Myohemerythrin from the sipunculid, Phascolopsis gouldii: purification, properties and amino acid sequence. Two previously unknown isoforms, labelled iso I and iso II, of the oxygen-carrying protein, myohemerythrin, have been isolated from carcasses of the sipunculid worm, Phascolopsis gouldii. The two isoforms have non-identical N-terminal amino acid sequences and slightly different absorption spectra in the met form. Far-ultraviolet circular dichroism shows that iso I contains approximately 69% alpha-helix. The complete amino acid sequence for iso I was obtained. The molecular weight calculated from this amino acid sequence and including the active site Fe-O-Fe unit, is 13,829. All of the physical and chemical properties of iso I noted above, including the amino acid sequence, are very similar to those of T. zostericola myohemerythrin. Except for the amino acid sequence, these properties are also very similar to that of a subunit in hemerythrin, the octameric analog found in hemerythrocytes. Only 58 of the 113 residues in P. gouldii hemerythrin are conserved in iso I. Sequence comparisons were used to help identify residues responsible for maintaining the common tertiary and diiron site structures in hemerythrin and myohemerythrin. The seven iron ligand residues previously identified in crystal structures of hemerythrin and myohemerythrin are conserved in iso I. However, none of the ten residue pairs previously identified as engaging in direct salt-bridge or hydrogen bond interactions between subunits in the hemerythrin octamer are conserved in iso I.


Inhibition of xanthine oxidase by uric acid and its influence on superoxide radical production. The inhibition of xanthine oxidase by its reaction product, uric acid, was studied by steady state kinetic analysis. Uric acid behaved as an uncompetitive inhibitor of xanthine oxidase with respect to the reducing substrate, xanthine. Under 50 microM xanthine and 210 microM oxygen, the apparent K(i) for uric acid was 70 microM. Uric acid-mediated xanthine oxidase inhibition also caused an increase in the percentage of univalent reoxidation of the enzyme (superoxide radical production). Steady-state rate equations derived by the King-Altman method support the formation of an abortive-inhibitory enzyme-uric acid complex (dead-end product inhibition). Alternatively, inhibition could also depend on the reversibility of the classical ping-pong mechanism present in xanthine oxidase-catalyzed reactions.


Partial purification and characterization of membrane-associated diacylglycerol kinase of Drosophila heads. A membrane-associated diacylglycerol kinase of Drosophila heads was purified to near homogeneity from the KCl extract of Drosophila heads. The purification procedure involved chromatography on Q-Sepharose, ammonium sulfate fractionation, Superose 12, hydroxyapatite and ATP-agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of fractions after the ATP-agarose column chromatography showed that only a 115 kDa protein correlated well with the enzyme activity. The apparent Km values of partially purified DG kinase were 220 microM for ATP and 540 microM for diolein, respectively. The activity of the DG kinase was inhibited by deoxycholate and was not activated by Ca2+.


Phosphatidic acid and polyphosphoinositide metabolism in rod outer segments. Differential role of soluble and peripheral proteins. The phosphorylation of endogenous diacylglycerol (DAG) and phosphoinositides by [tau-32P]ATP was studied in bovine rod outer segments (ROS) selectively depleted of soluble or peripheral and soluble proteins by treatment with moderate (100 mM) or low (5 mM) ionic strength medium, respectively. DAG kinase activity was similar in bleached and non-bleached ROS extracted with 100 mM medium, and amounted to 70% of that observed in the corresponding non-extracted ROS. Phosphatidic acid (PtdH) labelling in ROS extracted in the dark with low ionic strength medium was markedly lower than in those extracted in light. Thus, even when a major proportion of DAG kinase was associated to the membrane, a soluble form also occurred. Most of the membrane-bound fraction behaved as a peripherally associated protein, its binding to the membrane being modified by light. Ir ROS extracted at moderate ionic strength the labelling of inositides was similar to that in non-extracted ROS. A marked enhancement in polyphosphoinositide labelling was observed in ROS extracted in the dark with low ionic strength. Alkaline treatment of ROS also produced inhibition of polyphosphoinositide phosphorylation. A peripheral form of a type C phospholipase, or a peripheral protein-mediated activation of a particulate form thereof, is suggested. Labelled polyphosphoinositides were more actively hydrolyzed in the light and in the dark plus GTP tau S than in the dark-incubated membranes. The results of phosphorylation experiments in membranes where differential extraction of the alpha subunit of transducin was carried out suggest that alpha and beta tau subunits may play opposite modulating roles in PtdH and polyphosphoinositide metabolism.


Isolation and characterization of two sub-species of Lp(a), one containing apo E and one free of apo E. Lipoprotein Lp(a) was isolated by immunoaffinity chromatography using anti apolipoprotein B and anti apolipoprotein (a) immunosorbents. Besides apolipoproteins (a) and B, this fraction was shown to contain apolipoproteins C and E. Therefore, it was decided to further purify this crude Lp(a) into particles containing apolipoprotein E and particles free of apo E, using chromatography with an anti apolipoprotein E immunosorbent. Lp(a), free of apolipoprotein E was cholesterol ester rich and triacylglycerol poor and was found mainly in the LDL size range. In contrast, Lp(a) containing apolipoprotein E was triacylglycerol rich and was distributed mainly in the VLDL and IDL size range. Binding of these two fractions, one containing apo E and one free of it, to the apo B/E receptor of HeLa cells was studied. Both fractions bound to the receptor but the one containing apo E had a better affinity than the one free of apo E. Further studies are needed to identify the clinical importance of these two different entities.


Altered ganglioside composition in virally transformed rat embryo fibroblasts. The composition of gangliosides was examined in a normal rat embryo fibroblast cell line (REF52) and in two viral transformants: a polyoma transformant (REF52-PyMLV) and a simian viral 40 transformant (REF52-SV40). The distribution of gangliosides in the cell lines was determined using gas-liquid chromatography and high-performance thin-layer chromatography. N-acetylneuraminic acid was the predominant sialic acid species detected in the three cell lines. The total ganglioside concentration (microgram/100 mg dry weight of cells) in the normal, PyMLV, and SV40 lines was 144.7 +/- 10.4, 153.8 +/- 9.2, and 86.1 +/- 6.8, respectively. Gangliosides GM3, GM2, GM1, and GD1a were the major species in the normal and transformed lines. The distribution of these gangliosides, however, differed markedly between the normal and the transformed lines and also between the transformed lines themselves. The transformed cells also differed from the normal cells in growth rate, morphology, and social behavior. The cell line with highest GM3 content (PyMLV) formed islands, whereas the normal and SV40 cell lines, which had lower GM3 levels, grew as monolayers. The findings suggest that PyMLV and SV40 transformation can have multiple and different effects on cellular ganglioside distribution and growth behavior.


Glucose-induced activation of plasma membrane H(+)-ATPase in mutants of the yeast Saccharomyces cerevisiae affected in cAMP metabolism, cAMP-dependent protein phosphorylation and the initiation of glycolysis. Addition of glucose-related fermentable sugars or protonophores to derepressed cells of the yeast Saccharomyces cerevisiae causes a 3- to 4-fold activation of the plasma membrane H(+)-ATPase within a few minutes. These conditions are known to cause rapid increases in the cAMP level. In yeast strains carrying temperature-sensitive mutations in genes required for cAMP synthesis, incubation at the restrictive temperature reduced the extent of H(+)-ATPase activation. Incubation of non-temperature-sensitive strains, however, at such temperatures also caused reduction of H(+)-ATPase activation. Yeast strains which are specifically deficient in the glucose-induced cAMP increase (and not in basal cAMP synthesis) still showed plasma membrane H(+)-ATPase activation. Yeast mutants with widely divergent activity levels of cAMP-dependent protein kinase displayed very similar levels of activation of the plasma membrane H(+)-ATPase. This was also true for a yeast mutant carrying a deletion in the CDC25 gene. These results show that the cAMP-protein kinase A signaling pathway is not required for glucose activation of the H(+)-ATPase. They also contradict the specific requirement of the CDC25 gene product. Experiments with yeast strains carrying point or deletion mutations in the genes coding for the sugar phosphorylating enzymes hexokinase PI and PII and glucokinase showed that activation of the H(+)-ATPase with glucose or fructose was completely dependent on the presence of a kinase able to phosphorylate the sugar. These and other data concerning the role of initial sugar metabolism in triggering activation are consistent with the idea that the glucose-induced activation pathways of cAMP-synthesis and H(+)-ATPase have a common initiation point.


Platelet-activating factor-mediated synthesis of prostaglandins in rat Kupffer cells. Synthesis of prostaglandins was stimulated in rat Kupffer cells upon challenge with platelet-activating factor (PAF). PAF-mediated synthesis of prostaglandins was inhibited by the Ca2+ ion chelator (EGTA), the Ca2+ channel antagonist (nifedipine) and U66985, a structural analogue and antagonist of the biological effects of PAF in other cellular systems. Inhibitors of protein kinase C, staurosporine and polymixin B, did not affect PAF-induced prostaglandin synthesis. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, stimulated synthesis of prostaglandins in Kupffer cells; PAF and PMA exerted additive actions on this process. Both PAF- and PMA-stimulated prostaglandin production was inhibited by TMB-8. PAF-stimulated synthesis of prostaglandins also was inhibited upon treatment of Kupffer cells with pertussis toxin. Cholera toxin, in contrast, stimulated the production of prostaglandins in a concentration-dependent manner; cholera toxin and PAF together had an additive effect. These results suggest that PAF-induced synthesis of prostaglandins is stimulated via a specific receptor coupled to a pertussis toxin-sensitive G-protein, is dependent upon extracellular Ca2+ and is not influenced by protein Kinase C activation. Since PAF and prostaglandins are produced in the liver under conditions such as endotoxemia, PAF-mediated synthesis of these lipid autacoids may be of importance in the regulation of hepatic function during pathophysiological episodes.


Hormonal activation of the cGMP-inhibited low-Km cyclic AMP phosphodiesterase of rat adipocytes from different sites: influence of ovariectomy. Hormonal activation of the cGMP-inhibited low Km cyclic AMP phosphodiesterase isoenzyme (cGI.PDE) by effectors, acting either through the cAMP-independent (insulin) or through cAMP-dependent (isoproterenol, forskolin ACTH and 8Br-cAMP) mechanisms, were compared in parametrial (PM) and femoral subcutaneous (SC) adipocytes from sham-operated (SHAM) and ovariectomized (OVX) rats. In SHAM rats, the basal cGI.PDE activity was 50% higher in PM than in SC adipocytes. In OVX rats, the cGi.PDE activatory responses to all the effectors tested remained unchanged in SC, but were completely suppressed in PM adipocytes. The mechanism underlying these defective cGI.PDE activatory responses to cAMP-dependent effectors observed in PM adipocytes after OVX seems to involve protein kinase A, since a decreased activation of cGI.PDE by protein kinase A was also found in these cells. Treatment of OVX rats with both estradiol and progesterone reversed the defective cAMP-dependent activation of cGI.PDE, but not the refractoriness of this isoenzyme to insulin activation. Taken together with previous observations from this laboratory on the fat cell adenylate cyclase system (Lacasa et al. (1991) Endocrinology 128, 747-753), these results: (a) demonstrate that the influence of the ovarian status on the key enzymes controlling cAMP metabolism in fat cells depends on the anatomical origin of these cells, and; (b) provide a biochemical explanation to the insensitivity of the SC adipocyte lipolytic system to ovarian hormones.


Protection from pseudorabies virus challenge in mice by a combination of purified gII, gIII and gVI antigens. The antigens gII, gIII and gVI were purified from the lysates of the pseudorabies virus (PRV) -infected HmLu-1 cells using Sepharose 4 B coupled with MAbs against these antigens. Mice immunized with either gII, gIII, gVI antigen or a mixture of them were challenged intraperitoneally with 8.5 x 10(3) plaque forming units of PRV. All the mice immunized with 1.5 and 4.5 micrograms of the mixture and 4.5 micrograms of the gIII antigen survived. The sera of mice immunized with the mixture had virus neutralizing activity which was independent of complement, hemagglutination inhibition activity, and recognized major (93 kilodaltons) and minor (129, 74, 68 and 50 kilodaltons) PRV proteins under reducing conditions in western blotting. The serological activity levels in the lower survival group were not different from those in the complete survival. These results indicate that the mixture of each glycoprotein is more effective for eliciting of protective immunities in mice and that serological activity do not always correlate with protection.


Inhibition of mouse hepatitis virus multiplication by an oligonucleotide complementary to the leader RNA. An oligonucleotide complementary to a leader RNA of positive-stranded mouse hepatitis virus (MHV) was tested for the effect on the viral multiplication in mouse DBT cells. A 14-mer antisense oligonucleotide contained a sequence complementary to the conserved pentanucleotide sequence, UCUAA, of the leader RNA. A treatment of MHV-infected cells with the antisense oligonucleotide at concentrations from 5 to 25 microM had an inhibitory effect on the viral multiplication and reduced the synthesis of viral specific mRNA and proteins. No inhibitory effect was observed when the cells were treated with sense oligonucleotide and oligonucleotide which contained unrelated sequences at concentrations from 1 to 10 microM. These results showed that antisense oligonucleotide against the leader RNA reduced the multiplication of positive-stranded RNA virus, MHV.


Humoral immune responses of cats to feline infectious peritonitis virus infection. Immunoperoxidase antibody (IPA) method as a titrating method of feline infectious peritonitis (FIP) virus (FIPV) was developed for titrating antibody to FIPV (IPA-titer). By this method the immune responses of the cats that had been infected with FIPV, were traced. The infected cats could be grouped into three types by their immune response to FIPV and clinical appearances. Type I cats lived for a long time, formed a major group among infected cats, had 160 to 1 x 10(4) IPA-titers, and showed healthy appearances without any changes both on autopsy and histopathologically. From among type I cats, type II cats appeared sporadically with rapid elevation of IPA titers to 3.2 x 10(5) and showing clinical signs of FIP, and died. Type III cats lived healthily for a long time with gradual elevation of IPA-titers to a plateau of about 1 x 10(5), then showed neuronal disorder of hind leg paralysis with the descending IPA-titers to 2 x 10(4), and died. Thus, typical FIP appeared as a hyper-immune disease. Other related problems are discussed.


Altered surface antigen expression on peripheral blood mononuclear cells in cats infected with feline immunodeficiency virus. Expression of CD4, CD8, IL-2 receptor alpha chain (IL-2R alpha), and MHC class II (MHC-II) on peripheral blood mononuclear cells were examined in cats infected with feline immunodeficiency virus (FIV). CD4/CD8 T cell ratio in FIV-infected cats was slightly decreased, as compared with that in specific-pathogen-free (SPF) cats. However, there was no statistical differences between them. The number of circulating IL-2R alpha+ cells in FIV-infected cats was higher than that in healthy cats, whereas induction of IL-2R alpha expression by concanavalin A (Con A) stimulation was depressed in FIV-infected cats. By using two-color cytofluorometry, Con A-induced enhancement of IL-2R alpha expression was found to be reduced in both CD4+ and CD8+ populations in PBMC from FIV-infected cats. The circulating MHC-II+ cells were also increased in FIV-infected cats. Furthermore, the induction of IL-2R alpha expression on PBMC after Con A-stimulation significantly depressed by FIV inoculation in vitro. These results suggest that FIV activates PBMC in vivo via direct and/or indirect mechanisms, leading to the unresponsive state of T cells to further stimuli in vitro.


Characterization of Japanese isolates of Aujeszky's disease virus by restriction endonuclease cleavage patterns, virulence in mice and thymidine kinase activity. Twenty four cloned isolates of Aujeszky's disease virus collected from outbreaks of Aujeszky's disease from 1981 through 1989 in Japan were characterized by their restriction endonuclease (RE) cleavage patterns, virulence for mice and thymidine kinase (TK) activity. All of the isolates belonged to Type II of the four types classified by Herrmann et al. (1984). Based on the number and migration rate of the restriction fragments, the isolates were divided into 7 groups with Bam HI, 9 groups with Kpn I, 3 groups with BstE II and 2 groups with Sal I. The results indicate that the RE analysis, especially with Bam HI and Kpn I, provides useful epidemiological information about field isolates of Aujeszky's disease virus. All of the isolates showed virulence for mice ranging from 6.9 to 63.0 (PFU/LD50). It was interesting that the Nagano S87 strain, which had the highest virulence for the mouse, showed unique RE cleavage patterns with four enzymes. On the other hand, ara-T-resistant, TK-negative strain, was avirulent for mice (greater than 10(6.4) PFU/LD50). All of the isolates investigated in this study showed TK activity by the thymidine plaque autoradiography.


Characterization of pseudorabies viruses recently isolated in Japan by restriction endonuclease assay. A total of 148 Japanese isolates of pseudorabies virus (PrV) collected in 1987 to 1990 were examined for the cleavage patterns of their genomes by a restriction endonuclease (RE) assay using BamHI and KpnI. Basically, there was no large difference in the cleavage patterns of viruses recently isolated in Japan. All of them were considered as belonging to BamHI cleavage pattern type II as well as strain Yamagata-S81 that is the first isolate of PrV in Japan, suggesting that no remarkable variations occurred in PrVs spreading in Japan since the first outbreak in 1981. However, considerable variations that are probably due to the gain and/or loss of cleavage sites, and to the addition and/or deletion of nucleotide sequences were detected in the repeat, conjunction and left end regions of genome. Some of those variations were similar to one another among the viruses isolated in the same geographical areas or farms at the same times, and from the epidemiologically related outbreaks, indicating that the RE assay on PrV genome is one of useful tools for the epidemiological studies on Aujeszky's disease.


The prevalence of types I and II feline coronavirus infections in cats. The types of feline coronaviruses that are prevalent throughout Japan were determined by competitive enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (MAb) to feline infectious peritonitis virus (FIPV) Type II and neutralizing test using Type II FIPV as challenge virus. A total of 1,079 cat serum samples were tested by indirect fluorescent antibody (IFA) assay for FIPV Type II antigen, all 42 sample from natural cases of FIP, 138 of 647 (21.3%) from cases with some chronic diseases and 57 of 390 (14.6%) from apparently non-diseased cases were positive. Of the 42 cases with FIP, 29 (69%) and 13 (31%) were found to have infection with FIPV Types I and II, respectively. Of the cases with chronic diseases, 111 (80.4%) were shown to have infection with FIPV or FECV Type I, while 14 (10.1%) with FIPV or FECV Type II. All of the 57 apparently non-diseased cases seemed to have been infected with FIPV or FECV Type I. These results indicated that feline coronavirus Type I is more high prevalent in Japan.


Cerebellar and frontal cortical benzodiazepine receptors in human alcoholics and chronically alcohol-drinking rats. Postmortem cerebellar and frontal cortical membrane homogenates from human alcoholics, control subjects without neurological or psychiatric illnesses, and rats that chronically drank alcohol were studied to determine the binding characteristics of an imidazobenzodiazepine, [3H]Ro 15-4513. This ligand binds to classical gamma-aminobutyric acidA (GABAA)/benzodiazepine receptors, as well as to a "diazepam-insensitive" site associated with the GABAA receptor complex in the cerebellar granule cell layer. There were no differences in the density of the binding sites between alcoholics and their controls, between alcohol-drinking AA rats that had a choice between 10% alcohol or water for about 10 weeks and their controls, or between Wistar rats that had been given 20% alcohol as their only fluid for 4 months and their controls, which were pair-fed isocalorically with sucrose. The affinity for the cerebellar binding of [3H]Ro 15-4513 was higher in the alcoholics than the controls. No differences were observed in the frontocortical binding. No affinity differences were observed in the rat models. There were no differences between the groups in the characteristics of [3H]Ro 15-4513 binding to human cerebellum in the presence of micromolar diazepam, thus revealing the diazepam-insensitive binding. When this component was subtracted from the total cerebellar binding, to reveal the diazepam sensitive binding, both the KD and Bmax were lower in the alcoholic than the control group. The binding of [3H]muscimol, a GABAA agonist, tended to be higher in the frontal cortices of alcoholics; a similar trend for greater effects was observed in the alcoholics for the GABA inhibition of [3H]Ro 15-4513 binding. These results suggest that no drastic changes occur through chronic alcohol abuse in the numbers of cerebellar and frontocortical benzodiazepine receptors in humans and rodent models; however, the data indicate that the alcoholics have either acquired or innate differences in classical benzodiazepine recognition sites of the cerebellum and in the coupling of these sites to GABAA sites in the frontal cortex, without any differences in cerebellar granule cell-specific diazepam-insensitive [3H]Ro 15-4513 binding sites.


[Renal-urinary calculosis in children. Its etiology, pathogenesis, pathological anatomy, clinical picture and diagnosis]. The paper points the etiological, pathogenetical and anatomo-pathological aspects of children's urolithiasis. A special attention was made a clinical symptomatology and a diagnosis scheme.


[The neuropsychic manifestations of HIV infection in children]. The authors analyse the frequency, the significance for prognosis and the localisation of the neuropsychological manifestation concerning the steps of evolution of the HIV infection in children. The pathogenetic aspects, the principal histopathological lesions and clinical corresponding aspects are also presented. The text refers to the modern neuroimaging investigations and to the new treatment possibilities.


[The indications for intravenous immunoglobulins in pediatric practice]. Current techniques allow the manufacture of intravenous immunoglobulin that fully retain their antibody content and function. Immunoglobulins have been show to be effective therapy for immune thrombocytopenia, neonatal allo(iso)immune thrombocytopenia, agammaglobulinemia, acquired immunodeficiency., premature neonates, burn patients, prophylaxis and treatment of infection in high risk and other conditions.


[Antiviral chemotherapy in pediatrics--its present and prospects]. Authors discuss the general aspects of viral infections and the principles of antiviral chemotherapy. First section deals with the stages, the types of viral infections and the basic principles for action of antiviral drugs. Authors review also the up-to-date situation of antiviral therapy in pediatrics, the problem of resistant strains and formulate the expected progress in the field of antiviral chemotherapy.


[The diagnostic algorithm in neonatal infections]. Authors review the principles of diagnosis in neonatal bacterial infections (local and systemic), in congenital, peri- and postnatal viral infections and also in Candida spp. and other mycotic infections of the neonatal period. They try to delineate the clinical and epidemiological criteria of suspicion and modalities of confirmation of the neonatal infections by specific paraclinical methods. Attention is focused on modern diagnostic methods (such as immunofluorescent techniques, counterimmunoelectrophoresis and so on), which are important for the early etiological diagnosis and for thr rapid initiation of specific therapy. Authors made a practical diagnostic algorithm for the most frequent encountered neonatal infections. They also focused on the recent changes in the etiology of neonatal infections and their therapeutic significance.


Controlled release of hydrophilic compounds by resorbable and biodegradable ceramic drug delivery devices. Hydroxyapatite (HA), aluminum-calcium-phosphorous oxide (ALCAP), bone meal (BM), and tricalcium phosphate (TCP) ceramic implants are biodegradable and nontoxic to the host. The purpose of this study was to investigate the capability of these ceramics to deliver the catecholamine, epinephrine (EPI) in a sustained and controlled manner. The ceramic powder (less than 38 um particle size) was prepared in our laboratory using standard procedures. Sixteen cylinders were prepared (1 g each) from each of the four ceramic materials. All cylinders were pressed at a compression load of 615 Kg and sintered for 36 hours. ALCAP (group I) and BM (group II) cylinders were sintered at 1400 degrees C and HA (group III) and TCP (group IV) ceramic capsules were sintered at 1150 degrees C. Three capsules from each group were loaded with 30 mg EPI. Capsules containing EPI and control (empty) capsules were each suspended in a serum bottle containing 100 ml of phosphate buffered saline (pH 7.4). The amount of EPI released from each capsule was determined by spectrophotometric methods. Data collected from this study showed that the rate of release of EPI from ALCAP, HA, BM and TCP was 6.14 +/- 0.3 3.55 +/- 0.29, 2.07 +/- 0.26 and 1.17 +/- 0.04 mg/day, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


[Ultrastructural changes in the gastric mucosa in chronic gastritis in children]. Studying a series of 30 children with chronic gastritis (diagnosed by optic fiber gastroscopy and by mucosal biopsy), authors have founded important changes of the superficial mucosa and gastric glands. The histologic changes could explain the well-known functional alterations in chronic gastritis. They discussed also a possible immuno-pathogenic mechanism for these mucosal ultrastructural changes.


Pressure modulation of cytochrome-to-cytochrome electron-transfer. Models and enzyme reactions. The kinetics of electron-transfer involved in reactions of reduction of 2,6-dichlorophenol indophenol and Fe(CN)3-(6) by L-ascorbic acid and reduction of ferric cytochrome c by both L-ascorbic acid and reduced hydroxylamine oxidoreductase were studied as a function of three parameters: ionic strength, pressure (1-2000 bar) and temperature (4-20 degrees C) using the high-pressure stopped-flow method. From measurements, the thermodynamic parameters of activation volume (delta V++), and, when possible, activation enthalpy and entropy (delta H++ and delta S++) have been calculated. We found, for these four systems, that the pressure has revealed solvation effects involved in electron-transfer. For the reduction of ferric cytochrome c by reduced hydroxylamine oxidoreductase (a cytochrome-to-cytochrome electron-transfer), we have not obtained evidence for a conformational change.


[Ultrasonography in the pre- and perinatal periods. The indications and risks]. The echographic diagnosis is now wide-spread used in Pediatrics, including the prenatal period. Its use has some well-known advantages, as lack of invasiveness, reproducibility, informative value and the low cost. The author reviewed the up-to-date indications of the echographic methods in Pediatrics. The attention is focused on the biologic side-effects of the ultrasounds in children, including the prenatal period. Based on actual dates from the pediatric literature, it seems that echography in Pediatrics has minor if any long-term side effects.


Prevention of calcium overload and down-regulation of calcium channels in rat heart by SR 33557, a novel calcium entry blocker. The effect of SR 33557, a novel calcium entry blocker, on calcium overload, and regulation of calcium channels and beta-adrenergic receptors was investigated in the rat heart. Calcium overload and infarct-like lesions were produced by a large dose of isoproterenol (40 mg/kg, subcutaneously) to rats. Calcium overload was maximal 8 hours after administration of isoproterenol (control: 5.7 mmol Ca2+/kg dry weight, isoproterenol: 34.9 mmol Ca2+/kg dry weight). At that time, a decrease in the total number of beta-adrenergic receptors (-27%) and calcium channels (-20% and -23%) was observed. Intravenous injection of SR 33557 (0.5-10 mg/kg), 30 minutes before administration of isoproterenol, attenuated the increase in calcium content in a dose-related manner, such that 5 mg/kg SR 33557 reduced calcium overload by 50%. At this dose, SR 33557 had no effect on the number of beta-adrenergic receptors but prevented the decrease in the number of calcium channels. The total number of binding sites and the dissociation constants of each radioligand were estimated from saturation isotherms. The dissociation constants were unchanged when animals given isoproterenol or SR 33557 and isoproterenol were compared to the control group. The results indicate that SR 33557 is able to protect against the calcium overload induced by sympathetic over-stimulation. This over-stimulation of the sympathetic system causes a down-regulation of the number of active beta-adrenergic receptors and calcium channels. The down-regulation of calcium channels is selectively reduced by earlier administration of SR 33557.


[Jaundice with indirect bilirubinemia via the mother's milk]. Among the clinical entities included in the differential diagnosis of the prolonged jaundice at the end of neonatal period and in the small infant is the jaundice due to mother's milk, whose incidence is of 0.5-2% in the naturally fed infants. The authors present two cases in which the diagnosis was reached by exclusion and confirmed by the therapeutically positive test. Physiopathologic considerations are made on the basis of the literature data, and the nosologic background of the affection is presented. This clinical entity, considered to have an excellent prognosis, presented a special evolution in one of the cases.


Alterations in sarcolemmal adrenergic receptors due to intracellular Ca2+ overload in the myocardium. To investigate the effect of an overload of intracellular Ca2+ on the adrenergic receptors, rat hearts perfused with Ca(2+)-free medium for 5 or 10 minutes were reperfused (Ca(2+)-paradox) with a medium containing 1.25 mM Ca2+. The status of beta-adrenergic and alpha-adrenergic receptors in a purified sarcolemmal preparation was studied by measuring the specific binding of [3H]dihydroalprenolol and [3H]prazosin, respectively. Reperfusion with Ca(2+)-containing medium increased the density of both beta- and alpha-adrenoceptors. While the hearts perfused with Ca(2+)-free medium for 5 minutes did not show any change in the Kd values upon reperfusion, the hearts perfused with Ca(2+)-free medium for 10 minutes showed an increase in the Kd value for beta-receptors and a decrease in the Kd value for alpha-receptors. The alterations in adrenergic receptors upon reperfusion in the hearts perfused with Ca(2+)-free medium for 5 minutes were not evident if the medium contained a low concentration of Na+ (35 mM) or was maintained at a low temperature (21 degrees C) to prevent the occurrence of an overload of intracellular Ca2+. These results suggest that an overload of intracellular Ca2+ might play an important role in changing the characteristics of adrenergic receptors in diseased hearts.


Fatty acid composition of phosphoinositides in cultured cardiomyocytes: effects of docosahexaenoic acid and alpha 1-adrenoceptor stimulation. Using cultures of beating cardiomyocytes from neonatal rats, we have studied the fatty acid composition of phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate and the effect of supplementing the culture medium with docosahexaenoic acid on the fatty acid composition of the three phosphoinositides. Docosahexaenoic acid was incorporated into the phosphatidylinositol fraction of the supplemented cells, but not into the phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate fractions. At complete confluence, the cardiomyocytes were stimulated with an alpha 1-agonist (phenylephrine). This altered the acidic pattern of the phosphoinositides in both control and supplemented cells. The differences observed between the polyphosphorylated classes and the phosphatidylinositol fraction suggest the existence of different mechanisms of selection of fatty acids in the biosynthesis of phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate.


[Nifedipine poisoning in a child].

[Nifedipine poisoning in a child]. The present paper concerns the problem of nifedipine poisoning at a 2-year- [correction of 1-year-] and 4-month-old boy, developed after ingestion of 15 tablets with 150 mg active substance. The medical care was represented by: a) gastric lavage with evacuation of stomach content and b) repeated administration of calcium with permanent monitoring of cardiac rhythm, electrocardiography and blood pressure. The evolution was very good with total disappearance of clinical signs in first 6 hours after admission.


Transferrin binding of bone marrow cells and metabolic activity of erythrocytes after 5 Gy irradiation. The effect of total body irradiation (5 Gy) on functional mouse erythroid lineage has been studied. The transferrin binding capacity by bone marrow cells and the activity of glycolytic regulatory enzymes and intracellular levels of 2,3 bisphosphoglycerate in peripheral blood erythrocytes have been determined. Results obtained along one year post-irradiation period suggest a complete recovery in the erythroid cell lineage with respect to the biological endpoints investigated.


Characterization of a 12-mer duplex d(GGCGGAGTTAGG).d(CCTAACTCCGCC) containing a highly reactive (+)-CC-1065 sequence by 1H and 31P NMR, hydroxyl-radical footprinting, and NOESY restrained molecular dynamics calculations. The solution structure of the GC-rich non-self-complementary DNA 12-mer duplex (I), which contains a (+)-CC-1065 highly reactive bonding sequence 5'AGTTA* (where * denotes the [formula: see text] covalent modification site), has been examined thoroughly by one- and two-dimensional proton and phosphorus NMR spectroscopy, hydroxyl-radical footprinting, and NOESY restrained molecular mechanics and dynamics calculations. The assignments of the nonexchangeable proton resonances (except some of the H5' and H5" protons due to severe resonance overlap), phosphorus resonances, and the exchangeable resonances (except amino protons of adenosine and guanosine) of this 12-mer duplex have been made. The results show that this 12-mer duplex maintains an overall B-form DNA with all anti base orientation throughout in aqueous solution at room temperature. Hydroxyl-radical footprinting experiments on a 21-mer sequence that contains this 12-mer duplex used for NMR studies showed that the minor groove is somewhat narrowed at the 7G-8T and 17A-18C steps, as indicated by the inhibition of cleavage at these locations. Although both high-field NMR and hydroxyl-radical footprinting experiments supported a bent-like structure for this 12-mer duplex, nondenaturing gel electrophoresis on the ligated 21-mer sequence that contains this 12-mer duplex did not show the abnormally slow migration characteristic of a bent DNA duplex. Analysis of the NMR data sets reveals several local structural perturbations similar to those found on an (A)n tract DNA duplex. For example, the existence of a propeller twist was detected within the A.T-rich region for both the 12-mer and the (A)n tract DNA duplexes. The 18CH5 aromatic resonance that is directly adjacent to the 3' side of the 5'TAA segment was significantly shifted upfield with a chemical shift of 5.10 ppm, which is almost within the region normally associated with sugar H3' protons. The sugar geometries for 18C and 7G, which are located to the 3' side of the 5'TAA segment, are proposed to be in the neighborhood of C3'-endo and O1'-endo in equilibrium C3'-endo, respectively. We propose that this unusually upfield-shifted resonance signal for 18CH5 and the average C3'-endo sugar geometry for 18C nucleotide on the 12-mer duplex is connected with the peculiar conformation, possibly a transient kink, within the 5'AC/GT step. The results of the NOESY restrained molecular mechanics and dynamics calculations on the 12-mer sequence reveal two kinks, which are located on either side of the 18C nucleotide that has an average C3'-endo sugar geometry.(ABSTRACT TRUNCATED AT 400 WORDS)


Nucleotide sequence of diatom plasmids: identification of open reading frames with similarity to site-specific recombinases. We have determined the nucleotide sequence of two small circular DNA plasmids, pCf1 and pCf2 [22], from the marine diatom Cylindrotheca fusiformis. pCf1 is 4273 bp, and pCf2 is 4079 bp in size. In each plasmid, all of the major open reading frames (ORFs) are encoded on the same DNA strand. Two ORFs are similar, comparing the two plasmids. ORF218 (pCf1) and ORF217 (pCf2) share 80% amino acid identity and ORF482 (pCf1) and ORF484 (pCf2) share 54% amino acid identity. ORF218/217 shows significant similarity (28-31% amino acid identity) to the Tn3 class of resolvases. Resolvases are most commonly found in bacterial transposons. However, two other features found in the Tn3 class of transposon are missing in the plasmids; an ORF encoding a transposase and terminal inverted repeat sequences. This, and data mapping the portions of the plasmids that hybridize to genomic chloroplast DNA, suggest that the plasmids do not contain active transposons. By analogy with the R46 plasmid from Enterobacter [5, 6], another potential role for the resolvases encoded by pCf1 and pCf2 is the conversion of multimeric forms of the plasmid to monomers. The similarity of ORF218/217 to resolvases documents the first identification of a potential coding function in an algal plasmid.


Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. The use of dichlorofluorescin (DCFH) as a measure of reactive oxygen species was studied in aqueous media. Hydrogen peroxide oxidized DCFH to fluorescent dichlorofluorescein (DCF), and the oxidation was amplified by the addition of ferrous iron. Hydrogen peroxide-induced DCF formation in the presence of ferrous iron was completely inhibited by deferoxamine and partially inhibited by ethylenediaminetetraacetic acid, but was augmented by diethylenetriaminepentaacetic acid. Iron-peroxide-induced oxidation of DCFH was partially inhibited by catalase but not by horseradish peroxidase. Nonchelated iron-peroxide oxidation of DCFH was partially inhibited by several hydroxyl radical scavengers, but was independent of the scavenger concentration, and this suggests that free hydroxyl radical is not involved in the oxidation of DCFH in this system. Superoxide anion did not directly oxidize DCFH. Data suggest that H2O2-Fe(2+)-derived oxidant is mainly responsible for the nonenzymatic oxidation of DCFH. In addition, peroxidase alone and oxidants formed during the reduction of H2O2 by peroxidase oxidize DCFH. Since DCFH oxidation may be derived from several reactive intermediates, interpretation of specific reactive oxygen species involved in biological systems should be approached with caution. However, DCFH remains an attractive probe as an overall index of oxidative stress in toxicological phenomena.


Expression in transgenic tobacco of the bacterial neomycin phosphotransferase gene modified by intron insertions of various sizes. A plant selectable marker gene consisting of cauliflower mosaic virus expression signals and the protein-coding sequence of bacterial neomycin phosphotransferase was modified by insertion of an intron sequence from a storage protein gene, phaseolin. Correct and efficient splicing of the resulting mosaic RNA was observed in transgenic tobacco plants. The insertion of various linkers or gradual increase of intron size by addition in both orientations of internal intron sequences from another plant gene (parsley, 4-coumarate ligase) had little or no effect on the precision of slicing. The gene activity measured by selectability assay in the protoplast transformation showed that only introns enlarged to 1161 bases and longer caused decreased selectability. The suitability of such mosaic marker genes for studies of RNA splicing, DNA recombination and early events after infection of plants with Agrobacterium is discussed.


Effect of solution ionic strength on lipid peroxidation initiation by the perhydroxyl (xanthine oxidase-derived) and peroxyl radicals. The role of solution ionic strength in perhydroxyl (HOO.) and peroxyl (ROO.) radical initiated lipid peroxidation has been defined and investigated. Xanthine oxidase activity was used as the source of superoxide (O2-) and its conjugate acid (HOO.) in these experiments. While the enzyme's activity varied with changes in ionic strength, the effect could be factored out of the lipid peroxidation studies. Both HOO.- and ROO.-initiated peroxidations of linoleic acid were promoted by increases in solution ionic strength: the inclusion of 0.1 M of various alkali metal salts in the reaction resulted in up to a 4-fold increase in the overall peroxidation rate. Significant differences between alkali metal cations (Li+, Na+, K+, Cs+) and halogen anions (F-, Cl-, Br-) were not observed. Thus, the increased rates of lipid peroxidation were attributable to changes in solution ionic strength rather than specific ion-reaction interactions. Ionic stimulation of lipid peroxidation occurred only in the presence of preexisting fatty acid hydroperoxides (LOOHs), which provided additional support for the hydrogen atom transfer mechanism previously proposed [Aikens, J., and Dix, T. A. (1991) J. Biol. Chem. 266, 15091] for the HOO./LOOH initiation process. Physiologically appropriate salt concentrations were used in these studies, hence the results may have biological significance.


Cytokines as positive and negative regulators of tumor promotion and progression. Cytokines can have both negative and positive effects on cells undergoing carcinogenesis. The promotion and progression phases of carcinogenesis may be affected by autocrine loops involving cytokines with growth factor activities such as IL-1, IL-2, low molecular weight B cell growth factor, TNF, IL-3, GM-CSF, M-CSF and IL-9. Aberrations in cytokine receptors such as the truncated EGF receptor present in v-erB promotes the growth of neoplastic cells. Aberrant signaling mechanisms, as found with spleen focus-forming virus, which mimics the ligand that activates the erythropoietin receptor, can also contribute to proliferation of preneoplastic and neoplastic cells. In contrast, cytokines such as interferons, LIF, TGF-beta, TNF and leukoregulin, with antiproliferative or differentiating activities, are sometimes capable of inhibiting carcinogenesis. Transfection of tumor cells with cytokine genes, such as IL-2, IL-4 and TNF, can cause suppression of in vivo tumor cell growth by mobilizing host immune and inflammatory cell responses. Thus cytokines and their receptors may play a direct role in early stages of tumor cell development and growth.


Neural regulation of respiration. Physiology and pathophysiology. The central nervous system links the body's metabolic demands with the neuromuscular act of breathing. Automatic and voluntary control systems function together to adjust ventilation in response to chemical and somatic signals. Excitatory or inhibitory outputs from higher brain centers descend the spinal cord and influence the motoneurons to the respiratory muscles. Lesions at any level can disrupt the normal respiratory pattern and may or may not produce alveolar hypoventilation. Several neurologic diseases of importance in veterinary practice may affect the respiratory system. Ventilatory insufficiency tends to occur late in the progression of these disease. Prompt localization of the lesion and correction of the underlying dysfunction is often critical to patient survival.


[Role of imbalance of sex hormones and gonadotropins in the development and course of essential hypertension in women]. Overall 146 females suffering from essential hypertension (borderline, stage I and II according to WHO, 1980) were examined for basal concentrations of estradiol, estriol, progesterone, testosterone, cortisol, ACTH, prolactin in plasma, LH and FSH in blood and urine. The patients and 69 control normotensive females were reproductive, premenopausal, menopausal, postmenopausal, postcastration. Latent hypertension was elicited at bicycle exercise. It was found that relationships between sex hormones and gonadotropins both in hypertensive and normotensive women did not differ noticeably in reproductive period and in menopause. Hormone defects due to primary and secondary infertility, castration, menopause did not raise the risk of arterial hypertension onset. An-Unfavorable course of hypertension was observed in premenopause, following operative castration, in climacteric syndrome. The severity of AH at reproductive age was associated with hyposecretion of progesterone, estrogens and gonadotropins LH, FSH. This relationship was absent in menopausal women. A conclusion is proposed on miner significance of sex hormones imbalance and gonadotropins in the onset of arterial hypertension in females varying by generative activity.


Self-reports by alcohol and drug abuse inpatients: factors affecting reliability and validity. The reliability and validity of self-report data regarding substance abuse has often been questioned. To determine how best to enhance the veracity of self-report, three factors which might affect self-report veracity were examined: alcohol status at time of interview; level of cognitive functioning; and method of self-report data collection. Subjects were 234 admissions to an inpatient substance abuse treatment unit. Self-report data were collected via both personal interview on the day of admission and and questionnaire within the first week of stay. Self-reports concerned use of alcohol, cocaine, and marijuana in the days preceding admission. Test-retest reliability for the questionnaire data produced reliability coefficients of 0.88, 0.91, and 0.88, for alcohol, cocaine, and marijuana, respectively. Variation in inter-test interval had virtually no effect upon reliability coefficients. Interview data were compared to toxicologic analyses of blood and urine samples collected on admission. Overall, this comparison showed self-reports to be valid, with a 97% agreement between verbal report and laboratory data for alcohol, 93% for cocaine, and 84% for marijuana. The comparison of interview data with questionnaire responses also showed self-reports to be valid: 90% agreement for alcohol, 93% for cocaine, and 81% for marijuana. Level of cognitive function did not influence the validity of self-reports for any of the three substances. Recent consumption of alcohol also had no statistically significant effect on the validity of self-reported marijuana use, regardless of the operational form of validity tested. However, BAC-negative subjects produced a significantly greater validity coefficient for self-reported cocaine use (kappa = 0.87) than did BAC-positive patients (kappa = 0.43), when interview data were compared with toxicologic measures. A similar finding was not uncovered when interview and questionnaire data were compared. An interaction between admission alcohol status and cognitive function was uncovered for cocaine self-reports when interview data was compared with toxicologic measures. The rate of agreement for alcohol-negative subjects is quite high for both cognitively impaired and unimpaired subjects (M = 93% and M = 94%, respectively) as well as for alcohol-positive, cognitively unimpaired subjects (M = 94%), but not for alcohol-positive, cognitively impaired subjects (M = 67%). Results are discussed in terms of threats to the validity of self-report and strategies for the optimization of response accuracy.


The structure and function of Na+ channels. The past year has seen major advances in our understanding of voltage-gated ion channels through a powerful combination of patch-clamp and molecular biological techniques. These approaches have identified regions (in some cases single amino acid residues) that are essential for voltage-dependent activation and inactivation, lining of the pore, and regulation of channel function.


Ca2+ channels: diversity of form and function. The past year has seen some significant advances in our understanding of the structural and functional properties of neuronal voltage-gated Ca2+ channels. Molecular cloning and protein purification studies have identified structural components, and expression studies are beginning to define the biophysical and pharmacological properties of the cloned channels. A number of studies of native Ca2+ channels show that the concept of channel modulation includes gating by both voltage and ligands.


The role of protein phosphatases in synaptic transmission, plasticity and neuronal development. In the past year significant advances have been made in our understanding of the role of protein dephosphorylation in the control of neuronal function. Molecular cloning has identified a large number of serine/threonine and tyrosine protein phosphatases in the nervous system. Many of these enzymes are selectively enriched in the nervous system, some are localized to specific neurons, and yet others are expressed only during specific periods of neuronal development. The availability of purified protein phosphatases and selective inhibitors has facilitated the analysis of these enzymes and their role in the regulation of neurotransmitter receptors and ion channels.


Trans-synaptic regulation of gene expression. Neurotransmitters regulate gene expression through second messenger cascades that transmit the signal from the plasma membrane to the nucleus of the postsynaptic cell. Ca2+ and cAMP are two of the second messengers that regulate gene expression in response to neurotransmitters. The Ca2+ and cAMP signals induce expression of a class of genes, termed immediate early genes, within minutes of neurotransmitter receptor activation. Many of these genes encode transcription factors that regulate the expression of late response genes. The results of recent experiments have elucidated mechanisms by which neurotransmitter-induced Ca2+ and cAMP signals regulate immediate early gene expression.


The recognition of hypothalamo-neurohypophysial functions by developing T cells. Neuropeptide signals and specific neuropeptide receptors have been described in the thymus supporting the concept of a close dialogue between the neuroendocrine and the immune systems at the level of early T-cell differentiation. In this paper, we review recent data about neurohypophysial (NHP)-related peptides detected in the thymus from different species. We suggest that we are dealing in fact with other member(s) of the NHP hormone family, which seems to exert its activity locally through a novel model of cell-to-cell signaling, that of cryptocrine communication. This model involves exchange of signals between thymic epithelial cells and developing thymocytes. The NHP-related peptides have been shown to trigger thymocyte proliferation and could induce immune tolerance of this highly conserved neuroendocrine family.


V beta gene family usage in spontaneous lymphomas of AKR mice: evidence for defective clonal deletion. T-cell receptor (TCR) beta-chain usage and expression of the CD3, CD4, and CD8 differentiation antigens were analyzed in 14 spontaneous AKR lymphomas. Lymphoma cells massively infiltrated and/or proliferated in the organs analyzed (thymus, spleen, and mesenteric lymph nodes), giving rise to a loss of organ structure. One lymphoma occurred only in the thymus, and failed to express CD3, CD4, and CD8. All other lymphomas expressed the CD3/TCR complex. With respect to CD4 and CD8 expression, the lymphomas were either double-negative (DN), double-positive (DP), or single-positive (SP). The frequency of DP (CD4+8+) lymphomas was low compared to the frequency of DP thymocytes in a normal AKR thymus. A substantial heterogeneity was seen in the intensity of CD4 and CD8 expression among various lymphomas, which was independent of the level of CD3 expression. Considering TCR V beta gene family usage, 2 out of 14 lymphomas expressed V beta 6. Normally, V beta 6+ thymocytes are deleted from the thymocyte pool at the immature DP stage of T-cell development in AKR mice. These data support the hypothesis that the lymphocytes in the immature DP stage of T-cell development are susceptible to the induction of AKR lymphomagenesis. The presence of V beta 6+ lymphoma cells indicates that the lymphomagenesis is accompanied by a defective clonal deletion of cells expressing a possible autoreactive TCR.


Basic fibroblast growth factor production and growth factor receptors as potential targets for melanoma therapy. A number of growth factors can stimulate the proliferation of human malignant melanoma cell lines. We investigated the effects of exogenous growth factors including basic fibroblast growth factor (bFGF), epidermal growth factor, transforming growth factor-alpha, insulin, insulin-like growth factor-I (IGF-I), acidic fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta and nerve growth factor on six human metastatic melanoma cell lines. The mitogenic activity of each growth factor was tested using the [3H]thymidine incorporation assay. There was a variable response of the different cell lines to most growth factors. All of the melanoma cell lines tested responded to IGF-I. Furthermore, the effects of growth factors were additive, a combination of bFGF and IGF-I having the greatest effect on three melanoma cell lines tested. The quantitative radioimmunoassay for bFGF and [125I]bFGF binding assay revealed that all of these melanoma cell lines produced bFGF and expressed high affinity receptors for bFGF. A 20-mer antisense oligodeoxynucleotide against the AUG initiation site of the bFGF coding region inhibited the proliferation of Mel-Tang by 40% (p less than 0.0001) and that of SK-MEL-5 by 20% (p less than 0.005), suggesting that these cell lines are at least under partial autocrine control of proliferation by bFGF. The presence of bFGF receptors on a high percentage of melanoma cell lines makes these a potential target for melanoma therapy.


MSH receptor expression and the relationship to melanogenesis and metastatic activity in B16 melanoma. In this study we have compared the effects of different pro-opiomelanocortin (POMC) peptides on melanogenesis and metastasis and their relationship to MSH receptor expression in B16F1 melanoma cells. All peptides, apart from beta-endorphin, increased melanogenesis and the order of potency was Nle4DPhe7-alpha-MSH greater than alpha-MSH greater than ACTH[1-39] greater than des-acetyl alpha-MSH greater than ACTH[1-24]. A similar order of potency was found for metastasis, except for ACTH [1-24], which had a relatively greater effect on metastasis. These findings suggest that the effects on melanogenesis and metastasis are mediated via the same receptor. The results of ligand binding studies also indicated the presence of a single receptor with a KD value for Nle4DPhe7-alpha-MSH of 62 +/- 16pM. This was consistent with crosslinking studies using [125I] Nle4DPhe7-alpha-MSH which produced a single 50-55 kD band on analysis by SDS-PAGE. However, the relative binding affinities of the different peptides, measured by displacement of [125I]-Nle4DPhe7-alpha-MSH, did not closely correlate with the relative potencies in stimulating melanogenesis and metastasis. This suggests that receptor activation and the subsequent biological response is not determined solely by binding affinity.


Comparison between diploid and aneuploid hepatocellular carcinomas: a flow cytometric study. Nuclear DNA content of hepatocellular carcinoma (HCC) was estimated by flow cytometry after hepatic resection in 91 patients during the past 5 years. There were 53 diploid and 38 aneuploid tumours. Clinicopathological features were compared retrospectively between the patients with diploid and those with aneuploid HCC. DNA ploidy did not show any correlation with age, sex, alcohol abuse, hepatitis B virus, serum alpha-fetoprotein level or underlying liver disease. Histopathologically, the incidence of HCC less than 2 cm in diameter tended to be higher in the diploid group but no difference was seen for large tumours (greater than 5 cm). The grade of tumour differentiation also tended to be higher in this group of small HCC. The ploidy pattern did not influence the rate of capsule or daughter nodule formation, or venous invasion. There were no significant differences in survival rate or in the incidence and time of intrahepatic tumour recurrence between the two groups. This study may indicate that nuclear DNA ploidy is not a particularly predictive factor for the surgical treatment of HCC.


Role of congenital hypertrophy of the retinal pigment epithelium in the predictive diagnosis of familial adenomatous polyposis. A study was carried out to evaluate congenital hypertrophy of the retinal pigment epithelium (CHRPE) as a disease marker in a defined population with familial adenomatous polyposis (FAP). Indirect ophthalmoscopy was performed on 75 individuals from 25 known families with FAP, of whom 32 were known to be affected and 43 were at a 50 per cent prior risk of developing the disease. A further ten individuals from five families with hereditary non-polyposis colorectal cancer (HNPCC) were also tested. CHRPE was seen in 28 of the 32 affected individuals, 27 of whom met the criteria for a positive examination. Three individuals at risk of FAP also had positive examinations. Five individuals from the families with HNPCC also had CHRPE, although none met the criteria for a positive examination. Of four types of CHRPE analysed, one (small pigmented dots) was found to be more frequent in older family members (P = 0.012), suggesting that this type of lesion may proliferate with age. Compliance with ophthalmic screening was 97 per cent in families with FAP. Using a combined set of diagnostic criteria, CHRPE identified affected individuals with a specificity of at least 94 per cent and a sensitivity of 84 per cent. Results argue for a combined screening programme for FAP of DNA analysis, indirect ophthalmoscopy and bowel examination.


Carob pod (Ceratonia siliqua) meal in geese diets. 1. The apparent and true metabolisable energy values of carob pods meal for geese were measured to be 6.1 MJ/kg and 6.6 MJ/kg respectively. 2. Performance from 5 to 12 weeks was examined in geese fed on four diets containing 0, 100, 200 and 300 g/kg of carob pods meal. 3. The inclusion of carob pods meal up to 200 g/kg in geese diets did not affect the performance. 4. At 300 g/kg performance was highly depressed. 5. The digestibility of protein in the diets decreased linearly with an increase in the level of inclusion of carob pods meal. 6. The length of small intestine, large intestine and caeca and the weight of gizzard expressed per kg of body weight increased with an increase in the level of carob pods meal, which is rich in fibre, in the diets.


Association of the major histocompatibility complex with avian leukosis virus infection in chickens. 1. Association of the B blood group, the major histocompatibility complex (MHC) in chickens, with avian leukosis virus (ALV) infection shown by shedding of group-specific (gs) antigen was studied in an Australorp line selected for short oviposition interval to improve egg production. Three haplotypes (B8a, B9a and B21) were segregating in this line at frequencies of 66.7, 15.6 and 17.8%, respectively, averaged over three generations. 2. The relative risk (odds ratio) of a hen becoming a gs-antigen shedder was calculated for progenies of the dams shedding gs-antigen and those of non-shedding dams separately and pooled over three generations. In the progenies of shedding dams, the relative risk was not significantly different from 1.0 for the three haplotypes. In contrast, in the progenies of non-shedding dams, the relative risk was 0.67, 0.48 and 2.53 for B8a, B9a and B21, respectively, with the last two ratios being significantly different from 1.0. 3. The average effect of haplotype substitution on probability of shedding was estimated from a linear logistic model. The estimates (relative to zero for B8a) for B9a and B21, respectively, were -0.26 and 0.03 among the progenies of shedding dams, and -0.16 and 0.87 among the progenies of non-shedding dams. The last estimate only was highly significant. 4. These results suggest that the three haplotypes were similar in susceptibility to congenital infection through hatching eggs, but differed in susceptibility to post-hatching infection from other infected birds.


Assessment of the nervous system.

Assessment of the nervous system. This article describes the examination of the neurologic and ophthalmic systems in ruminants. Neuroanatomy with respect to lesion localization is reviewed. Pertinent age and species differences are addressed. The practical aspects of assessing the neurologic system are stressed, and the reader is directed to other sources of information for assistance in determining the differential diagnosis and therapy of neurologic disease.


Inactivation of coliphage MS-2 and poliovirus by copper, silver, and chlorine. The efficacy of electrolytically generated copper and silver ions (400 and 40 micrograms/L, respectively) was evaluated separately and in combination with free chlorine (0.2 and 0.3 mg/L) for the inactivation of coliphage MS-2 and poliovirus type 1 in water at pH 7.3. The inactivation rate was calculated as log10 reduction/min: k = -(log10 Ct/C0)/t. The inactivation of both viruses was at least 100 times slower in water containing 400 and 40 micrograms/L copper and silver, respectively (k = 0.023 and 0.0006 for MS-2 and poliovirus, respectively), compared with water containing 0.3 mg/L free chlorine (k = 4.88 and 0.036). Significant increases in the inactivation rates of both viruses were observed in test systems containing 400 and 40 micrograms/L copper and silver, respectively, with 0.3 mg/L free chlorine when compared with the water systems containing either metals or free chlorine alone. Poliovirus was approximately 10 times more resistant to the disinfectants than coliphage MS-2. This observation suggests either a synergistic or an additive effect between the metals and chlorine for inactivation of enteric viruses. Use of copper and silver ions in water systems currently used in swimming pools and spas may provide an alternative to high levels of chlorination.


Blood lactate response to the Canadian Aerobic Fitness Test (CAFT). This study evaluated the blood lactate (LA) response to stepping exercise, specifically the Canadian Aerobic Fitness Test (CAFT). It also compared the correlation between either LA or heart rate (HR) at a given stage of the CAFT and directly measured maximal aerobic power (VO2max). A total of 137 male Canadian Forces (CF) personnel between the ages of 18 and 53 years participated in this study. The LA concentration after each stage of the CAFT was measured in all subjects from blood sampled from the fingertip. Seventy-eight of these subjects also had their VO2max measured directly during a maximal treadmill run. The results showed that increasing stages of the CAFT were associated with exponentially increasing LA. At Stage 5, LA concentration ranged from 1.0 to 7.0, with a mean of 2.6 mmol.L-1, while HR ranged from 72 to 192 with a mean of 132 beats. When the LA and HR measures at Stage 5 of the CAFT were compared for their ability to predict VO2max, the correlation between lactate and VO2max was -0.71 (p less than .001); between HR and VO2max it was -0.36 (p less than .01). These results suggest that LA is a better predictor of VO2max than HR.


Imagery and the acquisition of motor skills. This paper examines the relationship between imagery and the acquisition of motor skills. Since most of the research in the motor domain has considered imagery under the topic of mental practice, a comparison between imagery and mental practice is first drawn. Then the basic mental practice paradigm is outlined and research on the effects of imagery is summarized. Factors influencing the use of imagery are considered, including the task, the imagery instructions, and individual imagery abilities. Implications for employing imagery in the teaching of motor skills are discussed and, finally, an approach to studying imagery and motor skills is put forward.


Velocity specificity of training in bodybuilders. This study investigated the effects of many years of bodybuilding on muscular strength and endurance. Eight bodybuilders (BB), 4 males and 4 females, and 8 controls (C), 4 males and 4 females, performed a muscle fatigue test (MFT) consisting of 25 maximal leg extensions at angular velocities of 180 and 300 degrees/s. The results for strength showed that at both the slow and fast contraction speeds, BB were significantly stronger than C and males were significantly stronger than females. For muscular endurance the results showed that at the slow contraction speed the torques and torque decline were greater in the BB than in the C, and greater in the males than the females. At the faster contraction speed, torque decline was similar in all groups while torques for the BB and male-C were similar and significantly greater than the torques for the female-C. The ratio of torques (fast/slow) for both strength and muscular endurance showed a main effect due to training status which supported the velocity specificity hypothesis. However, when the relative torques over the 25 contractions at both speeds were looked at, no velocity specificity occurred for muscular endurance. The data suggest a definite velocity specificity for the strength factor in BB whereas the same conclusion is not as clear for the muscular endurance factor.


Eccentric and concentric torques of knee and elbow extension in young and older men. The purpose of this study was to compare the strength of knee extensors and elbow extensors in young and older men under conditions of eccentric loading and concentric contractions. Twelve men ages 23 to 32 years and 12 ages 60 to 75 years were tested at two angular velocities of movement, 90 and 180 degrees.s-1, on a KinCom isokinetic dynamometer. Compared to young men, older men had lower concentric peak torque values for elbow (31%) and knee (32%) extensors (p less than 0.05). The older group showed lower eccentric peak torques on all elbow comparisons (21% lower), and for knee extension at the slower velocity (20%). At the faster velocity, knee extension eccentric peak torque did not differ from that of younger men. Differences between the age groups were significantly less for the eccentric muscle action than for the concentric one. The observation of maintained eccentric strength in older men, particularly at fast velocity, warrants research in defining the mechanism.


Returning injured athletes to competition: a role and ethical dilemma. This study investigated the extent to which decisions by coaches and athletic trainers concerning the return of injured athletes to competition were influenced by the player's status and the game situation. Samples were drawn from Oregon high school and Canadian university basketball teams. Coaches and athletic trainers completed a questionnaire in which they decided whether an injured player should be returned to competition. The scenarios included winning, losing, or close games and involved a starter, first substitute, or bench player. Chi-square analyses revealed that coaches made decisions based on both a player's status and the game situation. In contrast, athletic trainers' decisions were not significantly influenced by these variables. Since the role of the coach seemingly makes it difficult to make health related decisions concerning injured players, athletic trainers should be responsible for such decisions.


Mechanical analysis of the Nautilus leg curl machine. The present study assessed the capability of the Nautilus leg curl machine to reflect changes in the isokinetic resistance torque offered to the user commensurate with the human torque pattern generated by the knee flexor muscle group. An averaged isokinetic torque pattern was determined from the exercise machine and from a subject pool (N = 20) of physically active men performing prone knee flexion at two angular velocities (30 degrees/s and 60 degrees/s). The torque patterns of the exercise machine and the subject pool were expressed mathematically. Analysis of the linear regression coefficients established that the resistance torque pattern of the exercise machine was not similar to that of the subject pool (p less than .001). It was concluded that the present exercise machine system did not adequately alter the weight-stack load to provide a resistive torque suited to the biomechanical capabilities of the knee flexors under the two isokinetic conditions studied.


Reliability and validity of a fitness assessment for epidemiological studies. A pilot study was conducted to investigate the applicability of the Canadian Aerobic Fitness Test (CAFT) for use in epidemiological studies. Thirty subjects ranging in age from 18 to 65 were evaluated for cardiorespiratory fitness on four separate visits. Protocols used included maximal treadmill testing, maximal step testing, and the CAFT. Results from these evaluations suggested that (a) habituation to the CAFT was negligible; (b) prediction of VO2max from the CAFT in fit subjects remains a problem and further equation development for this group may be necessary; (c) maximal step-test protocols do not result in unequivocal VO2max determinations and may lead to misclassification of fitness level; and (d) although the CAFT correlates highly to treadmill VO2max (r = 0.90), a relatively large standard error may result in as high as a 13% error in estimating VO2max and may lead to problems in classifying fitness in some populations (e.g., older unfit).


Physiologic and perceptual responses to exercise on a new cycle ergometer. This study observed oxygen consumption (VO2), heart rate (HR), and rating of perceived exertion (RPE) during both maximal and submaximal exercise tests on a conventional Monark cycle ergometer and a new Velodyne ergometer. The Velodyne uses the subject's own bicycle attached to an electrically braked roller, which regulates power output. Trained male cyclists performed maximal exercise tests on both ergometers (N = 7) and two submaximal tests on each of three identical Velodynes and a Monark (N = 6). VO2, HR and RPE were measured during the submaximal and maximal tests. Data were analyzed via multiple repeated-measures ANOVA. No differences were found across cycles during the maximal or submaximal tests. The results indicate that the Monark and the Velodyne ergometers elicited similar physiologic and perceptual responses and that the Velodyne can be a viable alternative to the conventional Monark ergometer.


Travel and the home advantage.

Travel and the home advantage. The purpose of the present study was to examine the relative contributions of various travel related variables to visiting team success in the National Hockey League. A multiple regression design was used with game outcome as the dependent variable. The independent variables of interest included, as main effects and interactions, number of time zones crossed, direction of travel, distance traveled, preparation/adjustment time, time of season, game number on the road trip, and the home stand. Visiting team success was negatively associated with the interaction of number of time zones crossed and increased preparation time between games, and was positively associated with game number on the road. It was concluded that only a small portion of the variance in the home advantage/visitor disadvantage can be explained by travel related factors.


The effects of a hockey-specific training program on performance of Bantam players. Few studies have attempted to identify the effects of training on performance measures related to ice hockey. The present study was designed to examine the effects of a 7-week hockey-specific training program on the on- and off-ice test performance scores of 14- and 15-year-old (Bantam) hockey players. Pre- and post-training tests of percent fat (ultrasound), center of gravity location, 40-yard dash, vertical jump, and on-ice tests of top speed, acceleration, and concerning ability were completed on 28 male subjects (16 in a training group, 12 in a control group of summer league participants). The training group showed significant improvements (p less than .01) in percent fat, top speed, acceleration, and cornering test performance whereas only percent fat was significantly improved for the control group. The results suggest that performance on tests related to ice hockey can be improved by training specifically for hockey but that performance is not affected by summer league play alone.


Dexamethasone iontophoresis: effect on delayed muscle soreness and muscle function. The purpose of this study was to evaluate the pain alleviating effect of dexamethasone iontophoresis on delayed onset muscle soreness (DOMS) produced via an eccentric exercise bout, and to determine the effect on muscle function. Baseline data were collected on 18 female subjects for maximum isometric knee extension contraction (MVC), knee extension peak torque (PT), knee extension work (W), and muscle soreness perception (SP). All values were subsequently reassessed 24 and 48 hours after a 10-min bout of bench stepping. Immediately following the 24-hr reassessment, the experimental (E) (N = 6) and placebo (P) (N = 6) groups received iontophoresis treatment while the control (C) group (N = 6) received no treatment. Percent deviation from baseline of SP was significantly less at 48 hours (p less than 0.05) for the E group compared to P and C groups. However, MCV, PT, and W were no different between the three groups at 48 hours post muscle soreness bout.


Incidence of ras oncogene activation in lung carcinomas in Hong Kong. In Hong Kong, lung carcinomas contribute to the majority of cancer deaths among Chinese. Point mutational activation of ras oncogenes has been observed in several populations. The incidence of these mutations in Hong Kong lung carcinomas was investigated. Lung resections obtained from 52 Chinese patients whose conditions were newly diagnosed as non-small cell lung cancer, paraffin sections from 29 Chinese patients with previously diagnosed adenocarcinoma of the lung, and paraffin sections from 49 squamous cell carcinomas were examined for the presence of point mutations in Ki-ras codon 12, N-ras codon 61, and Ha-ras codon 12 oncogenes by allele-specific hybridization after specific amplification of appropriate regions of the DNA using the polymerase chain reaction. Among the 130 lung carcinomas investigated, Ki-ras point mutations were detected in seven cases, of which six were adenocarcinomas and one a squamous cell carcinoma. No mutations were detected in the N-ras and Ha-ras codons. The incidence of Ki-ras codon 12 point mutational activation in Chinese patients with adenocarcinomas was 6 of 63 (9.5%). The incidence of Ki-ras 12 point mutational activation among men with lung adenocarcinomas in Hong Kong (6 of 32 patients, 18.8%) is significantly different from that in women in Hong Kong (0 of 31 patients, 0%). Although ras oncogenes are implicated as having a role in the development of lung adenocarcinomas, especially among smokers, it is clear from these data that they are not associated with the unusually high incidence of lung adenocarcinomas among women in Hong Kong.


Small cell neuroendocrine carcinoma of the vagina. A 65-year-old woman with a history of recurrent vaginal intraepithelial neoplasia was found to have small cell carcinoma (SCC). Exfoliative cytology was instrumental in the discovery of each episode of vaginal neoplasia. Thorough examination of the patient established the tumor as being primary to the vagina, and immunohistochemistry confirmed it to be a neuroendocrine SCC. Eleven patients with neuroendocrine SCC of the vagina have been reported previously. Morphologic characteristics and histogenesis are discussed within the context of the embryology and natural history of extrapulmonary-genital SCC. They have been classified in the amine precursor uptake and decarboxylation family of neoplasms. Originally, a neuroectodermal origin was proposed, but derivation now is thought to be from multipotential epithelial stem cells of the genital tract. Neuroendocrine SCC tends to be an aggressive neoplasm with a propensity for early spread. Long-term survival for patients with vaginal SCC has not been documented. Therapeutic decisions regarding SCC from this site have been based on information gained from the treatment of these tumors elsewhere. Combined modality therapy using initial surgery and adjuvant treatment, including systemic chemotherapy and local exposure to radiation, has produced an apparent complete response in our patient.


Lethal midline granuloma (peripheral T-cell lymphoma) after lymphomatoid papulosis. A Japanese woman with an 8-year history of lymphomatoid papulosis (LP) had lethal midline granuloma (LMG) develop at the age of 51 years. There were histologic similarities between LP and LMG seen in this patient. Surface phenotypic studies on nasal and cutaneous lesions demonstrated a population of T-cells expressing CD2, CD4, CD25, CD30, and histocompatibility antigen-DR (HLA-DR). Genotypic analyses of nasal and skin biopsy specimens disclosed a clonal rearrangement of the beta T-cell receptor gene with the same rearrangement pattern. These data indicate that this patient had LMG characterized by clonal peripheral T-cell lymphoma, which probably resulted from progression of the LP.


p53 gene mutations in human gastric cancer: wild-type p53 but not mutant p53 suppresses growth of human gastric cancer cells. To further investigate the role of p53 gene inactivation in gastric tumorigenesis, the mutational status of the p53 gene in primary human gastric cancer samples was examined. Reverse transcriptase polymerase chain reaction and subsequent direct sequencing of the p53 gene from gastric cancer samples revealed frequent point mutations of the p53 gene: some of these coincided with those previously identified in gastric cancer cell lines. In addition, both allelic deletion analysis using pYNZ 22 and polymerase chain reaction-restriction fragment length polymorphism analysis demonstrated an allelic deletion of the p53 gene in cancer tissue which contained a point mutation of the p53 gene in the remaining allele. Transfection of the wild-type or mutant p53 genes into gastric cancer cells showed that the wild-type but none of the mutated p53 genes suppressed the colony formation of gastric cancer cells. Furthermore, the incorporation of thymidine into DNA was reduced in cancer cells expressing the wild-type p53 gene. The glutathione S-transferase-wild type p53 fusion protein bound to simian virus 40 large T antigen in COS-1 cell lysate. None of the p53 fusion proteins containing mutations at codons 143, 175, 248, or 273 bound to simian virus 40 large T antigen. By contrast, two different mutant p53 fusion proteins containing mutations specifically observed in gastric cancer bound to simian virus 40 large T antigen. These results indicate that inactivation of the p53 gene through mutations and the allelic deletion may play an important role in gastric tumorigenesis. These mutations may cause a conformational change in the p53 protein resulting in the loss of the suppression by p53 of the growth of gastric cells, partly through disruption of the association of p53 protein with a cellular component.


Anoxia-inducible endonuclease activity as a potential basis of the genomic instability of cancer cells. Normal rat fibroblasts exhibit a staged response to anoxia which in several respects parallels processes activated in malignant tumor cells. We describe here a new element of the anoxic response, the induction by anoxia of a sequestered endonuclease activity. Such activity is elevated approximately 3-fold within anoxic fibroblasts and during Hirt DNA isolation is able to digest chromatin to produce a nucleosomal ladder. However, DNA is not measurably affected within intact cells, and cells retain complete viability as the endonuclease is induced. The anoxia-inducible endonuclease acts without specificity for DNA sequence. Trace leakage of this endonuclease into the nucleus has obvious potential to underlie the known propensity of anoxic cells to undergo amplification and may be associated with the break-related genomic instability of cancer cells.


In vitro activation of distinct molecular and cellular phenotypes after induction of differentiation in a human neuroblastoma cell line. In this report we provide evidence for the activation of distinct differentiation pathways during treatment of the neuroblastoma cell line SMS-KCNR with 1 mM dibutyryl cyclic AMP (dbcAMP) and/or 5 microM retinoic acid (RA). Our results show that the adrenal gland specific gene pG2 is induced only during dbcAMP treatment, while RA induces a neuronal phenotype and expression of all neural related genes while decreasing the expression of many chromaffin related genes. Furthermore dbcAMP does not affect the DNA content distribution of SMS-KCNR [G1 = 61.8 +/- 4.1% (SD); S = 20.3 +/- 6.3%; G2-M = 18 +/- 5.4%] despite morphological and molecular signs of cellular differentiation. Conversely, RA arrests cell growth causing a decrease in cells in the growth fraction (S + G2 + M = 15.6 +/- 6.1%) and an increase in cells in G1 (G1 = 84.3 +/- 5%). Using cyclic AMP and RA in combination, we found that RA inhibited expression of adrenal gland specific gene pG2 and induced a neuronal phenotype. Since dbcAMP does not cause a significant G1 block in SMS-KCNR cells we propose that this agent may be able to induce SMS-KCNR only to an intermediate stage of chromaffin differentiation in which cells retain their proliferative potential.


Preferential expression of immunoreactive fucosylceramide in adenocarcinoma of the lung. The expression of fucosylceramide (PC47H antigen) in 97 lung cancers and 4 extrapulmonary squamous cell carcinomas was examined with the use of a novel monoclonal antibody, PC47H, recognizing fucosylceramide specifically. The observed variation in fucosylceramide content was dependent on the degree of glandular differentiation in adenocarcinoma of the lung. Fucosylceramide was abundantly expressed in well differentiated adenocarcinoma of the lung and poorly expressed in poorly differentiated adenocarcinoma. Some squamous cell carcinomas of the lung reacted with this monoclonal antibody weakly, but the reaction was noted only at the periphery of the epithelial sheets. Extrapulmonary squamous cell carcinoma and small-cell carcinomas did not react with monoclonal antibody PC47H. Interestingly, large cell carcinomas of uncertain cell origin were all positive for fucosylceramide, which accumulated in the cytoplasm. At the ultrastructural level, fucosylceramide was located in the plasma membrane and unit membrane of the rough endoplasmic reticulum. On the other hand, carcinoembryonic antigen as an adenocarcinoma-associated tumor marker was expressed significantly in squamous cell carcinomas as well as adenocarcinomas. Taken together, fucosylceramide seems to be expressed preferentially in adenocarcinomas, and is closely linked to glandular differentiation. Thus it may be a better tumor marker than carcinoembryonic antigen.


Induction of uterine cervical neoplasias in mice by human papillomavirus type 16 E6/E7 genes. We constructed a recombinant retrovirus containing the E6/E7 genes of human papillomavirus (HPV) 16 (ZE67) and examined the morphological changes in the cervicovaginal epithelium induced by inoculation of this virus into the vagina of mice. The ZNeo virus without HPV genes was used as a negative control. Moreover, cotreatment with phorbo-13-myristate-12-acetate or N-methyl-N'-nitro-N-nitrosoguanidine and these viruses was carried out. At the end of the observation period (9 months for CD-1 nu/nu and 15 months for C57BL/6J), of 31 CD-1 nude mice treated with ZE67, 7 and 19 had low-grade and high-grade dysplasia, respectively, while 5 of 22 mice treated with ZNeo had low-grade dysplasia (rank sum test, P less than 0.001). Similarly, 4 and 3 of 8 C57BL mice treated with ZE67 had low-grade and high-grade dysplasias, respectively, whereas 3 of the 8 control mice had low-grade dysplasias (P = 0.049). ZE67 plus phorbol-13-myristate-12-acetate and ZE67 plus N-methyl-N'-nitro-N-nitrosoguanidine cotreatments induced cervical cancers in 2 of 13 CD-1 nude mice and 6 of 15 C57BL mice, respectively. On the contrary, none of 6 CD-1 and 2 of 10 C57BL mice had cancer in the control groups (P = 0.0142 for phorbol-13-myristate-12-acetate treatment; P = 0.0173 for N-methyl-N'-nitro-N-nitrosoguanidine treatment). In addition, the existence and expression of HPV 16 E6/E7 genes were detected in the lesions induced by ZE67 but not in the lesions of the control mice by analysis by polymerase chain reaction and mRNA in situ hybridization. The present results suggest that HPV 16 E6/E7 genes induce dysplastic changes but require additional promoting or mutagenic stimulation for the development of cancer.


Regulation of intracellular pH in tumor cell lines: influence of microenvironmental conditions. The effect of microenvironmental factors on the regulation of intracellular pH (pHi) in MGH U1 cells and EMT-6 cells was studied using the fluorescent pH probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. Na+/H+ exchange and Na(+)-dependent Cl-/HCO3- exchange were found to be present in both cell types. The activity of both exchangers was dependent on pHi, with low levels of activity at neutral pH and an increase in activity as pHi fell. The level of extracellular pH (pHe) also influenced the operation of the exchangers, with a fall in activity as pHe was reduced over the range 7.4-6.6. This effect was more marked for the Na(+)-dependent Cl-/HCO3- exchanger than for the Na+/H+ antiporter, suggesting that under conditions of reduced pHe the Na+/H+ antiporter is the major mechanism for regulation of pHi. Neither 6 h of radiobiological hypoxia nor variations in the extracellular [Ca2+] over the range 1-6 mM had an effect on the regulation of pHi, while extracellular lactate (5-10 mM) caused a small, concentration-dependent decrease in the combined activity of both exchangers. We conclude that under the microenvironmental conditions found in some regions of tumors, Na+/H+ exchange may be the major method of regulation of pHi.


Amsacrine and etoposide hypersensitivity of yeast cells overexpressing DNA topoisomerase II. Increasing the cellular concentration of DNA topoisomerase II in yeast by expressing constitutively a plasmid-borne TOP2 gene encoding the enzyme greatly increases the sensitivity of the cells to amsacrine and etoposide (VP-16). This increased drug sensitivity at a higher intracellular DNA topoisomerase II level is observed in both RAD52+ repair-proficient strains and rad52 mutants that are defective in the repair of double-stranded breaks. These results provide strong support of the hypothesis that the cellular target of these drugs is DNA topoisomerase II, and that these drugs kill cells by converting DNA topoisomerase II into a DNA damaging agent.


Rational design and molecular effects of a new topoisomerase II inhibitor, azatoxin. Azatoxin [NSC 640737-M; 5.R,11aS-1H,6H,3-one-5,4,11,11a-tetrahydro-5-(3,5-dimethoxy-4-hydr oxyphenyl) oxazolo (3',4':1,6)pyrido-(3,4-b)indole] was rationally designed from a model for the pharmacophore of drugs with topoisomerase II inhibition activity. This pharmacophore has at least 2 domains: a quasiplanar polycyclic ring system proposed to bind between the DNA base pairs and a pendant substituent proposed to interact with the enzyme and/or to the DNA grooves. The present study shows that, in cell free systems, azatoxin induces a large number of double strand-breaks in linear Simian virus 40 and human c-myc DNA. These breaks yield cleavage patterns that are different from those of well established topoisomerase II inhibitors (epipodophyllotoxins, amsacrine, mitoxantrone). Azatoxin also inhibits the catalytic activity of purified topoisomerase II, and is a nonintercalator. The structure-activity relationship of 3 isomers and 6 derivatives of azatoxin shows a stringent stereochemical requirement for activity. The effects of azatoxin pendant ring substitution on topoisomerase II mediated DNA cleavage activity were similar to the relationship observed for etoposide.


Collagen-induced activation of the M(r) 72,000 type IV collagenase in normal and malignant human fibroblastoid cells. Although the M(r) 72,000 type IV collagenase (matrix metalloproteinase 2) has been implicated in a variety of normal and pathogenic processes, its activation mechanism in vivo is unclear. We have found that fibroblasts from normal and neoplastic human breast, as well as the sarcomatous human Hs578T and HT1080 cell lines, activate endogenous matrix metalloprotease 2 when cultured on type I collagen gels, but not on plastic, fibronectin, collagen IV, gelatin, matrigel, or basement membrane-like HR9 cell matrix. This activation is monitored by the zymographic detection of M(r) 59,000 and/or M(r) 62,000 species, requires 2-3 days of culture on vitrogen to manifest, is cycloheximide inhibitable, and correlates with an arborized morphology. A similar activation pattern was seen in these cells in response to Concanavalin A but not transforming growth factor beta or 12-O-tetradecanoylphorbol-13-acetate. The interstitial matrix may thus play an important role in regulating matrix degradation in vivo.


Correlation of serum metalloproteinase levels with lung cancer metastasis and response to therapy. Cancer cells elaborate metalloproteinases which may play a role in invasion and metastasis. The serum level of the M(r) 72,000 type IV collagenase (MMP-2) was measured in 87 lung cancer patients. Stage IV cancer levels were significantly elevated (P less than 0.0001) compared to normal sera. A significant difference (P less than 0.01) was found between enzyme levels in the presence versus the absence of distant metastasis. For 29 patients treated with combination chemotherapy, a positive relationship was noted between response failure and elevated enzyme levels. Serum metalloproteinase levels may provide information relevant to prognosis as well as treatment decisions.


Amplification and/or overexpression of platelet-derived growth factor receptors and epidermal growth factor receptor in human glial tumors. Analysis of genomic organization and expression of platelet-derived growth factor receptors (PDGFR) and epidermal growth factor receptor (EGFR) in human malignant gliomas showed amplification and overexpression of both receptors in distinct subsets of tumors. Amplification of the alpha PDGFR was detected in 4 of 50 glioblastomas (8%). EGFR was amplified in 9 of the 50 tumors (18%). Western blot analysis showed elevated expression of alpha PDGFR and EGFR proteins in 4 (24%) and 3 (18%), respectively, of 17 tumor specimens analyzed. Increased production of alpha PDGFR as well as EGFR proteins was observed in the presence or absence of gene amplification. Three of the 4 tumors with elevated levels of alpha PDGFR also overexpressed the beta PDGFR, which was present as a single copy gene in all 50 tumors analyzed. Our findings suggest that the amplification and/or overexpression either of EGFR or of the alpha PDGFR along with the coordinate overexpression of the beta PDGFR can contribute to the malignant phenotype of distinct subsets of human glioblastoma.


Phospholipase C-beta 1 is a GTPase-activating protein for Gq/11, its physiologic regulator. Purified M1 muscarinic cholinergic receptor and Gq/11 were coreconstituted in lipid vesicles. Addition of purified phospholipase C-beta 1 (PLC-beta 1) further stimulated the receptor-promoted steady-state GTPase activity of Gq/11 up to 20-fold. Stimulation depended upon receptor-mediated GTP-GDP exchange. Addition of PLC-beta 1 caused a rapid burst of hydrolysis of Gq/11-bound GTP that was at least 50-fold faster than in its absence. Thus, PLC-beta 1 stimulates hydrolysis of Gq/11-bound GTP and acts as a GTPase-activating protein (GAP) for its physiologic regulator, Gq/11. GTPase-stimulating activity was specific both for PLC-beta 1 and Gq/11. Such GAP activity by an effector coupled to a trimeric G protein can reconcile slow GTP hydrolysis by pure G proteins in vitro with fast physiologic deactivation of G protein-mediated signaling.


Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit. Purified bovine brain phosphatidylinositol 3-kinase (Pl3-kinase) is composed of 85 kd and 110 kd subunits. The 85 kd subunit (p85 alpha) lacks Pl3-kinase activity and acts as an adaptor, coupling the 110 kd subunit (p110) to activated protein tyrosine kinases. Here the characterization of the p110 subunit is presented. cDNA cloning reveals p110 to be a 1068 aa protein related to Vps34p, a S. cerevisiae protein involved in the sorting of proteins to the vacuole. p110 expressed in insect cells possesses Pl3-kinase activity and associates with p85 alpha into an active p85 alpha-p110 complex that binds the activated colony-stimulating factor 1 receptor. p110 expressed in COS-1 cells is catalytically active only when complexed with p85 alpha.


The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. A cDNA clone encoding a novel, widely expressed protein (called growth factor receptor-bound protein 2 or GRB2) containing one src homology 2 (SH2) domain and two SH3 domains was isolated. Immunoblotting experiments indicate that GRB2 associates with tyrosine-phosphorylated epidermal growth factor receptors (EGFRs) and platelet-derived growth factor receptors (PDGFRs) via its SH2 domain. Interestingly, GRB2 exhibits striking structural and functional homology to the C. elegans protein sem-5. It has been shown that sem-5 and two other genes called let-23 (EGFR like) and let-60 (ras like) lie along the same signal transduction pathway controlling C. elegans vulval induction. To examine whether GRB2 is also a component of ras signaling in mammalian cells, microinjection studies were performed. While injection of GRB2 or H-ras proteins alone into quiescent rat fibroblasts did not have mitogenic effect, microinjection of GRB2 together with H-ras protein stimulated DNA synthesis. These results suggest that GRB2/sem-5 plays a crucial role in a highly conserved mechanism for growth factor control of ras signaling.


Genetic analysis of genomic imprinting: an Imprintor-1 gene controls inactivation of the paternal copy of the mouse Tme locus. The Thp deletion on mouse chromosome 17 is lethal when inherited from the mother, because it deletes the T-associated maternal effect (Tme) locus, the paternal copy of which is inactivated by genomic imprinting. We have found a paternally nonimprinted Tme variant in crosses of Thp females with Mus m. musculus males. The data are consistent with the existence of a single Tme-unlinked gene, Imprintor-1 (Imp-1), with two alleles, one of which only causes imprinting at the Tme locus. Imp-1 is unlinked to the gene for cation-dependent Man-6-P receptor and acts prezygotically. Although Tme and Igf2r were thought to be identical, they show different patterns of imprinting in interspecies hybrids. The apparent nonequivalence of the Igf2r gene and Tme results in occurrence of viable mice lacking an active Igf2r gene. These mice are bigger at birth than their normal littermates, in accord with the proposed function of the IGF-II/Man-6-P receptor.


Immunohistochemical localization of estradiol, progesterone, and progesterone receptor in human salivary glands and salivary adenoid cystic carcinomas. Immunohistochemical analyses of estradiol, progesterone and progesterone receptor were carried out in human salivary gland and salivary adenoid cystic carcinoma. Immunoreactivity to estradiol and progesterone was found in cytoplasm of the cells of the excretory duct system within normal salivary glands, whereas the progesterone receptor was restricted to nuclei of the cells where both sex steroids were positive. In addition, we demonstrated the presence of both sex steroids and the receptor for progesterone in salivary adenoid cystic carcinomas. These data indicate that the human salivary gland is one of the target tissues of estrogen. This also suggests the good possibility that tumors which express progesterone receptors will respond to endocrine therapy.