Transforming growth factor-beta enhances calcitonin-induced cyclic AMP production and the number of calcitonin receptors in long-term cultures of human umbilical cord blood monocytes in the presence of 1,25-dihydroxycholecalciferol. Transforming growth factor-beta (TGF-beta) is a multifunctional polypeptide, abundant in bone, that regulates both proliferation and differentiation of a wide variety of cells, but its role in osteoclast differentiation remains controversial. We have recently shown that long-term cultures of human cord blood monocytes, in the presence of 1,25 dihydroxycholecalciferol (1,25-(OH)2D3), give rise to cells that express two markers of the osteoclast phenotype, namely, the vitronectin receptor (VNR) and the calcitonin receptor (CTR). TGF-beta enhanced the proportion of cells expressing the VNR. In the present study, we investigated the effect of TGF-beta on the expression of CTR in cord blood monocytes cultured during 3 weeks in the presence of 1,25-(OH)2D3. When added within the first 2 weeks of culture, TGF-beta (500 pg/ml) significantly decreased the cell protein content. TGF-beta alone did not stimulate basal cAMP production. The 10 nM-sCT-stimulated cAMP production was enhanced by increasing TGF-beta concentrations from 50 pg/ml to 1,000 pg/ml: for 500 pg/ml TGF-beta, it was 294 +/- 28% vs. 140 +/- 25% for control cultures (p less than 0.01). The sCT dose-response curves showed a higher cAMP production from 10(-9) M to 10(-7) M of sCT in the presence of 500 pg/ml TGF-beta than in control cultures. The increase was 325 +/- 36% in the presence of TGF-beta and 195 +/- 13% in the absence of TGF-beta, for 10(-7) M sCT (p less than 0.01). This effect of TGF-beta on cAMP production was not observed either when it was added to monocyte cultures the last day or 2 hours before the end of the culture or in MCF7, a human breast cancer cell line that expresses CTR. [125I]-sCT binding studies performed on confluent cells showed similar Kd in control and TGF-beta-treated cells. By contrast, the CTR number was significantly increased in the presence of TGF-beta: 6.1 +/- 2 x 10(4) receptors per cell in control cultures and 28.8 +/- 8.1 x 10(4) receptors per cell in TGF-beta-treated cultures (p less than 0.05). It is thus suggested that TGF-beta increases the number of CTR of these cells that have other features of preosteoclasts. The role of this cytokine on the process of osteoclast differentiation and in bone resorption is thus emphasized.


Macrophage membrane glycoprotein binding of Griffonia simplicifolia I-B4 induces TNF-alpha production and a tumoricidal response. Thioglycollate-elicited macrophages (m phi), upon binding the lectin Griffonia simplicifolia IB4 (GSIB4) at the plasma membrane, are induced to secrete several low molecular weight proteins. In this investigation, results from specific ELISA and immunoprecipitation analysis of these molecules confirmed that the cytokine, tumor necrosis factor-alpha (TNF-alpha), belongs to the group of elicited proteins. This specific m phi response is directly influenced by the dose of GSIB4 used and the time in contact with the cells. At 40 micrograms/ml GSIB4, the maximum dose of lectin used, the m phi activity was equal to that achieved when the cells were incubated with an interferon-gamma/lipopolysaccharide (IFN/LPS) stimulus alone. Moreover, the data showed that TNF-mediated tumoricidal activity was significantly influenced by GSIB4 binding to the m phi membrane. When the lectin was incubated alone or in sequence with IFN/LPS, this ligand-receptor binding promoted the lysis of WEHI 164 tumor target cells. However, concurrent incubation of both IFN/LPS and GSIB4 with m phi significantly diminished the tumoricidal response. This suggested that one of the metabolic pathways utilized subsequent to receptor-ligand binding was altered by these interactions. When cyclic AMP (cAMP) and inositol triphosphate (IP3) levels were examined, the results showed that the concentration of cAMP was unchanged despite the fact that IP3 levels were significantly enhanced upon m phi-GSIB4 binding. Collectively, the data show that GSIB4 binding to specific glycoproteins in the m phi membrane induces TNF-alpha production and facilitates TNF-alpha dependent tumoricidal responses. It also appears that the transduction of the signal, in part, at least utilizes the phosphatidyl inositol pathway. Finally, it is noteworthy that m phi activity is influenced by the sequence in which GSIB4 is presented to the m phi relative to the IFN/LPS treatment.


Transforming growth factor-beta 1 inhibitory effect of platelet-derived growth factor-induced signal transduction on human bone marrow fibroblasts: possible involvement of protein phosphatases. Transforming growth factor-beta 1 (TGF-beta 1) is a potent growth inhibitor for many cell types. On fibroblasts, TGF-beta 1 has been shown to inhibit human platelet-derived growth factor (PDGF)-induced mitogenicity. The mechanism implicated in this growth inhibition is unknown. In this work, we show on human bone marrow fibroblasts that TGF-beta 1, which inhibited PDGF-BB mitogenicity, was able to block PDGF-BB-induced early events such as polyphosphoinositide (PtdIns 4,5-P2, PtdIns 4-P, and PtdIns) breakdown and Ins 1,4,5-P3 formation. No significant modification by TGF-beta 1 of PDGF-BB binding (n1 = 200,000 vs. n2 = 195,000 sites per cell with TGF-beta 1; Kd1 = Kd2 = 0.5 x 10(-9) M) and of internalization kinetics was observed. In addition, TGF-beta 1 was shown to inhibit PDGF-BB receptor autophosphorylation either in intact cells or in partially isolated membranes and to partially inhibit PDGF-R tyrosine kinase activity. Since a dephosphorylation mechanism through protein phosphatases could be implicated, we used okadaic acid, a potent inhibitor of type 1 and 2A serine/threonine phosphatases and showed that okadaic acid restored PDGF-receptor autophosphorylation on tyrosine residues. Based on these data, we suggest that an alternative regulatory mechanism of PDGF tyrosine phosphorylation seems to involve serine/threonine phosphatase activation.


Dissociation between parathyroid hormone-stimulated cAMP and calcium increase in UMR-106-01 cells. We used the osteogenic sarcoma cell line, UMR-106-01, to determine whether the rise in free cytosolic Ca2+ concentration ([Ca2+]i) and cellular cAMP following PTH stimulation are able to be regulated independently. For this purpose, we compared the effect of a PTH antagonist, stimulation of protein kinase C, augmentation by prostaglandins, and the time course of desensitization of the two cellular responses. Two x 10(-7) M of the PTH antagonist 8,18Nle 34Tyr-bPTH(3-34) amide ([Nle,Tyr]bPTH(3-34)A) was required to inhibit 10(-9) M bPTH(1-34)-stimulated cAMP generation by 50%. 10(-7) M bPTH(1-34) completely overcame the inhibition induced by 10(-6) M [Nle,Tyr]bPTH(3-34)A. Only 7 x 10(-8) M and 2.7 x 10(-7) M [Nle,Tyr]bPTH(3-34)A were required to half maximally inhibit the [Ca2+]i increase evoked by 3 x 10(-8) and 10(-7) M bPTH(1-34), respectively. In addition, dissociation between [Ca2+]i and cAMP signals was observed when modulation by protein kinase C and prostaglandins was tested. Preincubation of the cells with 10 nM TPA for 5 minutes markedly inhibited the PTH-evoked [Ca2+]i increase. Short incubation with PGF2 alpha augmented the PTH-evoked [Ca2+]i increase. Similar pretreatments had no effect on the PTH-stimulated cAMP increase. Finally, preincubation with 1.5 x 10(-9) M bPTH(1-34) for 20 minutes almost completely blocked the effect of 10(-7) M bPTH(1-34) on [Ca2+]i, while preincubation with 5 x 10(-9) M bPTH(1-34) for 4 hours was required to inhibit the effect of 10(-8) M bPTH(1-34) on cAMP production by 50%. The differences in the regulation of the two PTH-stimulated cellular signaling systems, in particular, the response to antagonists and the time course of desensitization, could be at the level of the PTH receptor(s) or at a postreceptor domain.


Autophagic vacuoles rapidly fuse with pre-existing lysosomes in cultured hepatocytes. Autophagic vacuoles (AVs) arise when membranes of the ER sequester parts of the cytoplasm, forming a new, double-membraned vacuole, to which lysosomal enzymes are then delivered. To investigate the mechanism of lysosomal enzyme delivery to nascent AVs, amino acid (AA) starvation and glucagon treatment were used to induce autophagy in a cultured cell system using rat hepatocytes (Fu5C8 cells). The induction of autophagy was assayed using biochemical, morphometric and immunocytochemical techniques. In these cells, AA starvation resulted in a fivefold increase in total cellular proteolysis, and sixfold and 4.5-fold increases in the volume and surface densities of AVs, respectively. Using an antibody against the mannose 6-phosphate receptor (MPR) and two sizes of colloidal gold to label separately and track the endosomal and lysosomal compartments, the time course of endosomal and lysosomal fusion with AVs was analyzed in detail. On the basis of these experiments, we found that AVs rapidly fuse with pre-existing lysosomes, but seldom with a prelysosomal compartment (PLC). Using immunoperoxidase, staining for the MPR was infrequently observed in association with any AVs. However, at early times following the induction of autophagy (less than 2 h), many autophagic vacuoles stained positively for the lysosomal enzyme cathepsin D. Consistent with these results, treatment of cells with tunicamycin had no effect on autophagy-induced proteolysis. We conclude that lysosomal enzyme delivery to nascent AVs occurs primarily by the fusion of pre-existing mature lysosomes, with a much smaller contribution by MPRs or the PLC.


In situ localisation of single-stranded DNA breaks in nuclei of a subpopulation of cells within regenerating skeletal muscle of the dystrophic mdx mouse. Degeneration of muscle fibres during the early stages of Duchenne Muscular Dystrophy (DMD) is accompanied by muscle fibre regeneration where cell division and myoblast fusion to form multinucleate myotubes within the lesions appear to recapitulate the events of normal muscle development. The mechanisms that govern the expression of genes regulating differentiation of myoblasts in regenerating skeletal muscle are of great interest for the development of future therapies designed to stimulate muscle regeneration. We show here that single-stranded breaks in DNA are localised in nuclei, using an exogenously applied medium containing labelled deoxynucleotides and the Klenow fragment of DNA polymerase I. The nuclei of a sub-population of cells lying in the inflammatory infiltrate of lesions in the skeletal muscle of the muscular dystrophic mouse (mdx), a genetic homologue of DMD, were labelled in this fashion. By contrast, labelled cells were completely absent from the muscles of normal non-myopathic animals (C57BL/10) and non-lesioned areas of mdx muscles. Cells expressing the muscle-specific regulatory gene, myogenin, were also found within mononucleate cells and myotubes within similar mdx muscle lesions. While we cannot yet say that the cells labelled by the DNA polymerase reaction are in fact differentiating, they were found only in significant numbers within mdx muscle lesions where new muscle fibres appear, providing strong circumstantial evidence that they are intimately associated with the regenerative process. Using a range of nucleases and different DNA polymerases, we show that the DNA polymerase-labelling reaction observed was DNA-dependent and most probably due to infilling of naturally occurring single-stranded gaps in DNA. Since the regenerative process in human Duchenne Muscular Dystrophy is apparently less effective than that seen in mdx mice, continued study of single-stranded DNA breaks may help to elucidate further the mechanisms controlling the expression of genes that characterise the myogenic process during skeletal muscle regeneration. Such findings might be applied in the development of future therapies designed to stimulate muscle regeneration in human dystrophies.


Regional cerebral blood flow after marijuana smoking. Regional CBF was measured with the 133Xe inhalation technique before and thrice after smoking marijuana of two strengths and placebo in 20 physically and mentally healthy male volunteers with a previous history of exposure to marijuana. They were drug-free at the time of the study. Blood pressure, pulse rate, end-tidal carbon dioxide, end-tidal carbon monoxide, and forehead skin perfusion were quantified during the CBF measurements. Blood samples were drawn for quantification of plasma levels of delta 9-tetrahydrocannabinol (THC) before and during the 2 h after smoking marijuana or placebo. Drug-induced intoxication and changes in mood were quantified with rating scales. Marijuana smoking was associated with bilateral CBF increase, which was maximal 30 min later. Greater CBF increases were seen in the frontal region and right hemisphere. No significant CBF changes were seen after placebo. Pulse rate and respiration increased significantly after marijuana but not placebo. Both marijuana and placebo smoking were associated with increased end-tidal carbon monoxide. CBF increase in both hemispheres correlated significantly with degree of intoxication, plasma levels of THC, and pulse rate.


Ion homeostasis in rat brain in vivo: intra- and extracellular [Ca2+] and [H+] in the hippocampus during recovery from short-term, transient ischemia. Changes in intra- and extracellular [Ca2+] and [H+], together with alterations in tissue PO2 and local blood flow, were measured in areas CA1 and CA3 of the hippocampus during recovery (up to 8 h) after an 8-min period of low-flow ischemia. Restoration of blood supply was followed by an immediate rise in flow and tissue PO2 above normal, with large fluctuations in both persisting for up to 4 h. In area CA1, [Ca2+]i decreased rapidly from an ischemic mean value of 30 microM to a control mean level of 73.1 nM in 20-30 min, whereas normalization of [Ca2+]e took approximately 1 h. Recovery of [Ca2+]i was accelerated by preischemic administration of a calcium antagonist, nifedipine, and a free radical scavenger, N-tert-butyl-alpha-phenylnitrone (PBN), but not by MK-801, a blocker of N-methyl-D-aspartate receptors. There was a secondary rise in [Ca2+]i in many cells beginning approximately 2 h after reperfusion. This was attenuated somewhat by PBN but not clearly influenced by either nifedipine or MK-801. Changes of [Ca2+]i in area CA3 were much smaller and slightly slower than in area CA1 and were not affected by the drugs mentioned above. In both areas CA1 and CA3, pHe and pHi fell during ischemia to an average value of 6.2, from which there was a rapid initial recovery in the first 5-10 min when blood flow was restored. Thereafter tissue pH rose slowly and did not reach control levels for approximately 1 h, and in some microareas not at all. It is concluded that (a) effective mechanisms for restoring normal [Ca2+]i remain intact after 8 min of low-flow ischemia; (b) in neurons of area CA1, some insidious change in the homeostasis of calcium triggers a secondary rise in its free cytosolic concentration, which may be causally related to activation of irreversible cell damage; and (c) the changes in [Ca2+]i and [Ca2+]e during and following 8 min of ischemia can be adequately accounted for by movements of a fixed pool of Ca between intra- and extracellular compartments, and possible mechanisms are discussed.


Enhanced calcium uptake by CA1 pyramidal cell dendrites in the postischemic phase despite subnormal evoked field potentials: excitatory amino acid receptor dependency and relationship to neuronal damage. After 6-12 h of recovery from transient cerebral ischemia, the pyramidal cells of the hippocampal CA1 region take up excessive amounts of calcium upon electrical stimulation, which has been suggested to be important for the development of delayed neuronal death. The aim of this study was to further characterize this enhanced calcium uptake with respect to time-course of development, relationship to neuronal damage, and amplitude of evoked field potentials as well as the dependency on N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Adult Wistar rats were used and calcium-sensitive microelectrodes were placed in the stratum radiatum of the CA1 hippocampus for recording of the extracellular calcium concentration ([Ca2+]ec) during 20 min of ischemia and for 6 h of reflow. High-frequency stimulation of the perforant pathway elicited burst firing in CA1 and a transient decrease in [Ca2+]ec which reflects neuronal uptake. Shifts in [Ca2+]ec could not be evoked 0-1 h after ischemia. However, from 1-2 h burst firing could be evoked and the accompanying shift in [Ca2+]ec increased thereafter in amplitude with prolonged reflow, exceeded preischemic levels after 4 h, and reached 250 +/- 116% (mean +/- SD) of control after 6 h of reflow (p less than 0.05). The extracellular reference potential shift during electrical stimulation and the amplitude of evoked field potentials were still subnormal after 6 h [85 +/- 25% and 83 +/- 25%, respectively (mean +/- SD)]. There was a significant correlation between the degree of stimulated calcium uptake at 6 h postischemia and the extent of CA1 damage evaluated 7 days after the ischemic insult (r = 0.849; p less than 0.001). The shifts in [Ca2+]ec were reduced by the NMDA antagonist MK-801 (0.5-2 mg/kg, i.v.) to approximately 50% of the initial level during both control and postischemic conditions (p less than 0.01). The non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[F]quinoxaline (NBQX) (42 +/- 13 mg/kg, i.p.; mean +/- SD) decreased the amplitude of the evoked field potentials (to 30 +/- 28% of control, p less than 0.05) and completely abolished the evoked shifts in [Ca2+]ec. In conclusion, the uptake of calcium into CA1 pyramidal cells during electrical stimulation was enhanced already 4 h after ischemia in spite of the fact that other measures of excitability were subnormal. This calcium uptake correlated to the extent of CA1 pyramidal cell damage and was dependent on both NMDA and non-NMDA receptor activation.


Global forebrain ischemia results in decreased immunoreactivity of calcium/calmodulin-dependent protein kinase II. Previous studies utilizing crude brain homogenates have shown that forebrain ischemia results in inhibition of calcium/calmodulin-dependent protein kinase II (CaM kinase II) activity without large-scale proteolysis of the enzyme. In this report, a monoclonal antibody (1C3-3D6) directed against the beta- (60-kDa) subunit of CaM kinase II that does not recognize ischemically altered enzyme was utilized to further investigate the ischemia-induced inhibition of CaM kinase II. Immunohistochemical investigations showed that the ischemia-induced decreased immunoreactivity of CaM kinase II occurred immediately following ischemic insult in ischemia-sensitive cells such as pyramidal cells of the hippocampus. No decrease in CaM kinase II immunoreactivity was observed in ischemia-resistant cells such as granule cells of the dentate gyrus. The decreased immunoreactivity was observed for CaM kinase II balanced for protein staining and calmodulin binding in vitro. In addition, autophosphorylation of CaM kinase II in the presence of low (7 microM) or high (500 microM) ATP did not alter immunoreactivity of the enzyme with 1C3-3D6. The data demonstrate the production of a monoclonal antibody that recognizes the beta-subunit of CaM kinase II in a highly specific manner, but does not recognize ischemic enzyme. Together with previous studies, the data support the hypothesis that rapid, ischemia-induced inhibition of CaM kinase II activity may be involved in the cascade of events that lead to selective neuronal cell loss in stroke.


Treatment of HIV infection: the antiretroviral nucleoside analogues. Nucleoside analogues: combination therapy. Combination antiretroviral therapy is an important development in the management of HIV infection. AZT with ddC is the first such combination to be approved for clinical use. Several issues remain, however, including the precise clinical benefit and toxicity of combination therapy, its effect on drug resistance, and the development of ever more effective therapeutic strategies.


Treatment of HIV infection: the antiretroviral nucleoside analogues. Nucleoside analogues: adverse effects. The three approved nucleoside analogues are fairly well tolerated, and only a few patients have to discontinue therapy permanently. However, because clinical experience is limited, especially with ddI and ddC, delayed toxicities are likely to emerge over time. In addition, physicians should be vigilant for overlapping toxicities from combination therapy with these and other medications.


Treatment of HIV infection: the antiretroviral nucleoside analogues. Nucleoside analogues: monotherapy. At present, the nucleoside analogues are the cornerstone of therapy for HIV infection. Of the three that have been approved for clinical use, AZT is the only one that has clearly proved to prolong survival. ddI is indicated for patients who develop toxicity or resistance to AZT. Current data do not support ddC monotherapy as first-line treatment.


A novel semi-automated paramagnetic microparticle based enzyme immunoassay for hepatitis C virus: its application to serologic testing. A new rapid serologic enzyme immunoassay for antibodies to hepatitis C virus (HCV) is described. The assay combines synthetic peptide and recombinant antigens representing putative structural and non structural HCV gene products with paramagnetic microparticle assay (MP assay) technology. Assay readout is based upon an enzymatically generated fluorescent product which is quantified with a novel semi-automated washer/reader instrument system. Assay sensitivity and specificity was determined to be greater than the first generation HCV C-100 EIA using a non-A, non-B hepatitis disease panel, an HCV performance panel, an HCV seroconversion panel, dilutions of HCV reactive sera, and random volunteer blood donor specimens.


K+ current stimulation by Cl- in the midgut epithelium of tobacco hornworm (Manduca sexta). I. Kinetics and effect of Cl(-)-site-specific agents. Goblet cells in the midgut epithelium of the tobacco hornworm (Manduca sexta larva, 5th instar) actively secrete K+. This can be measured as short-circuit current (Isc) when the tissue is mounted in an Ussing chamber and bathed in K(+)-rich standard saline containing 32 mmol K+.l-1. Isc depends strictly on basolateral (i.e. haemolymph side) K+ and is therefore termed K+ current, IK. Basolateral, but not apical, chloride, bromide and iodide stimulate IK when compared to the baseline current recorded with gluconate-, nitrate- or thiocyanate-containing salines. So-called "Cl(-)-specific" transport inhibitors (frusemide, 9-anthracene carboxylic acid, diphenylamine carboxylic acid and 4,4'-diisothiocyana-to-stilbene-2,2'-disulphonic acid) reduce IK when added to the basolateral bath, whether Cl- or gluconate is the principal ambient anion. Cl- stimulates IK according to saturation kinetics. The Michaelis-Menten-type, K+ concentration-dependent, saturation of IK is altered in a highly specific manner when gluconate is replaced by Cl-: maximal K+ current, as well as the apparent Michaelis constant, are increased by a factor of 4. Since IK develops in these conditions exclusively via basolateral, Ba(2+)-blockable K+ channels, these results can be understood if it is assumed that haemolymph Cl- interferes with the K+ channel by simultaneously lowering the binding affinity for K+ ions and increasing their subsequent transfer rate across the basolateral goblet cell membrane.


Arachidonic acid regulates the phosphoinositide signal transduction pathway in submandibular acinar cells. Modulation of the phosphoinositide signal transduction pathway by arachidonic acid (AA) in collagenase-dispersed rat submandibular acinar cells was investigated. The muscarinic agonist, carbachol, stimulated PIP2 hydrolysis and the generation of IP3 to five-fold the control levels. This response was inhibited by 75% on pre-treatment of cells with AA. The AA inhibitory effect was not duplicated by a range of prostaglandins and leukotrienes and was not reversed by blockers of the cyclo-oxygenase and lipoxygenase synthetic pathways, indicating that AA action was not mediated by eicosanoid metabolites. Additional experiments confirmed that the enzyme, protein kinase C, was also not a mediator of the AA effect. Arachidonic acid did not affect the uptake of radioactive inositol into acinar cells, but it did inhibit the incorporation of inositol into inositol phospholipids of the phosphoinositide cycle. In studies on inositol phospholipid turnover, AA alone reduced the level of PIP2 but not of PIP or PI. Under conditions of PI cycle stimulation with carbachol, AA significantly lowered PIP2 and PIP but not PI. These findings suggest that arachidonic acid may regulate the phosphoinositide response by inhibiting the synthetic phase of the cycle at a locus distal to PI generation.


Subsurface demineralization in dental enamel and other permeable solids during acid dissolution. Subsurface demineralization of dental enamel during acid dissolution has been reported many times, but its cause remains obscure. At first, the phenomenon was thought to result from the physical structure of enamel. More recent studies have shown that subsurface demineralization occurs in other permeable solids, indicating that there must be more fundamental factors involved in this curious effect. In order for this phenomenon to be investigated, dissolution experiments were carried out by means of real-time scanning microradiography in various systems, including enamel, or aggregates of hydroxyapatite (calcium, strontium, or barium), or hydroxides (calcium or magnesium). These were chosen to discriminate between effects of structure and composition. It was found that it was not possible for the demineralization observed in these systems to be attributed to a common feature. From this, it is concluded that subsurface demineralization in enamel and other mineralized tissues should not be ascribed to a single cause.


Examination by X-ray photoelectron spectroscopy of the adsorption of chlorhexidine on hydroxyapatite. X-ray photoelectron spectroscopy (XPS) was used for determination of the effects of chlorhexidine (CHX) solutions (0.2% and 1% solutions of the digluconate salt) on the elemental composition of hydroxyapatite surfaces. So that the nature of the adsorbed species after they were washed with water could be identified, comparisons were made with reference spectra for CHX obtained from a CHX digluconate film and CHX dichloride powder. The XPS results clearly indicated the retention of CHX moieties, which could be ascertained from the spectra by the presence of N and Cl, features unique to CHX. The spectral envelopes were virtually identical to those obtained from the reference spectra. High-resolution C 1s spectra also gave support for the retention of CHX; however, the spectra differed from those of the CHX digluconate film in that no feature attributable to the C-OH of the gluconate anion was present, consistent with the view that the CHX cation remains behind to form an electrostatic bond with the phosphate groups of the hydroxyapatite. The N:Cl ratio for the washed samples was found to be higher than that for the reference samples and may be indicative of partial decomposition of the CHX. Decomposition was also seen to be induced by x-ray exposure. While the high-resolution spectra presented here do not directly address the controversy on the mechanism for the anti-plaque efficacy of CHX, they do provide the necessary basis for the application of XPS to future in vitro studies on the retention of CHX to dental surfaces.


Auditory-nerve action potentials form a nonrenewal point process over short as well as long time scales. The firing patterns of auditory-nerve action potentials exhibit long-term fractal fluctuations that do not arise from the distribution of the interevent intervals, but rather from the ordering of these intervals. Using the serial interevent-interval correlation coefficient, the Fano-factor time curve, and shuffling of interevent intervals, it is shown that adjacent intervals for spontaneous firings exhibit significant correlation. The events are therefore nonrenewal over short as well as long time scales.


Na+K+ATPase activity and ouabain binding sites in erythrocytes in hyperthyroidism before and after treatment. Erythrocyte sodium pump is decreased in hyperthyroid patients. We described the effect of untreated hyperthyroidism on Na+K+ATPase activity, ouabain binding sites and intracellular sodium concentration. We found a reduction in Na+K+ATPase activity and in number of ouabain binding sites with a concomitant increase in intracellular sodium. B-blockade therapy failed to restore normal pump activity and sodium concentration, where only thionamide treatment was successful when it was able to decrease free T3.


Effects induced by olive oil-rich diet on erythrocytes membrane lipids and sodium-potassium transports in postmenopausal hypertensive women. Since we have observed that monounsaturated fatty acids (MUFA) enriched diet modifies red cell membrane lipids and cation transport systems in normotensive subjects, we similarly evaluated a group of hypertensive patients undergoing an analogous dietary modification. In a group of 18 moderately hypertensive women, the diet was supplemented for two months with olive oil (about 45 g/day), which replaced an equal amount of seasoning fats. Before and after this period, red cell fatty acid composition was evaluated by gas-chromatography in order to verify diet compliance: a significant increase in oleic acid was observed, while the content of saturated and polyunsaturated fatty acids remained unchanged. After olive oil, maximal rates of Na-K pump (5580 +/- 329 vs 6995 +/- 390, p less than 0.001) and Na-K cotransport (Na-COT 544 +/- 52 vs 877 +/- 46, p less than 0.001: K-COT 790 +/- 76 vs 1176 +/- 66, p less than 0.001), cell Na content (9.58 +/- 0.4 vs 10.61 +/- 0.6, p less than 0.03) and passive permeability for Na (936 +/- 74 vs 1836 +/- 102, p less than 0.001) rose significantly. Although the reduction in maximal rate of the Li-Na CT after olive oil was not significant, it was the only cation transport parameter being correlated with the variations of membrane lipids, namely negatively with UFA (r = -0.528, p less than 0.05) and positively with SFA (r = 0.482, p less than 0.005). The change in maximal rate of Li-Na CT was also correlated with the variation of systolic and diastolic BP (r = 0.50, p less than 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)


Intracranial dissemination of an ACTH secreting pituitary neoplasm--a case report and review of the literature. A 52-year-old woman developed recurrent hypercortisolism 3 yr after successful surgical treatment of Cushing's disease. At postmortem, eosinophilic pituitary tissue showing positive ACTH immunohistochemical staining was present in the frontal lobe and cerebellum but there was no tumor in the sella. In the absence of a pituitary tumor, extrasellar ACTH producing tissue could arise from seeding of the cerebrospinal fluid with tumor cells at the time of operation or from an atypical pituitary carcinoma. In this report we review the mechanisms of intracranial dissemination of pituitary tissue and ACTH-secreting pituitary carcinomas, including parasellar invasion, meningeal seeding, and cerebrospinal and hematogenous spread.


Prevalence of markers of hepatotropic viruses among drug addicts in Warsaw, Poland. We studied 100 unselected parenteral drug abusers for infection with hepatitis C, B, A and D virus (HCV, HBV, HAV and HDV). Seventy-six percent had serological evidence of HCV infection. 12% were positive for HBsAg and at least one marker of HBV infection was present in 69%. These results were significantly higher than in a matched control population. Compared to controls, the prevalence of anti-HAV (65%) was not significantly increased in drug addicts. Of the anti-HCV-positive drug addicts, 80.3% had at least one marker of HBV infection compared to 33.3% of anti-HCV-negative cases (p less than 0.001). No such correlation was found between the prevalence of HCV or HBV infection markers and the presence of anti-HAV. Antibodies against HDV were detected in 16 (16%) of the samples from drug addicts. No significant association was found between antibodies to HCV and gender, age and duration of drug abuse. The risk of HBV infection increased significantly with years of drug abuse but was not associated with age and sex. The presence of anti-HAV was related to age only. Sixteen (16%) of the subjects were definitely positive for anti-HIV-1, but at the time of the study they were asymptomatic. No significant association was found between the presence of anti-HIV and the prevalence of serological markers of HBV, HCV, HAV and HDV infection.


Prevalence of markers of hepatitis viruses in out-patient alcoholics. The prevalence of HCV, HBV and HAV markers was investigated in unselected patients attending an outpatient alcoholic clinic. Anti-HCV were detected in 35 (24%) of 144 patients studied, and at least one marker of HBV infection was present in 72 (50%). These results are significantly higher than in a matched control population. The presence of anti-HCV was related to previous blood transfusions and familial history of alcoholism. We conclude that alcoholics should be considered a high risk group for both HCV and HBV infection.


Hepatitis C virus in kidney recipients. Epidemiology and impact on renal transplantation. In an attempt to evaluate the prevalence, kinetics and impact of HCV infection in renal transplantation, we analyzed 140 kidney recipients according to the histopathological status of the liver. Thirty-three HBsAg-negative patients had chronic active hepatitis, 73 HBsAg-negative patients had a normal liver, 21 HBsAg-negative kidney recipients had minimal pathological changes and 13 patients had HBsAg-positive cirrhosis. Serum antibodies to HCV were detected using the ELISA from Ortho Diagnostic and confirmatory tests using the Ortho recombinant-based immunoblot assays. The overall prevalence of antiHCV antibodies was 23.6%. AntiHCV were more frequently present in HBsAg-negative patients with chronic active hepatitis (60.6%) than in HBsAg-negative patients with normal livers (8.2%) (p less than 0.0001) or minimal liver changes (33.3%) (NS) or in HBsAg-positive patients with cirrhosis (0%) (p less than 0.001). The recombinant-based immunoblot assays confirmed antiHCV-positive ELISA results in 86.7% of patients. Among the 27 antiHCV-positive kidney recipients who had serial serological follow-up, 10 (37.0%) were already positive at transplantation and remained antiHCV-positive during follow-up. Eleven patients (40.8%) acquired antiHCV an average of 95 months after renal transplantation, while antiHCV disappeared an average of 111 months after transplantation in six (22.2%), who had antiHCV prior to transplantation. The kinetics of antiHCV antibodies did not differ according to liver histology. Patient and graft survival were not different in antiHCV-positive and antiHCV-negative kidney recipients irrespective of liver histology, and there was no difference in survival between antiHCV-positive and antiHCV-negative patients with chronic hepatitis.(ABSTRACT TRUNCATED AT 250 WORDS)


Hepatitis C virus testing in primary biliary cirrhosis. We retrospectively investigated anti-HCV prevalence in a series of 160 consecutive patients with primary biliary cirrhosis who presented between 1980 and 1989. Of these, 19 (12%) were positive for anti-HCV by C-100 ELISA. Serum IgG levels were significantly higher in anti-HCV-positive patients and correlated to optical density values. A serum sample was again collected from all the patients from the same series who were seen in 1990 for follow-up, after a median period of 32 months. Anti-HCV positivity was found to be substantially unchanged in this subgroup of patients when the freshly drawn blood samples were retested with C-100 ELISA, while it increased from 10% to 17% when second generation ELISA was used. Three of the C-100 ELISA positive samples were C-100 RIBA reactive, and six of the second generation ELISA positive samples were 4-RIBA reactive. The HCV genome was not detected in any of the seven anti-HCV C-100 ELISA and second generation ELISA positive sera which were studied by polymerase chain reaction, including four cases confirmed by 4-RIBA. Life expectancy, as determined by survival analysis, did not differ significantly between anti-HCV-positive and -negative patients. These findings suggest that anti-HCV positivity does not influence the clinical presentation and course of primary biliary cirrhosis.


Central mu-opioid receptors are down-regulated in a rat model of cholestasis. Ameliorations of the pruritus of cholestasis by opioid antagonists are consistent with this form of pruritus being centrally mediated by the opioid system. To determine whether the central opioid system is altered in cholestasis, the specific binding of a selective mu-opioid receptor ligand, 3H-DAMGO, to mu-opioid receptors was studied in rats with acute cholestasis due to bile duct resection. Using whole brain membranes and subcellular mitochondrial-synaptosomal fractions the density of mu-receptor sites was 30% (p less than 0.01) and 22% (p = 0.03) less in bile-duct-resected rats than in sham-resected rats. Using membranes from individual brain regions specific binding of 3H-DAMGO was reduced by 43-53% in the cerebral cortex, hippocampus and caudate nucleus of bile-duct-resected rats. Thus mu-opioid receptors in the brain are down-regulated in a classical model of cholestasis. This alteration of the central opioid system could be a consequence of increased exposure of opioid receptors to endogenous opioids in cholestasis and may reflect an important mechanism in the pathogenesis of the pruritus of cholestasis.


Efficacy of low-dose captopril in addition to furosemide and spironolactone in patients with decompensated liver disease during blunted diuresis. The renin-angiotensin-aldosterone system is activated by diuretics and involved in the diuretic resistance of cirrhotic patients with ascites and oedema. In previous studies relatively high doses of captopril (25-400 mg daily) were unsuccessful in promoting diuresis and natriuresis in these patients. We analyzed the efficacy of a low dose of captopril in eight patients with massive ascites resistant to therapy of salt/fluid restriction and increasing doses of spironolactone and furosemide. Mean duration of diuretic use was 73 days (range 7-240 days). After at least 3 days of observation on 80 mg furosemide and 100 mg spironolactone only, captopril was added. Four out of eight patients responded with an increase in natriuresis and diuresis; daily dose of captopril was 20.6 mg in responders and 26.5 mg in non-responders. After the addition of captopril the mean weight change was -7.5 kg in responders and +0.25 kg in non-responders. Mean urinary sodium output in responders increased from 72.8 (S.D. = 35.2) to 128.5 (63.5) mmol within 10 days. Increased diuresis in responders made diuretic reduction necessary: mean furosemide from 80 to 53.3 mg, and mean spironolactone from 100 to 68.1 mg. Creatinine clearances remained stable. High levels of plasma renin activity, plasma aldosterone and angiotensin-II were found in all patients. Non-responders showed more severe hyponatremia and higher vasopressin levels. Natriuretic atrial factor (NAF) was in the upper-normal range or slightly elevated in both groups. In non-responders we noticed low levels of cGMP in 24-h urine, compared with responders.(ABSTRACT TRUNCATED AT 250 WORDS)


Cryptogenic chronic liver disease and hepatitis C virus infection in children. The clinical features of 'cryptogenic' chronic liver disease and the prevalence of antibody to hepatitis C virus (HCV) in serum have been investigated in 33 Italian children (mean age 5 years). The diagnosis was based on the persistence of increased alanineaminotransferase values for longer than 6 months after the exclusion of biliary diseases, of extra-hepatic causes of hypertransaminasemia, of infection with known hepatotropic viruses and of autoimmune or metabolic disorders. Five patients had been transfused early in life, three had undergone surgery and one girl's mother had had acute non-A, non-B hepatitis during pregnancy. The remaining patients had no history of overt parenteral exposure. At presentation only 11 patients were symptomatic, the others had been referred after a check-up for intercurrent diseases. Liver histology performed in 21 cases showed persistent or mild active hepatitis in 18 cases and severe hepatitis or cirrhosis in three cases. Anti-HCV antibodies were found in 48% of the cases, including 88% with obvious exposure and 33% of the remaining cases. During a mean follow-up period of 5 years (range 1-14 years) only 11% of the cases achieved sustained biochemical remission, although none developed signs of liver failure. There was no significant difference in the clinical features and outcome of the disease between anti-HCV-positive and -negative patients. The results of this study suggest that HCV is implicated in most cases of 'cryptogenic' chronic liver disease observed in Italian children with a history of parenteral exposure and in at least one-third of the cases without overt exposure.(ABSTRACT TRUNCATED AT 250 WORDS)


Minor lymphocyte stimulatory antigen-bearing stimulator cells require lipopolysaccharide activation to induce programmed cell death in T cell hybridomas. T cell hybridomas respond to conventional peptide Ag associated with self major histocompatibility restriction elements, as well as to alloantigens, activating lectins, and stimulatory forms of mAb by producing lymphokines and undergoing programmed cell death (PCD). We show here that the level of PCD and IL-2 production correlate well in responses to CD3 or allostimulation. The response to minor lymphocyte-stimulatory (Mls) Ag, members of the family of endogenous superantigens, however, are marked by divergence in the levels of the PCD and lymphokine responses. Specifically, PCD in response to Mls activation is achieved poorly despite vigorous IL-2 production. B lymphoma cell stimulators induced PCD in alloreactive T cell hybridomas but not in Mls-reactive T cell hybridomas. This suggests that the absence of PCD in the Mls response is a function of superantigen recognition rather than the stimulator cell type. LPS-preactivated Mls+ stimulators, either splenic B or B lymphoma cells, are shown to trigger PCD in the T cell hybridomas. These results imply that T cell interaction with Mls presented by untreated stimulator cells is not sufficient for induction of PCD and thus is distinct from interactions with conventional Ag.


Generation of anti-AKR/gross murine leukemia virus cytotoxic T lymphocytes (CTL). An analysis of precursor CTL frequencies in the AKR.H-2b and C57BL/6 mouse strains. C57BL/6 mice, after immunization and secondary in vitro restimulation with AKR/Gross murine leukemia virus (MuLV)-induced tumors, generate AKR/Gross MuLV-specific CTL. After similar immunization protocols, AKR-H-2b mice fail to generate CTL specific for AKR/Gross MuLV. The basis for nonresponsiveness in AKR.H-2b mice is unknown, however, unlike C57BL/6 mice, AKR.H-2b mice carry endogenous proviruses and express N-ecotropic viral Ag. Thus, clonal deletion of pCTL populations due to the expression of AKR/Gross MuLV-like Ag is a likely mechanism for the nonresponsiveness. To determine if nonresponsiveness is due to clonal deletion, limiting dilution cultures were performed to assess the presence of pCTL specific for AKR/Gross MuLV. Our study demonstrates that the frequencies of pCTL specific for AKR/Gross MuLV are similar in both the responder C57BL/6 and nonresponder AKR.H-2b strains. The observation that normal levels of AKR/Gross MuLV-specific pCTL exist in AKR.H-2b mice, suggests that clonal deletion of pCTL is not responsible for the inability of AKR.H-2b mice to generate anti-AKR/Gross virus-specific CTL.


Oncostatin M induces tyrosine phosphorylation in endothelial cells and activation of p62yes tyrosine kinase. Oncostatin M is a polypeptide cytokine produced by activated and transformed T lymphocytes that has diverse biologic effects, including growth inhibition of tumor cells and induction of IL-6 expression in cultured human endothelial cells (HEC). HEC are highly responsive to oncostatin M and express high levels of oncostatin M receptors relative to other cell types. Oncostatin M has previously been found to bind a specific receptor of 150 to 160 kDa. We have found through the use of anti-phosphotyrosine immunoblotting that oncostatin M induces tyrosine phosphorylation in HEC. Anti-phosphotyrosine antibodies specifically immunoprecipitated labeled oncostatin M cross-linked to its receptor, demonstrating that the oncostatin M receptor is either directly phosphorylated on tyrosine after ligand binding or is tightly associated with a phosphotyrosyl protein in these cells. The tyrosine kinase inhibitor herbimycin A blocked the induction of IL-6 by oncostatin M in HEC. In addition, immune complex kinase assays showed that oncostatin M markedly increased the activity of the p62yes tyrosine kinase with a small increase in p59fyn but no increase in p56lyn tyrosine kinase activity in HEC. We conclude that oncostatin M utilizes a tyrosine phosphorylation signal transduction pathway in HEC involving the activation of the p62yes tyrosine kinase, and that this tyrosine phosphorylation pathway leads to the induction of IL-6 expression.


[Reduction of neutrophil activation by prostaglandin E1 in open heart surgery]. This study was designed to demonstrate the effect of prostaglandin E1 (PGE1) on neutrophil activation in open heart surgery. Twenty adult patients undergoing cardiopulmonary bypass (CPB) for various cardiac operations were divided into 2 groups. PGE1 group consisted of 10 patients (7 males and 3 females) and the control group consisted of 10 patients (6 males and 4 females). In PGE1 group patients, 20-50 ng/kg/min of PGE1 was administered intravenously from the induction of anesthesia to the completion of CPB. Blood samples were taken before, during, after CPB, and in the morning of the first postoperative day. Differential counts of white blood cells, plasma neutrophil elastase (PNEL) activity, serum complements activity (C3a, CH50) and superoxide production of neutrophils were measured. Superoxide production by isolated neutrophils was evaluated utilizing luminol dependent chemiluminescence. After the initiation of CPB complements were activated markedly, and PNEL activity increased significantly in both groups. Although after CPB PNEL activity turned to decrease, it was still significantly higher on the first postoperative day than the preoperative value. There were no significant differences between two groups as for complements activation and PNEL activity. The total number of white blood cells unchanged during CPB and neutrophilia appeared after CPB, but no significant difference between two groups. Superoxide production of neutrophils relatively decreased during CPB and significantly increased after CPB in the control group. However, in PGE1 group superoxide production was reduced after CPB, especially on the first postoperative day. These results showed that PGE1 reduced neutrophil-mediated superoxide production in open heart surgery. In conclusions, PGE1 is useful agent to reduce the hazardous effects of neutrophils after CPB.


Role of complement in endotoxin/platelet-activating factor-induced lung injury. C receptor-1 is a protein involved in the regulation of C3 and C5-convertases. Recombinant human soluble C receptor-1 has recently been produced and shown to reduce infarct size in a rat model of myocardial ischemia/reperfusion injury. The present study aimed to investigate whether recombinant human soluble C receptor-1 exerts any protective effect on pulmonary injury produced in a rodent model of adult respiratory distress syndrome. In this model, Escherichia coli endotoxin (LPS, 0.1 microgram/kg) combined with platelet-activating factor (1 pmol/kg/min over 60 min, n = 10) caused microvascular lung injury characterized by elevation of myeloperoxidase activity, deposition of C3 and C5b-9 on the endothelium of pulmonary vessels, and pulmonary edema. Furthermore, bronchoalveolar lavage revealed increased neutrophil count and elevated protein concentration. These pulmonary responses were associated with elevated serum TNF-alpha. Pretreatment (10 min, i.v.) with recombinant human soluble C receptor-1 at 10 mg/kg (n = 13), but not at 1 mg/kg, prevented the LPS/platelet-activating factor-induced pulmonary edema (p less than 0.01) and changes in the bronchoalveolar lavage fluid cell count (p less than 0.01) and protein concentration (p less than 0.05), and attenuated the deposition of C3 and C5b-9 to lung vessels. There was no effect on lung myeloperoxidase activity and serum TNF-alpha. Also, C depletion by cobra venom factor (500 U/kg, i.v.) eliminated the pulmonary edema and elevated leukocyte count in bronchoalveolar lavage fluid, but had no effect on lung myeloperoxidase activity and serum TNF-alpha. These data suggest that C factors may play an important role in the pathophysiology of adult respiratory distress syndrome.


[New reconstruction method for defect of chest wall using composite graft]. In the case of a 64-year-old man squamous cell carcinoma in the left lung with metastasis to the rib, we excised the affected portion of the chest wall and used a composite graft, a combination hydroxyapatite (HAP) filler, for reconstruction with good results. HAP filler is a ceramic that combines the calcium phosphate apatite hydroxide with 3-calcium phosphate. The Dacron fabric used was made of polyethylene terephthalate fiber. Two layers of Dacron fabric were laid together, stitched to the rib with nylon thread, and the resulting tubular cavity packed with HAP filler to create an artificial rib. The intercostal muscle was sandwiched between the layers of Dacron fabric and the ends of the natural rib laid across those of the artificial one. We used the periosteum and intercostal muscle to integrate the graft and complete reconstruction. Postoperative recovery was good, and there was no visible sign of deformation at the graft site. Even in palpation we were unable to tell that the rib was artificial.


[Detection of human papillomavirus DNA in uterine cervix by polymerase chain reaction (PCR) method]. In recent years, human papillomavirus (HPV) infection in female genital organs has attracted increasing attention because of its association with lesions in the uterine cervix, especially cervical carcinoma. In this study, the author attempted to determine the presence or absence of HPV infection in the cervical region by means of the polymerase chain reaction (PCR) method, which has become practical thanks to the development of thermostable DNA polymerase, and compared this method with traditional Southern blotting. Moreover, we conducted PCR after detecting DNA by two methods: target punch biopsy and cervicovaginal lavage, and compared the results in terms of detection. 1. The results of HPV detection from the isolated tissue were compared with those of traditional Southern blotting and PCR combined with subsequent hybridization under high stringency (PCR-H). Among the cases that showed negative response with the former method, two of six specimens of cervical carcinoma tissue, and four of eight specimens of normal cervical tissue yielded a positive response as a result of hybridization under low stringency after PCR (PCR-L). HPV was investigated in 56 clinical specimens by PCR. PCR-L was positive in 50.0% and 20.0% of the specimens of cervical carcinoma and normal cervical tissue, respectively. With PCR-H, the positive rates were 37.5% and 7.5% in cervical carcinoma and normal cervical tissue, respectively. 2. Among CIN patients who were followed up at the outpatient clinic, PCR was conducted in specimens obtained by cervicovaginal lavage and target punch biopsy, and the rates of HPV detection were compared.(ABSTRACT TRUNCATED AT 250 WORDS)


[The pathogenesis and etiology of giant cell tumor of bone from a viewpoint of bone resorptive factors]. The pathogenesis and etiology of giant cell tumor of bone was studied by analysing the bone resorptive factors in the conditioned culture medium. In the primary culture characteristic multinucleated giant cells and mononuclear cells were coexisted. The values of interleukin 1 (IL-1) and prostaglandin E2 (PGE2) in the conditioned medium obtained from the primary culture were high. In the primary culture, an immunohistochemical technique revealed the presence of IL-1 both in mononuclear cells and in giant cells. When the medium obtained from the primary culture was tested for proteolytic activity by zymography with SDS/polyacrylamide containing gelatin, multiple gelatinolytic activities were observed. In subcultures, multinucleated giant cells were not persisted and only stromal cells were visible. In subcultures, the values of IL-1 and PGE2 were much lower. Proteolytic activities were similarly weak. However, the exposure of the passaged stromal cells to the medium containing IL-1 stimulated the stromal cells to produce PGE2 and proteolytic enzymes. Immunofluorescent localization technique revealed the expression of the proteolytic enzymes in the stromal cells. These findings demonstrated that coexistence of multinucleated giant cells with mononuclear cells should be needed for the tumor to express the original phenotype. In the presence of multi-nucleated cells, mononuclear cells seem to be stimulated to produce PGE2 and proteolytic enzymes, which accelerate the bone resorption. These factors are considered to act synergetically in the resorption of bones.


The inhibition of Na, K-ATPase, and Mg-ATPase by timolol maleate in cultured non-pigmented epithelial cells of the ciliary body. Bovine, non-pigmented, ciliary body epithelial cells were isolated and grown in culture to determine whether timolol maleate might affect the activity of their plasma membrane ATPases. The possible effects were tested in drug concentrations in a range of 5 x 10(-19) to 5 x 10(-5) M over an incubation period of 30 min at 37 degrees. Assays of specific activity showed that the drug significantly (p less than .001 for most concentrations) inhibited both Na,K-ATPase and Mg-ATPase. However, the inhibition was partially reversed in concentrations greater than 10(-6) M for Na,K-ATPase and 10(-5) M for Mg-ATPase. The latter enzyme also indicated a second partial reversal in activity at concentrations between 10(-12) and 10(-9) M. These reversals in activity suggest that more than one binding site is involved in the inhibition of both enzymes. Since Na,K-ATPase in non-pigmented, ciliary body cells is responsible for the generation of aqueous fluid and the intraocular pressure (IOP), this inhibition demonstrates a possible mechanism for the pharmacological action of timolol maleate in lowering IOP.


Effect of topical and intracameral methysergide on calcitonin gene-related peptide-induced irritative changes in the rabbit eye. Calcitonin gene-related peptide (CGRP) is a neuropeptide localized in the ocular sensory nerves. It is responsible for most of the irritative changes in the rabbit eye in neurogenic inflammation, namely vasodilation in the anterior uvea, breakdown of the blood-aqueous barrier and increase in the intraocular pressure. In the present study, intracameral methysergide inhibited the CGRP-induced irritative changes in the rabbit eye. Provided that sufficient concentration of methysergide could be reached in the anterior chamber after topical application, it might be possible to use locally administered methysergide to limit different pathophysiological conditions in the eye in which CGRP is involved.


Opiate receptors: a review of analgesic properties and pharmacological side effects. Pain and suffering are the most common, and distressing, complaints of patients. Opiates remain the drugs of choice for severe pain. A review of the current information known about these drugs is essential not only for any anesthesiologist, but for any physician. The term "opioid" refers to any peptide that binds stereospecifically to opioid receptors, regardless of whether it occurs naturally or is chemically synthesized. There are five basic classes of opioid receptors that have been discovered. Understanding each of the receptor's activities and side-effects is essential for proper use of available opiates. This review will provide current information of the opioid receptors. Not only will understanding result in better patient care but it will encourage greater research in this field.


Differential effects of interleukin-4 on superoxide anion production by human alveolar macrophages stimulated with lipopolysaccharide and interferon-gamma. The effect of interleukin-4 (IL-4) on the activation state of human alveolar macrophages (AMs) and blood monocytes induced by lipopolysaccharide (LPS) or recombinant interferon-gamma (IFN-gamma) was investigated on the basis of their ability to produce superoxide anion (O2-). AMs were obtained from healthy donors by bronchoalveolar lavage, and O2- productions of these cells were assayed by a cytochrome c reduction method after incubation with stimulants for 24 h. AMs produced more O2- than autologous blood monocytes when stimulated with LPS. IL-4 alone had little effect on O2- production by unstimulated AMs but down-regulated O2- production by LPS-stimulated AMs in a dose-dependent manner. IL-4 also suppressed O2- production by AMs induced by the synergistic actions of muramyl dipeptide (norMDP) and IFN-gamma. Maximum suppression by IL-4 of O2- production by AMs was observed when IL-4 was added within 1 h after initiation of LPS stimulation. AMs also showed high O2- production when stimulated with IFN-gamma alone. In contrast to its suppression of O2- production by LPS-stimulated AMs, IL-4 enhanced O2- production by AMs stimulated with IFN-gamma. These data suggest that IL-4 is an important regulator of O2- production by macrophages through different pathways depending on the stimulus.


Macrophage function in response to PGE2, L-arginine deprivation, and activation by colony-stimulating factors is dependent on hematopoietic stimulus. Different macrophage preparations were compared for functional capacity in conditions of high prostaglandin E2 (PGE2) or low L-arginine concentrations. Macrophages derived in vitro from bone marrow progenitor cells (bone marrow-derived macrophages, BMDMs) using colony-stimulating factor 1 (CSF-1) as the myelopoietic stimulus displayed a greater sensitivity to PGE2-induced suppression of tumor necrosis factor alpha (TNF-alpha) secretion than did macrophages derived using granulocyte-macrophage colony-stimulating factor (GM-CSF). Neither BMDM population was inhibited by PGE2 for the direct cytolysis of L929 cells (TNF-alpha sensitive), and only GM-CSF-derived macrophages showed decreased killing of TNF-alpha-resistant K562 targets. Exogenous cAMP inhibited TNF-alpha secretion, but not nitrite secretion, by both BMDM populations. GM-CSF-derived macrophages accumulated less cAMP following PGE2 treatment than did CSF-1-derived macrophages. Removing L-arginine from the medium did not inhibit cytotoxicity or PGE2 secretion, but the listeriacidal activity specific to interferon-gamma plus lipopolysaccharide (LPS)-activated GM-CSF-derived macrophages was blocked by removal of L-arginine. Treatment with CSF-1 or GM-CSF alone did not activate the macrophages, but GM-CSF efficiently primed both BMDM populations for augmented TNF-alpha secretion in response to secondary stimulation using LPS. However, GM-CSF augmented the LPS-induced production of nitrite and PGE2 by CSF-1-derived macrophages only. These results demonstrate the potential for differential macrophage function within inflammatory sites based on the hematopoietic stimulus under which the macrophage is derived and the specific conditions present in the lesion.


Characteristics of alveolar macrophages in experimental septic lung. We investigated the pathogenesis of lung injury in sepsis (septic adult respiratory distress syndrome) by focusing on the functional changes of alveolar macrophages (AMs). Sepsis was induced in male WK rats by cecal ligation and puncture. Histological examination of the lungs from this experimental model revealed edematous change at 24 h after the surgery. The protein and endotoxin concentrations in the bronchoalveolar lavage fluid (BALF) increased with time after the surgery. The time course studies of AM function after surgery indicated that AMs from septic rats were activated by endotoxins. Specifically, this was suggested by the finding that AM adherence to and spreading on a plastic dish had increased. On stimulation, these AMs enhanced generation of superoxide anions and increased release of lysosomal enzymes, such as beta-glucuronidase. On the other hand, AMs in sepsis generated much smaller amounts of arachidonate lipoxygenase metabolites, such as leukotriene B4 (LTB4) and 12- and 5-hydroxyeicosatetraenoic acids (HETEs), on stimulation than did AMs from sham rats or untreated rats. However, the concentrations of immunoreactive LTC4 in the BALF of septic rats seemed to be higher than in untreated rats. It is suggested that the AMs of septic rats released lipoxygenase metabolites in alveoli and that these AMs could not be stimulated in vitro. These functional changes in the AMs of septic rats progressed along with the sepsis. These results implicate AMs in the development and progression of septic lung injury by releasing superoxide anions, beta-glucuronidase, and arachidonate metabolites. Furthermore, we speculate that reduced production of LTB4 by septic AMs may increase host susceptibility to severe pulmonary infection during septic ARDS.


Platinum-diaminobenzidine reaction and its contribution to the quantitation of cytochrome oxidase activity. The platinum-diaminobenzidine (Pt-DAB) reaction, which yields a black, electron-dense, insoluble, Pt-containing reaction product at the active site of cytochrome oxidase without postosmification, has been developed. This reaction permits us to quantitate the oxidase activity in an individual mitochondrion or in its constitutent part by the energy dispersive X-ray analysis (EDAX), based on the platinum atoms incorporated in the product.


Detection of neuromuscular junctions on isolated branched muscle fibers: application of nitric acid fiber digestion method for scanning electron microscopy. Three-dimensional morphology and neuromuscular junctions of the branched muscle fibers of the rat skeletal muscles isolated from the rat were examined by application of the nitric acid digestion method for scanning electron microscopy. Treatment with fifteen percent nitric acid and a supersonic wave bath were effective in isolating the branched muscle fibers and for detecting their innervations by scanning electron microscopy. Mean numbers of total and branched fibers in the rat plantaris muscles were 11,130 +/- 314 and 32.4 +/- 4.8 (n = 12), respectively. Bifurcation of these fibers resembled either the letter X (X type) or letter Y (Y type). The Y type fibers were most frequently seen (84% of total branched fibers), and X types accounted for 14%. The remaining 2% of the fibers were subtypes of X and Y with overlapping bifurcation. These branched fibers were differentiated by their length from the bifurcated point, i.e., short, medium and long branches. Neuromuscular junctions on these branched fibers were located almost at the center of their length. The number of innervations was decided by the number of bifurcating fibers across the central part of the length.


Simultaneous quantitation of delta-9-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in serum by GC/MS using deuterated internal standards and its application to a smoking study and forensic cases. A new procedure for the simultaneous detection of delta-9-tetrahydrocannabinol (THC) and its major metabolite, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in serum has been evaluated. The method combines rapid, efficient, solid-phase extraction and simple derivatization by methylation. Analysis and quantitation is performed by gas chromatography/mass spectrometry (GC/MS) using deuterated cannabinoids as internal standards (IS). Reproducibility and sensitivity of the method are good. The procedure is applied to serum specimens collected from a smoking study with 24 volunteers and 212 forensic cases. Results are interpreted based upon the current knowledge about THC metabolism and pharmacokinetics.


Uneven distribution of mitochondrial DNA mutation in MERRF dizygotic twins. A new family of myoclonic epilepsy with ragged-red fibers (MERRF) was studied at clinical, histological, biochemical and molecular genetic levels. There was a remarkable variation in the age of onset, the clinical presentation and the severity of symptoms. Multiple defects affecting respiratory chain complexes I, III and IV were detected in 2 patients. The point mutation at 8344 of the mitochondrial genome was found in all the maternal lineage with a relatively narrow range of variation in the percentage of mutant mitochondrial genomes. The one exception was represented by a set of dizygotic twins, one clinically affected and showing high proportions of mutant mitochondrial DNAs (mtDNAs) in blood cells, while the other was asymptomatic and showed very small amounts of mutant mt-DNAs in blood and skin. This could suggest an early segregation of the mitochondrial genome during ovogenesis.


Mitochondrial DNA deletions and cytochrome c oxidase deficiency in muscle fibres. We have studied cytochrome c oxidase (COX) deficient muscle fibre segments in 6 patients with mitochondrial myopathy and deletions of mitochondrial DNA (mtDNA). The distribution of transcripts of normal and mutated mtDNA in skeletal muscle sections was studied by in situ hybridization. The results were compared with the enzyme histochemical activity of COX and the immunohistochemical distribution of mtDNA encoded and nuclear DNA encoded subunits of COX. In all cases a proportion of the muscle fibres (less than 1-30% of the fibres in cross-sections) had low COX activity and high activity of succinate dehydrogenase (COX deficient muscle fibres). Transcripts of normal and deleted mtDNA showed the same distribution within the tissue as the corresponding mtDNA, indicating that the deleted mtDNA is transcribed. The COX deficient muscle fibres showed accumulation of transcripts of deleted mtDNA, which had a similar distribution as the accumulated mitochondria within these fibres. With few exceptions, there was a low level of transcripts of normal mtDNA in these COX deficient fibres. Immunohistochemical analysis revealed low levels of immunoreactive material using antiserum to the mtDNA encoded subunits II/III as well as the nuclear DNA encoded subunit IV of COX in all COX deficient muscle fibres. The fraction of deleted mtDNA in muscle ranged from 43 to 87%. There was no correlation between the proportion of COX deficient muscle fibres and the fraction of deleted mtDNA. In 2 cases the deletion did not involve any COX gene. One of these cases had 87% deleted mtDNA but less than 1% COX deficient muscle fibres.(ABSTRACT TRUNCATED AT 250 WORDS)


Immunohistological study of grumose degeneration of the dentate nucleus in progressive supranuclear palsy. The grumose degeneration observed in the dentate nuclei of 7 cases of progressive supranuclear palsy (PSP) was studied with a panel of antibodies which included 2 neurofilaments, Tau and ubiquitin. Dentate nucleus neurons were negative with all antibodies except ubiquitin which showed a slightly positive homogeneous pattern of staining. The amorphous material surrounding swollen or normal neurons was strongly positive for neurofilament and subunits and numerous torpedoes were observed in the granular layer of the cerebellar cortex. Our results confirm that grumose degeneration consists in degeneration of terminal axons of Purkinje cells in the dentate nucleus. The positivity of dentate nucleus neurons for ubiquitin may support the concept of synaptic dysfunction between Purkinje cells and dentate nucleus neurons.


Clonal composition of glioblastoma multiforme. Glioblastoma multiforme, the most common and most lethal primary central nervous system neoplasm, is noted for its phenotypic and biological heterogeneity. This heterogeneity may result from genetic alterations accumulated by a single transformed astrocyte as it evolves into a monoclonal tumor. Alternatively, it may be attributed to the presence of multiple biologically and genetically distinct astrocytic populations within a polyclonal tumor. To address the issue of clonal composition of glioblastoma multiforme the authors used two independent approaches: analysis of X-chromosome inactivation and analysis of chromosomes 10 and 17 for tumor-specific somatic deletions. The analysis included 10 tumors from nine female patients with glioblastoma multiforme (eight primary and two recurrent tumors), who were heterozygous at either of two X-chromosome genes (hypoxanthine phosphoribosyl-transferase or phosphoglycerate kinase). Nine glioblastomas multiforme demonstrated a monoclonal pattern on X-chromosome analysis; contamination with normal tissue obscured the analysis in one tumor. Somatic deletions on chromosomes 10 and/or 17 occurred in nine tumors, supporting a monoclonal composition for these tumors. These data suggest that glioblastoma multiforme is a monoclonal neoplasm, derived from the clonal expansion of a single transformed astrocyte that has, as a fundamental step in tumorigenesis, sustained a critical genetic alteration on chromosome 10 and/or 17.


Effect of pectin on formyl methionyl-leucyl-phenylalanine (FMLP)-injured intestinal mucosa of rat. (1) We investigated the trophic effect of pectin on the intestinal mucosa injured by formyl methionyl-leucyl-phenylalanine (FMLP), a chemoattractant produced by the intestinal bacterial flora. (2) We first demonstrated that oral administration of FMLP for 7 days reduced the disaccharidase activities and increased the permeability, measured by fluorescein-isothiocyanate-conjugated dextran, of rat small intestine. (3) After 7 days of FMLP administration, rats were divided into fiber-free group which was given liquid elemental diet (Elental) and the pectin group which was given Elental supplemented with 2.5% pectin. (4) After 3 days of feeding (Day 3), the maltase activities of the pectin group was significantly greater than that of the fiber-free group and than that of the initial level just after the 1 week administration of FMLP. At Day 7, there was no difference of maltase activity between the two groups. The sucrase activity of the pectin group was also significantly greater than that of fiber-free group at Day 3. (5) Plasma enteroglucagon was significantly increased in the pectin group. We conclude that pectin-supplemented diet promoted the recovery of disaccharidase activities in the FMLP-injured intestinal mucosa which may be mediated by enteroglucagon.


Evaluation of a low-temperature calcium phosphate particulate implant material: physical-chemical properties and in vivo bone response. A study was conducted to evaluate the osteoconductive ability of a particulate, low-temperature hydroxylapatite (HA(LT)) material (OsteoGen; Impladent, Holliswood, NY). An implantable chamber model was used to determine the ability of this material to encourage bone ingrowth into channels lined with either rough-surfaced titanium or rough-surfaced plasma-sprayed hydroxylapatite. The HA(LT) material increased bone ingrowth into the titanium-lined channels comparable with that in plasma-sprayed hydroxylapatite-coated channels. It was incorporated into ingrowing bone without intervening soft tissue, with the bone bonding directly to the material surface in much the same fashion as it bonds at the plasma-sprayed hydroxylapatite surface. Mechanical testing of the ingrown bone showed no weakness because particles were incorporated. At 12 weeks, the particles began to show signs of dissolution. It was concluded that the HA(LT) material is a biocompatible, osteoconductive material that conducts bone ingrowth in much the same way as high-temperature particulate hydroxylapatite ceramics. This material has the additional desirable property of being slowly resorbable, a beneficial characteristic for many bone-filling applications.


E-test: a new technique for antimicrobial susceptibility testing for periodontal microorganisms. The purpose of the study was to validate the Epsilometer test (E-test) method for antimicrobial susceptibility testing of selected periodontopathic microorganisms using the agar dilution method as a standard. The E-test has been developed to provide a direct quantification of antimicrobial susceptibility of microorganisms. The device consists of a predefined, continuous, and exponential gradient of antibiotic concentrations immobilized along a rectangular plastic test strip. After 48 hours incubation a drop-shaped inhibition zone intersects the graded test strip at the inhibitory concentration (IC) of the antibiotic. Twenty-two subgingival plaque samples from periodontitis sites were plated on trypticase soy agar supplemented with 5% rabbit blood or 5% sheep blood and trypticase soy agar supplemented with vancomycin and bacitracin. A total of 60 strains of key periodontal pathogens (Prevotella intermedia, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Eikenella corrodens, Campylobacter rectus, and Capnocytophaga) isolated from 22 sites of 12 patients were studied. The antibiotics tested were clindamycin, metronidazole, tetracycline, ciprofloxacin, cefoxitin, and ampicillin at concentrations above and below the achieved blood or gingival crevicular fluid levels. As a standard reference the minimal inhibitory concentrations (MICs) were determined using the agar dilution method. MICs were compared with ICs determined using the E-test method. The results showed an agreement ranging from 67% to 100%; sensitivity ranging from 75% to 100%; predictability ranging from 56% to 100% and specificity ranging from 33% to 96%. The E-test ICs for ampicillin, cefoxitin, and metronidazole against the Gram-negative capnophilic and microaerophilic rods and the black-pigmented anaerobic rods ICs for ampicillin, clindamycin, metronidazole, and tetracycline showed a high percentage of agreement with the agar dilution MICS.(ABSTRACT TRUNCATED AT 250 WORDS)


Development of a rapid latex agglutination test for periodontal pathogens. The studies reported here describe the development, characterization, and initial application of latex agglutination assays for periodontal pathogens. Latex reagents were prepared by sensitization of latex microspheres with rabbit IgG antibodies to either Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, or Prevotella intermedia. The protein concentration utilized for sensitization and microsphere size were optimized. One reagent was prepared to A. actinomycetemcomitans and a second combination reagent was prepared by mixing latex sensitized with antibodies to P. gingivalis and latex sensitized with antibodies to P. intermedia. The sensitivity of both latex reagents in the traditional wet and a dried format was evaluated. In addition, sensitivity and specificity with homologous and heterologous bacterial suspensions were evaluated. The reagents were found to demonstrate both specificity and sensitivity. Initial studies with subgingival human plaque demonstrated the ability of these reagents to detect the specific organisms in plaque.


Southern blot detection of human papillomaviruses (HPVs) DNA sequences in gingival tissues. The highly sensitive and specific methods of molecular biology emphasize the frequency of subclinical infections in the genital tract tissues by the human papillomaviruses (HPVs). The purpose of this work was to investigate occult viral infections by the HPV type 6, 11, 16, and 18 in the gingival tissues. The Southern blot method with 32P-radiolabeled DNA probes applied under stringent conditions to 20 interproximal gingival papilla specimens revealed homologous viral sequences in 1 of 6 cases of adult periodontitis (HPV 16), 1 of 2 cases of rapidly progressive periodontitis (RPP) (HPV 6/HPV 11), 2 of 2 cases of acute gingivitis in psychiatric institutionalized patients (HPV 6; HPV 6/HPV 11), and 2 of 10 cases of acute gingivitis in AIDS patients (HPV 6/HPV 11/HPV 16; HPV 6). No periodontal or extra-periodontal specimen hybridized with the HPV 18 probe. Simultaneous hybridization with two or three HPV types was common (3/6 cases). The present detection of HPV 6, 11, 16 DNAs or related-DNAs in periodontal tissues without obvious clinical signs of viral infection suggests that the gingival epithelium may act as a reservoir.


Synergistic effect of pyrophosphate and sodium dodecyl sulfate on periodontal pathogens. Our previous studies have shown that pyrophosphate (PPi), the anticalculus component of tartar-control dentifrices, inhibits the growth of organisms associated with coronal and root surface caries. The purposes of this investigation were to: 1) determine if periodontal pathogens are similarly susceptible to the growth-inhibitory properties of PPi; and 2) determine if combinations of pyrophosphate-sodium dodecyl sulfate (PPi-SDS) inhibit growth synergistically. Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Eikenella corrodens, and Campylobacter rectus (formerly Wolinella recta) were cultured in appropriate enriched media under anaerobic conditions. Inhibition assays were performed in tubes containing media supplemented with PPi and/or SDS. A range of concentrations of PPi and SDS in 2-fold increments was employed, with each concentration assayed in triplicate. Minimal inhibitory concentration (MIC) analyses revealed all of the bacteria were susceptible to PPi and SDS, with MICs of 0.67% (25 mM) and 0.01% w/v respectively. Combination studies with PPi-SDS showed much greater growth inhibition against P. gingivalis and A. actinomycetemcomitans than achieved with the agents individually. Determination of fractional inhibitory concentration indices indicated a synergistic growth-inhibitory effect. Under the constraints of the conditions employed, these studies demonstrate the efficacy of PPi-SDS combinations in inhibiting the growth of periodontal pathogens. It is conceivable that these compounds may have clinical benefit as a subgingival irrigant.


A preliminary study on the effects of the Nd:YAG laser on root surfaces and subgingival microflora in vivo. The purpose of this study was to examine the effects of root preparation using the pulsed Nd:YAG laser, either alone or in combination with manual instrumentation. Study specimens consisting of 18 teeth with associated periodontal pockets from 8 different patients were treated as follows: 5 specimens were root planed with curets followed by laser exposure for 3 minutes using energy settings of 3.0 W at 20 pps; 2 specimens were root planed and then laser treated for 3 minutes using settings of 2.25 W and 20 pps; 4 specimens were treated by laser for 1 minute at settings of 1.75 W and 20 pps followed by root planing; 4 specimens were treated by laser only for 1 minute using settings of 1.75 W and 20 pps; and the remaining 3 teeth served as untreated controls. Both prior to and after completion of the laser and root planing treatments, microbiological samples were obtained from the treated pockets and submitted to a commercial laboratory for analysis of levels of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia. With the exception of two 7-day specimens, all others were extracted immediately post-therapy and processed for SEM examination. All treated specimens, regardless of treatment sequence, exhibited some degree of laser-induced root surface alteration. Notably, laser-treated calculus deposits were free of their characteristic surface layer of microbial plaque. Microbial sampling indicated a post-therapy reduction in levels of all 3 putative microbial pathogens compared to pre-treatment samples and control specimens. However, SEM examination revealed residual deposits of plaque and calculus in all treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)


Normalization of the light/dark rhythm of melatonin after prolonged subcutaneous administration of interleukin-2 in advanced small cell lung cancer patients. It has been demonstrated that antitumor immune response is an IL-2-dependent phenomenon. Moreover, experimental results suggest the existence of interactions between IL-2 and the pineal gland, which also plays a role in the control of immunity and cancer growth. Alterations of both IL-2 and melatonin secretion have been reported in cancer patients. To further investigate pineal/IL-2 relationships in humans with cancer, we evaluated the melatonin rhythm in seven advanced small cell lung cancer patients, before and at weekly intervals during immunotherapy with IL-2, given subcutaneously at a daily dose of 3 x 10(6) IU/m2 twice daily for 5 days/week for 4 weeks. Before IL-2, no patient showed a light/dark rhythm of melatonin. IL-2 administration induced a normalization of the melatonin circadian rhythm, with the appearance of a night time peak in 4/7 patients. This effect, however, disappeared with IL-2 interruption in 3/4 patients. This preliminary study, by showing that IL-2 may restore a normal melatonin rhythm, suggests that the anomalous pineal function in cancer may depend at least in part on the altered endogenous IL-2 production.


Interaction of melatonin with human lymphocytes: evidence for binding sites coupled to potentiation of cyclic AMP stimulated by vasoactive intestinal peptide and activation of cyclic GMP. Melatonin binding sites were characterized in human blood lymphocytes. The specific binding 2-[125I]iodo-melatonin ([125I]MEL) to human lymphocytes was dependent on time and temperature, stability, saturation, and reversibility. Moreover, guanine nucleotides decreased the specific binding of [125I]MEL to crude membranes of human lymphocytes, suggesting the coupling of these binding sites to a guanosine nucleotide binding regulatory protein(s). In competition studies, the specific binding of [125I]MEL to lymphocytes was inhibited by increasing concentrations of native melatonin. Scatchard analysis showed that data were compatible with the existence of two classes of binding sites: a high-affinity site with a Kd of 5.20 +/- 0.79 nM and a binding capacity of 50.6 +/- 11.0 fmol/10(7) cells, and a low-affinity site with a Kd of 208.5 +/- 50.2 nM and a binding capacity of 2691 +/- 265 fmol/10(7) cells. However, concentration-dependent binding of [125I]MEL to lymphocytes was saturable and resulted in a linear Scatchard plot, suggesting binding to a single class of binding sites. The Kd for the single site was 1.02 +/- 0.34 nM with a binding capacity of 10.1 +/- 1.6 fmol/10(7) cells. Their affinities closely correlated with the production of cyclic nucleotides, suggesting a physiological role for the melatonin binding sites. Thus, melatonin potentiated the effect of vasoactive intestinal peptide (VIP) on cyclic AMP production (ED50 = 1.9 nM) and stimulated cyclic GMP accumulation (ED50 = 125 nM). Results demonstrate the existence of two binding sites for [125I]MEL in human blood lymphocytes, with a high-affinity binding site coupled to the potentiation of the effect of VIP on cyclic AMP production and a low-affinity binding site coupled to activation of cyclic GMP production.


Effect of hygiene instrumentation on titanium abutments: a scanning electron microscopy study. Implant abutments can be exposed to a variety of oral prophylaxis procedures. In this in vitro study, titanium abutments were subjected to five oral prophylaxis treatment modalities; a metal scaler, a plastic scaler, a rubber cup, a rubber cup with tin oxide, and an air-powder abrasive. The abutment surfaces were then examined under both light and scanning electron microscopes. The metal scaler was seen to roughen the titanium surface. All other modalities tested appeared to smooth the titanium surface by removing surface debris and rounding off the sharp machined grooves present on the untreated abutment surface.


Orbital prosthesis following temporal muscle or forehead flap reconstruction: use of optics and illusions. Temporalis myocutaneous flap reconstruction of orbital defects may cause difficulty with fabrication of esthetic orbital prostheses because of limited space. Two patient treatments are presented to show the wide variation in defect size that may be encountered after surgical reconstruction. Methods for using optics and illusions to enhance the esthetic result are also presented.


Opioid receptors and their pharmacological profiles. Opioid receptors can be divided into three major classes, which are called mu, delta and kappa-receptors. The molecular basis of the receptors is discussed and a hypothesis of the binding of bivalent ligands to the receptor is presented. Furthermore the mechanism of action, the distribution and the probable function of these classes is overviewed. Increasing evidence is accumulating that the classical binding model cannot explain completely the interaction of opioids with their receptors. In addition to the mu-receptors, high affinity mu 1 binding sites have been demonstrated. Similarly, the delta receptors may be divided in delta 1 and delta 2. The significance of these subclasses is not yet fully understood. The high affinity mu 1-binding sites, however, represent probably an activated receptor complex, e.g. the complex between the receptor and the guanine-nucleotide-binding protein.


Human papillomavirus-associated lesions of the vagina and cervix. Treatment with a laser and topical 5-fluorouracil. Twenty women with cervical and vaginal human papillomavirus-associated lesions were treated with CO2 laser ablation followed by eight weekly applications of 5-fluorouracil. Viral subtyping in a majority of patients and histology were obtained before and after treatment. After treatment 88% (15 of 17) had normal vaginal biopsies, and 59% (10 of 17) had normal cervical biopsies. There were no treatment failures with subtype 6/11 infection of the cervix or vagina. All the failures were with viral subtypes 16/18 and 31/35/51. The protocol was effective in treating patients with cervical and vaginal human papillomavirus-associated lesions.


Extragenital metastases to uterine leiomyomata. A case report. Malignant extragenital neoplasms with metastases to the uterus are not common, and involvement of a uterine leiomyoma by an extragenital tumor is rare. A case of an infiltrating ductal carcinoma of the breast occurred, with widespread metastases that included a uterine myoma. This entity can be confused with a bizarre or symplastic leiomyoma.


Remission rates and significance of prognostic factors in gestational trophoblastic tumors. The clinical courses of 82 women with gestational trophoblastic tumors were evaluated in accordance with International Federation of Gynecologists and Obstetricians staging, traditional criteria and the modified World Health Organization prognostic scoring system. The overall remission rate was 79.3% (65/82), and the mortality rate was 20.7% (17/82). All the patients with nonmetastatic stage I disease and all with prognostic scores less than or equal to 4 attained remission, whereas patients at high risk and with stage IV disease had the lowest remission and survival rates. The results support the validity of each system. However, the World Health Organization scoring system, a combination of traditional criteria and International Federation of Gynecologists and Obstetricians staging seemed to be the most reliable for predicting treatment failures.


Conformational preferences of the kappa-selective opioid agonist U50488. A combined molecular mechanics and nuclear magnetic resonance study. The conformational preferences of the kappa-selective opioid agonist U50488 have been studied using MM2-87 calculations and nuclear magnetic resonance (NMR) spectroscopy. The calculations were performed for the protonated form with a dielectric constant of 80 and the unprotonated form with dielectric constants of 1.5 and 80. A systematic search found 72 stable conformers with certain consistent conformational preferences for some of the important dihedral angles. The preferred conformers proved to be compact structures stabilized by intramolecular attractive van der Waals interactions, though at least some of these appear to be electrostatically unfavorable. The conformation of U50488 was also examined in aqueous solution using one-dimensional (1D) and two-dimensional (2D) high-resolution 1H NMR techniques such as the interpretation of 1H-1H vicinal coupling constants, 1D and 2D nuclear Overhauser effect (NOE) experiments, and 2D correlated spectroscopy (COSY) experiments. Five crystallographic conformations were examined as well. There was generally good agreement between all three methods of conformational analysis. There appeared to be a reasonable geometrical agreement between the relatively rigid kappa-agonist (-)-ketazocine and a gauche conformer of U50488. The proposed pharmacophore is also consistent with other kappa-selective analogs of U50488 including one in which the peptide bond is incorporated into a lactam ring. The low affinity of U50488 for mu-receptors was attributed to its cyclohexane ring which occupies space not present in the nonselective (-)-ketazocine.


Omega-[(omega-arylalkyl)aryl]alkanoic acids: a new class of specific LTA4 hydrolase inhibitors. The synthesis and structure-activity profile of a new class of potent and specific LTA4 hydrolase inhibitors are described. Many compounds of this series of omega-[5-(omega-arylalkyl)-2-thienyl]- and omega-[4-(omega-arylalkyl)phenyl]alkanoic acids were found to be potent in vitro inhibitors of the LTB4 production by porcine leukocytes with IC50 ranging from 1 to 10 microM. The side-chain lengths were critical for an optimal activity. Substitutions on the terminal aromatic ring, in the benzene series, by lipophilic and electron-donating substituents substantially enhanced the LTA4 hydrolase inhibition potency. On the other hand, in the thiophene series, the effect of of such substitutions on the LTA4 hydrolase inhibition was rather small. Functionalization within the carboxylic acid side chain by a carbonyl or by a hydroxyl group led to less potent compounds. A metabolically stable LTA4 hydrolase inhibitor, RP64966, was obtained by insertion of an oxygen atom in the beta-position on the carboxylic acid side chain. After oral administration of RP64966 to rats, a plasma extract was found to display potent inhibition of the LTB4 biosynthesis (40% inhibition at 5 mg/kg, po).


Omega-[(omega-arylalkyl)thienyl]alkanoic acids: from specific LTA4 hydrolase inhibitors to LTB4 receptor antagonists. A series of omega-[(omega-arylalkyl)thienyl]alkanoic acid isomers was prepared and a structure-activity relationship was investigated. These compounds have displayed either LTA4 hydrolase inhibition activities or LTB4 receptor binding activities, or both, depending on the relative orientation of the two side chains on the thiophene ring. Whereas the 2,5-isomers specifically exhibited LTA4 hydrolase inhibition, 3,5-isomers displayed both activities. On the other hand, the "ortho-isomers" specifically inhibited the binding of the LTB4 to its receptor. The side-chain lengths were also important for an optimal inhibition or binding activity. Substitutions on the terminal aromatic ring or on the thiophene nucleus led to small changes in both activities. The most dramatic effect was obtained by substituting the carboxylic acid side chain in the alpha-position with one or two methyl groups, which substantially enhanced the LTB4 receptor binding activity. In the most favorable case, the alpha,alpha-dimethyl derivative RP66153 was found 20-fold more potent than its linear counterpart.


Benzothiazole hydroxy ureas as inhibitors of 5-lipoxygenase: use of the hydroxyurea moiety as a replacement for hydroxamic acid. A novel series of N-[(2-benzothiazolylthio)alkyl]-N'-hydroxyurea derivatives (9-25) was synthesized and evaluated for biological activity as inhibitors of 5-lipoxygenase both in vivo (mouse zymosan peritonitis assay) and in vitro (Ca2+ ionophore-stimulated human peripheral blood leukocyte model). The compounds of this series were based on the corresponding hydroxamic acid derivatives (1, 3, 4, and 5) which were moderately active in vitro but inactive in vivo. A number of compounds in the hydroxyurea series exhibited oral activity for 5-lipoxygenase inhibition. Results of studies relating structure to in vivo and in vitro 5-lipoxygenase activity are reported.


Antiviral and antitumor structure-activity relationship studies on tetracyclic eudistomines. The in vitro antiviral and antitumor activities of (-)-debromoeudistomin K (1a) and 10 structural analogues (1b-1j and 11) were evaluated. The synthesis was accomplished with an intramolecular Pictet-Spengler condensation reaction as the key step. This examination revealed some structural and stereochemical features that are important for both the antiviral and antitumor activities. The most striking points for activity are the necessity to have the correct natural stereochemistry at both C(1) and C(13b) and the presence of the C(1)-NH2 substituent. As was revealed before with naturally isolated eudistomins a substituent in the indole ring greatly influences the biological activity. The 5-OMe derivative 1h shows high potency in both antiviral and antitumor models.


New vitamin D3 derivatives with unexpected antiproliferative activity: 1-(hydroxymethyl)-25-hydroxyvitamin D3 homologs. Surprisingly, both of the synthetic 1-(hydroxymethyl)-25-hydroxyvitamin D3 diastereomers (-)-2 and (+)-3 retained the antiproliferative activity of natural calcitriol in murine keratinocytes. Preliminary studies indicated, however, that both of these synthetic diastereomers were less than 0.1% as effective as calcitriol for binding to the 1,25-(OH)2-D3 receptor and that they were less than 0.1% as potent as calcitriol for calbindin-D28K induction in organ-cultured embryonic chick duodenum. 1-(Hydroxymethyl)-25-hydroxyvitamin D3 homologs (-)-2 and (+)-3 were synthesized in a convergent manner by combining enantiomerically pure C,D-ring ketone 12 with highly enantiomerically enriched A-ring phosphine oxides (-)-11a and (+)-11b. These A-ring chirons were prepared starting from thermal [2 + 4] cycloaddition of 3-bromo-2-pyrone and acrolein.


4-Methyl-3-(arylsulfonyl)furoxans: a new class of potent inhibitors of platelet aggregation. A series of 4-methyl-3-(arylthio)furoxans were synthesized by oxidation of 1-(arylthio)-2-methylglyoxymes with dinitrogen tetroxide. Reduction with trimethyl phosphite of the furoxan derivatives afforded the corresponding furazans, while oxidation with an equimolar amount of 30% hydrogen peroxide in acetic acid or with an excess of 81% hydrogen peroxide in trifluoroacetic acid afforded the corresponding arylsulfinyl and arylsulfonyl analogues, respectively. All the furoxan and furazan derivatives showed activity as inhibitors of platelet aggregation. 4-Methyl-3-(arylsulfonyl)furoxans were the most potent derivatives of the series. 4-Methyl-3-(phenylsulfonyl)furoxan (10a), one of the most active derivatives, inhibits the AA-induced increase of cytosolic free Ca2+ and production of malondialdehyde. A primary action of the compound on cyclooxygenase is excluded, as a stable epoxymethano analogue of prostaglandin H2 does not reverse the inhibitory effect of 10a. This compound produces a significant increase in cGMP which is likely to cause inhibition at an early stage of the platelet activation pathway.


Positive regulation of the expression of the Escherichia coli pts operon. Identification of the regulatory regions. The pts operon of Escherichia coli is composed of the ptsH, ptsI and crr genes coding for three proteins central to the phosphoenolpyruvate dependent phosphotransferase system (PTS), the HPr, enzyme I and EIIIGlc proteins, respectively. We previously showed that transcription from the promoter region located upstream from the pts operon is regulated by two control circuits, which can occur independently from each other. Transcription of the pts operon is (1) stimulated by the CAP-cAMP complex and (2) enhanced during growth on glucose, a PTS substrate. The DNA regions involved in regulation of the expression of the pts operon have been identified. Two promoters, P0 and P1, separated by 100 bp are located upstream from the pts operon. In these promoter regions, we identified two sequences showing similarity with the consensus of CAP-binding sites, CAPa located near P0 and CAPb located in the -35 region of P1. In vivo experiments showed that binding of CAP-cAMP at the CAPa site stimulates transcription from the P0 promoter. The binding sites of CAP-cAMP and/or RNA-polymerase on a DNA fragment containing both P0 and P1 promoters as well as both CAPa and CAPb sites were examined by the technique of DNase I footprinting. These in vitro experiments suggested that CAP-cAMP binding at the CAPb site might also play a role in regulation of the pts operon expression. In addition, we showed that the DNA region carrying the CAPa site is important for regulation by glucose. We finally propose that the expression of the pts operon is controlled by two alternative positive regulatory mechanisms, which are designed to allow activation of the pts operon under a great variety of growth conditions.