Modulation of endogenous ADP-ribosylation in rat brain. Endogenous ADP-ribosylation of proteins was measured in homogenates, membranes, and cytosol from rat brain regions. Several proteins were ADP-ribosylated in homogenates, especially a 49 kDa protein. Sodium nitroprusside, a source of nitric oxide, particularly enhanced the ADP-ribosylation of 47 kDa and 39 kDa proteins. Levels of basal and sodium nitroprusside-stimulated ADP-ribosylated proteins were similar, but not identical, in homogenates from the cerebral cortex, hippocampus, striatum, thalamus and cerebellum. In neonatal cerebral cortex, ADP-ribosylation of an additional 110 kDa protein was detected and this was also enhanced by sodium nitroprusside. ADP-ribosylation of the 110 kDa protein was evident one and two days after birth, but not at five days and later. Each protein demonstrated unique sensitivities to sodium nitroprusside and rates of ADP-ribosylation. Cyclic GMP did not mimic the effects of sodium nitroprusside. Mg2+ inhibited ADP-ribosylation of the 49 kDa and 47 kDa proteins but had a smaller effect on the 39 kDa protein. ADP-ribosylation in the cytosol predominantly affected only a single protein of 39 kDa, and this was stimulated by sodium nitroprusside and by addition of cofactors necessary for activation of nitric oxide synthase. Several proteins in membranes were ADP-ribosylated and the 49 and 47 kDa proteins were released from the membranes coincidentally with ADP-ribosylation. The predominate substrates of endogenous ADP-ribosylation did not appear to be substrates for pertussis toxin-induced ADP-ribosylation. These and previously published results indicate that nitric oxide generated from sodium nitroprusside or endogenous sources may have modulatory effects through regulation of the endogenous ADP-ribosylation of proteins.


Distinct distributions of two bombesin receptor subtypes in the rat central nervous system. We have previously demonstrated the presence of two distinct bombesin receptor subtypes in the rat CNS and distinguished them as bombesin/gastrin-releasing peptide (BBS/GRP) and neuromedin B (NMB)-preferring binding sites. In the present study, we conducted a complete evaluation of the distribution of these binding sites throughout the rat brain using in vitro receptor autoradiography. The BBS/GRP-preferring binding sites were characterized as those that bound 125I-(Tyr4)BBS but not 125I-(D-Tyr0)NMB. At these sites 125I-(Tyr4)BBS binding was inhibited in the presence of 100 nM BBS but not by the same concentration of NMB. In contrast, NMB-preferring sites bound both radioligands and binding at these sites was inhibited in the presence of 100 nM NMB. Our results indicate that the distributions of BBS/GRP and NMB-preferring binding sites are widespread and distinct at all levels of the rat brain suggesting these peptides mediate separate functions in the rat central nervous system.


Brain Na+/K(+)-ATPase regulation by serotonin and norepinephrine in normal and kindled rats. In this work we confirmed the activation of rat brain Na+/K(+)-ATPase by norepinephrine (NE) and observed a variable response of the enzyme according to the brain region considered. In isolated neuronal or glial fractions from normal cerebral cortices, we studied the response of the enzyme to increasing concentrations of serotonin (5-HT) (10(-9)-10(-3) M). A dose-dependent response over basal values was present in glial fractions, beginning at 10(-6) M. No such response was obtained in the neuronal fractions. In amygdaloid kindled brains, the pattern of activation by NE was different than in controls: less pronounced (cortex, brainstem, and diencephalon), inhibition-activation (cerebellum), or no change (striatum). The activation of Na+/K(+)-ATPase by 5-HT observed in the control glial fraction was not present in the kindled glial fraction. In conclusion, 5-HT seems to activate Na+/K(+)-ATPase preferentially in glial cells, and the kindling process markedly modifies this regulation. The normal response to NE in brain homogenates is less altered by kindling than is the response to 5-HT in the same regions.


Asymmetric distribution of melatonin receptors in the brain of the lizard Anolis carolinensis. The pineal hormone melatonin may regulate seasonal reproduction and entrainment of circadian rhythms by binding to specific brain receptors. An analysis of melatonin receptor distribution in the lizard brain revealed an asymmetry of melatonin binding in the diencephalon. A high degree of melatonin binding was present in the left habenular nucleus, but no binding was observed in the habenulum of the right brain hemisphere. It is intriguing that the left habenular nucleus, in contrast to the right habenulum, both possesses a high density of melatonin receptors and receives primary photic input from the parietal eye. Similarly, the optic tectum, which receives primary visual input from the retina, is also rich in melatonin receptors. These observations suggest that the left habenulum is under dual control (neuronal and hormonal) of the parietal eye/pineal complex, and that melatonin may play a significant role in neural processing of visual information.


Quantitative autoradiographic analysis of muscarinic cholinergic and GABAA (benzodiazepine) receptors in the forebrain of rats flown on the Soviet Biosatellite COSMOS 2044. The quantitative autoradiographic analysis of muscarinic cholinergic and GABAA (benzodiazepine) receptors was performed on selected regions of the cerebral cortex and striatum of rats flown in the Soviet Biosatellite COSMOS 2044. An age- and strain-matched synchronous ground-based control group was employed for comparison. Muscarinic cholinergic receptor density was found to be significantly lower in the striatum of the flight animals as compared with that in the synchronous control group. No significant differences between flight and synchronous control groups were found in the other regions examined. GABAA (benzodiazepine) receptors showed no significant differences between the flight and control groups in any of the regions sampled. Although additional studies are needed to reach definitive conclusions, the decrease in muscarinic cholinergic receptors observed in the striatum suggests spaceflight-related alterations in motor activity.


Human Y-79 retinoblastoma cells express functional corticotropin-releasing hormone receptors. In human Y-79 retinoblastoma cells corticotropin-releasing hormone (CRH) produces a marked and rapid increase of adenylate cyclase activity. The concentration of the peptide producing half-maximal stimulation is 60 nM. The effect of CRH is significantly antagonized by the specific CRH receptor antagonist alpha-helical CHR 9-41 and is mimicked by sauvagine and urotensin I, two peptides displaying sequence homology with CRH. These results demonstrate the presence of functional CRH receptors in human Y-79 retinoblastoma cells and suggest that this cell line may be a suitable model in which to study the action of CRH on human retinal cell function.


cAMP-mediated expression of inwardly rectifying potassium channels in cultured mouse Schwann cells. Voltage-gated ionic currents were recorded from freshly dissociated or cultured mouse Schwann cells obtained from neonatal sciatic nerves by the whole-cell variation of the patch-clamp technique. Schwann cells virtually lost inwardly rectifying potassium (Kir) currents within 2 days after nerve transection or in culture conditions of neonatal sciatic nerves confirming the previous results that axonal signals were suggested to play an important role in the expression of functional Kir channels. To see the effects of adenosine 3',5'-monophosphate (cAMP) analogues or forskolin on the expression of Kir channels in cultured Schwann cells, these agents were added to the culture medium 4 days after the start of the culture, when Kir currents were almost eliminated from cultured Schwann cells. Cultured Schwann cells restored the expression of Kir currents by co-culture with agents which elevate intracellular cAMP level. The dose-response of 8-(4-chlorophenylthio) (CPT) cAMP for the incidence of the expression of Kir currents showed a steep increase in the percentage of cells with Kir currents between 0.02 and 0.1 mM of external CPT cAMP and approximately two thirds of cells had Kir currents in higher concentrations of more than 0.1 mM of CPT cAMP after 4 days of incubation. After removal of CPT cAMP from the culture media after 4 days of incubation, Kir currents disappeared from cells within 2 days. The simultaneous application of cycloheximide (1 microgram/ml), an inhibitor of protein synthesis, with CPT cAMP suppressed the expression of Kir currents for up to 6 days of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)


[Selective opioid agonists and inhibitors of enkephalin degradation enzymes: pharmacological and clinical values]. A recently developed series of highly selective and systemically active delta-agonists such as Tyr-X-Gly-Phe-Leu-Thr(OtBu), with X = D-Ser (OtBu) in BUBU and X = D-Cys(OtBu) in BUBUC, and complete inhibitors of enkephalin metabolism (Kelatorphan, RB 38A, RB 101) have enabled the major role played by mu-opioid receptors in supraspinal analgesia to be demonstrated. This is in agreement with the results of in vivo mu-receptor occupancy measured by taking into account the cross-reactivity of the delta-ligand for mu-sites. In contrast mu and delta binding sites seem to act independently to control pain at the spinal level. Strong analgesic effects can also be obtained by complete protection of tonically or phasically released endogenous enkephalins with mixed inhibitors. Chronic i.c.v. administration of the mu agonist DAMGO, led to a severe naloxone precipitated withdrawal syndrome whilst a weak dependence was seen with the delta agonist, DSTBULET or with RB 38A and none after repeated i.p. injection of RB 101, a systemically active mixed inhibitor. Moreover, chronic administration of RB 101 did not induce antinociceptive tolerance, a major side effect observed during chronic administration of opiates. These differences could be related to a more efficient and selective stimulation of opioid receptors by the endogenous enkephalins. This suggest that the large changes in receptor density, adenylate cyclase activity or phosphorylation of proteins following chronic morphine treatment is not significantly triggered by occupation of the opioid receptors by their natural ligands. All these data emphasize the interest in developing delta-agonists and mixed inhibitors with appropriate bioavailability for clinical evaluation.


Factors predicting the acute effect of pamidronate on serum calcium in hypercalcemia of malignancy. In this study we retrospectively reviewed results of the first 9 days of treatment with pamidronate at doses of 30 mg (n = 13), 45 mg (n = 9), and 90 mg (n = 13) in an attempt to see what factors influenced the response of serum calcium to pamidronate. The nadir of serum calcium obtained post treatment was correlated with pretreatment levels of nephrogenous cyclic adenosine monophosphate (NcAMP), the renal tubular threshold for phosphate reabsorption (TmPO4), and the renal tubular threshold for calcium reabsorption (TmCa). Using the post treatment serum calcium levels, patients were divided into "good" and "poor" responders depending on whether a normal serum calcium was obtained. Pretreatment NcAMP was significantly correlated with the magnitude of the response of serum calcium (r = 0.45, P = 0.0001). Pretreatment NcAMP was significantly higher in the poor responders (mean +/- SEM): 65.0 +/- 9.4 nmol/liter GF (poor responders) versus 29.6 +/- 6.3 (good responders), P = 0.004. NcAMP as a predictor of the acute response of serum calcium showed a sensitivity of 93% and a specificity of 72%. Pretreatment TmPO4 was negatively correlated with the serum calcium response post treatment (r = -0.41, P = 0.003). However, though TmPO4 tended to be lower in the poor responders, this was not statistically significant [0.65 mmol/liter GF +/- 0.09 (poor responders) versus 0.76 mmol/liter GF +/- 0.06 (good responders)]. As a predictor of the acute response of serum calcium, TmPO4 was less good with a sensitivity of 70% and specificity of 58%. No significant correlation was present between TmCa and the serum calcium response.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibition of cardiac sarcolemmal Na+/H+ antiporter by opioids. In our routine screening of chemicals that would inhibit cardiac sarcolemmal Na+/H+ antiporter, we discovered that some of the opioids produced inhibition of cardiac sarcolemmal Na+/H+ antiporter in micromolar concentrations. Using U-50,488H, a selective kappa-opioid agonist, we characterized the nature of interaction between opioids and the Na+/H+ antiporter. The inhibitory effect of U-50,488H on Na+/H+ antiporter was immediate and reversible, and was not mediated through the interaction with the opioid receptors but due to the direct interaction of U-50,488H with the Na+/H+ antiporter. The kinetic data show that in the presence of U-50,488H the Km for Na+ was increased from 2.5 +/- 0.2 to 5.0 +/- 0.3 mM, while the Vmax (52.0 +/- 5.0 nmol.mg-1.min-1) remained the same. These results suggest that U-50,488H and Na+ compete for the same site on the antiporter. When testing the effect of U-50,488H on other transport systems of cardiac sarcolemma, we found that U-50,488H also inhibited Na+/Ca2+ antiporter and Na+/K+ pump but at much higher concentrations suggesting that U-50,488H shows some degree of selectivity for cardiac sarcolemmal Na+/H+ antiporter. When we compared the inhibitory potency of U-50,488H with amiloride and its analog, namely 5-(N,N-hexamethylene)amiloride, we found that U-50,488H (IC50 = 100 +/- 15 microM) was threefold more potent than amiloride (IC50 = 300 +/- 20 microM) but it was three-fold less potent than the amiloride analog (IC50 = 30 +/- 10 microM) in inhibiting cardiac sarcolemmal Na+/H+ antiporter. These results show that although U-50,488H is more potent than amiloride, the inhibitory characteristics of U-50,488H on cardiac sarcolemmal Na+/H+ antiporter are similar to amiloride.


Vascular actions of thrombin receptor peptide. We have examined the action of the thrombin receptor-derived polypeptide, S42FLLRNPNDKYEPF55 (TRP 42-55), in rat and guinea pig aortic rings and helical arterial strips, and we have compared the actions of the peptide with those of thrombin. In rat preparations, both TRP 42-55 and thrombin caused a concentration-dependent endothelium-dependent relaxation that was blocked by N omega-nitro-L-arginine methyl ester; the relaxation response of the intact rat aortic strip preparation to concentrations of the peptide in the range 30-60 micrograms/mL (17-34 microM) was equivalent to the response to 0.03-0.1 U/mL of thrombin (about 0.3-0.9 nM), yielding a potency ratio (TRP 42-55:thrombin) of about 38,000:1. In contrast with the complete desensitization of thrombin-treated rat aortic preparations to a second administration of the enzyme, the rat aortic tissue was not desensitized by repeated exposures to TRP 42-55 and remained responsive to the peptide even after treatment of the tissue by thrombin. In contrast with the rat aortic tissue, in either intact or endothelium-free guinea pig aortic preparations both TRP 42-55 and thrombin caused a concentration-dependent endothelium-independent contraction. The contractile action of 60 micrograms/mL of receptor peptide (34 microM) in guinea pig aortic strip preparations was equivalent to the contractile action of 0.1-0.3 U/mL thrombin (0.9-3 nM), yielding a potency ratio of about 17,000:1. In guinea pig aortic preparations with an intact endothelium that were precontracted with noradrenaline, neither thrombin nor TRP42-55 caused relaxation, whereas substance P did so.(ABSTRACT TRUNCATED AT 250 WORDS)


Malignant fibrous histiocytoma coexistent with mucinous cystadenoma of the pancreas. A patient with malignant fibrous histiocytoma (MFH) coexistent with mucinous cystadenoma of the pancreas is reported. Primary MFH of the pancreas is rare, with only six patients reported in the world medical literature. A patient with MFH coexistent with a pancreatic mucinous epithelial tumor has not been reported previously, although two patients with pseudosarcomatous tumor associated with mucinous cystadenocarcinoma of the pancreas have been reported. Mural nodules of similar histologic appearance have been reported in ovarian mucinous tumors. The authors believe this to be the first report of the occurrence of MFH in mucinous cystadenoma of the pancreas.


Midline malignant B-cell lymphoma with leukemic transformation. This article presents a case of lethal midline granuloma on the palate of a 44-year-old woman, which had been identified histologically as B-cell lymphoma with leukemic transformation in the terminal stages. At the first visit, physical and laboratory examinations showed no remarkable findings except for a necrotizing ulcer of the palate, and the biopsy specimens only showed massive inflammatory cell infiltration and necrosis of the granulation tissue. There was a short-term resolution after treatment with cyclophosphamide and prednisolone, but the disease reactivated and the necrotic ulcerative lesion progressively advanced into the nasal cavity. Specimens from the third biopsy exhibited histologic features that were consistent with malignant lymphoma of the diffuse, mixed B-cell type. Chemotherapy with the regimen of cyclophosphamide, doxorubicin, vincristine, and prednisolone was adopted but was interrupted after a short time because of bone marrow suppression. Subsequently, large numbers of enlarged abnormal lymphocytes with a few vacuoles in the cytoplasm appeared in the circulating blood, indicating leukemic transformation of the midline lymphoma. The patient died on the seventh day after the initiation of chemotherapy. In the presentation of this case, the authors mention clinically important matters regarding midline lethal lymphoma and briefly discuss the pathophysiology and pathogenesis.


Peripheral T-cell lymphoma involving the placenta. Placental involvement by maternal malignant lymphoma is a rare complication of pregnancy. The case of a 29-year-old woman found to have placental involvement by peripheral T-cell lymphoma (PTCL) diagnosed by morphologic study, immunoperoxidase stains, and molecular genetic studies is reported. The lymphoma was pathologic Stage IVB and although there were no adverse consequences for the child, but the neoplasm progressed rapidly during the postpartum period. The clinical course suggested that the immunologic changes of the pregnancy exerted a lymphoma-suppressive effect on the lymphoma. The authors know of no other report of a patient with placental involvement by PTCL.


Anti-phosphoinositide auto-antibodies in sera of cancer patients: isotypic and immunochemical characterization. High levels of circulating anti-phosphatidylinositol (PtdIns) auto-antibodies (auto-Ab) have been previously found in sera of patients with malignant tumors. Using our ELISA test, a high statistical level of immunological binding directed against PtdIns was found in a large series of sera from patients with proliferative pathologies. In contrast to tumor markers, anti-PtdIns auto-Ab did not vary whatever the histological grades, TMN classification and patient's age. These anti-PtdIns auto-Ab were immunoglobulins (Ig) of G class. Their specificity was evaluated by competition experiments in ELISA and found to be rather high. An increase of these circulating auto-Ab reflected possible disturbances in PtdIns turnover and appearance of endogenous neo-antigen with PtdIns structure.


Abnormal chloride conductance in multidrug resistant HL60/AR cells. Chloride channel currents were measured in drug sensitive parental HL60 and multidrug resistant (MDR) subline HL60/AR cells, using a whole cell patch-clamp technique. In addition, the in vitro effects of 4,4' diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a Cl- channel blocker, on intracellular accumulation and sensitivity to daunorubicin and intracellular pH (pH(i)) in HL60 cells were examined. Baseline DIDS blockable Cl- currents were consistently lower in HL60/AR cells (0.9 pA/pF) as compared to HL60 cells (7.0 pA/pF). Similarly cAMP-activated Cl- currents were minimal in HL60/AR cells (0.2 pA/pF) as compared to HL60 cells (8 pA/pF). In vitro treatment of drug sensitive HL60 cells with DIDS resulted in concentration-dependent decreased accumulation and increased resistance to daunorubicin and decreased pH(i). These data show that altered Cl- permeability is associated with MDR and suggest that Cl- channels may play a role in MDR.


Aberrant expression of blood group H-antigen in Shope carcinoma cells. Blood group H-antigen was clearly demonstrated as glycolipids in Shope carcinoma cells and its culture cell lines by thin-layer chromatography immunostaining and immunohistochemical analyses using the anti-H monoclonal antibody. This antigen was not detected in normal epidermal and Shope papilloma cells of rabbits but was present in the outer sheath cells of hair follicles of normal rabbit skin. The H-antigen was also detected by cytofluorometric analysis on Shope carcinoma cell lines and the cells were lysed in vitro by the antibody and complement in a dose-dependent manner. These results suggested that the H-antigen was aberrantly synthesized in epidermal carcinoma cells.


Incessant automatic ventricular tachycardia complicating acute coxsackie B myocarditis. A 13-year-old girl presented with incessant ventricular tachycardia complicating acute Coxsackie B3 myocarditis. Electrophysiologic assessment revealed that the tachycardia could not be terminated, overdrive suppressed or accelerated by programmed electrical stimulation, but was transiently slowed by intravenous adenosine triphosphate and had marked spontaneous and sympathoautonomic-mediated fluctuation in the tachycardia cycle length. These features were atypical of reentry and triggered automaticity and suggested that abnormal automaticity was the likely tachycardia mechanism. Intravenous amiodarone slowed the ventricular tachycardia, but the patient eventually succumbed from rapidly progressive left ventricular failure. Postmortem pathohistologic examination confirmed the diagnosis of acute myocarditis.


Re-expression of alpha skeletal actin and regulation of alpha 1 adrenoceptor signalling in DOCA-salt hypertension in rats. To determine the effects of increased sympathetic nervous system activity in humoral hypertension on the regulation of surface alpha 1 adrenoceptors and signal transduction, deoxycorticosterone acetate (DOCA) salt hypertension was induced in rats and the animals killed three weeks following the initiation of hypertension. Experiments were performed on male Sprague-Dawley rats, weighing 250-300 g, divided into two groups: DOCA-salt (n = 75), and control (n = 60). Radioligand binding studies of the alpha 1 adrenoceptors, noradrenaline stimulated phosphoinositol turnover, ADP ribosylation of 41 kD substrate by pertussis toxin, and myocardial noradrenaline content were measured in the ventricular myocardium. The expression of sarcomeric actin isoforms was examined by northern blot and hybridisation with specific oligonucleotide probes. The density of alpha 1 adrenoceptors was decreased by 51% in DOCA-salt treated rats. However, noradrenaline stimulated phosphoinositol turnover in myocytes from DOCA-salt hearts was not diminished. The relative quantities of pertussis toxin labelled substrates did not differ in experimental and control hearts. Myocardial noradrenaline content was reduced by 60% in DOCA-salt hearts. Northern blots on RNA extracted from hypertrophic hearts of DOCA-salt treated rats showed an upregulation of alpha skeletal actin. The adrenergic state present in short term DOCA-salt hypertensive hypertrophy is characterised by enhanced signal transduction via the alpha 1 adrenoceptors and the re-expression of alpha skeletal actin in enlarging myocytes.


The effects of ageing on the morphology and function of the zonae fasciculata and reticularis of the rat adrenal cortex. The morphological counterpart of the well-known age-dependent marked impairment of glucocorticoid secretion of rat adrenals was investigated by use of morphometric techniques. For this purpose 4-, 8-, 16- and 24-month-old rats were studied. Despite the notable lowering of both basal and ACTH-stimulated production of corticosterone by collagenase-dispersed inner adrenocortical cells, ACTH and corticosterone plasma concentrations displayed significant increases with ageing. Zona fasciculata (ZF) and zona reticularis (ZR) showed a notable hypertrophy in aged rats, which was due to rises in both the average volume and number of their parenchymal cells. The hypertrophy of ZF and ZR cells was in turn associated with increase in the volume of the mitochondrial compartment and proliferation of smooth endoplasmic reticulum, i.e., the two organelles involved in steroid-hormone synthesis. All these morphologic changes, conceivably due to the chronic exposure to high levels of circulating ACTH, are interpreted as a response enabling ZF and ZR to compensate for their age-dependent lowering in glucocorticoid secretion. Stereology also demonstrated that ZF and ZR cells underwent a striking age-related lipid-droplet repletion. Lipid droplets are the intracellular stores of cholesterol esters, the obligate precursors of steroid hormones in rats. This finding is in keeping with the contention that the mechanism underlying the age-dependent decline in rat-adrenal glucocorticoid secretion mainly involves impairments of the utilization of intracellular cholesterol previous to its intramitochondrial transformation to pregnenolone.


Augmentation of phorbol ester-induced T cell proliferation by agents which raise intracellular cyclic adenosine monophosphate. Although raising intracellular cyclic adenosine monophosphate (cAMP) levels is generally considered to be inhibitory on the mitogen-induced T cell proliferation, in this study we have shown that the addition of either dbcAMP (50 microM) or cholera toxin (1 ng/ml) resulted in an increase in [3H]thymidine uptake in PBMC cultures stimulated with phorbol ester, 12-tetradecanoylphorbol 13-acetate (TPA), or with a combination of TPA plus anti-CD3 mAb (mAb 235). In contrast, under similar culture conditions, the phytohemagglutinin-P (PHA-P) response was inhibited by these agents as has been reported. The augmentative effect of dbcAMP in PBMC cultures was due to an increase in IL-2 production and not to increased in IL-2R-alpha chain expression. The enhancing effect of dbcAMP and CT observed with PBMC was monocyte dependent and not seen with purified T cell preparations. The addition of monocytes reconstituted the ability of intracellular cAMP elevating agents to augment the T cell response to TPA with and without mAb to CD3. The monocytes mediate their action via soluble factor(s) with molecular weight (m.w.) of more than 10 kDa. Neither rIL-1, rIL-6, nor rTNF-alpha have any augmentative effect as contrast with the supernatant from treated monocytes. Taken together, our results indicate that cAMP can play a positive regulatory role in T cell proliferation due to factor(s) secreted by dbcAMP-treated monocytes resulting in increased IL-2 synthesis in T cells.


Cap100, a novel phosphatidylinositol 4,5-bisphosphate-regulated protein that caps actin filaments but does not nucleate actin assembly. The fast and transient polymerization of actin in nonmuscle cells after stimulation with chemoattractants requires strong nucleation activities but also components that inhibit this process in resting cells. In this paper, we describe the purification and characterization of a new actin-binding protein from Dictyostelium discoideum that exhibited strong F-actin capping activity but did not nucleate actin assembly independently of the Ca2+ concentration. These properties led at physiological salt conditions to an inhibition of actin polymerization at a molar ratio of capping protein to actin below 1:1,000. The protein is a monomer, with a molecular mass of approximately 100 kDa, and is present in growing and in developing amoebae. Based on its F-actin capping function and its apparent molecular weight, we designated this monomeric protein cap100. As shown by dilution-induced depolymerization and by elongation assays, cap100 capped the barbed ends of actin filaments and did not sever F-actin. In agreement with its capping activity, cap100 increased the critical concentration for actin polymerization. In excitation or emission scans of pyrene-labeled G-actin, the fluorescence was increased in the presence of cap100. This suggests a G-actin binding activity for cap100. The capping activity could be completely inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and bound cap100 could be removed by PIP2. The inhibition by phosphatidylinositol and the Ca(2+)-independent down-regulation of spontaneous actin polymerization indicate that cap100 plays a role in balancing the G- and F-actin pools of a resting cell. In the cytoplasm, the equilibrium would be shifted towards G-actin, but, below the membrane where F-actin is required, this activity would be inhibited by PIP2.


Behavior of Dictyostelium amoebae is regulated primarily by the temporal dynamic of the natural cAMP wave. The instantaneous velocity plots of Dictyostelium discoideum amoebae responding to natural waves and simulated temporal waves of cAMP with periods of 7 min are highly similar. This similarity has been used to deduce the dynamics of a natural wave crossing an amoeba, and the behavior of amoebae has been characterized during the different phases of a natural wave with a computer-assisted dynamic image analyzing system. During the first approximately 150 sec of the front of a natural wave, cells move persistently toward the aggregation center, with high instantaneous velocity and a decreased frequency of lateral pseudopod formation. During the last 30 sec of the front of the wave and the first 30 sec of the back of the wave, there is a "freeze" in cell shape and a dramatic depression in cell motility, pseudopod formation, and intracellular particle movement. During the last 180 sec of the back of the wave, there is a rebound in pseudopod formation, but it is random in direction and leads to no net cellular translocation. The data suggest that all of the behavior of a cell but orientation during the translocation phase is mediated by the temporal dynamics of the wave. The data also suggest that orientation toward the aggregation center occurs early in the front of the wave and that, once oriented, cells move in a blind fashion during the translocation phase.


Relationships between various uses of antineoplastic drug-interaction terms. In in vitro testing, no pharmacologic synergism has been found for the combination of cisplatin and etoposide in P388 leukemia in contrast to the demonstration of therapeutic synergism in the same model. No pharmacologic synergism has been found for the same combination in the treatment of four small-cell lung-cancer cell lines, although clinical results obtained using this combination in small-cell lung cancer and other cancers suggest a therapeutic advantage. The popular concept of synergy, implying a therapeutic advantage, is different from the pharmacologic meaning, which generally implies that less drug is required in a combination for an equal effect. Therapeutic advantage may be obtained regardless of whether drugs are synergistic in the pharmacologic sense in the treatment of a tumor. To gain a more comprehensive insight into concepts of drug interaction, it is important to recognize that the type of drug interaction seen is dependent on the drug doses used and may vary with the treatment of different cell lines. All of these factors complicate the use of the word synergism, or any associated term, in a categorical manner to describe the effects of combinations of antineoplastic drugs.


The importance of schedule on diethyldithiocarbamate modulation of drug-induced myelosuppression. Sodium diethyldithiocarbamate (DDTC) has been investigated as a biochemical modulator of the toxicity associated with clinically used cancer chemotherapeutic agents. In the present study, we assessed the ability of DDTC to accelerate recovery of the granulocyte/macrophage progenitor cel (GM-CFC) population following treatment with the myelosuppressive drugs carboplatin (CBDCA), tetrachloro(d,1-trans)1,2-diaminocyclohexane platinum(IV) (tetraplatin), 5-fluorouracil (5-FU), and etoposide (VP-16) in B6D2F1 mice. Myelotoxicity was assessed 24 h after the injection of the anticancer drug using a GM-CFC clonogenic assay. In the case of all four anticancer drugs, the timing of DDTC administration appeared to be a critical parameter with regard to protection. A delay time of 1 h between the injection of the myelotoxic drug and treatment with DDTC (30 mg/kg) resulted in a significant reduction in cytotoxicity to GM-CFC, whereas a longer delay time did not. These results suggest that the timing of DDTC administration may be essential in modulating the myelosuppression associated with many chemotherapeutic regimens used in the clinic.


Dissociation of cytotoxicity and DNA cleavage activity induced by topoisomerase II-reactive intercalating agents in hamster-human somatic cell hybrids. Previous studies using the mutant Chinese hamster ovary cell line VpmR-5 indicate that its resistance to epipodophyllotoxins and intercalating agents is likely to be mediated through a qualitative change in type II topoisomerase that confers resistance to drug-stimulated DNA cleavage activity. In a further investigation of the genetic basis of drug resistance in VpmR-5 cells, we fused a hypoxanthine-guanine phosphoribosyl transferase-deficient subline of VpmR-5 (Vtgm-6) with normal human lymphocytes and analyzed the resultant hybrid lines (HL) for altered drug sensitivity. In all, 3 of 16 hybrid clones exhibited partial reconstitution of sensitivity to etoposide, mitoxantrone, doxorubicin, and 5-iminodaunorubicin while retaining complete resistance to m-AMSA. However, enhanced sensitivity to drug-induced DNA cleavage activity was observed only for etoposide. Biochemical and molecular-marker analysis of the hybrids failed to identify human chromosome 17 (the provisional location of TOP2) or any other human chromosome that is consistently and uniquely associated with drug sensitivity. We therefore sought to verify the chromosomal assignment of TOP2 by Southern blot hybridization of TOP2 cDNA on a human hybrid mapping panel and confirmed its location on chromosome 17. However, no hybridizing sequence to the TOP2 cDNA was found in any of the 16 Vtgm-6 hybrid lines. Efforts to select more directly for human chromosome 17 VpmR-5 hybrids using microcell fusion of mouse A9 cells carrying human 17 linked to pSV2neo were unsuccessful. None of the five hybrid clones thus obtained had 17q markers, including the gene for TOP2. Although the mechanism underlying partial reversion to a drug-sensitive phenotype in the original Vtgm-6 hybrid lines has yet to be defined, the data obtained in these lines indicate that anthracycline- and anthracenedione-induced cytotoxic effects can be dissociated from DNA cleavage activity. This suggests that pathways distal to cleavable-complex formation or, alternatively, independent of interactions with topoisomerase II that involve other intracellular targets are important in mediating the cytotoxicity produced by these drugs.


Potentiation of etoposide-induced cytotoxicity and DNA damage in CCRF-CEM cells by pretreatment with non-cytotoxic concentrations of arabinosyl cytosine. Pretreatment of the human lymphoblastoid cell line CCRF-CEM with 0.02 microM arabinosyl cytosine (ara C) enhances both the cytotoxic and the DNA-damaging effects of etoposide. This concentration of ara C is itself non-cytotoxic and results in no detectable DNA damage as measured by alkaline elution. Ara C pretreatment results in the synchronisation of cells, a 24-h pretreatment resulting in the accumulation of cells in the early S phase. The sensitivity of cells to etoposide-induced cytotoxicity was increased 2.5 times and DNA damage was enhanced 1.66 times by this pretreatment. Maximal potentiation of etoposide-induced DNA damage (2.06-fold increase) was observed after 48 h continuous treatment with ara C, but no further enhancement of cytotoxicity occurred. Cell-cycle analysis demonstrated that 48 h ara C treatment resulted in the accumulation of cells in the late S/G2M phase. Cells returned to a normal cell-cycle distribution within 24 h of the removal of ara C, and the potentiation of etoposide activity was then reduced to a 1.3- to 1.4-fold level. DNA damage induced by etoposide following ara C pretreatment was qualitatively identical to that produced by etoposide alone, suggesting a mechanism involving topoisomerase II. To investigate this possibility, we measured topoisomerase II protein levels by immunoblotting. Measurement of topoisomerase II levels in whole-cell lysates of ara C-pretreated cells showed a 3- to 5-fold increase in topoisomerase levels relative to total protein content. This suggests that elevated enzyme levels may be responsible for the increased sensitivity of ara C-pretreated cells to etoposide.


Pharmacodynamics of three daily infusions of etoposide in patients with extensive-stage small-cell lung cancer. The objectives of this study were to define the pharmacodynamics of etoposide and to develop potentially useful models (1) to estimate the plasma clearance using a limited number of samples and (2) to describe the relationship between clearance and the dose-limiting toxicity. A total of 17 patients with extensive-stage small-cell lung cancer were treated with 150 mg/m2 etoposide daily for 3 consecutive days and with 100 mg/m2 cisplatin on day 3 only. Both drugs were given intravenously over 1 h. Treatment was repeated every 21 days for up to six courses. All patients were newly diagnosed (no previous chemotherapy or irradiation) and had a performance status of 0-2. Six patients achieved a complete response as confirmed by repeat bronchoscopy and five patients showed a partial response, for an overall objective response rate of 65% (95% confidence interval, 38%-87%). The median survival was 8 months (range, 1-24+ months). The dose-limiting toxicity was neutropenia. Etoposide pharmacokinetics were measured during the first course and determinations were repeated during courses 3 or 4 and 6. Complete blood counts were obtained weekly. Correlations for etoposide clearance and hematologic toxicities were evaluated for 17 initial courses and for an overall number of 33 courses. Pharmacodynamic correlations were significant for graded hematologic toxicities, as well as nadirs of leukocytes, neutrophils, and platelets for the initial courses and for all courses. To reduce the requirement for numerous blood samples, a limited sampling model was developed to estimate the area under the concentration versus time curve (AUC) with the following equation: AUC = 15.45 + 3.86 x C2 + 7.10 x C4, where C2 and C4 represent the etoposide concentrations at 2 and 4 h, respectively. The total plasma clearance was calculated as the dose divided by the AUC; correlations with toxicity were better for clearance expressed in milliliters per minute than for that expressed in milliliters per minute per square meter of body surface area. The absolute neutrophil count at the nadir (ANCn) can be estimated by the following pharmacodynamic model, which is based on 33 courses: ANCn = -0.399 + 0.024 x Ecl, where Ecl represents the etoposide clearance expressed in milliliters per minute. Further studies are necessary to validate both models prospectively.


Effect of WR-2721 on the toxicity and antitumor activity of the combination of carboplatin and 5-fluorouracil. We evaluated the effects of WR-2721 on the toxicity and antitumor activity of the combination of 5-fluorouracil (5FU) and carboplatin (CBDCA) in BALB/c and C57B1/6 mice. On a weekly schedule, i.p. injection of 200 mg/kg WR-2721 at 5 min prior to the administration of this combination enabled us to increase the CBDCA dose from a nontoxic level of 45 mg/kg to a normally toxic dose of 60 mg/kg in non-tumor-bearing BALB/c mice while maintaining the 5FU dose at 100 mg/kg. When WR-2721 was given 30 min before this combination, the CBDCA dose could not be increased to 60 mg/kg without producing drug-related deaths. WR-2721 protected against CBDCA- and 5-FU-induced thrombocytopenia but did not prevent leukopenia or anemia in C57B1/6 mice. The antitumor activity of the combination against colon 26 tumors in BALB/c mice was increased by pretreatment with WR-2721, which facilitated elevation of the CBDCA dose to 60 mg/kg in combination with 100 mg/kg 5FU. These results reveal better therapeutic efficacy for the combination of 5FU and CBDCA following pretreatment with WR-2721.


Colposcopy findings of CIN and cancer-like lesions of the cervix in pregnancy. In the course of routine colposcopy evaluation of the cervix in all the cases examined and treated in our clinic we pay attention to the cervical lesions accompanying pregnancy, which are macroscopically and/or colposcopically, and even frequently histologically suspected of CIN and/or cancer. These lesions include findings connected with HPV-infection, i.e. fully developed papillomas, subclinical forms of this infection, as well as the decidual ectopy of the cervix. Fully developed papillomas estimated not only macroscopically and colposcopically, but sometimes also histologically yield falsely positive pictures of cancer, and the decidual transformations on the cervix can be misinterpreted as non-epithelial malignant changes. The incidence rate of cervical papillomas is about 1% in non-selected material; one half of these cases occurs in pregnancy. Decidual ectopy was diagnosed in our material in 6.2% of pregnant women, chiefly between 21 and 30 years of age, and only in single cases in non-pregnant women. On the basis of our routine colposcopy examinations of the cervix in pregnancy conducted for over 30 years, the authors present the colposcopy-clinical signs of these CIN and carcinoma-like changes of the cervix in pregnancy. These observations are colpophotographically documented and allow us to differentiate between these changes and pre- and neoplastic lesions of the cervix in pregnancy.


Development and characterization of a cloned rat pulmonary arterial smooth muscle cell line that maintains differentiated properties through multiple subcultures. Pulmonary hypertension is associated with abnormal pulmonary arterial contractility and growth. The mechanisms for these abnormalities are largely unknown. To study these processes, we sought to develop an in vitro system. Even though cultured aortic and pulmonary artery smooth muscle cells (SMCs) have been of considerable value in studying smooth muscle biology, one drawback of this system has been that these cells often lose differentiated properties in an unpredictable manner when they are passaged in culture. In addition, there appear to be significant differences in physiological and pathological responses between the systemic and pulmonary circulations, many of which could be directly related to the smooth muscle. We therefore established a cloned population of rat pulmonary arterial SMCs (PASMCs) that maintain differentiated properties through multiple subcultures. PASMCs were obtained initially by enzymatic dissociation from pulmonary arteries of adult Sprague-Dawley rats. From these cells, clones were isolated. The cloned cells retained expression of functional surface receptors for angiotensin II, norepinephrine, and alpha-thrombin and high levels of the smooth muscle isoforms of alpha-actin, myosin heavy chain, myosin regulatory light chain, and alpha-tropomyosin mRNAs even after multiple passages. The cells could also be transfected and processed exogenous transcripts in a smooth muscle-specific fashion. These cloned PASMCs retain many differentiated characteristics and should be valuable for future studies of pulmonary vascular smooth muscle cell biology.


Vulvar intraepithelial neoplasia. Epidemiological and clinical study of 45 cases. We observed 45 cases of Vulvar Intraepithelial Neoplasia (VIN) histologically diagnosed which came to our observation between 1986 and 1991. The average age of the patients and the grade of the VIN lesions were evaluated. We also examined the eventual association with Papillomavirus infection, with non-neoplastic epithelial disorders in the adjacent areas and with intraepithelial neoplasias of the cervix and/or the vagina (CIN and/or VAIN). These data were studied in relation to the vulvoscopic pictures and symptomatology presented by the patients at the moment of diagnosis.


Two cases of plantar epidermal cyst associated with human papillomavirus. HPV-associated epidermal cysts of the sole (HAECS) of the foot have been reported recently in Japan in which there is positive staining for papillomavirus antibody in the nuclei of the epithelial cells in the cyst wall and vacuoles in the stratified horny material inside the cysts. A causative association with a newly recognized HPV, HPV 60, has been recorded. The authors report two new cases. The possible mechanism of cyst formation in HPV 60-infected epithelium is discussed.


Chediak-Higashi lymphoblastoid cell lines: granule characteristics and expression of lysosome-associated membrane proteins. Chediak-Higashi syndrome (CHS) is characterized morphologically by the presence of giant lysosomal granules resulting from the dysregulated fusion of primary lysosomes. Lysosome-associated membrane proteins comprise a family of highly glycosylated proteins which are postulated to facilitate many aspects of normal lysosomal function. In this study, Epstein-Barr virus-transformed lymphoblastoid cell lines derived from a patient with CHS were analyzed for the presence of giant granules and the expression of the lysosome-associated membrane proteins lamp1 and lamp2. Giant myeloperoxidase positive granules typical of CHS, which had a complex structure when examined by electron microscopy, could be demonstrated in the lymphoblastoid cell lines. In situ immunofluorescence with antibodies directed against lamp1 and lamp2 demonstrated abundant expression of each of these proteins in the giant CHS granules. Lack of expression of lysosomal cathepsin G in these granules was also noted. These observations suggest that the lymphoblastoid cell lines provide a convenient model for the study of Chediak-Higashi granules and the lysosome-associated membrane proteins and provide additional evidence that CHS is a "lysosomal" disease. Further study will be necessary to delineate whether the function of these membrane proteins is altered in Chediak-Higashi syndrome.


HIV-1 expression in chimpanzees can be activated by CD8+ cell depletion or CMV infection. CD8+ cell antiviral activity and cytomegalovirus (CMV) were investigated in vivo as possible cofactors influencing the outcome of HIV-1 infection. The role of CD8+ cell suppression of HIV replication was evaluated by depleting CD8+ cells in two infected chimpanzees by inoculation with monoclonal anti-CD8 antibodies. Two other infected animals were injected with chimpanzee CMV (CCMV)-infected human fibroblasts to determine if exposure to this virus would induce HIV replication. Treatment with anti-CD8 antibody resulted in recovery of virus from the CD4+ lymphocytes of one animal at 1, 4, and 6 months, and from a second animal at 1 month postinoculation. In contrast, virus had been recovered only once or not at all from these infected chimpanzees for 4 years prior to treatment. Similarly, HIV was recovered from the CD4+ cells of the two animals 2 to 3 months after inoculation of CCMV-infected fibroblasts but not after inoculation of control uninfected fibroblasts. These studies suggest that CD8+ cell-mediated suppression and the presence of other viruses (such as CMV) could act as cofactors in influencing the extent of HIV-1 replication in vivo and, possibly, progression to disease.


Detection of ovine lentivirus in seronegative sheep by in situ hybridization, PCR, and cocultivation with susceptible cells. Serological surveys for ovine lentivirus (OvLV), a worldwide cause of pneumonia and chronic debilitation in sheep, have demonstrated a wide range of seroprevalence rates. This study analyzed OvLV infection in a purebred sheep flock with a history of OvLV disease (flock 1), and compared the prevalence with that of a flock lacking previous OvLV-associated disease (flock 2). Serological tests (ELISA and Western blot assay) indicated that 25% of sheep of all ages in flock 1 (Group A) and 33% of animals of all ages in flock 2 (Group B) had antibodies to OvLV. In situ hybridization, however, detected viral RNA in a much larger proportion of sheep (72 and 67%, respectively). Animals less than 1 year of age rarely had antibodies to OvLV, although most harbored viral RNA. Twenty animals in this age group from flock 1 (Group C) were therefore studied more closely for infection. These yearling animals were tested serologically by ELISA and their peripheral blood-derived macrophages were cultured for 14 days to amplify any infection in these target cells. The macrophages were then tested by in situ hybridization, PCR, and cocultivation with susceptible target cells. The results of these tests showed that while only 10% of animals in Group C were seropositive, 70% were positive by in situ hybridization, PCR, and cocultivation. These data suggest that latent OvLV infection is common in sheep and that infection is frequently undetected by serological tests.


Human EBV-transformed lymphocytes of patients with Schistosoma japonicum infection secrete idiotypically related immunoregulatory antibodies. Lymphocytes derived from the peripheral blood of individuals infected with Schistosoma japonica were transformed in vitro with Ebstein-Barr virus (EBV). Serological characterization of antibody molecules revealed both antigen reactive (idiotypic) and anti-idiotypic transformants. One idiotypic EBV transformant, LO2C2, describes a major cross-reactive idiotype associated with anti-antigen binding molecules. Other antibody populations expressing idiotypic cross-reactivity were derived from separate individuals showing shared idiotypy in S. japonicum field study populations in the Republic of Philippines. Both idiotypic and anti-idiotypic molecules suppressed parasite antigen-driven blastogenesis of heterologous human peripheral blood lymphocytes. The data show a serologically related immunoregulatory immune network in patients in the Republic of the Philippines which is serologically distinct from idiotypy expressed in other selected S. japonicum endemic areas in the Far East.


Characterization of two monoclonal antibodies directed against the 67 kDa high affinity laminin receptor and application for the study of breast carcinoma progression. Two monoclonal antibodies (mAbs), designated MLuC5 and MLuC6, were produced against a human small cell lung carcinoma cell line. They were found to exhibit a superimposable reactivity on different cell lines and on platelets. Moreover, they both immunoprecipitated a 67 kDa molecule from the membrane of the reference target cells. Immunodepletion and cross-inhibition tests indicated that the two mAbs recognize two epitopes closely localized on the same molecule. The MLuC5 mAb was further characterized for its reactivity on platelets. Immunoprecipitation and ELISA assays demonstrate that this mAb recognizes the 67 kDa high affinity laminin receptor. MLuC5 reactivity was evaluated by immunohistochemistry on a variety of normal and tumor tissues, in particular breast specimens including normal epithelium, dysplastic lesions, in situ carcinomas, invasive primary carcinomas and distant metastases. The laminin receptor was found to be strongly expressed in 50% of the infiltrating carcinomas, whereas in situ carcinomas and benign lesions, as well as the normal mammary epithelium, were only weakly and focally positive. In metastatic lesions MLuC5 reactivity was only found in 11% of the samples tested, independently of the site of origin of the lesion.


Localization of peroxidase activity in the retina and the retinal pigment epithelium of the Syrian golden hamster (Mesocricetus auratus). 1. Endogenous peroxidase was localized by electron microscopy within the retinal tissue of the golden Syrian hamster. 2. Peroxidase activity was found in the apical parts of rod outer segments, in the cytoplasm and phagosomes of the retinal pigment epithelium (RPE), and extracellularly between the basal infoldings of the RPE. 3. No reaction product was detected when either H2O2 or tetramethyl benzidine were omitted from the reaction medium. 4. The same was true when the tissue was osmicated before the incubation in the reaction medium or treated with 100 mM sodium azide. 5. These results indicate that the peroxidatic activity was enzymatically mediated.


A comparative study on tissue distribution and metabolic adaptation of IMP-GMP 5'-nucleotidase. 1. Activity of a cytoplasmic 5'-nucleotidase which preferentially hydrolyzes IMP and GMP (IMP-GMP 5'-nucleotidase) was determined by a specific immunochemical method in two species of birds and two species of mammals. 2. The activity was markedly high in avian liver, and it increased two-fold in response to a high protein diet in chicken liver. 3. In mammals, the activity was high in testis and spleen. In the rat, the activities in liver, kidney and heart extracts increased by about 30% in response to the high protein diet, while they increased three-fold in regenerating liver. 4. Low activities were detected in skeletal muscles and in erythrocytes of all the species studied.


Dolphin ceruloplasmin: the first proteolytically stable mammalian ceruloplasmin. 1. Ceruloplasmin, the blue protein of the plasma of vertebrates, was isolated from dolphin, a marine mammal. The protein showed overall physico-chemical parameters very similar to those of all other mammalian ceruloplasmins. The spectroscopic properties indicated a conservation of the copper binding sites. 2. Non-denaturing electrophoresis revealed a conformation similar to that of other mammalian ceruloplasmins. EPR spectroscopy and calorimetric analyses indicated a three-domain arrangement of the protein typical of "aged" ceruloplasmin. 3. Dolphin ceruloplasmin is the only mammalian ceruloplasmin insensitive to trypsin, plasmin or chymotrypsin. This property, however, does not result in a higher conformational stability of the molecule. Thus, susceptibility of ceruloplasmin to aging is not directly related to the lability to proteases, which is typical of all other mammalian ceruloplasmins so far studied.


Phosphorylation of endogenous protein in primate kidney. Effects of cyclic AMP. 1. Phosphorylation of endogenous proteins in response to cyclic AMP was assessed in membrane and cytosol from primate kidney. 2. Quantitative studies showed that cAMP significantly increased phosphorylation in baboon kidney membranes; in cytosol there was no effect. 3. Phosphorylation of specific proteins which had been electrophoretically separated showed that five major bands were intensified by cAMP in baboon membranes; in cytosol, three bands were intensified. Similar results were found in normal human kidney. 4. Photoaffinity labelling indicated that a 56 kDa band phosphorylated in cytosol may correspond to the regulatory subunit of type II cAMP-dependent protein kinase.


Compatibility between the spermicide nonoxynol 9 and mid-cycle human cervical mucus. The rate of diffusion of [125I]-nonoxynol 9 into mid-cycle human cervical mucus was measured and found to be negligible compared with D-glucose (used as an uncharged small molecule for comparison with nonoxynol 9). These data were confirmed by observation of the lack of inhibition of the movement of spermatozoa in mucus that had been in surface contact, for 3 hours, with a concentration 30 times greater than the ED100 of nonoxynol 9. These results show that the entry of nonoxynol 9 into mucus could be minimal under conditions that would exist in vivo and highlights a limitation of this compound as a vaginal contraceptive.


Effect of histamine on signal transduction in cultured human trabecular meshwork cells. Stimulation of cultured human trabecular meshwork cells by histamine caused time and dose related increases in inositol phosphates and intracellular free calcium. The increase in inositol trisphosphate (IP3) was immediate and calcium independent while that of inositol monophosphate (IP1) was gradual and calcium dependent. The rise in intracellular calcium was also rapid and occurred as a result of mobilization from intracellular stores and influx from external medium. Histamine also caused time and concentration related de novo synthesis of inositol phospholipids. Mepyramine but not cimetidine inhibited the action of histamine. These results indicate that histamine, via H1 receptor, evokes an early hydrolysis of inositol phospholipids and increase in intracellular free calcium, signals which may be involved with the function of the trabecular meshwork cells.


Intracranial tumors in children. An analysis of 2000 cases. Two thousand patients aged 15 years and below, who underwent surgical treatment for intracranial tumors in Beijing Tiantan Hospital from 1955 to 1989, were studied. All of the tumors in this series have been verified by operative findings and histological studies. This series accounts for 15.1% of all cases of intracranial tumors treated within the same period. The characteristics of the histological classifications, locations and clinical manifestations of the intracranial tumors in children are presented. Stresses are placed on the importance of making correct diagnoses in the early stage. Misdiagnoses had been made in 1/4 of the cases in this series before they came to our hospital, thus causing much delayed treatment in many of them. Malignant tumors make up a majority of intracranial tumors in children, and so the prognoses of intracranial tumors are poorer in children than in adults. The authors are of the opinion that adequate radiotherapy is needed after surgical intervention for many of the intracranial tumors.


Histopathological classification of 272 primary epithelial tumors of the lacrimal gland. 272 primary epithelial gland tumors were classified pathologically. These tumors were sampled from 70% of 390 lacrimal fossa lesions out of 1530 orbital tumors from one hospital. Benign mixed tumors ranked the first in frequency (52%) to be followed by adenocystic carcinomas (25%) and malignant mixed tumors (9%). From the other less common tumor types, we identified, for the first time 3 polymorphous low-grade adenocarcinomas, 2 spindle celled myoepitheliomas and one carcinosarcoma of the lacrimal gland. The diagnosis and differential diagnosis of polymorphous low-grade adenocarcinoma and spindle cell myoepithelioma were discussed.


Borrelia burgdorferi infection may be the cause of sarcoidosis. Serum antibody to Borrelia burgdorferi was measured in 33 patients with sarcoidosis which was confirmed clinically and pathologically. The results showed that 81.8% of the patients were positive for anti-B. burgdorferi antibody. In addition, a strain of B. burgdorferi was isolated from a patient's blood. Fifteen patients received ceftriaxone 2g per day or penicillin 12 million U per day. The antibody titers of the patients decreased to nearly normal levels rapidly. Serum angiotensin converting enzyme (SACE) turned to normal range after the treatment. According to the findings mentioned above, we consider that B. burgdorferi infection may be the cause of sarcoidosis and sarcoidosis might be a specific type of Lyme disease.


Spontaneous evolution of nuclear size and DNA content of non-small-cell-lung cancers grafted onto nude mice. We monitored the biological evolution of four human non-small-cell lung cancers labeled KLX6, KLX7, KLX9, and KLX14 that had been grafted onto nude mice. This monitoring was carried out by means of digital cell image analysis which assessed morphometric (nuclear area, NA) and densitometric (nuclear DNA content, DI) features on Feulgen-stained nuclei from imprint smears. In each of the four models, the biological evolution of the NA and DI was characterized through their progression during serial passaging onto nude mice. The results show that of the four lung cancer models analyzed, one (KLX6) remained definitively stable with respect to its DNA content (DI assessments), while the other three, i.e., KLX7, KLX9, and KLX14, varied significantly. As assessed by the morphometric parameter, i.e., the NA, the four xenografted lines also varied significantly over serial passaging. In conclusion, we show that digital cell image analyses of Feulgen-stained cell nuclei including morphometric and densitometric parameters are a powerful tool for monitoring the biological evolution of human lung cancers grafted onto nude mice. We think therefore that the ploidy level of a tumor might be dependent upon its age and/or its related clinical stage.


Molecular extinction coefficients of lead sulfide and polymerized diaminobenzidine as final reaction products of histochemical phosphatase reactions. Molar extinction coefficients of precipitated lead sulfide (PbS) and polymerized diaminobenzidine (polyDAB) have been determined at wavelengths of 450 nm and 480 nm, respectively, for quantitative histochemical analysis of phosphatase reactions. These values are essential for the conversion of cytophotometric (mean integrated) absorbance values to absolute units of substrate converted per unit time and volume of tissue. This conversion allows direct comparison of histochemical and biochemical data. The molar extinction coefficient of PbS at 450 nm was found to be 3,800 and therefore, per mole phosphate liberated, the molar extinction coefficient is 5,700 because 3 moles phosphate are captured by 2 moles lead at neutral or alkaline pH. Parallel experiments with the cerium-DAB method revealed that the molar extinction coefficient of polyDAB at 480 nm is 5,500 with respect to liberated phosphate. The molar extinction coefficients were applied for comparison of data from biochemical and histochemical assays of glucose-6-phosphatase activity in rat livers. A significant correlation was found between both sets of data. The values were in the same order of magnitude with histochemical values approximately 1.4 times higher than biochemical values.


Growth behavior of human pancreatic carcinoma xenograft correlates with nuclear features. Two human pancreatic ductal adenocarcinomas with different growth rates were serially transplanted into nude mice. Feulgen-stained 4 microns sections and imprints from the xenografts were studied with a VICOM automated image analysis system. After pooling the results from two passages, with three mice in each passage, it was shown that of 23 nuclear parameters measured the following were correlated with a fast tumor growth rate: in sections, a decrease in heterogeneity of the chromatin and an increase in perimeter and nuclear area; in imprints, an increase in lesser diameter, in mean grey level difference between second neighboring pixels, and in total integrated optical density (DNA content). Several parameters differed significantly between passages, and between animals in the same passage. These findings suggest that the growth speed of pancreatic tumors may be predicted by nuclear parameters.


Plasmids as epidemiologic markers in nosocomial gram-negative bacilli: experience in an intensive care unit. The authors have compared the antimicrobial resistance patterns and plasmid profiles of Gram-negative isolates in an intensive care unit over a 7-month period in order to identify epidemiologically related isolates. Bacterial plasmids were found to be valuable markers for the comparison of strains of nosocomial Gram-negative bacilli. Thirty-nine mechanically ventilated patients in an ICU were included. From bronchoaspiratus, the authors isolated 58 strains of Gram-negative bacilli (24 Ps. aeruginosa and 34 Enterobacteria). Common plasmids were found in most Enterobacteria. The interspecies plasmid exchange suggests that interstate spread of these strains may have occurred. Twenty-six Enterobacteria carried plasmids, 11 of which proved transmissible. The R-factors were transferred to other genera that were isolated in the hospital, thereby adding to the pool of multiresistant nosocomial isolates. Larger plasmids transferred ampicillin and carbenicillin resistance, while gentamycin and cephalotin resistance was carried by smaller plasmids. Only 4 Ps. aeruginosa carried plasmids, one of which was transmissible. Pseudomonas plasmid DNA is extracted with difficulty by the simple lysis method, due to the roughness of the colonies. All Pseudomonas isolates belonged to the same biotype which can be regarded as an epidemiological marker. Therefore, plasmid profiling is a useful tool for epidemiological surveillance of Enterobacteria and is a good method for determining the relatedness of isolates in a nosocomial environment.


Transgenic mouse eggs with functional hamster sperm receptors in their zona pellucida. Sperm receptors are located in the mammalian egg extracellular coat, or zona pellucida. Mouse and hamster sperm receptor glycoproteins, mZP3 (83 x 10(3) M(r)) and hZP3 (56 x 10(3) M(r)), respectively, have very similar polypeptides (44 x 10(3) M(r); 81% identical) that are glycosylated to different extents. Purified mZP3 and hZP3 can bind to mouse sperm, prevent them from binding to eggs and induce them to undergo exocytosis, the acrosome reaction, in vitro. A DNA construct that placed the hZP3 gene under the control of mZP3 gene 5'-flanking sequence was used in this report to produce two mouse lines that harbored the foreign sperm receptor transgene. In both lines, the transgene was expressed only by growing oocytes, at a level comparable to that of the endogenous mZP3 gene, and the developmental pattern of transgene expression resembled that of the mZP3 gene. In addition to mZP3, transgenic mouse oocytes synthesized and secreted a glycoprotein indistinguishable from hZP3, and incorporated both glycoproteins into a mosaic zona pellucida. Importantly, hZP3 purified from such zonae pellucidae exhibited both sperm receptor and acrosome reaction-inducing activities in vitro and, following fertilization of transgenic mouse eggs, was inactivated. These results demonstrate that a biologically active foreign sperm receptor can be synthesized and secreted by transgenic mouse oocytes, assembled into a mosaic zona pellucida, and inactivated following fertilization as part of the secondary block to polyspermy.


Differential distribution patterns of CRABP I and CRABP II transcripts during mouse embryogenesis. We have compared the transcript distribution of cellular retinoic acid binding protein (CRABP) I and II genes in mouse embryos at various stages of development. Both CRABP transcripts are present in embryonic structures from the earliest stages studied and exhibit specific patterns of distribution, suggesting that the two retinoic acid (RA) binding proteins perform different functions during mouse embryogenesis. The CRABP I transcript distribution correlates well with structures known to be targets of excess retinoid-induced teratogenesis (e.g. neural crest cells and hindbrain), suggesting that cells expressing CRABP I are those that cannot tolerate high levels of RA for their normal developmental function. The embryonic structures expressing CRABP II transcripts include those structures that have been shown to be adversely affected by excess of retinoids, such as limbs and hindbrain, but CRABP II transcripts are also found in structures not known to be specifically vulnerable to raised RA levels. The CRABP II gene is coexpressed with retinoic acid receptor (RAR)-beta and cellular retinol binding protein (CRBP) I genes in a number of tissues such as the gut endoderm, hypophysis and interdigital mesenchyme, all of which are devoid of CRABP I transcripts. Interestingly, the expression of the three genes, RAR-beta, CRABP II and CRBP I, is induced by retinoic acid, which suggests a link between the synthesis of RA from retinol and the control of expression of subsets of RA-responsive genes. The transcript distribution of CRABP I and II is discussed in relation to the teratogenic effects of RA, and compared to the RA-sensitive pattern of expression of other important developmental genes.


Using technetium-99m (V) dimercaptosuccinic acid to detect malignancies from single solid masses in the lungs. Fifty patients (43 male, 7 female, age 31-77 years) with single solid masses in their lungs based on the findings of a chest X-radiograph [40 malignancies: 5 small cell carcinoma (Ca), 17 epidermoid Ca, 12 adeno Ca, 6 undifferentiated large cell Ca] and 10 benign lesions underwent technetium-99m (V) dimercaptosuccinic acid [99m-(V)DMSA] scans to evaluate the usefulness of 99mTc-(V)DMSA in the detection of lung Ca with different cell types and benign lesions. Only 43% (17/40) of the malignancies in the lungs were detected by 99mTc-(V)DMSA, including 29% (5/17) epidermoid Ca, 50% (6/12) adeno Ca and 17% (1/6) undifferentiated large cell Ca of the lungs. However, all 5 cases of small cell Ca and 11 cases combined with bone metastasis were revealed by 99mTc-(V)DMSA. In addition, 3 of the 10 benign lesions (2 organizing pneumonias, 1 benign tumor) presented with an uptake of 99mTc-(V)DMSA. The diagnostic sensitivity, specificity and accuracy were 43%, 70% and 48%, respectively, in differentiating malignant from benign lesions for the single solid mass in the lungs. In conclusion, 99mTc-(V)DMSA is of little or no use in the differentiation of lung Ca from single solid masses in the lungs.


Evidence for analgesia mediated by peripheral opioid receptors in inflamed synovial tissue. Intra-articular morphine (5 mg in 25 ml) was administered to patients for post-operative analgesia following arthroscopic knee surgery. At various time intervals, 30 min to 4 h post morphine, venous blood samples were taken in order to determine plasma levels of morphine and its primary metabolites, morphine-3-glucuronide and morphine-6-glucuronide. Measurable amounts of morphine and morphine-3-glucuronide were found in the plasma of 7/10 patients whereas morphine-6-glucuronide was detected in only 2/10 patients. The plasma levels of morphine were lower than that regarded sufficient for post-operative analgesia in all but two patients, indicating a possibility of peripheral analgesia. In addition, synovial biopsy samples were assayed for the presence of opioid binding sites. Tissue samples from 11 different patients were analysed and 6/11 exhibited specific binding of [3H]naloxone, indicating the presence of opioid binding sites/receptors. The receptor type (i.e. mu-, delta- or k-) is at present unknown. Taken together, these data provide evidence that locally administered opiates can act on specific opioid receptors in the synovium to mediate analgesia.


Characteristics of depressing effect of cycloprotobuxine-A on the maximal velocity of depolarization in myocardium. The characteristics of the depressing effect of cycloprotobuxine-A on Vmax in isolated guinea pig myocardium were investigated with a standard microelectrode technique. Cycloprotobuxine-A 3-30 mumol/l produced a concentration-dependent decrease in Vmax, associated with a prolongation of the action potential duration. At 3 mumol/l, a small resting block of Vmax, but a pronounced use-dependent block was found. This use-dependent block increased progressively as stimulation frequency increased from 0.5 to 4.0 Hz. The time constant and rate for onset of use-dependent block were 2.0-7.3 s and 0.11-0.25 AP-1 (reciprocal action potential number), respectively. The time constant and half-life for recovery from use-dependent block were 11.8 and 8.1 s. Thus, the kinetics of the depressing effect of cycloprotobuxine-A on Vmax were intermediate. In muscles depolarized by high [K+]0, the resting block was enhanced slightly, while use-dependent block was greatly augmented. These results suggested that cycloprotobuxine-A could have a low affinity for resting Na+ channels but a much higher affinity for activated and/or inactivated Na+ channels, and that the use-, frequency- and voltage-dependent effects of cycloprotobuxine-A on Vmax may play an important role in preventing the development of cardiac arrhythmias.


Mechanism of relaxing action of the antiasthmatic drug, azelastine, in isolated porcine tracheal smooth muscle. Azelastine (1-300 microM) inhibited contractions of isolated porcine trachea induced by high K+, carbachol and endothelin-1 (ET-1) with a decrease in [Ca2+]cyt (as measured by fura-2-fluorescence). Verapamil (0.1-10 microM) also inhibited the high K(+)-induced increases in [Ca2+]cyt and contraction, although it only partially inhibited the responses evoked by carbachol or ET-1. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), carbachol induced a transient increase in [Ca2+]cyt and force by releasing Ca2+ from cellular stores. Azelastine (100 microns) completely inhibited these contransient changes. In the absence of extracellular Ca2+, carbachol and 12-deoxyphorbol 13-isobutyrate (DPB) induced small sustained contractions without increasing [Ca2+]cyt. Azelastine inhibited these contractions. In muscle permeabilized with alpha-toxin, Ca2+ (0.3-3 microM) induced contraction in a concentration-dependent manner. DPB (without GTP) and carbachol or ET-1 (with GTP) enhanced the Ca(2+)-induced contraction. Azelastine partially inhibited the contraction induced by 0.3 microM Ca2+ but not the contraction induced by 3 microM Ca2+, and strongly inhibited the potentiating effects of DPB, carbachol and ET-1. Azelastine had no effect on the content of cyclic AMP or cyclic GMP. These results suggest that azelastine inhibits smooth muscle contraction by (i) decreasing [Ca2+]cyt, by inhibition of Ca2+ channels, (ii) decreasing agonist-induced Ca2+ release, and (iii) direct inhibition of contractile elements.


High concentrations of the neuroleptic remoxipride block voltage-activated Na+ channels in central and peripheral nerve membranes. The actions of the neuroleptic compounds remoxipride, haloperidol and (-)-sulpiride on Na+ and K+ ion current flow were examined in rat CNS and frog PNS, using 86Rb+ ion flux and voltage-clamp techniques, respectively. By combining veratridine and high K(+)-evoked 86Rb+ efflux, it was determined that remoxipride blocked Na+ current flow in a concentration-dependent fashion in tissue pieces from either cerebral cortex or striatum (IC50 approximately 20 microM), leaving K+ current flow virtually unaffected. Similarly, haloperidol concentration dependently blocked Na+ current flow in both tissues (IC50 approximately 50 microM). (-)-Sulpiride did not have a significant effect. Direct actions of the compounds on voltage-gated Na+ and K+ channels were determined in voltage-clamp experiments. The findings confirmed the results of the ion flux measurements in that remoxipride (Kd approximately 300 microM) and haloperidol (Kd approximately 1.5 microM) reduced mainly the Na+ current, having little effect on the K+ current, whereas (-)-sulpiride did not have a measurable action. The relatively high concentrations of remoxipride or haloperidol needed to alter the Na+ current makes it unlikely that these actions are of importance at clinically relevant doses.


Phosphoprotein phosphatase 2A dephosphorylates eIF-4E and does not alter binding to the mRNA cap. The phosphorylation and dephosphorylation of the 25 kDa mRNA cap binding protein eukaryotic initiation factor-4E (eIF-4E) is regulated during different physiologic and pathophysiologic states that include cell growth and the late phase of adenovirus infection. We have found that okadaic acid is much more effective in increasing the phosphorylated fraction of eIF-4E than phorbol 12-myristate 13-acetate in Hep G2 cells. Phosphoprotein phosphatase 2A dephosphorylated eIF-4E isolated from both phorbol 12-myristate 13-acetate- or okadaic acid-treated cells, whereas alkaline and acid phosphatase were relatively ineffective. The ability of purified [35S]eIF-4E isolated from okadaic acid-treated cells to bind mRNA caps was compared to phosphoprotein phosphatase 2A-treated [35S]eIF-4E and found to be no different. This suggests that alternative explanations for the previously observed effects of eIF-4E phosphorylation on protein synthesis must be considered. In addition, our results indicate that the in vivo phosphorylation of eIF-4E is not catalyzed solely by protein kinase C.


Apamin-sensitive potassium channels mediate agonist-induced oscillations of membrane potential in pituitary gonadotrophs. In cultured rat pituitary gonadotrophs, gonadotropin-releasing hormone (GnRH) induces rapid hyperpolarization of the cell membrane and causes cessation of the spontaneous electrical activity present in non-stimulated cells. This initial response to GnRH is followed by slow oscillations of membrane potential (Vm) which often exhibit brief bursts of action potentials (AP) fired from the peak of the oscillations. The hyperpolarization waves are synchronous with GnRH-induced elevations of cytoplasmic Ca2+ concentration ([Ca2+]i), such that Vm maxima alternate with the peak values of [Ca2+]i. The Vm oscillations result from repetitive activation of apamin-sensitive K+ channels by cytoplasmic Ca2+. Thus, GnRH activation of Ca2+ mobilization can generate a bursting pattern of membrane potential through the activation of K+ channels against a background of spontaneous electrical activity.


Effect of temperature on the kinetics of electron transfer from the tetraheme cytochrome to the primary donor in Rhodopseudomonas viridis. The kinetics of electron transfer from the third highest potential heme (c-552, Em = +20 mV) to the primary donor (P-960) have been measured by flash absorption spectroscopy in isolated reaction centers of Rhodopseudomonas viridis between 300 K and 7 K. The data are analyzed on the basis of three exponential components with a very fast phase (t1/2 = 120 ns) dominating at high temperature and a very slow one (t1/2 = 1.2 ms) at low temperature. This multiphasic behavior is interpreted in terms of the existence of three states with a temperature-dependent population and a very limited effect of the temperature on the kinetics for each state.


Structural and functional correlates of a mutation in the malignant hyperthermia-susceptible pig ryanodine receptor. The skeletal muscle ryanodine receptor of malignant hyperthermia-susceptible (MHS) pigs contains a mutation at residue 615 that is highly correlated with various abnormalities in the regulation of sarcoplasmic reticulum (SR) Ca2+ channel activity. In isolated SR membranes the Arg615 to Cys615 ryanodine receptor mutation is now shown to be directly responsible for an altered tryptic peptide map, due to the elimination of the Arg615 cleavage site. Furthermore, trypsin treatment released 86-99 kDa ryanodine receptor fragments encompassing residue 615 from the SR membranes. We conclude that the 86-99 kDa domain containing residue 615 is near the cytoplasmic surface of the ryanodine receptor and likely near important Ca2+ channel regulatory sites.


Calcium signaling in lymphocytes and ELF fields. Evidence for an electric field metric and a site of interaction involving the calcium ion channel. Calcium influx increased during mitogen-activated signal transduction in thymic lymphocytes exposed to a 22 mT, 60 Hz magnetic field (E induced = 1.7 mV/cm, 37 degrees C, 60 min). To distinguish between an electric or a magnetic field dependence a special multi-ring annular cell culture plate based on Faraday's Law of Induction was employed. Studies show a dependence on the strength of the induced electric field at constant magnetic flux density. Moreover, exposure to a pure 60 Hz electric field or to a magnetically-induced electric field of identical strength resulted in similar changes in calcium transport. The first real-time monitoring of [Ca2+]i during application of a 60 Hz electric field revealed an increase in [Ca2+]i observed 100 s after mitogen stimulation; this suggests that the plateau phase rather than the early phase of calcium signaling was influenced. The hypothesis was tested by separating, in time, the early release of calcium from intracellular stores from the influx of extracellular calcium. In calcium-free buffer, 60 Hz field exerted little influence on the early release of calcium from intracellular stores. In contrast, addition of extracellular calcium during exposure enhanced calcium influx through the plasma membrane. Alteration of the plateau phase of calcium signaling implicates the calcium channel as a site of field interaction. In addition, an electric field exposure metric is mechanistically consistent with a cell-surface interaction site.


The amino-terminal fragment of gelsolin is cross-linked to Cys-374 of actin in the EGTA-resistant actin-gelsolin complex. It has been shown that the EGTA-resistant actin, one of the two actin molecules associated to gelsolin, can be predominantly cross-linked to gelsolin by benzophenone-4-maleimide (BPM), a photoaffinity-labeling reagent, which was conjugated to Cys-374 of actin prior to cross-linking (Doi, Y., Banba, M. and Vertut-Doï, A. (1991) Biochemistry 30, 5769-5777). When a chymotryptic digest of gelsolin containing the amino-terminal 15-kDa fragment was mixed with BPM-actin (42 kDa) and irradiated for cross-linking, a band of 58 kDa appeared on SDS-PAGE which was shown to contain actin molecule by using fluorescently labeled actin. The amino-terminal sequence of the 58-kDa complex was identical to that of gelsolin, confirming that the amino-terminal segment (residues 1-133) of pig plasma gelsolin lies closely to Cys-374 of actin in the EGTA-resistant complex.


Inhibition of calcineurin by cyclosporin A-cyclophilin requires calcineurin B. The interaction of the immunosuppressive complex cyclosporin A-cyclophilin (CsA-CyP) with the Ca2+/calmodulin-dependent protein phosphatase calcineurin is investigated using a recombinant form of the A subunit of calcineurin (rCNA). Only in the presence of purified calcineurin B (CNB) does rCNA show the response of native calcineurin, i.e. 50% inhibition of rCNA phosphatase activity at 6 nM human cyclophilin B and 0.6 microM human cyclophilin A using [32P]casein as substrate, yet stimulation of activity with p-nitrophenyl phosphate as substrate. This study demonstrates that the B subunit is necessary to confer sensitivity of calcineurin to CsA-CyP.


Full activation without calmodulin of calmodulin-dependent cyclic nucleotide phosphodiesterase by acidic glycosphingolipids: GM3, sialosylneolactotetraosylceramide and sulfatide. Among calmodulin-non-binding glycosphingolipids, GM3, sialosylneolactotetraosylceramide (LM1), and sulfatide potently activated calmodulin-dependent cyclic nucleotide phosphodiesterase with or without Ca2+ showing ED50 1-5 microM. In contrast to calmodulin-binding gangliosides, these glycosphingolipids activated the enzyme up to the maximum level achieved by Ca2+/calmodulin and did not inhibit the activity at higher concentrations. Competition studies with GD1b that bind both to calmodulin and the enzyme suggest that the calmodulin-non-binding glycosphingolipids activate the enzyme through interaction with the same site of the enzyme as GD1b interacts.


Interaction of human myeloperoxidase with nitrite. EPR (electron paramagnetic resonance) and optical spectroscopy show that human neutrophil myeloperoxidase is converted from ferric high-spin to low-spin by the addition of nitrite. The Soret peak shifts from 429 to 447 nm and new peaks appear in the visible region at 573 and 627 nm; the EPR g-values change from 6.84, 5.02, 1.95 to 2.55, 2.31, 1.82. Small differences are seen in the EPR (but, not optical) spectra of myeloperoxidase isoenzyme I compared to isoenzymes II and III. The reaction with nitrite is detectable by EPR in intact neutrophils and is thus a possible in vivo monitor of NO/nitrite production by these cells.


BisG10, a K+ channel blocker, affects the calcium release channel from skeletal muscle sarcoplasmic reticulum. The action of bisG10, a potent K+ channel inhibitor, was tested on the Ca2+ release from isolated sarcoplasmic reticulum vesicles of rabbit skeletal muscle. Using a rapid filtration technique, we found that the drug inhibited Ca(2+)-induced Ca2+ release elicited in the presence of extravesicular K+ as counter-ion. This inhibition was not reversed by the addition of valinomycin and still occurred when Cl- was used as co-ion, indicating that not only K+ channels are involved in the inhibiting effect. We found that bisG10 decreased the binding of ryanodine to sarcoplasmic reticulum vesicles, showing that bisG10 is able to block the sarcoplasmic reticulum Ca2+ release channel.


Inactivation of the Na,K-ATPase by modification of Lys-501 with 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS). The sodium pump or Na,K-ATPase, maintains the Na+ and K+ gradients across eukaryotic cell membranes at the expense of ATP. Incubation of purified canine renal Na,K-ATPase with 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) inhibited the ATPase activity. Both the labeling of the protein and the loss of ATPase activity were prevented by co-incubation with ADP (acting as an ATP analog) or KCl. Only the alpha-subunit was labeled by SITS. The alpha-subunit from the inhibited enzyme was extensively digested with trypsin, and SITS-labeled peptides were purified by reverse-phase HPLC and sequenced. The amino acid sequence determined, His-Leu-Leu-Val-Met-X-Gly-Ala-Pro-Glu, indicated that SITS modifies Lys-501 (X) on the alpha-subunit of Na,K-ATPase.


The effect of cAMP and some sulphur compounds upon the activity of mercaptopyruvate sulphurtransferase and rhodanese in mouse liver. The aim of the experiments was to evaluate the effect of administration of cysteine, methionine, thiocystine, and thiosulphate upon the activity of mercaptopyruvate sulphurtransferase (MPST) and rhodanese in mouse liver. It was found that rhodanese activity increased following thiocystine and methionine administration and showed a smaller increase after cysteine and thiosulphate. The MPST activity significantly increased after cysteine and to a lesser extent after thiocystine and thiosulphate. On the other hand, methionine seemed to exert no effect upon the enzymatic activity. The results suggested that methionine metabolic cycle in mouse liver proceeded from cysteine to sulphane sulphur as thiocystine and, therefore, these three compounds increased rhodanese activity. Thiosulphate seemed rather to be involved in metabolic changes related to maintaining the stability of the physiological thiosulphate level and activated both the enzymes.


The effects of two fixed hormonal replacement therapy protocols on blood lipid profile. Hormonal Replacement Therapy (HRT) is known to be accompanied by changes in blood lipid profile. The present prospective cohort study compared the blood lipid profile of healthy postmenopausal women treated with either (a) a preparation containing a fixed regimen of estradiol, estriol and norethisterone acetate (EENA, marketed under the trade name Trisequens); (b) a fixed protocol of conjugated equine estrogen and medroxyprogesterone acetate (CEEMPA, marketed under the trade name Premaril Plus); or with (c) a concurrent group which underwent no treatment. Blood lipid profiles (total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), and triglycerides (TG)) were performed at the beginning of the study and at 3-month intervals, for 9 months. The EENA-treated women showed a significant and profound decrease in TC and LDL-C when compared with their initial values, with the control group, and with the CEEMPA group. The CEEMPA group showed an increase in HDL-C values and a decrease in LDL-C values when compared with their initial values and with the control group, but no increase was shown when compared with an EENA group. There was a favorable decrease in the TC/HDL-C and LDL-C/HDL-C ratios in both treatment protocols. As the primary goal of prevention of coronary artery disease is total cholesterol reduction, the EENA protocol seems to be preferred.


Hyperglycaemia alters the physico-chemical properties of proteins in erythrocyte membranes of diabetic patients. 1. The dynamic properties of erythrocyte membranes in diabetic children and of control erythrocyte membranes subjected to in vitro glycation have been investigated by means of fluorescence quenching of membrane tryptophan residues and ESR spectroscopy. 2. The apparent distance separating the membrane protein tryptophan and the bound 1-anilino-8-naphthalenesulphonate (ANS) molecules was decreased in erythrocyte membranes from children with diabetes. This resulted in a significant increase of the maximum energy transfer efficiency in diabetic membranes. 3. The relevant alterations occurred in the above parameters due to the in vitro nonenzymatic glycosylation of control membranes. 4. These changes were accompanied by the decreased hw/hs parameter of MSL and the increased relative rotational correlation time (tau c) of ISL in diabetic membranes and in the membranes subjected to in vitro glycation. 5. The results suggest that the conformational changes in membrane proteins may occur at both the intrinsic and exposed thiol groups. 6. Both the in vivo and the in vitro data indicate that nonenzymatic glycosylation of membrane proteins may be the major factor attributable to the alterations in the dynamic properties of erythrocyte membrane in diabetic state.


A comparison of itraconazole and griseofulvin in the treatment of tinea corporis and tinea cruris: a double-blind study. A total of 40 patients with clinically and mycologically documented tinea corporis or tinea cruris were treated with 100 mg/day itraconazole (n = 19) or 500 mg/day griseofulvin (n = 21) for 15 days. Of the itraconazole-treated patients, 83.3% were healed or markedly improved, i.e. 'responders', after 15 days compared with 85.7% of griseofulvin-treated patients. At 15 days after the end of treatment, 88.2% of itraconazole- and 80.9% of griseofulvin-treated patients were classed as 'responders'. The mycological cure rate (both microscopy and culture negative) was generally lower than the clinical response rate. Both treatments were equally effective at the end of 15 days' treatment with 66.7% of patients cured, but itraconazole was superior to griseofulvin at the 15-day follow-up visit (77.8% of itraconazole-treated patients compared with 66.7% of griseofulvin-treated patients were cured). Both therapies were well tolerated; only one patient treated with itraconazole reported minor side-effects (dizziness, headache and gastro-intestinal disturbances). The results confirm those of earlier comparative trials and suggest that griseofulvin-treated patients are more at risk of relapse than are itraconazole-treated patients.


Distribution pattern of connexin 43, a gap junctional protein, during the differentiation of mouse heart myocytes. In the cardiac muscle, the electrical coupling of myocytes by means of gap (or communicating) junctions, allows the action potentials to be propagated. Connexin 43 (CX 43) is the major constitutive protein of the gap junctions in the mammalian myocardium. In this organ, the abundance of CX 43 and of its messenger, as well as the spatial expression of this protein, are developmentally regulated. These findings are complemented by the results presented in this article, which deals with the distribution of CX 43 in the ventricular myocytes of mouse heart during differentiation, between the 11 days post coitum embryo stage and adulthood. By immunoelectron microscopy experiments on ultrathin sections of cardiac ventricular tissue of one-week-old mouse, we have provided confirmation that the anti-CX 43 antibodies used here specifically recognized the gap junctions. Double labeling immunofluorescence experiments have been undertaken to localize, within the same cells, either CX 43 and desmin, or CX 43 and Con A or WGA receptor sites. From the earliest stage investigated (11 days post coitum) onwards, expression of CX 43 is always associated with desmin-positive cells, that is, with the myocytes. Up to birth, there is in the ventricular wall a gradient of expression of CX 43 which is superimposable on a gradient of expression of desmin. Immunoreactivity to anti-CX 43 and anti-desmin antibodies is high in the sub-endocardial trabeculae and low (or even undetectable for CX 43, in the early stages) in the sub-epicardial cell layers. In the embryonic stages, the expression sites of CX 43 are visible in the form of small dots, whose abundance increases as development proceeds. During these stages, the immunoreactive sites are distributed in a relatively homogeneous pattern throughout the membrane of the myocytes. One week after birth, the CX 43 expression is restricted to the two ends of the myocytes (where the intercalated discs develop), and the adjacent lateral regions. This polarization of CX 43 is more pronounced at the two and three weeks post natal stages and in the fully differentiated ventricular myocytes (adult stage) CX 43 is only present in the intercalated discs.


Mutations in c-K-ras 2 gene codon 12 during colorectal tumorigenesis in familial adenomatous polyposis. Colorectal carcinomas may be induced from adenomas, or they may occur de novo. To clarify the histogenesis of colorectal carcinomas, point mutations in codon 12 of the c-K-ras 2 gene in neoplasias of familial adenomatous polyposis patients were examined. Nineteen colorectal advanced carcinomas, 135 adenomatous polyps, 9 hyperplastic polyps, and 27 normal colonic mucosae were obtained from 48 patients. In 27 normal mucosae and 9 hyperplastic polyps, a mutation in the K-ras gene was not detected. Mutations were detected as follows: 0 of 24 in adenomas with mild atypia, 10 of 77 in adenomas with moderate atypia, and 24 of 34 in adenomas with severe atypia. The incidence of mutations in c-K-ras 2 codon 12 is correlated with the degree of atypia of adenomas. However, only 5 such mutations were detected in 19 advanced carcinomas, indicating that the mutation frequency in advanced carcinomas is much lower than that in adenomas with severe atypia. If a mutation of c-K-ras 2 gene is an important component in the formation of adenocarcinoma, these results did not confirm the successive development from adenomas with severe atypia to advanced carcinomas as the main route for colorectal carcinogenesis in familial adenomatous polyposis patients.


Fecal weight, colon cancer risk, and dietary intake of nonstarch polysaccharides (dietary fiber). Low fecal weight and slow bowel transit time are thought to be associated with bowel cancer risk, but few published data defining bowel habits in different communities exist. Therefore, data on stool weight were collected from 20 populations in 12 countries to define this risk more accurately, and the relationship between stool weight and dietary intake of nonstarch polysaccharides (NSP) (dietary fiber) was quantified. In 220 healthy U.K. adults undertaking careful fecal collections, median daily stool weight was 106 g/day (men, 104 g/day; women, 99 g/day; P = 0.02) and whole-gut transit time was 60 hours (men, 55 hours; women, 72 hours; P = 0.05); 17% of women, but only 1% of men, passed < 50 g stool/day. Data from other populations of the world show average stool weight to vary from 72 to 470 g/day and to be inversely related to colon cancer risk (r = -0.78). Meta-analysis of 11 studies in which daily fecal weight was measured accurately in 26 groups of people (n = 206) on controlled diets of known NSP content shows a significant correlation between fiber intake and mean daily stool weight (r = 0.84). Stool weight in many Westernized populations is low (80-120 g/day), and this is associated with increased colon cancer risk. Fecal output is increased by dietary NSP. Diets characterized by high NSP intake (approximately 18 g/day) are associated with stool weights of 150 g/day and should reduce the risk of bowel cancer.


Sodium transport in human intestinal basolateral membrane vesicles. The current investigation was aimed at characterizing transport pathways for Na+ in basolateral membrane vesicles (BLMV) isolated from organ donor jejunum and ileum. An outward proton gradient [pH inside, 5.5; pH outside, 7.5] led to a 4-5-fold stimulation of transport rates compared with the absence of proton-gradient conditions in both human jejunal and ileal BLMV. Voltage-clamping the vesicles (K+ inside = K+ outside + valinomycin) reduced the uptake of 22Na by 20%, indicating a minor conductive component of Na+ transport. Uptake of 22Na (1 mmol/L) in voltage-clamped BLMV was inhibited 70% by 2 mmol/L amiloride. Li+ and NH4+ inhibited transport of 22Na into voltage-clamped BLMV. Transport of Na+ exhibited saturation kinetics, and the Michaelis constant (Km) and Vmax values for jejunum and ileum were similar [Km, 27 +/- 3 mmol/L (jejunum) and 18 +/- 2 mmol/L (ileum); Vmax, 19 +/- 2 nmol.mg protein-1.min-1 (jejunum) and 16 +/- 1 nmol.mg protein-1.min-1 (ileum)]. Vmax values were < 15% of those reported for brush border membrane, whereas Km values were comparable. The results show that Na+ transport in human jejunal and ileal BLMV occurs via an Na+/H+ exchanger and a minor conductive pathway.