A mammalian homologue of the Drosophila retinal degeneration B gene: implications for the evolution of phototransduction mechanisms.

Comparative analysis of homologous genes in distantly related species provides important insights into the evolution of complex physiological processes. The Drosophila retinal degeneration B (rdgB) gene encodes a protein involved in phototransduction in the fly. We have isolated a human gene, DRES9, and its murine homologue (Dres9), which show a high degree of similarity to the Drosophila rdgB gene. RNA in situ hybridization studies performed on mouse-embryo tissue sections at various developmental stages revealed that Dres9 is expressed at very high levels in the neural retina and in the central nervous system (CNS), similar to its Drosophila counterpart. The high level of sequence conservation and similarities in the expression patterns of rdgB and DRES9 during development in Drosophila and mammals indicate that Dres9 is the orthologue of RdgB, and strongly suggest a possible functional conservation of these proteins during evolution. DRES9 encodes a phosphatidylinositol-transfer protein, suggesting that phosphatidylinositol may have a role as an intracellular messenger in vertebrate phototransduction. The identification of this gene and the study of its expression pattern in mammals will help shed new light on the evolution of vision mechanisms and suggest DRES9 as a candidate gene for human retinopathies.

Identification and mapping of human cDNAs homologous to Drosophila mutant genes through EST database searching.

Cross-species comparison is an effective tool used to identify genes and study their function in both normal and pathological conditions. We have applied the power of Drosophila genetics to the vast resource of human cDNAs represented in the expressed sequence tag (EST) database (dbEST) to identify novel human genes of high biological interest. Sixty-six human cDNAs showing significant homology to genes causing Drosophila mutant phenotypes were identified by screening dbEST using the "text string' option, and their map position was determined using both fluorescence in situ hybridization (FISH) and radiation hybrid mapping. Comparison between these genes and their putative partners in Drosophila may provide important insights into their function in mammals. Furthermore, integration of these genes into the transcription map of the human genome contributes to the positional candidate approach for disease gene identification.

Disruption by interferon-alpha of an autocrine interleukin-6 growth loop in IL-6-dependent U266 myeloma cells by homologous and heterologous down-regulation of the IL-6 receptor alpha- and beta-chains.

IL-6 is an autocrine growth factor for U266 myeloma cells and their growth is inhibited by IFN-alpha or IL-6 mAb. We asked, therefore, whether IFN-alpha-induced growth inhibition involved IL-6. IFN-alpha and mAb against IL-6, the IL-6R alpha-(gp80) or beta-chain (gp130) potently inhibited U266 cells. Remarkably, this effect occurred despite IFN-alpha-augmented secretion of endogenous IL-6. However, examining the IL-6R revealed that IFN-alpha drastically curtailed expression of the IL-6R alpha- and beta-chain. This effect occurred on two different levels (protein and mRNA) and by two different mechanisms (directly and indirectly through IL-6). First, IFN-alpha, but not IL-6, greatly decreased gp80 and, to a lesser extent, gp130 mRNA levels which resulted in a loss of IL-6 binding sites. Second, IFN-alpha-induced IL-6 predominantly down-regulated membrane-bound gp130. IFN-alpha-mediated decrease of gp80 levels was not detected on IL-6-independent myeloma (RPMI 8226) or myeloid cells (U937). We conclude that IFN-alpha inhibited IL-6-dependent myeloma cell growth by depriving U266 cells of an essential component of their autocrine growth loop, a functional IL-6R.

Distribution of nicotinic receptors in the human hippocampus and thalamus.

Neuronal nicotinic acetylcholine receptors consist of different subunits, alpha and beta, with different subtype arrangement corresponding to distinct pharmacological and functional properties. The expression of alpha 3, alpha 7 and beta 2 mRNA in the human brain was studied by in situ hybridization and compared to [3H]nicotine, [3H]cytisine and [125I]alpha-bungarotoxin binding in contiguous sections. The beta 2 probe showed a strong hybridization signal in the granular layer of the dentate gyrus and in the CA2/CA3 region of the hippocampus and in the insular cortex, and a signal of lower intensity in the subicular complex and entorhinal cortex. The alpha 3 probe showed strong hybridization in the dorsomedial, lateral posterior, ventroposteromedial and reticular nuclei of the thalamus, and a weak signal in the hippocampal region and in the entorhinal, insular and cingular cortex. The amount of alpha 7 mRNA was high at the level of the dentate granular layer and the CA2/CA3 region of the hippocampus, in the caudate nucleus and in the pulvinar and ventroposterolateral nuclei of the thalamus. [3H]Nicotine and [3H]cytisine binding appeared to be identical in anatomical distribution and relative intensity. It was high in the thalamic nuclei, the putamen and in the hippocampal formation in the subicular complex and the stratum lacunosum moleculare. The level of [125I]alpha-bungarotoxin binding was particularly high in the hippocampus and in the pyramidal cells of the CA1 region, but was relatively low in the subicular complex. Our data indicate that in the human brain nicotinic receptor subtypes have discrete distributions, which are in part different from those of other species.

Distribution of neuronal nicotinic receptor subunits in human brain.

Neuronal nicotinic acetylcholine receptors (nAchRs) are multimeric proteins constituted of two different subunits, alpha and beta, with different subtypes arrangement and different pharmacological and functional properties. nAchRs mediate neurotransmission in many central and peripheral synapses and appear to be affected in human degenerative disorders. We have studied the distribution of nAchR in human brain, particularly in the hippocampus and thalamus, by binding of 3H-nicotine and 3H-cytisine and by in situ hybridization with human alpha 3 and beta 2 nAchR subunits of mRNA. An alpha 3 probe shows a strong hybridization signal in the thalamus, while a beta 2 probe has a good signal at the level of the enthorinal cortex, hippocampus and in caudate and putamen. The alpha 3 and beta 2 mRNA localization is different from that described in other species. 3H-nicotine and 3H-cytisine binding were very similar in terms of anatomical distribution and comparable to the binding described in other animal species. The binding of the two ligands was distributed over the areas labeled by the alpha 3 and beta 2 probes and did not completely overlap with either of the subunits.

Splicing of an anti-sense Alu sequence generates a coding sequence variant for the alpha-3 subunit of a neuronal acetylcholine receptor.

In this report we demonstrate that an alpha-3 acetylcholine receptor subunit transcriptional variant originates through alternative splicing of a complementary sequence of the right arm of an Alu element. This element is located within the 5.1 Kb intron found between exons 5 and 6 of the alpha-3 acetylcholine receptor subunit gene. The transcriptional variant originates from the normal splicing process and carries an in-frame stop codon. If translated, it should encode for a peptide lacking the 4th transmembrane domain of the normal subunit.

Neuronal-type nicotinic receptors in human neuroblastoma and small-cell lung carcinoma cell lines.

A beta subunit of the neuronal nicotinic receptor, sharing 88% homology with the rat beta 4 subunit, has been cloned from a human neuroblastoma cell line. The gene encoding the human beta 4 subunit is expressed in association with the alpha 3 gene in neuroblastoma and small-cell lung carcinoma cell lines. Patch-clamp experiments and radioligand binding assays confirm that these neuroendocrine tumor cell lines express functional neuronal nicotinic receptors. We suggest that these receptors might play a crucial role in the control of neurotransmitter and hormone secretion from neurosecretory human tumors.

Pharmacological modulation of neuropeptides in peripheral mononuclear cells.

The concentrations of beta-endorphin and cholecystokinin were measured in fresh resting peripheral mononuclear cells obtained from rats and human subjects in basal conditions and after different pharmacological treatments. Both in the human and the rat, beta-endorphin concentrations in mononuclear cells, increased after treatment with serotoninergic agonists, decreased after dopaminergic or GABAergic drugs, while the respective antagonists exerted the opposite effect. In vitro, serotoninergic and GABAergic compounds confirmed their roles in the modulation of beta-endorphin in mononuclear cells. Cholecystokinin was never affected by the pharmacological treatments.

Effect of psychoactive drugs on lymphocyte neuropeptides.

The concentrations of beta-endorphin and cholecystokinin were measured in lymphocytes obtained from young or old rats and from humans at different ages. Both in rats and humans, beta-endorphin and cholecystokinin increase with age; also in vitro, after 48-h culturing, the concentrations of beta-endorphin and cholecystokinin in lymphocytes obtained from humans of different ages changed with the same pattern observed in ex vivo experiments. In the human, beta-endorphin in lymphocytes shows a circadian rhythm that shifts approximately 6 h when compared to plasma ACTH and cortisol rhythm. The HPLC analysis of the molecular forms of beta-endorphin in lymphocytes revealed the presence of N-acetyl-beta-endorphin, with a ratio of beta-endorphin to N-acetyl-beta-endorphin ranging from 1 to 2. The concentrations of beta-endorphin and cholecystokinin were also measured in lymphocytes obtained from rats and human subjects undergoing different pharmacological treatments. In rat, the serotonin receptor antagonist metergoline decreased basal concentrations of the opioid peptide and blocked the increase of beta-endorphin concentrations induced by the serotonin precursor 5-hydroxytryptophan and the tricyclic antidepressant chlorimipramine. Also in the human, the antidepressant drug chlorimipramine increased lymphocyte beta-endorphin concentrations. In contrast to what was observed for beta-endorphin, cholecystokinin concentrations were not affected by the modulation of the serotoninergic system. Chronic treatment of rats with the dopamine receptor antagonist haloperidol induced an increase of beta-endorphin concentrations in lymphocytes that was reversed by the concomitant treatment with the dopamine receptor agonist bromocriptine, which when given alone decreased the basal concentrations of the peptide. In the human, haloperidol increased concentrations of beta-endorphin after both 24 h and chronic treatment, while cholecystokinin was never affected. Finally, beta-endorphin, but not cholecystokinin, increases both in rat and human lymphocytes after treatment with the GABA agonist sodium valproate.

[Vectorcardiographic evolution after a valve replacement operation]. / Evoluzione vettorcardiografica dopo intervento di sostituzione valvolare.

The vectorcardiograms of 35 patients with valvular heart disease were recorded before and after prosthetic valve replacement. The spatial magnitude of the 40 msec and maximum QRS vectors were decreased postoperatively in patients with mitral insufficiency aortic stenosis and aortic insufficiency. 9 patients were studied postoperatively by right heart catheterization.

[Malfunction of disc prostheses. Echocardiographic study of 5 cases (author's transl)]. / Malfunzionamento di protesi a disco. Studio ecocardiografico di cinque casi.

78 patients who underwent disc prostheses replacement, 36 in mitral area, 58 in aortic area were studied by echocardiography. The Authors found 5 cases of malfunction, 3 in mitral area and 2 in aortic area. Regarding mitral malfunctions in 1 case a valve thrombosis was found; in 2 cases there was a partial leak. Regarding aortic malfunctions there was paravalvular leak. In mitral area malfunctions the Authors found alterations of the disc morphology during diastolic opening time associated with alteration of opening time. An increased diastolic closure velocity in 2 cases of paraprosthetic leak was found. A diagnostic element in the case with thrombosis was variability of maximal disc escursion during the same recording, because opening time variability never got over 10 m. seconds. In aortic area malfunctions the Authors found a constant fluttering of anterior mitral leaflet, a sinergic septal motion with the posterior wall and in 1 case the presence of disc opening before the first component of the first sound. The Authors underline the importance of simultaneous eco-phonocardiographic examination and the check-ups for the time to be.