A surface polysaccharide of Escherichia coli O111 contains O-antigen and inhibits agglutination of cells by O-antiserum

The repeating pentasaccharide of O-antigen from Escherichia coli O111 contains galactose, glucose, N-acetylglucosamine, and colitose, the latter representing the major antigenic determinant. Phenol extraction of this strain was previously shown to release two fractions (I and II) containing O-antigen carbohydrate, and both fractions were believed to be lipopolysaccharide. We have now characterized fractions I and II and conclude that only fraction II represents lipopolysaccharide. Fraction II contains phosphate, 2-keto-3-deoxyoctonate, beta-hydroxymyristic acid, and potent endotoxin activity, whereas fraction I was deficient in all of these properties of the lipid A and core oligosaccharide regions of lipopolysaccharide. Fractions I and II each represented 50% of the total cellular O-antigen, and both were present on the cell surface. Both fractions were metabolically stable, and no precursor-product relationship existed between them. Fraction II had a number-average molecular weight of 15,800, corresponding to an average of 12 O-antigen repeats per molecule. In contrast, fraction I had a number-average molecular weight of 354,000, corresponding to an average of 404 O-antigen repeats per molecule. Before heat treatment, cells of E. coli O111 are poorly agglutinated by O-serum; although this indicates the presence of a capsule, the corresponding K-antigen was never detected. We conclude that fraction I, when present on the cell surface, inhibits agglutination of unheated cultures of E. coli O111 by O-serum because: (i) a variant strain which lacks fraction I was agglutinated by O-serum without prior heating; (ii) erythrocytes coated with purified fraction I behaved like bacteria containing fraction I in showing inhibition of O-serum agglutination; and (iii) heat treatment released fraction I and rendered bacterial cells agglutinable in O-serum.     

Dichotomy in Growth and Invasion from Low- to High-Grade Glioma Cellular Variants

Glial dysfunction outraging CNS plasticity and integrity results in one of the most dangerous cancers, namely glioma, featuring little median survival period and high recurrence. The hallmark properties of proliferation, invasion and angiogenesis with the infiltrated macrophages in glioma are expected to be tightly coupled or cross-linked, but not properly related so far. The present study is aimed to find a relationship between this featured quadrangle from lower to higher grades (HG) of post-operative glioma tissues and their invading subsets. Elevated Ki67-associated proliferation in lower grades (LG) was supported with VEGF dependent angiogenic maintenance which found a decrease unlikely in HG. In contrast, MMP 2 and 9-associated invasions augmented high in HG with the dominant presence of CD204⁺ M2 polarized macrophages and a general increase in global DNMT1-associated methylation. Marked differences found in ECM invading cellular subsets of HG showing high proliferative capacity indicating rationally for recurrence, contrasting the nature of gross tumor tissue of the same grade. Thus in LG, the neoplastic lesion is more inclined to its growth while in higher grade more disposed towards tissue wreckage in support with cellular environmental milieu whereas the cellular variants and subsets of invaded cells showed different trends. Therefore, some operational dichotomy or coupling among cellular variants in glioma is active in determining its low- to high-grade transition and aggressive progression.

     

In vitro efficacy of 7-benzylamino-1-isoquinolinamines against Plasmodium falciparum related to the efficacy of chalcones

A series of 1,7-diaminoisoquinolinamines, that are expected to mediate antimalarial activity by the same mechanism employed by the chalcones, were produced. Six 7-benzylamino-1-isoquinolinamines were found to be submicromolar inhibitors in vitro of drug-resistant Plasmodium falciparum, with the best possessing activity comparable to chloroquine. Despite being developed from a lead that is a DHFR inhibitor, these compounds do not mediate their antimalarial effects by inhibition of DHFR.     

Role of protein kinase C isoforms in the regulation of interleukin-13-induced 15-lipoxygenase gene expression in human monocytes

We reported previously that interleukin-13 (IL-13) induces tyrosine phosphorylation/activation of Jak2 and Tyk2 kinases and Stats 1, 3, 5, and 6 in primary human monocytes. We recently revealed that p38 MAPK-mediated serine phosphorylation of both Stat1 and Stat3 is required for the induction of 15-lipoxygenase (15-LO) expression by IL-13. In this study, we present data indicating that another serine/threonine kinase, PKCdelta, is also required for IL-13-induced 15-LO expression. PKCdelta, a member of the novel protein kinase C (PKC) subclass, was rapidly phosphorylated and activated upon exposure to IL-13. Treatment of cells with rottlerin, a PKCdelta inhibitor, blocked IL-13-induced 15-LO mRNA and protein expression, whereas Go6976, an inhibitor of the conventional PKC subclass, had no inhibitory effects. Down-regulation of cellular PKCdelta protein levels by PKCdelta-specific antisense oligodeoxyribonucleotides also inhibited 15-LO expression markedly. IL-13-induced 15-LO expression resulted in significant inhibition of synthesis of the potent chemotactic factor leukotriene B4, and that process was reversed by rottlerin, presumably through the blockage of PKCdelta-dependent 15-LO expression. Furthermore, our data demonstrate that IL-13-mediated activation of PKCdelta and p38 MAPK are independent pathways, because inhibition of one kinase activity had no effect on the other, suggesting that the two pathways act in parallel to regulate the downstream targets necessary for 15-LO expression. Inhibition of PKCdelta activation by rottlerin also markedly attenuated IL-13-induced Stat3 DNA binding activity. Our findings indicate that PKCdelta plays an important role in regulating IL-13-induced 15-LO expression in human monocytes and subsequently modulates the inflammatory responses mediated by 15-LO products.     

Cys-113 and Cys-422 form a high affinity metalloid binding site in the ArsA ATPase

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB extrusion pump for the trivalent metalloids As(III) and Sb(III). ArsA, the catalytic subunit has two homologous halves, A1 and A2. Each half has a consensus signal transduction domain that physically connects the nucleotide-binding domain to the metalloid-binding domain. The relation between metalloid binding by ArsA and transport through ArsB is unclear. In this study, direct metalloid binding to ArsA was examined. The results show that ArsA binds a single Sb(III) with high affinity only in the presence of Mg(2+)-nucleotide. Mutation of the codons for Cys-113 and Cys-422 eliminated Sb(III) binding to purified ArsA. C113A/C422A ArsA has basal ATPase activity similar to that of the wild type but lacks metalloid-stimulated activity. Accumulation of metalloid was assayed in intact cells, where reduced uptake results from active extrusion by the ArsAB pump. Cells expressing the arsA(C113A/C422A)B genes had an intermediate level of metalloid resistance and accumulation between those expressing only arsB alone and those expressing wild type arsAB genes. The results indicate that, whereas metalloid stimulation of ArsA activity enhances the ability of the pump to reduce the intracellular concentration of metalloid, high affinity binding of metalloid by ArsA is not obligatory for transport or resistance. Yet, in mixed populations of cells bearing either arsAB or arsA(C113A/C422A)B growing in subtoxic concentrations of arsenite, cells bearing wild type arsAB replaced cells with mutant arsA(C113A/C422A)B in less than 1 week, showing that the metalloid binding site confers an evolutionary advantage.     

Bilateral Common Carotid Artery Ligation Transiently Changes Brain Lipid Metabolism in Rats

Brain lipid metabolism was studied in rats following permanent bilateral common carotid artery ligation (BCCL), a model for chronic cerebral hypoperfusion. Unesterified (free) fatty acids (uFA) and acyl-CoA concentrations were measured 6 h, 24 h, and 7 days after BCCL or sham surgery, in high energy-microwaved brain. In BCCL compared to sham rats, cytosolic phospholipase A(2) (cPLA(2)) immunoreactivity in piriform cortex, and concentrations of total uFA and arachidonoyl-CoA, an intermediate for arachidonic acid reincorporation into phospholipids, were increased only at 6 h. At 24 h, immunoreactivity for secretory phospholipase A(2) (sPLA(2)), which may regulate blood flow, was increased near cortical and hippocampal blood vessels. BCCL did not affect levels of brain IB(4)+ microglia, glial fibrillary acidic protein (GFAP)+ astrocytes, cyclooxygenase-2 (COX-2) immunoreactivity at any time, but increased cPLA(2) immunoreactivity in one region at 6 h. Thus, BCCL affected brain lipid metabolism transiently, likely because of compensatory sPLA(2)-mediated vasodilation, without producing evidence of neuroinflammation.     

Cytologic features of subependymal giant cell astrocytom: a review of 7 cases

OBJECTIVE: To describe the cytologic features of subependymal giant cell astrocytoma (SEGA) on smears and analyze cytomorphologic parameters that may help in reaching the diagnosis of SEGA. STUDY DESIGN: Cytologic smears of 7 cases of SEGA were reviewed and graded semi-quantitatively for 11 cytologic features: clustering, cytoplasmic fibrillary processes (fibrillarity), cellularity, small prominent nudcleoli, binucleation or multinucleation, "strap cells", spindle-shaped cells, mitoses, intranuclear inclusions, nuclear atypia and perivascular palisading/pseudorosettes. Corresponding histologic sections were also reviewed. RESULTS: The study included 5 male and 2 female patients with an average age of 8.3 years (range, 3-16) at surgery. Cytologic examination revealed loosely cohesive clusters of large cells possessing round to oval nuclei with no or minimal atypia; fine, evenly distributed chromatin; and abundant eosinophilic cytoplasm enmeshed in abundant thin, hairlike processes. Predominant features included hypercellularity, cell clustering, and fibrillarity. Binucleation or multinucleation; small, prominent nucleoli; and strap cells were often seen. Although common in histologic sections, perivascular palisading/pseudorosettes and spindled astrocytic cells were rarely noted on smears. CONCLUSION: The cytologic features of SEGA are highly characteristic and thus are of great use in supporting a diagnosis of SEGA and in excluding mimics, primarily gemistocytic astrocytoma and ependymoma.