Carcinogen metabolism in human lung tissues and the effect of tobacco smoking: results from a case--control multicenter study on lung cancer patients. Cigarette smoking is the strongest risk factor for lung cancer, but genetically determined variations in the activities of pulmonary enzyme that metabolize tobacco-derived carcinogens may affect individual risk. To investigate whether these enzymes (e.g., CYP1A-related) can serve as markers for carcinogen-DNA damage, lung tissue specimens were taken during surgery from middle-aged men with either lung cancer or non-neoplastic lung disease. Phase I [aryl hydrocarbon hydroxylase (AHH), ethoxycoumarin O-deethylase (ECOD)] and phase II (epoxide hydrolase, UDP-glucuronosyltransferase, glutathione S-transferase) enzyme activities, glutathione and malondialdehyde contents were determined in lung parenchyma and/or bronchial tissues; some samples were also analyzed for DNA adducts, using 32P-postlabeling. The data were then analyzed for the following: a) differences in metabolic profiles between bronchial and parenchymal lung tissue; b) the effect of recent exposure to tobacco smoke on enzyme inducibility and benzo[a]pyrene metabolism; c) differences in enzyme inducibility between lung cancer and non-lung cancer patients; d) the effect of smoking on metabolism of mutagens in vitro; e) pulmonary DNA adduct levels and AHH activity in lung parenchyma of smokers and ex-smokers; f) lipid peroxidation products in lung tissue from lung cancer and non-lung cancer patients, as related to smoking habits and degree of airway obstruction; and g) prognostic value of AHH pulmonary activity in lung cancer patients. The results demonstrate a pronounced effect of tobacco smoke on pulmonary metabolism of xenobiotics and prooxidant state and suggest the existence of a metabolic phenotype at higher risk for tobacco-associated lung cancer.


Integrated approach for evaluating species and interindividual differences in responsiveness to dioxins and structural analogs. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous environmental contaminant that is produced inadvertently during the synthesis of some organochlorine compounds, such as the chlorinated phenoxy pesticides. It is biologically and ecologically persistent, with an estimated half-life of 7 years in humans. It possesses high acute toxicity in rodents and is a carcinogen, teratogen, and immunotoxin. In chronic bioassays for carcinogenicity, TCDD at a dose of 10 ng/kg/day increases the incidence of liver tumors in female rats, making it one of the most potent animal carcinogens ever tested. A recent study in humans has shown an increase in the incidence of respiratory tract tumors in workers in chlorinated phenoxy herbicide plants. Considerable controversy and uncertainty remain, however, concerning its carcinogenic potency in humans and the reliability of using animal data to predict human risks. It is generally accepted that most, if not all, of the effects of TCDD require its binding to the Ah receptor. In addition to its toxic effects, TCDD produces a number of biochemical effects, such as induction of CYP1A1, downregulation of binding activity of the estrogen and epidermal growth factor (EGF) receptors, and changes in cytokine pathways. These effects suggest that the Ah receptor plays an important role in regulating the cell cycle. A number of structural analogs of TCDD, such as the polychlorinated dibenzofurans, also interact with the Ah receptor, and they produce the same spectrum of responses as TCDD in animal and cell models. The potency of these compounds is strongly correlated with their binding affinity to the Ah receptor.(ABSTRACT TRUNCATED AT 250 WORDS)


Immunohistochemical detection of pulmonary cytochrome P450IA and metabolic activities associated with P450IA1 and P450IA2 isozymes in lung cancer patients. The main polycyclic aromatic hydrocarbon-inducible cytochrome P450 was studied in lung tissue from 57 lung cancer patients by immunohistochemistry, using a monoclonal antibody (1-7-1) that recognizes P450IA1 and P450IA2 isozymes. The intensity of immunostaining was compared with the pulmonary activity of a P450IA1-dependent enzyme, aryl hydrocarbon hydroxylase (AHH), and with P450IA2-related metabolic activity estimated from the ratio of caffeine metabolites in urine. Immunostaining was not observed in peripheral lung tissue of nonsmokers or ex-smokers but was seen in the bronchiolar and alveolar epithelium of all patients who were smokers and had a peripheral carcinoma (16/16) and of 60% (10/17) of those who had a bronchial carcinoma. AHH activity was positively related to the intensity of immunostaining, and an almost 2-fold increase due to smoking was detected in the ratios of caffeine metabolites. These results demonstrate that tobacco smoke induces P450IA1 in the lung and probably P450IA2 in the liver, and suggest a role for certain metabolic phenotypes of P450IA1 in peripheral pulmonary carcinoma.


Biomarkers for individual susceptibility to carcinogenic agents: excretion and carcinogenic risk of benzo[a]pyrene metabolites. In rats exposed to a single intraperitoneal dose of 200 mg/kg of the environmental carcinogen benzo[a]pyrene (BP) in sunflower oil, significant individual variations in excretion of the BP activation (BP-7,8-diol) and deactivation (3-OH-BP) derivatives were found. Most rats developed peritoneal sarcomas. Only the levels of BP-7,8-diol excreted in the urine correlated directly with the latency of tumor formation. After a similar exposure to a dose of 100 mg/kg BP, Macaca fascicularis monkeys excreted smaller quantities than rats of both metabolites. After rats were given 10 intraperitoneal injections each of 10 mg/kg of BP in a water-lipid emulsion, the excreted levels of both metabolites after the first, fifth, and tenth injection were lower than those of the rats that received 200 mg/kg. BP metabolites were also detected in the urine of lung cancer patients who were heavy smokers. The applicability of monitoring the excretion of the BP metabolites to predicting individual cancer risk is discussed.


Improved isolation of salmonellae from faeces using a semisolid Rappaport-Vassiliadis medium. A modified semisolid Rappaport-Vassiliadis (MSRV) enrichment medium was evaluated as an alternative to Rappaport-Vassiliadis (RV) broth for culture of salmonellae from faeces. Faeces from 1544 subjects were cultured using MSRV medium, selective agars, and RV and selenite F enrichment broths. Of the 183 strains of Salmonella spp. isolated, 88% were recovered on MSRV medium, whilst only 59% were recovered using RV broth. When MSRV medium was combined with direct culture and selenite enrichment, 98.9% of salmonellae were recovered. The MSRV culture was found to be easy to read, and in most cases confirmation of organisms as Salmonella spp. could be made 24 hours after receipt of the faecal specimen.


Cytologic evidence of the association of different infective lesions with dysplastic changes in the uterine cervix. Cervical smears from 4055 women were examined and classified as per WHO criteria. 873 (21.53%) smears revealed dysplastic changes. The dysplastic smears were further examined cytologically for the presence of Chlamydia trachomatis, Herpes simplex virus type-2, Human papilloma virus, Trichomonas vaginalis and Candida albicans. Chlamydia was found to be the commonest micro-organism associated with cervical dysplasia followed by Herpes simplex virus type-2 and Human papilloma virus. The association of Trichomonas and Candida with cervical dysplasia was found to be insignificant. Cervical dysplasia associated with Herpes simplex virus type-2 commonly occurred in the early reproductive life. The data observed in this study provide useful baseline information for detecting the subjects harbouring the infective microbes in the cervical epithelium.


Tumor-suppressor genes: cardinal factors in inherited predisposition to human cancers. A predisposition to the development of certain specific and familial cancers is associated with the inheritance of a single mutated gene. In the best-characterized cases, this primary mutation is a loss of function mutation consistent with viability but resulting in neoplastic change consequent to the acquisition of a second somatic mutation at the same locus. Such genes are referred to as tumor-suppressor genes. Classical examples are the Rb-1 gene associated with the development of retinoblastoma and the p53 gene, which is associated with a wider range of neoplasms, including breast cancer. Other tumor-suppressor genes have been isolated which are associated with Wilms' tumor, neurofibromatosis, and inherited and sporadic forms of colorectal cancer. Some of these genes appear to act as negative regulators of mitotic cycle genes, and others may have different properties. The nature of these genes is discussed, as is the evidence for the involvement of tumor-suppressor genes in other inherited, and sporadic, forms of cancer. Some recent data on the Wilms' tumor gene, WT1, and on the involvement of the p53 gene in breast cancer are presented, and the importance of genomic imprinting in contributing to the excess of suppressor gene mutations in chromosomes of paternal origin is considered.


Krukenberg tumor. A report of six cases. The Authors present six cases of ovarian neoplasm with histological characteristics of typical Krukenberg tumor. Usually, Krukenberg Tumor is bilateral and often secondary to gastrointestinal carcinomas. Secondary ovarian neoplasms often become evident a short time after the diagnosis of primary tumor. Moreover, the post ovulatory rearrangement of the ovarian surface could favour the metastatic spread of cancerous cells. In four cases TAG 72 serum levels were useful in the monitoring of relapsing disease.


Pentoxifylline modulates deformability, F-actin content, and superoxide anion production of polymorphonuclear leukocytes from diabetic cats. Capillary occlusion is an early event in the development of diabetic retinopathy, and white blood cells have recently been shown to be involved. We have shown previously that pentoxifylline improves deformability and decreases F-actin content of unstimulated polymorphonuclear leukocytes from normal human subjects. The purpose of this study was to determine if pentoxifylline would improve three properties of unstimulated polymorphonuclear leukocytes from diabetic cats. The measured parameters were mechanical (whole cell deformability), structural (F-actin content) and biochemical (rate of superoxide anion production). Chronic hyperglycemia was induced in three cats by partial pancreatectomy, and they were kept in poor glycemic control for at least 6 months prior to the study. Polymorphonuclear leukocytes were isolated and the entry time of individual passive cells was measured during aspiration into a 4-micron micropipette under constant suction pressure (-15 cmH2O). Deformability was defined as the inverse of the entry time. F-actin content of passive cells was measured by NBD-phallacidin labeling followed by flow cytometry. The rate of superoxide anion production was measured spectrophotometrically by superoxide dismutase-inhibitable cytochrome c reduction. Following incubation for 15 min with 0.1, 1.0 and 10.0 mM pentoxifylline, the average entry time of passive polymorphonuclear leukocytes was reduced from control by 11 +/- 5% (P = 0.045), 17 +/- 6% (P = 0.007), and 36 +/- 5% (P < 0.001), respectively. The F-actin content decreased by 0%, 4 +/- 0.6% (P < 0.001), and 10 +/- 3% (P < 0.001), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Polarized budding of vesicular stomatitis and influenza virus from cultured human and bovine retinal pigment epithelium. The retinal pigment epithelium (RPE) is able to perform a variety of functions because of its high degree of plasma membrane polarity. Some aspects of this polarity such as the localization of the majority of Na-K ATPase to the apical membrane distinguish the RPE from kidney cells and most other transporting epithelia. The polarized budding of enveloped viruses such as vesicular stomatitis and influenza from the basolateral and apical membrane, respectively, has been used to study mechanisms underlying the domain-specific sorting of membrane proteins in cultured epithelial cell lines. These processes also serve as a useful index of the degree of polarization in epithelial cell cultures. Viral budding from apical and basolateral RPE membranes was used in this study to determine whether the sorting of viral envelope membrane proteins by the RPE is reversed in polarity from that of kidney cells and, if so, whether this might predict a fundamental difference in membrane protein sorting for RPE. The results clearly indicate that the polarity of viral membrane sorting and subsequent viral budding is the same in RPE as in other polarized epithelial cell lines examined to date.


Ubiquitin and ubiquitin conjugates in human lens. Ubiquitin, an 8.5 kDa polypeptide found almost universally in plants and animals, is a normal component in the lens. The best documented function for ubiquitin involves its conjugation to proteins as a signal to initiate degradation. Conjugates for ubiquitin-dependent degradation tend to be of very high molecular mass and are rapidly degraded. Another role of ubiquitin conjugation may be as a stabilizer during stress for protection of constituent proteins, resulting in ubiquitin conjugates that are long-lived. Examination of clear and cataractous human lenses of < 1 to > 50 years revealed the dramatic accumulation of ubiquitin and ubiquitin conjugates with age, beginning at approximately 10 years. Epithelial tissue contained predominantly conjugates of > 250 kDa, although ubiquitin conjugates were found at 98 and 40-60 kDa in tissues from older donors. The water-soluble, urea-soluble and urea-insoluble fractions of lens cortex and core also contain ubiquitin conjugates that accrue with age. High molecular mass conjugates (> 250 kDa) are particularly prominent in older lens tissue. Cataractous lenses, as compared with normal lenses of the same age, show more of these high molecular mass conjugates in the urea-soluble and urea-insoluble fractions of cortex and core. Heterogeneous conjugates in the 20-85 kDa range accumulate in an age-related fashion in all lens cortex and core fractions. While levels of free ubiquitin are significant in the epithelium and the water-soluble cortex and core for all ages, there is no detectable free ubiquitin in the urea-soluble and urea-insoluble fraction under conditions used in this study.(ABSTRACT TRUNCATED AT 250 WORDS)


Parvalbumin and calbindin D-28k immunoreactivities coexist within cytochrome oxidase-rich compartments of squirrel monkey area 18. Previous studies have demonstrated the presence of the calcium-binding proteins parvalbumin (PARV) and calbindin D-28k (CALB) in interdigitating neuronal systems of the primary visual cortex of primates (Celio et al. 1986; Hendry et al. 1989; Van Brederode et al. 1990). Since the processing of visual information takes place in the higher cortical areas (Hubel 1982), we wondered if complementarity of expression is maintained in the secondary visual cortex (area 18). We therefore examined tangential and coronal sections from the occipital lobe of squirrel monkeys using immunohistochemical techniques employing polyclonal antibodies against PARV and CALB. The pattern of PARV immunoreactivity is characterized by tangentially organized, alternating thick and thin stripes, separated by areas of lower immunoreactivity. Both the thick and thin stripes consist of PARV-immunoreactive neuropil. CALB immunoreactivity forms mainly thick stripes containing large numbers of labelled neurons. Thus in area 18, these zones of increased immunoreactivity coincide with the compartments revealing increased cytochrome oxidase activity, whereas the distribution of PARV and CALB is almost complementary in the subcortical visual centre and in the primary visual cortex (area 17) of New World monkeys.


Attenuation of platelet-activating factor induced bronchoconstriction by nedocromil sodium. We assessed the effect of nedocromil sodium on bronchoconstriction and airway responsiveness induced by platelet-activating factor (PAF) in eight normal subjects, in a double-blind, placebo-controlled cross-over study. Subjects inhaled PAF by a dosimeter method in 5 doses of 18 micrograms each, separated by an interval of 15 min, (total dose of 90 micrograms). Airway calibre was measured by partial expiratory flow at 30% of vital capacity (Vp30) before and at 1, 3, 5, 10 and 15 min after each dose of PAF. The bronchoconstrictor response was assessed by measuring the area under the curve of the percentage fall in Vp30 over time. There was a significant reduction in PAF-induced bronchoconstriction after nedocromil sodium (1,225 +/- 392 arbitrary units; mean +/- SEM) compared to placebo (2,395 +/- 598; p < 0.01). There was no significant difference in the fall in peripheral neutrophil count measured at 5 min after PAF with nedocromil sodium (48.5 +/- 9.5%) compared to placebo (43.3 +/- 6.8%). In conclusion, nedocromil sodium significantly attenuates PAF-induced bronchoconstriction but not the peripheral neutropenia in normal subjects. Since PAF is not a direct constrictor of human airway smooth muscle, this effect of nedocromil sodium may indicate inhibition of release of bronchoconstrictor mediators.


Effects of general anaesthetics on neuronal sodium and potassium channels. 1. The effects of clinical inhalation anaesthetics, such as halothane and methoxyflurane, and "model" anaesthetics, such as hydrocarbons and n-alkanols, on neuronal sodium and potassium channels are reviewed. 2. Lipid-based mechanisms for the actions of anaesthetics on the gating parameters of squid axon sodium and delayed rectifier potassium currents are considered in conjunction with evidence of more specific effects in other preparations, notably a fast inactivating potassium current in Helix neurones and a voltage-gated sodium current in rat dorsal root ganglion neurones. 3. The proconvulsant actions of some inhalation anaesthetics are discussed in relation to the induction of spontaneous firing of action potentials in the squid giant axon.


Regional differences of the contractile responses to acetylcholine and neurokinin A in rabbit airway: heterogeneous distribution of the metabolic enzymes. 1. Experiments were designed to determine whether differences exist in the sensitivity to muscarinic and tachykinin agonists in rabbit airways. 2. The rank order of sensitivity (pD2 value) to acetylcholine was: trachea > proximal bronchus > distal bronchus, whereas no regional difference was observed in the sensitivity to carbamylcholine which is resistant to acetylcholinesterase. 3. Acetylcholinesterase activity was greater in the distal than in the proximal airway. 4. In the absence of the peptidase inhibitor, phosphoramidon, the pD2 values of neurokinin A (NKA) and substance P (SP) in trachea were significantly greater than that in bronchus, whereas no regional difference was observed in the NK1 selective agonist, substance P methyl ester (SPOMe). 5. Application of phosphoramidon (10 microM) to avoid peptide degradation abolished the regional difference of the pD2 values of SP. 6. In conclusion, regional differences in sensitivities to acetylcholine and NKA in the rabbit airway were suggested to be due to distribution to the metabolic enzymes of these drugs.


Activation of 5-HT2 receptors inhibits the evoked release of [3H]noradrenaline in the rat spinal cord. 1. The participation of 5-HT2 receptors in the modulation of the evoked release of [3H]noradrenaline from rat spinal cord slices has been examined. 2. In rat spinal cord slices preincubated with [3H]noradrenaline, the alpha 2-receptor agonist clonidine (10(-6) mol/l) decreased the release of tritium evoked by field stimulation (600 pulses at 5 Hz, 20 mA, 2 msec), while the alpha 2-antagonist yohimbine (10(-6) mol/l) increased it. 3. The 5-HT2/5-HT1c agonist DOI (3 x 10(-7) mol/l) decreased the evoked release of tritium, an effect which was prevented by ketanserin (10(-7) mol/l). 4. It is suggested that in addition to presynaptic alpha 2-adrenoceptors, there are 5-HT2 receptors which modulate the release of noradrenaline in the rat spinal cord.


The noradrenergic component contributing to spinal fentanyl-induced antinociception is supraspinally mediated. 1. Male Sprague-Dawley rats were fitted with intrathecal (i.t.) and intracerebroventricular (i.c.v.) catheters. Fentanyl was injected either i.t. or i.c.v., and the antinociceptive efficacy of fentanyl was evaluated using the tail-flick analgesiometric assay. 2. Fentanyl dose-dependently elevated tail-flick latency (TFL) following i.c.v. or i.t. administration. The antinociceptive effects of fentanyl were reversed by naltrexone. 3. Experiments were also designed to evaluate the effects of serotonin and alpha-adrenoceptor antagonists on i.t. or i.c.v. fentanyl-induced elevations in TFL. 4. Phentolamine administered i.t. reversed both the spinal and supraspinal antinociceptive effects of fentanyl, whereas i.t. methysergide did not significantly alter the i.t. or i.c.v. effects of the mu agonist. 5. These data suggest that fentanyl-induced antinociception does not rely on local serotonergic neuronal activation. Due to the highly lipophilic nature of fentanyl, it is possible that the noradrenergic component contributing to spinal fentanyl-induced analgesia is supraspinally-mediated.


Inhibition in L-type Ca2+ channel by stimulation of protein kinase C in isolated guinea pig ventricular cardiomyocytes. 1. Electrophysiological effects of phorbol esters on the L-type Ca2+ current (ICa(L)) in isolated single ventricular cells from guinea pig hearts were investigated. 2. In whole-cell voltage-clamped myocytes, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) at 10(-7) M inhibited ICa(L). An antagonist of protein kinase C (PK-C), H-7, at 10(-5) M did not modify the TPA-induced inhibition. The time-course of inactivation process for ICa(L) was greatly slowed. 3. In cell-attached patch-clamp experiments, TPA (10(-7) M) also markedly decreased the opening of L-type Ca2+ channels. The conductance was unaffected. 4. Even H-7 (10(-5) M) alone inhibited the opening of the channels. Addition of TPA (10(-7)-10(-8) M) caused further decrease in the opening. 5. On the other hand, 4-alpha-phorbol-12,13-didecanoate (not a PK-C activator) had no effect on the Ca2+ channels. 6. These results indicate that the PK-C activation induced by TPA greatly depresses the opening of L-type Ca2+ channels in ventricular cell membranes.


Comparative mechanical and electrical actions of A23187 and X-537A in canine Purkinje fibers. 1. At concentrations lower than 10(-5) M, A23187 did not affect the contractile force in canine Purkinje fibers, but at 2 x 10(-5) M, decreased it significantly. 2. X-537A (10(-6) M) slightly increased the contractile force. The increase was not modified by 10(-7) M pindolol. 3. At 20 min after application both ionophores (2 x 10(-5) M) affected little or no changes in the action potential configurations, except for a marked shortening of the action potential duration. A delayed afterdepolarization and an aftercontraction occurred. 4. The electrophysiological effects were potentiated with an increase in extracellular Ca2+ and further by isoproterenol (1-3 x 10(-7) M). 5. A post-rest potentiation was depressed in the presence of these Ca2+ ionophores. 6. When the stimulation frequency was stepped up from 120 to 180 beats/min, the negative staircase of the contractile force was reversed to positive one in the presence of A23187, high Ca2+ and isoproterenol. 7. These results indicate that in canine Purkinje fibers, X-537A produces cellular Ca2+ overload, but A23187 alone does not cause it. Mechanical and electrophysiological effects induced by these ionophores are potentiated by addition of high Ca2+ and isoproterenol.


[3H]PN200-110 and [3H]glibenclamide binding in normal and cardiomyopathic hamsters. 1. We examined the binding of the Ca2+ channel ligand [3H]PN200-110 and the ATP-sensitive K+ channel ligand [3H]glibenclamide to brain and heart from cardiomyopathic hamsters and compared them to controls. 2. We found that [3H]PN200-110 binding site density was elevated in the heart, but not in the brain, of 30- and 180-day old cardiomyopathic hamsters when compared to controls. 3. [3H]Glibenclamide binding site density was greatly reduced in the heart of 180-day old cardiomyopathic animals compared with all other groups. 4. Quantitative autoradiography revealed that [3H]glibenclamide binding was elevated in several brain areas of 30-day old cardiomyopathic hamsters relative to controls. 5. It is concluded that alterations in both Ca2+ and K+ channels exist in the cardiomyopathic hamster.


On the mechanism of the interaction of ketamine and halothane in vitro. 1. Electrically induced contraction of guinea pig ileum myenteric plexus-longitudinal muscle was inhibited by ketamine and halothane with IC50s of 2.1 x 10(-4) M and 1.8 v/v% respectively. 2. The inhibitory action of ketamine was partially antagonized by naloxone and the selective kappa antagonist nor-binaltorphimine. 3. The actions of ketamine and halothane were synergistic at high levels of response (above 30% inhibition). 4. The actions of ketamine and halothane became antagonistic after treatment with pertussis toxin. 5. The interaction of ketamine and halothane was similar to the interaction of morphine and halothane.


Effects of RU33368, a low affinity ligand for neuronal benzodiazepine receptors, on rodent behaviours and GABA-mediated synaptic transmission in rat cerebellar slices. 1. The effects of the low affinity benzodiazepine receptor ligand RU33368 were studied on rodent behaviours and on GABA-mediated synaptic transmission in rat cerebellar slices. 2. RU33368 inhibited stress induced ultrasounds in rat pups without inducing marked muscle relaxation. RU33368 also enhanced operant responding in rats that had been suppressed by mild footshock. These effects of RU33368 in these two models of anxiety were both blocked by the benzodiazepine antagonist Ro15-1788 (flumazonil). 3. In cerebellar slices RU33368 enhanced stimulus-induced synaptic inhibition of Purkinje layer cells with a minimal effective concentration in the order of 1 microM. The classical benzodiazepine agonist RU32007 was approx. 10 times more potent. This action of RU33368 was blocked by Ro15-1788. 4. The minimal effective concentration of RU33368 fully blocked the effect of RU32007 in 2 of 4 cells tested and partially antagonized it in a third cell. 5. These data suggest that RU33368 is a partial agonist at benzodiazepine receptors and this, at least in part, explains its non-sedative anxiolytic behavioural profile.


Ethanol and the peripheral benzodiazepine receptor: in vivo and in vitro experiments. 1. The effect of chronic ethanol exposure on rat peripheral benzodiazepine receptors (PBR) was studied. 2. The binding of 3H-RO 5-4864 to PBR was increased (35.4%) in the kidneys of rats treated with 10% (v/v) ethanol for 12 weeks, but not in renal membranes isolated from rats exposed to ethanol 30% (v/v) during the same period. 3. Similarly, short-term administration of ethanol (4 weeks) did not alter the binding of 3H-RO 5-4864 to renal membranes. 4. To examine the possibility of a direct interaction of ethanol with PBR, in vitro experiments were carried out. Only high concentrations of ethanol (> 100 mM) caused a significant inhibition of 3H-RO 5-4864 binding in kidney, testis and cerebral cortex. 5. The results presented indicate that chronic ethanol exposure causes a time and dose-dependent increase in renal PBR.


A comparison of the effects of propofol with other anaesthetic agents on the centripetal transmission of sensory information. 1. A range of anaesthetic agents affect the centripetal transmission of sensory transmission by activating cortico-thalamic inhibitory mechanisms. 2. This transmission of information through thalamic sensory relay nuclei is impeded and the sensory flow to the cerebral cortex is considerably reduced. 3. In addition cortical transfer of information is blocked since cells in layers III and V show an additional sensitivity to anaesthetic agents. 4. Diprivan (2,6-diisopropylphenol, propofol, ICI) appears to exert an action on sensory transmission mainly by acting on cortical cells in layers III and V. 5. Its action on thalamic sensory relay cells is to increase their latency of discharge and disrupt their pattern of firing.


Inducible expression bovine papillomavirus shuttle vectors containing ferritin translational regulatory elements. The combination of transcriptional and translational control elements in an inducible expression vector suitable for use in stably transformed cell lines was explored. To this end, ferritin translational control elements have been inserted downstream from a mouse metallothionein (mMT-I) transcriptional promoter (PmMT-I), and upstream from various reporter protein-encoding open reading frames (ORFs), all carried on a bovine papillomavirus shuttle vector. Protocols which stimulate transcription (with zinc) and translation (with iron) were developed to optimize the induction of reporter protein synthesis. It was found that insertion of an iron regulatory element between the PmMT-I and a reporter ORF bestowed a sixfold inducibility of reporter protein synthesis with iron and a 90-fold inducibility with iron plus zinc in a classical superinduction protocol. Surprisingly, inclusion of other rabbit ferritin light chain sequences (rFL), including the ORF, enhanced reporter inducibilities to over 15- and 500-fold, respectively. These additional rFL sequences not only increased inducibility but also (i) increased the half-life of the mRNA and (ii) strongly inhibited translation of an ORF located downstream from the 5' proximal ORF. The maximum levels of reporter proteins attained in transformed cells after prolonged induction represented from 1% to 7% of total cellular protein. These inducible expression vectors should prove useful for the production and study of cytotoxic proteins.


Lack of evidence for proofreading mechanisms associated with an RNA virus polymerase. The in vitro fidelity of the virion-associated RNA polymerase of vesicular stomatitis virus was quantitated for a single conserved viral RNA site and the usual high in vitro base misincorporation error frequencies (approx. 10(-3)) were observed at this (guanine) site. We sought evidence for RNA 3'-->5' exonuclease proofreading mechanisms by varying the concentrations of the next nucleoside triphosphate, by incorporation of nucleoside[1-thio]triphosphate analogues of the four natural RNA nucleosides, and by varying the concentrations of pyrophosphate in the in vitro polymerase reaction. None of these perturbations greatly affected viral RNA polymerase fidelity at the site studied. These results fail to show evidence for proofreading exonuclease activity associated with the virion replicase of an RNA virus. They suggest that RNA virus replication might generally be error-prone, because RNA replicase base misincorporations are proofread very inefficiently or not at all.


Modified binary plant transformation vectors with the wild-type gene encoding NPTII. The defective gene encoding neomycin phosphotransferase (NPTII) present in the binary plasmid vector, pBin19, was replaced with the wild-type (wt) gene. Plasmid vectors analogous to pBin19, pBI121 and pBI101 were constructed carrying the gene encoding the wt NPTII enzyme activity.


Vitamin D and its metabolites inhibit cell proliferation in human rectal mucosa and a colon cancer cell line. Like calcium, vitamin D may protect against colorectal neoplasia as it reduces epithelial cell proliferation and induces differentiation. Although its therapeutic use is limited by its effects on calcium metabolism, analogues such as calcipotriol produce little hypercalcaemia. Stathmokinetic and immunohistochemical techniques were used to study the effect of 1,25 (OH)2 D3 and its analogues on cell proliferation in human rectal mucosa and a colon cancer cell line. Paired sigmoidoscopic biopsy specimens were obtained from 17 control patients and five patients with familial adenomatous polyposis. Explants were established in organ culture, with or without the addition of vitamin D. Proliferation was assessed using (1) metaphase arrest to determine the crypt cell production rate (CCPR) and (2) Ki-67 monoclonal antibody directed against an antigen present in proliferating cells. 1,25 (OH)2 D3 in concentrations of 1 microM-100 pM (10(-6)-10(-10) M) reduced the CCPR (cells/crypt/hour) from 4.74 to 2.15-2.67 (p < 0.001), and the Ki-67 labelling index from 7.28-3.74 (p < 0.01). Likewise, vitamin D2, 10 nM (10(-8) M) reduced the CCPR from 4.74-2.74 (p < 0.05) and calcipotriol from 4.86-2.38 (p < 0.05). In familial adenomatous polyposis patients 1,25 (OH)2 D3 100 pM (10(-10) M) halved the CCPR from 8.75-4.22. Calcipotriol (10(-5) M to 10(-9) M) produced a clearcut dose response inhibition of HT-29 cell growth. Thus, vitamin D and its metabolites inhibit proliferation in normal and premalignant rectal epithelium and suppress growth in a colorectal cancer cell line.


Pharmacological analysis of the cardiac effects of 5-HT and some 5-HT receptor agonists in the pithed rat. A pharmacological analysis of the effects of 5-HT on heart rate has been performed in the pithed rat. 5-HT induced a dose-dependent increase in heart rate whereas 5-HT1 receptor agonists--8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), 5-methoxy-N,N-dimethyl-tryptamine (5-MeODMT), 5-methoxy 3-(1,2,3,6-tetrahydro-4-piridinyl) 1H indole (RU 24969) and 1-(m-trifluoromethylphenyl)-piperazine (TFMPP)--failed to increase heart rate. The increase in heart rate induced by the selective 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodo-phenyl)-2-aminopropane (DOI) was not significant. The dose-response curve to 5-HT for its tachycardic effects was shifted two-fold to the right by ketanserin and LY 53857 and nine-fold to the right by methiothepin. The effects of high doses of 5-HT (higher than 100 micrograms/kg iv) were antagonized by methiothepin, (-)propranolol, 2-(2-[4(O-methoxyphenyl)-piperazine-1-yl]-ethyl)4,4-dimethyl-1,3 (2H-4H) isoquinoline-dione (AR-C 239) and by pretreatment with reserpine. The 5-HT1 receptor antagonists, pindolol and spiroxatrine, the 5-HT3 receptor antagonist MDL 72222 and the alpha 2-adrenoceptor blocking agent idazoxan failed to antagonize the tachycardia induced by 5-HT. It is concluded that in the pithed rat, the tachycardia induced by 5-HT remained unexplained (implication of 5-HT2 receptors probably different from the classical vascular 5-HT2 receptor, or implication of 5-HT1C receptors?). Moreover, at high doses (higher than 100 micrograms/kg iv), 5-HT may increase heart rate by releasing catecholamines.


The apolipoprotein (a) gene: a transcribed hypervariable locus controlling plasma lipoprotein (a) concentration. Lipoprotein(a) [Lp(a)] is a quantitative trait in human plasma. Lp(a) consists of a low-density lipoprotein and the plasminogen-related apolipoprotein(a) [apo(a)]. The apo(a) gene determines a size polymorphism of the protein, which is related to Lp(a) levels in plasma. In an attempt to gain a deeper insight into the genetic architecture of this risk factor for coronary heart disease, we have investigated the basis of the apo(a) size polymorphism by pulsed field gel electrophoresis of genomic DNA employing various restriction enzymes (SwaI, KpnI, KspI, SfiI, NotI) and an apo(a) kringle-IV-specific probe. All enzymes detected the same size polymorphism in the kringle IV repeat domain of apo(a). With KpnI, 26 different alleles were identified among 156 unrelated subjects; these alleles ranged in size from 32 kb to 189 kb and differed by increments of 5.6 kb, corresponding to one kringle IV unit. There was a perfect match between the size of the apo(a) DNA phenotypes and the size of apo(a) isoforms in plasma. The apo(a) DNA polymorphism was further used to estimate the magnitude of the apo(a) gene effect on Lp(a) levels by a sib-pair comparison approach based on 253 sib-pairs from 64 families. Intra-class correlation of log-transformed Lp(a) levels was high in sib-pairs sharing both parental alleles (r = 0.91), significant in those with one common allele (r = 0.31), and absent in those with no parental allele in common (r = 0.12). The data show that the intra-individual variability in Lp(a) levels is almost entirely explained by variation at the apo(a) locus but that only a fraction (46%) is explained by the DNA size polymorphism. This suggests further heterogeneity relating to Lp(a) levels in the apo(a) gene.


Detection of genital human papillomavirus (HPV) DNA by PCR and other conventional hybridisation techniques in male partners of women with abnormal Papanicolaou smears. To study the prevalence of human papillomavirus (HPV) infection, using several different hybridisation techniques, in men whose female sexual partners had cervical HPV and/or cervical intraepithelial neoplasia (CIN). The male genital area was examined colposcopically and areas suspicious of HPV changes were biopsied. Each biopsy was subjected to histological examination and HPV DNA analysis by conventional DNA analysis such as Southern, reverse and dot blot as well as with polymerase chain reaction (PCR). Colposcopic examination of men showed 133 to be normal whilst 82 (38%) had clinical or subclinical lesions. Of 55 colposcopically directed biopsies from the male lesions taken, detection of HPV DNA by hybridisation with conventional techniques and by PCR showed HPV DNA in 29 (53%) and 47 (85%) of biopsies respectively. Overall HPV types 6/11 were the predominant types. In 18 (33%) biopsies positive by PCR, multiple types were found. HPV DNA was present in the majority of biopsy specimens taken, with HPV 6/11 being the predominant type. Among methods for HPV DNA detection, PCR was the most sensitive and useful technique.


The value of chronic suppressive therapy with itraconazole versus clotrimazole in women with recurrent vaginal candidiasis. To determine the comparative efficacy of oral itraconazole versus intravaginal clotrimazole in suppressing recurrent episodes of vulvovaginal candidiasis. Prospective randomised open study of women with recurrent vulvovaginal candidiasis. Clinical and microbiological assessments were made monthly for 12 months. Women's Clinic of a University teaching hospital. Forty-four otherwise healthy, non-pregnant women, with at least four proven episodes of candida vaginitis in the last year were enrolled into the study. After an acute episode of candida vaginitis, 22 women received oral itraconazole 200 mg daily for five days, then 200 mg twice weekly for six months; and 22 women received intra-vaginal clotrimazole 200 mg ovules daily for five days, then 200 mg twice weekly for six months. Symptomatic recurrent clinical vulvovaginal candidiasis during the first six months of suppressive therapy was the major endpoint. A secondary endpoint was recurrent candida vaginitis within six months after completion of therapy. Six patients did not complete the study, one in the itraconazole group and five in the clotrimazole group. Of the evaluable patients, seven of 21 patients (33.3%) in the itraconazole group versus none (0%) of 17 patients on clotrimazole were failures on suppressive therapy, p = 0.02. Following discontinuation of suppressive therapy, recurrences of candida vaginitis were similar, 10 (47.6%) of patients on itraconazole (95% confidence interval (CI) 27-67%), versus 11 (64%) patients on clotrimazole (CI 41-87%), p = 0.15. Intermittent suppressive therapy with clotrimazole was more effective than itraconazole in preventing recurrent candida vaginitis, provided patients adhered to the regimen. Recurrence of vaginitis was common with both regimens after stopping suppressive therapy.


"In vitro" effect of interleukin-1 beta on human glioma cell lines: regulation of cell proliferation and IL-6 production. The human glioma cell lines U251 and HP591 were chosen as "in vitro" models for functional astrocytes. When cultured in the presence of IL-1 beta these cell lines demonstrated a marked increase in interleukin-6 production and in [3H]-thymidine uptake. The addition of dbcAMP could mimic the first effect of IL-1 beta but at the same time suppressed cell proliferation. These results suggest that IL-1 beta possibly exerts one of its biological effects (IL-6 synthesis) by means of the cyclic AMP pathway.


Asbestos-related pleural thickenings in Japanese sake brewers. Asbestos was long used as an additive material for the filtration of many kinds of alcoholic beverages. There has been, however, only one case report of a distiller in Italy showing pleural thickenings and lung parenchymal fibrosis due to exposure to asbestos. We report a retired Japanese sake brewer who showed bilateral calcified pleural plaques on chest X-ray films. X-ray diffraction analysis and energy disperse X-ray microanalysis demonstrated the additive material used for sake filtration to be almost pure chrysotile. Furthermore, 17 cases showing probably asbestos-related pleural thickenings were found on examination of 235 chest X-ray films of male workers at different sake breweries. These findings indicate that Japanese sake brewers should be listed as workers at possible risk of asbestos-related health problems.


Vibration perception thresholds in workers exposed to vibration. The vibration perception thresholds (VPT) at six frequencies from 16 to 500 Hz were examined in 77 workers exposed to hand-arm vibration and in 77 controls using a limits procedure. A dose-response relationship between VPTs and exposure to vibration was found, and the age-adjusted VPTs at each frequency were higher in workers exposed to vibration than in controls. Carpal tunnel syndrome (at 250 Hz) and consumption of alcohol (at 125 Hz) significantly increased the VPT, but vibration-induced white finger was not correlated with VPT. Indices for low (16 and 32 Hz) and high (63-500 Hz) VPT frequencies were calculated to evaluate the entire vibrogram, which consisted of several frequencies with two numbers. The results showed that hand-arm vibration disturbs first the high frequencies, and that the disturbance spreads thereafter to the low frequencies. The characteristics of the VPT test regarding vibration exposure and the association between VPT and nerve symptoms in the hand support the view that VPT is a useful measure for vibration-induced sensory nerve damage.


Single cell observation of calcium signals in class II specific killer T cells. A confocal fluorescence microscope with an argon ion laser and a digital imaging fluorescence microscope were used to study calcium signals in class II-specific killer T cells after interaction with target cells. Here, we used two kinds of I-Ak specific allogeneic killer T cell clones, QM11 (CD8+) and QM56 (CD4+), and target cells, B10.A spleen cells (I-Ak) and B cell hybridomas (TP67.21, I-Ak). After interaction with target cells, the intracellular free calcium ion concentration ([Ca2+]i) in QM11 cells (CD8+) increased rapidly with short lag-times (several seconds). The [Ca2+]i in QM56 cells (CD4+), however, increased with much longer lag-times (hundreds of seconds). The results were consistent with previous findings that the cytolytic activity of QM11 cells (CD8+) was much greater than that of QM56 cells (CD4+).


Influence of chronic ethanol consumption on the inositol phospholipid fatty acid composition of human peripheral blood lymphocytes. The breakdown of inositol phospholipids is an important event after the binding of antigens to the T-cell antigen receptor. In alcoholics, changes either in early or in late steps of lymphocyte activation have been documented, however no study on the role of phosphoinositide fatty acid composition in signal transduction has been reported. We have analyzed the fatty acid pattern of phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate from peripheral blood lymphocytes of alcoholic patients and healthy controls, in order to point out the possible compositional differences which could interfere with the signal transmission responsible for the early events in lymphocyte activation. In alcoholics, the arachidonic acid relative molar content in all the inositol phospholipid (PtdIns) fractions derived from lymphocytes was lower than in controls; all PtdIns classes appeared much more saturated than the corresponding fractions from control lymphocytes. The different fatty acid pattern of PtdIns in alcoholic patients could be responsible for an altered second messenger production, above all the production of a modified diacylglycerol which, in turn, could cause a different activation pattern of protein kinase C, with a consequent alteration in cell proliferation. The decrease in arachidonic acid molar content in the phosphoinositides and particularly in the phosphatidylinositol-4,5-bisphosphate fraction of PBL of alcoholic patients could lead to a reduced synthesis of prostanoids of the (n-6) series, and, as a consequence, to an alteration in the mitogenic response of the cells.


Beta-adrenergic receptors and salivary gland secretion during aging. Beta-adrenergic signal transduction is primarily responsible for the control of the protein secretions by salivary cells. To examine the relationship between beta-adrenergic signal transduction and beta-adrenergic agonist-stimulated salivary secretion, we simultaneously assessed beta-adrenergic receptor number and pilocarpine-isoproterenol-stimulated salivary flow and secreted proteins in parotid and submandibular glands from 3-, 12- and 24-month-old female NNIA F-344 rats. There were no age-related changes in the density of beta-adrenergic receptors in the parotid gland or in the submandibular gland. In the parotid gland there was a significant increase in saliva flow rate in the oldest age group and no changes in the amount of total proteins secreted over 30 min. However, when normalized to gland weight, flow rate was unchanged and the amount of total secreted proteins decreased with age. In the submandibular gland there were age-related increases in both absolute volume and total secreted protein, but when normalized to gland weight there were no longer changes with age. Changes in flow rate were paralleled by reciprocal changes in protein secretory function such that changes in the salivary protein concentrations for the most part were unchanged with age for both the parotid and the submandibular gland. These parameters were compared to our previous data on adenylate cyclase activity, and collectively, these data suggest that in the submandibular gland salivary secretory function does not correlate with changes in beta-adrenergic receptor density or isoproterenol-stimulated adenylate cyclase activity.


Phase II study of iproplatin (CHIP) in patients with cisplatin-refractory germ cell tumors; the need for alternative strategies in the investigation of new agents in GCT. Fifteen patients with advanced, cisplatin-refractory germ cell tumors (GCT) were treated with iproplatin (CHIP). No objective responses were noted in any of the patients treated. By restricting the entry criteria to heavily pre-treated patients, the identification of new active agents in phase II trials may be hindered. Alternative strategies for the investigation of new agents in patients with GCT should be considered, particularly when studying the efficacy and relative toxicity of platinum analogues.


Phase II trial of trimetrexate for unresectable or metastatic non-small cell bronchogenic carcinoma. We treated 34 chemotherapy-naive patients with stage IIIb or IV non-small cell lung cancer with trimetrexate 150-200 mg/m2 intravenously over 30 minutes every two weeks. Six of 31 evaluable patients (19%) achieved a partial response. The major toxic effects from this regimen were myelosuppression, nausea/vomiting, and skin rash. We conclude that this well-tolerated schedule of trimetrexate has significant activity as a single agent against non-small cell lung cancer.


Noradrenergic innervation of the human adrenal cortex as revealed by dopamine-beta-hydroxylase immunohistochemistry. Noradrenergic innervation of the human adrenal cortex was investigated using immunohistochemistry directed at dopamine-beta-hydroxylase. Nerves were present as slender trunks and individual varicose fibres in the capsule and all cortical zones except the inner zona reticularis. Some fibres were located adjacent to blood vessels and in the muscular tunics of arterioles; others were apparently adjacent to parenchymal cells. These results in the human confirm and extend previous animal studies and suggest a possible anatomical substrate for regulation of adrenal blood flow, and also for the direct action of noradrenaline on zona fasciculata cells to stimulate glucocorticoid secretion via beta-1-adrenoceptors.


[Survey of the antibody to HCV in National Leprosarium Suruga]. Blood specimens from 210 leprosy patients (average age 67.4 years old) and 84 staff members (average age 43.5 years old) in National Leprosarium Suruga were tested for anti-HCV antibody using Ortho's Ab ELISA system. Among the patients, 17 patients had chronic hepatic dysfunction as well as leprosy. Twenty of the 210 patients (9.5%) had anti-HCV antibody in their blood. Eleven of the 17 patients (65%) with chronic hepatic dysfunction were positive for anti-HCV antibody. Only one of the staff member was anti-HCV antibody positive. This high positive ratio of anti-HCV antibody in the leprosy patients is similar to the results of another research reported from National Leprosarium Oku Komyo-En. We, therefore, conclude that the prevalence of anti-HCV antibody in leprosy patients is higher than that of the general population and that anti-HCV antibody is related closely to chronic hepatic dysfunction. Some investigators have recently reported that there was an increased incidence of hepatocellular carcinoma in leprosy patients. And so, it is speculated that this is due to the high prevalence of the hepatitis C virus. However, the reason for this high prevalence of anti-HCV antibody in the sample is obscure.


Moderation of myocardial ischemia reperfusion injury by calcium channel and calmodulin receptor inhibition. Intracellular Ca2+ accumulation is implicated in the pathogenesis of myocardial reperfusion injury. To study approaches designed to modify Ca2+ uptake during coronary revascularization after acute infarction, a pig heart surgical infarct model (left anterior descending artery occlusion for 60 min) was subjected to 60 min hypothermic potassium cardioplegic arrest, followed by 60 min of global reperfusion. Four groups of six hearts each were studied in a randomized manner, i.e., cardioplegia alone (control), cardioplegia + 10 microM diltiazem (Ca2+ slow channel blocker), cardioplegia + 10 microM trifluoperazine (TFP), (a Ca(2+)-calmodulin antagonist), and cardioplegia+diltiazem (10 microM) + TFP (10 microM). Left ventricular contractility (global and segmental), metabolism (coronary blood flow and O2 consumption), and creatine kinase generation were measured during reperfusion. Both the Ca2+ channel blocker, diltiazem, and the calmodulin antagonist, TFP, improved myocardial global and regional function as well as myocardial metabolism. While diltiazem better restored global and regional contractility, trifluoperazine had a greater effect on coronary blood flow and myocardial oxygen consumption. Enzyme release and lipid peroxidation were equally moderated by both drugs. From this study it can be concluded that Ca2+ influx does play a role in ischemic and reperfusion injury. The mechanisms of its effect are complex, but can be successfully antagonized by Ca2+ blockers as well as by calmodulin antagonists, with improved myocardial preservation.


Cardiac muscle fiber force versus length determined by a cardiac muscle crossbridge model. A mathematical model incorporating Huxley's sliding filament crossbridge muscle model coupled with parallel and series elastic components was simulated to examine force-length relations under different external calcium concentrations. Several researchers have determined experimentally in both papillary muscle preparations and in situ heart experiments that the calcium concentration (or effective concentration from inotropic agents) will affect the strength and convexity of the cardiac muscle fiber force-length relations. Simulations were performed over a several-order-of-magnitude range of calcium concentrations in isometric contractions and these showed that the force-length curve convexity was changed. Simulation results demonstrated that increasing the stiffness in the model contractile element or series elasticity element did not change the force-length convexity. Increasing the series elasticity element stiffness did slightly change the shape of the force-length curve. The model predicts that the curve convexity changes as a result of the calcium-troponin interactions.


Comparative clinical profiles of triazolam versus other shorter-acting hypnotics. The efficacy, safety, and performance of triazolam was compared with those of other shorter-acting hypnotics acting on the gamma-aminobutyric acid (GABA) receptor--zopiclone, zolpidem, midazolam, brotizolam, temazepam, lormetazepam, and loprazolam. In all, 5506 patients participated in 38 clinical and epidemiologic studies, of whom 2462 were treated with triazolam in parallel-design and crossover studies. To provide clinically relevant comparisons, only studies using comparator agents in doses equipotent to the triazolam doses were included. Two general findings emerged. First, "serious" central nervous system side effects, such as excitement and violence, were not demonstrated for any of the hypnotic agents, including triazolam. Other central nervous system side effects, such as depression and irritability, were reported with equal frequencies for all the hypnotics reviewed. Rebound insomnia, reported intermittently with most of these agents, was short-lived and not clinically significant. So-called early morning insomnia was noted only once and does not appear to be a valid clinical entity. Daytime anxiety was not observed in large numbers of triazolam-treated subjects studied, which is contrary to claims that the drug is anxiogenic. Second, a remarkable similarity was found among all of these shorter-acting agents in terms of efficacy, side effects, and performance-related effects. This was particularly of note for zopiclone and zolpidem. Although claims have been made suggesting differences, evaluation of the studies herein showed that these nonbenzodiazepine hypnotics were indistinguishable from triazolam and other benzodiazepine hypnotics in their clinical and pharmacologic activity. Thus, different chemical structures did not a priori predict different clinical profiles when drugs share a similar mechanism of action.


Human osteoblasts in culture synthesize collagenase and other matrix metalloproteinases in response to osteotropic hormones and cytokines. Collagenase production by rodent osteoblasts in response to calciotropic hormones has led to the hypothesis that bone cells play a major role in bone resorption by degrading the surface osteoid layer, thereby exposing the underlying mineralized matrix to osteoclastic action. Many studies suggest, however, that this model might not apply to bone resorption in the human. Human osteoblasts have been shown to produce gelatinase-A (72 kDa) and TIMP-1 (tissue inhibitor of metalloproteinases), but previous investigators have been unable to demonstrate the synthesis of collagenase by human osteoblasts either constitutively or in response to bone resorptive agents. In the present study the ability of human osteoblasts to produce the matrix metalloproteinases (MMPs) collagenase, gelatinase and stromelysin, and their specific inhibitors TIMPs-1 and 2, was examined using highly sensitive and specific antisera and by zymography. Semi-quantitative histomorphometric data showed that cells cultured on either glass or a type I collagen substratum constitutively synthesized gelatinase-A and TIMP-1. On type I collagen, however, a small proportion of unstimulated cells produce both collagenase (7%) and gelatinase-B (95 kDa; 3%). Treatment of cells with either parathyroid hormone (PTH), 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), or partially purified mononuclear cell conditioned medium (MCM), stimulated the synthesis of collagenase, gelatinase-B and stromelysin; MCM was 2- to 3-fold more potent than either PTH or 1,25(OH)2D3. Zymography using SDS/PAGE on conditioned media from cells cultured on type I collagen films revealed the presence of active gelatinase-A and that MCM stimulated progelatinase-B synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)


Disorganization of the Golgi complex and the cytoplasmic microtubule system in CHO cells exposed to okadaic acid. A combination of immunocytochemical and electron microscopic methods was used to study the effects of okadaic acid, a specific inhibitor of protein phosphatase types 1 and 2A, on the Golgi complex and the microtubule system of interphase CHO cells. At a concentration of 0.25 microM and within 2-3 h of exposure, okadaic acid caused a reversible disorganization of the Golgi complex, observed as a disintegration of the stacks of cisternae and formation of clusters of tubules and vesicles dispersed in the cytoplasm. At the same time, staining for mannosidase II was shifted from the Golgi stacks to the endoplasmic reticulum, whereas the clusters of tubules and vesicles for the main part were negative. This change in localization of the enzyme was not blocked by cycloheximide and thus not dependent on ongoing protein synthesis. The changes in the morphology of the Golgi complex were coordinated in time with a remodelling of the microtubule system, observed as a reduction in the number of microtubules, a tendency of the remaining microtubules to arrange in an aster-like pattern, and an increased sensitivity to low concentrations of the microtubule-disruptive drug nocodazole. After removal of the drug, the microtubule system was rapidly normalized (1-2 h) and subsequently also the Golgi complex (4-8 h). The results suggest that okadaic acid induces a redistribution of the Golgi stacks into the endoplasmic reticulum, leaving the trans-most elements behind as tubules and vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)


The efficiency and timing of plasmid DNA replication in Xenopus eggs: correlations to the extent of prior chromatin assembly. Injection of the circular plasmid FV1 (derived from type I bovine papilloma virus) into Xenopus eggs before the start of the first cell cycle dramatically increases the efficiency of plasmid replication once eggs are chemically activated. We call this the preloading effect and report kinetic and quantitative characterization of this phenomenon here. The timing and the amount of FV1 synthesis were measured by both BrdUTP density labelling and an optimized method of selective enzymatic digestion of replicated and unreplicated molecules using the three methyladenosine-sensitive isoschizomers, DpnI, MboI and Sau3a. DpnI in 100 mM NaCl proved particularly useful for distinguishing and quantitating unreplicated, once-replicated, and repeatedly replicated molecules accumulated over several cell cycles. Our results reveal that both the amount of DNA replicated and the timing of synthesis during the first S-phase correlate with the length of the preloading period. Longer preloading leads to larger amounts of DNA being replicated sooner. In fact, up to 30-50% of 1 ng injected plasmid can replicate in a semiconservative cell cycle-dependent manner during the first S-phase. But such high levels of synthesis during the first cell cycle appear to limit the egg's ability to rereplicate this material in subsequent cell cycles. The preloading effect does not depend on synthesis of either viral or egg proteins, but does appear to correlate with the extent of plasmid assembly into chromatin before the start of the cell cycle. We postulate that each plasmid molecule must achieve a critical degree of chromatin assembly before it can proceed along the replication pathway. These observations illuminate some of the difficulties inherent in building a vector for gene insertion into Xenopus embryos, but also suggest an experimental strategy toward this aim.


Ligand location and biochemical productivity of silica-based immunoaffinity adsorbents. Ligand location within particles, detected by immunogold labelling, was shown to influence the biochemical productivity of a silica-based solid phase, Sorbsil C-500, using a model ligand-biomolecule system (immobilised human immunoglobulin G-anti-human immunoglobulin G monoclonal antibody). The distribution of the ligand was in turn affected by the initial ligand challenge used to prepare the immunoadsorbents. Maximal productivity was achieved with adsorbents prepared with an initial challenge of about 3 mg human immunoglobulin G per ml: the ligand in these cases was shown to be more uniformally distributed within the adsorbent particles than adsorbents, exhibiting low productivity, prepared with either low (1 mg/ml) or high (9 mg/ml) concentrations of human immunoglobulin G. The ligand in the latter was restricted to the periphery of the particles.


Study of affinity supports based on reactive polymers immobilized on silica: affinity constant determination from isocratic zonal elution. Polymers bearing benzamidine moieties have been prepared from reactive copolymer containing chloroformate functions and deposited on porous silica matrices. These high-performance affinity chromatography supports were characterized by quantitative methods, which analyse the zonal elution behaviour of trypsin in the presence of a soluble competitor (L-arginine). The column loading capacity for trypsin was measured by the zonal elution method in mass overload conditions. On the basis of a Langmuir isotherm, the influence of the protein capacity and the concentration of the soluble ligand on the elution volume was studied for the determination of the binding constants. The plate heights determined for silica supports of various porosities and particle diameters show that the strong affinity interactions between trypsin and p-aminobenzamidine are mainly responsible for the low efficiencies observed.


Separation of immunoglobulin G by high-performance pseudo-bioaffinity chromatography with immobilized histidine. I. Preliminary report on the influence of the silica support and the coupling mode. High-performance liquid affinity chromatography with immobilized histidine as a pseudo-biospecific ligand has been used for the fractionation of human immunoglobulin G (IgG). Histidine was immobilized onto silica in two different modes: directly onto silica after epoxy activation or using an intermediate amino derivatization of silica and then coupling histidine using water-soluble carbodiimide. The behaviours and capacities of the obtained affinity supports as well as the influence of pH, silica type, pore diameter and coupling mode have been studied. IgG was effectively separated from human plasma and high maximal binding capacities were obtained.


Clinical pharmacokinetics of vinorelbine alone and combined with cisplatin. Vinorelbine (NVB) is a new anticancer drug belonging to the vinca alkaloid family that shows activity as a single-agent treatment in advanced non-small cell lung cancer (NSCLC). Preliminary results show a better response rate with a combination of cisplatin (CDDP) and NVB than with NVB alone. To assess whether this increased activity is secondary to a pharmacokinetic interaction, the authors compared the pharmacokinetic profiles of NVB alone and combined with CDDP in previously untreated inoperable NSCLC patients. Five patients received NVB (30 mg/m2) by short intravenous infusion, and four patients received CDDP (80 mg/m2) 1 hour after the NVB infusion (30 mg/m2). Serum NVB was assayed using a specific high-performance liquid chromatography method. The mean serum concentrations in both groups were similar. In addition, the areas under the curve calculated from 1 hour (beginning of CDDP administration) to 72 hours were 414 ng.hour/mL (standard deviation [SD]: 94) (group NVB alone) and 407.1 ng.hour/mL (SD: 32.9) (group NVB + CDDP). In conclusion, the increased activity observed with the combination NVB + CDDP is not the result of a pharmacokinetic interaction.


Hepatitis C virus infection, HBsAg carrier state and hepatocellular carcinoma: relative risk and population attributable risk from a case-control study in Italy. In 1990, a case-control study was conducted in Italy to investigate the possible association between HCV infection and hepatocellular carcinoma (HCC). Serum samples from 65 subjects with newly diagnosed hepatocellular carcinoma and 99 hospital control subjects were tested for the presence of anti-HCV by second-generation ELISA test; positive sera were assayed by RIBA anti-HCV second-generation test. In addition, samples were tested for hepatitis B surface antigen (HBsAg), antibodies to the hepatitis B core antigen (anti-HBc), and antibodies to HBsAg (anti-HBs). The presence of HCV and/or HBsAg serologic markers was significantly associated with hepatocellular carcinoma risk: the relative risk (RR) of HCC was 21.3 (95% CI = 8.8-51.5) for anti-HCV positivity in the absence of HBsAg; the relative risk of HCC was 13.3 (95% CI = 5.5-32.2) for the presence of HBsAg in the absence of anti-HCV. A higher risk (77.0) was observed when both markers were present. These findings indicate that HCV and HBsAg are independent risk factors for HCC. The results of multivariate analysis showed that the adjusted RR linking anti-HCV and HCC was 26.9 (95% CI = 9.9-72.5), the adjusted RR linking HBsAg and HCC was 11.4 (95% CI = 3.1-41.4), whereas no association (RR 1.5; 95% CI = 0.6-3.6) was found to link HCC with anti-HBc and/or anti-HBs positivity. Through the computation of population attributable risk we estimate that 25% of HCC cases occurring in Italy could be attributed to anti-HCV positivity alone and 20% to HBsAg carrier state alone.(ABSTRACT TRUNCATED AT 250 WORDS)


Ascites dynamics in cirrhosis. Proposal and validation of a methylene blue dilution test. In order to investigate ascites dynamics, we examined a simple compartmental model based on the analysis of the peritoneal clearance rate of methylene blue in 58 patients with cirrhosis and ascites. After abdominal injection of 10-50 mg methylene blue, the ascitic concentration of the dye progressively decreased, following an exponential trend. The dye distribution volume (7.1 + 0.61, mean + SE) and its peritoneal clearance (87.6 + 5.0 ml/min) were determined by mathematical analysis. The accuracy of volume determinations was controlled in 5 subjects by total paracenteses. In 6 patients, methylene blue clearances were also compared with free-water peritoneal clearances, estimated by a deuterium oxide dilution technique. The decrease in the ascitic concentrations of both tracers followed a corresponding exponential decay in all patients, and the parameters of ascites dynamics determined by the two techniques (peritoneal volumes and clearance values) showed no statistical difference. We therefore suggest the use of the methylene blue dilution test to estimate free-water transperitoneal dynamics, which may be useful in the evaluation and control of cirrhotic patients with ascites.


Replication of hepatitis C virus in peripheral blood mononuclear cells. Effect of alpha-interferon therapy. Hepatitis C virus (HCV) is a positive-stranded RNA virus which replicates through a negative-stranded RNA intermediate. Using a PCR procedure to detect positive and negative strands, we investigated the existence of HCV replication in lymphoid cells. Both positive and negative strands were found in the peripheral blood mononuclear cells (PBMC) of all patients (n = 10) with untreated chronic hepatitis C. No HCV sequences were detected in PBMC in any of the 8 healthy controls. Fifteen patients with chronic hepatitis C were studied at the end of a 12-month course of alpha-interferon therapy. The positive strand was detected in PBMC in all 9 non-responder patients, and the negative strand in 7. In contrast, in PBMC from responder patients (n = 6) the positive strand was found in 4 and the negative strand in only 2 cases. These results demonstrate that HCV can infect PBMC and replicate in these cells and that interferon seems to exert an inhibitory effect on this process. Persistence of HCV-RNA in PBMC may help explain disease relapse after successful interferon therapy.


[Study on local recurrence in patients undergoing curative surgical resection of lung cancer]. Clinical features of recurrence in patients of lung cancer undergoing curative surgical resection were examined with special reference to the local recurrence. Subjects were 308 patients, consisting of 160 adenocarcinomas, 121 squamous cell carcinomas, 15 large cell carcinomas, 12 small cell carcinomas. They underwent curative resection in our department between 1973 and 1987. Local recurrence developed in 54 patients (18%). There was no significant difference in the incidence of local recurrence among the four histological types of carcinoma. According to the pathological stage, the incidence was 9% in stage I, 15% in stage II, and 35% in stages IIIA+IIIB. The local recurrence was subdivided into the following patterns: 1) lymphatic metastases to the hilar, mediastinal, or supraclavicular sites, 2) recurrence at the surgical margin, 3) malignant pleuritis or pericarditis, 4) so-called "endobronchial metastasis". The incidence of recurrence according to the patterns was 15%, 2%, 2% and 1%, respectively. The incidence of recurrence due to lymphatic metastases was correlated significantly with the pN factor but not with the pT factor. Patients with central type lung cancer showed a significantly higher incidence of lymphatic recurrence than patients with the peripheral type. Patients having postoperative radiotherapy to prevent local recurrence showed a lower incidence of lymphatic recurrence than patients having no radiotherapy. In conclusion, lymphatic recurrence was the most frequent pattern in local recurrence after curative resection of lung cancer, and therefore improvements in the operative procedure of lymphatic dissection, as well as in postoperative adjuvant therapy including radiotherapy will be urgently required for the purpose of reducing local recurrence.


[A clinical study on hemostatic fluctuation during and after cardiopulmonary bypass using hemostatic molecular markers]. Heparin is indispensable anticoagulant for cardiopulmonary bypass, but the dose of heparin is even now under discussion. In this study, hemostatic fluctuation was analyzed during and after the bypass using hemostatic molecular markers. The subjects were 16 adult cases of open heart surgery, 12 males, 4 females. The average age was 55.0 year. Operations were aortocoronary bypass in 12, valvular surgery in 3 and ASD patch closure in one with moderate hypothermic cardiopulmonary bypass. At the beginning of cardiopulmonary bypass, 3 mg/kg heparin was administered and the equivalent amount of protamine sulfate was used for neutralization at the end of the bypass. Platelet count, hematocrit, antithrombin III (ATIII), beta-thromboglobulin, platelet factor 4, fibrinopeptide A, thrombin antithrombin III complex, FDP, D dimer FDP, plasmin alpha 2 plasmin inhibitor complex, tissue plasminogen activator (t-PA), and thrombomodulin (TM) were measured through the operation up to two weeks after surgery. ATIII decreased to 50% of control value all through the bypass. Platelet markers increased immediately, and the activated state continued 3 hours after the bypass. Coagulation markers increased markedly after the aortic declamping, and reached at its peak by three times as control value, immediately after the protamine neutralization and continued for 3 hours. During the bypass, fibrinogenolysis caused by t-PA which was stimulated by non-physiological circulation and stimulating substances, was observed. Fibrinolysis occurred following the hypercoagulability after the neutralization. TM was within normal range before the aortic declamping. But increased gradually after the declamp, and reached twice as much as the base line. It could be concluded that hypercoagulability and high platelet activation might play a role of perioperative thrombosis. Hypercoagulability and increase of serum TM would be related to reperfusion of the lung. The increasing of TM would reflect broad injury of vessel walls after the bypass, because plasma TM increased following the generalized injury of endothelial cells.


[Surgery for the patients whose total pulmonary vascular resistance was over 700 dyne during unilateral pulmonary artery occlusion test]. To determine the indication for surgery from the point of pulmonary function, we have performed the unilateral pulmonary artery occlusion test in lung cancer patients. In these patients, we gave a surgery in 13 cases whose total pulmonary vascular resistance were over 700 dyne.sec.cm-5/M2 per body surface area. The pulmonary hemodynamics before and during pulmonary artery occlusion were 549 +/- 82 and 798 +/- 78 dyne.sec.cm-5/M2 in total vascular resistance, 18.7 +/- 3.8 and 27.2 +/- 4.3 cmHg in mean pulmonary arterial pressure and 2.69 +/- 0.36 and 2.73 +/- 4.3 L/min/M2 in cardiac index respectively. In 7 cases out of 13, we performed selective pulmonary artery occlusion test. In pulmonary function test, 10 cases had a chronic obstructive diseases, 2 cases had a disturbance of diffusion and one case has a contractive disease. We gave a lobectomy in 12 cases and completion pneumonectomy in one case. Two of lobectomy cases died in the early phase: 42 and 72 days after surgery due to pulmonary complications. One of these cases needed an additional completion pneumonectomy because of bronchial fistel. Surgical complications were seen in 12 cases. These 13 cases used to be recognized that they have no indication of surgery because of low pulmonary function, however it revealed that we can give a lobectomy through a high intensive care after surgery.


The processing of beta-endorphin and alpha-melanotrophin in the pars intermedia of Xenopus laevis is influenced by background adaptation. beta-Endorphin- and alpha-melanotrophin (alpha-MSH)-related peptides were extracted from the pars intermedia of Xenopus laevis maintained for 2, 4 or 6 weeks on a white background and for the same periods on a black background. The peptides were resolved under dissociating conditions by gel exclusion chromatography on Sephadex G-50 and they were detected by radioimmunoassay with antibodies to beta-endorphin, alpha,N-acetyl beta-endorphin and alpha-MSH. The beta-endorphin-related peptides separated into two fractions of different molecular size. Further purification of the peptides in each fraction was by ion exchange chromatography on SP-Sephadex C-25 and by high-pressure liquid chromatography. The alpha-MSH-related peptides were resolved by gel exclusion and ion exchange chromatography. The purified beta-endorphin- and alpha-MSH-immunoreactive peptides were identified by comparison of their chromatographic properties with the corresponding peptides from porcine pituitary or by comparison with synthetic peptides. The major form of beta-endorphin in the pars intermedia of the frog adapted to a white background was identified as alpha,N-acetyl beta-endorphin (1-8); it was accompanied by a small quantity of acetylated peptides with molecular size similar to beta-endorphin. In contrast, the pars intermedia of the frogs adapted to a black background contained approximately equal amounts of alpha,N-acetyl beta-endorphin (1-8) and the larger forms of beta-endorphin. The higher molecular weight forms were identified as the alpha,N-acetyl derivatives of beta-endorphin (1-26), (1-27) and (1-31); however after 6 weeks of white adaptation the sole remaining peptide in this group was the 26-residue peptide. An additional beta-endorphin immunoreactive peptide, provisionally identified as beta-endorphin (10-26), was present in both black- and white-adapted animals; the amounts of this peptide increased during white adaptation. Major differences in the processing of alpha-MSH were also observed. In the frogs adapted to a black background des-acetyl alpha-MSH greatly predominated over the acetyl form whereas after 6- weeks adaptation to a white background the acetylated peptide proved to be the principal component. The results demonstrate that the proteolytic processing of beta-endorphin and the acetylation of alpha-MSH in Xenopus laevis are influenced by background adaptation. The formation of beta-endorphin (1-8) appears to reflect the action of an endopeptidase that acts at the single arginine residue present at position 9.(ABSTRACT TRUNCATED AT 400 WORDS)


Voltage-activated currents in somatic muscle of the nematode parasite Ascaris suum. 1. Voltage-activated currents in cell bodies of the somatic muscle cells of Ascaris suum were studied using a two-microelectrode voltage-clamp technique. Cells recorded from had resting membrane potentials around -35 mV and had input conductances in the range 1-10 microS. 2. In cells bathed in artificial perienteric fluid, depolarizing steps from a holding potential of -35 mV elicited outward currents at a threshold of -15 mV. These currents had inwardly directed inflections on the rising phase, suggesting the presence of more than one current. Hyperpolarizing steps did not activate current. 3. Tetraethylammonium (TEA+, 69 mmol l-1) blocked the outward currents and allowed a voltage-dependent inactivating Ca2+ current to be observed. The peak current-voltage relationship was U-shaped with a threshold around -15 mV and peak at +5 mV. The reversal potential of the Ca2+ current was estimated by extrapolation to be +45 mV. 4. The permeability of the voltage-activated outward currents was studied by examining reversal potentials of tail currents. The reversal potentials were linearly dependent on the logarithm of the extracellular potassium concentration if extracellular [K+] was greater than 10 mmol l-1. The Na+/K+ permeability ratio of the currents was 0.04. 5. Inactivation, seen as a decline following the peak of the K+ current, was produced by maintained depolarization. The recovery from inactivation was complex and could be described by the sum of two exponentials with time constants of 0.67 s and 20.1 s. Steady-state inactivation of the K+ currents was observed at a range of holding potentials. Only a proportion (34%) of the total K+ current was inactivated by holding potentials more positive than -20 mV. 6. Extracellular application of 5 mmol l-1 4-aminopyridine (4-AP) selectively abolished an early fast component of the K+ current (the peak). The 4-AP-sensitive current decayed quickly with a time constant of around 10 ms; a Boltzmann fit to its activation curve had a half-maximal activation voltage of +14 mV and a 'slope' of 10.5 mV. The 4-AP-resistant current decayed with a time constant of around 1 s; a Boltzmann fit to its activation curve had a half-maximal activation voltage of +29 mV and a 'slope' of 12 mV. 7. Depolarization activates a Ca2+ current and two K+ currents: the K+ currents were separated into lower-threshold, fast-inactivating (Ia-like) and higher-threshold, slowly inactivating (Ik-like) currents.


Virulence plasmid pJM1 prevents the conjugal entry of plasmid DNA into the marine fish pathogen Vibrio anguillarum 775. Studies involving the introduction of cloned homologous genes into Vibrio anguillarum revealed that several plasmids could not be conjugally introduced into V. anguillarum 775(pJM1), but were transmissible to the pJM1-cured derivative H775-3. Recombinant pBR322 plasmids containing V. anguillarum genomic DNA inserts were mobilized from Escherichia coli donors, using pRK2013, into V. anguillarum H775-3 recipients at frequencies of 10(-6) to 10(-5) per recipient. When identical matings were performed with V. anguillarum 775(pJM1) recipients, the infrequent exconjugants recovered carried the pBR322-based plasmid but had lost the large virulence plasmid pJM1. Similar studies were carried out with plasmid RP4 and with recombinant derivatives of the closely related broad-host-range plasmid pRK290. While RP4 was transmissible from E. coli to V. anguillarum H775-3 at frequencies of 6.7 x 10(-2) per recipient, transmission to V. anguillarum 775(pJM1) recipients occurred at frequencies of only 2.5 x 10(-7). When pRK290 contained V. anguillarum DNA inserts, the only exconjugants recovered had lost pJM1, or contained pJM1 and a deletion derivative of the recombinant pRK290 plasmid where all of the DNA insert had been deleted. The use of Dam-, Dcm-, or EcoK- methylation-deficient E. coli donor strains failed to result in appreciable numbers of V. anguillarum 775(pJM1) exconjugants that contained the desired transferred plasmids. Following UV mutagenesis, a derivative of V. anguillarum 775(pJM1) was isolated that would accept conjugally transferred plasmid DNAs at frequencies similar to those observed when using V. anguillarum H775-3 recipients. These data suggest that virulence plasmid pJM1 mediates a restriction system that prevents conjugal transmission of plasmid DNA from E. coli donors into V. anguillarum 775(pJM1). This putative restriction system appears not to be directed towards Dam-, Dcm-, or EcoK-methylated DNA, and appears not to involve a Type II restriction endonuclease.


TPK gene products mediate cAMP-independent thermotolerance in Saccharomyces cerevisiae. Incubation of Saccharomyces cerevisiae with the plant cytokinin N6-(delta 2-isopentenyl)adenine (2iP) resulted in an induction of thermotolerance similar to that induced by sublethal temperatures. Intracellular cAMP levels did not change significantly either during incubation at a sublethal temperature or in the presence of 2iP or ethanol. This suggested that stress-induced thermotolerance is triggered by a mechanism independent of cAMP activation. However, measurement of stress-induced thermotolerance in two mutant strains (tpk1, tpk2, TPK3; tpk1, TPK2, tpk3) each deficient in two of the catalytic subunits of the cAMP-dependent protein kinase (cAPK), revealed that sublethal heat induces thermotolerance by a mechanism part-mediated by the catalytic subunits of cAPK. In contrast, 2iP and ethanol induced thermotolerance by a mechanism fully dependent on the catalytic subunits of cAPK for expression. Therefore, this implies there must be an alternative novel mechanism, other than cAMP, for activating cAPK during stress. Sublethal heating resulted in large increases in intracellular trehalose levels which correlated with the induction of thermotolerance. However, incubation in 2iP or ethanol had no significant effect. This suggests trehalose synthesis is either coincidental with heat stress or that different stress factors induce thermotolerance by alternative mechanisms. Incubation with protein synthesis inhibitors reduced the levels of trehalose synthesized during sublethal heating, suggesting that synthesis of trehalose-6-phosphate synthase during heat stress could be accounting for the increased trehalose levels.


PCR-mediated search for herpes simplex virus DNA in sections of brain from patients with multiple sclerosis and other neurological disorders. Herpes simplex virus (HSV) has been shown to cause central nervous system demyelination in experimental animals and several studies have implicated HSV in the aetiology of multiple sclerosis (MS). We have used the polymerase chain reaction to look for DNA of both type 1 HSV (HSV-1) and type 2 HSV (HSV-2) in formalin-fixed paraffin-embedded brain tissues from patients with MS and other neurological diseases. Primers which amplify a fragment of the normal cellular gene c-myc were included in the reactions to assess the preservation of DNA in the tissue samples. 77 plaques of demyelination from 23 patients with MS were examined. HSV-1 DNA was amplified from only one plaque. This plaque involved the trigeminal root entry zone in the pons and it is suggested that the presence of viral DNA was related to the site examined rather than to the demyelination per se. HSV-2 DNA was amplified from none of the plaques. As expected, HSV-1 DNA was detected in the brains of 6 patients who died of HSV-1 encephalitis and HSV-2 DNA was amplified from the brain of a neonate with congenital HSV-2 infection. In sections of brain from 39 patients with a wide range of other neurological diseases HSV-1 DNA was detected in the pons of only 1 patient, who had AIDS associated with cytomegalovirus ventriculitis; subsequent investigation confirmed the presence of concomitant HSV-1 brain stem infection.(ABSTRACT TRUNCATED AT 250 WORDS)


Expression of CD45RC and Ia antigen in the spinal cord in acute experimental allergic encephalomyelitis: an immunocytochemical and flow cytometric study. We performed immunocytochemical studies to analyze the inflammatory infiltrate and major histocompatibility complex class II (Ia) antigen expression in the spinal cord of Lewis rats with acute experimental allergic encephalomyelitis (EAE) induced by inoculation with myelin basic protein and adjuvants. Using antibodies to lymphocyte markers and other monoclonal antibodies we found that during clinical episodes the inflammatory infiltrate was chiefly composed of T lymphocytes and macrophages. The majority of cells in the inflammatory infiltrate were stained by the W3/25 antibody to CD4 and a proportion was stained by OX22 which labels the high molecular weight form of the leucocyte common antigen (CD45RC). CD8+ T cells were sparse and B cells were not detected. There was minimal staining with the OX39 antibody to the interleukin-2 receptor. Presumptive microglia, identified by their dendritic morphology, expressed Ia antigen during the clinical episodes and after recovery. The prominence of Ia antigen expression after recovery could indicate that this Ia expression was associated with downregulation of the encephalitogenic immune response. We also performed flow cytometry studies on cells extracted from the spinal cord of rats before and during attacks of EAE. With flow cytometry, we found that in established disease a mean of 83(SD, 23)% of CD2+ cells were CD4+, and a mean of 27(SD, 12)% of CD2+ cells were CD45RC+. In rats sampled on the first day of signs, a mean of 43(SD, 22)% of CD2+ cells were CD45RC+. In the cells extracted from the spinal cord of rats with established disease a mean of 47(SD, 32)% of macrophages were CD45RC+. Our study has combined an immunocytochemical assessment of tissue sections with quantitative flow cytometry assessment of cells extracted from the spinal cord of rats with acute EAE. We have shown that the majority of T lymphocytes in the spinal cord are CD45RC-. We have also found prominent Ia expression on dendritic cells in acute EAE and after clinical recovery.


The metabolism of gluconate in Escherichia coli: a study in continuous culture. The gluconate metabolism in Escherichia coli involves duplicate activities of transport and phosphorylation for gluconate. In both cases, these activities can be differentiated in vitro by their different affinities for the substrate. In addition, the two gluconokinases can be differentiated by their heat sensitivities. The technique of continuous culture was used to investigate the influence of the growth rate on this metabolism in an E. coli HfrG6 strain during gluconate-limited growth under conditions of high and low oxygen concentrations. The transport and phosphorylation for gluconate, induced when the cells are cultivated in media with gluconate were differently influenced by the culture dilution rate. These activities were induced under the two conditions investigated; however, the low affinity transport system for gluconate and the thermosensitive gluconokinase were not detected under conditions of high and low oxygen concentrations, respectively. The induction of the dehydratase was favoured under conditions of low oxygen concentration. The experimental data suggest that induction and repression work together to regulate the levels of these activities during gluconate-limited growth conditions. Furthermore, that an effector molecule distinct from gluconate might be involved in the induction of the dehydratase.


Mo-MuLV nucleotide sequence exhibits three levels of oligomeric repetitions, suggesting a stepwise molecular evolution. An exhaustive computer-assisted analysis of the Moloney murine leukemia virus nucleotide sequence shows numerous deviations in the oligomeric distribution, suggesting three overlapping levels of a stepwise duplicative evolution. (1) The sequence fits the universal rule of TG/CT excess which has been proposed as the construction principle of all sequences, and maintains some degree of symmetry between the two complementary strands. (2) Oligomeric repeating units share a core consensus regularly scattered throughout the sequence. This consensus is not merely predictable from the doublet frequencies and codon usage, but could correspond to an intermediary stage in a so-called periodic-to-chaotic transition. (3) Probable stepwise local duplications could be accounted for by slippagelike mechanisms. Comparison with the human spumaretrovirus (HSRV) shows similar segments in the overrepresented oligomers of the two sequences. The intermediary stage of transition oligomeric repeating units is not so clearly suggested in HSRV, perhaps because of numerous stepwise local duplications. In any case, a common evolutionary origin for the two viruses is not ruled out.


A protocol for foscarnet administration. Recently the Food and Drug Administration approved the drug foscarnet for treatment of cytomegalovirus retinitis. As with many other medications, foscarnet has some serious side effects and needs to be administered according to a standardized protocol. This article presents a protocol that was developed for the safe and appropriate delivery of foscarnet.


Borage or primrose oil added to standardized diets are equivalent sources for gamma-linolenic acid in rats. The aim of this study was to evaluate the effect of different doses and sources of dietary gamma-linolenic acid (GLA) on the tissue phospholipid fatty acid composition. Rats fed four different levels of GLA (2.3, 4.6, 6.4 and 16.2 g of GLA/kg diet) in the form of either borage oil or evening primrose oil during 6 wk were compared with animals fed corn oil. The levels of dihomo-gamma-linolenic acid (DHLA) and GLA showed a significant dose-related increase in liver, erythrocyte and aorta phospholipids. Moreover, the arachidonic acid/DHLA ratios in tissues decreased with increasing intake of dietary GLA. There was no significant difference in tissue GLA and DHLA levels within groups given equal amounts of dietary GLA either as borage oil or evening primrose oil. The amount of dietary GLA administered did not significantly influence prostaglandin E2 production in stimulated aortic rings and thromboxane B2 levels in serum; however, an increase in prostaglandin E1 derived from DHLA was observed in the supernatants of stimulated aorta.


Hepatobiliary complications of oral contraceptives. Complications secondary to the use of oral contraceptive agents are rare. Hepatobiliary complications, while often dramatic in presentation, occur infrequently. In a patient without predisposing conditions to complications, the benefits achieved with estrogen/progesterone products outweigh the risks. Those conditions that would absolutely and relatively contraindicate the use of oral contraceptives are listed in Table 4. Patients with a past history of liver disease in whom liver function tests have returned to normal may tolerate the introduction of oral contraceptives. They need to be monitored closely for adverse reactions. Patients who have experienced cholestatic jaundice of pregnancy should avoid all contraceptives because of a high risk of disease recurrence. Women whose first-degree relatives have experienced cholestasis of pregnancy or oral contraceptive-induced cholestasis may be at increased risk and should be closely monitored while taking birth-control pills. Women with current or previous benign or malignant hepatic tumors should not take oral contraceptives. Active hepatitis is an absolute contraindication to using birth control pills, although patients with a past history of hepatitis and no evidence of active disease can have a trial of these drugs with close follow-up. A final group of women who should avoid oral contraceptives is those with familial defects of biliary excretion, including the Dubin-Johnson syndrome, Rotor's syndrome, and benign intrahepatic recurrent cholestasis. Dubin-Johnson syndrome is often asymptomatic and may manifest only during pregnancy or during the use of oral contraceptives. The reduction in hepatic excretory function induced by the sex steroids can transform the mild hyperbilirubinemia into frank jaundice. Oral contraceptive agents are the most widely used reversible means of birth control currently available. Fortunately, the complications associated with these drugs are infrequent and may be decreasing due to lower-dose products. Complications still occur, however, and need to be recognized by the general internist as medication-induced problems so the offending drugs can be discontinued and appropriate treatment and follow-up initiated. In addition, patients at risk for the development of complications need to be recognized and advised prior to the introduction of oral contraceptives.


Increased globotriaosylceramide in familial dysautonomia. Familial Dysautonomia (FD) is an autosomal recessive disease of unknown etiology, occurring primarily in Ashkenazi Jews. Patients are neurologically impaired, with deficits primarily in autonomic and sensory functions. The biochemical and genetic defects have remained elusive, precluding carrier detection and prenatal diagnosis. High-performance liquid chromatography data indicated up to a threefold increase in the neutral glycosphingolipid globotriaosylceramide in Dysautonomic fibroblasts and lymphoblasts. Total ganglioside values, measured by colorimetric, fluorometric or specific sodium borohydride incorporation, were decreased. Affected fibroblasts exhibited a range of pleomorphic phenotypes, such that the usual swirl-like confluent growth pattern of normal fibroblasts was distorted to varying degrees, suggesting abnormalities in the FD plasma membrane, possibly affecting cell-cell contacts. The glycosphingolipid increase could not be accounted for on the basis of markedly decreased alpha-galactosidase activity, as in Fabry's disease, where patients also display decreased autonomic function.


Lipoprotein receptor mediated metabolism of [14C]arachidonic acid labeled chylomicron remnants by Hep G2 cells. During lipolysis of chylomicron triacylglycerol by lipoprotein lipase, arachidonic acid (AA) esters are hydrolyzed at a slower rate than the predominant 16-18 carbon fatty acid esters. The further metabolism of the AA that is hereby enriched in the chylomicron remnant acylglycerols has not been investigated. In the present study, we examined the low density lipoprotein (LDL) dependent and independent metabolism of [14C]AA present in chylomicron remnants in the human hepatoma cell line Hep G2. Mesenteric duct cannulated rats were fed [14C]AA and [3H]cholesterol in corn oil, and the chyle obtained was injected intravenously into hepatectomized rats to form chylomicron remnants labeled with [14C]AA in the triacylglycerol (TG) and with 3H in the cholesteryl ester portion. The remnants were then incubated with Hep G2 cells. The uptake of [14C]AA within 2-4 h was similar to that of [3H]cholesteryl ester. After uptake into the cells, [14C]AA was preferentially incorporated into phospholipids, a high proportion being found in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. [14C]AA and [3H]cholesteryl ester uptake were influenced to similar extents by factors unknown to regulate the LDL receptor and by an anti-LDL receptor antibody. Addition of compactin thus increased the uptake of [14C]AA by 50% in 4 h and mevalonolactone decreased the uptake by 86%. Using an anti-LDL receptor antibody, 25.0% of [3H]cholesterol/cholesteryl ester and 37.7% of [14C]AA binding to the cells at 4 degrees C were blocked. There was no lipolysis of [14C]TG or [14C]diacylglycerol by lipase secreted into the medium during incubations.(ABSTRACT TRUNCATED AT 250 WORDS)


Fecal bile acid excretion and composition in response to changes in dietary wheat bran, fat and calcium in the rat. The effect and possible interactive influence of different dietary amounts of wheat bran, fat and calcium on the fecal excretion, concentration and composition of bile acids was studied in Fischer-344 rats. The fecal bile acids were analyzed using gas-liquid chromatography. Dietary wheat bran increased both total bile acid excretion and fecal weight without changes in fecal bile acid concentration. The proportion of fecal hyodeoxycholic acid decreased with increasing dietary fiber, whereas that of lithocholic and deoxycholic acids increased significantly with fiber intake. The percent content of fecal chenodeoxycholic acid did not change. Increasing dietary fat led to an increase in bile acid excretion without changes in either fecal weight or bile acid concentration. In contrast, the level of dietary calcium did not affect the total excretion of bile acids. However, since calcium increased the fecal weight, it consequently diluted bile acids and decreased their fecal concentration. Dietary fat and calcium had no influence on fecal bile acid composition. There were no interactive effects of wheat bran, fat and calcium on fecal bile acids. The finding in this study that dietary fiber, fat and calcium induce significant changes in fecal bile acids may be of relevance to the potential of bile acids to promote carcinogenesis.


Glucose repression of pigment production in atypical isolates of Aeromonas salmonicida responsible for goldfish ulcer disease. The effect of glucose and other carbohydrates on the pigmentation of three atypical isolates of Aeromonas salmonicida indicated that the addition of D-glucose at a concentration of 0.1% (w/v) or more, inhibited pigment production and caused a reduction in the size of colonies. The addition of cAMP reversed the inhibitory effects of D-glucose on pigment production.