Dominant-negative c-Jun inhibits rat cardiac hypertrophy induced by angiotensin II and hypertension.

Cardiac activator protein-1 (AP-1), composed of c-Jun, is significantly activated by hypertension or angiotensin II (AngII). This study was undertaken to elucidate whether c-Jun could be the potential target for treatment of cardiac hypertrophy. We constructed recombinant adenovirus carrying dominant-negative mutant of c-Jun (Ad.DN-c-Jun). Using catheter-based technique of adenoviral gene transfer, we achieved global myocardial transduction of DN-c-Jun in rats, to specifically inhibit cardiac AP-1. (1) AngII (200 ng/kg/min) infusion in rats caused cardiac hypertrophy, increased cardiac p70S6 kinase activity by 1.3-fold (P<0.05) and enhanced the gene expression of cardiac hypertrophic markers. Ad.DN-c-Jun, which was transferred to the heart 2 days before AngII infusion, prevented cardiac hypertrophy (P<0.01), decreased p70S6 kinase phosphorylation (P<0.05), and suppressed cardiac gene expression of brain natriuretic peptide, collagen I, III, and IV, monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) (P<0.01). (2) In genetically hypertensive rats with cardiac hypertrophy, cardiac gene transfer of Ad.DN-c-Jun, without affecting hypertension, regressed cardiac hypertrophy (P<0.05), and suppressed p70S6 kinase phosphorylation by 20% (P<0.05) and suppressed the enhanced expression of collagen I, III, and IV, MCP-1 and PAI-1. These results provided the first evidence that in vivo blockade of cardiac c-Jun inhibits pathologic cardiac hypertrophy.

A chimeric CYP11B1/CYP11B2 gene in glucocorticoid-insuppressible familial hyperaldosteronism.

Although a chimeric gene combining the 11beta-hydroxylase gene (CYP11B1) and the aldosterone synthase gene (CYP11B2) explains the pathophysiology of familial hyperaldosteronism (FH) type I, the contribution of this abnormality to FH type II has not been tested. We screened genomic DNA from a Japanese family with FH type II for the CYP11B1/CYP11B2 gene. The index patient was a 27-year-old woman with hypertension. Hypokalaemia, elevated plasma aldosterone and suppressed plasma renin activity suggested primary aldosteronism. Though computed tomography failed to reveal an adrenal tumour, left adrenalectomy was indicated due to a high aldosterone concentration in left adrenal venous blood. The resected adrenal gland contained an adenoma. As her mother had also been diagnosed with primary aldosteronism due to an adenoma, we administered oral dexamethasone to our patient before the operation and observed the response of the blood pressure and plasma aldosterone concentration for 2 weeks. Both parameters remained elevated during the treatment period, confirming the diagnosis of FH type II. Total DNA was isolated from blood cells of the index patient, her mother, and an unaffected brother. Samples were amplified by polymerase chain reaction using specific primers from CYP11B1 and CYP11B2. Unique DNA fragments of 1.4 kb were obtained from the index patient and her mother, but not from the healthy subject. The CYP11B1/CYP11B2 chimeric gene was found in a Japanese family with FH type II.

Genetic analyses of feline foamy virus isolates from domestic and wild feline species in geographically distinct areas.

To know the genetic diversities and phylogenetic relationship among feline foamy virus (FeFV) isolates from domestic cats (Felis catus) and FeFV-related viruses from the Iriomote cats (Felis iriomotensis) and leopard cats (Felis bengalensis) in geographically distinct areas, we sequenced a partial gag-pol region of 17 strains and a partial env region of nine strains, and the U3 region of long terminal repeat of three strains of the viruses. FeFV-related viruses from the feral cats were quite similar to the FeFV from domestic cats in the sequenced regions. In the partial gag region, the identities of nucleotide sequences among the isolates were from 94 to 99%. In the partial env gene, the isolates were divided into two distinct genotypes (F17- and FUV-types) as reported by Winkler et al. (Virology 247 (1999) 144-151). More than 94% nucleotide identities were observed in the env region within a particular env genotype and about 75% nucleotide identities were noted between the two genotypes.

Diurnal variation of hemodynamic indices in non-dipper hypertensive patients.

The purpose of this study was to elucidate the underlying mechanisms of blunted nocturnal blood pressure reduction in non-dipper hypertensive patients. We studied the diurnal variations in systemic hemodynamic indices and baroreflex sensitivity. In 45 subjects with essential hypertension (24 men; mean age, 49+/-1 years), intra-arterial pressure was monitored telemetrically. Non-dippers were defined as those with a nocturnal reduction of systolic blood pressure of less than 10% of daytime systolic blood pressure. Stroke volume was determined using Wesseling's pulse contour method, calibrated with indocyanine green dilution. Baroreflex sensitivity was calculated as deltapulse interval/deltasystolic blood pressure on spontaneous variations. The mean values of the hemodynamic parameters were calculated every 30 min. Twenty-six subjects were classified as non-dippers. Daytime blood pressure was not significantly different between dippers (149+/-4/87+/-3 mmHg) and non-dippers (147+/-3/82+/-2 mmHg), while the nighttime blood pressure was significantly reduced in dippers (131+/-3/77+/-2 mmHg) but not in non-dippers (145+/-3/80+/-2 mmHg). Nocturnal decreases in both cardiac index and stroke index were smaller in non-dippers (-12.0+/-1.2% and 1.5+/-1.0%) than in dippers (-17.5+/-1.4% and -2.2+/-1.1%). Baroreflex sensitivity significantly increased at nighttime both in dippers (6.5+/-0.6 to 8.0+/-0.7 ms/mmHg) and in non-dippers (5.1+/-0.3 to 6.4+/-0.4 ms/mmHg). Neither daytime nor nighttime baroreflex sensitivity was significantly different between the groups. We conclude that the hemodynamics of non-dipper essential hypertension are characterized by an inadequate nocturnal decrease in cardiac index and stroke index, suggestive of relative volume expansion or malsuppressed sympathetic activity.

Molecular cloning of the cDNAs encoding the feline B-lymphocyte activation antigen B7-1 (CD80) and B7-2 (CD86) homologues which interact with human CTLA4-Ig.

We cloned the cDNAs encoding the feline homologues of B-lymphocyte activation antigens B7-1 (CD80) and B7-2 (CD86). We expressed recombinant feline CD80 and CD86 molecules by the baculovirus expression system, and demonstrated their binding ability to human CTLA4-murine immunoglobulin fusion protein.

Identification of the products of the equine herpesvirus type 4 gI and gE genes.

In order to identify the products of the equine herpesvirus type 4 (EHV-4) gI and gE genes, we have constructed recombinant vaccinia viruses containing the putative gI or gE genes. These recombinant viruses synthesized EHV-4 gI and gE with apparent molecular masses of 75 and 80kDa, respectively. Antibodies raised against both recombinant viruses detected a 75 kDa gI and a 95 kDa gE in EHV-4-infected cells. The results also suggest that the EHV-4 gI and gE would form a complex like in other herpesviruses.

Molecular cloning and sequencing of a cDNA encoding the feline cytotoxic T-lymphocyte-associated protein 4 (CTLA4) homologue.

The feline CTLA4 cDNA encodes a transmembrane protein which shares 87.9% sequence identity with the human CTLA4 molecule. The cytoplasmic region of the feline CTLA4 is identical to that of humans and mice, which suggests conserved function(s) such as the regulation of cell-surface expression of this molecule.

Molecular cloning and sequencing of the cDNA encoding the feline FcgammaRIIIA (CD16) homologue.

We amplified the cDNA encoding the feline FcgammaRIIIA (CD16) homologue from peripheral blood mononuclear cells by polymerase chain reaction and cloned two forms of FCGR3A cDNA. Sequencing analysis revealed that the open reading frame of feline FCGR3A cDNA consists of 750 or 747 base pairs encoding 250 or 249 amino acid residues, respectively. Comparison of the predicted amino acid sequence of feline FCGR3A cDNA with those of other mammalians' homologues revealed that the extracellular domain has a relatively low homology. However, the cytoplasmic domain contained an 8-amino acid motif, Leu-Phe-Val-Val-Asp-Thr-Gly-Leu, which was considered to interact with an accessory molecule such as the gamma chain of Fc receptors for IgE to form heterodimeric complexes.

Identification of the Marek's disease virus serotype 2 genes homologous to the glycoprotein B (UL27), ICP18.5 (UL28) and major DNA-binding protein (UL29) genes of herpes simplex virus type 1.

We determined the nucleotide sequence of non-pathogenic Marek's disease virus serotype 2 (MDV2) strain HPRS24 glycoprotein B (gB) (UL27), ICP18.5 (UL28) and major DNA-binding protein (MDBP) (UL29) genes homologous to herpes simplex virus type 1 (HSV-1). The sequence data revealed that important motives in the proteins are conserved in MDV2 ICP18.5 and MDBP, however the sequence of viral DNA replication origin which exists in the regions between the UL29 and UL30 genes of other alphaherpesviruses was not found in the regions of the MDV2 genome. By northern blot analyses, we also demonstrated that 8.9, 5.0 and 2.6 kb transcripts were actually transcribed from the sequenced region in MDV2-infected cells. The MDV2 UL28 and UL29 genes have not been reported in other serotypes of MDV.

Development of restriction fragment-length polymorphism method to differentiate five subtypes of feline immunodeficiency virus.

Feline immunodeficiency virus (FIV) isolates have been classified into five subtypes (A to E) based on the sequences of the env variable V3 to V5 region. In this study, we sequenced a partial gag region of 4 and 3 isolates belonging to subtypes C and E, respectively. Phylogenetic analysis revealed that the branching pattern based on the region was similar to that based on the env V3 to V5 region. Here, we propose a protocol to differentiate five subtypes by polymerase chain reaction amplifying 329 bp within the region followed by restriction fragment-length polymorphism analysis using four restriction enzymes.

Identification and sequence analysis of the Marek's disease virus serotype 2 homologous genes of the herpes simplex virus type 1 UL25, UL26 and UL26.5 genes.

We identified and determined the nucleotide sequence of Marek's disease virus serotype 2 (MDV2) UL25, UL26 and UL26.5 homologous genes of herpes simplex virus type 1 (HSV-1). The UL25, UL26 and UL26.5 genes of HSV-1 encode virion proteins (UL25 and UL26.5) and serine protease (UL26). The deduced amino acid sequences of the three proteins show a high degree of homology to counterparts of HSV-1. By northern blot analyses we found that four transcripts whose sizes are 4.9, 3.9, 2.0 and 1.3 kb are transcribed from the domains of MDV2 genome containing the three genes. This is the first report dealing with UL25, UL26 and UL26.5 homologues of HSV-1 in MDV serotypes.

Identification and DNA sequence analysis of the Marek's disease virus serotype 2 genes homologous to the herpes simplex virus type 1 UL20 and UL21.

We determined 3,135 bp of the nucleotide sequence located in an 8.5 kb EcoRI-E fragment in the unique long (UL) genome region of Marek's disease virus serotype 2 (MDV2), and identified UL20 and UL21 homologous genes of herpes simplex virus type 1 (HSV-1). The UL20 and UL21 homologous genes of MDV2 are arranged colinearly with the prototype sequence of HSV-1. In addition, an open reading frame (MDV2 ORF 273), which has been identified within the UL21 homologous gene of MDV2, has no apparent relation to any other known herpesvirus genes. Northern blot analysis and reverse transcriptase polymerase chain reaction confirmed the existance of RNA transcripts related to the UL20 and ORF 273 genes in MDV2-infected cells, except no transcript related to the UL21 gene being detected. The putative protein product of the MDV2 UL20 gene had a relatively low homology but that of the MDV2 UL21 gene had a moderate homology among herpesviruses. Further, the possible functions and features of the predicted proteins encoded within the sequenced region are discussed.

Identification and sequence analysis of the Marek's disease virus serotype 2 gene homologous to the herpes simplex virus type 1 UL52 protein.

The gene of Marek's disease virus serotype 2 (MDV2) homologous to the UL52 gene of herpes simplex virus type 1 (HSV-1) was identified and characterized. The MDV2 UL52 homologous gene encodes 1,071 amino acids with a molecular weight of 118.7 kDa, which includes putative metal-binding site and overlapping region with the UL53 homologous gene. Although a putative polyadenylation signal sequence was found in the downstream of the MDV2 UL52 gene, a MDV2 UL52 DNA probe reacted only with the polycistronic 6.3 kb transcript, representing the UL52 and the downstream genes of UL53 and UL54. Transcriptional pattern of this region of MDV2 was somewhat different from corresponding regions of HSV-1 and infectious laryngotracheitis virus.

Characterization of feline CD56 molecule expressed on insect cells by the baculovirus expression system.

Recently we cloned 140 kDa form of feline CD56 cDNA. In this study, we expressed the feline CD56 molecule by the baculovirus expression system. We found that the molecule was expressed on the cell surface when examined by the indirect immunofluorescence assay using an anti-human CD56 monoclonal antibody. Immunoblotting analysis revealed that the molecular weight of the major expressed product was 140 kDa. Interestingly we found that the insect cells expressing the feline CD56 molecule aggregated, indicating that the expressed molecule mediates homophilic adhesion.

Absence of plasma protease-antiprotease imbalance in the formation of saccular cerebral aneurysms.

OBJECTIVE: We examined the hypothesis that a plasma protease-antiprotease imbalance contributes to the formation of saccular cerebral aneurysms and the suggestion that the assay of these enzymes might be a screening tool for people at higher risk for aneurysm formation. METHODS: From June 1997 through May 1998, the plasma leukocyte elastase, which is an important proteolytic enzyme, and alpha-antitrypsin and alpha2-macroglobulin, which are important antiproteolytic enzyme plasma proteins, were examined in 18 patients with ruptured aneurysms, 9 patients with unruptured aneurysms, and 22 controls. RESULTS: The elastase:alpha1-antitrypsin ratio and the elastase:alpha2-macroglobulin ratios were significantly higher in patients with ruptured aneurysms within 24 hours after subarachnoid hemorrhage (SAH) than in the controls. The protease-antiprotease imbalance depended on the elevation of the elastase level, which might be correlated with leukocytosis after SAH. The elastase level decreased to the control level 3 months after the onset of SAH. No significant difference in the elastase:alpha1-antitrypsin and elastase:alpha2-macroglobulin ratios was observed between the patients with unruptured aneurysms and the controls. CONCLUSION: These results do not support the hypothesis that a plasma protease-antiprotease imbalance is a potential marker to predict the formation of saccular cerebral aneurysms. The increase in plasma elastase levels in patients with ruptured aneurysms might be attributable to leukocytosis after SAH.

Molecular cloning and sequence analysis of the phosphoprotein (P) gene of the lapinized rinderpest virus.

We determined the nucleotide sequence of the coding region for the phosphoprotein (P) gene of the L strain of rinderpest virus (RPV). The gene encodes two overlapping open reading frames of 1521 and 531 nucleotides. Use of the first ATG would produce a P polypeptide of 507 amino acids, while use of the second ATG would produce a C polypeptide of 177 amino acids. In addition, the insertion of an extra G residue at the editing site generates an alternative mRNA potentially encoding the V protein of RPV. Homology comparisons of the P, C and V proteins among various viruses suggest that RPV is closer to measles virus (MV) than to canine distemper virus (CDV). Alignment of the sequences unique to the V protein revealed that the cysteine residues are well conserved among RPV, MV and CDV, and form a "zinc finger"-like motif.