Characterization of the sequences of the human cytomegalovirus enhancer that mediate differential regulation by natural and synthetic retinoids.

Evidence exists to suggest that human cytomegalovirus (hCMV) may opportunistically use retinoic acid (RA) to advance its own replication, in which transcriptional activation of the viral major immediate-early promoter is a crucial control point. We demonstrate that the enhancer of the viral promoter contains three RA-response-elements that cooperate in mediating RA activation. These elements are direct repeats of two sequence motifs separated by 2 bp (DR2 site, REa) and 5 bp (DR5 sites, REb and c). DNA-binding experiments revealed that each of these elements bind RA receptor (RAR)-retinoid X receptor (RXR) heterodimers more efficiently than either homodimer. Apparent equilibrium dissociation constants of RAR-RXR heterodimers for sites REa, REb, and REc were estimated to be 5 nm, 10 nm, and 20 nm, respectively. The level of contribution of each of these elements to RA inducibility correlated with the strength of binding by RAR-RXR heterodimers to each site. These experiments demonstrate that RAR and RXR are necessary for RA responsiveness of the viral promoter. Using synthetic RA analogs, which selectively activate RARs and RXRs, the RAR partner within the heterodimeric complex appeared to be sufficient while the RXR partner was insufficient to independently activate transcription. However, joint activation of RARs and RXRs indicated that RXRs (in the presence of a transcriptionally active RAR) could contribute to transactivation. This restricted co-dependent ligand activation of RXR varied depending on the particular response element and the cell context. These studies further indicate that signaling of retinoid receptors (in particular RAR) by RA plays an important role in modulating hCMV infection.

Synthesis and structure-activity relationships of novel retinoid X receptor-selective retinoids.

Two series of potent retinoid X receptor (RXR)-selective compounds were designed and synthesized based upon recent observation that (E)-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1- propenyl]benzoic acid (TTNBP) binds and transactivates only the retinoic acid receptor (RAR) subtypes whereas (E)-4-[2-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydro-2-naphthalenyl)-1-propenyl]benzoic acid (3-methyl TTNPB) binds and transactivates both the RAR and RXR subfamilies. Addition of functional groups such as methyl, chloro, bromo, or ethyl to the 3 position of the tetrahydronaphthalene moiety of 4-[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)carbonyl]benzoic acid (5a) and 4-[1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2- naphthyl)ethenyl]benzoic acid (6a) results in compounds which elicit potent and selective activation of the RXR class. Such RXR-selective compounds offer pharmacological tools for elucidating the biological role of the individual retinoid receptors with which they interact. Activation profiles in cotransfection and competitive binding assays as well as molecular modeling calculations demonstrate critical structural determinants that confer selectivity for members of the RXR subfamily. The most potent compound of these series, 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]ben zoi c acid (6b), is the first RXR-selective retinoid (designated as LGD1069) to enter clinical trials for cancer indications.

Design and synthesis of potent retinoid X receptor selective ligands that induce apoptosis in leukemia cells.

Structural modifications of the retinoid X receptor (RXR) selective compound 4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2- naphthyl)ethenyl]benzoic acid (LGD1069), which is currently in phase I/IIA clinical trials for cancer and dermatological indications, have resulted in the identification of increasingly potent retinoids with > 1000-fold selectivity for the RXRs. This paper describes the design and preparation of a series of RXR selective retinoids as well as the biological data obtained from cotransfection and competitive binding assays which were used to evaluate their potency and selectivity. The most potent and selective of the analogs is 6-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2- yl)cyclopropyl]nicotinic acid (12d; LG100268). This compound has proven useful for investigating RXR dependent biological pathways including the induction of programmed cell death (PCD) and transglutaminase (TGase) activity. Our studies indicate that the induction of PCD and TGase in human leukemic myeloid cells is dependent upon activation of RXR-mediated pathways.

A new class of analogues of the bifunctional radiosensitizer alpha-(1-aziridinylmethyl)-2-nitro-1H-imidazole-1-ethanol (RSU 1069): the cycloalkylaziridines.

A series of compounds related to alpha-(1-aziridinylmethyl)-2-nitro-1H-imidazole-1-ethanol (RSU 1069, 1) were synthesized and evaluated as selective hypoxic cell cytotoxic agents and as radiosensitizers. The aziridine moiety was replaced with a number of other potential alkylating groups including cycloalkylaziridines and azetidines. The data indicated that modification of the aziridine of 1 resulted in a substantial decrease in the ability of the compounds to selectively kill hypoxic cells. However, these modifications did not affect the compounds' in vitro radiosensitizing activity since many of the derivatives were as potent as 1. All of the compounds that were evaluated in vivo were less toxic than 1, and several members of this series had significant activity. The best compound was trans-alpha-[[(4-bromotetrahydro-2H-pyran-3-yl) amino]methyl]-2-nitro-1H-imidazole-1-ethanol (18), which, due to its activity and log P value, is a candidate for additional in vivo studies.

Hemocytes of Pomacea canaliculata: I. Reversible aggregation induced by Ca2+.

Using a platelet aggregometer, factors involved in hemocyte aggregation of a prosobranch snail Pomacea canaliculata were investigated. Aggregation-inducing tests revealed that extracellular Ca2+ is required to provoke the cellular response which is reversible. The aggregation-dispersion response can be induced by adding Ca2+ and EDTA alternately. Aggregation was inhibited by a calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) in a dose-dependent manner, and by a protein kinase-C inhibitor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7). Thus, calmodulin and protein kinase-C may contribute to the calcium-mediated aggregation of hemocytes, which resembles a phenomenon that occurs in vertebrate leucocytes. Unlike the aggregation of mammalian leucocytes, no stimulus other than Ca2+ is required to induce the response of Pomacea hemocytes. Thus, this system may serve as a simple model for analysing cellular aggregation responses.

Effect of PD 128763, a new potent inhibitor of poly(ADP-ribose) polymerase, on X-ray-induced cellular recovery processes in Chinese hamster V79 cells.

The modifying effects of PD 128763 (3,4-dihydro-5-methyl-1(2H)-isoquinolinone), a potent inhibitor of poly(adenosine-diphosphate (ADP)-ribose) polymerase, on radiation-induced cell killing were examined in Chinese hamster V79 cells. This compound has an IC50 value against the purified enzyme approximately 50X lower than 3-aminobenzamide (3-AB), a widely used specific inhibitor of the enzyme. Exposure of exponentially growing cells to a noncytotoxic concentration (0.5 mM) of PD 128763 for 2 h immediately following X irradiation increased their radiation sensitivity, modifying both the shoulder and the slope of the survival curve. When recovery from sublethal damage and potentially lethal damage was examined in exponential and plateau-phase cells, respectively, postirradiation incubation with 0.5 mM PD 128763 was found not only to inhibit both these processes fully, but also to enhance further the level of radiation-induced cell killing. This is in contrast to the slight effect seen with the less potent inhibitor, 3-AB. The results presented suggest that the mechanism of radiosensitization by PD 128763 is related to the potent inhibition of poly(ADP-ribose) polymerase by this compound.

The potential of subtype-selective neuronal nicotinic acetylcholine receptor agonists as therapeutic agents.

Neuronal nicotinic acetylcholine receptors (NAChRs) are pentameric ligand-gated ion channel receptors which exist as different functional subunit combinations which apparently subserve different physiological functions as indicated by molecular biological and pharmacological techniques. It is possible to design and synthesize novel compounds that have greater selective affinities and efficacies than nicotine for different NAChRs, which should translate into different behavioral profiles and therapeutic potentials. Examples of NAChR agonists studied are nicotine, SIB-1508Y, SIB-1553A and epibatidine. These compounds have different degrees of selectivity for human recombinant NAChRs, different neurotransmitter release profiles in vitro and in vivo and differential behavioral profiles. Preclinical studies suggest that SIB-1508Y is a candidate for the treatment of the motor and cognitive deficits of Parkinson's disease, whereas SIB-1553A appears to have potential as a candidate for the treatment of Alzheimer's disease. Epibatidine has a strong analgesic profile, however the ratio between pharmacological activity and undesirable effects is so low that it is difficult to envisage the use of this compound therapeutically. Nicotine has a broad profile of pharmacological activity, for instance demonstrating activity in models for cognition and analgesia. As for epibatidine, the adverse effects of nicotine severely limits its therapeutic use in humans. The discovery of subtype-selective NAChR agonists such as SIB-1508Y and SIB-1553A provides a new class of neuropsychopharmacological agents with better therapeutic ratios than nonspecific agents such as nicotine.

Immunoglobulins specific to mosquito salivary gland proteins in the sera of persons with common or hypersensitive reactions to mosquito bites.

Using the immunoblot technique, we analyzed the quality and quantity of IgG, IgG4, and IgE specific to mosquito salivary gland (hereafter abbreviate as SG) components of Aedes albopictus in the sera of volunteers with common reactions and of 3 patients with severe reactions. In the volunteers with delayed reactions only or with both delayed and immediate reactions, IgG against SG components of A. albopictus formed several faint or moderately stained bands. Those with immediate reactions showed several intense bands and many other weak bands. In volunteers, who had been bitten by Aedes sp. frequently but had no skin reaction, and in severe cases, many intense IgG bands were observed. IgG4 bound to SG components were found in the sera of the common reaction group at the levels of 24 and 48 kD, but, in one severe case, no bands were observed, although the total IgG was very high. IgE levels specific to SG components were much higher in severe cases than in the volunteers. These results indicate that high titers of specific IgG and IgE and lack of IgG4 for particular components of SG may lead to severe allergic reactions in severe cases. Immunoblotting analysis of the antibodies also verified the possibility of developing in vitro tests to identify causative species of the mosquito for severe cases.

[An experimental model of intravitreal neovascularization in rabbits].

The author developed a consistent model of intravitreal neovascularization in the eyes of pigmented rabbits by incomplete posterior vitreous detachment after air injection followed by implantation of basic fibroblast growth factor (b-FGF; 250 ng or 1 microgram) in the vitreous and simultaneous intravitreal injection of DL-alpha-aminoadipic acid in physical saline solution (1 mg/kg). Newly formed vessels were observed in the proliferative fibrous membrane that surrounded the b-FGF pellet. In fluorescein angiography, we observed fluorescein leakage from the newly formed vessels. Histologically, the newly formed vessels showed fenestration of endothelial cells. This method provides an easy and consistent model to study neovascularization.

[Prevalence of diabetic ocular complications and systemic factors].

The prevalence of diabetic ocular complications and the correlation between diabetic retinopathy and systemic factors were examined in 2,300 cases (4,600 eyes) with non-insulin-dependent diabetes mellitus. The prevalence of cataract was 66.7%, of retinopathy 37.0%, of refractive and accommodative change 6.2%, of glaucoma 1.9% (rubeotic glaucoma was 1.0%), of rubeosis iridis 1.5%, of iridocyclitis 0.8%, of extraocular muscle palsy 0.2%, and of ischemic optic neuropathy 0.1%. Duration of diabetes mellitus, HbA1C value, methods of diabetic control, age, diabetic nephropathy, diabetic neuropathy, hypertension, systolic blood pressure, diastolic blood pressure, and arteriosclerosis obliterans were related with diabetic retinopathy. We suggest that the management of diabetic patients needs sufficient attention in the cases with oral administration of medication, insulin therapy, and diabetic nephropathy.

Studies on the venomous spicules and spines of moth caterpillars. I. Fine structure and development of the venomous spicules of the Euproctis caterpillars.

Scanning and transmission electron microscopy (SEM and TEM) revealed unique structures and development of the venomous spicules of tussock moth caterpillars of the genus Euproctis: (1) Flower-like structure at the distal end and a longitudinal minute depression on the proximal subapical wall of these spicules were observed by SEM. This depression was revealed to be a small hole by TEM. (2) During molting, observed were cytoplasmic processes of several trichogen cells penetrating the cytoplasm of a tormogen cell to form the spicules with the holes at their subapical portions. A papilla was formed by a tormogen and several epidermal cells. (3) After the molting, the cytoplasmic process in a spicule disappeared and the spicule cavity was replaced by electron-dense materials secreted apparently from the trichogen cell. (4) It was considered that the electron-dense materials were the main toxic or precursory substances in the Euproctis spicules.

Characterization of ehrlichial organisms isolated from a wild mouse.

An infectious agent was isolated from the enlarged spleen of a wild mouse, Eothenomys kageus, by intraperitoneal inoculation of the spleen homogenate into laboratory mice. The laboratory mice developed splenomegaly, and the agent was maintained by serial passage of spleen homogenates in laboratory mice. The agent in the spleen homogenate was inactivated after incubation at 37 or 50 degrees C. Tetracyclines were effective in preventing infection of mice with this agent, but penicillin and sulfonamides were ineffective. Cytoplasmic inclusion bodies were observed in the peritoneal macrophages of infected mice. Electron microscopy revealed numerous small pleomorphic cocci within membrane-lined vacuoles in the cytoplasm of splenic macrophages. Morphologically similar to the ehrlichial organisms, each organism was surrounded by a distinct plasma membrane and rippled outer cell membrane without a distinct peptidoglycan layer. The agent did not grow in chicken embryos, and the Weil-Felix test result was negative. In the indirect fluorescent-antibody test, the agent reciprocally cross-reacted with Ehrlichia canis and cross-reacted somewhat with Ehrlichia sennetsu but did not cross-react with Ehrlichia risticii, Neorickettsia helminthoeca, Rickettsia tsutsugamushi, or Chlamydia spp. The mouse antiserum against this agent reacted with 64-, 47-, 46-, 44-, and 40-kDa proteins of E. canis by Western blotting (immunoblotting). Since E. canis and closely related Ehrlichia chaffeensis and Ehrlichia ewingii are not known to proliferate or cause splenomegaly in mice, these results suggest that the agent is a new species within the tribe Ehrlichieae of the family Rickettsiaceae. The finding suggests that wild rodents may serve as reservoirs for pathogenic ehrlichiae.

New Ehrlichia species closely related to Ehrlichia chaffeensis isolated from Ixodes ovatus ticks in Japan.

Seven Ehrlichia strains (six HF strains and one Anan strain) that were obtained from laboratory mice by intraperitoneally inoculating homogenates of adult Ixodes ovatus collected in Japan were characterized. 16S rRNA sequences of all six HF strains were identical, and the sequences were 99.7, 98.2, and 97.7% identical to those of Anan strain, Ehrlichia chaffeensis (human monocytic ehrlichiosis agent), and E. muris, respectively. Partial GroEL amino acid sequencing also revealed that the six HF strains had identical sequences, which were 99.0, 98.5, and 97.3% identical to those of E. chaffeensis, the Anan strain, and E. canis, respectively. All HF strains were lethal to mice at higher dosages and intraperitoneal inoculation, whereas the Anan or E. muris strain induced only mild clinical signs. Light and electron microscopy of moribund mice inoculated with one of the HF strains revealed severe liver necrosis and the presence of numerous ehrlichial inclusions (morulae) in various organs. The study revealed that members of E. canis genogroup are naturally present in Ixodes ticks. HF strains that can cause severe illness in immunocompetent laboratory mice would be valuable in studying the pathogenesis and the roles of both cellular and humoral immune responses in ehrlichiosis caused by E. canis genogroup.

Ehrlichia muris sp. nov., identified on the basis of 16S rRNA base sequences and serological, morphological, and biological characteristics.

The 16S rRNA gene of a new infectious agent, strain AS145T (T = type strain), which was isolated from a wild mouse in Japan, was amplified by using the PCR. The amplimers were directly sequenced by dideoxynucleotide methods with Taq DNA polymerase. Sequence comparisons with other members of the tribe Ehrlichieae and related species revealed that the infectious agent isolated from the mouse is a new species of the genus Ehrlichia that is most closely related to Ehrlichia chaffeensis (level of sequence similarity, 97.9%), an agent of human ehrlichiosis in the United States. This result was consistent with the results of an immunoblot analysis performed with immune sera against different ehrlichiosis agents. On the basis of these findings and other morphological, biological, and serological characteristics of the organism, we propose that ehrlichiae with these properties belong to a new species, Ehrlichia muris.

Impaired antigen specific responses and enhanced polyclonal stimulation in mice infected with Ehrlichia muris.

The immune status of BALB/c mice infected by intraperitoneal inoculation with Ehrlichia muris was examined. The level of E. muris infection in both peritoneal cavity and spleen was greatest at day 10 postinoculation (PI). Thereafter, the infection level was dramatically reduced while the organism persisted for up to 400 days PI. The greatest intraperitoneal infiltration of leukocytes, splenomegaly, and leukocytosis were observed on days 10, 15, and 20 PI, respectively. Infected mice developed marked hypergammaglobulinemia of IgG and IgM that peaked at day 20 PI; however, IgA plummeted at day 15 PI. Of IgG, G2a and G3 increased while G1 and G2b remained constant. Despite hypergammaglobulinemia, both IgG and IgM antibody titers against E. muris were very low throughout the 30-day study. Antibody development and plaque-forming cells against sheep red blood cells (SRBC) were abolished when the antigen was inoculated on day 10 PI. IgM antibody development against SRBC was more severely inhibited than IgG antibody development. However, when mice were immunized with SRBC prior to E. muris infection, antibody development against SRBC was not reduced. Delayed type hypersensitivity reaction to dinitrofluorobenzene was also maximally inhibited when the antigen was administered on day 10 PI. The IFN-gamma level in the blood was maximal at day 10 PI. These results indicate that although the vigorous polyclonal activation and protective IFN-gamma responses occurred by day 10 PI- which cleared most of the ehrlichial infection-antigen-specific immune stimulation was impaired primarily at the level of antigen-priming at peak parasitemia.

Retinoid activation of retinoic acid receptors but not of retinoid X receptors promotes cellular differentiation and replication of human cytomegalovirus in embryonal cells.

The susceptibility of human embryonal cell line NT-2/D1 to replicate human cytomegalovirus (hCMV) is dependent on retinoic acid (RA) stimulation. Physiological responses to retinoic acid involve two distinct subfamilies of nuclear receptors, the RA receptors (RARs) and retinoid X receptors (RXRs), which function by activating transcription as heterodimeric or RXR homodimeric complexes from cis-acting DNA response elements. At present, it is not clear whether the association between these two classes of receptors can lead to multiple distinct induction pathways by signalling one or both receptor partners. Here we have determined, by selectively activating endogenous receptors with novel synthetic ligands specific for either RARs or RXRs, what ligand interaction is physiological in the retinoid receptor pathways necessary for inducing replication of hCMV in differentiated embryonal cells. We show that ligand binding to RAR alone is sufficient and that exclusive ligand activation of RXR is insufficient for inducing replication of hCMV. We also find that differentiation and inhibition of NT-2/D1 cell growth are promoted by compounds that signal the RAR pathway. These results provide direct evidence that RAR ligand-mediated physiological responses are separable and distinct from RXR ligand activation functions. Moreover, our results provide insight into a hormone response pathway for cellular differentiation that might be coopted by hCMV in the host.

Comparison of Ehrlichia muris strains isolated from wild mice and ticks and serologic survey of humans and animals with E. muris as antigen.

In metropolitan Tokyo, the Ehrlichia muris seropositivity rate of 24 wild mice was 63% in Hinohara Village, but in the surrounding areas, it was 0 to 5%. This finding suggests that the reservoir of E. muris is focal. Among the 15 seropositive mice, ehrlichiae were isolated from 9 Apodemus speciosus mice and 1 A. argenteus mouse, respectively. Five ehrlichial isolates were obtained from 10 ticks (Haemaphysalis flava) collected in Asuke Town, Aichi Prefecture, where the E. muris type strain had been isolated. These new isolates were compared with the E. muris type strain. The mouse virulence and ultrastructure of the new isolates were similar to those of the type strain, and all of them were cross-reactive with each other, as well as with the type strain, by indirect immunofluorescent-antibody test. The levels of similarity of the base sequences of the 16S rRNA gene of one of the A. speciosus isolates and one of the tick isolates to that of the E. muris type strain were 99.79 and 99.93%, respectively. We suggest that all of these isolates are E. muris; that E. muris is not limited to Eothenomys kageus but infects other species of mice; and that E. muris is present at locations other than Aichi Prefecture. It appears that H. flava is a potential vector of E. muris. Twenty (1%) of 1803 humans from metropolitan Tokyo were found to be seropositive for E. muris antibodies. A serological survey revealed that exposure to E. muris or organisms antigenically cross-reactive to E. muris occurred among dogs, wild mice, monkeys, bears, deer, and wild boars in Gifu Prefecture, nearby prefectures, and Nagoya City, central Japan. However, human beings and Rattus norvegicus rats in this area were seronegative. These results indicate broader geographic distribution of and human and animal species exposure to E. muris or related Ehrlichia spp. in Japan.

Western immunoblot analysis of Haemobartonella muris and comparison of 16S rRNA gene sequences of H. muris, H. felis, and Eperythrozoon suis.

Infectious agents were isolated from the spleens of three wild mice (Apodemus argenteus) by intraperitoneal inoculation of the spleen homogenate into laboratory mice. The laboratory mice developed clinical signs and splenomegaly, and three isolates were maintained by passage in mice. Tetracyclines were effective in preventing infection of mice with these agents, but streptomycin and penicillin were ineffective. The agents did not grow in bacterial growth media or chicken embryos. In smears of blood from infected mice stained by the Giemsa or the indirect immunofluorescence method, numerous organisms were found on the surfaces of erythrocytes. Electron microscopy revealed cell wall-less pleomorphic cocci of 350 to 700 nm in diameter. On the basis of these results, the isolates were identified as Haemobartonella muris. There was no antigenic cross-reactivity with Rickettsia or Ehrlichia spp. or other related organisms. Western immunoblot analysis of three strains of H. muris with mouse antisera to H. muris revealed identical major antigens of 118, 65, 53, 45, and 40 kDa. By heteroduplex analysis of the three PCR-amplified segments of the 16S rRNA genes, the three strains of H. muris were found to be identical. The 16S rRNA genes of one of the H. muris strains, four strains of H. felis, and two strains of Eperythrozoon suis were sequenced and compared. The sequences of two strains of H. felis from cats in California were identical, as were the sequences of a strain from a cat in Ohio and a strain from a cat in Florida, but the similarity of sequences between the California and the Ohio-Florida strains was only 85%. The sequence of an H. muris strain was unique and was more closely related to that of the Ohio-Florida strain of H. felis (89%) than to that of the California strain of H. felis (84%). The sequence of E. suis from a pig in Illinois was identical to that from another pig from Taiwan. The similarity of the 16S rRNA gene sequence of E. suis with those of three Haemobartonella strains was 84 to 92%, with that of E. suis being most similar to that of the H. felis strain from California. In the phylogenetic analysis based on 16S rRNA gene sequences, the Haemobartonella spp. and E. suis formed a distinct clade more closely related to Mycoplasma spp. (79 to 83% similarity) than to Anaplasma marginale (72 to 75% similarity). Our results suggest that the Haemobartonella spp. and E. suis may be reclassified in the same genus in the family Mycoplasmataceae.