Tumor necrosis factor-alpha-induced changes in insulin-producing beta-cells

The migration of macrophages and lymphocytes that produce cytokines such as tumor necrosis factor-alpha (TNF-alpha) causes beta-cell death, leading to type 1 diabetes. Similarly, in type 2 diabetes, the adipocyte-derived cytokines including TNF-alpha are elevated in the circulation, causing inflammation and insulin resistance. Thus, the studies described in this article using TNF-alpha are relevant to furthering our understanding of the pathogenesis of diabetes mellitus. We used RINr1046-38 (RIN) insulin-producing beta-cells, which constitutively express calbindin-D(28k), to characterize the effect of TNF-alpha on apoptosis, replication, insulin release, and gene and protein expression. Western blots of TNF-alpha-treated RIN cells revealed a decrease in calbindin-D(28k). By ELISA, TNF-alpha-treated beta-cells had 47% less calbindin-D(28k) than controls. In association with the decline in calbindin-D(28k), TNF-alpha treatment of RIN cells led to a 73% greater increase in changes in intracellular calcium concentration (Delta[Ca(2+)](i)) in TNF-alpha-treated cells as compared to that in control RIN cells upon treatment with 50 mM KCl; caused a greater increase in the [Ca(2+)](i) following the addition of 5.5 microM ionomycin; increased by more than threefold the apoptotic rate, expressed as the percentage of TUNEL-positive nuclei to total nuclei; decreased the rate of cell replication by 36%; and increased and decreased selectively the expression of specific genes as determined by microarray analysis. The subcellular localizations of Bcl-2, an antiapoptotic protein, and Bax, a proapoptotic protein, within RIN cells were altered with TNF-alpha treatment such that the two were colocalized with mitochondria in the perinuclear region. We conclude that the proapoptotic action of TNF-alpha on beta-cells is manifested via decreased expression of calbindin-D(28k) and is mediated at least in part by [Ca(2+)](i).     

A novel autosomal recessive non-syndromic deafness locus (DFNB35) maps to 14q24.1-14q24.3 in large consanguineous kindred from Pakistan

Autosomal recessive nonsyndromic deafness is one of the most frequent forms of inherited hearing impairment. Over 30 autosomal recessive nonsyndromic hearing loss loci have been mapped, and 15 genes have been isolated. Of the over 30 reported autosomal recessive nonsyndromic hearing loss (NSHL) loci, the typical phenotype is prelingual non-progressive severe to profound hearing loss with the exception of DFNB8, which displays postlingual onset and DFNB13, which is progressive. In this report we describe a large inbred kindred from a remote area of Pakistan, comprising six generations and segregating autosomal recessive nonsyndromic prelingual deafness. DNA samples from 24 individuals were used for genome wide screen and fine mapping. Linkage analysis indicates that in this family the NSHL locus, (DFNB35) maps to a 17.54 cM region on chromosome 14 flanked by markers D14S57 and D14S59. Examination of haplotypes reveals a region that is homozygous for 11.75 cM spanning between markers D14S588 and D14S59. A maximum two-point LOD score of 5.3 and multipoint LOD score of 7.6 was obtained at marker D14S53. The interval for DFNB35 does not overlap with the regions for DFNA9, DFNA23 or DFNB5.     

Genomic Regions That Underlie Soybean Seed Isoflavone Content

Soy products contain isoflavones (genistein, daidzein, andglycitein) that display biological effects when ingested byhumans and animals, these effects are species, dose and agedependent. Therefore, the content and quality of isoflavones insoybeans is a key to their biological effect. Our objective wasto identify loci that underlie isoflavone content in soybeanseeds. The study involved 100 recombinant inbred lines (RIL) fromthe cross of ‘Essex' by ‘Forrest,' two cultivars that contrastfor isoflavone content. Isoflavone content of seeds from each RILwas determined by high performance liquid chromatography (HPLC).The distribution of isoflavone content was continuous andunimodal. The heritability estimates on a line mean basis were79% for daidzein, 22% for genistein, and 88% for glycitein.Isoflavone content of soybean seeds was compared against 150polymorphic DNA markers in a one-way analysis of variance. Fourgenomic regions were found to be significantly associated withthe isoflavone content of soybean seeds across both locations andyears. Molecular linkage group B1 contained a major QTLunderlying glycitein content (P = 0.0001, R² = 50.2%), linkagegroup N contained a QTL for glycitein (P = 0.0033, R² = 11.1%)and a QTL for daidzein (P = 0.0023, R² = 10.3%) and linkagegroup A1 contained a QTL for daidzein (P = 0.0081, R² = 9.6%).Selection for these chromosomal regions in a marker assistedselection program will allow for the manipulation of amounts andprofiles of isoflavones (genistein, daidzein, and glycitein)content of soybean seeds. In addition, tightly linked markers canbe used in map based cloning of genes associated withisoflavone content.     

Urachal malignant fibrous histiocytoma: a case report and review of the literature

Pathologic processes involving the urachus are usually related to inflammatory or sinofistular conditions. Neoplasms rarely arise within this structure, and when they do occur, they are typically epithelial, with mucinous adenocarcinoma being the most common. Mesenchymal lesions, both benign and malignant, have rarely been described in this location. We report the case of a 66-year-old white man who presented with a primary urachal malignant fibrous histiocytoma and died of metastatic disease 20 months after the initial diagnosis. This is an unusual case of malignant fibrous histiocytoma arising in a urachal remnant.     

Accumulation of allelic losses on chromosome 10 in human gliomas at recurrence

Aims—To elucidate the implications of allelic loss on chromosome 10 in the malignant progression of human gliomas.     

Mast cell activation markers for in vitro study

ABSTRACT

Mast cells (MCs) are well known for their role in allergic conditions. This cell can be activated by various types of secretagogues, ranging from a small chemical to a huge protein. Mast cell activation by secretagogues triggers the increase in intracellular calcium (iCa²⁺) concentration, granule trafficking, and exocytosis. Activated mast cells release their intra-granular pre-stored mediator or the newly synthesized mediator in the exocytosis process, in the form of degranulation or secretion. There are at least three types of exocytosis in mast cells, which are suggested to contribute to the release of different mediators, i.e.,, piecemeal, kiss-and-run, and compound exocytosis. The status of mast cells, i.e., activated or resting, is often determined by measuring the concentration of the released mediator such as histamine or β-hexosaminidase. This review summarizes several mast cell components that have been and are generally used as mast cell activation indicator, from the classical histamine and β-hexosaminidase measurement, to eicosanoid and granule trafficking observation. Basic principle of the component determination is also explained with their specified research application and purpose. The information will help to predict the experiment results with a certain study design.     

Increased production of human immunodeficiency virus (HIV) in HIV-induced syncytia formation: An efficient infection process

Syncytia or multinucleated giant-cell formation is one of the major cytopathic effects induced by human immunodeficiency virus (HIV) infection. Cell fusion results from the strong interaction of CD4 molecules on the surface of the uninfected T cells and gp120, an external envelope glycoprotein of HIV on the infected T cells. We studied the production of HIV in fusion cells between MOLT-4 and virus-infected MOLT-4/HIV cells and found that HIV production was enhanced up to three- to fivefold, which showed a good correlation with the appearance and extent of syncytia formation. Blocking the fusion by monoclonal antibody against a binding epitope of CD4 molecule to gp120 decreased the HIV production significantly. Enhancement of HIV production was observed by more than five-fold in comparison with chronically infected cells, which were fusion free 20 hr postcocultivation. Electron microscopic observation also showed the presence of abundant HIV particles inside the fused cells and on the outer surface. AZT blocked the HIV augmentation of fused cells in coculture completely. Southern blot analysis revealed that both integrated and unintegrated HIV DNA were highly accumulated in fusion cells, as compared with fusion-free MOLT-4/HIV cells. Among unintegrated DNA, circular and linear DNA were accumulated to a similar degree. Northern blot hybridization showed that rapid enhancement of all three species of HIV-specific RNA containing genomic (9.2 kb) and subgenomic (4.3 and 1.9 kb) RNAs were found 20 hr postinfection in fusion cells. These data suggest that syncytia formation is an extremely active infection process of HIV, by which multiple rounds of reinfection might take place.     

The Cd40 ligand

For several years, the primary function of CD40 ligand (CD40L) has been believed to be in regulation of contact-dependent, CD40-CD40L-mediated signals between B-and T-cells, which are essential for the regulation of thymus-dependent (TD) humoral immune responses. Recently, a flurry of reports indicate that CD40 is expressed by variety of cell types other than B-cells that include dendritic cells, follicular dendritic cells, monocytes, macrophages, fibroblasts, and endothelial cells. These studies show that CD40-CD40L interactions are important in inflammatory process. For the past few years, through the availability of CD40L-knockout mice, new data have emerged to support the belief that CD40L has many more functions than its role in TD humoral immunity. CD40L-deficient mice have provided significant information towards our understanding of the in vivo role of CD40L. The current picture that emerges indicates that CD40-CD40L interactions mediate many cell-mediated immune responses and T-cell-mediated effector functions that are required for proper functioning of the host defense system. This article focuses on the in vivo role of the CD40L in regulation of cell-mediated effector functions.     

Bone formation using novel interconnected porous calcium hydroxyapatite ceramic hybridized with cultured marrow stromal stem cells derived from Green rat

The clinical use of cultured marrow stromal stem cells (MSCs) has recently attracted attention in the field of tissue engineering. For the clinical use of the MSCs, a prominent scaffold is needed. A scaffold hybridized with MSCs is transformed into a "bioactive bone substitute," and this provides good osteoconduction. In this study, a novel calcium hydroxyapatite ceramic with an interconnected porous structure (IP-CHA) was used as a scaffold. MSCs were harvested from Green rats containing Green Fluorescent Protein (GFP), and then these hybrids were implanted into the tibias of Sprague-Dawley rats. The purposes of this study were to examine the osteogenic ability of these hybrids without coculture, and to evaluate whether the resulting bone formation originated from the grafted MSCs or the recipient's cells. The hybridized group showed excellent bone formation compared with the IP-CHA-only implant group. Observation of the implanted MSCs revealed that they survived 8 weeks after surgery, and differentiated into osteoblast-like cells, thus providing bone formation. This implantation of the MSCs/IP-CHA composite provides excellent osteoconduction, and is expected to have extensive clinical applications.