity, residual volume, compliance, and Pao2. The mean value of the constant k was 0.08 +/- 0.06. When expressed as a percent of normal values, 25 patients exhibited values of k lower than 70% of predicted; of the remaining 8 patients whose values were above 70% of predicted, 7 had PBD and only one IPF. On morphologic analysis, 19 patients displayed more than 50% fibrosis. No significant correlations were found between the extent of the lesion or severity of lung fibrosis and the conventional pulmonary function tests. By contrast, a moderate but significant correlation was found between k and the severity of lung fibrosis (r = -0.38, p < 0.05). These findings show that the shape of the LPVC, represented by the constant k, predicts the degree of lung fibrosis and could be useful in the clinical assessment and follow-up of patients with ILD.     

Role of relaxin and estrogen in the control of eosinophilic invasion and collagen remodeling in rat cervical tissue at term

The modified fluorescence method was used to determine the accumulation of norfloxacin by Mycobacterium aurum A+ and Mycobacterium smegmatis mc(2)155. By using an exogenous norfloxacin concentration of 10 microg/ml, a steady-state concentration (SSC) of 160 to 180 ng of norfloxacin/mg of cells was obtained for M. aurum, and an SSC of 120 to 140 ng of norfloxacin/mg of cells obtained for M. smegmatis. For both species of mycobacteria, the SSC was achieved within 5 min. The silicon oil method was investigated and gave higher SSCs than the modified fluorescence method. Further studies on the mechanism of norfloxacin accumulation by M. aurum were performed. An increase in the pH of the wash buffer from 7.0 to 9.0 did not significantly affect the final SSC obtained. Accumulation was nonsaturated over a norfloxacin concentration range of 0 to 100 microg/ml, and the proton motive force inhibitor 2,4-dinitrophenol (1 and 2 mM), whether it was added before or after norfloxacin was added, had no effect on the final SSC obtained. 2,4-Dinitrophenol also had no effect on norfloxacin accumulation by M. smegmatis. Furthermore, norfloxacin accumulation by M. aurum was unaffected by the presence of either Tween 80 or subinhibitory concentrations of ethambutol in the growth medium. Therefore, it is proposed that norfloxacin accumulation by mycobacteria occurs by simple, energy-independent diffusion.     

Reversal by desferrioxamine of tau protein aggregates following two days of treatment in aluminum-induced neurofibrillary degeneration in rabbit: implications for clinical trials in Alzheimer''s disease

Concern about possible transmission of bloodborne pathogens during medical procedures is growing among patients and healthcare workers alike. This fear has primarily been focused on nosocomial transmission of human immunodeficiency virus (HIV), but other bloodborne infectious agents may also be transmitted during procedures. Chief among these are the hepatitis viruses, particularly hepatitis B virus (HBV) and hepatitis C virus (HCV), both of which are significantly more widespread than HIV. Although radiology is not traditionally thought of as a field with significant risk for exposure to or transmission of pathogens, the expanding role of interventional procedures in recent years belies that perception. The potential for exposure to blood or other possibly infectious material exists in virtually any invasive radiological procedure, from arteriography to image-guided biopsy. Fortunately, the risk of such exposure is low, and the risk of actual transmission of a bloodborne pathogen, whether from patient to healthcare worker or vice versa, is even lower. Nevertheless, it is important for all radiologists who perform invasive procedures to be aware of these risks and to observe pertinent safety and infection control recommendations. This article will review these topics.     

The ontogeny of the expression of K+ channel-like gene (CHIF) in the rat kidney papilla

A whole genome scan was undertaken in a granddaughter design comprising 1158 progeny-tested bulls in order to map QTL influencing milk yield and composition. In this paper we report the identification of a locus on the centromeric end of bovine Chromosome (Chr) 14, with major effect on fat and protein percentage as well as milk yield. The genuine nature of this QTL was verified using the grand2-daughter design, that is, by tracing the segregating QTL alleles from heterozygous grandsires to their maternal grandsons and confirming the predicted QTL allele substitution effect.     

Molecular characterization of Borrelia burgdorferi sensu lato from Slovenia revealing significant differences between tick and human isolates

The aim of the study was to evaluate the morphological and functional status of the liver in acute, oral cholinesterase inhibitors poisoning using static scintigraphy, hepatography and measurements of chosen enzymes activity. Considering the different clinical picture of cholinesterase inhibitors poisonings in people, it was necessary to estimate the poisoning severity and its dependence on the frequency and intensification of the liver lesion. Under examination there were 37 cholinesterase inhibitors orally poisoned patients, treated at the Department of Toxicology in the years 1992-1995. The examined group comprised 7 women (19%) and 30 men (81%). Organophosphate compounds poisoning was noted in 14 patients, and carbamates poisonings in 23 patients. The reference group comprised 30 healthy men aged 24 to 59 years not exposed to hepatotoxic agents. More than 90% of patients were classified as severe poisoned. Any fatal case was not noted. A differently intensified pathological changes of the liver dependent on age and poisoning severity were found in 97.2% of patients and their frequency was significantly higher than in the control group. Hepatographic picture revealed in 96.6% of cases the liver lesion. Hepatographic picture of the liver was also dependent on poisoning severity. The higher activity of AST, ALT, AP and higher bilirubin concentration in blood were noted in poisoned men compared to the control group. Control scintigraphic examination revealed a considerable improvement in the intensification of the liver scintigraphic picture in 40% of the patients and a higher intensification in 13% of the subjects. In 46.6% of the patients the intensification of scintigraphic changes remained at the same level. Considering arbitrary criteria for the degree of the liver lesion, the improvement in the intensification of hepatographic changes was noted in 42.8% of the patients; the intensification of the liver lesion was not noted even in one case. Analyzing the percentage of the liver lesion for each individual patient, improvement was noted in 92.8% of the examined patients, and the changes with the same level of intensification in 7.2%. Deterioration was not noted at all. Conclusion: The liver scintigraphy and hepatography combined with biochemical analysis allows to assess the liver condition in acute cholinesterase inhibitors poisoning.     

Molecular cloning and functional characterization of murine sphingosine kinase

Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison with Saccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylated D-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydrosphingosine or N, N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger.     

pp56Lck mediates TCR zeta-chain binding to the microfilament cytoskeleton

The TCR zeta-chain (zeta) on mature murine T lymphocytes binds to the microfilament cytoskeleton in response to Ag receptor ligation. Here, we report the role of Src family kinases in zeta-cytoskeletal binding, using mutant mice and a cell-free model system. Binding of zeta to actin in the cell-free system has a specific requirement for ATP and divalent cations, with an apparent Michaelis-Menton constant for ATP in the millimolar range, and can be disrupted by either EDTA or the microfilament poison, cytochalasin D, suggesting that microfilaments provide the structural framework for an active process involving cellular kinases. Indeed, tyrosine-phosphorylated zeta is a predominant form of the zeta-chain bound to polymerized actin, while challenge with alkaline phosphatase prevents zeta-chain association in solution and releases zeta-chain from the bound state. Phosphorylated Src-family kinase pp56Lck also associates with membrane skeleton upon TCR engagement and is a component of the reconstituted cytoskeletal pellet. Zeta-chain phosphorylation and zeta-cytoskeletal binding are abrogated in cell lysates with reduced levels of pp56Lck and in activated mutant murine T cells lacking pp56Lck, implicating pp56Lck as the kinase involved in zeta-chain tyrosine phosphorylation and zeta-cytoskeletal binding. Finally, recombinant Lck Src homology 2 domain preferentially inhibits reconstituted zeta-cytoskeleton association, suggesting that zeta-microfilament binding is dependent on interactions between phosphorylated tyrosine residues in zeta-chain activation motifs and the Src homology 2 domain of the Lck protein tyrosine kinase.     

Pigmented uveal tumours in a transgenic mouse model

Lectins are sensitive probes which bind carbohydrate structures specifically. In this study, we modified the lectin staining procedure for sensitive detection of carbohydrate structures in formalin-fixed, paraffin-embedded sections of normal and heterologous serum-induced fibrotic livers. The liver sections were heated in hot distilled water at 100 degrees C for 10 min (thermo-treatment: TT), and then stained with 24 different lectins. In comparison with the results from sections without TT (nonTT), enhanced and/or alternated staining patterns of 19 lectins were demonstrated in sections with TT, and enhanced staining of Vicia villosa agglutinin seen in Kupffer cells was noted. Interestingly, no positive staining was seen with Dolichos biflorus agglutinin, peanut agglutinin or soybean agglutinin (SBA), which recognize O-linked carbohydrate chains, in Kupffer cells of non-TT sections, but strong positive staining was demonstrated in those of TT sections. SBA-positive staining in the cytoplasm of some scattered hepatocytes located in the periportal and perifibrous zones and central zone of pseudolobules was demonstrated only in the fibrotic liver sections with TT. Such findings indicate the heterogeneity of hepatocytes in the liver with fibrosis. Formalin fixation causes masking of lectin binding sites, especially O-linked carbohydrate chains, and TT may recover such masking reactions. TT improved the staining reactions for many lectins in formalin-fixed, paraffin-embedded liver sections, and new staining patterns appear after TT. Modified TT staining procedures may be useful for the diagnosis and prognosis of liver fibrosis.