Effects of mepartricin (S-160) on spontaneous canine benign prostatic hyperplasia.

OBJECTIVE: The effects of mepartricin (S-160) on spontaneous canine benign prostatic hyperplasia (BPH) were investigated by histological, histochemical and biochemical analysis. METHODS: Aged beagle dogs (5-9 years old) with spontaneously developed BPH were treated orally with a placebo or S-160 (5, 10 or 20 mg/kg/day) for 8 weeks. The methodology included measurement of prostatic volume by transrectal ultrasonography, qualitative evaluation of prostatic morphology, determination of plasma and intraprostatic estradiol level by radioimmunoassay and immunohistochemical detection of estrogen receptors and androgen receptors in the prostate. RESULTS: S-160 significantly reduced the prostatic volume and regressed histologically the hyperplastic grade of prostate, and also fairly decreased the plasma and intraprostatic estradiol concentration and the estrogen and androgen receptors in the prostate. CONCLUSIONS: These results suggest that the reduction of estradiol and estrogen receptors in the prostate may play a crucial role in the regression of BPH by S-160.

Induction of manganese-superoxide dismutase in MRC-5 cells persistently infected with an alphavirus, sindbis.

Sindbis virus (SV), a single-stranded positive-sense RNA virus, multiplies in a variety of cells and causes various outcomes of infection. As we described acute infection of SV induces stress response of small heat shock protein HSP27 and activation of mitogen-activated protein kinase (MAP) signaling pathway (Nakatsue, T., et al., Biochem. Biophys. Res. Commun. 253, 59-64, 1998). In contrast to lytic infection in Vero cells, MRC-5 cells, a human fetus lung cell line, resulted in persistent infection by SV. Here we investigated a cellular factor involved in persistent infection of MRC-5 cells infected with SV. Partial sequence analysis of a 25 kilodalton (kDa) protein, accumulated in large amounts in the cells, showed that manganese-superoxide dismutase (Mn-SOD) was induced during the infections. When Mn-SOD was overexpressed in Vero cells, 20% of the cells survived more than one month, in contrast with the death of 99% of the vehicle-transfected Vero cells at 48 h after infection with SV. These data strongly suggest that a cellular factor which regulates the oxidative pathway modulates the outcome of SV infection.

Effects of mepartricin, a polyene macrolide agent, on fecal excretion and serum concentration of estrogen and number of prostatic estrogen receptors in immature rats.

BACKGROUND: Mepartricin, an antifungal agent, was investigated for effects on fecal excretion and serum concentration of sex steroids and the number of sex steroid prostatic receptors in immature rats. METHODS: Mepartricin was orally administered at 2.5, 5, and 10 mg/kg once daily for 2 weeks. Fecal estrogen and testosterone excretions, serum estrogen, testosterone and luteinizing hormone concentrations, and numbers of prostatic estrogen and androgen receptors were assayed. Prostate weight was also monitored. RESULTS: Fecal estrogen excretion showed a dose-dependent increase, which was significant for the two higher dosages. Conversely, the serum estrogen concentration and prostatic estrogen receptors were significantly decreased. No significant changes in fecal testosterone excretion, serum testosterone and luteinizing hormone concentrations, and prostatic androgen receptors were observed. Prostate weight was significantly reduced at 5 mg/kg, but we did not observe dose-dependency. CONCLUSIONS: Mepartricin increases fecal excretion of estrogen by binding with it in the intestinal tract, which results in reducing the serum estrogen concentration and number of prostatic estrogen receptors.

Ubiquitin is covalently attached to the p6Gag proteins of human immunodeficiency virus type 1 and simian immunodeficiency virus and to the p12Gag protein of Moloney murine leukemia virus.

Host proteins are incorporated into retroviral virions during assembly and budding. We have examined three retroviruses, human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and Moloney murine leukemia virus (Mo-MuLV), for the presence of ubiquitin inside each of these virions. After a protease treatment to remove exterior viral as well as contaminating cellular proteins, the proteins remaining inside the virion were analyzed. The results presented here show that all three virions incorporate ubiquitin molecules at approximately 10% of the level of Gag found in virions. In addition to free ubiquitin, covalent ubiquitin-Gag complexes were detected, isolated, and characterized from all three viruses. Our immunoblot and protein sequencing results on treated virions showed that approximately 2% of either HIV-1 or SIV p6Gag was covalently attached to a single ubiquitin molecule inside the respective virions and that approximately 2 to 5% of the p12Gag in Mo-MuLV virions was monoubiquitinated. These results show that ubiquitination of Gag is conserved among these retroviruses and occurs in the p6Gag portion of the Gag polyprotein, a region that is likely to be involved in assembly and budding.

Acute infection of Sindbis virus induces phosphorylation and intracellular translocation of small heat shock protein HSP27 and activation of p38 MAP kinase signaling pathway.

In general, viral infection is supposed to induce stress responses in the host cell. However, very few detailed observations about virus-induced stress responses have been reported. Here we investigated specific stress responses in Vero cells infected with Sindbis virus (SV), a single-stranded RNA virus, acute infection with which is known to cause apoptotic cell death in the host cells. Prior to the onset of apoptosis, p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinases (JNKs) were activated. Subsequently, a 27-kDa heat shock protein (HSP27) became phosphorylated, and intracellular distribution of HSP27 was changed from the cytoplasm to the perinuclear region. These results indicate that the cellular signaling cascades activated by pro-inflammatory cytokines and environmental stresses are also activated as a result of lytic infection with SV. These responses may contribute to the delayed onset of apoptosis in the host cells and the facilitation of viral replication.

Alterations of the cytoskeletal organization in tumor cell lines by a cardiotonic drug, vesnarinone, through protein tyrosine phosphorylation.

We describe nonspecific and moderate activation of cellular protein tyrosine phosphorylation by a chemical compound, vesnarinone, which results in enhanced synthesis and/or assembly of cytoskeletal proteins and morphological alterations in several transformed cells. In A431 cells, vesnarinone induced tyrosine phosphorylation of the overexpressed epidermal growth factor receptors (EGFR) as well as other unidentified proteins, increased the synthesis of cytokeratins, and caused amplification of the intermediate filament networks and cell flattening. The drug effects were abolished by tyrphostin, a protein tyrosine kinase inhibitor. Two other cell lines responded to the drug with increased synthesis of a cell type-specific cytoskeletal protein: vimentin in QG56 human lung carcinoma cells and alpha-tubulin in NIH3T3 cells transformed with v-src. In all cell lines tested, the drug-induced tyrosine phosphorylation was localized in cell-cell and cell-substrate contacts as detected by immunofluorescent staining. Responsive protein substrates and their sensitivity to the drug varied from one cell line to another as observed by immunoblot analysis. Vesnarinone exerted neither activating nor inhibitory effect on in vitro enzyme reactions including EGFR tyrosine kinase, v-src kinase, and protein tyrosine phosphatases. This suggests that vesnarinone indirectly activates tyrosine phosphorylation of membrane proteins related to cell adhesion, which influences a signaling pathway linked to the stress fiber assembly in certain cell lines. The possible mechanism by which vesnarinone induces the cellular responses is discussed.

How the developing septo-preoptic medical basal hypothalamus stimulates the development of placode-derived LHRH neurons.

We examined the effects of the developing cerebral cortex (CC) and septo-preoptic medial basal hypothalamus (S-MBH) on the development of LHRH neurons in vitro. The serum-free basal culture medium (BCM) was supplemented with CC or S-MBH extracts prepared from 18.5-day-old embryos or from 2-day-old newborns, and the olfactory placode (NAP) of 12-day-old embryos was cultured. The migration of LHRH neurons was found on Day 3 in the cultures supplemented with the embryonic S-MBH extract (Group 3), where the cell development proceeded showing a numerical increase of the cells and the elongation of neurites. In cultures supplemented with the newborn S-MBH extract (Group 5), the cell development was less intensive in comparison with that of Group 3, while in cultures which had no brain extracts (Group 1), the neurons failed to survive a long term culture. The effects of the CC were less than of S-MBH extracts. Analysis of the protein composition of the extracts by electrophoretic and immunoblotting examinations demonstrated a protein spot of 70-kD in the embryonic S-MBH extract. Because the protein spot was identified to be alpha-fetoprotein (AFP), we further examined the effects of AFP. When the anti-AFP immunoglobulin was added to the Group 3 culture, the stimulative effects of the embryonal extract were inhibited, and the addition of AFP to Group 1 cultures did not show stimulative effects. We conclude that the developing S-MBH, the migrating target of LHRH neurons, contains some essential factors for the development of LHRH neurons, but further analysis is needed to determine the chemical natures of these factors.

Entrapment and inhibition of human immunodeficiency virus proteinase by alpha 2-macroglobulin and structural changes in the inhibitor.

The inhibitory effect of alpha 2-macroglobulin (alpha 2M), a major plasma proteinase inhibitor, on human immunodeficiency virus (HIV) proteinase was investigated. The activity of HIV proteinase toward the Moloney murine sarcoma virus-derived gag protein (a high-molecular-mass substrate) was found to be inhibited by alpha 2M at pH 5.5-7.4. On the other hand, the activity toward the B chain of oxidized insulin (a low-molecular-mass substrate) was scarcely inhibited. The complex of alpha 2M and HIV proteinase was isolated by gel filtration and the enzyme was shown to be significantly protected by the complex formation from autoinactivation under nonreducing conditions. The stoichiometry of the complex formation was found to be 2:1 (enzyme: alpha 2M, mol/mol). These results demonstrate the entrapment and concomitant inhibition of HIV proteinase by alpha 2M.

Bovine leukemia virus RNA sequences involved in dimerization and specific gag protein binding: close relation to the packaging sites of avian, murine, and human retroviruses.

In vitro detection of a specific complex of the bovine leukemia virus (BLV) MA(p15) protein and the 5'-terminal RNA dimer led to the hypothesis that the NH2-terminal domain of retrovirus gag protein precursor is involved in the selective viral RNA packaging mechanism. Here we describe mapping of the BLV RNA for dimer-forming and MA(p15)-binding abilities by a simple cDNA probing method followed by mutation analyses with the reactive U5-5' gag RNA. The RNA dimerization is mediated by the region harboring U5, the primer binding site (PBS), and the 30 bases immediately downstream of PBS. This conclusion is supported by computer-assisted RNA secondary-structure analysis which predicted a multibranched stem-loop folding throughout the dimer region determined. Another region from PBS to the 5'-terminal 60 residues of the gag gene, partially overlapping the dimer region, likely provides essential elements for the MA(p15) binding reaction, although the presence of either the 3' or 5' neighboring sequences increases the complex-forming efficiency significantly, and each of the substructures predicted within the core region has, if any, only very weak affinity to MA(p15). These in vitro characterizations of the BLV RNA may reflect general features of the specific protein-RNA interaction in the packaging events of various retroviruses. 5'-terminal folded structures of retroviral RNA molecules and their biological activities are discussed.

The role of the Maternal and Child Health Handbook system in reducing perinatal mortality in Japan.

OBJECTIVE: The purpose of this paper is to demonstrate the direct and indirect roles of the Maternal and Child Health (MCH) Handbook in promoting overall improvement in maternal health and child care and to attempt to clarify the relationship between the use of the MCH Handbook and the reduced perinatal mortality in Japan. Another important objective is to propose possible future applications of the MCH Handbook, especially with respect to the networking function of providing client-care provider feedback, and the exchange of health data between the authorities and relevant medical societies. RESULTS: Japan has achieved a decline in neonatal mortality in the 30-year period from 1960 to 1990, from 17.0 to 2.6 per 1,000 live births. There is a correlation between the ratio of the number of Handbooks distributed and the actual number of births and the perinatal mortality. CONCLUSIONS: The wide use of the MCH Handbook system seems to have played an important role in bringing about this reduction and in maintaining the figure as one of the lowest in the world. The reduction of perinatal mortality through the use of the MCH Handbook in this country suggests a similar possibility for application in other nations. The Handbook could aid in the early recognition of high-risk pregnancy and thus reduce inappropriate use of medical resources. The system, with the establishment of a feedback system between the client and the authorities via the care provider, may improve health care in such areas as maternal mortality, toxemia of pregnancy, and diabetes mellitus.

In vitro accurate transcription from the cap site of bovine leukemia virus (BLV) dependent on the BLV-infected cell nuclear lysate.

The cell-free transcriptional system initiating from the cap site in bovine leukemia virus (BLV) LTR by RNA polymerase II was constructed. The transcription was completely dependent on the template DNA and the nuclear lysate isolated from BLV-infected bat lung cells (TB1Lu). The relative transcriptional rates estimated using several deletion mutants around the promoter sequence in BLV LTR as templates closely corresponded to that obtained by transient expression assay in cultured cells using these plasmids and tax-producing plasmid. The partial purification of the factor(s) involving to the transcriptional activation from the nuclear lysate suggested that the factor(s) was different from tax and rex, the regulatory factors encoded on viral genome. The transcription from the caps site of adenovirus E3 was also stimulated in the presence of the nuclear lysate from BLV-infected cells.

[Quality management in healthcare. Evaluation and improvement].

Quality in healthcare is a concept ultimately determined by the satisfaction of the patient, or more broadly stated, society's needs. Improvement in quality begins with the review of health care organizations, in the degree to which their current role and function can and do meet these needs. Recent trends in quality evaluation have been along the lines of patients satisfaction, as well as structure, process, and outcome oriented aspects of health care delivery. Quality entails not only the science of medicine itself, but also health care delivery, as well as social and individual concerns. In 1990, Japan Hospital Quality Assurance Society was founded. The secretariat is located at this department. Currently, more than 60 hospitals participate for the development of standards and survey to the hospitals. The quality improvement effort has slowly begun to put the concept into practice. The public's growing concern is directed toward holding healthcare organizations accountable for the services they provide. The healthcare field, in turn, is recognizing the needs and merit of voluntary commitment to quality, and are placing emphasis on identifying pressing society needs, and developing effective leadership. Moving the entire healthcare field in the direction of continuous quality improvement will be the key to the survival into the 21st century.

Bovine leukemia virus matrix-associated protein MA(p15): further processing and formation of a specific complex with the dimer of the 5'-terminal genomic RNA fragment.

The retrovirus precursor protein has an arrangement of several characteristic domains with which it achieves selective and efficient packaging of the genome RNA during particle assembly. In this study, we analyzed the composition of the bovine leukemia virus (BLV) gag proteins and examined their RNA-binding properties in gel mobility shift assays, using various genomic RNA probes synthesized in vitro. Results obtained in amino acid sequence and composition analyses indicate that the matrix-associated protein MA(p15) is further processed by the BLV protease (PR) to generate MA(p10), a short peptide of seven amino acid residues, and p4. The gag precursor is now mapped as NH2-MA(p10)-p4-CA(p24)-NC(p12)-COOH. MA(p15) formed a specific complex with the dimer RNA of the U5-5' gag region presumed to contain the BLV packaging signal but not with other RNAs. The NH2-terminal cleavage product, MA(p10), bound all RNA fragments tested, while the COOH-terminal peptides with a sequence common to mammalian type C retroviruses had little affinity for RNA. The nucleocapsid protein NC(p12) bound to RNAs nonspecifically and randomly in the presence or absence of zinc ions. These results suggest a possible interaction of the NH2 terminus of the gag precursor with the 5' terminus of the genomic RNA in an early phase of particle assembly, when the conserved structure between the MA and CA domains might be involved.

Retrovirus protease characterized as a dimeric aspartic proteinase.

Retroviruses and retroviruslike elements have a protease for specific cleavage of their polyprotein precursors. On the basis of amino acid sequences conserved among species and the sensitivity to protease inhibitors, it was proposed that the retrovirus protease could be classified as an aspartic proteinase. Since the virus protease molecule is comparable to a single domain of aspartic proteinases having two symmetrical domains, we hypothesized and examined the dimer formation of the protease. The results of biochemical molecular mass determination and cross-linking experiments demonstrated that the virus protease molecules self-assemble into dimers. An inhibitory effect of fragmented protease molecules suggests the possibility that the intermolecular association is required for their activity. Other experiments of chemical inactivation suggest a close resemblance of the catalytic features of retrovirus and aspartic proteinases. Characterizations of these bovine and avian virus proteases would provide basic knowledge for the design of retrovirus protease-specific inhibitors, which is one of the possible strategies against human immunodeficiency virus infection.

Bovine leukemia virus trans-activator p38tax activates heterologous promoters with a common sequence known as a cAMP-responsive element or the binding site of a cellular transcription factor ATF.

The genome of bovine leukemia virus (BLV) encodes a transcriptional trans-activator p38tax (also referred to as pXBL-I) which amplifies the virus gene expression driven by its long terminal repeat (LTR). It was proposed that activation of cellular gene expression by p38tax might be involved in the mechanism of B-cell transformation caused in vivo by BLV infection. Here, we report that the U3 region of BLV LTR contains multiple regulatory elements responsive to p38tax. A core element composing the p38tax-inducible U3 structure is suggested to be a heptanucleotide motif of 5'TGACGTCA3', the consensus sequence proposed for a cAMP-responsive element (CRE) and for the binding sites of a cellular transcription factor (ATF). Adenovirus-5 E3 and E4, c-fos and somatostatin regulatory regions containing CRE/ATF-element exhibited responsiveness to p38tax in a chloramphenicol acetyltransferase transient expression assay. These suggest that in BLV-infected cells, cellular gene expression might be induced abnormally by the virus trans-activator through ATF or ATF-like factors.