Shunji Ohsako

The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain

Hairy-related proteins include the Drosophila Hairy and Enhancer of Split proteins and mammalian Hes proteins. These proteins are basic helix-loop-helix (bHLH) transcriptional repressors that control cell fate decisions such as neurogenesis or myogenesis in both Drosophila melanogaster and mammals. Hairy-related proteins are site-specific DNA-binding proteins defined by the presence of both a repressor-specific bHLH DNA binding domain and a carboxyl-terminal WRPW (Trp-Arg-Pro-Trp) motif. These proteins act as repressors by binding to DNA sites in target gene promoters and not by interfering with activator proteins, indicating that these proteins are active repressors which should therefore have specific repression domains. Here we show the WRPW motif to be a functional transcriptional repression domain sufficient to confer active repression to Hairy-related proteins or a heterologous DNA-binding protein, Ga14. This motif was previously shown to be necessary for interactions with Groucho, a genetically defined corepressor for Drosophila Hairy-related proteins. Here we show that the WRPW motif is sufficient to recruit Groucho or the TLE mammalian homologs to target gene promoters. We also show that Groucho and TLE proteins actively repress transcription when directly bound to a target gene promoter and identify a novel, highly conserved transcriptional repression domain in these proteins. These results directly demonstrate that Groucho family proteins are active transcriptional corepressors for Hairy-related proteins and are recruited by the 4-amino acid protein-protein interaction domain, WRPW.

Ca2+/calmodulin-dependent protein kinase II in the rat cerebellum: An immunohistochemical study with monoclonal antibodies specific to either α or β subunit

Abstract Monoclonal antibodies specific to either α or β subunit of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) of the rat brain were produced and the distribution of each subunit in the rat cerebellum was examined immunohistochemically. Each antibody detected solely the corresponding subunit in immunoblot analysis of crude homogenates of the rat forebrain and cerebellum, and purified CaM kinase II from the rat forebrain. Immunoreactivity for α subunit was present selectively in Purkinje cells: perikarya, dendrites with their spines, axons and their terminal-like structures in the cerebellar cortex, cerebellar nuclei and lateral vestibular nucleus. Many of these α subunit-immunoreactive axons from the cerebellum were traced only through the inferior cerebellar peduncle. β Subunit was detected in perikarya and dendrites of a limited number of Purkinje cells, many granule cells and neurons in the cerebellar nuclei. Thus, different distributions of α and β subunits of CaM kinase II in the cerebellum were demonstrated.

Receptor-mediated regulation of calcium mobilization and cyclic GMP synthesis in neuroblastoma cells

In neuroblastoma N1E 115 cells, carbachol, histamine and PGE1 elevated cyclic GMP content and, induced the efflux of preloaded 45Ca2+, the release of membrane-bound Ca2+ measured by fluorescent CTC, and the increase in [Ca2+]i as measured by Quin 2 fluorescence. The time course of the responses, the absolute requirement of extracellular Ca2+, the inhibition by receptor blockers, and the concentration dependency on histamine were all similar between these responses. The observation indicates that the mobilization of Ca2+, especially the increase of [Ca2+]i, may be intimately linked to the synthesis of cyclic GMP in the cells.

Phosphatidic acid mimicks the muscarinic action of acetylcholine in cultured bovine chromaffin cells

In cultured bovine chromaffin cells, acetylcholine as well as muscarine stimulated the 32Pi incorporation into phosphatidic acid, induced the efflux of 45Ca2+ from prelabelled cells, and, in parallel, elevated intracellular cyclic GMP content. Phosphatidic acid added to the medium also stimulated the efflux of 45Ca2+ and the synthesis of cyclic GMP in the cells in the same fashion as muscarinic agents, whereas it did not induce the secretion of catecholamines indicating that the effect of phosphatidic acid is specific to muscarinic action. The result supports the hypothesis that phosphatidic acid produced during phosphatidylinositol turnover is linked to the regulation mechanism of Ca2+ mobilization and cyclic GMP synthesis by muscarinic stimulation.

In situ hybridization demonstrating coexpression of urotensins I, II-α, and II-γ in the caudal neurosecretory neurons of the carp, Cyprinus carpio

Abstract In order to examine the coexpression of different urotensins in the same caudal neurosecretory neurons, in situ hybridization with synthetic oligonucleotide probes specific for mRNAs for urotensins I, II-α, and II-γ (UI, UII-α, and UII-γ) was applied to the caudal neurosecretory system of the carp, Cyprinus carpio, together with immunohistochemistry. Almost all identifiable caudal neurosecretory neurons were labeled with the oligonucleotide and cDNA probes for UI mRNA. Further, any two of the probes for UI, UII-α, and UII-γ mRNAs labeled the same neurons in serial sections. These results suggest that essentially all caudal neurosecretory neurons of the carp coexpress UI, UII-α, and UII-γ. Although most neurons were densely labeled with oligonucleotide probes, only a small portion of the neurons were intensely immunoreactive. This suggests that most caudal neurosecretory neurons actively synthesize all three hormones, but that in some neurons, all or some of the hormones are rapidly transported to the urophysis, resulting in low or undetectable immunoreactivity in the perikarya.

Intracapsular Organization of Ciliary Zonules in Monkey Eyes

Ciliary zonules are responsible for changing the curvature of a lens in the dioptric focus of an eye. Present established theory is based on the relaxation of zonular superficial fasciculi affixed to the capsular surface, thereby inducing the change of anterior‐ and posterior lens curvature causing spontaneous liquid movement of lens material. To achieve precise focusing at any distance, a more active functional organization should exist. The present studies were performed to determine not only the surface attachment but also the intracapsular affix of zonules on monkey eyes. In addition, the development of focusing in newborn and presbyopia is analyzed. Histology was prepared by conventional and molecular immunofluorescence stainings on the compositions of zonules with fibrillin‐1 (FBN 1) and lens capsule with collagen IV (COL IV), and in situ hybridization (ISH) analyses on frozen sections. Superficial circumferential attachments of zonule were found radially oriented between ciliary processes and anterior/posterior lens capsules forming a triangular figure. Two functional intralayer integrations were found above them; anterior‐posterior crossed fibers over the equator and radial fibers distributed toward the anterior or posterior polar areas. These fibers were bound tightly to the deep layer connective tissues close to the lens epithelium. Fine zonular fibers were aggregated, gradually forming bundles and bifurcated again on the way to the capsule. The lateral striped staining pattern in bundles suggested their elastic nature. Response of α‐helixes of collagen IV immunostaining was more positive on α‐1,2,4 than α‐3,5,6 on anterior‐ and posterior lens capsules. Newborn eyes revealed not fascicular but fine membranous zonules on the lens surface and small ciliary processes. ISH analysis revealed high synthetic expression of FBN 1 mRNA in cytoplasm of nonpigmented epithelial cells of ciliary processes. The synthetic expression of FBN 1 declined with aging. According to the mechanism of accommodation, active dynamic movement of anterior or posterior capsules play the main role of changing the lens configuration by two intralayer zonular integrations, including anterior‐posterior crossed fibers over the equator and radial fibers toward anterior or posterior polar areas acting with coordinated contraction of circular or longitudinal ciliary muscles. The developmental change on focusing is brought about by synthesis of FBN 1 in the newborn eye. Anat Rec 293:1797–1804, 2010.

Production of an antiserum using a fusion protein produced by a cDNA for rat choline acetyltransferase

A fusion protein produced by a cDNA for rat choline acetyltransferase (ChAT) inserted into a translation vector was used for immunization of rabbits. An antiserum was obtained that could recognize a single protein band in immunoblot analysis of a partially purified enzyme preparation of the rat brain. The antiserum revealed ChAT immunoreactivity in the motoneurons and terminal-like structures in the neuropil of the ventral horn in cryostat sections of the cervical spinal cord of the rat. This antiserum may be of particular use to study the development of the cholinergic neuron.

Subcellular distribution of α, and β subunit proteins of Ca2+ /calmodulin-dependent protein kinase II expressed in Chinese hamster ovary cells

When two cDNAs respectively encoding the entire coding regions of α and β subunits of Ca2+ /calmodulin‐dependent protein kinase II (CaM kinase II) were introduced into Chinese hamster ovary cells, the expressed α and β subunits were differently associated with subcellular structure. Although α subunit was loosely associated with subcellular structure, about 80% of CaM kinase II acitivity of α subunit was found in soluble fraction. More than 50% of the β subunit bound to the membrane, and the remainder was soluble but was loosely associated with subcellular structure. The relative rate of phosphorylation for substrate proteins of the β subunit bound to membrane was significantly different from that of the soluble form.

Expression of a catalytically active polypeptide of calmodulin-dependent protein kinase II α subunit in Escherichia coli

Two cDNAs, one containing the entire coding region of alpha subunit of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and the other containing only its protein kinase domain, were separately ligated into the bacterial expression vector pET3a and expressed in Escherichia coli. The activity of the recombinant alpha subunit protein was dependent on Ca2+/calmodulin, whereas the activity of the recombinant protein containing only the protein kinase domain (recombinant alpha-I protein) was absolutely independent of Ca2+/calmodulin. These proteins showed similar enzymatic properties to brain CaM kinase II with some minor differences. These results directly demonstrated that the protein kinase domain alone without the rest of the subunit was sufficient to exhibit its activity.

Expression and characterization of hydroxyindole O-methyltransferase from a cloned cDNA in Chinese hamster ovary cells

We have used two kinds of expression systems to test whether the cloned cDNA encoding hydroxyindole O-methyltransferase (HIOMT) of the bovine pineal gland was functional or not. First, when mRNA was synthesized in vitro by the SP6 system and injected into Xenopus oocytes, the enzymatic activity was expressed in the oocytes. Second, the cloned cDNA was recombined to a vector under the control of the simian virus 40 early promoter and transfected to Chinese hamster ovary (CHO) cells. The enzymatic activity of the crude supernatant of transfected cells (CHO-HT2) reached to 400 pmol melatonin formed per min per mg of protein, which value was approximately 9% of that of bovine pineal supernatant. The amounts of enzyme protein estimated by immunoblotting were proportional to the enzymatic activity in both CHO and pineal gland. The content of HIOMT protein was 8- to 30-fold larger in pineal gland compared to CHO cells. On the other hand, the content of mRNA encoding the enzyme measured by dot hybridization with [32P]cDNA, was in the same range in both CHO cells and pineal glands. These data suggest that the 11-fold higher enzymatic activity in pineal gland is due to an accumulation of the enzyme protein, not to a high level of the mRNA and also indicate that the cloned cDNA can express an intact hydroxyindole O-methyltransferase enzyme in CHO cells.