Increased expression of mitochondrial genes in human alcoholic brain revealed by differential display

Polymerase chain reaction (PCR)-based differential display was used to screen for alterations in gene expression in the mesolimbic system of the human alcoholic brain. Total RNA was extracted from the nucleus accumbens of five alcoholic and five control brains. A selected subpopulation of mRNA was reverse-transcribed to cDNA and amplified by PCR. A differentially expressed cDNA fragment was recovered, cloned, and sequenced. Full sequence analysis of this 467 bp fragment revealed 98.2% homology with the human mitochondrial 12S rRNA gene. Dot-blot analysis showed increased expression of this gem in nucleus accumbens and hippocampus, but not in the superior frontal cortex, primary motor cortex, caudate, and pallidus/putamen In a total of eight human alcoholic brains, compared with seven control brains. A similar increased expression was observed by dot-blot analysis, using RNA from the cerebral cortex of rats chronically treated with alcohol vapor. Hybridization of a 16S rRNA oligonucleotide probe indicated that the expression of both rRNAs genes was significantly increased in nucleus accumbens. These results indicate that chronic alcohol consumption induces alteration in expression of mitochondrial genes in selected brain regions. The altered gene expression may reflect mitochondrial dysfunction In the alcohol-affected brain.

A cell type-specific constitutive point mutant of the common beta-subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors requires the GM-CSF receptor alpha-subunit for activation

The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMR alpha) and a common signal-transducing beta-subunit (hpc) that is shared with the interleukin-3 and -5 receptors, We have previously identified a constitutively active extracellular point mutant of hpc, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287), This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMR alpha (mGMR alpha) subunit, since introduction of mGMR alpha, but not hGMR alpha, into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence, Experiments utilizing mouse/human chimeric GMR alpha subunits indicated that the species specificity lies in the extracellular domain of GMRa. Importantly, the requirement for mGMR alpha correlated with the ability of I374N (but not wild-type hpc) to constitutively associate with mGMRa. Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMR alpha surface expression. Taken together, these findings suggest a critical role for association with GMR alpha in the constitutive activity of I374N.

p75 neurotrophin receptor-mediated neuronal death is promoted by Bcl-2 and prevented by Bcl-x(L)

The p75 neurotrophin receptor (p75NTR) has been shown to mediate neuronal death through an unknown pathway. We microinjected p75NTR expression plasmids into sensory neurons in the presence of growth factors and assessed the effect of the expressed proteins on cell survival. We show that, unlike other members of the TNFR family, p75NTR signals death through a unique caspase-dependent death pathway that does not involve the death domain and is differentially regulated by Bcl-2 family members: the anti-apoptotic molecule Bcl-2 both promoted, and was required for, p75NTR killing, whereas killing was inhibited by its homologue BcI-x(L). These results demonstrate that Bcl-2, through distinct molecular mechanisms, either promotes or inhibits neuronal death depending on the nature of the death stimulus.

Isolation of a cDNA for an octopamine-like, G-protein coupled receptor from the cattle tick, Boophilus microplus

Octopamine is a biogenic amine neurotransmitter of invertebrates that binds to a G-protein coupled receptor that has seven transmembrane domains. Formamidine pesticides like amitraz are highly specific agonists of the octopamine receptor. Amitraz is used extensively to control the cattle tick, Boophilus microplus, and many other ticks but now there are strains of ticks that are resistant to amitraz. We have isolated a cDNA from the cattle tick, B. miciroplus, that belongs to the biogenic amine family of receptors. The predicted amino acid sequence from this cDNA is most similar to octopamine receptors from insects. The nucleotide sequence of this gene from amitraz-resistant and amitraz-susceptible cattle ticks was identical. Thus, a point mutation/s did not confer resistance to amitraz in the strains we studied. Alternative explanations for resistance to amitraz in B. microplus are discussed. (C) 1999 Elsevier Science Ltd. All rights reserved.

Gene transfer and expression of a non-viral polycation-based vector in CD4(+) cells

CD4-selective targeting of an antibody-polycation-DNA complex was investigated The complex was synthesized with the anti-CD4 monoclonal antibody B-F5, polylysine(268) (pLL) and either the pGL3 control vector containing the luciferase reporter gene or the pGeneGrip vector containing the green fluorescent protein (GFP) gene. B-F5-pLL-DNA complexes inhibited the binding of I-125-B-F5 to CD4(+) Jurkat cells, while complexes synthesised either without B-F5 or using a non-specific mouse IgG1 antibody had little or no effect Expression of the luciferase reporter gene was achieved in Jurkat cells using the B-F5-pLL-pGL3 complex and was enhanced in the presence of PMA. Negligible luciferase activity was defected with the non-specific antibody complex in Jurkat cells or with the B-F5-pLL-pGL3 complex in the CD4(-) K-562 cells. Using complexes synthesised with the pGeneGrip vector, the transfection efficiency in Jurkat and K-562 cells was examined using confocal microscopy. More than 95% of Jurkat cells expressed GFP and the level of this expression was markedly enhanced by PMA. Negligible GFP expression was seen in K-562 cells or when B-F5 was replaced by a nonspecific antibody. Using flow cytometry, fluorescein-labelled complex showed specific targeting to CD4(+) cells in a mixed cell population from human peripheral blood. These studies demonstrate the selective transfection of CD4(+) T-lymphoid cells using a polycation-based gene delivery system. The complex may provide a means of delivering anti-HIV gene therapies to CD4(+) cells in vivo.

Identification of a new ligand binding domain in the al subunit of the inhibitory glycine receptor

Four discontinuous extracellular sequence domains have been proposed to form the ligand binding sites of the ligand-gated ion channel receptor superfamily. In this study, we investigated the role of 12 contiguous residues of the inhibitory glycine receptor that define the proposed loop A ligand binding domain; Using the techniques of site-directed mutagenesis and patch-clamp electrophysiology, four of the 12 residues were shown to have impaired ligand binding. Three mutants, I93A, A101H, and N102A, resulted in significant (17-44-fold) increases in the agonist EC50 values as compared with the wild-type glycine receptor, whereas Hill coefficients, I-max values, and antagonist affinity remained largely unaffected. Consideration of receptor efficacy values indicates that these residues are involved in ligand binding rather than channel activation. A fourth mutant, W94A, failed to give rise to any glycine-activated currents, although cell-surface expression was observed, suggesting that this residue may also be involved in agonist binding. These data provide the most extensive characterization of the loop A ligand binding domain available to date and define two new residue locations, Ile(93) and Asn(102), as contributing to the four-loop model of ligand binding.

Expression of galectin-1 in the olfactory nerve pathway of rat

The olfactory neuroepithelium is characterised by the mosaic distribution of primary olfactory neurons that express different odorant receptors and cell surface glycoconjugates. Carbohydrates are believed to form a glycocode that mediates sorting out and fasciculation of primary olfactory axons through interactions with carbohydrate-binding proteins such as galectin-1. In the present study, we describe in detail the expression pattern of galectin-1 in the developing and adult rat olfactory system. We demonstrate that galectin-1 is expressed by olfactory ensheathing cells both in olfactory nerve and within the nerve fibre layer of the olfactory bulb of the embryonic and adult rat. In the adult rat, galectin-1 was preferentially expressed by olfactory ensheathing cells in the nerve fibre layer of the ventromedial and lateral surfaces of the olfactory bulb. Galectin-1 was also expressed by subsets of periglomerular cells and granule cells, particularly in the ventromedial region of the olfactory bulb. In adult rat, the galectin-1 ligand, N-acetyl-lactosamine, was expressed by primary olfactory axons that terminated in glomeruli present in the ventromedial and lateral olfactory bulb. These results suggest that expression of galectin-1 may provide a mechanism for the sorting of subpopulations of axons in the nerve fibre layer of the olfactory bulb during development as well as play a role in the postnatal maintenance of specific glomerular connections. (C) 1999 Elsevier Science B.V. All rights reserved.

Myeloproliferative disorder and leukaemia in mice induced by different classes of constitutive mutants of the human IL-3/IL-5/GM-CSF receptor common beta subunit

Several constitutively active mutant forms of the common β subunit of the human IL-3, IL-5 and GM-CSF receptors (hβc), which enable it to signal in the absence of ligand, have recently been described. Two of these, V449E and I374N, are amino acid substitutions in the transmembrane and extracellular regions of hβc, respectively. A third, FIΔ, contains a 37 amino acid duplication in the extracellular domain. We have shown previously that when expressed in primary murine haemopoietic cells, the extracellular mutants confer factor-independence on cells of the neutrophil and monocyte lineages only, whereas V449E does so on all cell types of the myeloid and erythroid compartments. To study the in vivo effects and leukaemic potential of these mutants, we have expressed all three in mice by bone marrow reconstitution using retrovirally infected donor cells. Expression of the extracellular mutants leads to an early onset, chronic myeloproliferative disorder marked by elevations in the neutrophil, monocyte, erythrocyte and platelet lineages. In contrast, expression of V449E leads to an acute leukaemia-like syndrome of anaemia, thrombocytopaenia and blast cell expansion. These data support the possibility that activating mutations in hβc are involved in haemopoietic disorders in man.

Expression of EphA5 during development of the olfactory nerve pathway in rat

The olfactory neuroepithelium is a highly plastic region of the nervous system that undergoes continual turnover of primary olfactory neurons throughout life. The mechanisms responsible for persistent growth and guidance of primary olfactory axons along the olfactory nerve are unknown. In the present study, we used antibodies against the Eph-related receptor, EphA5, to localise EphA5, and recombinant EDhA5-IgG fusion protein to localise its ligands. We found that although both EphA5 and its ligands were both expressed by primary olfactory neurons within the embryonic olfactory nerve pathway, there was no graded or complementary expression pattern. In contrast, the expression patterns altered postnatally such that primary olfactory neurons expressed the ligands, whereas the second-order olfactory neurons, the mitral cells, expressed EphA5. The role of EphA5 was analysed by blocking EphA5-ligand interactions in explant cultures of olfactory neuroepithelium using anti-EphA5 antibodies and recombinant EphA5. These perturbations reduced neurite outgrowth from explant cultures and suggest that intrafascicular axon repulsion may serve to limit adhesion and optimise conditions for axon growth. (C) 2000 Wiley-Liss, Inc.

The role of disulfide-linked dimerization in interleukin-3 receptor signaling and biological activity

Cysteine residues 86 and 91 of the beta subunit of the human interleukin (hIL)-3 receptor (h beta c) participate in disulfide-linked receptor subunit heterodimerization. This linkage is essential for receptor tyrosine phosphorylation, since the Cys-86 --> Ala (Mc4) and Cys-91 --> Ala (Mc5) mutations abolished both events. Here, we used these mutants to examine whether disulfide-linked receptor dimerization affects the biological and biochemical activities of the IL-3 receptor. Murine T cells expressing hIL-3R alpha and Mc4 or Mc5 did not proliferate in hIL-3, whereas cells expressing wild-type h beta c exhibited rapid proliferation. However, a small subpopulation of cells expressing each mutant could be selected for growth in IL-3, and these proliferated similarly to cells expressing wild-type h beta c, despite failing to undergo IL-3-stimulated h beta e tyrosine phosphorylation. The Mc4 and Mc5 mutations substantially reduced, but did not abrogate, IL-3-mediated anti-apoptotic activity in the unselected populations. Moreover, the mutations abolished IL-3-induced JAK2, STAT, and AKT activation in the unselected cells, whereas activation of these molecules in IL-3-selected cells was normal. In contrast, Mc4 and Mc5 showed a limited effect on activation of Erk1 and -2 in unselected cells. These data suggest that whereas disulfide-mediated cross-linking and h beta c tyrosine phosphorylation are normally important for receptor activation, alternative mechanisms can bypass these requirements.

A new small molecule C5a receptor antagonist inhibits the reverse-passive arthus reaction and endotoxic shock in rats

C5a is implicated as a pathogenic factor in a wide range of immunoinflammatory diseases, including sepsis and immune complex disease, Agents that antagonize the effects of C5a could be useful in these diseases. We have developed some novel C5a antagonists and have determined the acute anti-inflammatory properties of a new small molecule C5a receptor antagonist against C5a- and LPS-induced neutrophil adhesion and cytokine expression, as well as against some hallmarks of the reverse Arthus reaction in rats. We found that a single i.v. dose (1 mg/kg) of this antagonist inhibited both C5a- and LPS-induced neutropenia and elevated levels of circulating TNF-alpha, as well as polymorphonuclear leukocyte migration, increased TNF-alpha levels and vascular leakage at the site of immune complex deposition. These results indicate potent anti-inflammatory activities of a new C5a receptor antagonist and provide more evidence for a key early role for C5a in sepsis and the reverse Arthus reaction. The results support a role for antagonists of C5a receptors in the therapeutic intervention of immunoinflammatory disease states such as sepsis and immune complex disease.

Role of neurotrophin receptor p75NTR in mediating neuronal cell death following injury

1. The neurotrophin receptor p75NTR has been shown to mediate neuronal cell death after nerve injury. 2. Down-regulation of p75NTR by antisense oligonucleotides is able to inhibit both sensory and motor neuron death and this treatment is more effective than treatment with growth factors. 3. p75NTR induces cell death by a unique death signalling pathway involving transcription factors (nuclear factor kappa B and c-jun), Bcl-2 family members and caspases.

Characterization of peroxisome proliferator-activated receptor alpha in normal rat mammary gland and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mammary gland tumors from rats fed high and low fat diets

Normal Sprague-Dau ley rat mammary gland epithelial cells and mammary gland carcinomas induced by 2-amino-1 -methyl-6-phenylimidazo[4,5-b]pyridine, a carcinogen found in the diet, were examined for the expression of peroxisome proliferator-activated receptor alpha (PPAR alpha). PPAR alpha mRNA and protein was detected in normal and tumor tissue by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. By quantitative RT-PCR, carcinomas had a 12-fold higher expression than control mammary glands, a statistically significant difference. PPAR alpha expression was examined in carcinomas and normal tissues from rats on high fat (23.5/% corn oil) and low fat (5% corn oil) diets. Although neither carcinomas, nor control tissues showed statistically significant differences between the two diet groups, PPAR alpha expression was the highest in carcinomas from rats on the high fat diet. The expression of PPAR alpha in normal mammary gland and its significant elevation in mammary gland carcinomas raises the possibility of its involvement in mammary gland physiology and pathophysiology. (C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.

Mutant GABA(A) receptor gamma 2-subunit in childhood absence epilepsy and febrile seizures

Epilepsies affect at least 2% of the population at some time in life, and many forms have genetic determinants(1,2). We have found a mutation in a gene encoding a GABA, receptor subunit in a large family with epilepsy. The two main phenotypes were childhood absence epilepsy (CAE) and febrile seizures (FS), There is a recognized genetic: relationship between FS and CAE, yet the two syndromes have different ages of onset, and the physiology of absences and convulsions is distinct. This suggests the mutation has age-dependent effects on different neuronal networks that influence the expression of these clinically distinct, but genetically related, epilepsy phenotypes. We found that the mutation in GABRG2 (encoding the gamma2-subunit) abolished in vitro sensitivity to diazepam, raising the possibility that endozepines do in fact exist and have a physiological role in preventing seizures.

Estradiol-regulated expression of the immunophilins cyclophilin 40 and FKBP52 in MCF-7 breast cancer cells

The immunophilins, cyclophilin 40 (CyP40) and FKBP52, are associated with the unactivated estrogen receptor in mutually exclusive heterocomplexes and may differentially modulate receptor activity, We have recently shown that CyP40 and FKBP52 mRNA's are differentially elevated in breast carcinomas compared with normal breast tissue. Other studies suggest that such alterations ill the ratio of immunophilins might potentially influence steroid receptor function. Studies were therefore initiated to investigate the influence of estradiol on CyP40 and FKBP52 expression in MCF-7 breast cancer cells. Over a 24-h-treatment period with estradiol, CyP40 and FKBP52 mRNA expression was increased approximately five- and 14-fold, respectively. The corresponding protein levels were also elevated in comparison to controls. The antiestrogen, ICI 182,780, was an antagonist for CyP40 and FKBP52 mRNA induction. Cycloheximide treatment did not inhibit this increased immunophilin expression, suggesting that estradiol-mediated activation is independent off de novo protein synthesis. Treatment of MCF-7 cells with estradiol resulted in an increased half-life of both CyP40 and FKBP52 mRNA, as determined by actinomycin D studies. These results suggest that estradiol regulates CyP40 and FKBP52 mRNA expression through both transcriptional and posttranscriptional mechanisms. (C) 2001 Academic Press.