Functional analysis of a dominant mutation of human connexin26 associated with nonsyndromic deafness.

Cx26 has been implicated in dominant (DFNA3) and recessive (DFNB1) forms of nonsyndromic sensorineural deafness. While most homozygous DFNB1 Cx26 mutations result in a simple loss of channel activity, it is less clear how heterozygous mutations in Cx26 linked to DFNA3 cause hearing loss. We have tested the ability of one dominant mutation (W44C) to interfere with wild-type human Cx26 (HCx26wt). HCx26wt induced robust electrical conductance between paired oocytes, and facilitated dye transfer between transfected HeLa cells. In contrast, oocyte pairs injected with only W44C were not electrically coupled above background levels, and W44C failed to dye couple transfected HeLa cells. Moreover, W44C dramatically inhibited intercellular conductance of HCx26wt when co-expressed in an equal ratio, and the low levels of residual conductance displayed altered gating properties. A nonfunctional recessive mutation (W77R) did not inhibit the ability of HCx26wt to form functional channels when co-injected in the same oocyte pairs, nor did it alter HCx26wt gating. These results provide evidence for a functional dominant negative effect of the W44C mutant on HCx26wt and explain how heterozygous Cx26 mutations could contribute to autosomal dominant deafness, by resulting in a net loss, and/or alteration, of Cx26 function.

Expression of interleukin-1 genes and interleukin-1 receptors in the mouse brain after hippocampal injury.

In order to evaluate the role of IL-1 production in post-traumatic brain, transcripts for IL-1 (alpha, beta, RA) have been quantified following RT-PCR, in hippocampus and cortex after injury of either hippocampus (Hip) or striatum (Stri). Moreover, 125I IL-1alpha binding sites have been directly quantified using binding experiments on brain sections and quantitative autoradiography. Under basal conditions, levels of PCR products were very low. On day 1, IL-1RA transcripts only were strongly increased in the hippocampus after Hip-lesions and in cortex after Stri lesion. Transcripts were back to control values on day 7 post-lesion. IL-1 receptor densities in the hippocampus (dentate gyrus) were decreased at day 1 around the site of the lesion (but not on the contralateral side) and were back to controls on day 7 indicating a transient and local IL-1 production in the surroundings of the lesion. No changes were found following Stri lesion. This study provides further evidence of the role of the IL-1 molecules family, notably IL-1RA, in the brain reaction to trauma.

Interleukin-1 receptor defect in autoimmune NZB mouse brain.

Interleukin-1 receptors (IL-1R type I and II) have been characterized in murine nervous structures (hippocampus and frontal cortex), in vascular structures (vessels, choroid plexus), and in the anterior pituitary. Because interleukin-1 (IL-1), injected or induced in the brain, is a powerful regulator of the stress axis and immune functions, it was of interest to investigate IL-1Rs and IL-1 in autoimmune mice. In control mice, bacterial lipopolysaccharide (LPS), administered i.p. or i.c.v., induces a sharp decrease in available brain IL-1 receptors, in spite of a moderate increase in mRNAs for both receptor types. This is concomitant with an increase in IL-1 alpha, beta, and ra mRNA. Ligand production clearly overcomes receptor turnover. In autoimmune mice (NZB and NZB/NZW F1), a strong defect in IL-1R (type I) is demonstrated in the dentate gyrus. This tissue-specific defect cannot be explained by increased occupancy by endogeneous ligands as for LPS-treated mice. The transmission of the defect is Mendelian and suggests the involvement of a single gene. However patterns of IL-1R mRNAs (evaluated by RT-PCR) are similar in NZB and in controls, suggesting a translational or post-translational abnormality. The contribution of this genetic disorder in the development of autoimmunity remains to be clarified. Because the brain IL-1 system sends inhibitory signals towards immune functions, this lack of functional IL-1 binding sites might participate in the disregulations observed in NZB autoimmune mice.

Interleukin-1 receptor deficiency in the hippocampal formation of (NZB x NZW)F2 mice: genetic and molecular studies relating to autoimmunity.

Interleukin-1 receptor (IL-1R) deficiency has been previously described in the dentate gyrus of autoimmune NZB and (NZB x NZW) F1 (or BWF1) mice. In this study, the genetic and molecular characterization of this defect were investigated in BWF2 mice in relation to anti-DNA antibody production and microsatellite D1Nds4 (near the IL1r1 gene) polymorphism. IL-1R density was quantified in the brain, spleen and pancreas, using in vitro quantitative autoradiography with recombinant human [125I]-IL-1alpha as the ligand. This study of the dentate gyrus of F2 mice revealed three phenotypes: NZW-like, NZB-like and F1-like, which occurred in a ratio of 1:1:2, with IL-1R densities of 100%, 17% and 59%, respectively as compared to control NZW mice (100%). In contrast, IL-1R densities observed in the choroid plexus and peripheral organs were similar. Moreover a high production of IgG2a anti-DNA antibodies was observed in F2 mice, as in their parents, particularly those with the NZB-like phenotype. Microsatellite mapping of D1Nds4 revealed polymorphism in both parents and BWF2 mice in relation to the level of IL-1R density in the dentate gyrus. In spite of the acute defect in IL-1 binding in the dentate gyrus of NZB mice, molecular analysis of IL-1R mRNA (type I, II and accessory protein) showed similar amounts of mRNA, measured following RT-PCR amplification, in the hippocampal formation of both NZB and control C3H/He mice. In conclusion, the transmission of the IL-1R defect in the dentate gyrus of NZB mice is monofactorial and the defect appears to be at the post-transcriptional level of IL-1R synthesis. The lack of IL-1R in the dentate gyrus seems to correlate with some autoimmune characteristics. Correlation of D1Nds4 polymorphism with the level of IL-1R density suggests that it could be a predisposing gene to disease or a marker for other closely linked predisposing genes.

Analysis of brain mRNA by reverse transcription-polymerase chain reaction and hybridization with digoxigenin-labeled DNA probe.

The interleukin-1 family of polypeptides (IL-1 alpha, IL-1 beta and IL-1 receptor antagonist (RA)) induces various centrally mediated host defense responses to infectious pathogens. Considerable interest has focussed on IL-1 as a mediator in disease and in the production of systemic acute phase responses. We have recently studied the effects of a peripheral stimulation by intraperitoneal (i.p.) administration of lipopolysaccharide (LPS) on the mRNAs expressions of IL-1 (alpha, beta, RA) and their receptors (IL-1 receptor type I and type II (IL-1R1, IL-1R2)) in the central nervous system (CNS). The levels of these expressions being very low in the CNS, the reverse transcription-polymerase chain reaction (RT-PCR) techniques are required for these studies. RT-PCR is a developed method of identifying mRNAs in very small amount of nucleic acid. We have previously developed a method to choose specific PCR primers. The detection of specific PCR products is extremely important. Since amplifications with these specific PCR primers can be achieved under the same conditions (buffers and temperatures) reliable results can be obtained. Characterization of a PCR product requires the use of a specific DNA probe that hybridizes to the region of interest. In addition to providing specificity of detection, the use of labeled DNA probes provides increased sensitivity over ethidium bromide staining. We have previously described a method of synthesis of non-radioactive probe labeled with digoxigenin by nested PCR. Moreover the major advantage to the use of non-radioactive label is that it does not have a short half-life and can last for weeks or even months. A quantification of the PCR products can be obtained using a method based on the analysis of photographic negatives of agarose gels.

Interleukin-1 receptor accessory protein transcripts in the brain and spleen: kinetics after peripheral administration of bacterial lipopolysaccharide in mice.

Receptors for IL-1, type I (IL-1R1) and type II (IL-1R2), have been characterized by pharmacological and molecular techniques in the mouse brain. High densities are mainly found in the cortex, dentate gyrus and choroid plexus. It was therefore of interest to investigate the expression of mRNA IL-1 receptor accessory protein (IL-1R AcP), which is a part of the IL-1 receptor complex and has been shown to interact specifically with IL-1R1. IL-1R AcP transcripts were detected under basal conditions following RT-PCR amplification in the mouse brain, as well as in the pituitary, spleen, adrenal and liver. IL-1R AcP transcripts were found in higher amounts than IL-1R1 transcripts in all tissues except the spleen, where their expression was minor. Following bacterial lipopolysaccharide (LPS) stimulation (3-48 h), IL-1R AcP transcripts were not changed in the brain, while IL-1R1 transcripts were increased for 3-6 h. In the spleen, a slight increase in IL-1R AcP and IL-1R1 was observed during the first hours following LPS stimulation. In conclusion, IL-1R AcP mRNA is expressed in the brain and in other tissues where IL-1R1 transcripts are found. However, the regulation of its expression is distinct from IL-1R1. The high level of expression and the lack of regulation of IL-1R AcP transcripts in the brain under inflammatory conditions suggest that the protein might be constitutively expressed in excess.

Interleukin-1 receptors type I and type II in the mouse brain: kinetics of mRNA expressions after peripheral administration of bacterial lipopolysaccharide.

The expression of transcripts for Interleukin-1 (IL-1) type I and type II receptors (IL-1R1, IL-1R2) was investigated in the mouse brain and spleen using reverse transcription-polymerase chain reaction techniques under basal conditions and following injection of endotoxin (LPS, i.p., 4 mg/kg). Under basal conditions, mRNAs for both receptor types were found in various parts of the brain, in pituitary as well as in spleen. Following LPS stimulation, mRNA expressions were increased in all studied tissues. IL-1R1 mRNAs were predominant in the brain and pituitary while, IL-1R2 mRNAs were more abundant in the spleen. The maximal quantity of transcripts (IL-1R1, IL-1R2) was obtained 6 h after LPS injection in all studied tissues. The decrease to basal level was observed within 48 h in the brain. In the spleen, IL-1R1 mRNAs remained elevated 48 h after LPS while IL-1R2 mRNAs had already reached basal level. These results indicate a LPS-induced stimulation of IL-1 receptors mRNAs in the brain and a differential expression of IL-1R1 and IL-1R2 transcripts in brain and immune tissues.

Expression of interleukin 1 alpha, interleukin 1 beta and interleukin 1 receptor antagonist mRNA in mouse brain: regulation by bacterial lipopolysaccharide (LPS) treatment.

Lipopolysaccharide (LPS) stimulation is known to induce interleukin-1 (IL-1) mRNA expression in various immune cell types. Since IL-1 synthesis has been suggested to occur locally in brain tissue, we investigated the expression of IL-1 (alpha and beta) and IL-1 receptor antagonist (IL-1ra) mRNAs in various structures of the central nervous system, as well as in the spleen, following intraperitoneal injection of LPS (100 micrograms/mouse). After RNA extraction and amplification by the reverse transcription-polymerase chain reaction (RT-PCR), the PCR products were separated on an agarose gel, transferred and hybridized with digoxigenin-labeled probes synthetized by nested PCR. Glyceraldehyde phosphate deshydrogenase mRNA was used as an internal control. Under basal conditions the expression of IL-1 alpha, IL-1 beta and IL-1ra mRNAs in the brain was extremely low for the three cytokines; in the spleen these mRNAs were clearly detectable. Following LPS stimulation, mRNAs were strongly increased in all the tested tissues (cortex, hippocampus, hypothalamus, cerebellum, pituitary and spleen). The kinetics of mRNAs expressions in the brain were similar for all the tested regions, with a maximum at 6 h and a decrease up to 24 h after LPS administration. In the spleen the maximum was observed as soon as 1 h following stimulation. In conclusion, peripheral LPS stimulation induces a strong and transient expression of IL-1 alpha and IL-1 beta mRNAs in the brain. IL-1ra mRNA is also stimulated by LPS in various regions of the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Active metabolism of arachidonic acid by Kaposi sarcoma cells cultured from lung biopsies (KS-3); identification by HPLC and MS/MS of the predominant metabolite secreted as the 11,12-epoxy-eicosatrienoic acid.

The development of long-term culture of AIDS-KS cells has allowed us to investigate further a possible vascular origin of Kaposi sarcoma. Taking into account the relative specificity of arachidonic acid (AA) metabolism according to cell type, the AA 'cascade' was analyzed in cultured KS-3 cells established from lung biopsies and compared to human umbilical venous endothelial (H-UVE) cells and human myometrial smooth muscle (H-MSM) cells, under basal conditions and after stimulation with vasoactive agents such as histamine or thrombin. Considering strictly the 'prostaglandin' profile given by RIAs, the metabolism of AA was closer, whilst not identical, to H-UVE than to H-MSM cells. However, evaluation of all the eicosanoids released from [3H]AA labeled KS-3 cells revealed that the predominant metabolite was not prostacyclin (PGI2), as suggested from PG RIAs, but an epoxy-eicosatrienoic acid (EET), identified as the 11, 12 isomer by HPLC and MS/MS. The synthesis of this EET is probably cytochrome P-450 monooxygenase dependent. Its potential role in the development of the KS tumor cells is under investigation.

Plasma membrane lipids of bovine adrenal chromaffin cells.

The lipid composition of plasma membranes isolated from bovine adrenal chromaffin cells has been determined. Choline and ethanolamine phosphatides were predominant; the level of lyso compounds was very low. The amount of cholesterol and the cholesterol/phospholipid molar ratio was low compared to those of the other subcellular fractions of chromaffin cells. A complex pattern of neutral glycolipids was observed in contrast to that of gangliosides.

Triiodothyronine receptors in developing mouse neuronal and glial cell cultures and in chick-cultured neurones and astrocytes.

The evolution of L-triiodothyronine (T3) receptors was studied in developing cultures of cells dissociated from cerebral hemispheres of 14-day-old mouse embryos, which present successive distinct periods of cell proliferation and/or maturation. These periods are characterized essentially as neuronal from 1 to 12 days in vitro (DIV) and glial between 12 and 60 DIV. Furthermore myelin-related membranes are produced in this culture system. Binding capacities of the T3 nuclear receptors increased from 1 to 6 DIV, when it reached a maximum (16 fmol/100 micrograms DNA). A similar increase of the DNA content of the cell was observed until 8 DIV. Thereafter a sharp fall of receptor concentration leading to a 5-fold decrease in the binding capacity occurred until day 15, a period at which neurones disappeared from the cultures. From 25 to 50 DIV (coinciding with the glial period), the concentration of receptor remained more or less constant (1-2 fmol/100 micrograms DNA). In parallel, the DNA content did not vary greatly between 30 and 50 DIV. Scatchard analysis revealed the presence of a single class of receptors at 6 and 20 DIV, representative of 'neuronal' and 'glial' periods, respectively. The equilibrium dissociation constant (Kd) of the nuclear receptor from cells at 6 DIV (2 X 10(-10) M) was similar to that found at 20 DIV. These results were confirmed using pure cultured neurones and astrocytes prepared from embryonic chick brain. The effect of T3 on the cellular gangliosides used as an index of neuronal cell maturation, and on cerebroside sulfotransferase (CST), an enzyme involved in the production of myelin sulfatides, was studied to determine a possible correlation between the binding capacity of the T3 nuclear receptor and the response of the cultured cells to thyroid hormone. Our data demonstrate that T3 had no significant effect either on the content of gangliosides or on their developmental pattern, while it increased the level of CST activity by 75% between 18 and 25 DIV. These results show that, although the concentration of T3 receptors per 100 micrograms DNA in glial cells was lower than that in neurones, it was nevertheless sufficient to elicit a response in oligodendrocytes.

Membrane lipids of hepatocytes, Kupffer cells and endothelial cells.

The membrane lipid composition of isolated hepatocytes, Kupffer cells and endothelial cells was determined. The hepatocytes are characterized by a lower quantity of gangliosides, cholesterol, sphingomyelin and a reduced cholesterol/phospholipid molar ratio when compared to the two other liver cell types. The main gangliosides of Kupffer and endothelial cells are the GM3 species, and those of hepatocytes are of the polysialogangliosides species.

Regional distributions of gamma-aminobutyric acid (GABA), glutamate decarboxylase (GAD), and gamma-aminobutyrate transaminase (GABA-T) in the central nervous brains of C57/BR, C3H/He, and F1 hybrid mice.

The distributions of gamma-aminobutyric acid (GABA), glutamate decarboxylase (GAD), and gamma-aminobutyrate transaminase (GABA-T) have been studied in various brain areas of mice. These neurochemical markers, which are related to inhibitory neurotransmission, were investigated in different inbred strains of mice (C3H/He, C57/BR, and their F1 hybrids). The regional distributions of GABA, GAD activity, and GABA-T activity in adult mice of these three strains were quite similar. No significant differences were found in any brain area for GAD or GABA-T activity. However, significant differences in GABA level were found in several brain areas among these strains of mice, especially in hypothalamus, hippocampus, olfactory bulb, and occipital cortex. These results provide further information to the possible influence of the GABAergic system in these brain areas.

Comparative study of miscellaneous properties of cysteine sulfinate transaminase and glutamate oxaloacetate transaminase in chick retina homogenate.

The activity, properties, and developmental pattern of cysteine sulfinate transaminase (CSA-T) were studied in chick retina and compared with the activity, properties, and developmental pattern of glutamate oxaloacetate transaminase (GOT). Their optimum pH is identical whereas the effect of pyridoxal phosphate seems to be different. Developmental patterns are also different. The Km and Vm of CSA-T and GOT were determined in chick retina homogenate. These results suggest that two different enzymes are responsible for the transamination of cysteine sulfinate (CSA) and aspartate.