Role of mRNA stability and translation in the expression of cytochrome c oxidase during mouse myoblast differentiation: instability of the mRNA for the liver isoform of subunit VIa.
(65/10201)
The role of mRNA stability and translation in mediating the expression of selected subunits of cytochrome c oxidase (COX) was examined during the differentiation of mouse myoblasts into myotubes in cell culture. The expression of the liver (L) and heart (H) isoforms of COX VIa, which undergo an isoform switch during muscle development, as well as of the Va subunit, which is expressed in all tissues, was analysed. The translational efficiencies of COX Va, VIa-L and VIa-H, as well as of mitochondrially encoded COX mRNAs, were inferred from their distribution in polysome gradients. These experiments suggest that the translational efficiencies of these mRNAs do not change during myoblast differentiation, although the nuclear mRNAs for COX Va, VIa-L and VIa-H are translated more efficiently than the mitochondrial mRNAs. Analysis of mRNA stability using the tetracycline-repressible promoter system and/or actinomycin D indicates that COX VIa-L mRNA decays with a half-life of approximately 5-6 h in both myoblasts and myotubes, whereas COX VIa-H and Va mRNAs decay with half-lives of > 15 h in myotubes. This relative instability of COX VIa-L mRNA serves to limit the accumulation of COX VIa-L mRNA in these myogenic cells, as compared with mRNAs for other COX subunits. Deletion/replacement mapping experiments suggest that the COX VIa-L 3' untranslated region contains a destabilization element. Analysis of the rate of poly(A) tail shortening on COX VIa-L and stable alpha-globin mRNAs suggests that the overall rate of poly(A) shortening per se is not rate limiting for the degradation of COX VIa-L mRNA. (+info)
Identification of gamma-aminobutyric acid receptor subunit types in human and rat liver.
(66/10201)
GABA is a potent inhibitory neurotransmitter that binds to heterooligomeric receptors in the mammalian brain. In a previous study, we documented specific GABA binding to isolated rat hepatocytes that resulted in inhibition of hepatocyte proliferation. The purpose of the present study was to define the nature of hepatic GABA(A) receptors and to document their expression during rapid liver growth (after partial hepatectomy). PCRs with gene-specific primers derived from published sequences were performed with Marathon-ready human and rat liver cDNA. Two GABA(A) receptor subunit types (beta3 and epsilon) were expressed in human liver and one subunit type (beta3) in rat liver. PCR amplification of the human GABA(A) receptorbeta3-subunit produced a single product (molecular mass 53-59 kDa). In the case of the epsilon-subunit, two PCR products were identified. After partial hepatectomy, GABA(A) receptorbeta3-subunit expression inversely correlated with regenerative activity (r = -0.527, P = 0.006). In conclusion, these results indicate that in the human liver GABA(A) receptors consist of the beta3- and epsilon-subunit types, whereas in the rat liver only the beta3-subunit type is expressed. The results also support the hypothesis that GABAergic activity serves to maintain hepatocytes in a quiescent state. (+info)
Acrosome reaction of human spermatozoa is mainly mediated by alpha1H T-type calcium channels.
(67/10201)
The objectives of this study were: (i) to investigate the possible role of T-type Ca(2+) channels on the acrosome reaction (AR) of human spermatozoa; and (ii) to determine the sub-type of T-type calcium channels involved in the AR. The AR was induced in vitro by mannose-bovine serum albumin (BSA). The inhibitory effects of mibefradil (T-type Ca(2+) channel blocker), NiCl(2), or nifedipine (L-type Ca(2+) channel blocker) on the mannose-BSA induced AR were evaluated in capacitated human spermatozoa. The AR was sensitively inhibited by low micromolar concentrations of mibefradil (IC(50) = 1 micromol/l) in a dose-dependent manner. Low concentrations of Ni(2+) (IC(50) = 40 micromol/l) also inhibited the mannose-BSA induced AR. On the contrary, higher concentrations of nifedipine were required to block AR (IC(50) = 60 micromol/l). Reverse transcription-polymerase chain reaction (RT-PCR) was performed to identify the sub-types of T-type channels present in human testes. Analysis of PCR products showed that only alpha1H subunits are expressed in testes. The expression of the alpha1H subunit may be tissue specific since its mRNA was not detected in the human ovary. The present study suggests that the AR of human spermatozoa is highly associated with T-type Ca(2+) channels and is mainly mediated by calcium influx through alpha1H T-type Ca(2+) channels. (+info)
A positive role for the PP2A catalytic subunit in Wnt signal transduction.
(68/10201)
Protein phosphatase-2A (PP2A) is a multisubunit serine/threonine phosphatase involved in intracellular signaling, gene regulation, and cell cycle progression. Different subunits of PP2A bind to Axin and Adenomatous Polyposis Coli, components of the Wnt signal transduction pathway. Using early Xenopus embryos, we studied how PP2A functions in Wnt signal transduction. The catalytic subunit of PP2A (PP2A-C) potentiated secondary axis induction and Siamois reporter gene activation by Dishevelled, a component of the Wnt pathway, indicating a positive regulatory role of this enzyme in Wnt signaling. In contrast, small t antigen, an antagonist of PP2A-C, inhibited Dishevelled-mediated signal transduction, as did the regulatory PP2A-B'epsilon subunit, consistent with the requirement of PP2A function in this pathway. Although Wnt signaling is thought to occur via regulation of beta-catenin degradation, PP2A-C did not significantly affect beta-catenin stability. Moreover, the pathway activated by a stabilized form of beta-catenin was sensitive to PP2A-C and its inhibitors, suggesting that PP2A-C acts downstream of beta-catenin. Because previous work has suggested that PP2A can act upstream of beta-catenin, we propose that PP2A regulates the Wnt pathway at multiple levels. (+info)
Interaction between the unphosphorylated receptor with high affinity for IgE and Lyn kinase.
(69/10201)
Chinese hamster ovary fibroblasts previously transfected with the high affinity receptor for IgE (FcepsilonRI) were further transfected with the alpha subunit of the receptor for interleukin 2 (Tac) or with chimeric constructs in which the cytoplasmic domain of Tac was replaced with the C-terminal cytoplasmic domain of either the beta subunit or the gamma subunit of FcepsilonRI. Whereas native Tac failed to affect the aggregation-induced phosphorylation of FcepsilonRI, both chimeric constructs substantially inhibited this reaction. Alternatively, the FcepsilonRI-bearing fibroblasts were transfected with two chimeric constructs in which the cytoplasmic domain of Tac was replaced with a modified short form of Lyn kinase. The Lyn in both of the chimeric constructs had been mutated to remove the sites that are normally myristoylated and palmitoylated, respectively; one of the constructs had in addition been altered to be catalytically inactive. The catalytically active construct enhanced, and the inactive construct inhibited, aggregation-induced phosphorylation of the receptors. All of the chimeric constructs were largely distributed outside the detergent resistant microdomains, and whereas aggregation caused them to move to the domains in part, their aggregation was neither necessary nor enhanced their effects. These results and others indicate that the receptor and Lyn interact through protein-protein interactions that neither are dependent upon either the post-translational modification of the kinase with lipid moieties nor result exclusively from their co-localization in specialized membrane domains. (+info)
Alternative splicing in intracellular loop connecting domains II and III of the alpha 1 subunit of Cav1.2 Ca2+ channels predicts two-domain polypeptides with unique C-terminal tails.
(70/10201)
Novel splice variants of the alpha(1) subunit of the Ca(v)1.2 voltage-gated Ca(2+) channel were identified that predicted two truncated forms of the alpha(1) subunit comprising domains I and II generated by alternative splicing in the intracellular loop region linking domains II and III. In rabbit heart splice variant 1 (RH-1), exon 19 was deleted, which resulted in a reading frameshift of exon 20 with a premature termination codon and a novel 19-amino acid carboxyl-terminal tail. In the RH-2 variant, exons 17 and 18 were deleted, leading to a reading frameshift of exons 19 and 20 with a premature stop codon and a novel 62-amino acid carboxyl-terminal tail. RNase protection assays with RH-1 and RH-2 cRNA probes confirmed the expression in cardiac and neuronal tissue but not skeletal muscle. The deduced amino acid sequence from full-length cDNAs encoding the two variants predicted polypeptides of 99.0 and 99.2 kDa, which constituted domains I and II of the alpha(1) subunit of the Ca(v)1.2 channel. Antipeptide antibodies directed to sequences in the second intracellular loop between domains II and III identified the 240-kDa Ca(v)1.2 subunit in sarcolemmal and heavy sarcoplasmic reticulum (HSR) membranes and a 99-kDa polypeptide in the HSR. An antipeptide antibody raised against unique sequences in the RH-2 variant also identified a 99-kDa polypeptide in the HSR. These data reveal the expression of additional Ca(2+) channel structural units generated by alternative splicing of the Ca(v)1.2 gene. (+info)
The role of structural intersubunit microheterogeneity in the regulation of the activity in hysteresis of ribulose 1, 5-bisphosphate carboxylase/oxygenase.
(71/10201)
Many enzymes are composed of subunits with the identical primary structure. It has been believed that the protein structure of these subunits is the same. Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) comprises eight large subunits with the identical amino acid sequence and eight small subunits. Rotation of the side chains of the lysine residues, Lys-21 and Lys-305, in each of the eight large subunits in spinach RuBisCO in two ways produces microheterogeneity among the subunits. These structures are stabilized through hydrogen bonds by water molecules incorporated into the large subunits. This may cause different effects upon catalysis and a hysteretic, time-dependent decrease in activity in spinach RuBisCO. Changing the amino acid residues corresponding to Lys-21 and Lys-305 in non-hysteretic Chromatium vinosum RuBisCO to lysine induces hysteresis and increases the catalytic activity from 8.8 to 15.8 per site per second. This rate is approximately five times higher than that of the higher-plant enzyme. (+info)
Monoclonal antibodies recognizing surface residues of the beta subunit of Escherichia coli F(1) ATPase: functional importance of the epitope residues.
(72/10201)
Two monoclonal antibodies, beta 208 and beta 210, against the beta subunit of the F(1) ATPase from Escherichia coli reacted with an intact beta subunit and also a peptide corresponding to a portion of beta between residues 1 and 145. Mutations at Ala-1, Val-15, Glu-16, Phe-17, Leu-29, Gly-65, or Leu-66, and His-110 or Arg-111 for beta 210 and beta 208, respectively, caused decreased antibody binding to beta, suggesting that these residues form the epitopes and are thought to lie close together on the surface of the beta subunit. The topological locations of the corresponding residues in the atomic structure of the bovine beta subunit agree well with these expectations, except for Ala-1 and Leu-29. beta 210 binds to two beta strands including the epitope residues that are 50 residues apart, indicating that this antibody recognizes the tertiary structure of the N-terminal end region. Mutations in the epitope residues of beta 210 do not affect the F(1) ATPase activity, suggesting that surfaces of the two beta strands in the amino-terminal end region are not functionally essential. To analyze the functional importance around His-110 recognized by beta 208 we introduced site specific mutations at residues His-110 and Ile-109. Ile-109 to Ala or Arg, and His-110 to Ala or Asp caused defective assembly of F(1). However, the His-110 to Arg mutation had no effect on molecular assembly, suggesting that Ile-109 and His-110, especially the positive charge of His-110 are essential for the assembly of F(1). The His-110 to Arg mutation caused a large decrease in F(1)-ATPase activity, suggesting that a subtle change in the topological arrangement of the positive charge of His-110 located on the surface of beta plays an important role in the catalytic mechanism of the F(1)-ATPase. (+info)