Fugu (Takifugu rubripes) sexual differentiation: CYP19 regulation and aromatase inhibitor induced testicular development.

In order to assess the involvement of aromatase CYP19 isoforms and endogenous sex steroids in gonadal sex differentiation and development of the Japanese fugu (Takifugu rubripes), an aromatase inhibitor (AI, fadrozole) was administered to developing fishes from the 'first feeding' till the 100th day after hatching. It was observed that ovarian cavity formation was inhibited by fadrozole at doses of 500 and 1000 microg/g diet, which was followed by testicular differentiation in all treated fugu. In the non-treated fugu, CYP19A was predominantly expressed in the ovary and CYP19B in the brain (in both sexes), although both were expressed interchangeably at low levels. An exceptionally high expression of CYP19B was also evident in testis throughout the study period. Both forms of CYP19 mRNA showed low levels of expression in brain and gonad with no significant differences between the two AI treatments. AI treatment inhibited CYP19A mRNA in trunk during the crucial period of ovarian cavity formation and CYP19B in gonad and brain by the end of gonadal sex differentiation. An elevation of testosterone and 11-ketotestosterone was observed which can be associated with the down-regulation of the circulating 17beta-estradiol production during the AI treatment period. After stopping AI treatment, both circulating estrogen and androgen were normalized. The current results suggest that suppression of CYP19A before and during morphological sex differentiation inhibits ovarian cavity formation in fugu. Furthermore, non-detectable limits of 17beta-estradiol and high testosterone levels by the end of the gonadal differentiation period can be ascribed to inhibition of CYP19B, suggesting that conversion of 17beta-estradiol from testosterone is plausibly regulated by CYP19B, and that this factor (CYP19B) may play an important role in AI-induced testicular development after gonadal sex differentiation through regulation of the testosterone-17beta-estradiol balance in fugu.

Hydrometallurgical recovery of zinc from ashes of automobile tire wastes.

Study has been performed on the investigation of metal leaching behavior for fly and bottom ashes from automobile tire wastes using acid and alkaline solutions from both viewpoints of environmental protection and resource utilization. The two ashes were found to contain substantial amounts of zinc and iron along with small quantities of cobalt, manganese, magnesium, copper, titanium and aluminum. The fly ash contained a much larger amount of zinc than the bottom ash, and seems to be a promising secondary source for the metal. Effects of such experimental parameters as temperature, time and solid-liquid ratio on the leaching behavior were investigated. Using three mineral acids and citric acid, selective leaching of zinc was successfully attained; the concentration of zinc in the leach liquors from the fly ash reached as high as 20 g l(-1) while the iron leaching was much suppressed. Selective separation of zinc was also attained in the leaching with alkaline solutions, though the percent leaching was lower than that in the acid leaching. Moreover, solvent extraction and precipitation were applied to the metal-loaded leach liquors as downstream processing to evaluate the feasibility of zinc recovery.

Enhancement of phase separation using a drop coalescer in an aqueous two-phase system.

The effect of a drop coalescer on phase separation in a PEG/salt aqueous two-phase system (ATPS) in the absence and presence of protein has been investigated. Raschig rings of ceramic, PTFE and glass were used as a drop coalescer in order to separate the mixture into two phases. Among the three materials PTFE is the most effective in coalescing the dispersed drops, with the throughput with PTFE being twice that without the coalescer. Random packing gives good results for phase separation. Two types of fiber mesh coated with PTFE were also used as drop coalescers, one in a spirally folded form and the other in a three-dimensional lattice-form. Throughput in the PEG/salt system with the three-dimensional lattice-form is 1.2 times as high as that with the spirally folded form. Throughput with the coalescer formed by compiling PTFE Raschig rings and fiber mesh in lattice form is 1.6 and 1.2 times as high as the case of separate use of the fiber mesh and the PTFE Raschig rings, respectively. The hydrophobic surface of PTFE in the compiled coalescer has no significant effect on the recovery fraction of the protein in ATPS.

Budding of fowlpox and pigeonpox viruses at the surface of infected cells.

Chick embryo fibroblasts and chorioallantoic membranes infected with fowlpox virus (FWPV) or pigeonpox virus (PPV) were examined by transmission and scanning electron microscopy. Immature virus particles were observed in finely granular areas, i.e. virus factories, of the cytoplasm. These particles had various forms depending on their stages of development. Many tubular structures were also seen in these regions. Mature virus particles with ellipsoidal or brick-shaped forms enclosing electron-dense cores were detected throughout the cytoplasm. Notably, there was a high frequency of virus budding at the cell surface, but only occasional virus wrapping in the cytoplasm. Another remarkable feature of the infected cells was accumulation of many virions just beneath the plasma membrane, indicating that this phenomenon is closely related to virus budding. Based on the observed frequency of budding, this mechanism seems to be the predominant way for FWPV and PPV to exit the cell.

Titanocene-catalyzed carbosilylation of alkenes and dienes using alkyl halides and chlorosilanes

A new method for regioselective carbosilylation of alkenes and dienes has been developed by the use of a titanocene catalyst. This reaction proceeds efficiently at 0 degrees C in THF in the presence of Grignard reagents by the combined use of alkyl halides (R'-X, X = Br or Cl) and chlorotrialkylsilanes (R3''Si-Cl) as the alkylating and silylating reagents, respectively. Terminal alkenes having aryl or silyl substituents (YRC=CH2, Y = Ar or Me3Si, R = H or Me) afford addition products YRC-(SiR''3)-CH2R' in good yields, whereas 1-octene and internal alkenes were sluggish. When 2,3-disubstituted 1,3-butadienes were used instead of alkenes, alkyl and silyl units are introduced at the 1- and 4-positions giving rise to allylsilanes in high yields under similar conditions. The present reaction involves (i) addition of alkyl radicals toward alkenes or dienes, and (ii) electrophilic trapping of benzyl- or allylmagnesium halides with chlorosilanes. The titanocene catalyst plays important roles in generation of these active species, i.e., alkyl radicals and benzyl- or allylmagnesium halides.

Monitoring of human herpesvirus-6 and -7 genomes in saliva samples of healthy adults by competitive quantitative PCR.

Human herpesviruses-6 and -7 (HHV-6 and HHV-7) are thought to be transmitted during early infancy through saliva. However, the kinetics of the virus shedding in saliva of healthy adults, from whom children are assumed to acquire the viruses, is mostly unknown. This study was conducted to determine how many copies of the genome are secreted in saliva of healthy adults and to clarify the relationship between viral DNA load and virus isolation of HHV-6 and HHV-7. Competitive PCR was performed using primer sets in the U42 gene of each viral genome. In saliva samples from 29 healthy adults, HHV-6 and HHV-7 DNA was detected in 41.4% and 89.7%, respectively. The average copy number of the HHV-7 genome in the positive samples was higher than that of the HHV-6 genome. Follow-up studies of six seropositive individuals for 3 months showed that the amount of HHV-7 DNA was constant in each individual and that "high producers" and "low producers" could be distinguished. By contrast, the amount of HHV-6 DNA varied drastically over time in each individual. Although HHV-6 was never isolated from the saliva of any of the six individuals during the follow-up period, HHV-7 was isolated from each individual several times. The amount of HHV-7 DNA tended to be higher at the times when the virus was isolated than at the times when the virus was not isolated. These data demonstrate a striking contrast between HHV-6 and HHV-7 in the kinetics of genome and virus shedding.

[Inhibition of corneal neovascularization--the possibility of inhibition by drug administration].

PURPOSE: The anti-angiogenic activity of FR 118487, a new synthetic analog of Scolecobasidium arenarium products, was examined in Japanese white rabbit cornea. METHODS: We studied both systemic and locally administered FR 118487 (ointment) in a keratoplasty model consisting of corneal neovascularization after implantation of a Wister rat cornea into a rabbit cornea. RESULTS: Two weeks after the implantation, the maximum length of neovascularization was 3.4 +/- 0.3 mm in control corneas, 0.1 +/- 0.0 mm with systemic FR 118487 administration (10 mg/day) (p < 0.01), 0.1 +/- 0.1 mm with 10% FR118487 ointment (p < 0.001), 1.0 +/- 0.2 mm with 3% FR 118487 ointment (p < 0.001), and 0.9 +/- 0.9 mm with 1% FR 118487 ointment (p < 0.02). CONCLUSION: FR 118487 had a significant effect on inhibition of corneal neovascularization.

Poxvirus virions: their surface ultrastructure and interaction with the surface membrane of host cells.

Virions of vaccinia and orf viruses were examined by ultrahigh-resolution scanning electron microscopy using a non-coating method. Intracellular mature particles of vaccinia virus appeared to be covered with a net and ultrastructurally their surface consists of many fine ridges and globules, while the surfaces of orf virus mature particles recovered from infected cells consist of spirally running protrusions. The ridge-like structures of vaccinia virus were presumed to correspond to surface tubules shown by negative staining of this virus, while the spiral protrusions of orf virus were presumed to correspond to spiral threads having a criss-cross appearance by the same staining. Using scanning electron microscopy in which the samples were prepared by the conventional method, we observed: (i) many virions, i.e. one or two hundreds, or occasionally more reaching about one thousand particles, of the IHD strain of vaccinia virus, (ii) many or a moderate number of virions, i.e. about one hundred or fewer particles, of the 58 strain of cowpox virus and (iii) rather few virions, i.e. several tens or fewer particles, of the Iwate strain of orf virus on the free surface of each cell infected with these viruses. It must be noted that the number of virions detected considerably differed in respective cells examined. Virus budding was frequently observed at the cell surface of monolayer cells infected with vaccinia virus but it was never detected with cowpox or orf virus, indicating a difference in the mechanism of virus release between vaccinia and the other two viruses. When whole cells infected with vaccinia virus were examined by a combination of high-voltage and scanning electron microscopies, virions on the cell surface and those inside the cells were clearly differentiated. All virions on the cell surface had an envelope, and some of the envelopes had a slack and/or one or more bulges.

Comparison of antiviral compounds against human herpesvirus 6 and 7.

Four classes of antiviral compounds were evaluated for inhibitory activity against two variants of human herpesvirus 6 (HHV-6A and -6B) and human herpesvirus 7 (HHV-7). These included: (1) a pyrophosphate analog, phosphonoformic acid (PFA); (2) beta-guanine analogs, 9-(2-hydroxyethoxymethyl)guanine (acyclovir or ACV), 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (ganciclovir or GCV) and 9-(4-hydroxy-3-hydroxy-3-hydroxymethylbutylyl)guanine (penciclovir or PCV); (3) acyclic nucleoside phosphonates, (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine [cidofovir or (S)-HPMPC] and its cyclic derivative (S)-cyclic-HPMPC (cHPMPC), 9-[[2-hydroxy-1-phosphonomethoxy)ethoxy]methyl]guanine (HPMEMG) and 9-[(2-phosphonylmethoxy)ethyl]-2,6-diaminopurine (PMEDAP), and the seven other related compounds; and (4) a series of benzimidazole ribonucleosides, including 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BDCRB). End-point inhibitory concentration (EPC) and 50% effective inhibitory concentration (EC50) values were determined by a dot-blot antigen detection method in cord blood mononuclear cells infected with HHV-6A, HHV-6B or HHV-7 at a multiplicity of infection of 0.004 CCID50/cell. (S)-HPMPC and cHPMPC had an EC50 value of approximately 0.3 microg/ml for HHV-6A, 1.2 microg/ml for HHV-6B and 3.0 microg/ml for HHV-7. These compounds were the most active of those tested against each virus. The EC50 value of GCV for HHV-6A was 0.65 microg/ml, 1.33 microg/ml for HHV-6B, and >7 microg/ml for HHV-7. The EC50 values of ACV and PCV were approximately 6-8 microg/ml for HHV-6A, 16-24 microg/ml for HHV-6B and 121-128 microg/ml for HHV-7. These drugs were the least active. The sensitivity of HHV-7 to the guanine analogs was different from HHV-6, suggesting a difference in selectivity of specific viral enzymes.

Development of a dot blot neutralizing assay for HHV-6 and HHV-7 using specific monoclonal antibodies.

To elucidate further immune responses to human herpesviruses 6 and 7 (HHV-6 and -7), a neutralizing antibody assay was established for these viruses using a dot blot method. Three monoclonal antibodies against HHV-6 and 12 monoclonal antibodies against HHV-7 were developed and characterized by radio-immunoprecipitation. One monoclonal antibody which recognizes the 135 kDa late polypeptide of HHV-6 and several which recognize the 125 kDa late polypeptide of HHV-7 were selected to monitor virus growth by a dot blot antigen-detection method. The dot blot method was then used for the assay of HHV-6 and -7 neutralizing antibodies in human serum samples. The neutralization endpoints determined by the dot blot were comparable to those determined by immunofluorescence (IF). The neutralizing antibody titers appeared to correlate with the antibody titers determined by the indirect IF antibody test. The dot blot neutralization assay is easy to perform, is highly reproducible and objective when compared with the conventional methods based on cytopathology or IF for determining neutralization endpoints.

Multiple endocrine neoplasia 2B with glaucoma associated with codon 918 mutation of the RET proto-oncogene.

PURPOSE: We report the first case of a 35-year-old Japanese man with multiple endocrine neoplasia (MEN) 2B de novo to be associated with primary open angle glaucoma. METHODS: DNA was extracted from the patient's circulating leukocytes, specimens of the resected cervical lymph nodes, the neuroma of the eyelid, and of the conjunctival epithelium. Mutation was assayed by PCR/restriction enzyme and direct sequencing. RESULTS: The glaucoma and MEN 2B were diagnosed at the same time, when the patient was 16 years old. The glaucoma was controlled by medical treatment. The codon 918 mutation (met918thr) in exon 16 of the RET proto-oncogene associated with MEN 2B was identified in all these tissues. CONCLUSION: This patient was found to have the germline mutation at codon 918 (met918thr) in the RET proto-oncogene. The association between the RET proto-oncogene and glaucoma remains unclear, since glaucoma is a rare manifestation of MEN 2B.

[Structure and assembly of human beta herpesviruses].

This review is concerned with the structure and assembly of HCMV, HHV6 and HHV7. A characteristic ultrastructural feature common to all these viruses is a distinct tegumentary coating of intracytoplasmic capsids. The tegument structure is also distinctly seen in the virions of HHV6 and HHV7. Morphologically, acquisition of the tegument was observed to have taken place in the cytoplasm. Immunoelectron microscopic studies of HCMV infected cells, however, have demonstrated the existence of a tegument protein, pp150, on the surface of intranuclear capsids as well as on capsids in the cytoplasm and in extracellular virions. In addition, another tegument protein, pp65 has been detected within the matrix of cytoplasmic and extracellular dense bodies but not in virions. The molecular mechanism of the assembly of beta herpesviruses was also discussed.

Transmission of human herpesvirus 7 through multigenerational families in the same household.

BACKGROUND: Human herpesvirus 7 (HHV-7) closely resembles HHV-6 and to a lesser degree cytomegalovirus. HHV-7 infection is usually acquired during early childhood. Primary infection can cause a roseola-like illness but in most cases it is only mildly symptomatic. The majority of adults are seropositive and in contrast to HHV-6 and cytomegalovirus infection, they continue to secrete the virus in their saliva for many years. The mode of intrafamilial transmission of this virus is not well-understood. METHODS: Saliva samples for virus isolation and DNA restriction analysis were obtained from all 47 members of 6 Japanese families, including 4 families with 3 generations living in the same household. RESULTS: HHV-7 was isolated from 43 of 47 saliva samples collected from children and adult members of the 6 families (91.5%). In one family the restriction patterns of the maternal grandmother, the mother and the children were similar, and the patterns of the paternal grandmother and the father were similar. In another family the patterns of the father and 5 of 6 children were similar, and those of the mother and the other child were similar. Altogether similar HHV-7 restriction profiles with his or her mother were found in 48% of offspring, and similar profiles with his or her father were found in 28% of offspring. CONCLUSIONS: The results strongly suggested horizontal transmission of HHV-7 from grandparents to parents to children through close contact within a household. Either parent could transmit HHV-7 to the children.

Infection of a human retinal pigment epithelial cell line with human herpesvirus 6 variant A.

A retinal pigment epithelial (RPE) cell line (K-1034) was examined for its susceptibility to human herpesvirus 6 variant A (HHV-6A). Exposure of K-1034 cells to HHV-6A induced the formation of multinucleated giant cells, which was suppressed by an inhibitor of viral DNA synthesis. In the giant cells, herpesvirus nucleocapsids were demonstrated by electron microscopy and the viral glycoprotein B was detected by immunofluorescence assay. These results indicate that K-1034 cells are susceptible to HHV-6A and suggest that HHV-6A has an ability to directly destroy epithelial cells.

Suppressive effects of human herpesvirus 6 on in vitro colony formation of hematopoietic progenitor cells.

Human herpesvirus 6 (HHV-6) has been reported to be involved in bone marrow failure after bone marrow transplantation (BMT). To elucidate the role of HHV-6 in the marrow failure, we examined the comparative effect of two variants of HHV-6 (HHV-6A and HHV-6B) and human herpesvirus 7 (HHV-7) on in vitro colony formation of hematopoietic progenitor cells in methylcellulose semi-solid media. Progenitor cells prepared from cord blood mononuclear cells (CBMNCs) were infected with one of these viruses at various multiplicity of infection (MOI), and were subjected to methylcellulose colony assay. Formation of both granulocyte/macrophage (CFU-GM) and erythroid (BFU-E) colonies was MOI-dependently suppressed after infection with the Z29 strain of HHV-6B. Although HHV-6A suppressed the formation of BFU-E colonies as efficiently as HHV-6B, the former did not exhibit significant suppressive effect on the formation of CFU-GM colonies at an MOI 1. HHV-7 had no effect on hematopoietic colony formation at all. Based on frequent positivity of viral DNA in single colonies obtained from HHV-6-infected progenitor cells by polymerase chain reaction and in situ hybridization, direct effects of HHV-6 on the hematopoietic progenitor cells are suggested as the cause of the suppression rather than indirect effects via accessory cells of the bone marrow.

[Inhibition of corneal neovascularization by a new analog of fungal product].

The anti-angiogenic activity if FR 118487, a new synthetic analogue of Scolecobasidium arenarium products, was examined in corneas of Japanese white rabbits. We studied locally administered FR 118487 with hydron pellets in two models; corneal neovascularization after implantation of a CuCl2 pellet (CuCl2-induced model) and a Wistar rat's cornea (keratoplasty model) into a rabbit cornea. In the CuCl2-induced model, maximum length of neovascularization was 0.08 +/- 0.10 (mean +/- standard deviation) mm with FR 118487 (control 2.84 +/- 0.13 mm) at 1 week after the implantation. In the keratoplasty model, maximum length of neovascularization was 0.05 +/- 0.05 mm with FR 118487 (control 3.33 +/- 0.18 mm) at 2 weeks after the implantation. In both models, FR 118487 had a significant (p < 0.01) effect on inhibition of corneal neovascularization.

High-resolution immuno-scanning electron microscopy using a non-coating method: study of herpes simplex virus glycoproteins on the surface of virus particles and infected cells.

The expression of two glycoproteins, i.e. glycoprotein C (gC) and glycoprotein D (gD), of herpes simplex virus type 1 (HSV-1) on the surface of extracellular particles of this virus was examined by immuno-scanning electron microscopy. Scanning electron microscopy specimens of infected cells immuno-labelled against the glycoproteins with colloidal gold particles were prepared by a conventional coating and a non-coating method. Surface ultrastructure of infected cells and gold particles were observed more clearly with specimens prepared by the non-coating method. The appearance of virus particles in association with glycoprotein expression on these particles and on the surface of infected cells was then studied. Progeny virus particles began to appear 6 h after infection, increased in number as the infection proceeded, and covered most of the cell surface by 16 h. Six to 24 h after the infection, the labelling density for each glycoprotein on virus particles remained constant. The labelling density for gD was always higher than that for gC. The patch-like distribution of gold-labelling against gD was often detected on infected cell monolayers at the exponential and late stage of one cycle of virus growth. The labelling density for gD on virus particles was the highest on these produced in Vero and L-929 cells, moderate in MRC-5, BHK-21 and FL cells, and the lowest in HEp-2 cells.

In vitro selection of variants of herpes simplex virus type 1 which differ in cytopathic changes.

To analyze the mechanisms for in vitro emergence of the syncytial variants of herpes simplex virus type 1 (HSV-1), several cell lines were infected with a mixture of equal amounts of two HSV-1 variants, one syncytial and the other non-syncytial, and changes in their relative abundance were monitored during passage. With a combination of two variants of the Miyama strain of HSV-1, the syncytial variant became dominant during passage in Vero, RK-13 and FL cells. On the other hand, the ratios of the two variants remained around 1:1 during the passage in HEp-2, MGC and HEL cells. In another set of variants of the SKO strain of HSV-1, the outcomes were different from those of the Miyama strain in the FL, MGC and HEp-2 cells. The ratios of the two variants remained around 1:1 during passage in FL cells, while the non-syncytial variant became dominant during passage in MGC and HEp-2 cells. In addition, we examined the effects of a complement and interferon-beta (IFN-beta) on the outcome of the selection. As a result, the complement slowed the selection of a syncytial variant, whereas IFN-beta facilitated it.

Molecular analysis of outer capsid glycoprotein (VP7) genes from two isolates of human group C rotavirus with different genome electropherotypes.

Nucleotide sequences for the VP7 gene of human group C rotavirus were determined for two strains isolated in Okayama, Japan, during a 1988-1990 epidemic. These isolates, OK118 and OK450, were selected as prototypes of two different electropherotypes, patterns I and II, respectively. The genes were identical in size (1,063 bp), and both contained singled open reading frames encoding 332 amino acids. The alignment of two sequences revealed 46 nucleotide substitutions, 11 of which were predicted to give amino acid changes. The deduced amino acid sequence of VP7 from OK118 was similar to published sequences of a Japanese isolate and three foreign isolates (more than 98.4% identity), whereas the VP7 sequence of OK450 revealed around 96% identity with these isolates and had nine unique amino acid substitutions. The VP7 genes of nine Okayama isolated were than analyzed by dot blot hybridization with the VP7 probes of OK118 and OK450. Under highly stringent conditions, the OK118 probe produced strong hybridization signals with the genes of five pattern I strains and one pattern II strain, while the OK450 probe strongly reacted only with those of three pattern II strains. Our results concluded that relative sequence diversity in the VP7 gene was observed between two different electropherotypes prevalent in a limited area.