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Biomedical subjects

R Shibata

Publications and source records attributed to R Shibata.

At least 109 records · Page 6Linked to original sources

Construction and characterization of an infectious DNA clone and of mutants of simian immunodeficiency virus isolated from the African green monkey.

We constructed a full-length molecular clone of simian immunodeficiency virus from an African green monkey. Upon transfection, this clone directed the production of virus particles cytopathic and infectious to human CD4+ leukemia cell lines. Mutations were introduced by recombinant DNA techniques into eight open reading frames of simian immunodeficiency virus from the African green monkey thus far identified. The phenotypes of mutant viruses, i.e., infectivity, cytopathogenicity, transactivation of gene expression controlled by a long terminal repeat, and viral RNA and protein syntheses, were examined by transfection and infection experiments. Three structural (gag, pol, and env) and two regulatory (tat and rev) gene mutants were not infectious, whereas vif, vpx, and nef were dispensable for infectivity and mutant viruses were highly cytopathic. In transient transfection assays, a rev mutant produced mainly small mRNA species and no detectable virus protein and particles. The transactivation potential of a tat mutant was about 10-fold less than that of wild-type DNA, generating small amounts of virus.

Animals↗

Functional comparison of transactivation by human retrovirus rev and rex genes.

The effect of rev-responsive element deletion on human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) gene expression was examined. The phenotypes of HIV-1 and HIV-2 provirus DNAs lacking the rev-responsive element, as determined by transfection experiments, were indistinguishable from those of virus DNAs carrying rev gene mutations. By using rev-response elements derived from these two viruses, we developed two monitoring systems to evaluate the functionality of HIV-1 rev, HIV-2 rev, and human T-lymphotropic virus type I rex. In both systems, HIV-1 rev and human T-lymphotropic virus type I rex transactivated HIV-2 very efficiently. On the contrary, HIV-2 rev and human T-lymphotropic virus type I rex were poor activators of HIV-1. No functional replacement of rex by HIV-2 rev was observed.

Blotting, Northern↗

Generation and characterization of infectious chimeric clones between human immunodeficiency virus type 1 and simian immunodeficiency virus from an African green monkey.

A series of chimeric clones of human immunodeficiency virus type 1 and simian immunodeficiency virus isolated from an African green monkey was constructed in vitro. In transient transfection experiments, all clones produced virion-associated reverse transcriptase, gag proteins, and env proteins. Eight out of 10 chimeric viruses clearly grew in the human CD4+ cell line C8166. Susceptibility of other CD4+ cell lines, MT-4, A3.01, and Molt4 clone 8, to infection with these viruses was also demonstrated.

Animals↗

Mutational analysis of the human immunodeficiency virus type 2 (HIV-2) genome in relation to HIV-1 and simian immunodeficiency virus SIV (AGM).

We constructed an infectious molecular clone of the human immunodeficiency virus type 2 (HIV-2) and generated nine frameshift mutants corresponding to nine open reading frames identified so far. Three structural (gag, pol, env) and two regulative (tat, rev) gene mutants were not infectious, whereas vif, vpx, vpr, and nef genes were dispensable for infectivity. All of the mutants except env and rev were cytopathic in CD4+ human leukemia cells. In transfection assays, the expression of HIV-2 long terminal repeat was activated by infectious clones of HIV-1, HIV-2, and simian immunodeficiency virus from African green monkey but not by the tat mutants. However, an HIV-2 tat mutant could produce small amounts of virus proteins and particles in contrast to a rev mutant, which directed no detectable synthesis of virus proteins and virions.

Blotting, Northern↗

Complementation of the rev gene mutation among human and simian lentiviruses.

The functional exchangeability of the rev gene was assessed in transient transfection experiments by using in vitro-constructed rev and gag mutants of the following three primate lentiviruses: human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus from the African green monkey (SIV AGM). Cotransfection into SW480 cells of the rev and gag mutants derived from the DNA of each infectious virus resulted in the generation of progeny particles as determined by reverse transcriptase assay. rev gene mutants of HIV-2 and SIV AGM were also complemented by all gag mutants derived from the three viruses. In contrast, no evidence of complementation was obtained following cotransfection of the HIV-1 rev mutant and the gag mutant of HIV-2 or SIV AGM.

Amino Acid Sequence↗

Regulation of IgE and IgG4 synthesis in patients with hyper IgE syndrome.

In order to investigate the imbalance of IgG subclasses and its relationship to IgE level, 14 patients with hyper IgE syndrome (HIE syndrome) were examined for serum IgG subclasses and IgE levels and five of these patients were studied for in vitro IgE and IgG subclass production by peripheral blood lymphocytes (PBL) in response to interleukin-4 (IL-4)/interferon-gamma (IFN-gamma). Serum IgE levels were highly correlative with serum IgG4 levels (r = 0.75, P less than 0.005), but not with IgG1, IgG2 or IgG3 levels (r = 0.21, 0.43 and 0.41, respectively). In an in vitro study, recombinant IL-4 enhanced not only spontaneous IgE synthesis but also IgG4 synthesis in cultures of PBL from patients with HIE syndrome as well as in healthy donors (P less than 0.01), and the effect of recombinant IL-4 on both IgE and IgG4 synthesis was inhibited by low concentrations of recombinant IFN-gamma (P less than 0.01). The disturbed regulation of IgE and IgG4 seen in patients with hyper IgE syndrome may be caused mainly by the disturbed regulation of both cytokines.

Adolescent↗

Mutational analysis of simian immunodeficiency virus from African green monkeys and human immunodeficiency virus type 2.

We constructed ten mutants of simian immunodeficiency virus isolated from African green monkey (SIVAGM), and nine mutants of human immunodeficiency virus type 2 (HIV-2) in vitro. Their infectivity, cytopathogenicity, transactivation potential, virus RNA, and protein synthesis were examined by transfection and infection experiments. Mutations in three structural (gag, pol, env) and two regulator (tat, rev) genes abolished the infectivity of both viruses, but vpx, vpr (HIV-2), and nef were dispensable and mutant viruses were indistinguishable phenotypically from wild type virus. A vif mutant of HIV-2 showed poor infectivity in cell-free condition, whereas SIVAGM mutants grew equally well with wild type virus. In transient transfection assays, rev mutants derived from both viruses produced mainly small mRNA species and no detectable virus proteins and particles. Transactivation potential of tat mutants originated from both viruses was about three- to ten-fold less than that of respective wild type DNAs, generating small amounts of virus.

Animals↗

Nucleotide sequence of E1 region of canine adenovirus type 2.

The nucleotide sequence of the leftmost EcoRI-C fragment (0 to 11.3%) of canine adenovirus type 2 (CAd2) which could transform rodent cells morphologically but required additional sequences from 10 to 32 map units (m.u.) for full expression of its oncogenic potential was determined. The EcoRI-C fragment contains 3609 nucleotide base pairs (bp) encoding E1A, E1B, and pIX genes. Although the nucleotide sequence of CAd2 E1 shows little homology to those of human Ads, the amino acid sequences of the E1 proteins predicted from nucleotide sequence of CAd2 E1 and those for human and simian Ads are partially conserved.

Adenoviridae↗

Mutational analysis of the human immunodeficiency virus vpr open reading frame.

Mutations were introduced by recombinant DNA techniques into the vpr open reading frame of an infectious molecular clone of human immunodeficiency virus type 1. The effect of these changes on the replicative and cytopathologic properties of the virus recovered from transfected cells was studied in several human CD4+ lymphocyte cell lines. In all cases, mutant viruses were infectious and cytopathic. However, when a low-input dose was used, mutants grew significantly more slowly than the wild-type virus. The growth kinetics of vpr mutants were distinct from those of vif and vpu mutants.

Cytopathogenic Effect, Viral↗

Intracellular distribution of cardiac beta-adrenoceptors in SHR and WKY.

To compare the intracellular distribution of beta-adrenoceptors in isolated myocytes of SHR with that of age-matched WKY and to examine changes in the distribution with aging, we measured the number of cell surface and total beta-adrenoceptors in 6-(young groups) and 37-week old (old groups) SHR and WKY. The number of surface beta-adrenoceptors was significantly lower in 6-week old SHR compared with that in age-matched WKY. But there was no difference in the number of total beta-adrenoceptors between the two groups. The number of surface beta-adrenoceptors in old groups was significantly reduced compared with that in the young groups, in both SHR and WKY. However, the number of total beta-adrenoceptors in the old groups did not show any difference from that in the young groups. Isoproterenol-stimulated c-AMP formation in 6-week old SHR was significantly lower than that of WKY of the same age. Isoproterenol-stimulated c-AMP formation in the old groups was significantly reduced than that of the young groups in both SHR and WKY. These results suggested that (1) there may be a difference in the intracellular distribution of cardiac beta-adrenoceptors between 6-week old SHR and WKY, (2) the intracellular distribution of cardiac beta-adrenoceptors may be changed with aging, and (3) intracellular distribution might be related to the difference in c-AMP formation to beta-agonist.

Adenylyl Cyclases↗

Adrenal medullary cells transmute into dopaminergic neurons in dopamine-depleted rat caudate and ameliorate motor disturbances.

Adrenal medullary cell suspensions, derived from newborn rats (postnatal day 1-6), were implanted into the head of the caudate nucleus in 35 rats with unilateral 6-hydroxydopamine (6-OHDA) lesions in the nigrostriatal dopamine (DA) pathway. Behavioral recovery from Met-amphetamine induced circling, cell growth and morphological features (tyrosine hydroxylase positive cells), and release of adrenaline (Ad), noradrenaline (NA), DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were investigated for 40 weeks after transplantation. Met-amphetamine induced circling decreased significantly in 43% (15/35) of the rats. The decrease was concurrent with transmutation of the tyrosine hydroxylase-like immunopositive (THLI) cells into mature neurons that had abundant elongated neurites with varicosities and synapses on neuronal elements in the host caudate. In the absence of behavioral recovery (57%, 20/35) THLI cells were very scant. DA, DOPAC and HVA were reduced more than 90% in perfusates collected by in vivo dialysis from the striata of the animals that were not improved by transplant. These levels recovered to 20-50% of controls in animals whose behavior recovered. Ad and NA were not detected in the perfusates of either recovered or non-recovered animals. The results suggest that some grafted adrenal medullary cells transform into dopaminergic neurons and the release of DA from these grafted cells functionally affects behavior improvement for at least 40 weeks.

Adrenal Medulla↗

Transforming genes of canine adenovirus type 2.

Genes of canine adenovirus type 2 (CAd2) involved in transformation were localized and their functions investigated. Cells transformed by CAd2 whole DNA or the BamHI A fragment (leftmost 47%) totally lacked contact inhibition and were serum-independent, anchorage-independent and tumorigenic in newborn rats. All the cells transformed by the XbaI D (0 to 15.2%) and the EcoRI C (0 to 11.3%) fragments were morphologically transformed, but were serum-dependent, anchorage-dependent and not tumorigenic in newborn rats after 150 and 190 days observation, respectively, when 5 x 10(6) cells per rat were injected. No transforming activities were detected by DNA fragments smaller than EcoRI C. By Northern blot hybridization, it was shown that a 1 kb mRNA was encoded in the 0 to 45% region of the genome, and 1.1 kb and 2 kb RNAs in the 4.5 to 11% region. It was therefore suggested that these gene products are required for morphological transformation, and other gene(s) or element(s), which have not been identified, may be involved in serum independence, anchorage independence and tumorigenicity of the transformed cells.

Adenoviridae↗

Genomic comparison of adenovirus type 3 isolates from patients with acute conjunctivitis in Japan, Australia, and the Philippines.

A total of 53 Ad3 isolates obtained from patients with acute conjunctivitis in Japan, Australia, and the Philippines during 1973 to 1986 were analyzed for their genome types with 4 restriction endonucleases, BamHI, BglII, HindIII, and SmaI. Two new genome types designated tentatively as Ad3f and Ad3g were identified by combination of BamHI and BglII in the isolates. The changes of restriction sites and sizes of restriction fragments in newly recognized Ad3f and Ad3g were located at the similar regions reported in other Ad3 genome types by O'Donnell et al (1986) on physical maps of the Ad3 prototype strain GB genome. In Japan, 46 Ad3 isolates obtained from 1983 to 1986 were either Ad3f or Ad3g. Yearly alternation of predominance of both genome types were observed in the northern part of Japan during the period. In Australia, two genome types Ad3p and Ad3f were found in 6 isolates, and the former was observed in the 3 isolates obtained before 1981 and the remaining 3 isolates were obtained after 1983. In the Philippines, the only isolate obtained in 1984 was Ad3p.

Adenoviridae Infections↗

Diurnal- and behaviour-related activity of ventromedial hypothalamic neurones in freely behaving rats.

1. Long-term (25-93 h) single-neurone activity of seventy-eight ventromedial hypothalamic (v.m.h.) neurones was analysed in freely behaving rats. 2. The activity of fifty-seven (73%) of the seventy-eight neurones tested depended on arousal and sleep, determined by electroencephalogram (e.e.g.), and varied diurnally. The activity of 39.8% (31/78) increased during e.e.g. arousal, and decreased during slow-wave sleep. Activity of 33.3% (26/78) varied inversely. 3. The activity of 21.8% (17/78) was independent of e.e.g. arousal and sleep. The activity of all except one of the seventeen increased in the dark period and varied diurnally. Activity of one increased during light stimulation. 4. The activity of the remaining 5.1% (4/78) was less than 0.5 spikes/s, and was not related to e.e.g. arousal or sleep or the diurnal cycle. 5. The effects of bilateral lesions of the suprachiasmatic nuclei (s.c.n.) were investigated on six v.m.h. neurones. All of these lost their diurnal variation; five retained their dependence on e.e.g. arousal and sleep, and one retained its independence of e.e.g. arousal and sleep. 6. There was no discernible relation between v.m.h. recording site and any of the tested characteristics of these neurones. 7. Of seventy-eight v.m.h. neurones tested, seventy-four were classified as either: (1) diurnal, sleep-arousal related, or (2) diurnal, sleep-arousal independent. Because s.c.n. lesions abolished the circadian rhythm of v.m.h. activity in every neurone tested, it was concluded that at least some v.m.h. diurnal variation is driven from the s.c.n. as a biological clock. It is clear also that responses related to e.e.g. are driven from some source other than the s.c.n.

Action Potentials↗

Feeding- and chemical-related activity of ventromedial hypothalamic neurones in freely behaving rats.

1. The activity of seventy-eight single neurones in the ventromedial hypothalamus (v.m.h.) was recorded in sixty-three freely behaving rats, and the effects of feeding, intraventricular (I.C.V.) administrations of noradrenaline, glucose, NaCl and ambient temperature on neuronal activity were analysed. If I.C.V. NaCl had an effect, intraperitoneal (I.P.) NaCl, mannitol and polyethylene glycol were also tested. 2. Neurones in the v.m.h. were classified into three groups according to diurnal variations and their relations to electroencephalogram (e.e.g.) and responses to feeding: diurnal-e.e.g. related (57/78, 73.1%); diurnal-e.e.g. independent (17/78, 21.8%); non-diurnal-e.e.g. independent (4/78, 5.1%). Of fifty-seven e.e.g.-related neurones, twenty-six decreased activity during feeding episodes. Of seventeen e.e.g.-independent neurones, eight increased activity gradually during feeding and sustained the increase after the feeding episode. The response magnitude of two e.e.g.-independent neurones depended on the kind of food available. 3. Of twenty-five e.e.g.-related neurones tested, twelve responded to I.C.V. noradrenaline, but not to I.C.V. glucose or NaCl. Neurones independent of e.e.g. responded variously to I.C.V. noradrenaline, glucose and NaCl. When I.C.V. NaCl had an effect, I.P. NaCl, mannitol and polyethylene glycol had the same effect. The activity of three neurones was increased by I.C.V. glucose and decreased by I.C.V. noradrenaline, but was not changed by I.C.V. NaCl. The activity of three was increased by I.C.V. glucose and decreased by I.C.V. NaCl and by I.C.V. noradrenaline. The activity of five was increased, and that of three was decreased by I.C.V. glucose, NaCl and noradrenaline. Collectively, fourteen of twenty-four tested neurones responded to I.C.V. glucose, twenty-six of forty-one tested neurones responded to I.C.V. noradrenaline and eleven of twenty-six tested neurones responded to I.C.V. NaCl. 4. Increase of ambient temperature changed the activity of five e.e.g.-independent neurones. Directions of these activity changes were the same as directions of responses to NaCl; two up, three down. 5. The results suggest two main neuronal groups in the v.m.h.: the e.e.g.-related group is involved in the processing of information about sleep-arousal. The e.e.g.-independent group contributes to the long-term processing of information concerned with the regulation of the internal environment such as glucose level, osmotic pressure, NaCl level, the trigger mechanism for feeding, ambient temperature, food preference, etc.

Action Potentials↗