A novel selective medium for isolation of Aggregatibacter (Actinobacillus) actinomycetemcomitans.

BACKGROUND AND OBJECTIVE: Conventional selective media have been used for the selection of Aggregatibacter (Actinobacillus) actinomycetemcomitans in clinical samples. The proportion of A. actinomycetemcomitans grown on the selective media in vitro may not reflect the true counts in vivo because of the low selectivity. A novel selective medium, designated AASM, was developed for the isolation of A. actinomycetemcomitans. MATERIAL AND METHODS: AASM was prepared by adding of 200 microg/mL of vancomycin and 10 U/mL of bacitracin to AAGM, which contains dextrose, sodium bicarbonate, trypticase soy, yeast extract and agar. Clinical efficacy was evaluated by the recovery, on AASM, of A. actinomycetemcomitans from subgingival samples of 44 periodontally healthy subjects and 76 patients with chronic periodontitis. RESULTS: All serotypes (a-f) of A. actinomycetemcomitans strains grew well, and the average growth recovery of A. actinomycetemcomitans on AASM medium was 94.4% (80.0-109.7%) of that on AAGM. The exclusive rate of other bacteria was 99.9% in clinical samples cultured on AASM. A. actinomycetemcomitans was not detected in periodontally healthy persons but was detected in 25 (32.9%) patients with chronic periodontitis. The predominant serotype was c, detected in 11 subjects. CONCLUSION: The new selective medium, AASM, was highly selective for A. actinomycetemcomitans, eliminated possible false-positive results and was useful for the isolation of A. actinomycetemcomitans from clinical samples.

Acute and chronic intracerebroventricular morphine infusions affect long-term potentiation differently in the lateral perforant path.

Experiments were performed to investigate the effects of acute and chronic intracerebroventricular (icv) morphine infusions via osmotic minipumps on long-term potentiation (LTP) in the lateral perforant path (LPP)-granule cell synapse of the rat dentate gyrus. Although significant antinociceptive activity was observed when morphine was infused (25 nmol/microl/h) for 30 min or 1 h, the activity was not observed in rats receiving morphine chronically for 72 h, and the tail-flick latency of this group was comparable to that of rats receiving saline. LTP induction was significantly attenuated after acute morphine infusion (1 h) in LPP-granule cell synapses of the dentate gyrus. In contrast, LTP induction was augmented after chronic morphine infusion for 72 h. Bath-perfused morphine augmented the baseline population spike (PS) amplitude in rats treated with saline, whereas it attenuated the LTP induced by chronic morphine infusion. Returning the LTP to the level of saline infusion after in vitro morphine perfusion suggests that enhancement of the LTP is a withdrawal-like phenomenon. These results suggest a difference between the effects of acute and chronic intracerebroventricular morphine infusions on synaptic plasticity in the LPP-granule cell synapses of the dentate gyrus.

Design of a unique PCR primer for detection of oral HPV infection.

Human papillomavirus (HPV) has been known as a pathogen of oral dysplasia. However, suitable PCR primers for the detection of oral HPV infection have not been reported. The aim of this study was to design unique consensus primers. The consensus primers were designed by homologues analyses between subtypes 2, 6, 11, 13, 16, 18, 30, 32 and 58, which frequently infect the oral membrane. PCR, with our designed primer, detected HPV DNA subtypes 2, 6, 11, 16, 18 and 58, and also showed PCR product from a clinical papilloma sample. These results indicate that our designed consensus primer can be used for the study of the relationships between oral disease and HPV infection.

Effect of NMDA receptor antagonists on protein kinase activated by chronic morphine treatment.

In a previous study we indicated the involvement of the N-methyl-D-aspartate (NMDA) receptor in the development of morphine dependence as assessed by naloxone-induced rise in norepinephrine release in chronically morphine-treated rats. In the present experiments, we studied (1) the possible role of protein kinases in the increased norepinephrine release occurring after naloxone injection and (2) the effects of NMDA receptor antagonists on chronic morphine exposure-induced changes in protein kinase activity. The naloxone-induced rise in norepinephrine release was attenuated by concomitant administration of a protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine hydrochloride (H-7) or an NMDA receptor antagonist, (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine hydrogen maleate (dizocilpine, MK-801) with morphine. Both cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), which mediate neurotransmitter release, were clearly activated in the cytosol of the pons/medulla, but not in that of the hippocampus, in chronically morphine-treated rats. This activation of PKA and PKC by chronic morphine treatment was inhibited by infusion of dizocilpine or D(-)-2-amino-5-phosphonopentanoic acid (AP-5), an ionotropic glutamate receptor antagonist, together with morphine. These results suggest that NMDA receptor antagonists inhibit the increase in protein kinase activity produced by chronic morphine treatment, thus suppressing the naloxone-induced rise in norepinephrine release.

Inhibitory effect of the NMDA receptor antagonist, dizocilpine (MK-801), on the development of morphine dependence.

We investigated the effect of a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imin ehydrogen maleate (dizocilpine, MK-801), on hippocampal norepinephrine release in morphine-treated rats in order to clarify the relationship between NMDA receptors and the development of morphine dependence. Naloxone hydrochloride injected subcutaneously (s.c.) into morphine-dependent rats, induced an immediate increase in hippocampal norepinephrine release, which was associated with a typical morphine withdrawal syndrome. The increased norepinephrine levels persisted for at least 2 hr, even after the disappearance of the behavioral withdrawal syndrome. This striking effect of naloxone on hippocampal norepinephrine release was dependent on the duration of the intracerebroventricular (i.c.v.) morphine infusion. Pretreatment with dizocilpine (s.c.) before naloxone challenge reduce the rate of the rise in hippocampal norepinephrine release induced by naloxone in morphine-treated rats. Concurrent infusion (i.c.v.) of dizocilpine and morphine decreased the level of hippocampal norepinephrine release after a naloxone challenge. Both pretreatment with dizocilpine (s.c.) before naxolone injection and infusion (i.c.v.) of dizocilpine suppressed rearing and teeth-chattering signs, but not wet-dog shakes in morphine-treated rats. These results suggest that dizocilpine attenuates the development of morphine dependence through NMDA receptors, and thus that interaction between opioid receptors and NMDA receptors may be involved in the development of morphine dependence.

Effects of repeated administration of a kappa-opioid agonist on phorbol ester binding to membrane-bound protein kinase C in rat brain.

The dissociation constant (KD) of [3H]phorbol 12,13-dibutyrate (PDBu) binding to protein kinase C (PKC) in membranes of rat cortex and midbrain was significantly decreased with no change in the receptor density (Bmax) following 7-d treatment with a kappa-opioid agonist, U-50,488 (20 mg/kg/d, i.p.). Neither the Bmax nor KD values in pons/medulla were altered by repeated U-50,488 treatment. These results suggest that repeated administration of a kappa-opioid agonist increases the affinity for PDBu binding to the membrane-bound PKC in rat cortex and midbrain, but not in pons/medulla.

Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.

An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with Eco T22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.

Induction of antibodies against structural proteins of hepatitis C virus in mice using recombinant adenovirus.

Replication-deficient recombinant adenoviruses expressing structural proteins of hepatitis C virus (HCV) were constructed. Each recombinant lacks adenoviral E1A and E3 genes and bears expression units for HCV structural proteins. The expression units contain HCV cDNAs coding for either the protein or core, one of two envelopes (E1 and E2) or all of these structural proteins (core, E1 and E2) under the control of the SR alpha promoter. In HeLa or HepG2 cells, the recombinants can express efficiently HCV genes after infection without replication of the recombinants. We detected 22-kDa core, 35-kDa E1 and 58-kDa E2 proteins of HCV in these cells. The recombinant expressing all three HCV structural proteins was inoculated into mice. Antibodies to each of the three HCV proteins were detected in all of the ten mice tested. The results indicate that the recombinant adenoviruses efficiently express HCV genes and induce specific antibody against the expressed HCV proteins in animals.

Influence of chronic opioid treatment on low Km GTPase activity in rat brain: evidence for the involvement of G-proteins in opioid tolerance.

To investigate G protein function during the initial state of opioid tolerance, low Km GTPase activity was measured following chronic treatment with morphine (mu agonist) and butorphanol (mu/delta/kappa mixed agonist) in rats. Chronic opioid administration (20 mg/kg, IP) was performed once a day for 7 consecutive days. Under these conditions, antinociceptive tolerance to morphine but not butorphanol was developed. Chronic morphine treatment enhanced basal low Km GTPase activity in the pons/medulla, but not in the cortex and midbrain. On the other hand, chronic butorphanol treatment had no effect on basal low Km GTPase activity. These results suggest that chronic in vivo treatment of rats with mu agonists leads to an increase in the hydrolysis of GTP to GDP, by a basal low Km GTPase activity of G-proteins in the pons/medulla and that an enhancement of GTPase activity in this specific area may contribute to the development of antinociceptive tolerance to mu agonists.

A protein kinase inhibitor, H-7, inhibits the development of tolerance to opioid antinociception.

To investigate the effect of a protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), on the development of tolerance to antinociception induced by morphine (mu-opioid receptor agonist) and butorphanol (mu/delta/kappa-mixed opioid receptor agonist), rats were infused i.c.v. with morphine, butorphanol and H-7 through osmotic minipumps for 3 days. Concomitant infusion of H-7 dose dependently inhibited the development of tolerance to i.c.v. morphine- and butorphanol-induced antinociception. These results suggest that protein kinases may play an important role in the development of opioid tolerance.

Repeated administration of opioids alters characteristics of membrane-bound phorbol ester binding in rat brain.

Scatchard analysis of saturation binding data indicated that dissociation constant (KD) of [3H]phorbol 12,13-dibutyrate (PDB) binding to the membrane-bound protein kinase C of rat cortex and midbrain was significantly decreased following systemic repeated administration of morphine (mu-opioid receptor agonist) and butorphanol (mu/delta/kappa-mixed opioid receptor agonist). No change in the receptor density (Bmax) of [3H]PDB binding was found following repeated treatment with morphine and butorphanol. On the other hand, neither the Bmax nor KD values in pons/medulla were altered in these rats. These results suggest that systemic repeated opioid treatment, such as morphine and butorphanol leads to an increased affinity for phorbol ester binding to the membrane-bound protein kinase C in rat cortex and midbrain.

Enhancement of the rate of GTP hydrolysis in rat brain by repeated kappa-opioid treatment.

To investigate the G protein and protein kinase C (PKC) systems during the initial state of kappa-opioid tolerance, the low Km GTPase and PKC activities were measured following repeated treatment of rat with the kappa-agonist, U-50,488. In behavioral studies, antinociceptive tolerance to U-50,488 was developed following 7-day treatment with U-50,488. Under these conditions, repeated administration of U-50,488 significantly enhanced the basal low Km GTPase activity in the pons/medulla but not in the cortex and midbrain regions. On the other hand, repeated U-50,488 treatment had no effect on PKC activity in cytosol and membrane fractions under the calcium-chelating conditions. These results indicate that repeated administration of kappa-agonist, U-50,488, increases in the basal hydrolysis of GTP to GDP in rat pons/medulla but not PKC activity which was observed in the case of repeated administration with morphine in rats.

Influence of chronic morphine treatment on protein kinase C activity: comparison with butorphanol and implication for opioid tolerance.

The aim of this study was to determine whether chronic opioid treatment could influence the protein kinase C (PKC) activity in the rat brain. Chronic morphine (microns agonist) but not butorphanol (mu/delta/kappa mixed agonist) treatment enhanced cytosolic PKC activity in the pons/medulla, but not in the cytosolic fractions of cortex and midbrain regions. Concomitant administration of the opioid receptor antagonist, naloxone, blocked the PKC upregulation by chronic morphine. Chronic administration of morphine and butorphanol produced no change in the membrane PKC activity. Antinociceptive tolerance to morphine but not to butorphanol was developed under these conditions. These results suggest that chronic morphine administration leads to an upregulation of the cytosolic PKC activity in the pons/medulla through repeated activation of mu opioid receptors and that the PKC upregulation in this specific area may contribute to the morphine tolerance.

A simple and efficient method for purification of infectious recombinant adenovirus.

Recently, the adenovirus expression vector attracts much attention for the application to gene therapy and the method to purify and concentrate adenovirus without loss of infectivity has become very important, especially for animal experiments and gene therapy of humans. In this report, we show a simple and efficient method for purifying infectious adenovirus. The method consists of sequential centrifugation in CsCl step gradients without loss of infectivity and can be completed in one day. The method maintained the viral infectivity after 10-fold concentration and seemed to remove more than 99.9% of carried-over proteins. We showed also that the buffers for dialyzing the purified virions influenced the stability of infectivity. The buffers of 10 mM HEPES-1 mM EDTA-10% glycerol and PBS(-)-10% glycerol resulted in higher stability than did 10 mM HEPES-1 mM MgCl2-10% glycerol. The method is may be useful in many applications of recombinant adenovirus.

Involvement of kappa-opioid receptors in opioid dependence/withdrawal: studies using butorphanol.

The dependence liability of a class of opioid agonist/antagonist analgesics, e.g. pentazocine, butorphanol, and buprenorphine, is widely recognized. However, the relative involvement of mu-, delta-, and kappa-opioid receptors mediating physical dependence on these compounds is not completely known. In the present study, butorphanol dependence was produced by continuous intracerebroventricular (i.c.v.) infusion of butorphanol (26 nmol/h) for 3 days in male Sprague-Dawley rats. Nor-binaltorphimine, a long-acting kappa-opioid receptor antagonist, and naloxone, a nonspecific antagonist, were administered i.c.v. to precipitate withdrawal in butorphanol-dependent animals, so as to investigate the involvement of central kappa-opioid receptors in opioid dependence. ED50 ratios (naloxone/nor-binaltorphimine) for eliciting withdrawal signs were: teeth-chattering (1.25), yawning (2.13), and ejaculation (0.72). Our data indicate that nor-binaltorphimine precipitated withdrawal behaviors similar to those precipitated by naloxone. It appears that central kappa-opioid receptors may play a major role in the development of butorphanol dependence in rats.

Characterization of HCV structural proteins expressed in various animal cells.

Hepatitis C virus (HCV) is a main causative agent for transfusion-associated and sporadic cases of non-A, non-B hepatitis throughout the world. HCV has a positive-strand RNA of about 9,400 nucleotides as its genome, whose organization is similar to those of animal pestiviruses or human flaviviruses. In spite of the lack of an effective replication system in tissue culture cells, genes coding for viral proteins of HCV have been identified. The putative nucleocapsid (p22) and envelope (gp35 and gp60) proteins have been expressed in cells by different vectors under various foreign promoters. Furthermore, a truncated core protein and association of envelope proteins with nonstructural proteins have also been observed. These synthesized viral proteins have been shown to be useful for diagnostic assays.

[Inhibitory effect of bithionol on NADH-fumarate reductase in ascarides].

To elucidate the mechanism of anthelmintic action of bithionol, the inhibitory effect of the drug on NADH-fumarate reductase (NADH-FR) of Ascaris lumbricoides suum was examined. NADH-FR, an enzyme of anaerobic carbohydrate metabolic pathway was solubilized from the mitochondria of the worm's muscle with deoxycholate, and then partially purified with the monoethanolamine-Sepharose 4B column chromatography. Rhodoquinone (RQ), which is required for the electron transfer from NADH to fumarate, was separated from the enzyme protein and phospholipids. Although the enzyme protein fraction eluted from the above column did not show NADH-FR activity, this enzyme was reactivated by the addition of purified RQ and phosphatidylcholine. The IC50 value of bithionol for reconstituted NADH-FR was 18 +/- 2 microM. The inhibition type was competitive to RQ. Bithionol inhibited at most 30% NADH-ferricyanide reductase, which did not require RQ, even at high concentration of 150 microM. These results suggest that the pharmacological action of bithionol, a phenolic anthelmintic, depends on the inhibition of the electron transport system by the competition with RQ.

Inhibitory effects of NaCl and guanyl-5'yl-imidodiphosphate (GppNHp) on [3H]naloxone binding to kappa-opioid receptors in guinea pig cerebellum.

Studies were performed to characterize the opioid receptors in guinea pig brain using the radiolabeled opioid antagonists, [3H]naloxone and [3H]diprenorphine and the kappa-agonist [3H]U-69593. The binding of [3H]U-69593 to guinea pig cerebellar membranes was reduced by NaCl, guanyl-5'yl-imidodiphosphate (GppNHp) and NaCl+GppNHp, and [3H]naloxone binding to cerebellar membranes was also reduced by NaCl and GppNHp. In the guinea pig cerebral cortex and striatum and the rat cerebellum, [3H]naloxone binding was not affected significantly by GppNHp in the presence or absence of 100 nM [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) and [D-Ala2, D-Leu5]enkephalin (DADLE). Guinea pig cerebellar [3H]diprenorphine binding was not affected by NaCl, GppNHp or NaCl+GppNHp. Furthermore, [3H]naloxone binding was reduced after pretreating cerebellar membranes with N-ethylmaleimide (NEM), which also attenuated GppNHp-induced inhibition of cerebellar [3H]naloxone binding. These results suggest that the properties of [3H]naloxone binding in guinea pig cerebellum differ from those in other brain regions and rat cerebellum, and that the interaction of [3H]naloxone and [3H]U-69593, but not [3H]diprenorphine, with guinea pig cerebellar opioid receptors is associated with a G-protein.

Effects of nor-binaltorphimine on butorphanol dependence.

Butorphanol has been shown to act on mu-, delta-, and kappa-opioid receptors. However, the relative involvement of different opioid receptor subtypes in butorphanol dependence is not known. In the present study, nor-binaltorphimine, a long-acting non-peptide kappa-opioid receptor antagonist, was employed to mask central kappa-opioid receptors before and during the induction of butorphanol dependence in rats, so that the involvement of kappa-opioid receptors could be elucidated. The results revealed that treatment with nor-binaltorphimine markedly blocked naloxone-precipitated withdrawal signs of escape behavior, teeth-chattering, wet shakes, ptosis, body weight loss, and hypothermia at all doses tested, and attenuated the withdrawal symptoms of forepaw tremors (24 nmol: P < 0.001) and diarrhea (12 nmol: P < 0.05; 24 nmol: P < 0.01). In contrast, nor-binaltorphimine had no effect on yawning, ejaculation, nor urination in butorphanol-infused rats undergoing withdrawal. Three days of butorphanol infusion significantly increased KD values (in the cortex and striatum), decreased Bmax (in the cortex only) of [3H]U-69,593 binding, and shifted Ki of nor-binaltorphimine against [3H]U-69,593 (4.5 nM) binding in the cortex by more than 10-fold. Treatment with nor-binaltorphimine blocked the effects of butorphanol on kappa-opioid receptors. It is therefore concluded that kappa-opioid receptors are involved in mediating escape behavior, teeth-chattering, wet shakes, forepaw tremors, ptosis, diarrhea, weight loss, and hypothermia in butorphanol-dependent rats undergoing withdrawal. Furthermore, kappa-opioid receptors become desensitized to agonists (in the cortex and striatum), down-regulated (in the cortex), and supersensitive to antagonists in butorphanol-dependent rats.