Exclusive expression of transketolase in the vanadocytes of the vanadium-rich ascidian, Ascidia sydneiensis samea.

Ascidians, especially those belonging to the Ascidiidae, are known to accumulate extremely high levels of vanadium in vanadocytes, one type of blood (coelomic) cell. Vanadium, which exists in the +5 oxidation state in seawater, is accumulated in the vanadocytes and reduced to the +3 oxidation state. We have been trying to characterize all of the polypeptides specific to vanadocytes and to specify the proteins that participate in the accumulation and reduction of vanadium. To date, we have localized three enzymes in vanadocytes: 6-phosphogluconate dehydrogenase (6-PGDH: EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6PDH: EC 1.1.1.49), and glycogen phosphorylase (GP: EC 2.4.1.1), all of which are involved in the pentose phosphate pathway. In the current study, we cloned a cDNA for transketolase, an essential and rate-limiting enzyme in the non-oxidative part of the pentose phosphate pathway, from vanadocytes. The cDNA encoded a protein of 624 amino acids, which showed 61.8% identity to the human adult-type transketolase gene product. By immunocytochemistry and immunoblot analyses, the transketolase was revealed to be a protein that was expressed only in vanadocytes and not in any of the more than ten other types of blood cell. This finding, taken together with the localized expression of the other three enzymes, strongly supports the hypothesis that the pentose phosphate pathway functions exclusively in vanadocytes.

Human Ca2+/calmodulin-dependent phosphodiesterase PDE1A: novel splice variants, their specific expression, genomic organization, and chromosomal localization.

We report here the identification of novel human PDE1A splice variants, their tissue distribution patterns, genomic structure, and chromosomal localization of the gene. We identified one N-terminus (N3) and one C-terminus (C3) by cDNA library screening and dbEST database search. These N- and C-termini, including the reported N-termini (N1 and N2) and C-termini (C1 and C2), combined to generate nine different PDE1A cDNAs. N1 and N2 are similar to the 5' ends of the bovine PDE1A proteins of 61 kDa and 59 kDa, respectively, and C1 and C2 are the 3' ends of the reported human PDE1A variants. The results of PCR and Southern blot analysis show that nine PDE1A splice variants exhibit distinctive tissue distribution patterns by the difference of the N-terminus. PDE1As with N2 were widely expressed in various tissues, mainly in the kidney, liver, and pancreas. On the other hand, PDE1As with N1 and N3 were particularly expressed at a high level in the brain and testis, respectively. These findings suggest that the distinct expression patterns among PDE1A variants depend on the several promoters situated upstream of exons encoding 5' ends of the variants. The PDE1A gene spans over 120 kb of genomic DNA, and consists of at least 17 exons and 16 introns. The PDE1A gene was located on human chromosome 2q32 by fluorescent in situ hybridization analysis.

Identification of a conserved residue responsible for the autoinhibition of cGMP-dependent protein kinase Ialpha and beta.

We isolated a constitutively active form of cGMP-dependent protein kinase Ialpha (cGK Ialpha) by PCR-driven random mutagenesis. The replacement of Ile-63 by Thr in the autoinhibitory domain results in the enhancement of autophosphorylation and the basal kinase activity in the absence of cGMP. The hydrophobicity at position 63 is essential for the inactive state of cGK Ialpha, and Ile-78 of cGK Ibeta is also required for the autoinhibitory property. Furthermore, cGK Ialpha (Ile-63-Thr) is constitutively active in vivo. These findings suggest that a conserved residue in the autoinhibitory domain was involved in the autoinhibition of both cGK Is.

Characterization and effects of methyl-2- (4-aminophenyl)-1, 2-dihydro-1-oxo-7- (2-pyridinylmethoxy)-4-(3,4, 5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), a novel potent inhibitor of cGMP-binding cGMP-specific phosphodiesterase (PDE5).

An isoquinolone derivative, methyl-2-(4-aminophenyl)-1, 2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4, 5-trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032), was found to be a novel potent inhibitor of cyclic GMP (cGMP)-binding cGMP-specific phosphodiesterase (PDE5). We investigated the inhibitory effects of T-1032 on six PDE isozymes isolated from canine tissues. T-1032 specifically inhibited the hydrolysis of cGMP by PDE5 partially purified from canine lung, at a low concentration (IC(50) = 1.0 nM, K(i) = 1.2 nM), in a competitive manner. In contrast, the IC(50) values of T-1032 for PDE1, PDE2, PDE3, and PDE4 were more than 1 microM. T-1032 also inhibited PDE6 from canine retina with an IC(50) of 28 nM, which is of the same order of magnitude as the IC(50) of sildenafil. cGMP hydrolytic activities of two alternative splice variants of canine PDE5 expressed in COS-7 cells were inhibited by this compound to a similar extent. T-1032 increased the intracellular concentration of cGMP in cultured rat vascular smooth muscle cells in the presence and absence of C-type natriuretic peptide, an activator of membrane-bound guanylate cyclase, whereas the compound did not change cyclic AMP levels. These data indicated that T-1032, which belongs to a new structural class of PDE5 inhibitors, is a potent and selective PDE5 inhibitor. This compound may be useful in pharmacological studies to examine the role of a cGMP/PDE5 pathway in tissues.

6-Phosphogluconate dehydrogenase is a 45-kDa antigen recognized by S4D5, a monoclonal antibody specific to vanadocytes in the vanadium-rich ascidian Ascidia sydneiensis samea.

We previously prepared a monoclonal antibody, S4D5, specific to vanadocytes, vanadium-containing blood cells, in the vanadium-rich ascidian Ascidia sydneiensis samea. Here, we demonstrate that a 45-kDa antigen recognized by S4D5 is 6-phosphogluconate dehydrogenase (6-PGDH), an enzyme of the pentose phosphate pathway, based on cDNA isolation of RNA samples from blood cells of the ascidian. Western blot analysis confirmed an abundance of 6-PGDH protein in the vanadocytes and localization of 6-PGDH in the soluble extract of the blood cells. Soluble protein exhibited a correspondingly high level of 6-PGDH enzymatic activity. Ascidians are known to selectively accumulate high levels of vanadium in vanadocytes, and the highest recorded concentration of accumulated vanadium is 350 mM, which is 10(7) times the concentration in sea water. Almost all vanadium ions are reduced to the +3 oxidation state via the +4 oxidation state in vanadocytes, indicating that reducing agents must participate in the accumulation. On the other hand, vanadium ions in the +5 oxidation state are reduced to the +4 oxidation state by the presence of NADPH in vitro. Together, these observations suggest that NADPH produced in the pentose phosphate pathway may conjugate the reduction of vanadium from the +5 oxidation state through the +4 oxidation state in vanadocytes of ascidians.

Localization of N-ethylmaleimide-sensitive fusion protein in pinealocytes.

N-ethylmaleimide-sensitive fusion protein (NSF), a protein necessary for vesicular docking and/or fusion, was detected immunohistochemically in pinealocytes. NSF was distributed similarly to synaptophysin and vacuolar-type H(+)-ATPase (V-ATPase), marker proteins for synaptic-like microvesicles (MVs) abundantly present in pinealocytes. A subcellular fractionation study indicated that .> 95% of NSF was present as a membrane-bound form and that some NSF was associated with MVs. Like neuronal NSF, the protein was not solubilized from membranes with either 2 mM Mg-ATP or 2% sodium carbonate, suggesting that NSF was tightly bound to the membranes. NSF was also detected in purified MVs from bovine posterior pituitaries. Since MVs are the organelles in which transmitters are stored, these results suggest that NSF is involved in the MV-mediated exocytosis of transmitters from endocrine cells.

Glucose-6-Phosphate Dehydrogenase in the Pentose Phosphate Pathway Is Localized in Vanadocytes of the Vanadium-Rich Ascidian, Ascidia sydneiensis samea.

Ascidians are sessile marine animals known to accumulate high levels of vanadium selectively in vanadium-containing blood cells (vanadocytes). Almost all the vanadium accumulated in the vacuoles of vanadocytes is reduced to the +3 oxidation state via the +4 oxidation state, although vanadium is dissolved in the +5 oxidation state in sea water. Some of the reducing agents that participate in the reduction have been proposed. By chemical study, vanadium in the +5 oxidation state was reported to be reduced to the +4 oxidation state in the presence of NADPH. The present study revealed the existence of glucose-6-phosphodehydrogenase (G6PDH), the first enzyme to produce NADPH in the pentose phosphate pathway, in vanadocytes of a vanadium-rich ascidian. The results suggested that G6PDH conjugates the reduction of vanadium from the +5 through to the +4 oxidation state in vanadocytes of ascidians.

Finding of the same antigens in the polychaete, Pseudopotamilla occelata, as those in the vanadium-rich ascidian, Ascidia sydneiensis samea.

The polychaete Pseudopotamilla occelata is the first animal revealed to contain high levels of vanadium besides ascidians. The present experiment disclosed that P. occelata has the same antigens with those in the ascidian Ascidia syndneiensis samea, which were recognized by two types of antibodies, a polyclonal antibody against vanadium-associated proteins extracted from blood cells and a monoclonal antibody against vanadocytes in the vanadium-rich ascidian A. sydneiensis samea. There is, therefore, a possibility that similar mechanism works on the accumulation of vanadium between the Polychaeta and the Ascididae.

Identification of a vanadium-associated protein from the vanadium-rich ascidian, Ascidia sydneiensis samea.

Ascidians are known to accumulate vanadium in their blood cells (vanadocytes) at extremely high levels which correspond to about 10(6) to 10(7) times the levels of vanadium ions in seawater. The route for the accumulation of vanadium ions from the outside environment into the blood system in ascidians has not yet been discovered. In the present experiments, using a combined technique of anion exchange column and atomic absorption spectrometry, we first extracted a vanadium-associated protein (VAP) from the blood cells of the ascidian Ascidia sydneiensis samea. VAP was estimated to associate with vanadium at an approximate ratio of 1 mol:16 mole. SDS-PAGE and a polyclonal antibody against VAP (anti-VAP) revealed that VAP is composed of at least two types of peptides estimated to be 12.5 kDa and 15 kDa with a minor peptide of 18 kDa and that VAP is localized in the cytoplasm of the vanadocytes.

Subunit C of the vacuolar-type ATPase from the vanadium-rich ascidian Ascidia sydneiensis samea rescued the pH sensitivity of yeast vma5 mutants.

A vanadium-accumulating ascidian, Ascidia sydneiensis samea, expresses vacuolar-type H(+)-ATPases (V-ATPases) on the vacuole membrane of the vanadium-containing blood cells known as vanadocytes. Previously, we showed that the contents of their vacuoles are extremely acidic and that a V-ATPase-specific inhibitor, bafilomycin A(1), neutralized the contents of the vacuoles. To understand the function of V-ATPase in vanadocytes, we isolated complementary DNA encoding subunit C of V-ATPase from vanadocytes because this subunit has been known to be responsible for the assembly of V-ATPases and to regulate the ATPase activity of V-ATPases. The cloned cDNA was 1443 nucleotides in length, and encoded a putative 384 amino acid protein. By expressing the ascidian cDNA for subunit C under the control of a galactose-inducible promoter, the pH-sensitive phenotype of the corresponding vma5 mutant of a budding yeast was rescued. This result showed that the ascidian cDNA for subunit C functioned in yeast cells.

Accumulation of vanadium during embryogenesis in the vanadium-rich ascidian, Ascidia gemmata.

It is a remarkable and previously unrecognized fact that ascidians, which are known to contain high levels of vanadium in their blood cells, begin to accumulate vanadium during embryogenesis. This study revealed that the accumulation starts quite dramatically 2 wk after fertilization, and 2 mo later, the amount of vanadium in larvae is 600,000 times higher than that in the unfertilized egg. These results were obtained by neutron activation analysis, a highly sensitive method for determining levels of vanadium, in the Ascidia gemmata, the ascidian that contains the highest known levels of vanadium and accumulates vanadium at 150 mM in its blood cells, a concentration that corresponds to 4,000,000 times the concentration in seawater.

Identification of differentially expressed genes in psoriasis using expression profiling approaches.

To identify differentially expressed genes which play causal roles in pathogenesis and maintenance for psoriasis, we used BodyMapping and introduced amplified fragment length polymorphism approaches. From the BodyMap database, we selected 2007 genes which specifically expressed in epithelial tissues. Among 2007 genes, we surveyed genes which differentially expressed in involved or uninvolved psoriatic lesional skin samples compared with atopic dermatitis, mycosis fungoides, and normal skin samples. As a result of surveying 2007 genes, 241 genes were differentially expressed only in involved psoriatic skin but not in the other samples. Hierarchical cluster analysis of gene expression profiles showed that 13 independent psoriatic-involved skin samples clustered tightly together, reflecting highly similar expression profiles. Using the same 2007 gene set, we examined gene expression levels in five serial lesions from distal uninvolved psoriatic skin to involved psoriatic plaque. We identified seven genes such as alpha-1-microglobulin/bikunin precursor, calnexin, claudin 1, leucine zipper down-regulated in cancer 1, tyrosinase-related protein 1, Yes-associated protein 1, and unc-13-like protein (Coleonyx elegans) which show high-expression levels only in uninvolved psoriatic lesions. These seven genes, which were reported to be related to apoptosis or antiproliferation, might have causal roles in pathophysiology in psoriasis.

The accumulation of manganese from the environmental medium by the egg of Oryzias latipes.

The manganese content of the egg and embryo of the Medaka, Oryzias latipes was determined by activation analysis. A remarkable increase in the amount of manganese in the egg was observed within one hour after fertilization. The rate of increase was reduced by the gastrula stage and the concentration of manganese remained unchanged at a later stage. The accumulation of manganese by the Oryzias egg was discussed in relation to the effect of manganese on respiratory enzyme systems.

Purification of vanadium binding substance from the blood cells of the tunicate, Ascidia sydneiensis samea.

Vanadium binding substance has been partially purified through chromatographies on Sephadex G-25 and SE-Cellulose at pH 2.3. The binding substance was colorless, relatively stable and maintained vanadium ion. The vanadium ion in the substance existed in vanadyl form (VO(IV)). Furthermore, the substance had an apparent affinity for exogenous vanadium ion(V) and contained a reducing sugar.

EPR characterization of vanadyl (IV) species in blood cells of ascidians.

EPR spectra due to vanadyl species of blood cells of Ascidia ahodori collected from different places in Japan (Ushimado and Asamushi) were measured. The EPR spectral pattern as well as their EPR parameters for these two species were found to be different from each other. EPR analysis correlating g parallel and A parallel indicated that vanadyl ion in the animals from Ushimado was in a much stronger ligand field than those from Asamushi. Vanadyl species in the Ushimado animals was comparable to the stable complexes such as the vanadyl-ATP complex prepared at pH 12.

Vanadobin, a Vanadium-Binding Substance, Extracted from the Blood Cells of an Ascidian, Can Reduce Vanadate(V) to Vanadyl(IV).

Ascidians specifically accumulate high levels of vanadium from seawater in their blood cells. Almost all of the vanadium is present in a reduced form in the blood cells, although the metal exists in a +5 oxidation state in seawater. It has, therefore, been assumed that agents that cause the reduction of vanadate(V) to vanadyl(IV) must be present within ascidian blood cells. In this regard, we have extracted a vanadium-binding substance, which we have called vanadobin, from the vanadocytes of ascidians. We examined whether vanadobin is involved in the reduction of vanadate(V) accumulated from seawater. Data obtained by spectrophotometry and ESR spectrometry revealed that not only a crude homogenate of vanadium-rich blood cells but also a purer form of vanadobin eluted from a column of Sephadex G-15 could reduce vanadate(V). Our experiments demonstrate that vanadobin, a vanadium-binding substance extracted from ascidian blood cells, can reduce vanadate(V) to vanadyl(IV) and maintain it in the reduced form.

Immunological detection of a vacuolar-type H(+)-ATPase in vanadocytes of the ascidian Ascidia sydneiensis samea.

Ascidians belonging to the family Ascidiidae are known to accumulate vanadium from seawater in their blood cells, concentrating vanadium by a factor of 10(7). Among several different types of blood cell, the signet ring cells have both high levels of vanadium and a low pH. These observations suggest the possibility that proton ions concentrated by a H(+)-ATPase are energetically linked to the accumulation of vanadium. In the present experiments, therefore, we made an immunological search for a H(+)-ATPase in the vacuolar membranes of the signet ring cells, as a first step in our attempts to clarify the energetics of the accumulation of vanadium by these cells. Antibodies raised against the 72-kDa and 57-kDa subunits of a vacuolar-type H(+)-ATPase from bovine chromaffin granules reacted with the vacuolar membranes of signet ring cells. Immunoblotting analysis confirmed that specific antigens in ascidian blood cells actually reacted with the antibodies. Furthermore, addition of bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibited the uptake of protons by the vacuoles of signet ring cells. Thus, the addition of bafilomycin A1 inhibited the pumping function of the vacuoles of signet ring cells, with resultant neutralization of the contents of the vacuoles.

Stage sensitivity of eggs of the teleost Oryzias latipes to cadmium exposure.

The stage sensitivity of eggs of the teleost Oryzias latipes to cadmium exposure was examined. First, using eggs at the blastula stage, the proper concentration of cadmium to which the eggs should be exposed was estimated. The eggs were, therefore, exposed to cadmium solutions ranging from 10.0 to 300.0 mg Cd/liter for 1 hr, and then they were transferred to cadmium-free medium. The cumulative mortality of the eggs was estimated by counting dead eggs within 24 hr. Mortality (1.1 +/- 0.7%) indistinguishable from that of the control (1.0 +/- 0.2%) was obtained in the solution of 10.0 mg Cd/liter. Relatively higher mortalities of 13.2 +/- 1.5, 18.0 +/- 1.0, and 20.7 +/- 3.1% could be seen in the solutions containing 50.0, 100.0, and 150.0 mg Cd/liter, respectively. Moreover, the exposure of the eggs to 200.0 and 300.0 mg Cd/liter increased mortality rates to 32.0 +/- 0.8 and 34.7 +/- 2.4%, respectively. Consequently, the concentration of 200.0 mg Cd/liter was selected for examining the stage sensitivity. Eggs at 11 different developmental stages were exposed to cadmium at a concentration of 200.0 mg Cd/liter for 1 hr. The mortalities obtained at the 4- to 16-cell stage, 32-cell stage, and morula stage were 99.2 +/- 1.0, 97.4 +/- 2.6, and 89.6 +/- 10.6%, respectively. With the progress of embryonic development, the eggs became more resistant to cadmium toxicity. After the morula stage, the mortalities decreased abruptly. In order to ascertain whether the change in mortalities of the eggs with development was related to the amount of cadmium combined with the eggs, the cadmium content was determined. In contrast to the remarkable change in the stage sensitivity to cadmium, the curve of cadmium content in the eggs remained constant at about 520 ng/egg throughout the experimental period.