Calcium-activated potassium channels expressed from cloned complementary DNAs.

Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the slo locus of Drosophila melanogaster. Many cDNAs were found that encode closely related proteins of about 1200 aa. The predicted sequences of these proteins differ by the substitution of blocks of amino acids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches showed potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increased steeply both with depolarization and with increasing calcium concentration. The single-channel conductance was 126 pS with symmetrical potassium concentrations. The mean open time of the channels was clearly different for channels having different substituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels.     

Functional expression and CNS distribution of a beta-alanine-sensitive neuronal GABA transporter.

The synaptic action of gamma-aminobutyric acid (GABA) is terminated by high affinity, Na(+)-dependent transport processes in both neurons and glia. We have isolated a novel GABA transporter cDNA, GAT-B, which encodes a high affinity (Km = 2.3 microM), Na(+)- and Cl(-)-dependent GABA transport protein that is potently blocked by beta-alanine, a compound generally considered a selective inhibitor of glial transport. However, in situ hybridization studies indicate that GAT-B mRNA is expressed predominantly within neurons. These data indicate that the neuronal-glial distinction of GABA transporters based on inhibitor sensitivities must be reconsidered and suggest a greater diversity of GABA transporters than has been predicted by previous pharmacologic studies.     

Proteins and glycosaminoglycans in the intercellular matrix of the human cumulus-oophorus and their effect on conversion of proacrosin to acrosin.

Human cumuli-oophori were cultured in vitro in the presence of radioactive protein and polysaccharide precursors. The time course of the cumulus cell secretion was traced by histoautoradiography. Matrix solubilization, and sodium dodecyl sulphate polyacrylamide gel electrophoresis and high-performance liquid chromatography showed that proteoglycan (Mr greater than 1,700,000) was the main cumulus cell product that was prevailingly deposited in the cumulus intercellular matrix and partly released into the culture medium. It was capable of accelerating the conversion of proacrosin to acrosin and this activity was abolished by enzymatic removal of chondroitin sulphate, the predominant glycosaminoglycan of this proteoglycan fraction. None of the other fractions, including a proteoglycan of Mr 80,000-90,000, containing heparan sulphate, accelerated the conversion of proacrosin to acrosin under the conditions used. The results suggest that chondroitin sulphate is the active component of the high-Mr proacrosin activator of the human cumulus-oophorus.     

Lysophosphatidylserine enhances the transfer of 22:6n3 to lysophosphatidic acid in rat brain microsomes.

Although the acyl groups of phosphatidylserine in brain are uniquely enriched in docosahexaenoic acid (22:6n3), the mechanism for this enrichment is not well understood. When rat brain homogenates and microsomes were incubated in the presence of lysophosphatidylserine (LPS) together with [14C]22:6n3 and cofactors for activation to its acylCoA, very little radioactivity was incorporated into phosphatidylserine (PS). On the other hand, [14C]20:4n6 was more actively incorporated into PS. Addition of LPS (1-10 uM), however, resulted in a 2-5 fold enhancement of the transfer of labeled 22:6n3 and 20:4n6 to phosphatidic acid (PA). Kinetic analysis indicated the ability of LPS to lower the Km and increase the Vmax of the lysophosphatidic acid (LPA) acyltransferase reaction. Among other lysophospholipids tested, lysophosphatidylserine was most effective in enhancing PA biosynthesis. Since PA is an important intermediate for de novo biosynthesis of phospholipids, these results reveal a novel mechanism for promoting synthesis of PA enriched in polyunsaturated fatty acids in brain.     

The electrophysiological actions of neurotensin in the central nervous system

The endogenous neuropeptide, neurotensin (NT) alters the firing frequencies of certain neurons in the central nervous system (CNS). This is one of the findings that support the hypothesis that NT is a neurotransmitter substance. The direct application of NT on CNS neurons causes predominantly excitatory effects. These effects occur in a dose-related fashion via a calcium-dependent postsynaptic mechanism. The C-terminal hexapeptide fragment, NT 8-13 exerts similar electrophysiological effects to NT, while the N-terminal octapeptide fragment, NT 1-8 is devoid of such activity. NT produces a significant increase in the firing rates of individual neurons in the substantia nigra (SN), ventral tegmental area (VTA), medial prefrontal cortex (MPF), hypothalamus, and periaqueductal grey (PAG). This excitation occurs with a rapid onset and is readily reversible after cessation of NT application. In contrast, NT has no effect or weak inhibitory effects on the firing rates of neurons in the locus coeruleus (LC) and cerebellum. These electrophysiological actions of NT appear to be unique and not shared by other neurotransmitter and neuropeptide receptor antagonists and agonists that have been studied via direct co-application. NT attenuates dopamine (DA)-induced inhibition associated with direct application onto neurons in the SN and VTA both in vivo and in vitro. Intracellular recordings suggest that direct application of higher concentrations of NT appears to produce 'depolarization block' on individual neurons in the SN, VTA, MPF, and hypothalamus. The electrophysiological consequences of NT application not only show similarities to clinically efficacious antipsychotic medications, but also demonstrate the ability of NT to modulate the activity of dopamine (DA) neurons at the cellular level via specific NT binding sites. These findings further underscore the possibility that NT may play a pre-eminent role in the pathogenesis of, and psychopharmacological management of neurological and psychiatric disorders purportedly related to perturbation of CNS DA systems including schizophrenia.     

The effect of cyclophosphamide and gamma irradiation on adenosine deaminase and purine nucleoside phosphorylase in mice

Changes in ADA and PNP activities in the spleens and thymuses of mice were studied after a single administration of cyclophosphamide (CY, 200 mg/kg) and after whole-body gamma irradiation (5.5 Gy), applied alone or three days after CY application. In the first days after the treatment the enzyme activities were significantly depressed (p less than 0.01) with the exception of ADA in the spleen, where a high elevation (220-380%) in relation to controls was observed. During the regeneration period a pronounced rise of PNP activity in the spleen occurred mainly after a combined application of CY and irradiation (270%). In the thymus the regeneration was manifested by a mild increase of both ADA and PNP activities towards control values. The findings suggest that the expressive changes of ADA and PNP activities, participating in the purine salvage pathway, may, after a cytotoxic treatment, influence the nucleotide pool and DNA synthesis in lymphoid organs.     

The hydrophobic membrane-spanning sequences of the gp52 glycoprotein are required for the pathogenicity of Friend spleen focus-forming virus.

Friend spleen focus-forming virus (SFFV) codes for a transport-defective envelope glycoprotein designated gp52, which is responsible for the leukemogenic properties of the virus. gp52 is a monotopic integral membrane protein anchored in the membrane by a stretch of hydrophobic amino acid residues located near the carboxy terminus of the molecule. We have constructed a mutant SFFV envelope gene in which the sequences that code for the hydrophobic membrane-spanning domain have been deleted, and we expressed this gene by using recombinant vaccinia virus vectors or retroviral vectors. The mutant SFFV envelope gene was found to encode a truncated glycoprotein (gp52t) which was also transport defective; a majority of gp52t remained cell associated, while a small proportion of the molecules underwent oligosaccharide processing. The processed form of gp52t was secreted from the cells. Retroviral vectors carrying the mutant SFFV envelope gene were found to be nonpathogenic in adult mice. These results indicate that the hydrophobic membrane-spanning region of gp52 is required for pathogenicity of SFFV and suggest that these sequences may play a role in signal transduction. The results also indicate that the transport defect of SFFV gp52 is due to structural features of the ectodomain of the molecule.     

Disturbed nonlinear multispecies models in ecology.

We analyze a disturbed form of the general Lotka-Volterra model of an ecosystem with m interacting species. The disturbances act on the intrinsic growth rates of the species and are assumed to be bounded but otherwise unknown. We employ a Lyapunov technique and the concept of "reachable set" from control theory to estimate the set of all possible population densities that are attainable as a result of the disturbances. To calculate estimates for this reachable set, a number of numerical methods that entail the solution to one or more global optimization problems are developed. Specific examples involving two, three, and four species are solved. We also derive an explicit analytical expression that represents an estimate for the reachable set in the m-dimensional case. The estimate is conservative but can be evaluated without carrying out any optimization procedure. We show that methods developed in this paper can be applied to certain other types of nonlinear ecosystem models.     

Characteristics of poliovirus strains isolated in Poland during 1981-1989 using monoclonal antibodies and determination of nucleotide sequence in viral RNA]

In this study currently used methods for analysis of poliovirus with monoclonal antibodies and determination of nucleotide sequence in viral RNA are presented. Twenty one strains of polioviruses isolated in Poland between 1981 and 1989 were tested. Nineteen of these strains were determined as to be derived from attenuated strains, including 10 strains isolated from 9 poliomyelitis patients. Two strains, one of type 1 isolated in 1984 and one of type 2 isolated in 1982 from children with meningitis were determined to be wild strains. The results obtained with monoclonal antibodies were confirmed by partial nucleotide sequence by dideoxy primer extension method. Analysis of poliovirus strains circulating in Poland in last 15 years suggest that the elimination of wild strains was achieved and sporadic isolation of wild strains is due most probably to imported strains.