Identification of the sequence responsible for the nuclear localization of human Cdc6

The Cdc6 is the essential protein for the initiation of DNA replication. Cdc6 is localized in the G1 nucleus, and abnormal nuclear localization of this protein induces irregular initiation of DNA replication. We identified here that amino acids K57 and R58 in the human Cdc6 protein play an important role in the nuclear localization of the protein. The fundamental features of the mechanism regulating the localization of Cdc6 seem to be maintained in yeast, Xenopus, and human, since the amino acid sequence surrounding K57 and R58, (S/T)PXKR(L/I), is conserved in these species. Substitution of amino acid residue S54 with E and not Q blocked partially the nuclear localization of the protein, implying that the phosphorylation at S54 is involved in the regulating mechanism of the cell cycle-dependent localization of Cdc6.     

Comparative characterization of the oah2 gene homologous to the oah1 of Thermus thermophilus HB8

The oah2 gene homologous to the oah1 of Thermus thermophilus HB8 was cloned and sequenced. It comprised 1,236 bp encoding a protein of 412 amino acid residues and was overexpressed. The gene product, also having O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity, was purified to homogeneity and characterized comparatively with the oah1 product. The two proteins shared many characteristics.     

Does the natriuretic peptide system exist throughout the animal and plant kingdom?

Natriuretic peptides (NPs) and their receptors have been identified in vertebrate species ranging from elasmobranchs to mammals. Atrial, brain and ventricular NP (ANP, BNP and VNP) are endocrine hormones secreted from the heart, while C-type NP (CNP) is principally a paracrine factor in the brain and periphery. In elasmobranchs, only CNP is present in the heart and brain and it functions as a circulating hormone as well as a paracrine factor. Four types of NP receptors are cloned in vertebrates. NPR-A and NPR-B are guanylyl cyclase-coupled receptors, whereas NPR-C and NPR-D have only a short cytoplasmic domain. NPs are hormones important for volume regulation in mammals, while they act more specifically for Na(+) regulation in fishes. The presence of NP and its receptor has also been suggested in the most primitive vertebrate group, cyclostomes, and its molecular identification is in progress. The presence of ANP or its mRNA has been reported in the hearts and ganglia of various invertebrate species such as mollusks and arthropods using either antisera raised against mammalian ANP or rat ANP cDNA as probes. Immunoreactive ANP has also been detected in the unicellular Paramecium and in various species of plants including Metasequoia. Furthermore, the N-terminal prosegments of ANP, whose sequences are scarcely conserved even in vertebrates, have also been detected by the radioimmunoassay for human ANP prosegments in all invertebrate and plant species examined including Paramecium. Although these data are highly attractive, the current evidence is too circumstantial to be convincing that the immunoreactivity truly originates from ANP and its prosegments in such diverse organisms. The caution that has to be exercised in identification of vertebrate hormones from phylogenetically distant organisms is discussed.     

AGAP1, an endosome-associated,phosphoinositide-dependent ADP-ribosylation factor GTPase-activating protein that affects actin cytoskeleton

We have identified three members of the AGAP subfamily of ASAP family ADP-ribosylation factor GTPase-activating proteins (Arf GAPs). In addition to the Arf GAP domain, these proteins contain GTP-binding protein-like, ankyrin repeat and pleckstrin homology domains. Here, we have characterized the ubiquitously expressed AGAP1/KIAA1099. AGAP1 had Arf GAP activity toward Arf1>Arf5>Arf6. Phosphatidylinositol 4,5-bisphosphate and phosphatidic acid synergistically stimulated GAP activity. As found for other ASAP family Arf GAPs, the pleckstrin homology domain was necessary for activity. Deletion of the GTP-binding protein-like domain affected lipid dependence of Arf GAP activity. In vivo effects of AGAP1 were distinct from other ASAP family Arf GAPs. Overexpressed AGAP1 induced the formation of and was associated with punctate structures containing the endocytic markers transferrin and Rab4. AP1 was redistributed from the trans-Golgi to the punctate structures. Like other ASAP family members, AGAP1 overexpression inhibited the formation of PDGF-induced ruffles. However, distinct from other ASAP family members, AGAP1 also induced the loss of actin stress fibers. Thus, AGAP1 is a phosphoinositide-dependent Arf GAP that impacts both the endocytic compartment and actin.     

A new method for enzymatic preparation of isopentenyladenine-type and<Emphasis Type="Italic"> trans</Emphasis>-zeatin-type cytokinins with radioisotope-labeling

We describe a new enzymatic reaction method for the preparation of the radioisotope-labeled cytokinins isopentenyladenine (iP), trans-zeatin (tZ), and their ribosides. The method is based on the three enzyme activities of an adenylate isopentenyltransferase (IPT; EC 2.5.1.27) from Arabidopsis thaliana, an alkaline phosphatase (EC 3.1.3.1) from calf intestine, and a purine-nucleoside phosphorylase (EC 2.4.2.1) from Escherichia coli. The A. thaliana IPT, AtIPT7, utilized both dimethylallyldiphosphate and 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate as isoprenoid donors. The dual specificity of the substrates enabled us to produce iP-type and tZ-type cytokinins separately in the same system simply by switching the substrates. Our method affords a much higher yield of the labeled products than the chemical reaction methods previously used. These labeled compounds will be useful tools for cytokinin research, such as receptor–ligand assays and cell metabolism studies.     

Relative potency of three homologous natriuretic peptides (ANP, CNP and VNP) in eel osmoregulation

Evidence has accumulated that atrial natriuretic peptide (ANP) plays important roles in sea-water adaptation in eels. However, the roles of the other two natriuretic peptides (CNP and VNP) in osmoregulation have not been examined yet. In the present study, the effects of homologous ANP, CNP and VNP were compared on plasma Na+ concentration (an indicator of plasma osmolality), hematocrit (an approximate indicator of blood volume) and drinking rate in freshwater- and seawater-adapted eels. In seawater eels, ANP and VNP, but not CNP, infused at 5 pmol/kg/min decreased plasma Na+ concentration and drinking rate and increased hematocrit. In freshwater eels, ANP and VNP failed to decrease plasma Na+ concentration but increased hematocrit to the same extent as in seawater eels. Inhibition of drinking was not detectable in freshwater eels because of little drinking before NP infusions. These results show that the effects of NPs on plasma Na+ concentration, drinking rate and hematocrit are mediated by NPR-A, since only ANP and VNP that bind with higher affinity to NPR-A are effective in seawater eels. The mechanisms of regulation of plasma Na+ concentration and hematocrit are unknown, but NPR-A is present in the responsible tissues for regulation of hematocrit in both freshwater and seawater eels. However, NPR-A may be absent in the tissues of freshwater eels that are responsible for regulation of plasma Na+ concentration.     

Role of the renin-angiotensin system in drinking of seawater-adapted eels Anguilla japonica: a reevaluation

The role of ANG II, a potent dipsogenic hormone, in copious drinking of seawater eels was examined. SQ-14225 (SQ), an angiotensin-converting enzyme inhibitor, infused intra-arterially at 0.01-1 microgram. kg(-1). min(-1), depressed drinking and arterial blood pressure in a dose-dependent manner. The inhibition was accompanied by a small decrease in plasma ANG II concentration, which became significant at 1 microgram. kg(-1). min(-1). After the infusate was changed back to the vehicle, the depression of drinking and arterial pressure continued for >2 h, although plasma ANG II concentration rebounded above the level before SQ infusion. By contrast, infusion of anti-ANG II serum (0.01-1 microgram. kg(-1). min(-1)) did not suppress drinking and arterial pressure, although plasma ANG II concentration decreased to undetectable levels. Plasma atrial natriuretic peptide and plasma osmolality, which influence drinking rate in eels, did not change during SQ or antiserum infusions. These results suggest that the renin-angiotensin system plays only a minor role in the vigorous drinking observed in seawater eels. The results also suggest that the antidipsogenic and vasodepressor effects of SQ in seawater eels are not due solely to the inhibition of ANG II formation in plasma.