Annexin XI may be involved in Ca2+ - or GTP-gammaS-induced insulin secretion in the pancreatic beta-cell

The aim of this study was to investigate possible involvement of annexin XI in the insulin secretory machinery. In fluorescence immunocytochemistry, annexin XI was found in the cytoplasm of pancreatic endocrine cells and a pancreatic beta-cell line, MIN6, in a granular pattern. MIN6 cells also possessed weak and diffused annexin XI immunoreactivity in the cytoplasm. Immunoelectron microscopy revealed annexin XI in the insulin granules. Insulin secretion from streptolysin-O-permeabilized MIN6 cells was inhibited by anti-annexin XI antibody, when the release was stimulated by either Ca2+ or GTP-gammaS, but not by a protein kinase C-activating phorbol ester. Inhibition of insulin release by anti-annexin XI antibody was reproduced in permeabilized rat islets. These findings suggest that annexin XI may be involved in the regulation of insulin secretion from the pancreatic beta-cells.     

Serological Surveillance Development for Tropical Infectious Diseases Using Simultaneous Microsphere-Based Multiplex Assays and Finite Mixture Models

Background

A strategy to combat infectious diseases, including neglected tropical diseases (NTDs), will depend on the development of reliable epidemiological surveillance methods. To establish a simple and practical seroprevalence detection system, we developed a microsphere-based multiplex immunoassay system and evaluated utility using samples obtained in Kenya.

Methods

We developed a microsphere-based immuno-assay system to simultaneously measure the individual levels of plasma antibody (IgG) against 8 antigens derived from 6 pathogens: Entamoeba histolytica (C-IgL), Leishmania donovani (KRP42), Toxoplasma gondii (SAG1), Wuchereria bancrofti (SXP1), HIV (gag, gp120 and gp41), and Vibrio cholerae (cholera toxin). The assay system was validated using appropriate control samples. The assay system was applied for 3411 blood samples collected from the general population randomly selected from two health and demographic surveillance system (HDSS) cohorts in the coastal and western regions of Kenya. The immunoassay values distribution for each antigen was mathematically defined by a finite mixture model, and cut-off values were optimized.

Findings

Sensitivities and specificities for each antigen ranged between 71 and 100%. Seroprevalences for each pathogen from the Kwale and Mbita HDSS sites (respectively) were as follows: HIV, 3.0% and 20.1%; L. donovani, 12.6% and 17.3%; E. histolytica, 12.8% and 16.6%; and T. gondii, 30.9% and 28.2%. Seroprevalences of W. bancrofti and V. cholerae showed relatively high figures, especially among children. The results might be affected by immunological cross reactions between W. bancrofti-SXP1 and other parasitic infections; and cholera toxin and the enterotoxigenic E. coli (ETEC), respectively.

Interpretation

A microsphere-based multi-serological assay system can provide an opportunity to comprehensively grasp epidemiological features for NTDs. By adding pathogens and antigens of interest, optimized made-to-order high-quality programs can be established to utilize limited resources to effectively control NTDs in Africa.     

Formation of phospholipid hydroperoxides and its inhibition by alpha-tocopherol in rat brain synaptosomes induced by peroxynitrite

Peroxynitrite resulted from the reaction of nitric oxide and superoxide anion has been implicated in the genesis of neurotoxicity. In this study, the oxidation of phospholipids in rat brain synaptosomes induced by peroxynitrite generated from 3-morpholinosydnonimine (SIN-1) was studied in vitro. The formation and accumulation of phospholipid hydroperoxides, including phosphatidylcholine hydroperoxide (PCOOH) and phosphatidyl-ethanolamine hydroperoxide (PEOOH) in rat brain synaptosomes induced by peroxynitrite, were observed. PEOOH and PCOOH were formed rapidly and SIN-1 concentration-dependently. The hydroperoxides formed in synaptosomes were unstable and it was suggested that phospholipase A2 played a role in degradation of the hydroperoxides. The endogenous alpha-tocopherol acted as a potent antioxidant. It was oxidized very rapidly and concentration-dependently by SIN-1 to alpha-tocopheryl quinone. Furthermore, uric acid was found to be an effective antioxidant in inhibiting oxidative damage to synaptosomal lipids induced by SIN-1. The results provide direct evidence to show that peroxynitrite can not only deplete alpha-tocopherol, but also cause production of phospholipid hydroperoxides resulting in disrupted brain tissue.     

Isolation and Characterization of Tonoplast from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L

Tonoplasts were isolated in a high purity from etiolated young seedlings of Vigna radiata L. (mung bean) utilizing a sucrose density gradient system. The excised hypocotyls were homogenized in a sorbitol-buffer system and the 3,600 to 156,000g pellets obtained after the differential centrifugations were suspended in a sorbitol medium and loaded on a linear sucrose density gradient. After centrifugation at 89,000g for 2 hours, tonoplasts were banded at the sample load/sucrose interface. Assessed by electron microscopy and marker enzymes, the purity and the quantity were found to be sufficient for biochemical and biophysical analyses. The tonoplasts were associated with NO₃⁻-sensitive and vana-date-insensitive ATPase. The tonoplast ATPase was stimulated by proton ionophores such as carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone and gramicidin D, suggesting a proton-pumping enzyme. In the presence of ATP and Mg²⁺, a proton gradient was formed in the isolated tonoplast vesicles as assessed by fluorescence quenching of quinacrine. The tonoplasts contained several kinds of mannosylated or glycosylated glycoproteins and a major protein (65 kilodaltons) which was unique to the membranes.     

Developmental analysis of the grandchildless (gs(1)N26) mutation in Drosophila melanogaster: abnormal cleavage patterns and defects in pole cell formation

This article describes developmental analysis of gs(1)N26 mutation. gs(1)N26 is a temperature-sensitive maternal-effect mutation affecting the formation of the germ line (Y. Niki and M. Okada, Wilhelm Roux's Arch. Dev. Biol. 190, 1-10, 1981). At 25 degrees C, the cleavage nuclei do not divide synchronously and show various degrees of retarded migration to the posterior region. Blastoderm nuclei show antero-posterior mitotic waves; posterior yolk nuclei also are reduced in number at this stage. Pole cells form only when the cleavage nuclei migrate directly to the posterior pole. In fact, the posterior region of young eggs presents the usual ultrastructural features, and it is also able to participate in the formation of pole cells, as was proven by cytoplasmic transfer experiments. Therefore the defects in blastogenesis, in particular in the formation of pole cells of gs(1)N26 embryos, appear to result from the delayed migration of cleavage nuclei to the posterior pole.     

Characterization of the smtA gene encoding an S-adenosylmethionine-dependent methyltransferase of Escherichia coli

Abstract The mukB operon is located at 21 min on the Escherichia coli chromosome and seems to consist of four genes, orf30 ( smtA ), mukF , mukE , and mukB . Based on sequence similarity, the promoter-proximal gene, orf30 ( smtA ), could encode an S-adenosylmethionine-dependent methyltransferase. The smtA gene is not essential for cell growth and its expression is positively regulated by H-NS, an Escherichia coli histone-like protein.     

Carboxyl terminal region of the MukB protein in Escherichia coli is essential for DNA binding activity

Abstract The purified MukB protein of Escherichia coli has DNA binding activity and nucleotide binding activity. We have isolated a mutation, mukB1013 , causing a substitution of valine at position 1379 to leucine. This mutant MukB protein was defective for DNA binding, while the ATP binding activity remained unaffected. A truncated MukB protein that is short of 109 amino acids from the C-terminus failed to bind DNA.     

Cell membrane permeability and respiratory activity in chilling stressed callus

To improve our understanding of the mechanism of chilling injuryin chill-sensitive callus (Cornus stolontfera), early changesin cell permeability and respiratory activity were studied.Partial leakage of amino acids and an abrupt increase in permeationand oxidation of added dopamine were characteristic of chilledcallus in the late stage of chilling at 0?C (48 hr), when mostof the callus sustained severe injury. However, little or nochange in cell permeability was observed in the early stageof chilling (within 24 hr), when calli retained their viabilityfor growth after transfer to a warm temperature. These resultssuggest that changes in the cell membranes per se are by nomeans the primary step in cell injury. Temporary depressionof respiratory activity was detected soon after chilling for12 hr, but activity appeared to return to the original levelon further chilling up to 24 hr. An irreversible dysfunction,however, occurred in the respiratory system on prolonged chillingup to 48 hr. This implies that irreversible impairment of mitochondrialfunctions may not be involved in the early stage of the cellinjury. A possible relationship between these observed changesand ultrastructural changes in chilled cells is discussed. ¹Contribution No. 2153 from the Institute of Low TemperatureScience. (Received June 6, 1979; )     

Interleukin-1 Receptor-associated Kinase-4 (IRAK4) Promotes Inflammatory Osteolysis by Activating Osteoclasts and Inhibiting Formation of Foreign Body Giant Cells

Formation of foreign body giant cells (FBGCs) occurs following implantation of medical devices such as artificial joints and is implicated in implant failure associated with inflammation or microbial infection. Two major macrophage subpopulations, M1 and M2, play different roles in inflammation and wound healing, respectively. Therefore, M1/M2 polarization is crucial for the development of various inflammation-related diseases. Here, we show that FBGCs do not resorb bone but rather express M2 macrophage-like wound healing and inflammation-terminating molecules in vitro. We also found that FBGC formation was significantly inhibited by inflammatory cytokines or infection mimetics in vitro. Interleukin-1 receptor-associated kinase-4 (IRAK4) deficiency did not alter osteoclast formation in vitro, and IRAK4-deficient mice showed normal bone mineral density in vivo. However, IRAK4-deficient mice were protected from excessive osteoclastogenesis induced by IL-1β in vitro or by LPS, an infection mimetic of Gram-negative bacteria, in vivo. Furthermore, IRAK4 deficiency restored FBGC formation and expression of M2 macrophage markers inhibited by inflammatory cytokines in vitro or by LPS in vivo. Our results demonstrate that osteoclasts and FBGCs are reciprocally regulated and identify IRAK4 as a potential therapeutic target to inhibit stimulated osteoclastogenesis and rescue inhibited FBGC formation under inflammatory and infectious conditions without altering physiological bone resorption.