A case of maxillary sarcoma in a chimpanzee (Pan troglodytes).

Oral malignancy is rare in chimpanzees. A 34-year-old female chimpanzee (Pan troglodytes) at Kumamoto Sanctuary, Japan, had developed it. Treatment is technically difficult for chimpanzees while malignant neoplasm is seemingly rising in captive populations. Widespread expert discussion, guidelines for treatment, especially for great apes in terminal stages is urgently needed.

Enzastaurin has anti-tumour effects in lung cancers with overexpressed JAK pathway molecules.

BACKGROUND: Enzastaurin, an oral serine-threonine kinase inhibitor, was initially developed as an ATP-competitive selective inhibitor against protein kinase Cß. However, the mechanism by which enzastaurin contributes to tumourigenesis remains unclear. METHODS: We analysed the anti-tumour effects of enzastaurin in 22 lung cancer cell lines to ascertain the potential for enzastaurin-based treatment of lung cancer. To identify molecules or signalling pathways associated with this sensitivity, we conducted a gene, receptor tyrosine kinases phosphorylation and microRNA expression profiling study on the same set of cell lines. RESULTS: We identified eight genes by pathway analysis of molecules having gene-drug sensitivity correlation, and used them to build a support vector machine algorithm model by which sensitive cell lines were distinguished from resistant cell lines. Pathway analysis revealed that the JAK/STAT signalling pathway was one of the main ones involved in sensitivity to enzastaurin. Overexpression of JAK1 was observed in the sensitive cells by western blotting. Simultaneous administration of enzastaurin and JAK inhibitor inhibited enzastaurin-induced cell growth-inhibitory effect. Furthermore, lentiviral-mediated JAK1-overexpressing cells were more sensitive to enzastaurin than control cells. CONCLUSION: Our results suggested that the JAK1 pathway may be used as a single predictive biomarker for enzastaurin treatment. The anti-tumour effect of enzastaurin should be evaluated in lung cancer with overexpressed JAK pathway molecules.

Comparison of susceptibility to SIVmac239 infection between CD4(+) and CD4(+)8(+) T cells.

CD4-bearing T cells are the primary targets for human immunodeficiency virus type 1(HIV-1)/simian immunodeficiency virus (SIV) infection. However, it is unclear whether the susceptibility of CD4-bearing T cells including CD4 single positive and CD4/8 double positive T cells to HIV/SIV infection is the same or not. In this study, we compared the susceptibility to SIV infection between CD4(+) and CD4(+)8(+) T cells, using Herpesvirus saimiri (HVS)-transformed CD4(+) and CD4(+)8(+) T cells established from peripheral blood mononuclear cells (PBMC) of rhesus macaques. Although there was little difference between the two CD4-bearing T cell population in the expression level of CD4 molecules and chemokine receptors such as CXCR4 and CCR5, SIV replicated more efficiently in CD4(+)8(+) T cells than in CD4(+) T cells. Moreover, we found that reverse transcription initiated more efficiently in CD4(+)8(+) T cells than in CD4(+) T cells and that the cell lysates from CD4(+) T cells impaired the RT activity more strongly than that from CD4(+)8(+) T cells. These findings suggest that intracellular environment in CD4(+) 8(+) T cells is better for reverse transcription and that the infection of those CD4(+)8(+) T cells might play critical and different roles in HIV-1/SIV infection and dissemination.

DNA vaccination of macaques by a full genome HIV-1 plasmid which produces noninfectious virus particles.

In this study, we tried a DNA vaccination regime in rhesus macaques using a full genome HIV-1 plasmid. The HIV-1 genome is under the control of its original LTR promoter, but has a mutated zinc finger motif gene in the nucleocapsid region. Due to the lack of genomic RNA packaging, the plasmid produces only noninfectious viral particles. We repeatedly injected four macaque monkeys intramuscularly with the naked DNA over a period of 40 weeks. To evaluate the humoral and cell-mediated immunity provided by this DNA vaccination, no other booster or other recombinant viral vectors were used. Immunological responses against HIV-1 were elicited in all of the vaccinated monkeys: stable anti-HIV-1 Env antibodies were raised in two monkeys and CTL activities were induced in the other monkeys. The macaques were intravenously challenged at 54 weeks with 100 TCID(50) of SHIV-NM-3rN, which possesses an envelope gene homologous to the one in the vaccinated plasmid. In all of the vaccinated macaques, the peak plasma viral loads induced by the challenge virus were two to three orders of magnitude lower than those of the naive controls. These results suggest that a DNA vaccination regime with a full genome plasmid alone is potentially efficacious and provides a new possibility for the development of an AIDS vaccine.

Protective immunity of gene-deleted SHIVs having an HIV-1 Env against challenge infection with a gene-intact SHIV.

We constructed three simian-human immunodeficiency viruses (SHIVs) lacking regulatory gene(s) and analyzed their induction of protective immunity against challenge infection with gene-intact SHIV in rhesus macaques. Inoculation of SHIV-dn lacking nef and SHIV-drn lacking nef and vpr induced transient viremia, while that of SHIV-dxrn lacking nef, vpr, and vpx induced no viremia. The SHIVs with fewer deletions were more effective in inducing neutralizing antibodies and cytotoxic T lymphocyte responses. When these macaques were challenged with parental gene-intact SHIV-NM-3rN, all the SHIV-dn-vaccinated macaques and two of the four SHIV-drn-vaccinated macaques showed complete resistance. The other two SHIV-drn-vaccinated macaques and all SHIV-dxrn-vaccinated macaques did not show complete resistance, but they did show suppression of replication of the challenge virus. These results suggested that as more genes were deleted, protective immunity was decreased.

Protection of macaques against a SHIV with a homologous HIV-1 Env and a pathogenic SHIV-89.6P with a heterologous Env by vaccination with multiple gene-deleted SHIVs.

To evaluate the potential of SHIVs as anti-HIV-1 live vaccines, we constructed two gene-deleted SHIVs, designated SHIV-drn and SHIV-dxrn. The former lacks vpr/nef and the latter lacks vpx/vpr/nef. Four macaques that had been vaccinated with SHIV-drn were challenged with SHIV-NM-3rN, which has an HIV-1 Env that is the same as that of SHIV-drn. No challenge virus was detected by DNA PCR in, or recovered from, two of the macaques. In the other two, challenge virus was detected once and twice, respectively. Plasma viral loads were much lower than those in unvaccinated controls. Another four macaques were vaccinated with SHIV-dxrn. These macaques showed resistance but less than that of SHIV-drn-vaccinated macaques. When the two SHIV-drn-vaccinated macaques were challenged with pathogenic SHIV-89.6P, which has an HIV-1 Env that is antigenically different from that of SHIV-drn, replication of the challenge virus was restricted, and the usual decrease in the number of CD4(+) cells was prevented. In this protection, it is noteworthy that protection involved not only neutralizing antibodies and killer cell activity, but also other unknown specific and nonspecific immunity elicited by the infection.

Synthesis of (1-azabicyclo[3.3.0]octanyl)methyl-substituted aromatic heterocycles and their muscarinic activity.

In our development of drugs effective against Alzheimer's disease, we have researched a series of aromatic compounds having a characteristic cyclic amine, 1-azabicyclo[3.3.0]octane ring. In this report, we describe synthesis of a series of aromatic heterocycles with the 1-azabicyclo[3.3.0]octane ring and their pharmacological evaluation. 3-Amino-5-(1-azabicyclo[3.3.0]octan-5-yl)methyl-1,2,4-oxadiazole (2b) showed the highest M1 selectivity.

Synthesis and biological activity of the metabolites of N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-2-nitroaniline fumarate (SK-946).

Three metabolites of N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-2-nitroaniline fumarate (SK-946), a novel central muscarinic cholinergic receptor agonist, were prepared to confirm their proposed structures, and tested for muscarinic receptor affinity in vitro.

Synthesis and muscarinic activity of novel aniline derivatives with a 1-azabicyclo[3.3.0]octane moiety.

In order to develop drugs effective against Alzheimer's disease, we synthesized a series of aniline derivatives having a characteristic cyclic amine, 1-azabicyclo[3.3.0]octane ring, and evaluated their binding affinity for the central muscarinic cholinergic receptor. Among these compounds which showed high affinity to the M1 receptor, N-[2-(1-Azabicyclo[3.3.0]octan-5-yl)ethyl]-2-nitroaniline (9f fumarate, SK-946) showed the highest affinity. The ability of this compound to improve cognitive function was assessed using the passive avoidance test in scopolamine-induced dementia mice. Some anilines with a 1-azabicyclo[3.3.1]nonane ring were also synthesized by the ring expansion of the 1-azabicyclo[3.3.0]octane ring, and showed a high affinity for the central muscarinic cholinergic receptor.

Effects of SK-951, a benzofuran derivative, as a prokinetic agent in rats and dogs.

The gastrokinetic activity of SK-951 ((-)4-amino-N-[2-(1-azabicyclo[3.3.0]octan-5-yl)ethyl]-5-chloro-2,3-dihy dro-2-methylbenzo[b]furan-7-carboxamide hemifumarate), a benzofuran derivative with 5-hydroxytryptamine (5-HT)4-receptor agonist activity, was studied in rats and dogs. The effects of SK-951 were also investigated in a model of vagotomy-induced gastroparesis in comparison with cisapride. In rats, both SK-951 and cisapride enhanced gastric emptying of liquids (phenol red) at a dose of 1-100 mg/kg, p.o. Gastric emptying of liquid (acetaminophen) in fasted beagle dogs was enhanced significantly by SK-951 (1.0 mg/kg, i.v.), whereas the effect of cisapride (0.2-1.0 mg/kg, i.v.) was not statically significant. Similar results were found when radiopaque markers were given with standard meal to dogs with vagotomy-induced gastroparesis. The delayed gastric emptying of radiopaque markers by vagotomy was reversed by SK-951 (1.0 mg/kg, i.v.), whereas cisapride showed no effect at doses from 0.1 to 1.0 mg/kg, i.v. These results indicated that oral and intravenous administration of SK-951 accelerates gastric emptying of both liquids and solids in animal models. Thus, SK-951 may be a highly potent and useful prokinetic agent in comparison to cisapride.

Characterization of the neurochemical effects of N-[2-(1-azabicyclo[3,3,0]octan-5-yl)ethyl]2-nitroaniline fumarate (SK-946) as a cognition activator.

The neurochemical effects of SK-946, N-[2-(1-azabicyclo[3,3,0]octan-5-yl)ethyl]2-nitroaniline fumarate, were investigated in an attempt to elucidate cognition activating properties. In rat brain cortical membranes and in cloned human receptors expressed in Sf9 cells, SK-946 had submicromolar affinities for muscarinic receptors with a moderate selectivity for M1 (m1) sites. Although no increase in the antagonist (N-methylscopolamine)/agonist (oxotremorine-M) binding ratio was observed, SK-946 exhibited a partial agonistic effect on receptor-stimulated phosphoinositide hydrolysis in primary cultured rat fetal hippocampal cells. Heterogeneous, reversible and concentration-dependent excitations of the hippocampal neuronal cells treated with SK-946 (10(-5)-10(-3) M) were confirmed as increases in cytosolic Ca2+ concentrations measured in Fura-PE3 preloaded cells. Furthermore, SK-946 (>10(-5) M) increased [3H]myo-inositol uptake into the hippocampal cells. On the other hand, SK-946 had no effect on adenylate cyclase activities in primary cultured rat heart cells, and showed a weak antagonistic effect on carbachol-induced adenylate cyclase inhibition, suggesting that it is an M2 antagonist. Using in vivo microdialysis, it was found that relatively low concentrations (<10(-7) M) of SK-946 increased acetylcholine release and decreased choline content in that hippocampal area in rats. These results suggest that SK-946 accelerates muscarinic neuronal transmission in the central nervous system.

Purification and biological properties of a cell growth-stimulating factor from Clostridium perfringens FERM P-14028.

The active component that stimulates fibroblast growth was purified from cultured Clostridium perfringens FERM P-14028, and a quantitative assay method for the biological activity was established. The active component was named cell growth-stimulating factor (CGSF), and the molecular weight of this factor was estimated to be 420 kDa on gel permeation chromatography. CGSF(50-100 ng/ml) stimulated the growth rate of BHK-21 (C-13) cells in the logarithmic growth phase, and shortened the doubling time by 16-18%, but had no effect on confluent cells. These actions were indicated only with medium containing fetal bovine serum. These results suggest that CGSF is a novel protein that regulates cell growth via a mechanism of action different from that of other growth factors.

Test for interaction between treatment and stratum with ordinal responses.

A measure of interaction between treatments and stratum is proposed when there are two treatments and the response variable is measured on an ordered categorical scale. A test of the hypothesis of no interaction and a test of the hypothesis of no treatment effect are given and approximate expressions for the powers of these tests are obtained. Monte Carlo simulation is performed to examine the Type I error rate and the accuracy of the approximate power formulae of the tests. The loss of power of the tests due to combining categories is also examined. The simulation study shows that our methods perform well even in samples of size 20, and that the dichotomization results in appreciable loss of power.

Physiological correlation between nucleoside-diphosphate kinases and the 21-kDa guanine-nucleotide binding proteins copurified with the enzymes from the cell membrane fractions of Ehrlich ascites tumor cells.

The physiological correlation between nucleoside-diphosphate kinases (NDP-kinases) and the 21-kDa guanine nucleotide-binding proteins (G1 and G2) which are copurified with the enzymes from the cell membrane fractions of Ehrlich ascites tumor cells has been biochemically investigated in vitro. We found that: incubation of the phosphoenzyme (enzyme-bound high-energy phosphate intermediate) of NDP-kinases (F-I and F-II) with one of the nucleoside 5'-diphosphates in the presence of 1 mM Mg2+ or 0.25 mM Ca2+ results in the rapid formation of nucleoside 5'-triphosphates without strict base specificity; GDP on the guanine nucleotide-binding proteins (G1, G2 and recombinant v-rasH p21) acts as a phosphate acceptor for the high-energy phosphates of the phosphoenzyme in the presence of 0.25 mM Ca2+; and [32P]GTP is preferentially formed from the 32P-labelled phosphoenzyme F-I and GDP-bound G1 or GDP-bound recombinant v-rasH p21 protein, even if any other nucleoside 5'-diphosphates are present in the reaction mixture. Although [32P]GTP formed was bound with the guanine nucleotide-binding proteins, it was immediately hydrolyzed by the proteins themselves in the presence of 5 mM Mg2+, but not in the presence of 0.25 mM Ca2+. Available evidence suggests that NDP-kinase may be responsible for the activation of the guanine nucleotide-binding proteins (G1, G2 and p21 proteins) through phosphate transfer by the enzyme.

Physiological correlation between nucleoside-diphosphate kinase and the enzyme-associated guanine nucleotide binding proteins.

The physiological correlation between NDP-kinase and the enzyme-associated guanine nucleotide binding proteins (G1 and G2) has been studied in vitro. It was found that incubation of the phosphoenzyme (enzyme-bound high-energy phosphate intermediate) of NDP-kinases with one of the nucleoside 5'-diphosphates (NDPs) in the presence of divalent cations (Mg2+ and Ca2+) results in the formation of nucleoside 5'-triphosphates (NTPs) within 40 sec even at low temperatures (below 4 degrees C) without strict base-specificity; and high-energy phosphates on the phosphoenzyme can transfer preferentially to GDP on the guanine nucleotide binding proteins (G1, G2 and r-p21 protein) in the presence of 0.25 mM Ca2+ or 1 mM Mg2+ even if any other NDPs are present in the reaction mixtures. These observations suggest that NDP-kinase may be responsible for the phosphate-transfer between GDP on the guanine nucleotide binding proteins and its phosphoenzyme.

Complete recovery of the phosphoenzyme-forming activity of nucleoside-diphosphate kinases after reconstitution of their subunits.

Two distinct subunits [alpha-subunit (Mr 21 000, pI 7.6) and beta-subunit (Mr 19 000, pI 6.5)] of nucleoside-diphosphate (NDP) kinases highly purified from HeLa S3 cells can be separated by FPLC using a Mono P column in the presence of 6 M urea and 1% pharmalyte (pH range between 5.0 and 8.0). Comparatively high [32P]phosphate incorporation was detected when these two subunit fractions were reconstituted in vitro. Available evidence suggests that these two enzyme subunits are necessary for the formation of phosphoenzyme, which functions as an intermediate in NDP kinase action.

Phosphatidylinositol-hydrolyzing enzymes in chicken liver cells.

Phosphatidylinositol-hydrolyzing activities were found in mitochondrial, lysosomal, microsomal, and cytosol fractions of chicken liver. At least two different activities were detected; cytosolic activiti(es) was maximally exhibited around pH 6.0, activated by Ca2+, and inhibited by EDTA; whereas lysosomal activiti(es) had a optimal pH 5.0, being unaffected by Ca2+ or EDTA.