NHE blockade inhibits chemokine production and NF-kappaB activation in immunostimulated endothelial cells

Na⁺/H⁺exchanger (NHE) activation has been documented to contribute toendothelial cell injury caused by inflammatory states. However, therole of NHEs in regulation of the endothelial cell inflammatoryresponse has not been investigated. The present study tested thehypothesis that NHEs contribute to endothelial cell inflammationinduced by endotoxin or interleukin (IL)-1. NHE inhibition usingamiloride, 5-(N-ethyl-N-isopropyl)-amiloride, and5-(N-methyl-N-isobutyl)amiloride as well as thenon-amiloride NHE inhibitors cimetidine, clonidine, and harmalinesuppressed endotoxin-induced IL-8 and monocyte chemoattractant protein(MCP)-1 production by human umbilical endothelial vein cells (HUVECs). The suppressive effect of amiloride on endotoxin-induced IL-8 production was associated with a decreased accumulation of IL-8 mRNA.NHE inhibitors suppressed both inhibitory (I)B degradation andnuclear factor (NF)-B DNA binding, suggesting that a decrease inactivation of the IB-NF-B system contributed to the suppression of HUVEC inflammatory response by NHE blockade. NHE inhibition decreased also the IL-1-induced HUVEC inflammatory response, becauseamiloride suppressed IL-1-induced E-selectin expression on HUVECs.These results demonstrate that maximal activation of the HUVECinflammatory response requires a functional NHE.

     

Microarray Analysis of Gene Expression in Rat Hippocampus After Chronic Ethanol Treatment

It is thought that changes in gene expression in the brain mediate chronic ethanol-induced complex behaviors such as tolerance, dependence, and sensitization, and also relate to ethanol-induced brain toxicity. Using high-density filter-based cDNA microarrays (GeneFilters), we analyzed the expression of over 5000 genes in the dorsal hippocampus of rats treated with 12% ethanol or tap water for 15 months. Ethanol-induced changes in gene expression were particularly prominent in two groups of genes. One group consisted of oxidoreductases, including ceruloplasmin, uricase, branched-chain alpha-keto acid dehydrogenase, NADH ubiquinone oxidoreductase, P450, NAD⁺-isocitrate dehydrogenase, and cytochrome c oxidase, which may be related to ethanol-induced oxidative stress. The other group of genes included ADP-ribosylation factor, RAS related protein rab10, phosphatidylinositol 4-kinase, dynein-associated polypeptides, and dynamin-1, which seem to be involved in membrane trafficking. The results may reveal some of the pathways involved in ethanol-induced pathophysiological changes.     

Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes

To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks.     

Chaperones and aging: role in neurodegeneration and in other civilizational diseases

Chaperones are highly conserved proteins responsible for the preservation and repair of the correct conformation of cellular macromolecules, such as proteins, RNAs, etc. Environmental stress leads to chaperone (heat-shock protein, stress protein) induction reflecting the protective role of chaperones as a key factor for cell survival and in repairing cellular damage after stress. The present review summarizes our current knowledge about the chaperone-deficiency in the aging process, as well as the possible involvement of chaperones in neurodegenerative diseases, such as in Alzheimer’s, Parkinson’s, Huntington- and prion-related diseases. We also summarize a recent theory implying chaperones as “buffers” of variations in the human genome, which role probably increased during the last 200 years of successful medical practice minimizing natural selection. Chaperone-buffered, silent mutations may be activated during the aging process, which leads to the phenotypic exposure of previously hidden features and might contribute to the onset of polygenic diseases, such as atherosclerosis, cancer, diabetes and several neurodegenerative diseases.     

Shedding of the interleukin-6 (IL-6) receptor (gp80) determines the ability of IL-6 to induce gp130 phosphorylation in human osteoblasts

Human osteoblasts produce interleukin-6 (IL-6) and respond to IL-6 in the presence of soluble IL-6 receptor (sIL-6R), but the cell surface expression of IL-6R and the mechanism of sIL-6R production are largely unknown. Three different human osteoblast-like cell lines (MG-63, HOS, and SaOS-2) and bone marrow-derived primary human osteoblasts expressed both IL-6R and gp130 as determined by flow cytometry and immunoprecipitation. However, the membrane-bound IL-6R was nonfunctional, as significant tyrosine phosphorylation of gp130 did not occur in the presence of IL-6. Phorbol myristate acetate induced a dramatic increase of both IL-6R shedding (i.e. the production of sIL-6R) and IL-6 release in osteoblast cultures, but the cell surface expression of gp130 remained unchanged. IL-6 complexed with sIL-6R, either exogenously introduced or derived from the nonfunctional cell surface form by shedding, induced rapid tyrosine phosphorylation of gp130. This effect was inhibited by neutralizing antibodies to either sIL-6R or gp130, indicating that the gp130 activation was induced by IL-6/sIL-6R/gp130 interaction. Protein kinase C inhibitors blocked phorbol myristate acetate-induced and spontaneous shedding of IL-6R resulting in the absence of sIL-6R in the culture medium, which in turn also prevented the activation of gp130. In conclusion, human osteoblasts express cell surface IL-6R, which is unable to transmit IL-6-induced signals until it is shed into its soluble form. This unique mechanism provides the flexibility for osteoblasts to control their own responsiveness to IL-6 via the activation of an IL-6R sheddase, resulting in an immediate production of functionally active osteoblast-derived sIL-6R.     

SCide: identification of stabilization centers in proteins

SUMMARY: SCide is a program to identify stabilization centers from known protein structures. These are residues involved in cooperative long-range contacts, which can be formed between various regions of a single polypeptide chain, or they can belong to different peptides or polypeptides in a complex. The server takes a PDB file as an input, and the result is presented in graphical or text format. AVAILABILITY: SCide is available on the web at http://www.enzim.hu/scide. The source code can be obtained from the authors on request.     

Apoptosis,necrosis and cellular senescence: chaperone occupancy as a potential switch

Chaperone function plays a key role in repairing proteotoxic damage and in the maintenance of cell survival. Here we compare the regulatory role of molecular chaperones (heat shock proteins, stress proteins) in cellular senescence, apoptosis and necrosis. We also review the current data on chaperone level and function in aging cells, and list some possible therapeutic interventions. Finally, we postulate a hypothesis, that increasing chaperone occupancy might be an important event which forces cells out of the normal cell cycle towards senescence. In the case of severe stress, this may lead to apoptosis or, following lethal stress, to cell necrosis.     

Description of two new actinosporean types from a brook of Fuji Mountain,Honshu, and from Chitose River,Hokkaido, Japan

Actinospore infection of oligochaetes living in the mud of 3 freshwater biotopes in Japan was studied. Using the cell-well plate method, a new aurantiactinomyxon type was found in 0.77% of the examined Tubifex tubifex oligochaete specimens from a brook near Yamanashi Prefectural Fisheries Experimental Station on Fuji Mountain. In 0.14% of Lumbriculus variagetus collected from Chitose River, near Chitose Salmon Hatchery, a new siedleckiella type was found, while at the same time 8.1% of the Lumbriculus spp. oligochaetes released triactinomyxons of Myxobolus arcticus. Of the examined Rhyacodrilus komarovi oligochaetes collected from the Mena River system, Hokkaido, 0.2, 0.6, 0.5 and 0.8% were infected with echinactinomyxon, neoactinomyxum and 2 types of triactinomyxon spores, respectively, and described in our previous paper. The oligochaetes released actinospores for several weeks. Actinospore infection showed high intensity in positive oligochaetes in the case of all the actinosporean types. Two of the actinospore types (aurantiactinomyxon and siedleckiella) presented here have not been previously described.