Molecular cloning and expression of human cGMP-binding cGMP-specific phosphodiesterase (PDE5)

A laboratory mutant of Escherichia coli stably resistant to more than 36,000 U ml-1 of polymyxin B was isolated. The mutant exhibited moderate increases in minimum inhibitory concentration to fluoroquinolones and bacitracin but high levels of cross-resistance to beta-lactams and aminoglycosides. However, it remained susceptible to tetracycline, nalidixic acid and novobiocin. Changes were observed in the outer membrane proteins and lipopolysaccharide profile leading to a decrease in permeability as evident from reduction in the following: (i) minimum inhibitory concentration values in the presence of Tween 80, (ii) uptake of 1-N-phenyl naphthylamine and norfloxacin, (iii) hydrolysis of beta-lactams and (iv) diffusion of lactose and cefazolin into proteoliposomes reconstituted with outer membrane proteins. We therefore suggest that the novel pattern of cross-resistance of our isolate is due to the decrease in its permeability.     

Isolation and characterization of cDNAs encoding PDE5A, a human cGMP-binding, cGMP-specific 3'',5''-cyclic nucleotide phosphodiesterase

An attempt has been made to suppress the ethanol-induced formation of megamitochondria (MG) in the rat liver by 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-OH-TEMPO), a free radical scavenger, and by allopurinol (AP), a xanthine oxidase inhibitor. Changes observed in the liver of animals given ethanol (EtOH) for 1 month were remarkable decreases both in the body weight gains during the course of the experiment and in the liver weight at the time of sacrifice compared to those of the control; remarkable increases in the level of thiobarbituric acid reactive substances and lipid soluble fluorophores both in microsomes and mitochondria; decreases in the content of cytochrome a+a3 and b and lowered phosphorylating ability of mitochondria; and formation of MG in the liver. A combined treatment of animals with EtOH plus 4-OH-TEMPO completely suppressed the formation of MG in the liver induced by EtOH and distinctly improved the changes caused by EtOH, as specified above, while AP partly suppressed the MG formation. Results described herein provide additional insight into chronic hepatotoxicity of EtOH besides that previously reported. A novelty of the present work is that we were able for the first time to demonstrate reversibility of EtOH-mediated ultrastructural changes of the liver by a simple administration of aminoxyl-type free radical scavenger, 4-OH-TEMPO. Our results suggest that free radicals may be involved in the mechanism of the formation of MG induced by EtOH.     

Isolation and characterization of PDE9A, a novel human cGMP-specific phosphodiesterase

We have cloned and characterized the first human isozyme in a new family of cyclic nucleotide phosphodiesterases, PDE9A. By sequence homology in the catalytic domain, PDE9A is almost equidistant from all eight known mammalian PDE families but is most similar to PDE8A (34% amino acid identity) and least like PDE5A (28% amino acid identity). We report the cloning of human cDNA encoding a full-length protein of 593 amino acids, including a 261-amino acid region located near the C terminus that is homologous to the approximately 270-amino acid catalytic domain of other PDEs. PDE9A is expressed in all eight tissues examined as a approximately 2. 0-kilobase mRNA, with highest levels in spleen, small intestine, and brain. The full-length PDE9A was expressed in baculovirus fused to an N-terminal 9-amino acid FLAG tag. Kinetic analysis of the baculovirus-expressed enzyme shows it to be a very high affinity cGMP-specific PDE with a Km of 170 nM for cGMP and 230 microM for cAMP. The Km for cGMP makes PDE9A one of the highest affinity PDEs known. The Vmax for cGMP (4.9 nmol/min/microg recombinant enzyme) is about twice as fast as that of PDE4 for cAMP. The enzyme is about twice as active in vitro in 1-10 mM Mn2+ than in the same concentration of Mg2+ or Ca2+. PDE9A is insensitive (up to 100 microM) to a variety of PDE inhibitors including rolipram, vinpocetine, SKF-94120, dipyridamole, and 3-isobutyl-1-methyl-xanthine but is inhibited (IC50 = 35 microM) by zaprinast, a PDE5 inhibitor. PDE9A lacks a region homologous to the allosteric cGMP-binding regulatory regions found in the cGMP-binding PDEs: PDE2, PDE5, and PDE6.     

Characterization of human and mouse rod cGMP phosphodiesterase delta subunit (PDE6D) and chromosomal localization of the human gene

Unmethylated bacterial DNA containing a high frequency of the CpG motif, is mitogenic and induces T-cell independent, murine B-cell proliferation. These stimulatory effects are also induced by synthetic oligonucleotides that contain one or more unmethylated CpG dinucleotides (CpG oligo). Such mitogenicity is not seen with highly methylated vertebrate DNA, which has a lower prevalence of the CpG motif than bacterial DNA. Due to their stimulatory effects, CpG oligo have been proposed for use as vaccine adjuvants. In order to determine if a synthetic CpG oligo that was stimulatory for B-cell proliferation could augment the murine antibody response to protective bacterial polysaccharide epitopes (Pseudomonas aeruginosa LPS-O polysaccharide side chain; high-molecular-weight polysaccharide or high-MW PS), BALB/c mice were injected with mitogenic doses of CpG oligo simultaneously with high-MW PS, and antibody titers were measured by ELISA weekly for 4 weeks. Controls received PBS, a nonstimulatory control oligo plus PS, CpG alone, or PS alone. Despite evidence of B-cell mitogenicity and an increase in total IgM in CpG oligo-treated mice, CpG oligo treatment plus PS significantly decreased the high-MW PS antibody response compared to PS alone. The blunting of the anti-PS antibody response could be eliminated by vaccinating the animals with PS prior to CpG oligo. We conclude that despite in vitro and in vivo evidence of B-cell proliferation, this CpG oligo reduces PS-specific antibody responses in an animal model when given simultaneously with a bacterial polysaccharide. Based on results in this model, oligonucleotides containing stimulatory unmethylated CpG dinucleotides may not be useful adjuvants when given simultaneously with bacterial PS vaccines.     

Potential roles of conserved amino acids in the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase

The known mammalian 3':5'-cyclic nucleotide phosphodiesterases (PDEs) contain a conserved region located toward the carboxyl terminus, which constitutes a catalytic domain. To identify amino acids that are important for catalysis, we introduced substitutions at 23 conserved residues within the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (cGB-PDE; PDE5). Wild-type and mutant proteins were compared with respect to Km for cGMP, kcat, and IC50 for zaprinast. The most dramatic decrease in kcat was seen with H643A and D754A mutants with the decrease in free energy of binding (DeltaDeltaGT) being about 4.5 kcal/mol for each, which is within the range predicted for loss of a hydrogen bond involving a charged residue. His643 and Asp754 are conserved in all known PDEs and are strong candidates to be directly involved in catalysis. Substitutions of His603, His607, His647, Glu672, and Asp714 also produced marked changes in kcat, and these residues are likely to be important for efficient catalysis. The Y602A and E775A mutants exhibited the most dramatic increases in Km for cGMP, with calculated DeltaDeltaGT of 2.9 and 2.8 kcal/mol, respectively, that these two residues are important for cGMP binding in the catalytic site. Zaprinast is a potent competitive inhibitor of cGB-PDE, but the key residues for its binding differ significantly from those that bind cGMP.     

Genomic structure and chromosomal localization of the human interleukin 15 gene (IL-15)

The morphologic and functional characteristics of cultured hair follicle dermal papilla (DP) cells, dermal sheath (DS) cells and interstitial dermal fibroblasts (DF cells) derived from human scalp tissue are compared. DP and DS cells, but not DF cells, showed aggregative behavior at a preconfluent density. All three types of cells stained positive for type I collagen, type IV collagen, laminin and heparan sulfate proteoglycan. Only DP and DS cells expressed smooth muscle alpha-actin. DP and DS cells also synthesized more glycosaminoglycans (GAG) than DF cells, while there was no significant difference between DP and DS cells in GAG synthesis. Ultrastructurally, 7 out of 10 strains of DP and 2 out of 10 strains of DS cells were found to form intranuclear rodlets, while none of the 10 strains of DF cells examined formed intranuclear rodlets. The conditioned medium of the three types of cells was collected and tested for the presence of interleukin (IL)-1 beta, tumor necrosis factor (TGF)-beta 2, IL-6, platelet-derived growth factor-AB, epidermal growth factor, b-FGF, GM-CSF, insulin-like growth factor (IGF)-1 and HGF (hepatocyte growth factor) by ELISA or RIA. Among the tested cytokines and growth factors, TGF-beta 2, IL-6 and IGF-I were detectable in at least some conditioned media. The others were undetectable. There was no significant difference in the production of IL-6 and IGF-I among the three types of cells. In contrast, DP cells produced the highest levels of TGF-beta 2, DS cells produced intermediate levels of TGF-beta 2, and DF cells produced the lowest levels of TGF-beta 2. DP and DS cells are morphologically and functionally different from the nonfollicular, interstitial DF cells. Moreover, the presence of some minor biologic differences between DP and DS cells suggests that they represent follicular mesenchymal cells in different functional or differentiation states.     

Gene and locus structure and chromosomal localization of the protease-activated receptor gene family

Protease-activate receptors (PARs) mediate activation of platelets and other cells by thrombin and other proteases. Such protease-triggered signaling events are thought to be critical for hemostasis, thrombosis, and other normal and pathological processes. We report here the structure of the mouse and human PAR3 genes as well as the organization of a PAR gene cluster encompassing the genes encoding PARs 1, 2, and 3. We also report the structure of the mouse and human PAR4 genes, which map to distinct chromosomal locations and encode a new thrombin receptor. PARs 1-4 are all encoded by genes with the same two exon structure. In each case, exon 1 encodes a signal peptide, and exon 2 encodes the mature receptor protein. These are separated by an intron of variable size. The genes encoding PARs 1-3 all map to chromosome 13D2 in mouse and chromosome 5q13 in human. In mouse, all three genes are located within 80 kilobases of each other. The PAR1 gene is located centrally and is flanked upstream by the PAR3 gene and downstream by the PAR2 gene in both species. The proximity of the PAR1 and PAR3 genes suggests the possibility that these genes might share regulatory elements. A comparison of the structures of the PAR amino acid sequences, gene structures, locus organization, and chromosomal locations suggests a working model for PAR gene evolution.     

Genomic structure and chromosomal localization of processed pseudogenes for human RBP-Jk

The functional gene for human recombination signal sequence-binding protein (RBP-Jk) and corresponding processed psudogenes have been isolated from various species, such as Drosophila, Xenopus, mouse, and human. Here we report the isolation of another two genomic pseudogenes of human RBP-Jk, named K2 and K7, from a cosmid library of Hela cells. The nucleotide sequences of both genes exhibited more than 95% homology to the functional human gene for RBP-Jk. Moreover, they did not contain any intron sequences and were interrupted by several stop codons in all frames. In situ hybridization demonstrated that the pseudogenes, K2 and K7, were localized at chromosomes 9p13 and 9q13, respectively. Their physical maps differed from those of the true functional gene and of the pseudogenes reported previously by Amakawa et al. (1993).     

Characterization of gene expression, genomic structure, and chromosomal localization of Hells (Lsh)

We studied the development of multibank rod retinae by monitoring the size-related addition of new layers of rod inner and outer segments in four species of deep-sea fishes and found two different growth paradigms. In the mesopelagic Chauliodus sloani, new banks of rod inner and outer segments are added as long as the fish increases in size, as observed earlier by Locket (1980). By contrast, in three bathybenthic species (Antimora rostrata, Corvphaenoides (Coryphaenoides) guentheri, and Coryphaenoides (Nematonurus) armatus), the final complement of banks is reached when the specimens have grown to between 20 and 47% of their maximal size, suggesting that the visual system is mature only after this stage. Increase in retinal area, density of rod nuclei, and densities of rod inner and outer segments were also studied in these and additional species. Taken together with previous data on rod proliferation patterns and outer segment membrane synthesis, our findings indicate that at least in species with no continual addition of new banks, there is no major functional difference between the innermost and outermost banks of rod inner and outer segments. While Chauliodus spends all its life in the mesopelagic environment, the three bathybenthic species live in this environment during early development and descend towards greater depths only upon maturation. We speculate that this coincides with the stage when the full complement of rod banks is formed in the retina, as a possible prerequisite for a life outside the reach of sunlight.     

Dihydropyrimidinase deficiency: structural organization, chromosomal localization, and mutation analysis of the human dihydropyrimidinase gene

Dihydropyrimidinase (DHP) deficiency (MIM 222748) is characterized by dihydropyrimidinuria and is associated with a variable clinical phenotype. This disease might be associated with a risk of 5-fluorouracil toxicity, although no cases have been reported. We present here both the molecular characterization of the human DHP gene and, for the first time, the mutations causing DHP deficiency. The human DHP gene spans >80 kb and consists of 10 exons. It has been assigned to 8q22, by FISH. We performed mutation analysis of genomic DNA in one symptomatic and five asymptomatic individuals presenting with dihydropyrimidinuria. We identified one frameshift mutation and five missense mutations. Two related Japanese adult subjects were homozygous for the Q334R substitution, whereas two other, unrelated Japanese infant subjects were heterozygous for the same mutation, but this mutation is not common in the Japanese population. A Caucasian pediatric patient exhibiting epileptic attacks, dysmorphic features, and severe developmental delay was homozygous for W360R. Using a eukaryotic expression system, we showed that all mutations reduced enzyme activity significantly, indicating that these are crucial DHP deficiency-causing mutations. There was no significant difference, in residual activity, between mutations observed in the symptomatic and those observed in the asymptomatic individuals.     

Structural organization and chromosomal localization of the human ribosomal protein L9 gene

Monoclonal antibodies (MoAbs) raised against Trypanosoma cruzi microsomal fraction (Mc) and cross-reactive with mammalian tissues were used to evaluate the ability of cross-reactive T. cruzi antigens to induce an immune response in Chagas' disease. Thus, we studied the ability of sera from Chagas' disease patients (CDP) with different degrees of cardiac dysfunction to block the immune recognition of these MoAb to the target antigen determining for each serum an inhibition index (II). By means of this approach we inferred that blocking of monoclonal antibody binding to T. cruzi microsomes by subjects' serum represents antibodies with the same reactivity. After serological and medical examinations, individuals were separated into the following groups: Chagas' disease patients without manifest cardiac involvement (CDP-0), CDP with suspected or borderline cardiac disease (CDP-1), CDP with moderate myocardial dysfunction (CDP-2), CDP with overt cardiac dysfunction (CDP-3) and controls including healthy subjects (HS) and patients with idiopathic myocarditis (IMP). The reactivity between MoAb 5F2 and its target antigen was significantly (p < 0.05) inhibited by sera from CDP irrespective of the clinical stage [CDP: n = 46, 50 +/- 20, mean II +/- SD: control: n = 16, 18 +/- 8]. Moreover, 5F2 was able to distinguish (p < 0.05) sera from CDP with mild disease (CDP clinical grade 0/1: n = 26, 34 +/- 18) from that of CDP with severe disease (CDP clinical grade 2/3: n = 20, 67 +/- 7). Moreover, the inhibitory capacity of sera from asymptomatic CDP (CDP-0) correlated with patients age (r = 0.66, p < 0.05). CDP-0 below or equal 40 years of age had results (n = 15, 25 +/- 13) comparable (p > 0.05) to that of controls while mean inhibition of CDP-0 over 40 years of age (n = 5, 60 +/- 5) was indistinguishable (p > 0.05) from that of patients with severe disease. Competitive assay with MoAb 5A9B11 also showed significant differences (p < 0.05) between sera from CDP (n = 46, 46 +/- 24) and controls (n = 13, 5 +/- 5). On the contrary, the differences observed between CDP with different cardiac involvement was not significant (mild: n = 26, 31 +/- 22; severe: n = 20, 66 +/- 11). However a thorough study of data from asymptomatic sera revealed the existence of two levels of reactivity, with low and high capacity to inhibit the reaction of 5A9B11 against Mc. On the contrary, CDP sera showed a blocking activity for 1A10C11 comparable to that of controls (CDP: n = 25, 19 +/- 9; control: n = 12, 14 +/- 6). Some cross-reactive MoAbs recognized epitopes partially composed of carbohydrates. Interestingly, 5F2 and 5A9B11 epitopes did not appear to have carbohydrates moieties. In summary, immunoinhibition assays revealed differences in the immune response of chronic chagasic patients against parasite epitopes. These results have opened the possibility to identify a prognosis marker of the disease suggesting the clinical utility of monitoring levels of these anti-Mc antibodies in patients with chronic Chagas' disease.     

Genomic structure and chromosomal localization of the murine LIM class homeobox gene Lhx1

Catecholamine-induced cardiomyopathy is a rare complication of pheochromocytoma. We present a case of pheochromocytoma that developed preoperative heart failure. Left ventricular dilation and severe hypokinesia were demonstrated by echocardiography. Heart failure was successfully treated with digitalis, diuretics and captopril. There were no surgical complications and the follow up showed and improvement on the systolic function evaluated by echocardiography and isotope ventriculography, 3 and 6 months after surgery. We review the pathophysiology and evolution of catecholamine induced cardiomyopathy. Preload reserve can be one of the adaptive mechanisms of the ventricle in catecholamine-induced cardiomyopathy. Conventional therapy of hypertension and heart failure can be effective to correct the symptoms of cardiac dysfunction.     

cDNA cloning, expression, mutagenesis, intracellular localization, and gene chromosomal assignment of mouse 5-lipoxygenase

5-Lipoxygenase of mouse macrophages and bone marrow-derived mast cells (BMMC) was investigated. Indirect immunocytofluorescence combined with confocal microscopy provided evidence for distinct intracellular expression patterns and trafficking of 5-lipoxygenase upon cellular activation. In resting BMMC, 5-lipoxygenase was found within the nucleus co-localizing with the nuclear stain Yo-Pro-1. When BMMC were IgE/antigen-activated the 5-lipoxygenase immunofluorescence pattern was changed from nuclear to perinuclear. The absence of divalent cations in the incubation medium, or calcium ionophore A23187 challenge, altered the predominantly nuclear expression pattern to new sites both cytosolic and intranuclear. The cDNA for murine macrophage 5-lipoxygenase was cloned by the polymerase chain reaction and would predict a 674 amino acid protein. Using control cells obtained from 5-lipoxygenase-deficient mice it was determined that a single isoform accounts for both soluble and membrane-bound and nuclear and cytosolic-localized enzyme in macrophages and BMMC. A mutation at amino acid 672 (Val-->Met) introduced serendipitously during the cloning process was found to completely abolish 5-lipoxygenase enzyme activity when the enzyme was expressed in human embryonic kidney 293 cells. This subtle change is proposed to affect the ability of the COOH-terminal isoleucine to coordinate the essential non-heme iron atom. In macrophages and BMMC obtained from 5-lipoxygenase-deficient mice, compensatory changes in expression of genes involved in the biosynthesis of leukotriene B4 were investigated. 5-Lipoxygenase-activating protein expression was reduced by 50%, while leukotriene A4 hydrolase expression was unaltered. The 5-lipoxygenase gene was mapped to the central region of mouse chromosome 6 in a region that shares homology with human chromosome 10 by interspecific backcross analysis. These studies provide a global picture of the murine 5-lipoxygenase system and raise questions about the role of 5-lipoxygenase and leukotrienes within the nucleus.     

Identification and characterisation of a human calmodulin-stimulated phosphodiesterase PDE1B1

A cDNA encoding a calmodulin-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE) was isolated from a human brain cDNA library. The cDNA, designated HSPDE1B1, encoded a protein of 536 amino acids that shared 96% sequence identity with the bovine "63 kDa" calmodulin-stimulated PDE. The recombinant protein had cyclic nucleotide phosphodiesterase activity that was stimulated approximately 2-fold by Ca2+/calmodulin and preferred cGMP as substrate. In addition, the enzymatic activity of HSPDE1B1 was inhibited by phosphodiesterase inhibitors with potencies similar to that displayed toward the bovine PDE1 enzymes: IBMX approximately equal to 8-methoxymethyl-IBMX > vinpocetine approximately equal to zaprinast > cilostamide > rolipram. HSPDE1B1 mRNA was found predominantly in the brain. Lower mRNA levels were found in heart and skeletal muscle. In situ hybridisation of brain revealed expression of HSPDE1B1 predominately in neuronal cells of the cerebellum, hippocampus and caudate. The HSPDE1B1 gene was mapped to human chromosome 12. A partial genomic sequence of HSPDE1B1 was isolated and shown to contain two splice junctions that are conserved in the rat PDE4 and the Drosophila dunce genes.     

Cloning, tissue expression, and chromosomal localization of the mouse IRS-3 gene

Insulin receptor substrate (IRS) proteins are key regulators of basic functions such as cellular growth and metabolism. They provide an interface between multiple receptors and a complex network of intracellular signaling molecules. Two members of this family (IRS-1 and IRS-2) have been identified previously. In this investigation, we analyzed a mouse expressed sequence tag clone that proved to be a new member of the IRS family. Sequence analysis of this clone and comparison with the sequences deposited in GenBank demonstrates this protein may be the murine homolog of rat IRS-3, recently purified and cloned from rat adipocytes. Accordingly, we have named our protein mouse IRS-3. The expressed sequence tag clone contains the complete coding sequence of 1485 bp, encoding a protein of 495 amino acids. Sequence alignment with the other members of the IRS family shows that this protein contains pleckstrin homology and phosphotyrosine-binding domains that are highly conserved. In addition, there is conservation of many tyrosine phosphorylation motifs responsible for interactions with downstream signaling molecules containing SH2 domains. The murine IRS-3 messenger RNA (2.4 kilobases in length) is expressed in many tissues, with highest levels in liver and lung. Mouse IRS-3 is highly expressed in the first part of the embryonic life, when IRS-1 messenger RNA is barely detectable. Unlike the genes encoding IRS-1 and IRS-2, the IRS-3 gene contains an intron (344 bp in length) in the region between the pleckstrin homology and the phosphotyrosine-binding domains. Fluorescent in situ hybridization localized the mouse IRS-3 gene on the telomeric region of chromosome 5G2. Cloning of the murine IRS-3 gene will make it possible to apply genetic approaches to elucidate the physiological role of this new member of the IRS family of proteins.     

Isolation and chromosomal localization of GPR31, a human gene encoding a putative G protein-coupled receptor

The screening of a human genomic library with a chemokine receptor-like probe allowed us to obtain a putative member of the G protein-coupled receptor gene (GPCR) family, designated GPR31. Its deduced amino acid sequence encodes a polypeptide of 319 amino acids that shares 25-33% homology with members of the chemokine, purino, and somatostatin receptor gene families. Amino acid sequence comparison reveals that the best match in the protein databases is with the human orphan GPCR called HM74 (33% identity). Southern genomic analysis of the GPR31 gene shows a hybridization pattern consistent with that of a single-copy gene. Using fluorescence in situ hybridization, we have determined the chromosomal and regional localization of the GPR31 gene at 6q27. The GPR31 mRNA is expressed at low levels by several human cell lines of different cellular origins. The phylogenetic analysis suggests that the GPR31 receptor may represent a member of a new GPCR subfamily.