Analysis of splicing of four mouse JNK/SAPKalpha variants.

The JNK/SAPK (c-Jun NH2-terminal kinase/stress-activated protein kinase) cascade is activated by a variety of stress stimuli and by the inflammatory cytokines interleukin-I (IL-I) and tumor necrosis factor alpha (TNFalpha). Four splice variants of the mouse JNK/SAPKalpha isoform, which differ in a region located in subdomains IX-X of the protein, were previously identified. Analysis of the sequence of the central region of the mouse JNK/SAPKalpha gene indicates that splice variants I and II are generated by a typical alternative splicing mechanism, while splice variants III and IV are generated by a less common mechanism, where alternative 3' splice sites located inside an exon (cryptic sites) are selected. The major splice variants alphaI and all have a wide and similar distribution in hippocampus, cerebral cortex, caudate-putamen, amygdala and the granule cell layer of cerebellum, although their expression is specifically regulated in certain cell types.

Cloning of chicken and mouse alpha 1b adrenergic receptor.

A partial cDNA encoding most of the third intracellular loop of the chicken alpha 1b adrenergic receptor subtype, obtained by reverse transcription-polymerase chain reaction (RT-PCR) techniques using degenerate primers derived from mammalian sequences, was used to isolate an alpha 1b adrenergic receptor cDNA from brain. The cDNA encodes a potential protein of 507 amino acids and Northern hybridization of poly(A)+ RNA from chicken brain of different developmental stages detected a single 3.5 kb transcript. Analysis of receptor expression indicated that the alpha 1b adrenergic receptor is widely distributed in chicken tissues, specially kidney and liver. cDNA and genomic clones encoding sequences of the mouse alpha 1b adrenergic receptor were also isolated.

The autoradiographic perspective of central benzodiazepine receptors; a short review.

1. We reviewed studies performed to characterize central benzodiazepine binding sites. 2. An overview of the different radioligands used to characterize BZ1 and BZ2 binding sites and a mapping of these central benzodiazepine sites are described. 3. Saturation studies carried out by autoradiogram quantification also are reviewed. 4. The specific use of the autoradiographic technique to carry out studies on ontogeny, development, and phylogeny is discussed, as well as studies performed using this technique on some diseases and experimental conditions, such as drug treatments or chemical and mechanical lesions.

Phosphorylation of the third intracellular loop of the mouse alpha1b-adrenergic receptor by cAMP-dependent protein kinase.

The third intracellular loop of adrenergic receptors has been implicated in their interaction with guanine nucleotide-binding proteins (G proteins). One of the mechanisms involved in the modulation of receptor function is the phosphorylation of specific residues by intracellular kinases. alpha1b-Adrenergic receptor is phosphorylated in vitro by cAMP-dependent protein kinase (PKA), although its physiological effect remains to be determined. We have produced fusion proteins formed by glutathione S-transferase and sequences of the third intracellular loop of mouse alpha1a-, alpha1b-, and alpha1d-adrenergic receptor subtypes, and used them as substrates for PKA. Only the fusion protein containing the alpha1b sequence was phosphorylated in vitro by this kinase. Site-directed mutagenesis of a serine (homologue to serine 278 of the rat sequence, RSS) to an alanine residue precluded phosphorylation by PKA.

Identification of a long huntingtin mRNA transcript in mouse brain.

Two mRNA species of the Huntington disease (HD) gene that share identical protein coding sequences but differ in their 3' untranslated region have been identified in human. Although a similar situation has been suggested to occur in mouse, only one cDNA has been isolated to date. We report the isolation of a novel partial cDNA of the mouse HD gene that is identical in its protein coding sequence to the previously reported cDNA, although it differs in the distal portion of the 3' untranslated region. Northern blotting assays indicate that this mRNA transcript is preferentially expressed in brain, with highest levels in cerebellum, cerebral cortex and striatum.

Identification of four splice variants of the mouse stress-activated protein kinase JNK/SAPK alpha-isoform.

Four splice variants (alpha I-IV) of the stress-activated protein kinase JNK/SAPK alpha-isoform have been identified in the mouse. One of them (alpha I) contains an open reading frame of 1269 bp encoding a potential protein of 423 amino acids, whereas the second variant (alpha II) differs in a region encoding 31 amino acids located in subdomain IX. alpha III lacks this region and also differs in the terminal portion of the 3' untranslated region (3' UTR). A fourth variant (alpha IV) which lacks a region of 41 amino acids located in subdomain IX has also been identified. These splice variants are differentially expressed in mouse tissues: alpha I is the most abundant in brain areas, whereas alpha II is mainly expressed in extracerebral tissues, such as liver; alpha III and alpha IV are present in brain and other tissues although in lower amounts.

Molecular cloning of alpha 1d-adrenergic receptor and tissue distribution of three alpha 1-adrenergic receptor subtypes in mouse.

A partial cDNA encoding most of the third intracellular loop of the mouse alpha 1d-adrenergic receptor subtype was amplified from hippocampus by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate oligodeoxynucleotide primers. This DNA fragment was used as a probe to isolate an alpha 1d-adrenergic receptor cDNA from a mouse brain cDNA library. The deduced amino acid sequence encodes a potential protein of 562 amino acids, and northern hybridization of poly(A)+ RNA isolated from mouse brain detected a single 3.0-kb transcript. Partial cDNA fragments of the alpha 1b- and alpha 1a-adrenergic receptor subtypes were also amplified from mouse brain and sequenced. Analysis of the mRNA expression by RT-PCR indicated that the alpha 1-adrenergic receptors are widely distributed in mouse tissues. The alpha 1d subtype is expressed in brain areas such as hippocampus, striatum, and brainstem and also in many extracerebral tissues, such as lung, liver, heart, kidney, and spleen. The alpha 1a subtype is also expressed in many tissues, whereas the alpha 1b subtype has a more restricted expression, with high levels in striatum, brainstem, and diencephalus.

Immunodetection of the large form of the gamma 2 subunit of mammalian GABAA receptor.

Two forms of the GABAA receptor gamma 2-subunit, named short (gamma 2S) and long (gamma 2L), and generated by alternative RNA splicing, have been identified in mammalian brain by molecular cloning techniques. We have produced antibodies against a synthetic peptide containing the 8-amino acid insertion present in the long form but not in the short one. Using the antipeptide serum, we have identified the gamma 2L subunit in membrane preparations of GABAA receptors from rat and mouse cerebellum and its cellular location in cerebellum.

Immunodetection of serotonin transporter from mouse brain.

A DNA fragment encoding amino acid sequences from the amino terminal region of the mouse serotonin transporter was isolated and sequenced. This transporter is widely distributed throughout the mouse brain, as deduced by heminested reverse transcription-polymerase chain reaction (RT-PCR) assays. To identify the serotonin transporter protein, we have developed specific antibodies against a fusion protein containing its amino terminal region, a domain which shows a low degree of homology between the different neurotransmitter transporters. Western blot analysis of mouse brain membranes detected the serotonin transporter as a 71 kDa polypeptide.

Effect of ethanol treatment on rate and equilibrium constants for [3H] muscimol binding to rat brain membranes: alteration of two affinity states of the GABAA receptor.

Equilibrium binding curves were biphasic in control and ethanol-treated rats. [3H]Muscimol binds to sites of high (KDA of approximately 10 nM) and low (KDB of approximately 0.3-0.4 microM) affinity. Chronic ethanol treatment produced a decrease in BmaxA value, and the hyperbolic binding profiles were progressively affected by the chronic and in vitro ethanol treatments, with most of this effect corresponding to the high-affinity site. IC50 and Ki values were calculated for several competing ligands, using membranes from both control and ethanol-treated animals. The association and dissociation curves were also biphasic, using a radioligand concentration precluding a significant occupancy of the low-affinity sites, which suggests the existence of two forms or affinity states of the monoliganded receptor. Chronic ethanol treatment did not produce changes in the values of the dissociation rate constants (fast and slow phases). By contrast, we report for the first time a decrease in the values of the association rate constants, with this decrease being higher for the slow phase. Consequently, the dissociation equilibrium constants are two times higher in chronically ethanol-treated animals for both phases.

Identification of a cyclic adenosine 3',5'-monophosphate-dependent protein kinase phosphorylation site in the carboxy terminal tail of human D1 dopamine receptor.

Each of the dopamine receptor subtypes contains several consensus sites for phosphorylation in their intracellular domains. We have used fusion proteins of the carboxy terminal tail of D1 and D5 dopamine receptors to study the phosphorylation of these proteins by cyclic adenosine 3',5' monophosphate (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC). The fusion protein of D1 dopamine receptor was efficiently phosphorylated by PKA, but not by PKC. Site-directed mutagenesis of serine 380 to an alanine residue precluded the phosphorylation by the kinase. No phosphorylation of the D5 dopamine receptor fusion protein was observed with either PKA or PKC, which indicates that these receptor subtypes might differ in their mechanisms of regulation.

Alternative splicing of a 51-nucleotide exon that encodes a putative protein kinase C phosphorylation site generates two forms of the chicken gamma-aminobutyric acidA receptor beta 2 subunit.

Complementary DNAs that encode two forms of the chicken gamma-aminobutyric acid type A (GABAA) receptor beta 2 subunit have been isolated. These polypeptides differ by the presence of (beta 2L) or absence (beta 2S) of 17 amino acids, which contain a possible target for phosphorylation by protein kinase C, in the large intracellular loop between the third and fourth membrane-spanning domains. The extra sequence in the chicken beta 2L subunit is not found in previously published GABAA receptor beta 2-subunit sequences. Analysis of genomic DNA has revealed that the two beta 2-subunit mRNAs arise by alternative splicing of a novel 51-nucleotide exon. Although the two beta 2-subunit transcripts appear to be present in 1-day-old chick brain at similar steady-state levels, we have been unable to detect an mRNA for the long form of the beta 2 subunit in either the bovine or the rat. Because the various GABAA receptor genes are thought to have arisen by duplication of a common ancestor, our data, taken together with that on the gamma 2 subunit, which occurs in two forms that arise by alternative splicing of a 24-nucleotide exons, suggest that the coding region of the primordial gene or one of its very early descendants contained 10 exons, not nine as previously thought.

Characterization of the maltase activity of glucosidase II from rat liver. Kinetic model.

Glucosidase II is a key enzyme in the processing of N-glycoproteins since it removes the two glucose residues from the protein-linked oligosaccharide Glc2Man9GlcNAc2-R. We have studied the kinetics of the purified enzyme, using maltose as substrate. Analysis of data fitting to single and double-hyperbolic equations and the Eadie-Hofstee profile indicate that the enzyme has two binding (active) sites for the hydrolysis of maltose. The Km and Vmax values for the high-affinity site were 0.43 mM and 691 mU/mg, respectively, whereas the values for the low-affinity site were 57.7 mM and 2888 mU/mg, respectively. The Vmax/Km ratios were 1607 and 50.1 ml/min per g for the high- and low-affinity sites, respectively. A new kinetic model for this enzyme is proposed from the equilibria corresponding to the partial competitive inhibition produced by maltose on p-nitrophenyl-glucosidase activity. The amino acid composition of the enzyme has been established.

Differential expression of the alpha 1c adrenergic receptor subtype in rat tissues.

Three alpha 1 adrenergic receptor subtypes have been identified by molecular cloning techniques. However, the detection of the mRNA for the alpha 1c subtype has not yet been reported in rodent tissues. We have isolated from rat lung, by polymerase chain reaction techniques, a cDNA fragment corresponding to the third intracellular domain of the alpha 1c adrenergic receptor subtype. The nucleotide and deduced amino acid sequences of the cloned fragment presented an 87% and 96% identity, respectively, with those from the bovine receptor. Analysis of the distribution of the receptor mRNA in brain shows highest expression levels in cerebellum and striatum, whereas in non-neural tissues the receptor is mainly expressed in lung and heart. It was also found that the gene sequence which codes for this domain is not interrupted by introns.

Differential effect of chronic ethanol treatment on barbiturate and steroid modulation of muscimol-binding to rat brain cortex.

We report the differential alterations produced by chronic ethanol treatment on the modulation, by the barbiturate thiopental and the steroid 5 beta-pregnan-3 alpha-ol-20-one, of the binding of [3H]muscimol to membrane preparations from rat brain cortex. We found a clear barbiturate- and steroid-promoted enhancement of muscimol-binding to membranes in both control and ethanol-treated animals. However, the enhancements were higher in control animals, using the barbiturate, and in ethanol-treated rats, using the steroid, Bmax and Kd values were also differentially affected in control and ethanol-treated animals by the presence of the barbiturate or the steroid.

Effect of chronic ethanol treatment on the gamma-aminobutyric acid-mediated enhancement of [3H]flunitrazepam binding in rat cortex and hippocampus.

The mechanism by which ethanol affects the gamma-aminobutyric acid (GABA)/benzodiazepine complex is not clear. It is known that ethanol enhances the Cl- influx mediated by the GABAA receptor complex, and although chronic ethanol administration does not change the KD or Bmax for [3H]flunitrazepam binding, some reports have suggested that it could modify the modulation of benzodiazepine binding produced by GABA. In the present work, we studied the effect of chronic ethanol treatment on the modulation by GABA of [3H]flunitrazepam binding, using light microscopic autoradiography. This technique allows the measurement of densities of benzodiazepine receptors in different brain areas, the visual cortex and hippocampus, which appear to constitute the anatomical support for the behavioral and physiological responses affected by ethanol. We found enhancement of benzodiazepine binding by GABA at concentrations of greater than 10(-6) M for the various cortical and hippocampal areas studied from both control and ethanol-treated animals; this enhancement peaked at 10(-4) M GABA but decreased at 10(-3) M GABA. We found a clear effect of ethanol treatment on the modulatory properties of GABAA receptor, in both cortex and hippocampus, although only in cortex were the differences statistically significant between control and ethanol-treated animals.

Effects of several alcohols on glycosidase activities in rat liver and brain.

1. beta-D-Galactosidase, beta-D-glucuronidase and beta-NAc-D-glucosaminidase spec. acts from liver displayed statistically significant differences after treatments with certain alcohols. 2. No statistically significant difference was found for the kinetic parameters (Km and Vmax) of the indicated glycosidase activities. 3. beta-D-Glucuronidase and beta-NAc-D-glucosaminidase spec. acts from brain tissue showed statistically significant differences after treatments with certain alcohols. 4. beta-D-Glucuronidase and beta-NAc-D-glucosaminidase activities from liver showed a statistically significant decrease in the presence of in vitro ethanol. Tolerance effects were not observed.

Characteristics of c-fos and jun B gene expression in A5 cells after beta-adrenoreceptor stimulation and during the cell cycle.

The beta-adrenoreceptor agonist isoproterenol elevates cAMP concentrations in the A5 rat salivary epithelial cell line and rapidly and transiently induces the expression of c-fos and jun B at 30 and 60 min following continuous stimulation of these cells. The induction of both genes is mediated by cAMP. We show here that the inducibility of these genes by isoproterenol or 8-BrcAMP is transcriptionally regulated and short (5 min) incubations of A5 cells with either agent is sufficient to trigger the induction of c-fos and jun B. We also have investigated the expression and inducibility of these genes during the A5 cell cycle. Both c-fos and jun B mRNA are elevated at the early phase of the cell cycle and are detectable throughout the cycle. At different stages of the cell cycle in synchronous A5 cells, both genes are as highly induced by isoproterenol or 8-BrcAMP as in asynchronous A5 cells. These studies provide the first evidence for the transcriptional regulation of c-fos and jun B by beta-adrenergic receptor stimulation or cAMP in an epithelial cell line (A5) and demonstrate the coordinate expression and inducibility of these genes at the different stages of the A5 cell cycle.

Purification and characterization of different "acid" beta-galactosidases from sheep kidney.

1. Two "acid" forms, Am and Al, of beta-galactosidase from sheep kidney have been isolated and purified 349- and 154-fold, respectively, with a recovery of about 8%. 2. Their mol. wts were about 450,000 and 230,000, respectively. Am seems to be a dimer of Al. The aggregation is stimulated by NaCl. 3. The "acid" beta-galactosidase has a pH optimum between 4.0 and 5.0 for both forms. They are located in the lysosomes. The optimal temperature is 37 degrees C and 40 degrees C for Al and Am forms, respectively. 4. Three peaks were detected by isoelectric focusing. After sialidase treatment, these peaks were obtained at higher pH values. 5. The activation energy values were 10.75 and 11.72 kcal/mol for Am and Al, respectively. 6. A variety of chemicals were tested as possible activators or inhibitors. The enzyme is strongly inhibited by gamma-D-galactonolactone, and the kinetic evidence suggests a competitive inhibition in all cases.

Selective labeling of alpha-bungarotoxin with fluorescein isothiocyanate and its use for the study of toxin-acetylcholine receptor interactions.

The main product of the reaction of fluorescein isothiocyanate (FITC) and bungarotoxin (Bgt) under near stoichiometric conditions is a monofluorescein derivative preferentially labeled at Lys 26, a highly conserved residue known to be involved in the binding (McDaniel, C.S., Manshouri, T., and Atassi, M.Z. (1987) J. Prot. Chem. 6, 455-461; Garcia-Borron, J.C., Bieber, A.L., and Martinez-Carrion, M. (1987) Biochemistry 26, 4295-4303) of postsynaptic neurotoxins specific for the nicotinic acetylcholine receptor (AcChR). The fluorescently labeled toxin retains a high affinity for the AcChR, and an unaltered specificity. Binding of FITC-Bgt to AcChR results in a significant decrease in the fluorescence intensity of the probe. This AcChR-mediated quenching of FITC-Bgt fluorescence allows for a continuous monitoring of the binding process. The quenching of free and bound FITC-Bgt by charged and neutral quenchers shows few fluorophore accessibility changes as induced by the toxin-bound state. The results are consistent with a model in which the positively charged concave surface of the toxin interacts with a negatively charged complementary surface in the receptor molecule.