Direct in vivo gene introduction into rat kidney. We established a simple and highly efficient method for in vivo gene transfer using HVJ (Sendai virus) and liposomes. Plasmid DNA and high mobility group 1 (HMG1) protein were co-encapsulated in liposomes by agitation and sonication and were co-introduced into cells by HVJ-mediated membrane fusion. pACT SVT DNA, as a reporter gene, was introduced into the kidney of intact rats through a cannula in the renal artery, and SV40 large T antigen was detected by enzyme immunohistochemistry in glomerular cells 4 days after its introduction. This newly developed kidney-directed gene transfer method should be useful not only in basic research but also in potential gene therapeutics of renal diseases.


Isolation and characterization of mitochondria from human B lymphoblastoid cell lines. Mitochondria were isolated from detergent-treated Epstein-Barr virus-transformed human lymphocytes to examine their potential use in the study of the functional expression of genetic disorders of the respiratory chain. The increase of cytochrome c oxidase activity in the mitochondrial fraction indicated a 6-fold purification of intact mitochondria. Polarographic and spectrophotometric studies revealed that the isolated mitochondria were functionally well preserved. Furthermore, the isolated mitochondria supported an active in organello protein synthesis, which was dependent on the presence of a respiratory substrate generating ATP and was essentially abolished by chloramphenicol or by a specific respiratory chain inhibitor, such as antimycin. Thus, B lymphoblastoid cell lines constitute a valuable source of mitochondria to investigate mitochondrial functions in patients affected by respiratory chain disorders.


Induction of thermotolerance in T cells protects nuclear DNA topoisomerase I from heat stress. In this study, we have demonstrated that topoisomerase I DNA relaxing activity is protected against a severe heat shock in T cells made thermotolerant by a prior modest heat treatment. However, following a severe heat-shock challenge and incubation at 37 degrees C, topoisomerase activity in the control population eventually returned to levels similar to those detected in thermotolerant cells. This recovery of topoisomerase activity appears to result from the renaturation of heat-inactivated enzyme rather than from synthesis of new protein because the rate of recovery of catalytic activity was not inhibited by the presence of the protein synthesis inhibitor, cycloheximide.


Pertussis toxin blocks activin A-induced production of inositol phosphates in rat hepatocytes. The present study was conducted to examine an involvement of G protein in the action of activin A in rat parenchymal liver cells. Activin A induced a dose-dependent increase in inositol phosphates in cells prelabelled with [3H]inositol. The effect of activin A was completely blocked by pretreatment of the cells with pertussis toxin. In contrast, pertussis toxin had little effect on angiotensin II-induced production of inositol phosphates. Both activin A and angiotensin II inhibited glucagon-mediated production of cAMP. Pretreatment of the cells with pertussis toxin blocked the inhibition induced by both activin A and angiotensin II. In permeabilized cells, activin A augmented production of inositol phosphates. Activin-mediated production of inositol trisphosphate was enhanced by GTP-gamma S and was attenuated by GDP-beta S. These results suggest that a pertussis toxin-sensitive G protein(s) may be involved in the action of activin A in hepatocytes.


Proton transport and Na+/H+ exchange in vesicles isolated from sockeye salmon (Oncorhynchus nerka) kidneys during migration from salt to fresh water. Renal epithelial function, proton flux and sodium stimulated proton flux, was observed in vesicles isolated from the brush border of the proximal tubule of Sockeye Salmon (Oncorhynchus nerka) during migration. Brush border membrane vesicles (BBMV) were isolated from the body kidney of Sockeye Salmon using aggregation/differential centrifugation techniques. Vesicle purity was tested using a series of epithelial and basal lateral markers including alkaline phosphatase, maltase, gamma-glutamyl transferase (GGTP), Mg(2+)-activated ATP-ase, Na(+)+K(+)-activated ATPase, and 5'-nucleotidase and the lysosomal marker acid phosphatase. An enrichment/depletion factor for each marker was determined by comparison of purified BBMV with kidney homogenate. Vesicles exhibit an enrichment factor for alkaline phosphatase, GGTP, maltase, Mg(2+)-activated ATP-ase, Na(+)+K(+)-activated ATPase, and 5'-nucleotidase. A depletion factor was observed for acid phosphatase. Vesicle integrity was tested by measuring the time course of proton flux in the presence of a pH gradient. Amiloride sensitive sodium stimulated proton flux was observed in these vesicles. The presence of sodium caused a saturable increase in the rate of proton flux, indicating the activity of a sodium/proton antiport protein in BBMV.


Analysis of the order of autophosphorylation of human insulin receptor tyrosines 1158, 1162 and 1163. Insulin receptor tyrosines 1158, 1162 and 1163 are the most rapidly autophosphorylated residues following insulin binding. Although progression of these tyrosines from a bis- to tris-phosphorylated state leads to activation of the receptor tyrosine kinase towards added substrates, rather paradoxically, a receptor with a Y1158F mutation has been reported to be capable of normal activation. In the present study we demonstrate that autophosphorylation of the insulin receptor probably initiates on either of tyrosines 1158 and 1162 while autophosphorylation of tyrosine 1163 occurs predominantly late in the autophosphorylation cascade. Our results are compatible with tyrosines 1162 and 1163 being the major determinants of kinase activity and explain why wild-type insulin receptors only become active after all three of tyrosines 1158, 1162 and 1163 have been phosphorylated.


cAMP-regulated chloride currents in CHO cells. We examined whether elevations in cAMP levels increase membrane chloride permeability in native CHO cells by measuring whole cell chloride currents and efflux of 125I and 36Cl. With 20 microM forskolin, no significant effect was seen on whole cell currents. However, 100 microM forskolin increased both whole cell chloride currents and the rate of 125I and 36Cl efflux. Forskolin-activated currents showed a linear current-voltage relationship in solutions with symmetrical chloride concentrations and reversal potential changed in the direction anticipated for a chloride-selective current when chloride was replaced with gluconate. These results indicate that native CHO cells exhibit cAMP-regulated chloride conductance pathways which become apparent only after large elevations in intracellular cAMP levels.


Level of ETB receptor mRNA is down-regulated by endothelins through decreasing the intracellular stability of mRNA molecules. Using ROS17/2 rat osteosarcoma cells as a model system, we examined the possibility that endothelin (ET)-induced down-regulation of ETB receptor was accompanied by a decrease in levels of ETB receptor mRNA. Northern blot analysis showed that low doses of ET-1 and ET-3 caused a transient decrease in ETB receptor mRNA in the cells. The maximum decrease in the levels of ETB receptor mRNA (80%) occurred after 2-4 h of exposure of the cells to ETs and was followed by a gradual recovery to control levels by 24 h. The effects were dose-dependent (EC50-1 nM), and ET-1 and ET-3 were almost equipotent in eliciting the response. The addition of either ionomycin, a Ca2+ ionophore, or phorbol dibutyrate, a protein kinase C activator, mimicked the effect of ETs. These results suggested that ETs-induced down-regulation of ETB receptor mRNA was mediated by the activation of ETB receptor and that it may have involved ETB receptor coupled second messenger pathways. We also showed that ETB receptor mRNA had a long intracellular life span which suggested that ETs-induced down-regulation of ETB receptor mRNA may have been due to a decrease in the stability of mRNA, rather than inactivation of the transcription of mRNA.


Invasion of brain tissue by primary glioma: evidence for the involvement of urokinase-type plasminogen activator as an activator of type IV collagenase. The immunocharacterization of a metalloproteinase isolated from rat glioma cell conditioned medium is described and confirms that the enzyme is identical to type IV collagenase. Free, active plasminogen activator (PA) and PA-PAI complexes were identified as being secreted by the same cells. Using affinity-purified metalloproteinase we demonstrate that the enzyme can be partially activated by u-PA but not by plasmin in vitro. On the basis of these findings and previous published work we propose a scheme for the proteolytic degradation of normal brain tissue during tumour invasion.


Role of Mg2+ in activation of NADPH oxidase of human neutrophils: evidence that Mg2+ acts through G-protein. The membrane fraction and three cytosolic proteins of neutrophils, p47-phox, p67-phox and a G-protein, are involved in the cell-free activation of the O2(-)-generating NADPH oxidase in the presence of SDS, though it has been controversial whether the G-protein is required or just enhancing the activity. We have used the three cytosolic factors, the solubilized membrane fraction, GTP gamma S and SDS, and found that both G-protein and GTP gamma S are essential for the activation of the NADPH oxidase. The effect of GTP gamma S is modified by Mg2+: the cations enhance the O2- generation at low concentrations of GTP gamma S, whereas they attenuate the activity at higher concentrations of GTP gamma S. In presence of 10 microM GTP gamma S, the maximal activity is observed at 0.1 microM Mg2+, which is several-fold higher than that at 1 mM Mg2+. The omission of Mg2+ followed by the chelation with EDTA results in loss of the activation, which is completely restored by the addition of Mg2+. Thus, Mg2+ seems to modulate the activation of the NADPH oxidase at the level of the G-protein.


Purification and characterization of S-modulin, a calcium-dependent regulator on cGMP phosphodiesterase in frog rod photoreceptors. S-modulin is a 26 kDa protein that regulates light sensitivity of cGMP phosphodiesterase in a Ca(2+)-dependent manner in frog rod outer segments (ROSs). In the present study, we purified S-modulin by taking advantage of a hydrophobic interaction between Phenyl Sepharose and S-modulin at high Ca2+ concentrations. The yield was greater than 90%. 45Ca(2+)-binding experiment showed that S-modulin is a Ca(2+)-binding protein. At high Ca2+ concentrations, S-modulin binds to ROS membranes. The binding target of the Ca2+/S-modulin complex is possibly a ROS membrane lipid(s), but it was difficult to identify. The binding was observed mainly at greater than 1 microM Ca2+. The amino acid sequence deduced from proteolytic fragments of S-modulin was approximately 80% and 60% identical to those of recovering and visinin, respectively.


Amplification of P450c21 expression in cultured mammalian cells. We describe in this paper an investigation of mammalian expression systems for P450c21 (21-hydroxylase). Four different promoters, the SV40 early and late promoters, MMTV-LTR, and CMV immediate early promoter were tested for their ability to drive the expression of P450c21 in cultured COS-1 cells. With the exception of MMTV-LTR, all drove the expression of similar levels of functional 21-hydroxylase. In addition, the Rat-1 cell line was tested and shown to be suitable for the stable expression of functional P450c21. We have established cell lines derived from Rat-1 either normal or mutant P450c21 stably expressed together with amplifiable markers. The expression of P450c21 was further increased by selection in methotrexate.


Reconstitution and characterization of the human neutrophil respiratory burst oxidase using recombinant p47-phox, p67-phox and plasma membrane. Human neutrophil respiratory burst oxidase (NADPH-oxidase) activity can be reconstituted in a cell-free system consisting of plasma membrane, cytosol and an anionic amphiphile [e.g., sodium dodecyl sulfate (SDS) or arachidonate]. Herein, we report reconstitution of oxidase activity using isolated neutrophil plasma membrane together with purified recombinant p47-phox and p67-phox which had been produced using a baculovirus expression system. Activity required an anionic amphiphile (SDS or arachidonate) and was potentiated by diacylglycerol and GTP gamma S. Serial washes of the plasma membrane failed to affect its ability to reconstitute activity, indicating that a dissociable membrane component was not present. The Km for NADPH, 43 microM, was the same as that determined using cytosol in place of recombinant factors. The EC50 values for p47-phox and p67-phox under optimal activation conditions were 220 nM and 80 nM, respectively, indicating a relatively high affinity of these components in an activation complex. Since neither cytosolic component contains a nucleotide binding consensus sequence, these data indicate that the NADPH binding component of the oxidase resides in the plasma membrane.


Insulin induced phosphorylation and activation of the cGMP-inhibited cAMP phosphodiesterase in human platelets. Insulin induced phosphorylation and activation of the cGMP inhibited cAMP phosphodiesterase (cGI-PDE) in human platelets were demonstrated after isolation of the enzyme with specific polyclonal cGI-PDE antibodies. The demonstration of this insulin effect required suppression of basal cGI-PDE phosphorylation, through the use of the protein kinase inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine). The human platelet insulin receptor beta-subunit, previously identified as a 97 kDa polypeptide, was detected with the use of wheat germ agglutinin chromatography and anti-phosphotyrosine antibodies. These results suggest that insulin, through phosphorylation/activation of cGI-PDE, could decrease cAMP/cAMP dependent protein kinase (cAMP-PK) activity and thereby make the platelets more sensitive towards aggregating agents.


Okadaic acid induces the rapid and reversible disruption of the neurofilament network in rat dorsal root ganglion neurons. Treatment of 15-17 day old dissociated cultures of rat dorsal root ganglia with 1 microM okadaic acid caused a reduction in the mobilities of neurofilament subunits on SDS-polyacrylamide gels, signifying an increase in their phosphorylation levels. When cultures were exposed to okadaic acid for 0.5 hrs and harvested in buffer containing Triton X-100, NF-H was nearly completely redistributed to the detergent- soluble fraction while NF-M and NF-L required a longer exposure to the drug before undergoing a similar shift. This redistribution of subunits corresponded with striking changes in the immunofluorescence staining pattern for neurofilaments. Upon removal of okadaic acid from the culture medium following a 0.5 hr treatment, NF-L and NF-M returned to the Triton X-100 insoluble fraction within 2 hrs while NF-H required 10 hrs for recovery.


Ga3+ inhibits parathyroid hormone release without interacting with the Ca2+ receptor of the parathyroid cell. Gallium nitrate is an antihypercalcemic agent with established actions on bone. The effects of Ga(NO3)3 on parathyroid hormone (PTH) release, cytoplasmic Ca2+ concentration ([Ca2+]i) and cAMP production of enzymatically dispersed parathyroid cells from bovine as well as normal and pathological human parathyroid glands have now been studied. Ga3+ at 200 microM inhibited PTH release whereas 600 microM NO3- had no effect. The inhibition was additive to that obtained by elevating extracellular Ca2+. Unlike Ca2+, Ga3+ failed to increase [Ca2+]i or reduce cAMP formation. The results indicate that Ga3+ inhibits PTH release by a mechanism other than activation of the cation receptor of the parathyroid cells. This mechanism may contribute also to inhibition by other cations.


Two distinct membrane-bound phosphatidylinositol-4-phosphate phosphatases in bovine brain. Solubilized phosphatidylinositol-4-phosphate 4-phosphatase from bovine brain resolved into two peaks of activity by ion exchange chromatography. Both exhibited substantial detergent binding characteristic of integral membrane proteins, and both appear specific for phosphatidylinositol-4-phosphate, but their pH optima differ: the earlier eluting fraction (peak 1) is optimally active between pH 5.5 and 6, whereas the later eluting fraction (peak 2) is most active around pH 8.5. Detergent inhibition studies suggest that peak 2, but not peak 1, interacts with phosphatidylinositol-4-phosphate in the context of a single mixed micelle. Further characterization of these activities should help shed light on the biological function of polyphosphoinositide phosphatases.


Product analyses in DNA strand scission by antitumor antibiotic elsamicin A. Elsamicin A is an antitumor antibiotic with fascinating chemical structure and a good candidate for pharmaceutical development. Molecular mechanism of DNA backbone cleavage mediated by Fe(II)-elsamicin A has been examined. Product analysis using DNA sequencing gels and HPLC reveals the production of damaged DNA fragments bearing 3'-/5'-phosphate and 3'-phosphoglycolate termini associated with formation of free base. In addition, hydrazine-trapping experiments indicate that C-4' hydroxylated abasic sites are formed concomitant with DNA degradation by Fe(II)-elsamicin A. The results lead to the conclusion that the hydroxyl radical formed in Fe(II)-elsamicin A plus dithiothreitol system oxidizes the deoxyribose moiety via hydrogen abstraction predominantly at the C-4' carbon of the deoxyribose backbone and ultimately produces strand breakage of DNA.


A parathyroid-related peptide induces transcaltachia (the rapid, hormonal stimulation of intestinal Ca2+ transport). PTH-related peptide (PTHrP) has been shown to be responsible for the hormonal hypercalcemia of malignancy. Previously, we demonstrated that both 1,25(OH)2-vitamin D3 and the N-terminal fragment of PTH (1-34) stimulates the rapid transport of Ca2+ (transcaltachia) in the perfused chick intestine. Since there is a sequence homology between these two hormones in the n-terminal fragment, in this study we examined the effect of PTHrP(1-40) on stimulation of transcaltachia in the perfused chick duodenum. The results indicate that the maximal stimulation of transcaltachia occurs at 50 pM PTHrP(1-40), and that the dose-response curve is biphasic in nature. Perfusion with 25 pM, 50 pM, 100 pM or 200 pM PTHrP(1-40) for 40 min increases the transport of Ca2+ in perfused intestine 1.8-, 3.0-, 2.4- and 1.6-fold, respectively. The response is rapid, occurring within 10 min of introduction of the PTHrP. The Ca2+ channel inhibitor nifedipine, which is known to abolish the transcaltachic effect elicited by 1,25(OH)2 vitamin D3, also inhibited the rapid transport of Ca2+ stimulated by PTHrP(1-40). The transcaltachic effect of PTHrP(1-40) may be mediated by a signal transduction pathway in which Ca2+ channels are activated.


Human lung adrenergic and muscarinic cholinergic receptors in cancer and previous airways diseases. In the present study adrenergic and muscarinic cholinergic receptors have been investigated in human lung parenchyma in cancer and previous airways diseases (chronic pneumonia, tuberculosis). It has been found that the number of muscarinic sites significantly increases in cancer and does not change both in chronic pneumonia and tuberculosis lung parenchyma in comparison with the normal tissue; the number of beta-adrenergic sites decreases in cancer, chronic pneumonia, as well as severe tuberculosis lung parenchyma. The important role of beta-adrenergic and muscarinic receptors in the formation of human lung adenocarcinoma is discussed.


Identification of multiple biological factors in rabbit serum that modulate dopamine-mediated aortic constriction. A bioassay was established using freshly prepared rabbit aortic strip to monitor the effects of various biological factors contained in rabbit serum that modulate vasoconstriction. Serotonin was shown to be a major vasoconstriction modulator in rabbit serum. A rapid procedure is described for its isolation from rabbit serum by sequential gel filtration chromatography and reversed-phase HPLC using C4, C18, and C2/C18 columns with an overall cumulative yield of about 20%. It was also noted that rabbit serum contains multiple biological factors that modulate dopamine-mediated aortic contraction other than serotonin. A potent vasoconstriction inhibitor was identified in rabbit serum which appears to be a novel regulator that mediates its effect via both alpha-adrenergic and serotonin receptors.


Differential effects of platelet-activating factor on superoxide anion production in human eosinophils and neutrophils. The effect of platelet-activating factor (PAF) on the generation of superoxide anion (O2-) in human eosinophils and neutrophils was examined. The presence of PAF potentiated O2- production in either opsonized zymosan- or formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated cells. The effect of PAF was prominent in opsonized zymosan-stimulated eosinophils and in FMLP-stimulated neutrophils. We also found that eosinophils generate substantial amounts of O2- when treated with PAF alone. The high responsiveness of unstimulated or opsonized zymosan-stimulated eosinophils to PAF to generate O2- may be relevant to the pathological changes at the loci of allergic reactions where eosinophils and PAF are crucially involved.


New chemotactic peptide analogs with high biological activity for human neutrophils. As a part of a research programme aimed at studying structure activity relationships in the field of chemotactic peptides, modified analogs of the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) were examined for their capacity to activate several functions of human neutrophils. 4-Aminotetrahydrothiopyran-4carboxylic acid (Thp) and 2-aminoindane-2-carboxylic acid (Ain) were chosen as achiral, conformationally restricted amino acids suitable for mimicking the external Met and Phe residues of FMLP-OMe. The replacement of both produces a high locomotion activity, greater than the parent peptide; in contrast, the two Thp-containing analogs induce neither superoxide production nor lysozyme release. From these results we can hypothesize two different signal transduction systems: one which provides for movement, the other for superoxide generation and granule enzyme release.


Modification of G regulatory protein mediated actions by the interaction of histocompatibility antigens with cardiac muscarinic cholinergic receptors. In this work we characterized the interaction of class I histocompatibility (HC) antigens (Ag) with cardiac cholinergic receptors by means of specific radioligand binding and by production of cholinergic-mediated cellular transmembrane signals. Alloimmune as well as anti-class I but not anti-class II antibodies were able to inhibit in an allosteric manner the binding of [3H]quinuclidinyl benzilate to cardiac membrane. Moreover, alloantibody could modify all of the muscarinic cholinergic effects mediated by a G regulatory protein, i.e. decrement of atria contractility, inhibition of cAMP stimulation, and activation of the turnover of phosphoinositides via phospholipase C. The cGMP production was not altered by the alloantibody. The data indirectly indicated that HC-Ag-muscarinic cholinergic interactions trigger all the cholinergic functions related to G proteins. The induction of intracellular second messengers by class I antigens and hormone-receptor interactions is discussed.


Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-para-chlorophenylthio-cGMP. 8-(p-Chlorophenylthio)-cGMP (8-pCPT-cGMP) and 8-bromo-cGMP were compared with respect to their chemical and biological properties in order to evaluate their potential as selective activators of cGMP-dependent protein kinase (cGMP-PK; EC 2.7.1.37) in intact human platelets. 8-pCPT-cGMP, 8-Br-cGMP and cGMP were shown to be potent and selective activators of purified bovine lung cGMP-PK and of cGMP-PK present in human platelet membranes when compared with the activation of cAMP-dependent protein kinase (cAMP-PK; EC 2.7.1.37). 8-pCPT-cGMP was not hydrolysed by the purified cGMP-stimulated phosphodiesterase (cGS-PDE), cGMP-inhibited phosphodiesterase (cGI-PDE) and Ca(2+)-calmodulin-dependent phosphodiesterase (CaM-PDE), whereas cGMP and, to a lesser extent, 8-Br-cGMP were hydrolysed by all three types of 3',5' cyclic nucleotide phosphodiesterases (EC 3.1.4.17) examined. Also, 8-pCPT-cGMP was not hydrolysed by a human platelet homogenate which contains a high level of the cGMP-specific cGMP-binding phosphodiesterase (cGB-PDE). Additionally, 8-pCPT-cGMP did not activate the cGS-PDE or inhibit the cGI-PDE, whereas half-maximal inhibition of cGI-PDE occurred at 8 microM 8-Br-cGMP. The apparent lipophilicity of 8-pCPT-cGMP was higher than that of 8-Br-cGMP. Extracellular application of 8-pCPT-cGMP to intact human platelets reproduced the pattern of protein phosphorylation induced by sodium nitroprusside (SNP), a cGMP-elevating inhibitor of platelet activation. Quantitatively, 8-pCPT-cGMP was more effective than 8-Br-cGMP in inducing phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein, a major substrate of cGMP-PK in intact platelets. As observed with SNP, pretreatment of human platelets with 8-pCPT-cGMP prevented the aggregation induced by thrombin. The results suggest that 8-pCPT-cGMP is a very potent and selective activator of cGMP-PK in cell extracts and in intact human platelets and, in this respect, is superior to 8-Br-cGMP and other cGMP analogs used for intact cell studies. The data also suggest that inhibition of platelet activation in intact human platelets by nitrovasodilators is mediated by cGMP-PK.


Effect of free fatty acids on GABAA receptor ligand binding. Phospholipase A2 (PLA2) treatment of synaptosomal membranes, which causes the release of fatty acids, particularly unsaturated fatty acids, inhibits the flux of chloride ions through the gamma-aminobutyric acid (GABA) benzodiazepine receptor ion channel in response to activation by agonists. PLA2 treatment has also been shown to affect ligand binding to the receptor. In the present study, we have investigated the effect of unsaturated free fatty acids, arachidonic acid and oleic acid and saturated free fatty acids, arachidic acid and stearic acid on various characteristics of GABAA receptor ligand binding. Only the unsaturated fatty acids showed any effect: arachidonic acid and oleic acid enhanced flunitrazepam binding and muscimol binding but inhibited tert-butylbicyclophosphorothionate (TBPS) binding in a dose-dependent manner. The effects on muscimol and TBPS binding were shown to be due to changes in receptor density by saturation analysis. Oleic acid and arachidonic acid also decreased the enhancement of flunitrazepam and muscimol binding by cartazolate and pentobarbital but did not affect GABA enhancement of flunitrazepam binding. These data indicate that unsaturated free fatty acids can mimic the effects of PLA2 treatment and underline the importance of the lipid microenvironment on ligand binding to the GABAA receptor.


Picroliv, picroside-I and kutkoside from Picrorhiza kurrooa are scavengers of superoxide anions. Picroliv, the active principle of Picrorhiza kurrooa, and its main components which are a mixture of the iridoid glycosides, picroside-I and kutkoside, were studied in vitro as potential scavengers of oxygen free radicals. The superoxide (O2-) anions generated in a xanthine-xanthine oxidase system, as measured in terms of uric acid formed and the reduction of nitroblue tetrazolium were shown to be suppressed by picroliv, picroside-I and kutkoside. Picroliv as well as both glycosides inhibited the non-enzymic generation of O2- anions in a phenazine methosulphate NADH system. Malonaldehyde (MDA) generation in rat liver microsomes as stimulated by both the ascorbate-Fe2+ and NADPH-ADP-Fe2+ systems was shown to be inhibited by the Picroliv glycosides. Known antioxidants tocopherol (vitamin E) and butylated hydroxyanisole (BHA) were also compared with regard to their antioxidant actions in the above system. It was found that BHA afforded protection against ascorbate-Fe(2+)-induced MDA formation in microsomes but did not interfere with enzymic or non-enzymic O2- anion generation; and tocopherol inhibited lipid peroxidation in microsomes by both prooxidant systems and the generation of O2- anions in the non-enzymic system but did not interfere with xanthine oxidase activity. The present study shows that picroliv, picroside-I and kutkoside possess the properties of antioxidants which appear to be mediated through activity like that of superoxide dismutase, metal ion chelators and xanthine oxidase inhibitors.


Acute effects of cadmium on the renin angiotensin system in rats. The effect of cadmium acetate (1 mg/kg i.p.) on the renin angiotensin system was examined in male Sprague-Dawley rats. Blood pressure, plasma renin activity (PRA) and serum angiotensin converting enzyme (ACE) levels were determined in the rats. Cadmium acetate produced a hypertensive response which was not associated with elevated PRA. However, ACE levels in the serum were significantly decreased in the cadmium-treated group as compared with normal controls (P less than 0.01).


Heparin: does it act as an antioxidant in vivo? Previous studies have shown that heparin antagonizes oxygen radical-mediated injury to endothelial cells, suggesting an antioxidant role of the drug. In the present investigation, the hypothesis that heparin exerts direct antioxidant effects was tested in several experimental models. We have found that 1, 3, 5, 10, 20, 40 and 80 U/mL of heparin do not scavenge superoxide anion, hydrogen peroxide, hydroxyl radical or the stable free radical 1,1-diphenyl-2-pycrylhydrazyl. Moreover, the drug is ineffective towards iron-driven linolenic acid peroxidation, autooxidation of brain homogenate and linolenic acid peroxidation mediated by human internal mammary artery homogenate. Specific studies on the potential iron-binding-inactivating capacity of heparin prove the drug to be totally ineffective. Finally, the loss of protein sulphydryls from human plasma induced by hypoxanthine-xanthine oxidase-generated oxygen radicals is not prevented by heparin. In conclusion, heparin, even at concentrations far higher than those usually used therapeutically, has no direct antioxidant properties. Thus, other mechanisms not strictly antioxidant-type must be involved in heparin-mediated cell protection against toxic oxygen metabolites.


Inhibition of gastric H+,K(+)-ATPase and acid secretion by cassigarol A, a polyphenol from Cassia garrettiana Craib. The effects of cassigarol A, a naturally occurring polyphenol, on gastric H+,K(+)-ATPase and gastric acid secretion were studied. Cassigarol A inhibited H+,K(+)-ATPase and K-stimulated p-nitrophenyl phosphatase from hog gastric mucosa with 50% inhibition of 1.2 x 10(-6) and 6.3 x 10(-6) M, respectively. The kinetic study showed that the inhibition of H+,K(+)-ATPase by cassigarol A was competitive with respect to ATP and non-competitive with respect to K+. Cassigarol A inhibited both H+,K(+)-ATPase-mediated proton transport and 2-deoxy-D-glucose-induced acid secretion. On the other hand, cassigarol A acetate, in which phenolic hydroxy groups are acetylated, was not effective in the inhibition of enzyme activity and acid secretion. These results indicate that cassigarol A is a potent inhibitor of gastric H+,K(+)-ATPase, that the anti-secretory activity of cassigarol A is related to the inhibition of H+,K(+)-ATPase and that an important moiety of cassigarol A in the interaction with the enzyme is the phenolic hydroxy groups.


Dissociation of the striatal D-2 dopamine receptor from adenylyl cyclase following 6-hydroxydopamine-induced denervation. Intracellular cyclic AMP accumulation following exposure to dopamine (DA) agonists and and antagonists was measured in striatal slices from rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion of the nigrostriatal pathway and which showed contralateral circling to apomorphine. Both DA (10-320 microM) and the D-1 agonist SKF 38393 (0.1-32 microM) increased cyclic AMP accumulation in striatal slices from the lesioned and intact hemispheres. The EC50 for DA to increase cyclic AMP accumulation in slices was greater in the 6-OHDA-lesioned striata compared to the intact striatum, but the EC50 for SKF 38393 was not affected. The D-1 antagonist SCH 23390 (10 microM) completely inhibited the ability of DA and SKF 38393 to increase cyclic AMP accumulation in striatal slices from both denervated and intact sides of the brain. In slices from the intact hemisphere the increase in DA-induced cyclic AMP accumulation was enhanced by the D-2 antagonist (+/-)-sulpiride (50 microM) but (+/-)-sulpiride had no effect on the DA response in slices from the lesioned side. Similarly, the ability of SKF 38393 to enhance cyclic AMP accumulation was blocked by the D-2 agonist quinpirole (10 microM) in striatal slices from the intact hemisphere but not in tissue from the lesioned side. The density of striatal D-1 and D-2 receptors assessed by [3H]SCH 23390 and [3H]spiperone binding did not differ between the hemispheres although there was an increase in the affinity of D-1 receptors for [3H]SCH 23390 in the lesioned striatum. After striatal deafferentiation there appears to be an uncoupling of the "inhibitory" D-2 receptor from the D-1 receptor-associated adenylyl cyclase.


Acute reserpine treatment induces down regulation of D-1 dopamine receptor associated adenylyl cyclase activity in rat striatum. Behavioural studies suggest a functional interaction between D-1 and D-2 systems in normal rat striatum to alter motor behaviour and which is disrupted by dopamine depletion induced by acute reserpine treatment. Consequently, we have investigated the effect of acute reserpine treatment on the biochemical interaction between D-1 and D-2 receptors present in rat striatal slices. Twenty-four hours following the administration of reserpine (5 mg/kg i.p.), striatal dopamine content was depleted by more than 73%; the density (B(max)) of D-1 receptor sites measured by the in vitro binding of [3H]SCH 23390 to striatal membranes was increased while the binding of [3H]spiperone to D-2 receptor sites was unaltered. Reserpine treatment had no effect on the affinity (Kd) of [3H]SCH 23390 or [3H]spiperone for D-1 and D-2 sites. Basal levels of cyclic AMP accumulation in striatal slices prepared from reserpine-treated rats were lower than those observed in control slices. In striatal slices prepared from normal rats, dopamine (10-320 microM) and the D-1 agonist SKF 38393 (0.1-3.2 microM) induced concentration-dependent increases in cyclic AMP accumulation. The D-1 antagonist SCH 23390 (10 microM) abolished the accumulation of cyclic AMP produced by dopamine or SKF 38393. The D-2 antagonist (+/-)-sulpiride (50 microM) enhanced the response to dopamine (10-320 microM) while the D-2 agonist quinpirole (10 microM) abolished the response to SKF 38393 (0.1-3.2 microM). However, 24 hr after reserpine treatment the ability of dopamine (10-320 microM) and SKF 38393 (0.1-3.2 microM) to elicit an increase in cyclic AMP accumulation was markedly reduced in striatal slices. SCH 23390 (10 microM) did not enhance the trend for an increase in cyclic AMP accumulation produced by dopamine. Also, quinpirole (10 microM) did not affect the response to SKF 38393 (0.1-3.2 microM) in striatal slices from reserpine pretreated rats. The data confirm the positive linkage between D-1 receptors and adenylyl cyclase and the inhibitory coupling to D-2 sites in striatal slices from normal, rats. Acute reserpine treatment appears to cause an uncoupling of D-1 receptors associated with adenylyl cyclase.


Biochemical and functional alterations associated with hypercholesterolemia in platelets from hypertensive patients. Hypercholesterolemia and hypertension are two of the major risk factors associated with increased atherosclerotic vascular disease. An abnormal platelet function is one of the mechanisms proposed to participate in atherogenesis. This study was undertaken to find out whether hypercholesterolemia in hypertensive patients can change platelet lipid composition and reactivity. Twenty-nine untreated hypertensive patients were distributed into 3 age, body mass index and blood pressure-matched groups according to their plasma cholesterol levels (normal, borderline or elevated, group NC, BC and HC respectively). Their platelet lipid composition, cytosolic Ca2+ concentration, cyclic AMP content and aggregating response to ADP and collagen were determined. Platelet from group HC patients were characterized by reduced cyclic AMP content (evaluated in the presence and absence of a platelet phosphodiesterase inhibitor) and aggregating responses to ADP and collagen, increased palmitic acid content and decreased arachidonic, eicosapentaenoic and docosatetraenoic and pentaenoic acid content, resulting in a lowered polyunsaturated to saturated fatty acid ratio (P less than 0.001). In contrast, platelet cytosolic Ca2+ concentration, DPH steady-state anisotropy and cholesterol to phospholipid molar ratio were not significantly changed. This indicates that hypercholesterolemia is accompanied in hypertensive patients by marked changes in platelet fatty acid composition, cyclic AMP content and response to aggregating agents. These changes, which clearly differ from those induced by in vitro cholesterol loading, could reflect not only the balance between LDL and HDL stimulation but also an adaptation to hemodynamic perturbations.


Role of central GABAB receptors in physiology and pathology. There are two major classes of gamma-aminobutyric acid (GABA)-sensitive receptors: GABAA and GABAB. The GABAA receptor, the better known of the two GABA receptors, is a heterooligomeric complex that forms a chloride channel. Multiple subtypes of the GABAA receptor result from the composition of different subunits. In contrast to the GABAA receptor, the GABAB receptor protein has not been isolated and purified to homogeneity. Various effector systems, however, have been identified for the GABAB receptor using a limited GABAB-specific pharmacologic reportoire. In almost all cases, activated GABAB receptors employ a guanosine triphosphate-binding protein to transduce a signal intracellularly. There may be multiple subtypes of the GABAB receptor. Because the responses elicited by activation of GABAB receptors are small in terms of their intensity and are considered to be modulatory, the role these receptors play in the central nervous system (CNS) may not be very obvious. However, it is our view that in a finely tuned instrument such as the brain, treatment with neuromodulators (drugs that produce slight changes in brain neurochemistry) may be safer than most current drugs. Moreover, neuromodulators may have far greater potential as pharmacotherapeutic agents for CNS disorders. Thus, in this article, we will review the pharmacologic characteristics of the GABAB receptor, known physiologic roles that this receptor plays in the CNS, and the importance of this receptor in certain disease states.


The expression of a putative insulin-like growth factor-I receptor gene in the liver of the developing chick. The biological activity of insulin-like growth factor-I (IGF-I) is mediated by a transmembrane glycoprotein (type-1 IGF receptor or IGF-I receptor) that shows considerable sequence homology with the insulin receptor. In order to detect the expression of this gene in chicken liver tissue, a plasmid was constructed containing a fragment of chicken IGF-I receptor cDNA. The cDNA fragment corresponded to nucleotides 326-599 of the human IGF-I receptor cDNA and showed 86.1 and 69.3% homology at the nucleotide level and 96.7 and 80.2% homology at the amino acid level with the human IGF-I receptor and insulin receptor respectively. The construct was used to generate an antisense RNA probe for the detection of IGF-I receptor mRNA transcripts in 1- and 4-week-old chick liver tissue. IGF-I receptor gene expression was initially detected by the reverse transcriptase polymerase chain reaction using synthetic chicken IGF-I receptor oligonucleotides. Amplified fragments of the correct size were detected in both RNA samples. Northern blots were also used to detect IGF-I receptor mRNA transcripts in the liver RNA samples. The results indicated that the amount of receptor mRNA decreased significantly between 1 and 4 weeks after hatch. In contrast, chicken beta-actin gene expression remained constant over this period. A major IGF-I receptor RNA transcript (11 kb) was observed in blots from 1-week-old livers, less abundant transcripts were also observed ranging in size from 8 to 9 kb.(ABSTRACT TRUNCATED AT 250 WORDS)


Evidence for two distinct hormone-sensitive [3H]phosphoinositide pools in bovine adrenocortical zona fasciculata/reticularis cells stimulated with angiotensin II. Bovine adrenocortical cells from the zona fasciculata/reticularis were isolated and their phosphoinositides labelled to a steady state with [3H]inositol in primary culture. Experiments performed on these cells in the presence of Li+ have shown that, over a period of 60 min, angiotensin II (AII; 10(-7) M) stimulated a linear increase in [3H]inositol phosphates that was sustained through the utilization of two hormone-sensitive subpools of prelabelled lipid (30% and 45% respectively), and a rapid resynthesis of [3H]phosphoinositide into one of these pools using cytosolic [3H]inositol. The 30% pool was used immediately on stimulation, and was sustained at a steady-state size of 10-15% during the first 30 min of stimulation through rapid resynthesis using cytosolic [3H]inositol. Only after 30 min, when the cytosolic [3H]inositol was depleted and resynthesis could no longer occur, did the additional 45% pool start to supply further substrate to the phospholipase C, thereby further sustaining the generation of [3H]inositol phosphates. Once this pool was depleted however (by approximately 60 min), [3H]inositol phosphate generation finally ceased. These findings establish the differential use of two metabolically distinct hormone-sensitive pools of phosphoinositide following AII stimulation in bovine adrenocortical cells, events which are dependent upon the availability of cytosolic inositol for phosphoinositide resynthesis.


Effects of cisplatin on human thyrocytes in monolayer or follicle culture. Cis-diamminedichloroplatinum (II) (cisplatin) is a widely used anticancer drug which induces many side-effects, but its action on the thyroid gland is still unknown. We have investigated the effects of this drug on human thyrocytes cultured in monolayers or in follicles and stimulated with 200 microU TSH/ml. After 72 h in culture, different concentrations of cisplatin (15, 30 and 75 microM) caused partial or total inhibition of cyclic AMP (cAMP), thyroglobulin (Tg) and tri-iodothyronine (T3) production, whereas thyroxine levels increased in the medium of thyrocytes cultured as follicles. Small doses of the drug did not affect thyrocyte production. Decreases in neutral-red uptake by thyroid cells and in intracellular lactate dehydrogenase, alpha-hydroxybutyryldehydrogenase and creatine phosphokinase activities were induced by 30 and 75 microM cisplatin. These data show that high concentrations of cisplatin had a cytotoxic effect on thyrocytes. Cisplatin also induced inhibition of the production of cAMP, Tg and T3.


[Drash's syndrome and its variants. A report of 3 cases]. Three cases of Drash syndrome (DS) are presented. Two of them were 2 years old, the third one a newborn. Two are autopsy cases, one of the patients is alive. All three of them had nephrotic syndrome and renal failure, with mesangial sclerosis. All three had ambiguous genitalia, abdominal testes with variable degrees of dysgenesis and bilateral gonadoblastoma. Two cases had 46XY karyotype, in one case it was not performed. Two patients had nephroblastoma, one of them after several months of nephropathy, in the other it was the cause of consultation. The patient without nephroblastoma died at the age of 26 days with renal failure. We think that the original triad of DS: nephroblastoma, pseudohermaphroditism and nephropathy should be more precisely described and expanded to include related alterations in each one of its 3 elements: nephroblastomatosis, hamartomas, probably mesoblastic nephroma and pyelocaliceal malformations in addition to Wilms' tumor, variable disorders of sexual differentiation, gonadal dysgenesis and neoplasia, in addition to pseudohermaphroditism and superficial cortical renal dystrophy and increased reniculi, in addition to mesangial sclerosis. The various clinical forms of the syndrome are combinations of the triad in which a component can be missing and/or other related malformations be present. Chromosome anomalies are possibly responsible for this phenomena. Splitting into separate syndromes should not be done until the chromosome alterations and the regions and genes involved are identified.


Regulation of the lymphocyte adenylate cyclase system by prostaglandin E2 infusions in resistant hypertensive subjects. The effects of prostaglandin E2 (PGE2) infusions on the beta 2-adrenoceptor-dependent adenylate cyclase (beta 2ARAC) system of lymphocytes were studied in 26 patients with resistant hypertension (RH). The density of beta 2-adrenoceptors and their affinity for l-isoproterenol were measured with 125ICYP, and adenylate cyclase activity was determined with alpha 32P-ATP in mononuclear lymphocytes of RH patients before and at 1, 7, and 14 days after the last PGE2 infusion. Plasma epinephrine and norepinephrine concentrations were analyzed using high-performance liquid chromatography. The patients were divided into two groups: 19 receiving clonidine (group I) and seven receiving four-drug antihypertensive therapy (group II) before and after PGE2 infusions. Resistance to antihypertensive drugs in patients of both groups was overcome by PGE2 infusions, and was correlated with a decrease in lymphocyte beta 2-adrenoceptor density, an increase in beta 2-receptor affinity for l-isoproterenol, and an increase in adenylate cyclase activation. The absence of a PGE2 effect was associated with an acute increase in plasma epinephrine content and a decrease in the sensitivity of the lymphocyte beta 2ARAC system.


Role of cyclic AMP in adenosine inhibition of intracellular calcium rise in human platelets. Comparison of adenosine effects on thrombin- and epinephrine-induced platelet stimulation. Thrombin-induced platelet aggregation is associated with an increase in intracellular calcium. Epinephrine provokes aggregation in the absence of a rise in intracellular calcium. Adenosine has been postulated as an endogenous inhibitor of platelet aggregation. In this study, the authors examine the effect of adenosine on the rise in intracellular calcium and on platelet aggregation, and the role of cyclic AMP (cAMP) in these actions. Human platelets were obtained from citrated plasma containing 5 micrograms/mL of indomethacin. Intracellular calcium was determined by fura-2 fluorescent dye. Adenosine inhibited thrombin-induced platelet aggregation and the rise in intracellular calcium in a dose-dependent manner. At a concentration of 100 mumol/L, adenosine completely inhibited thrombin-induced aggregation, but only partly inhibited the rise in intracellular calcium (55%). Adenosine also partially inhibited the rise in calcium produced by thrombin in both calcium-containing and calcium-free media, suggesting that adenosine inhibits both calcium influx and calcium mobilization. The effects of adenosine on intracellular calcium, as in the case of platelet aggregation, appear to be linked to adenylate cyclase, since they were prevented by the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (1-mmol/L) and were potentiated by phospho-diesterase inhibition with papaverine (1 mumol/L). Adenosine and dibutyryl-cAMP also inhibited epinephrine-stimulated platelet aggregation in a dose-dependent manner. Thus, it appears that adenosine may inhibit platelet aggregation independently of its ability to decrease cytosolic free calcium.


Alteration of beta-adrenoceptor function in hypertensive patients with different degrees of left ventricular hypertrophy. Previous assessment of beta-adrenoceptor function has shown alterations in essential hypertension (EH). In the present study, we compared lymphocyte beta-adrenoceptor density (Bmax) and adenylate cyclase (AC) activity stimulated by l-isoproterenol, Gpp(NH)p, Gpp(NH)p + l-isoproterenol, and forskolin in 46 patients with EH and in 17 normotensive subjects. The patients with EH were divided into two subgroups, one with left ventricular myocardial mass (LVMM) less than 200 g and the second with LVMM greater than 200 g (according to Teichholz' formula). There were no significant differences in Bmax or in AC activity [basal and stimulated by Gpp(NH)p and forskolin] between the patients and the normotensive subjects. Adenylate cyclase activity stimulated by l-isoproterenol was reduced (% from basal AC) in the patients (P less than .05), and Bmax was increased only in the patients with left ventricular hypertrophy (P less than .05). There were no differences in AC activity between the two patient subgroups, and Bmax and AC activity did not correlate with blood pressure in either the patients or the normotensive subjects. Correlations were found between Bmax and LVMM (r = 0.38, P less than .02) and between Bmax and interventricular septum thickness (r = 0.412, P less than .02) among the patients. Thus, beta-adrenergic-mediated AC sensitivity to catecholamines is reduced in patients with EH and may represent a generalized defect in beta-receptor function in EH. Increased Bmax is likely to characterize more pronounced involvement of the target organs in the pathologic process associated with EH than is a higher blood pressure level.


Pathobiological reactions of C-fibre primary sensory neurones to peripheral nerve injury. Mammalian primary sensory neurones display profound anatomical and chemical changes in response to injury of their peripheral processes. Peripheral nerve damage results in transganglionic degeneration of central terminals of A-fibre primary afferent neurones terminating in the deeper layers of the medullary or spinal dorsal horn. The depletion of neuropeptides and other neurone-specific macromolecules from neurones in sensory ganglia and from the superficial dorsal horn, the known central projection area of C-fibre primary afferents, is a salient phenomenon commencing after peripheral nerve lesions. A recently devised new experimental procedure, termed the capsaicin-gap method, permitted evaluation of the transganglionic degenerative phenomena which develop in C-fibre primary sensory neurones after a lesion is inflicted upon their peripheral branches. The experimental findings indicated that C-fibre primary afferent terminals underwent transganglionic degeneration following a perineural treatment with capsaicin, and that this was associated with, and probably resulted from, ganglionic cell degeneration. Studies on the effects of peripheral nerve section yielded similar results. It has therefore been suggested that the observed depletion of specific macromolecules, including sensory peptides, specific glycoconjugates and sensory neurone specific acid phosphatase, may be accounted for, at least in part, by an irreversible loss of sensory ganglion neurones. In contrast, many injured neurones express peptides, including vasoactive intestinal polypeptide, peptide histidine-isoleucine and galanin, which can be demonstrated in only a few neurones under normal conditions. These seem to be involved in dorsal horn regenerative and/or compensatory processes following peripheral nerve damage. There is suggestive evidence that the partial deafferentation caused by the transganglionic degeneration of C-fibre primary afferents creates favourable circumstances for an anatomical rearrangement of neuronal connections within the spinal cord dorsal horn. These changes may provide a morphological substrate of some of the functional alterations demonstrated after peripheral nerve lesions.


Osteoclasts from medullary bone of egg-laying Japanese quail do not express the putative calcium 'receptor'. The present study reports the contrasting effects of extracellular calcium ([Ca2+]e) elevation on cytosolic free calcium levels ([Ca2+]i) of osteoclasts, freshly isolated either from medullary bone of the egg-laying Japanese quail or from rat cortical bone. [Ca2+]i was measured in single osteoclasts using the Ca(2+)-sensitive fluorochrome, Indo-1. We found that elevation of [Ca2+]e failed to induce a rise of [Ca2+]i in quail osteoclasts, whilst causing an elevation of [Ca2+]i in rat osteoclasts. The calcium ionophore, ionomycin, led to a sustained elevation of [Ca2+]i in both cell types. These findings suggest that osteoclasts isolated from egg-laying quail do not possess the calcium sensor or 'receptor' that appears to be vital for the survival and function of rat osteoclasts.


Measurement of intracellular pH in isolated bovine articular chondrocytes. Cartilage is an avascular tissue, the cells of which are exposed to reduced extracellular pH. The synthesis of cartilage matrix by chondrocytes is pH-sensitive and therefore control of intracellular pH (pHi) is important. We have measured the pHi of chondrocytes in MOPS-buffered saline (pH 7.4) using the fluorescent dye 2',7'-bis-2-(carboxyethyl)-5(6)-carboxyfluorescein. Mean pHi was 7.10 +/- 0.04 (+/- S.E.M., n = 22), and intracellular buffering power was 30 +/- 4 mmol l-1 (pH unit)-1 when pHi was displaced over the range 7.4-7.8 (n = 13). Recovery from acid load was blocked by 100 microM-ethylisopropylamiloride or by removal of extracellular Na+, indicating that these cells possess the Na(+)-H+ antiporter.


Neurotrophic factors promote the maturation of developing sensory neurons before they become dependent on these factors for survival. We have studied the early development of chicken embryo sensory neurons in culture before they become dependent on neurotrophic factors for survival. During this period, they undergo a distinct change in morphology:initially they have small, spindle-shaped, phase-dark cell bodies, which become spherical and phase bright and extend long neurites. Although this maturational change occurs in isolated cells grown in chemically defined medium, it is accelerated by brain-derived neurotrophic factor (BDNF) or neurotrophin-3 and is retarded by antisense oligonucleotides that inhibit expression of the common, low affinity neurotrophic factor receptor (gp75NGFR) and by antisense BDNF oligonucleotides. We conclude that neurotrophic factors play a role in the earliest stages of sensory neuron development and suggest that they operate by an autocrine mechanism at this time.


Silencing the type II sodium channel gene: a model for neural-specific gene regulation. Neural-specific expression of a sodium channel mini-gene has been shown to be mediated by a 28 bp silencer element, RE1, located in the 5' flanking region of the gene. This element is active exclusively in cell lines that do not express the endogenous brain type II sodium channel gene, including fibroblast, skeletal muscle, and certain neuronal cell lines. All of these non-type II expressing cells contain RE1-binding complexes. On the basis of mutational analysis and in vivo "repressor trap" experiments, we propose that cell-specific RE1-binding proteins are responsible, at least in part, for restricting expression of the type II sodium channel gene to specific neurons in the vertebrate nervous system.


A common silencer element in the SCG10 and type II Na+ channel genes binds a factor present in nonneuronal cells but not in neuronal cells. We have localized a cell type-specific silencer element in the SCG10 gene by deletion analysis. This neural-restrictive silencer element (NRSE) selectively represses SCG10 expression in nonneuronal cells and tissues. The NRSE contains a 21 bp region with striking homology to a sequence present in a silencer domain of the rat type II sodium channel (NaII), another neuron-specific gene. We have identified a sequence-specific protein(s) that binds the SCG10 NRSE, as well as the homologous element in the NaII gene. A point mutation in the NRSE that abolishes binding of this neural-restrictive silencer-binding factor (NRSBF) in vitro also eliminates silencing activity in vivo. NRSBF is present in nuclear extracts from nonneuronal cells but not in extracts from neuronal cells, suggesting that the neuron-specific expression of SCG10 reflects, at least in part, the absence or inactivity of this protein. These data identify the NRSE as a potentially general DNA element for the control of neuron-specific gene expression in vertebrates.


Priming of associative long-term depression in the dentate gyrus by theta frequency synaptic activity. Associative long-term synaptic depression (LTD) was investigated utilizing negatively correlated activity patterns in the medial and lateral perforant path inputs to the dentate gyrus in anesthetized rats. Normally only nonassociative, or heterosynaptic, LTD is elicited in naive pathways. We report here, however, that associative LTD in the lateral path is readily induced after being "primed" by a brief period of lateral path synaptic activity at a theta rhythm frequency (5 Hz). Priming of associative LTD lasts at least 2 hr and is not seen following priming activity at non-theta frequencies (1 and 15 Hz). N-methyl-D-aspartate receptor activation is critical for establishing the priming effect, but not for the subsequent induction of the associative LTD. These data suggest that theta rhythm activity in the dentate gyrus may predispose the system to a specific form of synaptic plasticity, associative LTD.


P-type calcium channels in rat central and peripheral neurons. The peptide toxin omega-Aga-IVA blocked P-type Ca2+ channel current in rat Purkinje neurons (KD approximately 2 nM) but had no effect on identified T-type, L-type, or N-type currents in a variety of central and peripheral neurons. omega-Aga-IVA blocked a substantial fraction of high threshold Ca2+ channel current in neurons from the hippocampal CA1 region (mean 26%), visual cortex (32%), spinal cord (45%), and dorsal root ganglia (23%), but less in hippocampal CA3 neurons (14%) and none in sympathetic neurons. In all cases, omega-Aga-IVA block could be reversed by a brief train of strong depolarizations. There was no overlap between current blocked by omega-Aga-IVA and the fractions blocked by dihydropyridines and omega-conotoxin GVIA, but not all current resistant to dihydropyridines and omega-conotoxin was blocked by omega-Aga-IVA. The results suggest that omega-Aga-IVA is highly selective for P-type channels and that many central neurons and some peripheral neurons possess substantial P-type current.


Histopathologic and cytochemical characteristics of interval breast carcinomas from the Stockholm Mammography Screening Project. Combined morphological and cytochemical malignancy grading in addition to tumor stage was applied to assess the malignant potential of so-called interval carcinomas from the Stockholm randomized mammography screening study. Only interval carcinomas surfacing within two years from screening were included. Fifty-four percent of the tumors were in stage I and 46% of the carcinomas eligible for DNA analysis were diploid, i.e. low-malignant. An overrepresentation of prognostically unfavorable tumor characteristics was found only within the subgroup 'true' and 'early' interval cancer.


Thallium-technetium subtraction scintigraphy of enlarged parathyroid glands after calcitonin stimulation of parathyroid hormone secretion. To improve the sensitivity of thallium-technetium subtraction scintigraphy for preoperative localization procedure of enlarged parathyroid glands in primary hyperparathyroidism, we administered calcitonin intramuscularly 4 hours before the scintigraphy in 14 consecutive patients. Injection of calcitonin reduced plasma levels of ionized calcium from 1.47 +/- 0.10 mmol/l to 1.41 +/- 0.09 mmol/l (p less than 0.01). Concomitantly, serum levels of intact parathyroid hormone increased from 6.4 +/- 2.5 pmol/l to 7.9 +/- 2.6 pmol/l (p less than 0.001). The scintigram after calcitonin injection visualized 11 adenomas (sensitivity 78%) compared to only 9 (sensitivity 64%) in conventional scintigrams. In addition, 5 of the adenomas were more distinctly imaged in the scintigram after calcitonin injection, whereas in only one patient was the conventional scintigram better. Thus, the calcitonin injection improved the scintigram in 7 cases and was inferior in only one case (p = 0.031). We conclude that stimulation of parathyroid hormone secretion with calcitonin results in a better preoperative localization of enlarged parathyroid glands in primary hyperparathyroidism.


Hilar cholangiocarcinoma. An evaluation of subtypes with CT and angiography. Sixty-seven patients had hilar cholangiocarcinomas which were divided into 3 types based on tumor morphology as observed on cholangiography and CT. The pathology, vascularity, and pattern of tumor spread of these types were compared. Most of the infiltrative tumors (n = 44) were scirrhous adenocarcinomas, which on CT showed poor or no contrast enhancement with frequent lymph node metastases and liver atrophy. At angiography, there was vascular encasement in 52%, in rare cases neovascularity, and tumor stain. The exophytic type (n = 19) was divided into 2 subgroups depending on the main location of the tumor. The nodular subtype (n = 16) was mainly inside the liver and somewhat hypervascular similar to peripheral cholangiocarcinoma, often with intrahepatic metastases. The periductal subtype (n = 3) was hypovascular, similar to the infiltrative cholangiocarcinoma, and had a tendency to spread along the portal vein. The intraductal type (n = 4) was observed as a filling defect on cholangiography. CT revealed an intraluminal low density mass. Histologically, they were papillary adenocarcinomas. The radiologic types of hilar cholangiocarcinoma showed different characteristics with regard to pathologic findings, vascularity, and pattern of spread.


Effects of granisetron and haloperidol, alone and in combination, on psychometric performance and the EEG. 1. The effects of granisetron (160 micrograms kg-1 body weight) and haloperidol (3 mg) on psychometric performance and the EEG were investigated in a randomised, single-blind, crossover, placebo controlled study of 12 healthy male volunteers. 2. There was evidence that haloperidol impaired psychometric performance; however its effects on the EEG were not clear cut. In contrast there was very little evidence to suggest that granisetron had any impact on performance or the EEG. 3. The findings from this study suggest that granisetron does not impair daytime function and that it can be co-administered with haloperidol without producing unwanted synergistic effects.


Lack of effect of flunarizine on the pharmacokinetics and pharmacodynamics of sumatriptan in healthy volunteers. Twenty-four healthy subjects completed a double-blind, placebo controlled, parallel group study to evaluate the effect of treatment with flunarizine on the pharmacokinetics and pharmacodynamics of sumatriptan, a 5HT1-like agonist. Subjects received a single oral 200 mg dose of sumatriptan on the eighth day of a once daily treatment with either flunarizine 10 mg or matching placebo. There were no significant differences between treatments in relation to Cmax (82.3 ng ml-1 in the absence and 81.4 ng ml-1 in the presence of flunarizine), AUC (368 ng ml-1 h in the absence and 360 ng ml-1 in the presence) and elimination half-life (2.2 h in the absence and 2.4 h in the presence of flunarizine) of sumatriptan. Similarly pretreatment with flunarizine was not found to have any clinically significant effect on the pharmacodynamics of sumatriptan as measured by pulse rate, blood pressure and ECG.


Overexpression of three ubiquitin genes in mouse epidermal tumors is associated with enhanced cellular proliferation and stress. A mouse ubiquitin clone that recognizes multiple transcripts overexpressed in murine tumors compared to normal epidermis was isolated by differential screening of complementary DNA libraries from mouse squamous cell carcinomas. Coding region probes detected five ubiquitin transcripts. Oligonucleotides were designed for unique parts of three mouse ubiquitin gene complementary DNA clones. The overexpressed transcripts at 2.4, 2.8, 6.4, and 7.8 kilobases (kb) were detected by an oligonucleotide specific for a mouse UbC polyubiquitin clone. A 1.2-kb UbB overexpressed transcript was detected by an oligonucleotide for a mouse four-unit polyubiquitin, and a 0.7-kb UbA overexpressed transcript was recognized by an oligonucleotide for the mouse ubiquitin carboxyl-extension protein of 52 amino acids. All three classes of transcripts were induced in mouse skin by the hyperproliferative agent ethylphenyl propionate and by the tumor promoting agent 12-O-tetradecanoylphorbol-13- acetate. Heat shock of cultured keratinocytes induced both the 6.4- and 7.8-kb transcripts recognized by the UbC-specific oligonucleotide. Consistent with the overexpression of the ubiquitin transcripts, the level of free ubiquitin protein, as determined by Western analysis, was elevated in the tumors and proliferating epidermis as compared to normal epidermis. Our results indicate that the overexpression of ubiquitin genes could be related to a sustained state of proliferation and stress in the tumors compared to the normal resting epidermis.


Spatial and temporal coherence in cortico-cortical connections: a cross-correlation study in areas 17 and 18 in the cat. Visual cortical areas are richly but selectively connected by "patchy" projections. We characterized these connections physiologically with cross-correlograms (CCHs), calculated for neuron pairs or small groups located one each in visual areas 17 and 18 of the cat. The CCHs were then compared to the visuotopic and orientation match of the neurons' receptive fields (RFs). For both spontaneous and visually driven activity, most non-flat correlograms were centered; i.e. the most likely temporal relationship between spikes in the two areas is a synchronous one. Although spikes are most likely to occur simultaneously, area 17 spikes may occur before area 18 or vice versa, giving the cross-correlogram peak a finite width (temporal dispersion). Cross-correlograms fell into one of three groups according to their full-width at half peak height: 1-8 ms (modal width, 3 ms), 15-65 ms (modal width 30 ms), or 100-1000 ms (modal width 400 ms). These classificatory groups are nonoverlapping; the three types of coupling appeared singly and in combination. Neurons whose receptive fields (RFs) are nonoverlapping or cross-oriented may yet be coupled, but the coupling is more likely to be the broadest type of coupling than the medium-dispersed type. The sharpest type of coupling is found exclusively between neurons with at least partially overlapping RFs and mostly between neurons whose stimulus orientation preferences matched to within 22.5 deg. The maximum spatial dispersion observed in the RFs of coupled neurons compares well with the maximum divergence seen anatomically in the A18/A17 projection system. We suggest three different mechanisms to produce each of the three different degrees of observed spatial and temporal coherence. All mechanisms use common input of cortical origin. For medium and broad coupling, this common input arises from cell assemblies split between both sides of the 17/18 projection system, but acting synchronously. Such distributed common-input cell assemblies are a means of overcoming sparse connectivity and achieving synaptic transmission in the pyramidal network.


Broadband temporal stimuli decrease the integration time of neurons in cat striate cortex. We have studied the responses of striate cortical neurons to stimuli whose contrast is modulated in time by either a single sinusoid or by the sum of eight sinusoids. The sum-of-sinusoids stimulus resembles white noise and has been used to study the linear and nonlinear dynamics of retinal ganglion cells (Victor et al., 1977). In cortical neurons, we have found different linear and second-order responses to single-sinusoid and sum-of-sinusoids inputs. Specifically, while the responsivity near the optimal temporal frequency is lower for the sum-of-sinusoids stimulus, the responsivity at higher temporal frequencies is relatively greater. Along with this change in the response amplitudes, there is a systematic change in the time course of responses. For complex cells, the integration time, the effective delay due to a combination of actual delays and low-pass filter stages, changes from a median of 85 ms with single sinusoids to 57 ms with a sum of sinusoids. For simple cells, the integration times for single sinusoids range from 44-100 ms, but cluster tightly around 40 ms for the sum-of-sinusoids stimulus. The change in time constant would argue that the increased sensitivity to high frequencies cannot be explained by a static threshold, but must be caused by a fundamental alteration in the response dynamics. These effects are not seen in the retina (Shapley & Victor, 1981) and are most likely cortical in origin.


Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays utilizing formate as an alternate substrate for bicarbonate. Formate is an alternate substrate for bicarbonate in the reaction with PEP catalyzed by phosphoenolpyruvate carboxylase from Zea mays, producing formyl phosphate and pyruvate. The Km for formate is 25 +/- 2 mM, and the maximum velocity is 1% of that for bicarbonate at pH 8.0. Use of [18O]formate produces inorganic phosphate containing 1 equiv of 18O, but no label is incorporated into residual phosphoenolpyruvate. PEP carboxylase catalyzes the hydrolysis of phosphoglycolate or L-phospholactate 2000 times more slowly and D-phospholactate 4000 times more slowly than the reaction between bicarbonate and PEP.


Proximity between nucleotide/dinucleotide and metal ion binding sites in DNA-dependent RNA polymerase from Escherichia coli. In order to understand translocation in transcription, it is important to develop a continuous functional assay for RNA polymerase (RNAP) activity in vitro. Fluorescent derivatives of ATP, UTP, UpA, and CpA with aminonaphthalene-5-sulfonic acid (AmNS) attached to the nucleotide triphosphates via a gamma-phosphoramidate bond or to the dinucleotide monophosphates via a 5'-secondary amine linkage were synthesized [Tyagi, S.C., & Wu, F.Y.-H. (1987) J. Biol. Chem. 262, 10684-10688]. The fluorescent emission spectra of (5'-AmNS)UpA, (5'-AmNS)CpA, (gamma-AmNS)ATP, and (gamma-AmNS)UTP overlap the absorption spectrum of co-substituted RNA polymerase (Co-RNAP) and ensure fluorescence resonance energy transfer (FRET) between the fluorescent analog and Co(II) in Co-RNAP. The binding constants at a single site for (gamma-AmNS)ATP, (gamma-AmNS)UTP, (5'-AmNS)UpA, and (5'-AmNS)CpA were observed to be 7.11, 5.26, 0.52, and 0.61 microM, respectively, in Co-RNAP and 5.70, 3.42, 0.12, and 0.21 microM, respectively, in Zn-RNAP. (8-AmTEMPO)ATP, with the spin probe AmTEMPO attached to the C-8 position at ATP [Tyagi, S.C. (1991) J. Biol. Chem. 266, 17936-17940], and Mn(3'-OCH3)UTP were synthesized. Mn-(II)-substituted RNA polymerase (Mn-RNAP) is prepared. The single site binding constants for (8-Am-TEMPO)ATP and Mn(3'-OCH3)UTP were 3.58 and 2.35 microM in Zn-RNAP and 5.77 and 3.43 microM in Mn-RNAP, respectively. These results indicate that dinucleotides bind much more tightly than mononucleotides to RNAP and that the binding constants are roughly the same for both Co- and Zn-substituted RNAP.(ABSTRACT TRUNCATED AT 250 WORDS)


A 1H-NMR study of the DNA binding characteristics of thioformyldistamycin, an amide isosteric lexitropsin. The interaction of thioformyldistamycin, an amide isostere of the naturally occurring antibiotic distamycin A, with a self-complementary decadeoxynucleotide duplex, d(CGCAATTGCG)2, has been examined using a variety of high-field 1H-NMR techniques. The ligand exhibits two forms in solution arising from geometric isomerism due to restricted rotation around the thioformamide bond. Only the thermodynamically more stable Z-form is shown to bind to the oligonucleotide along its minor groove at the central 5'-AATT segment with the end groups of the ligand extending into the flanking GC regions but without any close contact at the amidinium terminus. Cross-peaks involving characteristic intra- and interresidue proton connectivities in the 2D experiments (COSY and NOESY) were employed to assign individual resonances of both strands in the asymmetric DNA-drug complex. The solution structure of the complex was constructed by molecular mechanics calculations based upon initial estimates of drug-DNA NOE contacts and further refined through energy minimization. These results complement previous structural studies on distamycin and other lexitropsins with oligonucleotides. The exchange of the ligand between two equivalent binding sites on the DNA sequence was estimated to occur at 40 s-1 with a free energy of activation of 16.5 kcal.mol-1 at 321-326 K. There was no evidence of formation of a 2:1 drug-oligomer complex, in contrast to the case of the natural product, which is attributed to steric demands of the larger sulfur atom.


Homo- and heteronuclear NMR studies of the human retinoic acid receptor beta DNA-binding domain: sequential assignments and identification of secondary structure elements. An 80 amino acid polypeptide corresponding to the DNA-binding domain (DBD) of the human retinoic acid receptor beta (hRAR-beta) has been studied by 1H homonuclear and 15N-1H heteronuclear two- and three-dimensional (2D and 3D) NMR spectroscopy. The polypeptide has two putative zinc fingers homologous to those of the receptors for steroid and thyroid hormones and vitamin D3. The backbone 1H resonances as well as over 90% of the side-chain 1H resonances have been assigned by 1H homonuclear 2D techniques except for the three N-terminal residues. The assignments have been confirmed further by means of 15N-1H heteronuclear 3D techniques, which also yielded the assignments of the 15N resonances. Additionally, stereospecific assignments of methyl groups of five valine residues were made. Sequential and medium-range NOE connectivities indicate several elements of secondary structure including two alpha-helices consisting of residues E26-Q37 and Q61-E70, a short antiparallel beta-sheet consisting of residues P7-F9 and S23-C25, four turns consisting of residues P7-V10, I36-N39, D47-C50, and F69-G72, and several regions of extended peptide conformation. Similarly, two helices are found in the glucocorticoid receptor (GR) DBD in solution [Härd et al. (1990) Science 249, 157-160] and in crystal [Luisi et al. (1991) Nature 352, 497-505], and in the estrogen receptor (ER) DBD in solution [Schwabe et al. (1990) Nature 348, 458-461], although the exact positions and sizes of the helices differ somewhat. Furthermore, long-range NOEs suggest the existence of a hydrophobic core formed by the two helices.


Inhibitors of protein phosphatase type 1 and 2A attenuate phosphatidylinositol metabolism and Ca(2+)-transients in human platelets. Role of a cdc2-related protein kinase. The addition of either okadaic acid or calyculin A desensitizes human platelets to thrombin. One objective of this study was to determine which step(s) leading to secretion reactions may be affected by these protein phosphatase inhibitors. In a dose-dependent manner, okadaic acid or calyculin A inhibits phosphatidylinositol metabolism and Ca(2+)-transients. In all cases, calyculin A was approximately 10-fold more potent than okadaic acid, and it had maximal effects at a concentration of 1 microM. Although thrombin-induced rises in [Ca2+]i were diminished, an increase in the phosphorylation state of myosin light chains (MLC) was still observed. Changes in this phosphorylation were diminished, however, following the addition of thrombin to calyculin A-treated platelets that were loaded with dimethyl-BAPTA. These data demonstrate that calyculin A and okadaic acid lower agonist-induced Ca(2+)-transients, which in turn prevents responses such as secretion reactions. Calyculin A/okadaic acid-induced phosphorylation events were not diminished in BAPTA-loaded platelets, suggesting that these phosphorylations are Ca(2+)-insensitive. Thus, a second objective of this study was to identify the protein kinase(s) that was(were) responsible for the calyculin A-induced phosphorylations. In a platelet lysate system, calyculin A caused an increase in the incorporation of [32P]phosphate into p50. This phosphorylation event was identical to that observed in the intact platelet and was not mimicked by cAMP, cGMP, Ca2+, or a Ca2+/phospholipid/diacylglycerol mixture. Kinase activity was removed after the lysate was incubated with p13suc1-Sepharose. This suggests that a p13suc1-sensitive protein kinase, e.g., a cell cycle-dependent protein kinase, is responsible for the calyculin A-sensitive phosphorylation events.(ABSTRACT TRUNCATED AT 250 WORDS)


Triple inhibitor titrations support the functionality of the dimeric character of mitochondrial ubiquinol-cytochrome c oxidoreductase. The ubiquinol-2 or duroquinol oxidoreductase activity of mitochondrial ubiquinol-cytochrome c oxidoreductase was titrated with combinations of antimycin, myxothiazol and N,N'-dicyclohexylcarbodiimide (DCCD). A statistical model has been developed that can predict the activity of the complex treated with all possible combinations of these inhibitors. On the basis of the measured titration curves the model had to accommodate interaction between the two promoters of the complex. The titrations confirm that treatment with DCCD results in the modification of a certain site in one of the two promoters of the bc1 dimer, thereby blocking one antimycin A binding site without inhibiting electron transfer. Modification of both antimycin A binding sites of the dimer is apparently required for inhibition of electron transfer through the complex, just as modification of both myxothiazol-binding sites is required for full inhibition. The conclusion can be drawn that mitochondrial ubiquinol-cytochrome c oxidoreductase is a functional dimer, consisting of electrically interacting protomers.


Structure and function of the bc-complex of Rhodobacter sphaeroides. The ubiquinol:cytochrome c2 oxidoreductase (bc-complex) of Rhodobacter sphaeroides has three main subunits, which bear the prosthetic groups, and contribute to three catalytic sites and internal electron transfer pathways which define the modified Q-cycle mechanism. In this paper, we report on progress in modelling the structure of the bc-complex, and experiments using site directed mutagenesis and biophysical assay to probe the structural and function consequences of specific modifications to these subunits.


Protonation states of the catalytic intermediates of cytochrome c oxidase. Protonation changes accompanying conversion of oxidised (O state) cytochrome c oxidase to the 2-electron-reduced P state, and 3-electron-reduced F state at pH 8.0 have been measured. It was found that 2 and 3 protons, respectively, were taken up. The fourth proton required for the reduction of O2 to H2O must therefore be consumed in the remaining F----O portion of the catalytic cycle.


Characterization of the major phosphofructokinase-dephosphorylating protein phosphatases from Ascaris suum muscle. In contrast to the mammalian enzyme, PFK from the nematode Ascaris suum is activated following phosphorylation (Daum et al. (1986) Biochem. Biophys. Res. Commun. 139, 215-221) catalyzed by a cAMP-dependent protein kinase (Thalhofer et al. (1988) J. Biol. Chem. 263, 952-957). In the present report, we describe the characterization of the major PFK dephosphorylating phosphatases from Ascaris muscle. Two of these phosphatases exhibit apparent M(r) values of 174,000 and 126,000, respectively, and are dissociated to active 33 kDa proteins by ethanol precipitation. Denaturing electrophoresis of each of the enzyme preparations showed two bands of M(r) 33,000 and 63,000. The enzymes are classified as type 2A phosphatases according to their inhibition by subnanomolar concentrations of okadaic acid, the lack of inhibition by heat-stable phosphatase inhibitors 1 and 2, and their preference for the alpha- rather than for the beta-subunit of phosphorylase kinase. Like other type 2A phosphatases, they exhibit broad substrate specificities, are activated by divalent cations and polycations, and inhibited by fluoride, inorganic phosphate and adenine nucleotides. In addition, we have found that PFK is also dephosphorylated by an unusual protein phosphatase. This exhibits kinetic properties similar to type 2A protein phosphatases, but has a distinctly lower sensitivity towards inhibition by okadaic acid (IC50 approx. 20 nM). Partial purification of the enzyme provided evidence that it is composed of a 30 kDa catalytic subunit and probably two other subunits (molecular masses 66 and 72 kDa). The dephosphorylation of PFK by protein phosphatases is strongly inhibited by heparin. This effect, however, is substrate-specific and does not occur with Ascaris phosphorylase a.


EPR and redox properties of Desulfovibrio vulgaris Miyazaki hydrogenase: comparison with the Ni-Fe enzyme from Desulfovibrio gigas. We have carried out a detailed redox titration monitored by EPR on the hydrogenase from Desulfovibrio vulgaris Miyazaki. Typical 3Fe and nickel signals have been observed, which are very similar to those given by Desulfovibrio gigas hydrogenase in all the characteristic redox states of the enzyme. This confirms that D. vulgaris Miyazaki hydrogenase is a Ni-Fe enzyme closely related to that from D. gigas, as was recently proposed on the basis of sequence comparisons (Deckers, H.M., Wilson, F.R. and Voordouw, G. (1990) J. Gen. Microb. 136, 2021-2028).


Proton nuclear Overhauser effect study of the heme active site structure of Coprinus macrorhizus peroxidase. Proton nuclear Overhauser effect and paramagnetic relaxation measurements have been used to define more extensively the heme active site structure of Coprinus macrorhizus peroxidase, CMP (previously known as Coprinus cinereus peroxidase), as the ferric low-spin cyanide ligated complex. The results are compared with other well-characterized peroxidase enzymes. The NMR spectrum of CMPCN shows changes in the paramagnetically shifted resonances as a function of time, suggesting a significant heme disorder for CMP. The presence of proximal and distal histidine amino acid residues are common to the heme environments of both CMPCN and HRPCN. However, the upfield distal arginine signals of HRPCN are not evident in the 1H-NMR spectra of CMPCN.


Use of dual-energy X-ray absorptiometry (DEXA) to follow mineral content changes in small ceramic implants in rats. Dual energy X-ray absorptiometry (DEXA) imaging was used to quantify bone ingrowth into hydroxyapatite (HA) ceramic implants in rats. HA cylinders implanted in the proximal tibiae were followed for 13 wk. The increase in alkaline phosphatase (ALP) activity within the implants preceded the increase in mineral content as measured non-invasively by the DEXA technique. This was consistent with the timing of ALP activity in respect of mineralization as they occur during fracture healing. The results show that DEXA imaging is useful in measuring bone ingrowth into small ceramic HA implants in vivo, despite the high mineral content background of the implant scaffold.


Evaluation of a high-velocity flame-spraying technique for hydroxyapatite. Titanium alloy sprayed with hydroxyapatite (HA) was developed by a high-velocity flame-spraying technique (HVFST). Biocompatible responses of bony tissues to high-velocity flame-sprayed HA (HVFS-HA) implanted into tibias of adult male rabbits was investigated 4 and 18 months after implantation by light and electron microscopy. Both light and electron microscope features in histological sections showed that inflammatory responses of tissues in situ were completely cleared and the interface between the bone and the implant was filled with newly formed bony substrate. This suggests that the HVFS-HA was sufficiently biocompatible to be adapted to the bone in situ.


Development of a degradable composite for orthopaedic use: in vivo biomechanical and histological evaluation of two bioactive degradable composites based on the polyhydroxybutyrate polymer. As a direct method for the evaluation of tissue bonding to two polyhydroxybutyrate (PHB) based composites, a mechanical push-out test was performed on implants in the femur of mature Japanese White rabbits. Three composites were tested. The first, a hydroxyapatite/PHB (HA/PHB) composite showed an increase in interfacial shear strength (ISS) up to 8 wk, after which the ISS decreased due to degradation of the implant. The second composite was an HA/glass/PHB (HGP) composite and this gave lower values for the ISS attributed to ion release from the glass causing a soft tissue reaction at the interface. The third composite was a carbon fibre reinforced polysulfone (CFRP) and this showed high interfacial shear strength values, which continued to increase with time. These conclusions were supported by contact microradiography (CMR) and histology which showed enhanced endosteal bone growth for the HA/PHB but for the HGP, no periosteal or endosteal activity was detected. Interposed soft tissue for the HGP composite was difficult to discern, histologically, but it was proposed that this was the reason for the low ISS values. It was concluded that the high ISS values for the carbon fibre control were due to surface morphology allowing deep ingrowth of soft tissue and this was confirmed by SEM.


Adsorption of complement proteins on surfaces with a hydrophobicity gradient. Activation of the complement system is recognized as one of the major problems with respect to biocompatibility of biomaterials. The binding of C3 (central component of complement) and B (factor B, an activator of C3), and H (factor H, an inhibitor of C3 activation) plays a crucial role in the activation of the alternative pathway of complement on the surfaces of biomaterials during extracorporeal procedures. Here we report on the adsorption of C3, B or H on to the silica surface with a hydrophobicity gradient. The amount of native 125I-C3 bound to both hydrophilic and hydrophobic surfaces was very similar (0.8 and 0.9 micrograms/cm2; 4 x 10(-12) mol/cm2). Neither factor H nor factor B was able to displace already adsorbed 125I-C3 from either of the surfaces. The extent of binding of factors B and H to preadsorbed C3 was a function of the surface hydrophobicity: more 125I-B or 125I-H was bound to C3 adsorbed at the hydrophilic end than at the hydrophobic end of the gradient surface. The binding of 125I-B or 125I-H to preadsorbed C3 appeared to be influenced by the availability of their binding sites on adsorbed C3 molecules rather than by the amount of surface-bound C3. At the hydrophobic end of the gradient surface the molar binding ratio of B/C3 was considerably smaller than the molar binding ratio of H/C3. It can be speculated that the hydrophobicity of the surface determines orientation and/or conformation of adsorbed C3 molecule; when adsorbed at the hydrophobic end of the gradient, C3 molecule predominantly exposes the binding site to which only factor H can bind.(ABSTRACT TRUNCATED AT 250 WORDS)


Genetic analysis of picornaviruses.

Genetic analysis of picornaviruses. During the past year, genetic studies of picornaviruses, vastly facilitated by the application of infectious picornaviral cDNAs and RNAs, have contributed to our understanding of the function of individual picornavirus polypeptides and to the genetic processes that operate in these small RNA viruses. Especially notable were the demonstrations that the RNA-dependent RNA polymerase may have a function in RNA synthesis as an uncleaved precursor polypeptide, and that a mutation in the polymerase can be complemented in trans, in contrast to data obtained from previously studied polymerase mutants. A new in vitro system, in which positive-strand synthesis, negative-strand synthesis and RNA packaging were all observed, will facilitate further studies into the mechanism of these processes.


Papillomaviruses and human oncogenesis.

Papillomaviruses and human oncogenesis. During the past year, significant advances have been made in understanding the functions of the oncoproteins, E6 and E7, of human papillomaviruses that are associated with malignant genital tumors. In addition, important new information is now available on the responses of both the keratinocyte and of the individual following papillomavirus infection.


Effects produced by acute and chronic treatment with granisetron alone or in combination with haloperidol on midbrain dopamine neurons. In this study, we examined the effects of acute and chronic administration of the 5-HT3 receptor antagonist granisetron (0.1, 1.0 or 10 mg/kg i.p.) alone or in combination with haloperidol (0.5 mg/kg i.p.) on the number of spontaneously active dopamine neurons in the substantia nigra pars compacta (SNC or A9) and the ventral tegmental area (VTA or A10). This was accomplished using the technique of extracellular single unit recording. Acute granisetron at 0.1 mg/kg, but not at higher doses, selectively decreased the number of spontaneously active A10 DA cells. Chronic administration of 0.1 or 1 mg/kg granisetron selectively decreased the number of spontaneously active A10 dopamine cells compared to controls, mimicking the effect produced by chronic treatment with the atypical neuroleptic drug clozapine. However, unlike the effect produced by neuroleptics, this granisetron-induced effect was not reversed by the systemic administration of apomorphine (50 micrograms/kg). These results suggest that the chronic granisetron-induced reduction of the number of spontaneous active dopamine cells is not the result of depolarization inactivation. Chronic coadministration of granisetron with haloperidol negates the effects produced by either compound alone. Acute coadministration of granisetron with haloperidol also attenuated or abolished haloperidol's action, particularly that on the A9 dopamine cells. Overall, it appears that at 0.1 and 1.0 mg/kg, chronic granisetron may possess atypical neuroleptic drug potential. However, the combination of haloperidol and granisetron nullifies changes in midbrain dopamine neurons observed with either agent alone.


The SV40 early transcriptional regulatory element is unable to direct gene expression in pituitary GH-3 cells. The SV40 early (SV40E) transcriptional regulatory element (TRE) is able to direct heterologous gene expression in a variety of eukaryotic cell lines. This ability is conferred, in part, by the presence of several cis-elements. Transfection studies, mutational analyses, and in vitro DNA binding assays have demonstrated that the SV40E TRE is capable of interacting with several cellular transcription (trans) factors. In the present study, we have investigated the inability of the SV40E TRE to direct gene expression in cultured rat anterior pituitary GH-3 cells. Gel shift analysis demonstrated that nuclear factors within these cells can recognize and specifically bind to DNA containing SV40 enhancer sequences. Surprisingly, we have found that both HeLa and GH-3 cells possess relatively equal quantities of Sp1-specific RNA; however, a dramatic decrease in Sp1 protein was seen in GH-3 cells. Transfection studies utilizing CAT reporter plasmids revealed that the intact SV40E TRE is inactive in these cells, and that subsequent deletion of a region(s) where nuclear factor binding occurs does not result in detectable levels of gene expression. Thus, removal of cis-sites potentially involved in repressor binding does not result in activation of the SV40E TRE in these cells. Subcloning an SV40 enhancer fragment upstream of a heterologous TK promoter yielded chimeric TREs that could direct high levels of gene expression in HeLa but not GH-3 cells. Therefore, the prototypic SV40 enhancer, in the context of GH-3 cells, cannot enhance gene expression.


A meristem-related gene from tomato encodes a dUTPase: analysis of expression in vegetative and floral meristems. A meristem-specific gene coding for deoxyuridine triphosphatase (EC 3.6.1.23) (dUTPase) in tomato was isolated, and its developmental expression in vegetative and floral apices was monitored. An 18-kD polypeptide, P18, was isolated as a consequence of its accumulation in arrested floral meristems of anantha mutant plants. The corresponding cDNA isolated from an expression library exhibited a 40 to 60% similarity with the pseudoprotease sequences of poxviruses, genes that have been suggested to encode dUTPases. Enzymatic tests and conservation of peptide motifs common to bacterial and viral genes verified that the P18 cDNA clone indeed represents a eukaryotic dUTPase. Immunogold localization and in situ hybridization experiments showed that polypeptides and transcripts of dUTPase are maintained at high levels in apical meristematic cells of vegetative and floral meristems. dUTPase gene activity is also high in the potentially meristematic cells of the provascular and vascular system. Its expression is lower in the immediate parenchymal derivatives of the apical meristematic cells, and this downregulation marks, perhaps, the transition from totipotency to the first differentiated state.


Molecular analysis of an auxin binding protein gene located on chromosome 4 of Arabidopsis. We have isolated a cDNA clone from Arabidopsis, At-ERabp1, for the Arabidopsis auxin binding protein located in the lumen of the endoplasmic reticulum (ER). This cDNA clone codes for a protein related to the major auxin binding protein from maize, Zm-ERabp1. A single open reading frame, 594 bases in length, predicts a protein of 198 amino acid residues and a molecular mass of 22,044 D. The primary amino acid sequence contains an N-terminal hydrophobic signal sequence of 33 amino acids. We demonstrated by in vitro studies that the At-ERabp1 protein is translocated into ER-derived microsomes. The protein was processed, and the cleavage site for the N-terminal signal peptide was determined by radiosequencing. The mature protein is composed of 165 amino acid residues, with a molecular mass of 18,641 D. The At-ERabp1 protein contains potential N-glycosylation sites (Asn46-Ile-Ser and Asn130-Ser-Thr). In vitro transport studies demonstrated cotranslational glycosylation. Retention within the lumen of the ER correlates with an additional signal located at the C terminus and represented by the amino acids Lys196-Asp-Glu-Leu, well known to be essential for active retrieval of proteins into the lumen of the ER. DNA gel blot analysis of genomic DNA revealed single hybridizing bands, suggesting that only a single At-ERabp1 gene is present in the Arabidopsis genome. Restriction fragment length polymorphism mapping indeed revealed a single locus mapping to chromosome 4.


Different localization of inositol 1,4,5-trisphosphate and ryanodine binding sites in rat liver. The distribution of inositol 1,4,5-trisphosphate and ryanodine binding sites between plasma membrane, microsomal, and mitochondrial fractions of rat liver were compared. IP3 bound mostly to the plasma membrane fraction (Kd = 6 nM; Bmax = 802 fmol/mg protein). Some IP3 binding sites were also present in the microsomal and mitochondrial fractions (Kd = 2.5 and 2.9 nM; Bmax = 35 and 23 fmol/mg protein respectively). The possibility that these binding sites are due to contamination of the fractions with plasma membrane cannot be excluded. Binding of IP3 to the plasma membrane was inhibited by heparin but not by either caffeine or tetracaine. High-affinity ryanodine binding sites were present mostly in the microsomal fraction (Kd = 13 nM; Bmax = 301 fmol/mg protein). Lower affinity binding sites were also found to be present in the mitochondrial and plasma membrane fractions. Binding of ryanodine to the microsomal fraction was inhibited by both caffeine and tetracaine but not by heparin. These data demonstrate that IP3 and ryanodine binding sites are present in different cellular compartments in the liver. These differences in the localization of the binding sites might be indicative of their functional differences.


The natural history of primary liver cell carcinoma: a study of 89 untreated adult Nigerians. A Nigerian series of 890 patients with primary liver cell carcinoma, seen during the recent three years, has been examined with a view to establishing the natural history of the tumour in untreated cases. There were 60 males and the mean age of all the patients was 50 years. Hepatitis B surface antigen was positive in 70 pc of tested patients and there were higher pathologic levels of aflatoxins in these patients when compared to normal controls. Liver cirrhosis was associated with 81 pc of patients. Alcohol and smoking were unlikely to be aetiologically important in these patients. The macroscopic type of tumour was mainly diffusely nodular and the commonest microscopic pattern was the characteristic trabecular pattern. Metastases were present in 52 pc of the patients and were mainly to the lungs. Due to late presentation and underlying cirrhosis, most patients were critically ill with high incidence of ascites, jaundice and hepatic precoma. The mean survival time of all patients was six months after onset of the initial symptoms to death and only three weeks after admission to death. The major causes of death were advanced cancer in 78 pc, hepatic failure in 48 pc and rupture of tumour, 39 pc. These observations clearly show that the prognosis of liver cancer is dismal in this environment, as elsewhere. Medical education on earlier presentation in hospital and early operative removal of the tumour should be emphasised. It is suggested that an attempt through immunisation should be employed to reduce the incidence of liver cancer in the population.


Giant calcified retro-peritoneal malignant fibrous histiocytoma. The case report of a patient with a giant calcified retro-peritoneal malignant fibrous histiocytoma is presented. Despite the size of the tumour, the patient was in good health and there was no evidence of metastatic disease. He did well following laparotomy and complete excision of the tumour. He received a full course of post-operative radiotherapy and remains well six months after discharge from hospital.