gamma-aminobutyric acid increases the water accessibility of M3 membrane-spanning segment residues in gamma-aminobutyric acid type A receptors

gamma-Aminobutyric acid type A (GABA(A)) receptors are members of the ligand-gated ion channel gene superfamily. Using the substituted cysteine accessibility method, we investigated whether residues in the alpha(1)M3 membrane-spanning segment are water-accessible. Cysteine was substituted, one at a time, for each M3 residue from alpha(1)Ala(291) to alpha(1)Val(307). The ability of these mutants to react with the water-soluble, sulfhydryl-specific reagent pCMBS(-) was assayed electrophysiologically. Cysteines substituted for alpha(1)Ala(291) and alpha(1)Tyr(294) reacted with pCMBS(-) applied both in the presence and in the absence of GABA. Cysteines substituted for alpha(1)Phe(298), alpha(1)Ala(300), alpha(1)Leu(301), and alpha(1)Glu(303) only reacted with pCMBS(-) applied in the presence of GABA. We infer that the pCMBS(-) reactive residues are on the water-accessible surface of the protein and that GABA induces a conformational change that increases the water accessibility of the four M3 residues, possibly by inducing the formation of water-filled crevices that extend into the interior of the protein. Others have shown that mutations of alpha(1)Ala(291), a water-accessible residue, alter volatile anesthetic and ethanol potentiation of GABA-induced currents. Water-filled crevices penetrating into the interior of the membrane-spanning domain may allow anesthetics and alcohol to reach their binding sites and thus may have implications for the mechanisms of action of these agents.     

Roles for transforming growth factor-alpha in gastric physiology and pathophysiology.

Transforming growth factor alpha (TGF alpha) is a 5.6 kd single-chain polypeptide that acts through binding to the epidermal growth factor receptor (EGFR). TGF alpha is produced in a wide range of normal as well as embryonic and neoplastic cells and tissues. TGF alpha and EGFR, but not EGF, are expressed in normal gastric mucosa. We have identified the following biological roles for TGF alpha in the stomach, using a variety of primate and rodent models: inhibition of acid secretion; stimulation of mucous cell growth; protection against ethanol- and aspirin-induced injury. This last effect is associated with a time- and dose-dependent increase in levels of insoluble gastric mucin. Based on these known biological actions of TGF alpha, we have examined TGF alpha production in Ménétrier's disease, a disorder characterized by foveolar hyperplasia, hypochlorhydria, and increased gastric mucin content. In four patients with Ménétrier's disease, there was enhanced TGF alpha immunostaining throughout the gastric mucosa. Furthermore, metallothionein (MT)-TGF alpha transgenic mice which overproduce TGF alpha in the stomach exhibit histopathological and biochemical features characteristic of and consistent with the diagnosis of Ménétrier's disease. Thus locally produced TGF alpha may mediate a number of biological processes in the stomach, and its altered production may participate in the pathogenesis of selected pathological states.     

Cytomegalovirus but not human T lymphotropic virus type III/lymphadenopathy associated virus detected by in situ hybridisation in retinal lesions in patients with the acquired immune deficiency syndrome.

Paraffin sections of retinal tissue from five patients who died from the acquired immune deficiency syndrome (AIDS) and retinopathy were examined by in situ hybridisation experiments with deoxyribonucleic acid (DNA) labelled with sulphur-35 of lentivirus, human T lymphotropic virus type III/lymphadenopathy associated virus (HTLV-III/LAV), and cytomegalovirus. HTLV-III/LAV ribonucleic acid (RNA) was not detected in any of the tissue sections. Cytomegalovirus RNA was identified, however, in three of the five patients. Retinopathy induced by cytomegalovirus may thus be one of the many syndromes potentiated by the immunosuppression caused by HTLV-III/LAV.     

Some Observations on Harpacticoid Populations,in Relationship to the Competitive Exclusion Principle

In the field of ecology of populations, two concepts are largely distributed and generally accepted:- the competitive exclusion principle states that organisms occupying similar ecological niches, come into competition and consequently cannot coexist.- the more two organisms are close to each other in phylogeny, the more they will tend to occupy similar niches.The results obtained from the study of populations of Harpacticoids colonizing artificial substrata are somehow an illustration of this principles.Indeed, these species which coexist belong most of the time to different genera or even to different families.     

Power frequency fields promote cell differentiation coincident with an increase in transforming growth factor-beta(1) expression.

Recent information from several laboratories suggest that power frequency fields may stimulate cell differentiation in a number of model systems. In this way, they may be similar to pulsed electromagnetic fields, which have been used therapeutically. However, the effects of power frequency fields on phenotypic or genotypic expression have not been explained. This study describes the ability of power frequency fields to accelerate cell differentiation in vivo and describes dose relationships in terms of both amplitude and exposure duration. No change in proliferation or cell content were observed. A clear dose relationship, in terms of both amplitude and duration of exposure, was determined with the maximal biological response occurring at 0.1 mT and 7-9 h/day. Because this study was designed to explore biological activity at environmental exposure levels, this exposure range does not necessarily define optimal dosing conditions from the therapeutic point of view. This study reports the stimulation by power frequency fields of transforming growth factor-beta, an important signalling cytokine known to regulate cell differentiation. The hypothesis is raised that the stimulation of regulatory cytokines by electromagnetic fields may be an intermediary mechanism by which these fields have their biological activity.     

Conversion of TNF alpha from antiproliferative to proliferative ligand in mouse intestinal epithelial cells by regulating mitogen-activated protein kinase.

The mechanisms regulating the balance between intestinal epithelial cell proliferation and differentiation are essential to maintaining an intact mucosal barrier. Mitogen-activated protein (MAP) kinases appear to be key transducers of extracellular signals in these pathways. The goal of this study was to investigate the regulation of MAP kinase by tumor necrosis factor alpha (TNFalpha) and epidermal growth factor (EGF) in intestinal epithelial cells. The young adult mouse colon cell line was studied for TNFalpha and/or EGF regulation of MAP kinase in the presence or absence of the MAP kinase kinase (MEK1) inhibitor PD 98059. Proliferation was determined by hemocytometry, and activated MAP kinase was identified by Western blot analysis, in vitro kinase assay, and confocal laser immunofluorescent microscopy. TNFalpha stimulated sustained nuclear MAP kinase activity, while EGF stimulated transient cytoplasmic MAP kinase activity. Changing TNFalpha's sustained MAP kinase activation to transient converted TNFalpha from an anti-proliferative to a proliferative ligand. These findings demonstrate that both TNFalpha and EGF activate MAP kinase in intestinal epithelial cells. The kinetics and subcellular distribution of this enzyme activity may be pivotal in the transduction of divergent cellular responses in the intestinal epithelium with implications for altered proliferative signals in inflammatory bowel disease.     

The mechanism of low serum T3 in the fetus: hepatic T4-5'-monodeiodinase versus tissue sulfhydryl content--a clarification

Using optimal tissue concentration, supplemented with a thiol-protecting agent, the fetal liver has lower T4 5'-monodeiodinating activity than maternal or neonatal tissue. This is a true deficiency and is not due to deficiency of sulfhydryl groups in fetal tissue as previously suggested. Evidence indicates a dissociation of the neonatal surge of serum T3 and the increase in hepatic T4 to T3 conversion activity, suggesting that the neonatal T3 surge is related more to thyroid gland stimulation than to T4 to T3 conversion in non-thyroid tissues.     

Cell density—dependent regulation of PLCγ1 tyrosine phosphorylation and catalytic activity in an intestinal cell line (IEC-6)

Administration of epidermal growth factor (EGF) to rats has been shown to induce both mitogenic and nonmitogenic responses in the intestine. The mechanisms to describe a multiplicity of hormonal responses within a single tissue are unclear but likely involve selectivity among receptor substrates. A nontransformed rat jejunal crypt intestinal epithelial cell line (IEC-6) was studied to determine if the regulation of receptor tyrosine kinase substrates is affected by cell population physiology. EGF stimulated a rapid increase in inositol trisphosphate in confluent but not subconfluent cells. Similarly, treatment of confluent IEC-6 cells with EGF provoked a significant increase in the hydrolysis of Ptdlns 4,5-P₂ by immunoisolated PLCγ1. The tyrosine phosphorlation state of PLCγ1 and the association of PLCγ1 with the EGF receptor were increased by EGF in confluent cells only. In contrast, the autophosphorylation state of the EGF receptor and the tyrosine phosphorylation state of another SH2-containing EGF receptor substrate SHC were increased by EGF regardless of cell density. Western blot analysis revealed equal protein expression of PLCγ1 in confluent and subconfluent cells. EGF receptor protein expression and ligand binding capacity were slightly increased in confluent compared to subconfluent cells. EGF regulation of PLCγ1, therefore, is regulated by physiological factors dependent on cell density in IEC-6 cells. © 1995 Wiley-Liss, Inc.     

Phylogenetic relationships of bolitoglossine salamanders: a demonstration of the effects of combining morphological and molecular data sets

We analyzed sequence data for 555 bp of the mitochondrial gene cytochrome b in plethodontid salamanders, taken from 18 ingroup (tribe Bolitoglossini) and 4 outgroup (tribe Plethodontini) taxa. There were 257 variable sites, of which 219 were phylogenetically informative. Sequence differences among taxa exceeded 20%, and there were up to 15% amino acid differences among the sequences. We also analyzed 37 morphological (including karyological) characters, taken from the literature. Data were analyzed separately and then combined using parsimony and likelihood approaches. There is little conflict between the morphological and DNA data, and that which occurs is at nodes that are weakly supported by one or both of the data sets. Treated separately, the morphological and DNA data provide strong support for some nodes but not for others. The combined data act synergistically so that good support is obtained for nearly all of the nodes in the tree. Recent divergences are supported by silent transitions, and older divergences are supported by a combination of morphological, karyological, DNA transversion, and amino acid changes. Eliminating silent changes from the DNA data improves the consistency index and improves some bootstrap and decay index values for several deeper branches in the tree. However, the combined data set with all characters included provides a better supported tree overall. Maximum likelihood and parsimony with all of the data give not only the same topology but also remarkably similar branch lengths. Results of this analysis support the monopoly of the supergenera Hydromantes and Batrachoseps, and of a sister group relationship of Batrachoseps and the supergenus Bolitoglossa (represented in this study one species of the genus Bolitoglossa).      

Hypoxia-inducible factor-2α and iron absorptive gene expression in Belgrade rat intestine

The divalent metal transporter (DMT1, Slc11a2) is an important molecule for intestinal iron absorption. In the Belgrade (b/b) rat, the DMT1 G185R mutation markedly decreases intestinal iron absorption. We used b/b rats as a model to examine the genes that could be compensatory for decreased iron absorption. When tissue hypoxia was assayed by detecting pimonidazole HCl adducts, the b/b liver and intestine exhibited more adducts than the +/+ rats, suggesting that hypoxia might signal altered gene expression. Total RNA in the crypt-villus bottom (C-pole) and villus top (V-pole) of +/+, b/b, and iron-fed b/b rats was isolated for gene array analyses. In addition, hepatic hepcidin and intestinal hypoxia-inducible factor-α (Hifα) expression were examined. The results showed that expression of hepatic hepcidin was significantly decreased and intestinal Hif2α was significantly increased in b/b and iron-fed b/b than +/+ rats. In b/b rats, the expression of Tfrc mRNA in the C-pole and of DMT1, Dcytb, FPN1, Heph, Hmox1, and ZIP14 mRNAs in the V-pole were markedly enhanced with increases occurring even in the C-pole. After iron feeding, the increased expression found in b/b rats persisted, except for Heph and ZIP14, which returned to normal levels. Thus in b/b rats depressed liver hepcidin production and activated intestinal Hif2α starting at the C-pole resulted in increasing expression of iron transport genes, including DMT1 G185R, in an attempt to compensate for the anemia in Belgrade rats.