Nonstructural 5A protein of hepatitis C virus regulates heat shock protein 72 for its own propagation.

We identified heat shock protein 72 (Hsp72) as a host factor that was differentially expressed in cells expressing nonstructural 5A (NS5A) protein. To investigate how NS5A modulates Hsp72 in hepatitis C virus (HCV) life cycle, we examined the role of Hsp72 in HCV replication and virus production. NS5A specifically interacted with Hsp72. Both Hsp72 and nuclear factor of activated T cells 5 (NFAT5) levels were increased in cells expressing NS5A protein. Treatments of N-acetylcysteine and glutathione markedly reduced protein levels of both NFAT5 and Hsp72. Knockdown of NFAT5 resulted in decrease in Hsp72 level in cells expressing NS5A. Importantly, silencing of Hsp72 expression resulted in decrease in both RNA replication and virus production in HCV-infected cells. These data indicate that NS5A modulates Hsp72 via NFAT5 and reactive oxygen species activation for HCV propagation.

Superior mesenteric artery syndrome in a tetraplegic patient, 11 years after a spinal cord injury: a case report.

STUDY DESIGN: Case report. OBJECTIVE: To report on the need to consider the possibility of the superior mesenteric artery syndrome (SMAS) even after a long time from the initial spinal cord injury. SETTING: Ulsan, South Korea. METHODS: A 41-year-old man with complete tetraplegia was evaluated for nausea and vomiting. He had a cervical cord injury 11 years previously and his body mass index was 18.6 on admission. The contrast-enhanced abdominal computed tomography (CT) showed intestinal obstruction at the third-portion of the duodenum. With frequent position change and intravenous electrolyte support, the symptoms resolved. There was no relapse of the symptoms with some lifestyle modifications. CONCLUSION: Patients with spinal cord injury may develop SMAS even long after their initial injury.

Effects of tramadol on T lymphocyte proliferation and natural killer cell activity in rats with sciatic constriction injury.

We investigated the effects of acute and chronic tramadol treatment on T lymphocyte function and natural killer (NK) cell activity in rats receiving chronic constriction injury (CCI) of the sciatic nerve. T lymphocyte function was evaluated based on concanavalin-A (ConA)- and phytohemagglutinin (PHA)-induced splenocyte proliferation. NK cell activity was measured by lactic acid dehydrogenase release assay. The effects of tramadol on thermal hyperalgesia were also assessed by measuring paw withdrawal latency (PWL) in the rats. PWL was dose-dependently reversed by tramadol after acute treatment (single subcutaneous injection) with 10, 20, and 30 mg/kg, respectively. There was no significant change among acute treatment groups in NK cell activity, whereas splenocyte proliferation induced by ConA and PHA was significantly suppressed starting from a dose of 20 mg/kg. The reversal of the thermal hyperalgesia persisted throughout a period of chronic tramadol treatment of 40 and 80 mg/kg per day, respectively, with continuous subcutaneous infusion for 7 days at a uniform rate via osmotic minipumps. No modulation of NK cell activity was found in either dose group. However, the activity of splenocyte proliferation was decreased in the 80 mg/kg per day group when compared with the saline and 40 mg/kg per day groups. These data suggest that tramadol treatment has an immunological profile different from pure mu-opioid agonists like morphine, which is known to suppress both NK cell activity and T lymphocyte proliferation at a subanalgesic dose in CCI rats. Considering analgesic and immunosuppressive effects, tramadol treatment may be a better choice than morphine for treatment of chronic neuropathic pain, particularly in patients with compromised immunity.

Nuclear factor kappa B-mediated kainate neurotoxicity in the rat and hamster hippocampus.

Administration of the excitotoxin kainate produces seizure activity and selective neuronal death in various brain areas. We examined the degeneration pattern of hippocampal neurons following systemic injections of kainate in the hamster and the rat. As reported, treatment with kainate resulted in severe neuronal loss in the hilus and CA3 in the rat. While the hilar neurons were also highly vulnerable to kainate in the hamster, neurons in the CA1 area, but not CA3, were highly sensitive to kainate. In both animals, immunoreactivity to anti-p50 nuclear factor kappa B antibody was increased in nuclei of the hilar neurons within 4 h following administration of kainate. Kainate treatment also increased the nuclear factor kappa B immunoreactivity in hamster CA1 neurons and rat CA3 neurons 24 h later. Neurons showing intense nuclear factor kappa B signal were stained with acid fuchsin. Kainate also increased DNA binding activity of p50 and p65 nuclear factor kappa B in the nuclear extract of the hippocampal formation as analysed by electrophoretic mobility shift assay in the hamster, suggesting that activation of nuclear factor kappa B may contribute to kainate-induced hippocampal degeneration. Administration of 100 nmol dizocilpine maleate 3 h prior to kainate attenuated kainate-induced activation of nuclear factor kappa B and neuronal death in CA1 in the hamster. The present study provides evidence that the differential vulnerability of neurons in the rat and the hamster hippocampus to kainate is partly mediated by mechanisms involving N-methyl-D-aspartate-dependent activation of nuclear factor kappa B.

A neuron-specific gene transfer by a recombinant defective Sindbis virus.

We examined the possibility that Sindbis virus, an alpha virus with a single-stranded RNA genome, would be applied for neuronal gene transfer. The recombinant defective Sindbis viruses were constructed by replacing the structural genes of Sindbis virus with genes encoding beta-galactosidase (rdSind-lacZ) or enhanced green fluorescent protein (rdSind-EGFP). In neuron-glia cocultures prepared from the neocortex, hippocampus, and striatum, EGFP or beta-galactosidase was expressed selectively in neurons 24 h after infection with rdSind-EGFP or rdSind-lacZ. Most cortical neurons were infected with rdSind-lacZ at a multiplicity of infection (M.O.I.) of 5 while glial cells were little infected. In addition, transient neuron-specific expression of beta-galactosidase was observed near injection sites over the next 3 d following administration of rdSind-lacZ in adult rat. In the cortical neurons infected with rdSind-EGFP, treatment with NMDA induced neuritic blebs and cell body swelling in a Na+-dependent manner. Therefore, recombinant defective Sindbis viruses can be used as an efficient and selective vector for gene transfer into neurons and applied to investigate biological role of target genes delivered into neurons in vitro and in vivo.

A ribozyme specifically suppresses transformation and tumorigenicity of Ha-ras-oncogene-transformed NIH/3T3 cell lines.

In this study, the efficacy of an anti-ras ribozyme in reversing a transformed phenotype was investigated. A murine NIH/3T3-derived cell line, designated 2-12, contains an inducible Ha-ras oncogene, which is regulated by the Escherichia coli (E. coli) lac operator/repressor system, and displays a transformed phenotype after isopropyl-beta-D-thiogalactoside induction. To reverse the transformed characteristics, the ribozyme, which specifically targets the Ha-ras oncogene at the codon 12 mutation site (GGC to GUC), was transfected into 2-12 cells. Two (ribZ4 and ribZ7) clones were subsequently selected and analyzed for their transforming features. Our results show that, in the transfectants, ribozyme gene expression was detected, and the target Ha-ras transgene was expressed at basal levels. Their phenotypic responses, including morphology, cell growth rate, colony-formation efficiency and tumorigenicity in mice with severe combined immunodeficiency were more similar to those of NIH/3T3 than 2-12 transformed cells. Directly injecting the ribozyme DNA into tumors induced by transformed 2-12 cells in BALB/c mice also caused tumor regression. The enzymatic cleavage products of the ribozyme acting on mutant Ha-ras mRNA in vivo were detected by primer-extension analysis. These results indicate that the ribozyme were designed exhibits a site-specific ribonuclease function that effectively abrogates Ha-ras-oncogene-induced transformation, and this unique anti-Ha-ras property should shed light on the development of strategies against the Ha-ras-oncogene-initiated malignancy.

Caenorhabditis elegans as an environmental monitor using DNA microarray analysis.

In order to assist in the identification of possible endocrine disrupting chemicals (EDC) in groundwater, we are developing Caenorhabolitis elegans as a high throughput bioassay system in which responses to EDC may be detected by gene expression using DNA microarray analysis. As a first step we examined gene expression patterns and vitellogenin responses of this organism to vertebrate steroids, in liquid culture. Western blotting showed the expected number and size of vitellogenin translation products after estrogen exposure. At 10(-9) M, vitellogenin decreased, but at 10(-7) and 10(-5), vitellogenin was increased. Testosterone (10(-5) M) increased the synthesis of vitellogenin, but progesterone-treated cultures (10(-5) M) had less vitellogenin. Using DNA microarray analysis, we examined the pattern of gene expression after progesterone (10(-5), 10(-7), and 10(-9) M), estrogen (10(-5) M), and testosterone (10(-9) M) exposure, with special attention to the traditional biomarker genes used in environmental studies [vitellogenin, cytochrome P450 (CYP), glutathione s-transferase (GST), metallothionein (MT), and heat shock proteins (HSP)]. GST and P450 genes were affected by estrogen (10(-5) M) and progesterone (10(-5) and 10(-7) M) treatments. For vitellogenin genes, estrogen treatment (10(-5) M) caused overexpression of the vit-2 and vit-6 genes (2.68 and 3.25 times, respectively). After progesterone treatment (10(-7) M), the vit-5 and vit-6 were down-regulated and vit-1 up-regulated (3.59-fold). Concentrations of testosterone and progesterone at 10(-9) M did not influence the expression of the vit, CYP, or GST genes. Although the analysis is incomplete, and low doses and combinations of EDC need to be tested, these preliminary results indicate C. elegans may be a useful laboratory and field model for screening EDC.

Thermal stresses reduce natural killer cell cytotoxicity.

The effects of different ambient temperatures (Ta) on the splenic natural killer (NK) cell activity, effector-target cell conjugation activity, and NK cell numbers were assessed in male inbred C3H/HeNCrj mice (7-10 wk old). The splenic NK cytotoxic activities were examined in a 4-h 51Cr release assay in mouse spleen cells that were obtained 1, 2, 4, 8, or 16 days after exposure to Ta of 22, 4, or 35 degrees C. The percentage of conjugating lymphocytes was calculated by counting the number of single lymphocytes bound to single target cells per 400 effector cells. The numbers of NK cells were expressed by the percentage of 5E6-positive cells. The 5E6 identifies only a subset of NK cells. It was found that the splenic NK cell activity, the effector-target cell conjugation activity, or the NK cell number began to fall 1 day after cold (Ta 4 degrees C) or heat (Ta 35 degrees C) stress. After a 16-day period of either cold or heat exposure, the fall in the splenic NK cell activity, the effector-target cell conjugation activity, or the number of 5E6-positive subsets of NK cells was still evident. Compared with those of the control group (Ta 22 degrees C), the cold-stressed mice had higher adrenal cortisol concentration and lower colonic temperature, whereas the heat-stressed animals had higher adrenal cortisol concentration and higher colonic temperature during a 16-day period of thermal exposure. However, neither cold nor heat stress affected both the body weight gain and the spleen weight in our mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms and sites of pyrogenic action exerted by staphylococcal enterotoxin A in rabbits.

The febrile responses induced by i.v. administrations of staphylococcal enterotoxin A (SEA) was mimicked by direct injection of SEA into the organum vasculosum laminae terminalis (OVLT) in unanesthetized rabbits. Compared with the febrile responses induced by i.v. injection of SEA, the OVLT route of injection required a much lower dose of SEA to produce a similar fever. Furthermore, the fever induced by intra-OVLT or i.v. injection of SEA was significantly attenuated by pretreatment with intra-OVLT injection of anisomycin (a protein synthesis inhibitor), indomethacin or diclofenac (inhibitors of cyclo-oxygenase (COX)), and aminoguanidine or dexamethasone (inhibitors of inducible nitric oxide synthase (iNOS)). These results suggest that COX or iNOS pathway in the OVLT mediate the SEA-induced fever in rabbits.

Pyrogenicity of polyadenylic.polyuridylic acid in rabbits.

Polyadenylic.polyuridylic acid injected intravenously into rabbits produced a rapid-onset, monophasic fever. Pyrogenic tolerance occurred in rabbits following daily injections of polyadenylic.polyuridylic acid. However, direct injection of the agent into the preoptic anterior hypothalamic region of rabbit's brain produced a markedly different fever. After an intrahypothalamic injection of polyadenylic.polyuridylic acid, fever was delayed in onset and persisted for a longer period. At room temperature, the fever was due to both increased metabolism and cutaneous vasoconstriction. In a colder atmosphere the fever was due solely to increased metabolism, whereas in the heat the fever was due to reduction in cutaneous blood flow and respiratory evaporative heat loss. In addition, the fever induced by intravenous polyadenylic.polyuridylic acid injection was reversed by a cyclooxygenase inhibitor, but not by a protein synthesis inhibitor. Polyadenylic.polyuridylic acid was shown to stimulate PGE2 production from rabbit's hypothalamus in vitro. The results reveal that this agent is a prostaglandin-dependent pyrogen.

5-Hydroxytryptamine receptors in the hypothalamus mediate thermoregulatory responses in rabbits.

1. The effects of microinjection of 5-hydroxytryptamine (5-HT) or its antagonists methysergide (a 5-HT1 receptor antagonist), cyproheptadine (a mixed 5-HT1/5-HT2 receptor antagonist), or ketanserin (a 5-HT2 receptor antagonist) into the preoptic anterior hypothalamus on thermoregulatory responses were assessed in conscious rabbits at different ambient temperatures (Ta). 2. Intrahypothalamic injection of 5-HT caused dose-dependent hypothermia in rabbits when the Ta was 2 degrees C and 22 degrees C. At 2 degrees C the hypothermia was due to decreased metabolism, whereas at 22 degrees C the hypothermia was due to increased peripheral blood flow and increased respiratory evaporative heat loss. 3. In contrast, administration of either cyproheptadine, methysergide or ketanserin into the 5-HT-sensitive sites in the preoptic anterior hypothalamus caused dose-dependent hyperthermia in rabbits when the Ta was 2 degrees C, 22 degrees C and 32 degrees C. At 2 degrees C the hyperthermia was due to increased metabolism, whereas at 32 degrees C the hyperthermia was due to decreased peripheral blood flow and decreased respiratory evaporative heat loss. At 22 degrees C, the hyperthermia was due to increased metabolism and decreased peripheral blood flow. 4. For a given intrahypothalamic dose (e.g. 15-20 micrograms), either methysergide, cyproheptadine or ketanserin produced the same degree of rectal temperature elevation (e.g. about 1.4 degrees C) in rabbits. Thus, there did not appear to be any association between hypothalamic 5-HT receptor types and thermoregulation. 5. However, the present results suggest that hypothalamic 5-HT receptors mediate thermoregulatory responses in the rabbit.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitumor effect of 2,6-di(2,3-epoxypropoxy)xanthone on tumor cell lines.

2,6-di(2,3-epoxypropoxy)xanthone (EPX), a newly synthesized xanthone derivative, is a potent antitumor agent, which is more cytotoxic than the antitumor drug mytomycin C. EPX also demonstrated stronger growth inhibition to T24 (bladder carcinoma with Ha-ras gene mutation) and 212 cells (a NIH/3T3 derivative, transformed by Ha-ras oncogene) than to PLC/PRF/S (hepatoma with normal Ha-ras gene) and NIH/3T3 cells. The preferential repression of EPX on the cell proliferation of 212 and T24 cells was further demonstrated by decreasing Ha-ras oncogene expression levels while EPX dosage increased. The drug concentrations for 50% inhibition (IC50) of cell growth, DNA synthesis Ha-ras oncogene expression and colony formation of T24 and 212 cells are in the same range and lower than the values for RNA and protein synthesis. Moreover, EPX irreversibly reversed 212 cell morphology from a transformed phenotype to a normal one. These data indicate that EPX probably suppresses tumor cell proliferation by inhibiting DNA synthesis and reverses the transformed properties by suppressing Ha-ras gene expression. The mechanisms of biochemical action and cytotoxicity of EPX remain to be determined. However, our data suggest that the EPX-mediated inhibition of cell proliferative capacity of 212 and T24 cells was preceded by a selective down-regulation of Ha-ras oncogene RNA levels. EPX may have the potential to be used broadly against diverse tumors or specifically against Ha-ras oncogene initiated malignancy.

Synthesis and cytotoxic effect of 1,3-dihydroxy-9,10-anthraquinone derivatives.

1,3-Dihydroxy-9,10-anthraquinone (4) was reacted with epichlorohydrin or 1,omega-dibromo-alkane to yield 1-hydroxy-3-(2,3-epoxypropoxy)-9,10-anthraquinone (5) and 1-hydroxy-3-(3-chloro-2-hydroxypropoxy)-9,10-anthraquinone (6) or 1-hydroxy-3-(omega-bromoalkoxy)-9,10-anthraquinone. Ring-opening of the epoxide (5) or 1-hydroxy-3-(omega-bromoalkoxy)-9,10-anthraquinones with appropriate amines, afforded various 1-hydroxy-3-(3-alkylamino-2-hydroxypropoxy)-9,10-anthraquinones. The synthetic compounds were tested in vitro inhibition of human T-24, Hep 3B, Hep G2, SiHa, HT-3, PLC/PRF/5 and 212 cells. Almost all compounds showed significant inhibitory activity against several different cancer cell lines. Structure-activity analysis indicated epoxidation of the hydroxyanthraquinone increased cytotoxicity against tumour cells, but ring-opening of the epoxide group with amine did not enhance the cytotoxic activity. The phosphatidylserine (PS) externalization and DNA fragmentation in SiHa cells were significantly observed after 48 h incubation with selected compound 19. The results show that 19 cause cell death by apoptosis.

Gamma-pyrone compounds as potential anti-cancer drugs.

The gamma-pyrones, artomunoxanthotrione epoxide, cyclocommunol, cyclomulberrin, and cyclocommunin exhibited potent inhibition of human PLC/PRF/5 and KB cells in-vitro. Dihydroisocycloartomunin showed significant and potent inhibition of human PLC/PRF/5 and KB cells in-vitro, respectively. Cyclomorusin, dihydrocycloartomunin and artomunoxanthone showed significant inhibition of KB cells in-vitro. Based on the above finding and the reported antileukaemic activity of xanthone psorospermin, a series of natural gamma-pyrones was prepared and the inhibition of human PLC/PRF/5 and KB cells in-vitro was measured. Structure-activity analysis indicated the epoxide group substituted at 3-hydroxyl and 2,6-; 3,6-; and 3,5-dihydroxyl xanthone enhanced the anti-tumour activity. The epoxide group substituted at the 6-hydroxyl group of 1,6-dihydroxyxanthone did not show anti-tumour activity.

Xanthone derivatives as potential anti-cancer drugs.

Xanthone derivatives have been shown to be potent inhibitors of tumour growth. Oxygenated xanthones and [3-(dialkylamino)-2-hydroxypropoxy]xanthones have been prepared and tested for in-vitro inhibition of human PLC/PRF/5, KB and 212 cells. Structure-activity analysis indicated epoxidation of the hydroxyxanthone increased cytotoxicity against tumour cells but ring-opening of the epoxide group with dialkylamine did not enhance the anti-tumour activity. Further evaluation of three of the most active compounds 2, 6-, 3, 6-, and 3, 5-di(2,3-epoxypropoxy)xanthone (compounds 10a, 11a, and 12a, respectively) in DNA, RNA and protein synthesis of tumour cells showed potent inhibitory activity. The 3,5-di(2,3-epoxypropoxy)xanthone also showed potent inhibitory activity against 212 cells, a Ha-ras oncogene-transformed NIH 3T3 cell line. The results indicated that compounds 10a and 12a are potent anti-tumour agents which not only suppressed cellular DNA, RNA and protein synthesis but also specifically inhibited the Ha-ras oncogene in 212 cells.

Interferon produces hyperthermic responses in rats.

The effects of administration of alpha human leukocyte interferon into the anterior hypothalamic preoptic area on metabolic, respiratory and vasomotor activities, as well as body temperatures, were assessed in unanesthetized rats at various ambient temperatures (Ta). Intrahypothalamic administration of interferon produced dose-dependent fever in rats at Ta = 8-30 degrees C. The interferon-induced fever was due to increased metabolism and/or cutaneous vasoconstriction (or decreased heat loss). Furthermore, the fever induced by interferon was antagonized by pretreatment of animals with indomethacin, an inhibitor of prostaglandin synthesis. The data indicate that the fever induced by intrahypothalamic interferon is due to the endogenous release of prostaglandins in the anterior hypothalamic preoptic area of rat's hypothalamus.

Targeting microglial activation in stroke therapy: pharmacological tools and gender effects.

Ischemic stroke is caused by critical reductions in blood flow to brain or spinal cord. Microglia are the resident immune cells of the central nervous system, and they respond to stroke by assuming an activated phenotype that releases cytotoxic cytokines, reactive oxygen species, proteases, and other factors. This acute, innate immune response may be teleologically adapted to limit infection, but in stroke this response can exacerbate injury by further damaging or killing nearby neurons and other cell types, and by recruiting infiltration of circulating cytotoxic immune cells. The microglial response requires hours to days to fully develop, and this time interval presents a clinically accessible time window for initiating therapy. Because of redundancy in cytotoxic microglial responses, the most effective therapeutic approach may be to target the global gene expression changes involved in microglial activation. Several classes of drugs can do this, including histone deacetylase inhibitors, minocycline and other PARP inhibitors, corticosteroids, and inhibitors of TNFα and scavenger receptor signaling. Here we review the pre-clinical studies in which these drugs have been used to suppress microglial activation after stroke. We also review recent advances in the understanding of sex differences in the CNS inflammatory response, as these differences are likely to influence the efficacy of drugs targeting post-stroke brain inflammation.

Reproductive endocrine disruption in a sentinel species (Chrysemys picta) on Cape Cod, Massachusetts.

Freshwater turtles (Chrysemys picta) were collected from two sites on Cape Cod, MA. One site (Moody Pond), adjacent to the Massachusetts Military Reservation (MMR), was considered potentially impacted by toxic agents deriving from contaminant point sources on the MMR. The second (reference) site (Washburn Pond), to the east of the MMR, was considered not impacted by these pollutants and was chosen as a control site. Plasma estradiol 17 beta and vitellogenin were significantly lower in female turtles from Moody Pond. Ovarian follicular analysis indicated a significant decrease in the >16.00-mm follicular cohort in Moody Pond female turtles compared with Washburn Pond animals. Although testicular weight was lower at the Moody Pond site, histology, plasma testosterone, and sperm number were similar to these parameters in Washburn Pond animals. The data suggest that in Moody Pond, the reproductive capacity of turtles may be negatively affected by contaminants from the MMR.

Alpha-methyl-p-tyrosine reduces poly I:C-induced augmenting interferon production and splenic natural killer cell activity in mice.

The present investigation was designed to extend our previous observations that alpha-methyl-p-tyrosine (AMPT; an inhibitor of norepinephrine synthesis) reduced splenic natural killer (NK) cell activity and to test whether NK augmentation or augmenting interferon (IFN) production induced by polyriboinosinic acid:polyribocytidylic acid (poly I:C; an IFN inducer) can be attenuated by AMPT in mice. Therefore, in the current study, the effects of AMPT (300 mg/kg i.p.) on splenic NK activity or plasma titers of IFN-alpha+beta were assessed in inbred C57BL/6 mice. It was found that treatment with AMPT reduced both poly I:C induced serum IFN and splenic NK activity in a dose- and time-dependent manner. These results suggest that AMPT inhibits IFN production and decreases splenic NK activity in vivo.

Endogenous pyrogen formation by human blood monocytes stimulated by polyriboinosinic acid:polyribocytidylic acid.

The pyrogenic response to supernatants from human blood monocytes stimulated with polyriboinosinic acid:polyribocytidylic acid (poly I:C) was characteristic of a response to endogenous pyrogen in that it was brief and monophasic, and was destroyed by heating the supernatants at 70 degrees C for 30 min. Pyrogen production was unimpaired when the incubations were carried out in the presence of cycloheximide (50 micrograms/ml; an inhibitor of protein synthesis) or indomethacin (50 micrograms/ml; an inhibitor of prostaglandin synthesis). Also, neither interferon, interleukins, tumor necrosis factor nor prostaglandin E2 were detectable in the supernatants from the poly I:C-stimulated human monocytes.