A new chick mitochondrial DNA-binding protein exhibits sequence-specific interaction near heavy-strand replication origin: cleavage activity, stimulation of mtDNA synthesis, and enhancement in transformed fibroblasts. We have identified a new, double-strand-dependent, mtDNA-binding protein in chick embryo fibroblast (CEF) mitochondria (and inner-membrane-matrix preparations) which demonstrates both an exclusive specific affinity for the displacement loop (D-loop) control region of chick mtDNA and intramitochondrial levels that reflect corresponding changes in mtDNA replication activity both in vivo and in vitro. This approximately 36 kDa protein (designated aMDP1, avian mitochondrial DNA-binding protein 1) was identified by elution and renaturation following SDS-polyacrylamide gel electrophoresis and by direct isolation from specific mtDNA-protein complexes excised from mobility shift gels. Analysis of the entire 16.7-kb mt genome determined that a MDP1 mediates cleavage of chick mtDNA in vitro at three H- and two L-strand sequence-specific target sites located within a 90-bp A + T-rich genomic tract, theoretically capable of forming stable secondary structures, approximately 200 bases upstream from the H-strand origin (OH) of replication. Furthermore, gel-isolated aMDP1 relaxes supercoiled mtDNA, and exogenous addition of the protein, in a permeabilized in vitro system, preferentially stimulates the synthesis of H-strand sequences which hybridize to OH-containing fragments. Oncogenic transformation of CEF by Rous sarcoma viruses results in a threefold elevated level of aMDP1, directly correlating with a similarly increased level of mtDNA replication in vivo. Heterologous chick-human cross-competition experiments showed that aMDP1 also selectively interacts with human (HeLa) D-loop region mtDNA, possibly reflective of an evolutionary importance for aMDP1 interaction in the region. Functionally, we hypothesize that aMDP1 may operate in conjunction with other mtDNA-binding proteins, important in replication and transcription, by potentiating duplex unwinding either prior to or during an initial stage of H-strand synthesis. Together, these results suggest that aMDP1 is a good potential candidate for a nucleus-encoded regulatory protein which communicates with the mt genome during the replication process.


Lectin-induced differentiation of transformed neuroretinal cells in vitro. The orderly course of chick neuroretinal cell differentiation was disrupted in vitro by infection with a temperature-sensitive strain of the Rous sarcoma virus (LA29). The resulting cell culture LA29NR remained mitotically active at 42 degrees C, yet rapidly adopted a transformed phenotype upon activation of the pp60v-src oncogene product at 37 degrees C. As a further indication of metabolic state, LA29NR cells expressed the protooncogene product c-Fos, as shown by Western blot analysis. Highly proliferative LA29NR cells proved refractory to standard differentiation agents such as cAMP, and prostaglandin E1. In our novel approach, succinylated concanavalin A (SCA), a nontoxic derivative of the lectin concanavalin A, induced dramatic, reversible morphological changes in LA29NR cells, including neurite outgrowth and increased cell-to-cell adhesion. Fluoresceinated SCA appeared to localize to Golgi and lysosomal structures. Cellular response to SCA treatment included decreased growth rate, reversible decrease in the phosphorylation state of a 41-kDa phosphoprotein, and induction of neuron-specific enolase. The glial marker vimentin was also evident in these cultures. These data suggest that SCA is an effective differentiation agent for cells of neuroectodermal origin, permitting neuronal as well as glial phenotypic expression within these cell populations.


Inhibition of platelet-derived growth factor-induced mitogenesis and tyrosine kinase activity in cultured bone marrow fibroblasts by tyrphostins. Tyrphostins, which block protein tyrosine kinase activity, were studied for their inhibitory action on platelet-derived growth factor (PDGF)-induced proliferation of human bone marrow fibroblasts. Of the seven tryphostins examined, tyrphostin AG370 was found to be the most potent blocker against PDGF-induced mitogenesis (IC50 = 20 microM). This PTK blocker also blocks mitogenesis induced by epidermal growth factor (IC50 = 50 microM) and human serum (IC50 = 50 microM), but with lower efficacy. In digitonin-permeabilized fibroblasts as well as in intact fibroblasts, tyrphostin AG370 inhibits PDGF receptor autophosphorylation and the tyrosine phosphorylation of intracellular protein substrates (pp120, pp85, and pp75) which coprecipitate with the PDGF receptor. In comparison to AG370, AG18, a potent EGF receptor blocker, was less efficient in inhibiting PDGF-induced proliferation of fibroblasts and phosphorylation of the intracellular protein substrates. Under the conditions in which AG370 inhibits PDGF-induced mitogenesis and phosphorylation, it does not affect [125I]PDGF internalization and enhance [125I]PDGF binding. These findings suggest that AG370, which is an indole tyrphostin, may serve as a model for developing analogues with a therapeutic potential for treatment of diseases which involve abnormal cellular proliferation induced by PDGF.


Synergistic induction of erythroid differentiation of mouse erythroleukemia (MEL) cells by inhibitors of topoisomerases and protein tyrosine kinases. In vitro erythroid differentiation of mouse erythroleukemia (MEL) cells was induced by combinations of topoisomerase and protein kinase inhibitors. Neither inhibitor alone exhibited inducing activity. Although inhibitors of topoisomerases I and II were equally effective in the synergistic induction of erythroid differentiation, only inhibitors of tyrosine kinases, not of serine/threonine kinases, exhibited synergistic activity. The erythroid differentiation induced by the combination of topoisomerase and protein tyrosine kinase inhibitors was distinguished from that induced by typical erythroid inducing agents such as DMSO or HMBA by (1) earlier hemoglobin accumulation in the cells and (2) insensitivity to specific inhibitors (dexamethasone and sodium orthovanadate) of MEL cell differentiation.


Receptor-mediated endocytosis and nuclear transport of a transfecting DNA construct. A soluble construct consisting of a plasmid carrying the gene of the SV40 large T-antigen and an insulin-poly-L-lysine conjugate is able to selectively transfect PLC/PRF/5 human hepatoma cells which possess insulin receptors. Transfection can be efficiently competed by excess free insulin. To examine intracellular transport of the construct, it was fluorescently labeled and its accumulation on and in cells visualized by video-enhanced microscopy and quantitative confocal laser scanning microscopy. After 2 h at 37 degrees C, the labeled construct was found predominantly in intracellular acidic compartments, with a substantial portion of fluorescence localized both near and in the cell nucleus. Binding, endocytosis, and nuclear localization of the labeled conjugate could all be competed by excess free insulin, thus indicating that entry of the conjugate into cells was specifically mediated by the insulin receptor.


Phorbol esters augment polyamine transport by influencing Na(+)-K+ pump in murine leukemia cells. In this report, we elucidate the role of Na(+)-K+ pump in the regulation of polyamine spermidine (Spd) transport in murine leukemia (L 1210) cells in culture. Ouabain, known to bind extracellularly to the alpha-subunit of the Na(+)-K+ pump, inhibits the pump activity. The L 1210 cells were found to possess ouabain binding sites at 7.5 fmol/10(6) cells. Ouabain significantly inhibited the Spd uptake in a dose-dependent manner. The maximum inhibition of Spd uptake by ouabain was observed beyond 200 microM. Spd transport was inversely correlated with the [3H]ouabain binding to L 1210 cells: an increase in the saturation of ouabain binding to L 1210 cells resulted in a decrease of the Spd uptake process. Treatment of L 1210 cells with protein kinase C activator phorbol esters increased the Spd transport and, also, ouabain-sensitive 86Rb+ uptake, a measure of the activity of the Na(+)-K+ pump. H-7, a protein kinase C inhibitor, significantly inhibited the ouabain-sensitive 86Rb+ uptake by L 1210 cells. Phorbol esters stimulated the level, but not the rate, of 22Na+ influx. Addition of H-7 to L 1210 cells inhibited the 22Na+ influx process. A concomitant phorbol ester-induced increase in 22Na+ influx, [14C]Spd uptake, together with the functioning of Na(+)-K+ pump, indicates the role of the "Na+ cycle" in the regulation of the polyamine transport process.


Receptor-mediated endocytosis of the intrinsic factor-cobalamin complex in HT 29, a human colon carcinoma cell line. A HT 29 cell line derived from human colonic carcinoma was shown to express the intrinsic factor receptor, with about 5000 binding sites per cell and an association constant of 20 x 10(9) 1/mol at pH 7.4 and 4 degrees C. The number of binding sites increased dramatically between 7 and 10 days of culture time. Endocytosis of the intrinsic factor-cobalamin-receptor complex was inhibited by two ways: at 4 degrees C and at 37 degrees C by incubating the cells with vinblastine, monensin and chloroquine. The plasma membrane receptor was cross-linked to [57Co]cobalamin-intrinsic factor and solubilized with Triton X-100. The cross-linked complex had a relative molecular mass of 330 kDa in native PAGE.


Structural characterization and expression of a brain specific gene encoding chick kainate binding protein. The gene encoding chick kainate-binding protein (c-KBP), a member of the non-NMDA ionotropic glutamate receptor family has been isolated and characterized. The c-KBP gene is at least 13 kilobases long and contains 11 exons interrupted by 10 introns. Primer extension and RNase protection studies identified a major transcription initiation site located 117 bases upstream from the initiation methionine codon ATG. Consensus TATA and CCAAT sequences were detected in the putative promoter region. The structure of the c-KBP gene is strikingly different from that of other members of neurotransmitter-gated ion-channels (cloned at present) although the topology of c-KBP consists of four membrane-spanning domains, a structural characteristic of ionotropic receptor subunits. The c-KBP gene was found to be expressed at high levels in chick cerebellar Bergmann glia and at extremely low levels in the forebrain. The limited expression of the c-KBP gene raises important questions concerning the mechanisms governing the regulation of c-KBP gene transcription.


Regulation of interleukin-1 receptors on AtT-20 mouse pituitary tumour cells. To study the cellular mechanisms of interleukin-1 (IL-1) in the pituitary corticotroph, we studied the properties of IL-1 receptors on a mouse pituitary ACTH-producing cell line, AtT-20. Scatchard plot analysis revealed a single type of receptor with a Kd (dissociation constant) of 93 pM, and 482 binding sites/cell. [125I]IL-1 alpha binding competed with IL-1 alpha and IL-1 beta in an equimolar fashion. A 24 h pre-incubation with either CRH, epinephrine or nor-epinephrine increased the [125I]IL-1 alpha binding sites in the AtT-20 cells and conversely, a similar pre-incubation with either dexamethasone or tumour necrosis factor-alpha (TNF alpha) decreased them without affecting the affinity of the receptors in either case.


Relationship between intracellular pH changes, activation of protein kinase C and NADPH oxidase in macrophages. Activation of the superoxide-generating NADPH oxidase by phorbol ester or zymosan induced a cytoplasmic acidification when liver macrophages were incubated in sodium-free media or in the presence of amiloride. Staurosporine or desensitization of protein kinase C inhibited phorbol ester- and zymosan-induced pH changes and generation of superoxide. The intracellular pH remained unchanged in cells incubated in physiological sodium media. Ionomycin and arachidonic acid did not induce a change in intracellular pH or a generation of superoxide. Fluoride, which has been shown to induce a translocation of protein kinase C in these cells, did not elicit superoxide generation but induced a decrease in intracellular pH. These experiments support (1) a role of the Na+/H+ antiporter in macrophages as a metabolic regulator of intracellular pH upon stimulation of the superoxide-generating NADPH oxidase, and (2) suggest an involvement of protein kinase C in this process.


Isolation and characterization of polyferredoxin from Methanobacterium thermoautotrophicum. The mvhB gene product of the methylviologen-reducing hydrogenase operon. The methylviologen-reducing hydrogenase operon of Methanobacterium thermoautotrophicum contains an open reading frame, mvhB, the product of which was predicted to have a molecular weight of 44 kDa and to contain as many as 48 iron atoms in 12 [4Fe-4S] clusters, and was therefore suggested to be a polyferredoxin. We have now, for the first time, isolated this polyferredoxin. Its identity with the mvhB gene product was evidenced by a comparison of the N-terminal amino acid sequence. The dark-brown protein of apparent molecular weight 44 kDa was found to contain 53 mol Fe and 43 mol acid-labile sulfur per mol. The UV/visible spectrum showed two maxima at 280 nm and 390 nm, and a shoulder at 308 nm. The A390/A280 ratio was 0.73. The molar extinction coefficient at 390 nm was 170,000 M-1.cm-1. In the dithionite reduced state the protein displayed an EPR spectrum like that of [4Fe-4S] clusters. The results indicate that the mvhB gene product is indeed a polyferredoxin.


Autocrine receptors for endothelins in the primary culture of endothelial cells of human umbilical vein. Human umbilical vein endothelial cells (HUVECs) in primary culture produced and secreted endothelin 1 (ET-1) actively. Specific binding of [125I]ET-1 to these cells was not detectable because of the saturation of ET receptors with endogenously produced ET-1. However, addition of phosphoramidon, an inhibitor of ET-converting enzyme, to the medium reduced the production of ET-1 and thus the receptors on HUVECs were made available for exogenously added [125I]ET-1. Binding studies using phosphoramidon-treated HUVECs indicated the existence of a non-isopeptide-selective type (ETB) of ET receptor with a Kd of 17 pM. This receptor is thought to be involved in ET-induced vasodilation in an autocrine manner in vivo.


The 93 kDa protein gephyrin and tubulin associated with the inhibitory glycine receptor are phosphorylated by an endogenous protein kinase. The 93 kDa protein gephyrin is a tubulin binding peripheral membrane protein that is associated with the inhibitory glycine receptor and has been implicated in its anchoring at central synapses. Here, we demonstrate that gephyrin as well as co-purifying tubulin are phosphorylated by a kinase activity which is endogenous to highly purified glycine receptor preparations. This kinase phosphorylates serine and threonine residues and utilizes ATP, but not GTP, as phosphate donor. Its activity is not affected by various activators and/or inhibitors of cyclic nucleotide-dependent kinases, calcium/calmodulin-dependent kinases, or protein kinase C. A five-fold stimulation of kinase activity was, however, observed in the presence of poly-lysine. Phosphorylation of gephyrin and/or tubulin might regulate receptor/cytoskeleton interactions at postsynaptic membrane specializations.


Sequence motif in control regions of the H+/K+ ATPase alpha and beta subunit genes recognized by gastric specific nuclear protein(s). A nuclear protein(s) from rat or pig stomach recognized a conserved sequence in the 5'-upstream regions of the rat and human H+/K(+)-ATPase alpha subunit genes. A gel retardation assay suggested that part of the binding site was located in the TAATCAGCTG sequence. No nuclear proteins capable of the binding could be detected in other tissues of rat (liver, brain, kidney, spleen and lung) or pig liver. The sequence motif (GATAGC) located 5'-upstream of the beta-subunit gene also seemed to be recognized by the same protein, because the binding of nuclear protein to the sequence motifs in the alpha and beta subunits was mutually competitive. Considering the sense-strand sequence of the binding motif in the alpha-subunit gene, we conclude that (G/C)PuPu(G/C)NGAT(A/T)PuPy is a core sequence motif for the gastric specific DNA binding protein (PCSF, parietal cell specific factor).


Concentration-dependent modulation of basic fibroblast growth factor action on multiplication and locomotion of human teratocarcinoma cells. A human teratoma cell line (Tera 2) was grown in serum-free medium, and the population multiplication was stimulated by the addition of 1-10 ng basic fibroblast growth factor (bFGF)/ml. The bFGF-effect was abrogated by the addition of protamine sulphate. When high concentrations of bFGF were added, a preferential effect on cell locomotion was observed. 100 ng bFGF/ml stimulated cell movement but only exerted a marginal effect on cell multiplication. These observed exogenous requirements for multiplication and locomotion were complemented by the expression of bFGF receptors. Scatchard analysis of binding data suggests the existence of a high-affinity and a low-affinity class of receptors.


A new cyclopyrophosphate as a bacterial antistressor? In a number of bacteria an unusual glycosyl pyrophosphate (31P NMR signal chemical shift at about -15 ppm) was detected when the cells were subjected to oxidative stress. This substance from Brevibacterium ammoniagenes has now been identified as 2-methyl-butan-1,2,3,4,-tetraol-2,4-cyclopyrophosphate, which is accumulated in the cell under certain conditions in concentrations of of about 50 mM. It is now suggested that this compound is the long sought after bacterial antistressor.


Developmental changes in erythropoietin receptor expression of fetal mouse liver. Erythropoietin (EPO) stimulates proliferation and differentiation of late erythroid precursor cells (CFU-E) and thereby determines the rate of erythropoiesis. Liver is the major erythropoietic site in a fetus. We dealt with developmental changes in CFU-E and EPO receptor (EPO-R) of fetal mouse liver. The affinity of the EPO-R to EPO was unchanged during fetal development. The population size of CFU-E, the number of EPO-R per liver cell, and EPO-R mRNA decreased as gestation proceeded, in a pattern indicating that the expression of EPO-R on erythroid precursor cells in fetal mouse liver is governed mostly by the process of mRNA production.


Okadaic acid strongly increases gene transcription, mRNA and protein level for the urokinase receptor in human A549 cells. The specific phosphatase inhibitor, okadaic acid, increases the level of mRNA for the receptor for urokinase-type plasminogen activator (u-PAR) in 8 out of 13 human cell lines. The strongest increase (90-fold) was observed in A549 lung carcinoma cells, in which it was partly traced back to an increased transcription of the u-PAR gene. There was a parallel but less pronounced increase in the u-PAR protein level. These findings indicate that u-PAR gene transcription is regulated by one or more factors that are constitutively phosphorylated and are dephosphorylated by okadaic acid-sensitive phosphatases. A lack of additivity of u-PAR induction by okadaic acid and by the protein kinase C activator, PMA, in the A549 cells suggests that the regulatory factors affected by okadaic acid are phosphorylated by protein kinase C.


Thromboxane receptor stimulation inhibits adenylate cyclase and reduces cyclic AMP-mediated inhibition of ADP-evoked responses in fura-2-loaded human platelets. Stimulation of human platelets with the thromboxane A2 analogue, U46619, after treatment with prostaglandin E1 or forskolin, reduced the inhibition of ADP-evoked Mn2+ influx and the release of Ca2+ from intracellular stores. U46619 decreased the elevated concentration of 3',5'-cyclic AMP in platelets that were pretreated with prostaglandin E1. These results suggest that occupation of prostaglandin H2/thromboxane A2 receptors, like those for other agonists, inhibits adenylate cyclase activity, which can contribute to the promotion of platelet activation.


Genomic organization and analysis of the 5' end of the porcine ryanodine receptor gene (ryr1). In this study we describe the isolation of genomic clones of the 5' region of the porcine ryanodine receptor gene, a candidate for malignant hyperthermia in pigs and humans. The recombinants were isolated from a porcine liver, genomic DNA library in phage EMBL3A after screening with PCR amplified DNA fragments. The exon/intron structure of the ryanodine receptor gene was determined by DNA sequencing. Based on the sequence data it was possible to develop a simple test for the detection of malignant hyperthermia susceptible and normal pigs.


Mercurial activation of human PMN leucocyte type IV procollagenase (gelatinase). Autoproteolytic activation and processing of human polymorphonuclear leucocyte (PMNL) type IV procollagenase (gelatinase) was initiated by HgCl2 and was investigated by kinetic analysis and N-terminal sequence determination of the reaction products. In the first instance the propeptide domain was lost by subsequent cleavage of the Asp15-Leu16, Glu40-Met41, Leu52-Leu53 and Ala74-Met75 peptide bonds. The PRCGVPD sequence motif (residues Pro78-Asp84), which is conserved in all metalloproteinases and expected to be relevant for latency, remained uncleaved at the activated enzyme. The generated intermediate was further processed by three C-terminal cleavages. The Glu666-Leu667, Ala506-Glu507 and Ala398-Leu399 bonds were hydrolysed successively. From the fragmentation products we were able to conclude that three released fragment peptides contained unpaired free cysteine with the residues Cys497, Cys653, Cys683. Cleavage of the first C-terminal peptide bond resulted in the loss of one of the conserved Cys residues of the hemopexin-like domain, whereas the Cys residue of the PRCGVPD motif was retained at the fully active enzyme. The possibility of an entirely different activation mechanism for PMNL type IV procollagenase is discussed.


Acute and long-term effects of insulin-like growth factor I on glucose transporters in muscle cells. Translocation and biosynthesis. Insulin-like growth factor I (IGF-I) rapidly (less than 10 min) stimulated glucose uptake into myotubes of the L6 muscle cell line, at concentrations that act specifically on IGF-I receptors. Uptake remained stimulated at a steady level for 1-2 h, after which a second stimulation occurred. The first phase was insensitive to inhibition of protein synthesis. Subcellular fractionation demonstrated that it was accompanied by translocation of glucose transporters (both GLUT1 and GLUT4) to the plasma membrane from intracellular membranes. Translocation sufficed to explain the first phase increase in glucose transport, and there was no change in the total cellular content of GLUT1 or GLUT4 glucose transporters. The second phase of stimulation was inhibitable by cycloheximide, and involved a net increase in either GLUT1 or GLUT4 transporter content, which was reflected in an increase in transporter number in plasma membranes. These results define a cellular mechanism of metabolic action of IGF-I in muscle cells; furthermore, they suggest that IGF-I has acute metabolic effects that mimic those of insulin, bypassing action on the insulin receptor.


Evidence for non-cysteinyl coordination of the [2Fe-2S] cluster in Escherichia coli succinate dehydrogenase. The consequences of replacing Cys65 in the FrdB subunit of Escherichia coli fumarate reductase by Asp or Ala have been investigated in terms of bacterial growth, enzymatic activity, and the ERP/redox properties of the [2Fe-2S] cluster. An aspartic acid residue occupies the equivalent position in E. coli succinate dehydrogenase, and the FrdBCys65Asp mutation has little effect on cell growth, enzyme activity or the physical properties of the Frd [2Fe-2S] cluster. In contrast, the [2Fe-2S] cluster was not observed in the FrdBCys65Ala mutant showing that a coordinating residue is required at this position for assembly of this cluster and significant levels of enzymatic activity. These results support the presence of one non-cysteinyl, oxygenic ligand for the [2Fe-2S] cluster in E. coli succinate dehydrogenase.


Evidence for a new extracellular peroxidase. Manganese-inhibited peroxidase from the white-rot fungus Bjerkandera sp. BOS 55. A novel enzyme activity was detected in the extracellular fluid of Bjerkandera sp. BOS 55. The purified enzyme could oxidize several compounds, such as Phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, ABTS and guaiacol, with H2O2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H2O2. With some substrates, a strong inhibition of the peroxidative activity by Mn2+ was observed. Phenol red oxidation was inhibited by 84% with only 1 mM of this metal ion. Because DMP oxidation by this enzyme is only slightly inhibited by Mn2+, this substrate should not be used in assays to detect manganese peroxidase. The enzyme is tentatively named 'Manganese-Inhibited Peroxidase'.


Changes in the expression of mRNAs for myogenic factors and other muscle-specific proteins in experimental autoimmune myasthenia gravis. The regulation of genes for acetylcholine receptor (AChR), myogenic factors and other muscle-specific proteins has been analyzed in experimental autoimmune myasthenia gravis (EAMG) and following denervation. The levels of the transcripts for the myogenic factors, MyoD1, myogenin and MRF4, were measured using Northern blot analysis. Myogenin and MRF4 transcript levels were observed to be 3.1- and 2.6-fold higher in muscle of rats with EAMG than in controls, respectively. MyoD1 levels, however, remained unchanged. The increases in AChR, myogenin and MRF4 mRNAs were one order of magnitude higher in 2-week denervated muscle than in the myasthenic muscle. The levels of muscle creatine kinase (MCK), alpha-actin and muscle dystrophin transcripts were also analyzed. Dystrophin levels were found to be 1.7- and 4.7-fold higher in EAMG and denervated muscle, respectively, than in controls; in contrast, MCK and alpha-actin levels remained unchanged.


Molecular link between membrane cholesterol and Na+/H+ exchange within human platelets. Incubation of human platelets with cholesterol-poor, cholesterol-normal and cholesterol-rich liposomes revealed that: (i) acquisition or depletion of platelet membrane cholesterol was highly selective; (ii) variation in membrane cholesterol was highly selective. Variation in membrane cholesterol content (cholesterol-to-phospholipid molar ratio from 0.15-1.2) with respect to values found in unmodified normal platelets, was paralleled by the observed changes in amiloride-sensitive cytoplasmic pH, as well as phospholipase A2 activity. However, a decrease in cytoplasmic pH was accompanied by an increase in phospholipase A2 activity; (iii) membrane cholesterol-modulated changes in intra-platelet pH, as well as phospholipase A2 activity, was completely inhibited when platelets were pretreated with quinacrine (a specific phospholipase A2 inhibitor) before exposure to various types of liposomes. Although exposure of platelets (pretreated with amiloride) with various types of liposomes resulted in the inhibition of Na+/H+ exchange it had no noticeable effect upon the observed phospholipase A2 activity. Based upon these results we suggest that membrane cholesterol-modulated phospholipase A2 activity may be the basic mechanism responsible for the nature of Na+/H+ exchanger activity observed in cholesterol-enriched platelets, leading these platelets to a hypersensitized state.


Functional evidence of a transmembrane channel within the Ca2+ transport ATPase of sarcoplasmic reticulum. Ca2+ efflux can be studied conveniently following dilution of sarcoplasmic reticulum (SR) vesicles preloaded with 45Ca2+ by active transport. The rates of efflux are highly dependent on ATPase substrates and cofactors (Pi, Mg2+, Ca2+ and ADP) in the efflux medium. On the other hand, phenothiazines stimulate efflux through a passive permeability channel with no coupled catalytic events. Efflux activation by manipulation of catalytically active ATPase ligands, as well as by the catalytically inactive phenothiazines, can be prevented by thapsigargin, which is a highly specific inhibitor of the Ca(2+)-ATPase. This demonstrates that the passive channel activated by phenothiazines is an integral part of the ATPase, and can operate either uncoupled or coupled to catalytic events.


Inhibitory effect of oryzacystatins and a truncation mutant on the replication of poliovirus in infected Vero cells. Poliovirus, a picornavirus family member, requires the processing of its poly-protein by its own cysteine proteinase for replication. Oryzacystatin-I and oryzacystatin-II, proteinaceous cysteine proteinase inhibitors (cystatins) of rice seed origin, were found to inhibit the replication of poliovirus effectively in infected Vero cells in vitro. Truncated oryzacystatin-I, which lacks the NH2-terminal 25 amino acid residues of the intact protein, is an even more effective inhibitor, eliciting its effect at concentrations of less than 0.25 nmol/ml. The low molecular weight cysteine proteinase inhibitors, E-64, E-64C and loxistatin, showed no anti-viral effect at any concentration investigated.


Endogenous Xenopus-oocyte Ca-channels are regulated by protein kinases A and C. Calcium entry into Xenopus oocyte occurs mainly through voltage-dependent calcium channels. These channels were characterized as belonging to a particular type of calcium channel insensitive to dihydropyridines, omega-conotoxin, and Agelenopsis aperta venom, but blocked by divalent cations (Co, Cd, Ni). Intracellular injection of cAMP, or bath application of phorbol ester, induced a marked increase in calcium current amplitude and a slowing of the inactivation time-course. Despite their different pharmacology, endogenous calcium channels, like cardiac or neuronal calcium channels, could be thus regulated by protein kinases A and C.


Expression of dihydropyridine-sensitive brain calcium channels in the rat central nervous system. We have localized dihydropyridine (DHP-sensitive calcium channels in rat brain by in situ hybridization and immunohistochemistry. The mRNA for the dihydropyridine-sensitive calcium channel alpha 1 subunit (DHPR-B) is prominently localized in neuronal cells in the olfactory bulb, dentate gyrus, hippocampus, arcuate nucleus, paraventricular nucleus, ventromedial nucleus, cerebral cortex, superior colliculus and the cerebellar Purkinje cell layer. Strong expression of DHPR-B mRNA was also found in the pituitary and pineal glands. DHP-sensitive calcium channel alpha 1 subunit distribution has also been examined immunohistochemically with polyclonal antibodies raised against synthetic peptides specific for the DHPR-B alpha 1 subunit protein. The results from immunohistochemistry were in good agreement with those from in situ hybridization. Thus, regional distribution and localization of DHPR-B mRNA and alpha 1 subunit protein in rat brain suggest that this type of DHP-sensitive brain calcium channel may play an important role in excitation-secretion coupling functions in the neuroendocrine system.


Molecular characterisation, expression and localisation of human neurokinin-3 receptor. The complete amino acid sequence of the human neurokinin-3 receptor was deduced by DNA sequence analysis of human genomic fragments. Comparison of the predicted primary structure with those for the human neurokinin receptors 1 and 2 shows a highly conserved pattern of seven hydrophobic regions with maximum divergence occurring at the amino- and carboxy-termini. The position of intron-exon junctions are identical to those in other reported neurokinin genes. Using a chimeric genomic-cDNA gene, the human NK-3 receptor was expressed in Xenopus laevis oocytes where it mediates membrane conductance changes in response to its agonist, neurokinin B. More significantly, expression of the gene in mammalian cells resulted in detection of receptor binding as well as neurokinin-stimulated calcium mobilization and arachidonic acid release, all displaying the pharmacological characteristics expected of a neurokinin-3 receptor. By using the polymerase chain reaction we have shown that mRNA for the human neurokinin-3 receptor is expressed predominantly in the central nervous system.


A possible biological role of the electron transfer between tyrosine and tryptophan. Gating of ion channels. Experiments have demonstrated that four tryptophan residues are located near the tetrodotoxin binding site in Na+ channels, and that conserved tyrosine and tryptophan residues are located in the pore-forming region of voltage-sensitive K+ channels. This paper proposes an activation mechanism involving electron transfer between these residues. The K+ channel may be closed by four tyrosine residues forming hydrogen bonds with each other. After electron transfer, these hydrogen bonds will be broken, thereby opening the channel. The Na+ channel could be activated by a similar mechanism. This idea can be tested directly by observing tyrosine or tryptophan radicals when the channels are in the open state.


Cycloheximide inhibits cellular, but not SV40, DNA replication. We have prepared extracts from cycloheximide-treated cells for the study of simian-virus-40 (SV40)-DNA replication in vitro. When supplemented with the viral initiator protein (large T antigen), these extracts fully supported SV40-DNA replication. We also determined that SV40-DNA replication in vivo is much more resistant to cycloheximide than cellular DNA replication. SV40 encodes its own initiator protein, T antigen, which also functions as a DNA helicase, but depends on cellular functions for all additional replication reactions. Therefore, it appears to be quite likely that cycloheximide affects cellular DNA replication by blocking the synthesis of (a) cellular function(s) that is(are) performed by T antigen in SV40-DNA replication. Indeed, DNA fiber autoradiography and alkaline sucrose gradient centrifugation of pulse-labeled cellular DNA showed that cycloheximide treatment almost completely suppressed replicon initiation and reduced the rate of replication fork movement to about one third of the control.


Two-dimensional crystals of membrane-bound gastric H,K-ATPase. Two-dimensional crystallization of membrane-bound H,K-ATPase (EC 3.6.1.36) in vesicle preparations from parietal cells of hog gastric mucosa was induced by an imidazole buffer containing Mg2+ and VO3- ions. A continuous reorganization of the protein molecules started within a few hours by the formation of linear arrays. At later stages confluent two-dimensional crystals were formed. Electron microscopy and image processing showed that these were of a single tetragonal type. The asymmetric unit consisted of one pear-shaped protein domain corresponding to a H,K-ATPase protomer. Through stain-deficient contact regions four adjacent protein units were connected forming a tetrameric structure.


Kinetic mechanism of pyruvate decarboxylase. Evidence for a specific protonation of the enzymic intermediate. Decarboxylation of pyruvate by pyruvate decarboxylase (EC 4.1.1.1) was performed in a reaction mixture containing 50% deuterium. The isolated product, acetaldehyde, was investigated directly by 1H NMR and by mass spectrometry after conversion to the 2,4-dinitrophenyl hydrazone. The protium content of 56% at acetaldehyde C1 demonstrates a specific protonation of the corresponding intermediate by the enzyme. Proton inventory studies and enzyme modification indicate the 4' amino group of the coenzyme, thiamine pyrophosphate, in an immonium structure being a possible proton donor. A 'partially concerted' mechanism is suggested for the reaction steps following the decarboxylation.


Endothelin stimulates both cAMP formation and phosphatidylinositol hydrolysis in cultured embryonic bovine tracheal cells. Embryonic bovine tracheal (EBTr) cells were found to possess receptors for endothelin (ET) of ET-1-selective (ETA) subtype with a Kd for ET-1 of 114 pM and a Bmax of 12.9 fmol/10(5) cells. Stimulation of EBTr cells with 100 pM to 100 nM ET-1 increased the contents of both inositol phosphates and cAMP in a concentration-dependent manner, indicating that the receptors are coupled to both phosphatidylinositol hydrolysis and cAMP formation in EBTr cells.


Prostaglandin F2 alpha decreases the affinity of epidermal growth factor receptors in Swiss mouse 3T3 cells via protein kinase C activation. Prostaglandin F2 alpha (PGF2 alpha) selectively decreases the binding of 125I-labelled epidermal growth factor ([125I]EGF) to intact Swiss 3T3 cells. Scatchard analysis reveals that PGF2 alpha decreases the number of high-affinity EGF binding sites without changing the total number of receptors. Prostaglandins E1 (PGE1), E2 (PGE2) or F2 beta (PGF2 beta) do not alter the EGF binding to these cells and do not enhance the PGF2 alpha effect. R-59022 and R-59949, two diacylglycerol kinase inhibitors, enhance the inhibitory effect of PGF2 alpha, whereas down-modulation of protein kinase C (PKC) abolishes the effect. These results indicate that PGF2 alpha decreases EGF binding in Swiss 3T3 cells via PKC activation.


Voltage-dependent calcium entry in confluent bovine capillary endothelial cells. Confluent bovine capillary endothelial cells display, when examined for voltage-dependent calcium entries using cell-attached channel recordings, two types of Ca2+ channels (4 and 23.5 pS in 110 mM Ba2+) both sensitive to the dihydropyridine Ca agonist BAY K 8644. In contrast to isolated cells, confluent cells display no T-type, low threshold activity, and Ca currents were typically only elicited at very depolarized potentials. In these cells, voltage-dependent calcium entries will only be made operative by substances able to shift their activation towards the resting potential.


The nuclear pore: at the crossroads.

The nuclear pore: at the crossroads. The nuclear pore complex is at the crossroads of macromolecular traffic across the nuclear envelope. Our knowledge of the mechanism whereby nuclear transport is mediated by the nuclear pore complex is also at a crossroads; a molecular understanding of this process has major implications for applied medical sciences. This becomes obvious with the realization that nuclear proteins are synthesized in the cytoplasm and yet function in the nucleus, and that RNA is transcribed in the nucleus but translated in the cytoplasm. Thus, control of macromolecular traffic across the nuclear membrane is an important means for altering the levels and activities of such molecules as steroid hormone receptors, transcription factors, and enzymes involved in DNA replication. Nuclear proteins have been found to contain nuclear localization sequences (NLS) rich in basic amino acids, which target them for transport through the nuclear pore to the nucleus. It is also clear that a group of novel glycoproteins having a unique carbohydrate modification are required for transport across the nuclear pore complex. However, the mechanism by which the NLS is recognized to mediate transport across the nuclear envelope is poorly understood. It is the aim of this brief review to attempt a synthesis of what is known of this mechanism and what may be newly inferred on the basis of current experimental data.


Platelet-activating factor receptor and signal transduction mechanisms. Platelet-activating factor (PAF) is the most potent phospholipid agonist known to date. Radioligand binding studies using [3H]PAF and structurally different PAF antagonists have provided the characteristics of PAF receptor(s) and its heterogeneity. Although efforts have been made to isolate the receptor, it was not until the recent cloning of the PAF receptor that the molecular architecture of the receptor can be visualized. The receptor shows homology to the G protein-coupled receptors with seven transmembrane spanning segments. Several serine, threonine, and tyrosine residues are present at the cytoplasmic side, which could serve as sites for phosphorylation. PAF activates GTPase, causes phospholipid turnover via phospholipases C, D, and A2 pathways and also activates protein kinase C and tyrosine kinase. Further, PAF stimulates Ca2+ mobilization some of which may occur via receptor operated channel. Second messengers generated by these multiple signalling pathways play role (or roles) in PAF responses and in the PAF induced expression of primary response genes. These recent developments throw light on the PAF receptor and its signal transduction mechanisms.


Receptor tyrosine kinases.

Receptor tyrosine kinases. A major process through which environmental information is transmitted into cells is via activation of protein tyrosine kinases. Receptor tyrosine kinases contain extracellular ligand recognition, single membrane spanning, and cytoplasmic protein tyrosine kinase domains. The cytoplasmic kinase core is flanked by regulatory segments, which in some family members are also inserted into the core kinase domain. Ligand binding initiates receptor signaling from the cell surface. Activated receptors autophosphorylate to remove alternate substrate/inhibitory constraints and to provide loci for assembly of proteins that contain SRC homology regions. Information is transmitted and diffused by tyrosine phosphorylation of the assembled proteins and of cellular substrates that include protein kinases with specificity for serine/threonine residues. Signaling, which is strictly ligand-dependent, is attenuated by down-regulation of receptors and by feed-back inhibitory loops that involve receptor phosphorylation by cellular kinases. The tyrosine kinase receptors are essential for normal growth, development, and reparative processes. Mutations that remove normal regulatory constraints on the approximately 290 amino acid kinase core of these large proteins result in constitutive function and cell transformation.


MR imaging of hepatocellular carcinoma at 1.5 Tesla. The authors investigated the magnetic resonance appearance of hepatocellular carcinoma using a 1.5-Tesla magnet. Twenty-four patients with pathologically proven hepatocellular carcinoma had magnetic resonance imaging (MRI) studies, which were retrospectively reviewed. All patients were imaged with at least two of the following techniques: (1) T1-weighted (T1W), (2) T1-weighted with Gd-DTPA enhancement (T1W-E), (3) T2-weighted (T2W), (4) proton density (PD), and (5) gradient-recalled echoes (GRE). T1W images were equal to T2W images for tumor detection using a grading system. T1W images were slightly better than T2W images for the total number of lesions detected. The other pulsing techniques (PD, T1W-E, and GRE) detected fewer lesions. Eight cases of hepatocellular carcinoma (33%) had nonhomogeneous increased signal intensity on both T1W and T2W images. The authors conclude that T1W images are equal to T2W images for detection of hepatocellular carcinoma. The authors also conclude that 33% of hepatocellular carcinomas have an imaging pattern with increased signal intensity on both T1W and T2W images. This pattern is atypical for most other hepatic masses and hence can be used to suggest the mass is hepatocellular carcinoma.


Hepatocellular carcinoma with extrahepatic primary neoplasms. We experienced seven cases of hepatocellular carcinoma (HCC) with extrahepatic primary malignant neoplasms (PMNs), which accounted for 7.5% of 93 HCC cases in our department during a 5.5-year investigation period. No common characteristics of the seven patients with extrahepatic PMNs in terms of age, sex, computed tomographic (CT) features of hepatocellular carcinoma, or presence of liver cirrhosis were evident. One of the seven cases had triple PMNs. Extrahepatic PMNs were found in various organs: stomach, colon, kidney, lung, larynx, and esophagus. Attention to extrahepatic organs should be undertaken in the management of HCC. Two patients in hepatic coma died 4 and 6 months after the diagnosis of HCC due to the advanced HCC, and accompanying extrahepatic malignancies were not the primary cause of death.


Contribution of CT to characterization of focal nodular hyperplasia of the liver. Our personal series of 20 cases of focal nodular hyperplasia (FNH) of the liver is presented. All lesions were studied with computed tomography (CT), 16 of which with surgical control. Retrospective evaluation of the CT features of the identified FNH, along with those of five hepatocellular adenomas (HCA) and 30 hepatocellular carcinomas (HCC), allowed the definition of specific patterns leading to a correct characterization of FNH in 78% of cases. This greatly reduced the diagnostic errors, with the sole exception of patients with fatty liver in whom nuclear medicine may eventually provide a correct characterization. Fine-needle biopsy is thus only necessary in the dubious cases. A precise diagnostic workup of FNH is necessary, since it may avoid the surgical intervention.


Receptors for 1,25-dihydroxyvitamin D3 in gynecologic neoplasms. To determine if gynecologic malignancies are candidates for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) therapy we measured vitamin D receptor (VDR) levels in 11 tumor specimens using a radiolabeled ligand-binding assay. VDR was demonstrated in 3 of 6 ovarian tumors and 1 of 1 uterine sarcomas, but not in endometrial tumors (2), cervical tumors (1), or Krukenberg tumors (1). Scatchard plots revealed that [3H]1,25(OH)2D3 was bound to a single class of high-affinity (Kd = 0.3 to 0.6 nM), saturable sites characteristic of authentic 1,25(OH)2D3 receptors. Specificity of binding activity for 1,25(OH)2D3, the active vitamin D3 metabolite, was demonstrated by failure of 25-hydroxy- and 24,25-dihydroxyvitamin D3 to compete effectively against 1,25(OH)2D3 binding in total cellular tumor extracts. The ovarian carcinoma cell line NIH:OVCAR3 was shown to possess VDR (binding capacity = 137 fmol/mg protein, Kd = 0.48 nM). A 3-day incubation of NIH:OVCAR3 cells with 100 nM 1,25(OH)2D3 resulted in 49% inhibition of cell growth. The growth inhibition of an ovarian carcinoma line and the observation that 36% of gynecologic tumors assayed were shown to be VDR-positive suggest that further study is warranted to delineate the mechanism and possible therapeutic aspects of 1,25(OH)2D3 action in gynecologic tumors.


The concomitant determination of different tumor markers in patients with epithelial ovarian cancer and benign ovarian masses: relevance for differential diagnosis. The serum levels of CA 125 (cutoff limit, 65 U/ml), CA19.9 (cutoff, 40 U/ml), CA 15.3 (cutoff, 32 U/ml), CA72.4 (cutoff, 3.8 U/ml), and TATI (cutoff, 22 ng/ml) were preoperatively measured in 90 patients with epithelial ovarian cancer and in 254 patients with benign ovarian pathology. CA125 had a sensitivity of 75.6%, a specificity of 86.6%, and a diagnostic accuracy of 83.7% for epithelial ovarian cancer; CA19.9 had a sensitivity of 35.6%, a specificity of 81.1%, and a diagnostic accuracy of 69.2%; CA15.3 had a sensitivity of 57.1%, a specificity of 93.9%, and a diagnostic accuracy of 84.6%; CA72.4 had a sensitivity of 70.7%, a specificity of 91.8%, and a diagnostic accuracy of 86.2%; and TATI had a sensitivity of 47.3%, a specificity of 95.3%, and a diagnostic accuracy of 82.9%. CA 125 was the most sensitive marker for nonmucinous tumors, while CA19.9 and CA72.4 were the antigens more frequently expressed by mucinous malignancies. The sensitivities of serum CA 125 (81.1% vs 50.0%; P = 0.01) and TATI (55.2% vs 18.8%; P = 0.02) were higher in patients above 50 years of age than in younger patients while specificities were quite similar in both age groups. The association of serum CA125 and CA19.9 had a significantly higher sensitivity (93.2% vs 81.1%; P = 0.03) and a slightly lowered specificity (78.9% vs 86.0%; P = 0.46) than CA125 assay alone in the differential diagnosis of ovarian masses in patients above 50 years of age.


[Estradiol inhibits dopamine mediated behavior in rats--an animal model of sex-specific differences in schizophrenia]. We found in a representative sample of 392 first hospital admissions for schizophrenia a higher mean age at onset in females by 3.2 to 3.9 years, whereas the lifetime risk was equal for both sexes. In males the rates of onset show a steep increase reaching the maximum value in the age group 15-24 years, followed then by a steady decrease. Females reach the first peak with a clear delay between 20 and 29 years. After the decrease a second smaller peak is observed consistently in females within the age group 45-49 years and over. After having excluded alternative explanations for this gender differences (for example, diagnosis artefacts, sociocultural factors), we hypothesized that the effect of oestradiol on the dopaminergic system enhances the vulnerability threshold for schizophrenia, which is lowered again during the menopause. Alternatively we assumed that testosterone reduces the vulnerability threshold and thus furthers the earlier onset of schizophrenia in males. We tested these hypotheses in animal models by investigating the effects of the gonadal hormones on haloperidol-induced catalepsy and on apomorphine-induced stereotypies in both neonatal and adult rats. Testosterone showed no clear influence on the tested dopamine-mediated behaviour. Oestradiol caused a significant reduction on both dopamine-agonist and dopamine-antagonist induced behaviour. These effects were stronger in neonatal animals. Since oestradiol caused a 2.8-fold reduction of dopamine receptor affinity for sulpiride, we assumed that the behavioural changes caused by oestradiol were accounted for by a down-regulation of the dopaminergic system.(ABSTRACT TRUNCATED AT 250 WORDS)


Detection of the minus strand of hepatitis C virus RNA by reverse transcription and polymerase chain reaction: implications for hepatitis C virus replication in infected tissue. The combination of reverse transcription and polymerase chain reaction is a very powerful tool for the detection of hepatitis C virus RNA in sera of patients with hepatitis C virus infection. However, when studying the presence of this virus in tissue using polymerase chain reaction, it may be difficult to distinguish between blood viral particles adhering to the tissue and viral RNA contained within the tissue. Because hepatitis C virus has a single-stranded RNA of positive polarity, a minus-strand RNA is expected to be found in hepatitis C virus-replicating tissues as a template for the synthesis of genomic RNA. To see whether the detection of the minus strand of hepatitis C virus RNA by polymerase chain reaction can be used for the determination of hepatitis C virus-replicating tissues, we examined the presence of the minus strand of hepatitis C virus RNA in the plasma, peripheral blood mononuclear cells and liver specimens of patients with hepatitis C virus infection. The plus-strand RNA was detected in the plasma, peripheral blood mononuclear cells and the liver specimens, but the minus-strand RNA was only detected in the liver. These results suggest that hepatitis C virus replicates in the liver but not in peripheral blood mononuclear cells. This detection method for the minus strand of hepatitis C virus RNA should be useful for determining hepatitis C virus replication in tissues other than liver tissue.


Lack of increase in heterozygous alpha 1-antitrypsin deficiency phenotypes among patients with hepatocellular and bile duct carcinoma. Homozygous alpha 1-antitrypsin deficiency (PiZZ phenotype) is known to be associated with increased risk of cirrhosis and primary liver cancer. Although a relationship between heterozygous alpha 1-antitrypsin deficiency and chronic liver disease was suggested recently, it is still a matter of controversy whether such patients are at increased risk of liver cancer. The goal of this study was to determine the prevalence of heterozygous alpha 1-antitrypsin deficiency of different phenotypes among patients with primary hepatobiliary cancers. We studied 82 patients with primary hepatobiliary cancer; 59 had hepatocellular carcinoma and 23 had bile duct carcinoma. alpha 1-Antitrypsin quantitation and phenotyping were performed in each patient using standard methods. The distribution of the various Pi phenotypes was compared with that found in a normal population and reported elsewhere. Odds-ratio and chi 2 tests were used to measure the relative risk and the significance of association, respectively, between primary hepatobiliary cancers and heterozygous alpha 1-antitrypsin deficiency. Four patients in each of the cancer groups were heterozygous. Among the hepatocellular carcinoma patients, three had the PiMS phenotype and one had the PiMZ phenotype. Of these four heterozygous patients, only two had cirrhosis; one had cryptogenic cirrhosis and the other had hepatitis B virus-related cirrhosis. One noncirrhotic patient with a PiMZ phenotype had a fibrolamellar carcinoma. Of the four patients with bile duct carcinoma, three had the PiMS phenotype and one had the PiMZ phenotype. Of the four heterozygous patients, two had primary sclerosing cholangitis without associated inflammatory bowel disease and one patient had had previous biliary operations.(ABSTRACT TRUNCATED AT 250 WORDS)


Contribution of no-reflow phenomenon to hepatic injury after ischemia-reperfusion: evidence for a role for superoxide anion. Controversy exists as to the role of oxygen-derived free radicals in tissue injury and the no-reflow phenomenon in reperfusion injury after ischemia. In this study using an experimental rat model, left hepatic lobar ischemia followed by reperfusion resulted in an increase of serum glutamic pyruvic transaminase at 30 min with concomitant histological evidence of hepatocellular necrosis at 24 hr. In the in vivo liver microcirculation, reperfusion after ischemia resulted in an initial transient return of blood flow, but stasis of blood flow later developed in the liver sinusoids. Thus a no-reflow phenomenon in the microcirculation was demonstrated. Intravenous administration of a long-acting form of superoxide dismutase (half-life 6 hr, dose 4 or 8 mg/kg) significantly decreased the hepatocellular necrosis and reduced the microcirculatory stasis in the liver sinusoids. These studies established the important contribution of the no-reflow phenomenon in ischemia-reperfusion injury to the liver and the participation of superoxide anions in mediating the no-reflow phenomenon.


Multicentric development of pancreatic intraductal carcinoma through atypical papillary hyperplasia. We report a case of multiple intraductal carcinomas of the pancreas associated with diffuse atypical papillary hyperplasia. A 67-year-old Japanese man with a complaint of epigastric pain was examined by endoscopic retrograde pancreatography, which demonstrated multiple dilated branches of the pancreatic duct in the body and tail of the pancreas. Histologic examination on the resected pancreas showed diffuse atypical papillary hyperplasia in multiple dilated ducts associated with multiple intraductal carcinomas. Histologic features are described and multicentric carcinogenesis through atypical papillary hyperplasia is discussed.


Inhibition of release of arachidonic acid, superoxide, and IL-1 from human monocytes by monoclonal anti-HLA class II antibodies: effects at proximal and distal points of inositol phospholipid hydrolysis pathway. Incubation of human elutriator-purified monocytes with anti-HLA-DR or DQ antibody inhibited the release of arachidonic acid induced by serum-treated zymosan (STZ), a phagocytic stimulus that is known to induce inositol phospholipid hydrolysis and Ca2+ influx. However, only anti-HLA-DR antibody partially inhibited STZ-induced inositol phospholipid hydrolysis and concanavalin-A-induced Ca2+ influx. Incubation with anti-HLA-DR or -DQ antibody inhibited phorbol ester-induced AA release as well as superoxide production and IL-1 release. Inhibition of monocyte function by anti-class II antibodies was not accompanied by cAMP elevation. Furthermore, addition of exogenous db-cAMP and other agents (forskolin, cholera toxin, or 3-isobutyl-1-methyl-xanthine) that increase cAMP levels through different mechanisms, alone or in combination with anti-HLA antibodies, had no inhibitory effect on factor release. Our results demonstrate that perturbation of class II molecules down-modulates cell activation at more than one point of the signal transduction pathway with dominant inhibition distal to inositol phospholipid hydrolysis. They also suggest that the inhibition by anti-HLA class II antibody is probably not mediated via cAMP elevation.


Colchicine and methotrexate reduce leukocyte adherence and emigration in rat mesenteric venules. Colchicine and methotrexate are commonly used in the treatment of gout and rheumatoid arthritis, respectively; however the mechanism(s) of action of these drugs remain(s) unknown. The objective of this study was to determine whether colchicine and methotrexate can modify the adhesion and emigration of leukocytes in postcapillary venules that are exposed to inflammatory mediators such as platelet-activating factor (PAF) and leukotriene B4 (LTB4). The rat mesentery was prepared for in vivo microscopic observation. Venules with internal diameters ranging between 25 and 35 microns were selected for study. Erythrocyte velocity, vessel diameter, leukocyte rolling velocity, and the number of adherent (stationary for greater than or equal to 30 sec) and emigrated leukocytes were measured during superfusion of the mesentery with bicarbonate-buffered saline (BBS). Repeat measurements of adhesive and hemodynamic parameters were obtained between 50 and 60 min of superfusion with either 100 nM PAF or 20 nM LTB4 added to the superfusate. In some experiments, 1 microM of either colchicine or methotrexate was added to the superfusate containing either PAF or LTB4. Both PAF and LTB4 caused increases in leukocyte adherence and emigration and reductions in leukocyte rolling velocity and venular shear rate. Colchicine effectively prevented all of the adhesive and hemodynamic alterations induced by both inflammatory mediators, while methotrexate was largely effective in preventing the responses elicited by PAF, but not LTB4. These results indicate that the therapeutic actions of colchicine and methotrexate may result from the ability of these agents to interfere with the adhesion and emigration of leukocytes from postcapillary venules.


In situ expression of the IL-1-alpha and TNF-alpha genes by Reed-Sternberg cells in Hodgkin's disease. The histopathologic pattern of tissues involved by Hodgkin's disease (HD) suggests excessive activation of environmental cells by cytokines released by Hodgkin-Reed-Sternberg (HRS) cells, which are considered as the neoplastic component of HD lesions. This hypothesis has been supported by many studies performed in vitro using HD cell lines. In this study, we have tried to demonstrate the cytokine-producing cells in an environment as close as possible to the in vivo conditions, using in situ hybridization onto frozen sections of HD samples. [35S]-labelled single-stranded RNA probes were prepared by transcribing human cDNA fragments of the TNF-alpha and IL-1 alpha genes subcloned into appropriate vectors. A total of 19 specimens of HD lesions, including 7 cases of nodular sclerosing (NS) type and 12 cases of mixed cellularity (MC), were tested with both types of probes. Clinical stages included stage I (6 cases), stage II (4 cases), stage III (6 cases) and stage IV (3 cases). TNF-alpha and/or IL-1 alpha expression was observed in 12 among 19 HD cases. However, neither the histological type nor the clinical status of the patients was correlated with the profile of cytokine secretion. Most of the cytokine-producing cells could be identified as HRS cells due to their morphological appearance. In 3 cases, simultaneous analysis by immunohistochemistry and in situ hybridization showed that IL-1 alpha/TNF-alpha mRNA-producing cells simultaneously expressed the CD30 antigen, thereby confirming the HRS nature of these cells.


Prevalence of human papillomavirus types 16 and 18 in squamous-cell carcinoma of the penis: a retrospective analysis of primary and metastatic lesions by differential polymerase chain reaction. Human papillomaviruses (HPV), particularly types 16 and 18, may be carcinogenic effectors in a variety of human lower-genital-tract malignancies. Using the highly sensitive technique of differential polymerase chain reaction (D-PCR) with amplimers from the E6 open reading frames of HPV types 16 and 18, a retrospective analysis of a 20-year institutional experience with squamous-cell carcinoma of the penis (SCCP) was performed to determine the prevalence of these HPV types in this malignancy. Paraffin-embedded surgical specimens of primary (N = 27), locally recurrent (N = 5), and metastatic deposits (N = 26) from 29 patients with invasive SCCP were analyzed, as well as primary (N = 3) and recurrent (N = 2) specimens from 2 patients with penile carcinoma in situ (CIS) (Bowen's disease). Nine of the 29 (31%) patients had invasive SCCP containing HPV 16 or 18 DNA, with HPV 16 found in 8 (28%) and HPV 18 in I (3%); no patient had both. In 7 patients in which only tissue from metastatic sites was available, 2 had HPV 16 detected in 2 separate metastatic sites each. Specimens from both primary and metastatic sites were available in an additional 6 patients, and HPV 16 was detected in specimens from 3 of these 6 patients. HPV was detected in comparable copy number at both sites in each patient, indicating that HPV DNA may be a stable component within cancer cells during disease progression. Of patients with CIS only, 1 of 2 was positive for HPV 16, and upon multifocal recurrence, showed persistence of the virus at 2 separate sites. Southern blotting was performed to confirm the presence of type-specific HPV DNA and showed complete concordance with D-PCR, but discordant hybridization intensities for HPV 18 were noted between the control and positive patient specimens; sequence analysis of the patient specimen revealed 4 point mutations in the HPV-18 target segment. Comparison of the HPV-positive (both HPV 16 and HPV 18) and HPV-negative groups revealed no statistical differences between groups in patients age or ethnic origin, tumor histologic grade, or incidence of nodal involvement. Kaplan-Meier analysis of both overall and cause-specific survival likewise was not different between groups. These data, particularly the presence of HPV in metastatic deposits, provide strong evidence for an etiologic role of HPV type 16 (and possibly 18) in a substantial sub-set of patients from the southeastern United States who developed SCCP.


Experimental studies on biochemical modulation targeting topoisomerase I and II in human tumor xenografts in nude mice. Topoisomerase (topo) I and II are nuclear enzymes which are novel targets of cancer chemotherapy. A new camptothecin (CPT) analog, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyl-oxy-CPT (CPT-11), is a topo-I inhibitor with a higher activity and less toxicity than CPT. To investigate topo-I and -II-targeting chemotherapy in an in vivo model, we studied the effect of sequential or co-treatment using CPT-11 and adriamycin (ADR) a topo-II inhibitor, in 6 human tumor xenografts (2 esophageal, 2 gastric and 2 colon tumor lines). In sequential treatment, adriamycin was administered i.v. 24 hr after CPT-11 treatment, and no antagonistic effect of this treatment schedule was observed. ADR cytotoxicity was potentiated significantly by CPT-11 pretreatment in the case of 2 esophageal and 2 gastric tumor lines and 1 colon tumor line. On the other hand, co-treatment abolished the sensitivity to CPT-11 and ADR in all 6 tumor lines. Moreover, CPT-11 did not significantly enhance the cytotoxicity of other agents tested, including mitomycin C (MMC) and cisplatin (CDDP). Flow cytometry and dot-blot analyses showed that CPT-11 pretreatment induced an increase in the S-phase cell population with an increase of topo-II mRNA expression after 24 and 48 hr, respectively, in the esophageal and colon tumor lines. These results suggest that CPT-11 can modulate topo-11 levels to enhance the effect of topo-II inhibitors in some human tumors, and this suggests a new clinical method of topo-I and -II targeting chemotherapy for human solid tumors.


Visualization of the plasmin receptor on carcinoma cells. In order to visualize the receptor for plasmin and plasminogen present on human carcinoma cells, SW1116 and MCF7-MF cells were incubated with biotinylated plasminogen or plasmin and fluoresceinated streptavidin, and counterstained with propidium iodide. It was first demonstrated that biotinylation did not alter the binding properties of plasminogen and plasmin, provided that the biotinylation rate was around 2. Specific staining of tumor cells was obtained using these reagents. Images with green fluorescence were clearly visible as grains or contours at the surface of tumor cells. The localization of fluorescent grains was analyzed more precisely using confocal microscopy. The percentage of stained cells varied from one experiment to another between 10 and 60%. In no experiment were all the cells observed to be positive. Mitotic cells were more frequently stained than non-mitotic cells, suggesting a relationship between the presence of plasmin receptors and cell proliferation. This was confirmed by the use of Ki67 monoclonal antibody (MAb), as B-Pg-binding tumor cells generally had their nucleus stained by this antibody. Other images indicated staining of the extracellular matrix. Finally, 2 rat tumor-cell sub-lines of colonic origin (DHD K12 Pro-b and Reg-b) were shown to bind human biotinylated plasminogen, confirming the strong interspecies reactivity of plasmin receptors.


Effect of TGF-beta 1 on the EBV-induced transformation of human lymphocyte cultures. We tested the effect of TGF-beta 1 on the EBV-induced activation and immortalization of human B lymphocytes. In lymphocyte cultures of EBV-seropositive individuals, T cells can inhibit the EBV-induced transformation of B cells. We found that in the presence of TGF-beta 1 the transformation was more efficient, due to the inhibition of this function. TGF-beta 1 inhibited the EBV-induced proliferation of purified B-cell cultures. Its effect was strongest when added at the beginning of the culture. Added later, the inhibition gradually decreased and was lost by the 4th day. After 10 to 14 days, in the cultures initiated with TGF-beta 1, the number of cells was higher and they formed larger aggregates as compared with control cultures. Thus, TGF-beta 1 modifies EBV-induced transformation in a complex way. It inhibits the activation of B cells but does not affect those already activated. Once they acquire the immortalized state, the B cells are even stimulated by TGF-beta 1.


Detection of simian T-lymphotropic virus type I using the polymerase chain reaction. To develop the polymerase chain reaction (PCR) for the detection of simian T-lymphotropic virus type I (STLV-I) infection, cell lines or peripheral-blood mononuclear cells (PBMC) from 2 non-human primate species [African green monkeys (AGM), Cercopithecus aethiops; baboon, Papio cynocephalus] were evaluated for their STLV-I status using oligonucleotide primer pairs and probes specific for the tax and pol gene regions of the closely related human T-lymphotropic virus type I (HTLV-I). These PCR results were compared with serologic (Western blot assay) and viral culture (p24-antigen capture assay) data. PCR products for both gene regions were detected in established baboon, Japanese macaque and rhesus macaque STLV-I-producing cell lines. STLV-I tax and pol products were also detected in PBMC from 4 of 4 infected AGM and 4 of 4 infected baboons, each of which were also Western-blot-positive and p24-antigen-capture-positive. Of the remaining AGM (n = 7) and baboon (n = 1) which were PCR-negative, each was also Western-blot-negative and p24-antigen-capture-negative. Two seronegative and virus-culture-negative AGM were classified as PCR indeterminate with weak reactivity using tax primers. These primer pairs failed to amplify DNA from uninfected human PBMC, an uninfected human lymphoid cell line, a simian immunodeficiency virus macaque (SIVmac251)-infected cell line and a simian-retrovirus-type-D(SRV-D)-infected cell line. HTLV-II-pol-specific primer pairs failed to amplify DNA from STLV-I-infected cell lines and PBMC from STLV-I-infected monkeys. Further, HTLV-I pol and tax primer pairs successfully amplified RNA from HTLV-I- and STLV-I-infected cell lines by reverse transcriptase (RT)-PCR. We have demonstrated excellent specificity in the detection of STLV-I by PCR using these HTLV-I-derived primers and probes. Additionally, our data suggest that the tax and pol gene regions are conserved between HTLV-I and STLV-I strains found among these diverse species of non-human primates.


TGF-beta stimulation of endometrial and breast-cancer cell growth. Growth of the human endometrial carcinoma Ishikawa cell line was stimulated by transforming growth factor-beta1 when cultured in serum-containing and chemically defined culture medium. This response was not unique to endometrial carcinoma cells, as the breast-cancer cell line, T47-D, was similarly stimulated by TGF-beta 1. TGF-beta 1 stimulated growth of MCF-7 breast-cancer cells in chemically defined medium but inhibited growth of this cell line in serum-containing medium. The data provide a demonstration of a positive growth response to TGF-beta 1 in oestrogen-receptor-positive cells and do not support the hypothesis that this growth factor is simply a negative growth regulator in epithelial-cancer cell lines.


Antibody to hepatitis C virus in selected groups of a Canadian urban population. In an anonymous survey, 433 sera from Canadian individuals of selected categories were tested for the presence of antibody to hepatitis C virus (HCV) using a recombinant antigen-based immunoassay. About 50% of intravenous drug abusers (IVDA), 10% of transfusion recipients and an overall average of 7.9% of male homosexuals were reactive for antibody to HCV. Individuals with jaundice and negative hepatitis B virus (HBV) serology were not reactive for antibody to HCV compared with 26.7% of those with positive HBV serology. Similarly 58% of male Federal prisoners with positive HBV serology were also HCV-antibody reactive compared with 15% of those with negative HBV serology. A prevalence of 1.2% was recorded for individuals not in any of the above groups. Of 433 sera, 92 were reactive and the discrimination in absorbance values between reactive and not reactive samples was good except for 13 sera, eight of which gave values considerably higher than the average negative value and five which were just above the positive threshold.


Effect of copper ion on collagenase release. Its implication in corneal vascularization. The involvement of leukocytes in corneal neovascularization has been known for a long time. Recent observations suggest that collagenase from leukocytes may be a common mediator for prostaglandin E1 (PGE1)- and copper-induced corneal neovascularization. This study was designed to investigate the effect of copper ion on collagenase activity from leukocytes and other sources and leukocyte infiltration in the corneal angiogenic process induced by PGE1. These results demonstrated that collagenase production from leukocytes was stimulated in a dose-dependent manner by copper ion but not by PGE1. Copper chloride 0.2 mM produced the highest stimulation. Copper ion had no effect on collagenase release from corneal fibroblasts and capillary endothelium. There were more polymorphonuclear leukocytes (PMN) in the prostaglandin E1 treated corneas than in the control. The time-course study showed that the appearance of PMN reached a peak on day 2 and new vessel growth could not be identified until day 4. These results supported an earlier suggestion that leukocytes play a role in corneal neovascularization and further suggested that copper in corneal neovascularization can stimulate the release of collagenase from leukocytes.


An ocular model of adenovirus type 5 infection in the NZ rabbit. Ocular adenoviral infections occur worldwide, and currently, there is no ocular animal model for evaluating new antivirals or studying pathogenesis. With a paired-eye design, an ocular model was developed in 32 New Zealand rabbits following topical and intrastromal inoculation with a clinical isolate of adenovirus type 5 (Ad5 McEwen). Clinical signs of infection--conjunctivitis, corneal edema, subepithelial infiltrates, and iritis--and seroconversion were evaluated. Replicating virus on the ocular surface was determined by serial ocular titers. Reproducible acute ocular infection was demonstrated in 32 of 32 infected eyes (100%), with mean viral replication lasting for 8.3 days. Peak ocular viral titers (10(3) plaque forming units/ml) were achieved on day three after inoculation and represented a 2 log increase (100 times) over day one. Ocular viral replication was associated with acute conjunctivitis (24/34 eyes, 75%), and delayed-onset presumed immune-mediated clinical disease was associated with: blepharoconjunctivitis (21/32 eyes, 66%), iritis (29/32 eyes, 91%), corneal edema (32/32 eyes, 100%), and subepithelial corneal infiltrates (30/32 eyes, 94%). Seroconversion was demonstrated in 26 of 31 rabbits (84%). The study concludes that a potentially useful animal model of adenoviral ocular infection can be attained.


Identification and partial characterization of TGF-beta 1 receptors on trabecular cells. By using two specific receptor assays, we identified and partially characterized receptors for transforming growth factor-beta 1 (TGF-beta 1) on porcine trabecular cells in vitro. Cultured trabecular cells were incubated with labeled TGF-beta 1 and analyzed by flow cytometry. Pretreatment with trypsin or preincubation with cold TGF-beta 1 or a neutralizing antibody to TGF-beta 1 inhibited the binding of labeled TGF-beta 1. 125I-TGF-beta 1 was cross-linked covalently to cell surface receptors on the trabecular cells. By SDS-PAGE and autoradiography, we identified three labeled macromolecular species of receptors, two of which had apparent molecular weights greater than 212 kDa and one of which had an apparent molecular weight of approximately 80-90 kDa under reducing conditions. The low and high molecular weight species probably represent type II and type III TGF-beta 1 receptors, respectively. At concentrations of 0.5 and 1 ng/ml, activated TGF-beta 1 caused retraction and a marked decrease in the rate of proliferation and in the motility of trabecular cells in vitro. Our findings implicate TGF-beta 1 in the modulation of the functional homeostasis of trabecular cells and suggest that the aqueous humor contains a level of TGF-beta 1 which, once activated, is sufficient to exert a biologic effect on the trabecular meshwork.


Prolactin localization, binding, and effects on peroxidase release in rat exorbital lacrimal gland. Prolactin immunoreactivity has been detected in human tears and in lacrimal glands, and it has been suggested that this hormone might be a modulator of lacrimal secretion as well as a component of lacrimal gland fluid. The present study was designed to confirm the immunocytochemical localization of prolactin in the rat lacrimal gland, to determine the source of the prolactin, and to evaluate the acute effects of prolactin on lacrimal secretory function. We have confirmed that prolactin-like immunoreactivity is present in secretory vesicles of acinar cells of male and female Sprague-Dawley rats. Prolactin message was present at detectable levels in RNA extracts of lacrimal glands from males, indicating that at least a component of the prolactin-like immunoreactivity was the product of synthesis within the lacrimal glands. Crude membrane fractions from acini isolated from males bound 43.1 +/- 3.2 femtomoles prolactin/mg protein (mean +/- standard error of the mean; n = 6), which was significantly (P less than 0.01) more than comparable fractions from females (15.4 +/- 2.4 fmoles/mg protein, n = 6). Preincubating membranes at 65 degrees for 20 min to release endogenous ligands increased prolactin binding to 84.8 +/- 20.8 fmoles/mg protein for males and 63.8 +/- 17.4 fmoles/mg protein for females (P greater than 0.1), suggesting that, on average, similar numbers of receptors are expressed in acinar cells of male and female rats but a larger fraction of the receptors is occupied by endogenous prolactin-like peptides in females. Because prolactin binding triggers prolactin receptor internalization in various cell types, we propose that the prolactin-like immunoreactivity in lacrimal acinar cells of females has been accumulated from the circulation, while the immunoreactivity seen in males results, at least in part, from de novo synthesis. Ovine prolactin at concentrations of 10-20 ng/ml inhibited carbachol-induced peroxidase release by 19.6% +/- 6.9% (n = 8, P less than 0.02) but failed to alter peroxidase release in the absence of carbachol. These observations suggest that prolactin might function as an endocrine, paracrine, or autocrine modulator in the lacrimal gland.


The importance of peroxide and superoxide in the X-ray response. Radiation produces a number of damaging radicals as well as peroxide. The chief cellular protection against these radicals, their secondary reactants and peroxide is the cellular glutathione (GSH), GSH peroxidase, GSH-S-transferase (GSHTase), and catalase enzymes. Inhibition of cellular catalase alone does not enhance the aerobic radiation response because cellular GSH peroxidase is equally effective in reducing peroxide. However, inhibition of GSHTase, and partial inhibition of peroxidase by L-buthionine sulfoximine (LBSO)-linked GSH depletion, results in an increased aerobic radiation response. The major pathway for peroxide reduction is the GSH peroxidase. The enzyme is accountable for 70% inactivation of low peroxide concentrations. Catalase accounts for the remaining inactivation. However, it is difficult to assess the relative contributions of GSHTase and peroxidase to the inactivation of radiation-produced hydroperoxides. Our data suggest that GSH depletion results in the inhibition of cellular GSHTase before it inhibits GSH peroxidase. Therefore, part of the increased aerobic radiation response maybe due to cellular inability to reduce hydroperoxides. Peroxide is not a substrate for GSHTase. However, total inhibition of peroxidase by L-BSO plus N-ethylmaleimide (NEM) treatment maximizes the aerobic radiation response. Total inhibition of GSH-S-transferase and peroxidase would block both peroxide and hydroperoxide reduction.


Magnetic resonance spectroscopic measurement of cellular thiol reduction-oxidation state. Chromatographic and magnetic resonance spectroscopic measurements of thiol reduction-oxidation state in chemically constructed samples show close analytical agreement. This result, coupled with the synthesis of new probe molecules allowing greater sensitivity and lower toxicity, supports the development of an NMR method for non-invasive thiol redox measurement, an important variable in the response of tumors to radiation and chemotherapy.


Protection by WR-2721 of the toxicity induced by the combination of cisplatin and 5-fluorouracil. We evaluated the effects of WR-2721 and its metabolite WR-1065 on in vitro growth inhibition by 5-fluorouracil (5FU) and cisplatin (CDDP) and the effect of WR-2721 on in vivo toxicity and antitumor effect of 5FU and CDDP. In cell culture both WR-2721 and WR-1065 were not able to reverse growth inhibition caused by either 5FU or CDDP. Administration of WR-2721 i.p. at 525 mg/kg to mice resulted in a severe temperature drop to 27 degrees C; at 200 mg/kg hypothermia was less severe. WR-2721 failed to prevent 5FU toxicity, but the maximum tolerated dose of CDDP in the combination with 5FU (at 100 mg/kg) could be increased from 3 to 7 mg/kg. CDDP at 7 mg/kg enhanced leukopenia caused by 5FU at 100 mg/kg to 20% and thrombocytopenia to 40%; WR-2721 reduced leukopenia and prevented thrombocytopenia induced by the combination. Combination of CDDP, 5FU, and WR-2721 resulted in an enhanced antitumor activity against the murine colon tumor Colon 26 compared to 5FU alone and to 5FU combined with CDDP at their maximum tolerated dose.


A phase I study of WR-2721 in combination with total body irradiation (TBI) in patients with refractory lymphoid malignancies. This Phase I study was designed to establish the maximum tolerated dose (MTD) of WR-2721 when given twice weekly with total body irradiation (TBI) in the treatment of patients with advanced refractory lymphoid malignancies and to define the toxicities of this combination and schedule. Patients eligible for this study had advanced recurrent indolent non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia (CLL). Patients had symptomatic or progressive disease, a performance status of 0, 1, or 2, and adequate bone marrow, hepatic, and renal function. Only patients failing one or two regimens of prior chemotherapy were eligible. Patients who had received prior extended field irradiation were ineligible. Patients received TBI twice weekly (Tuesday and Friday) to a total of 10 doses at 15 cGy/fx. WR-2721 was given intravenously over 15 min beginning 30 min before irradiation. The escalation of WR-2721 was Level 1: 740 mg/m2 and Level 2: 910 mg/m2. The MTD of WR-2721 was that dose which produced predictable and reversible toxicity and would not interfere with patient well-being. Seven patients were entered onto the study, three at 740 mg/m2 and four at 910 mg/m2. Five patients had CLL and two patients small lymphocytic NHL. No patient had hypotension or nausea requiring reduction in dose level or even interruption of infusion of WR-2721. At 740 mg/m2 no grade 3 or 4 toxicities related to WR-2721 were observed, but two patients could not complete treatment because of TBI-induced prolonged thrombocytopenia following treatments 5 and 8. One patient completed all 10 treatments. At 910 mg/m2 of WR-2721, two patients requested removal from study because of malaise, one after 5 cycles and one after 7 cycles. One patient completed all 10 treatments. One patient was treated with a modified schedule of 7 treatments of 20 cGy/fx and tolerated and completed all treatments but developed significant thrombocytopenia following completion of treatment. No patients had disease progression during treatment. The median survival was 11 months. This study indicates that WR-2721 given at 910 mg/m2 twice weekly with TBI is well tolerated for at least 5 treatments and that 910 mg/m2 of WR-2721 is the MTD with this regimen. In view of the importance of total radiation dose in achieving a response with TBI, a dose escalation study of TBI with 910 mg/m2 of WR-2721 should be performed in patients with indolent non-Hodgkin's lymphoma.


Modification of radiation-induced carcinogenesis in mice by misonidazole and WR-2721. The effects of the radiosensitizer misonidazole (MISO) and the radioprotector WR-2721 on radiation-induced carcinogenesis in C3Hf/Kam mice were investigated. The right hind legs were exposed to graded single doses of gamma-rays. MISO and WR-2721 were given i.p. 30 min before irradiation at a dose of 1 mg/g and 0.4 mg/g, respectively. The RCD50, or radiation dose inducing tumors in 50% of the irradiated legs, was determined 650 days after treatment. The same animals were also checked for the effect of these drugs on hair loss and radiation-induced leg contractures. MISO enhanced radiation carcinogenesis by a factor of 1.43, whereas WR-2721 reduced it by a factor of 1.75. These effects on carcinogenesis correlated well with the modifying effects of the two agents on radiation-induced hair loss (early damage) and leg contractures (late damage).


Interim analysis of a randomized trial of radiation therapy of rectal cancer with/without WR-2721. The objectives of this randomized trial are to define the maximum tolerated dose of radiation therapy, at curative dose levels, that can be delivered following WR-2721, and to observe the anti-tumor effects and normal tissue responses. One hundred patients with inoperable, unresectable, or recurrent rectal cancer were stratified and randomized to radiation only, or WR-2721 and radiation. The entire pelvis is treated with 4 portals 4 times a week to a total of 4500 cGy (first level dose) in 5 weeks. WR-2721, 340 mg/m2 was given 15 minutes before radiation to the combination group. Subsequently, both groups received a conedown of 720 cGy in 4 days to 144(2) cm portals APPA, and if originally inoperable or unresectable 720 cGy in four days to second conedown of 64(2) cm. Patients were observed from 3 to 18 months (median = 12 months). No significant hypotension or hematologic toxicity occurred in the WR-2721 treated group. Mild to moderate emesis occurred in 80% of the courses. (No antiemetics were used.) Moderate or severe acute toxicities to normal tissues were observed less frequently in the WR-2721 arm. No moderate or severe late toxicities to the skin, mucous membrane, urinary bladder or intestine was observed in the WR-2721 group, however, 5 patients treated with radiation alone experienced moderate or severe late toxicity to these organs. No evidence of tumor protection was observed.


mRNA levels of catalytic subunits of protein phosphatases 1, 2A, and 2C in hepatocarcinogenesis. The mRNA levels of three phosphoseryl/phosphothreonyl protein phosphatases, PP1, PP2A and PP2C, in rat liver have been determined by Northern blot analysis in various stages of rat chemical hepatocarcinogenesis using a Solt-Farber model. Five weeks after administration of diethylnitrosamine, the mRNA levels of PP1 alpha, PP2A and PP2C were elevated 8, 29 and 11 times, respectively, as compared to those of the control livers. However, in primary hepatoma induced according to the Solt-Farber model, the mRNA levels of all three protein phosphatases were dramatically decreased to normal levels or even to much lower levels, whereas the mRNA level of glutathione S-transferase placental form, a tumor marker protein, was greatly elevated as compared with that of the control livers. In a poorly differentiated hepatoma AH13, a line of rat ascites hepatoma, the mRNA level of PP1 alpha was 5.6 times higher than that of the control livers, whereas the mRNA lever of PP2C was almost the same as that of the control livers and the level of PP2A mRNA was distinctly lower than that of the control livers. These data appear to suggest some involvement of protein phosphatases in hepatocarcinogenesis.


Reduced levels of transforming growth factor-beta type I receptor in human gastric carcinomas. The expressions of transforming growth factor beta (TGF-beta) and its receptor and TGF-beta inhibitory element (TIE)-binding protein were examined on human gastric carcinomas by Northern blot hybridization, immunohistochemistry, affinity labeling and gel retardation analysis. TGF-beta mRNA was expressed in tumor and normal tissues at various levels. Immunohistochemically, TGF-beta expression was confirmed to be present within tumor cells. Out of the 17 human gastric carcinoma tissues, 14 (82%) showed a reduction in the level of type I receptor (65 kDa) for TGF-beta when compared to corresponding normal mucosas. Interestingly, in seven of the 14 tumors the level of TIE-binding protein in the tumor tissue was lower than that in normal mucosa. Human gastric carcinoma cell line TMK-1, whose growth was inhibited by TGF-beta, had only type I receptor for TGF-beta and showed a high level of TIE-binding protein. Conversely, MKN-1, a TGF-beta-resistant cell line, exhibited an extremely low level of TGF-beta receptor and had no TIE-binding protein. These results overall indicate that although human gastric carcinoma cells produced TGF-beta, they showed a reduction in TGF-beta type I receptor and a low level of TIE-binding protein, resulting in escape from growth inhibition by TGF-beta.


Insulin induces translocation of the alpha 2 and beta 1 subunits of the Na+/K(+)-ATPase from intracellular compartments to the plasma membrane in mammalian skeletal muscle. Unlike glucose transport, where translocation of the insulin-responsive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane is the principal mechanism underlying insulin stimulation, no consensus exists presently for the mechanism by which insulin activates the Na+/K(+)-ATPase. We have investigated (i) the subunit isoforms expressed and (ii) the effect of insulin on the subcellular distribution of the alpha beta isoforms of the Na+/K(+)-ATPase in plasma membranes (PM) and internal membranes (IM) from rat skeletal muscle. Western blot analysis, using isoform-specific antibodies to the various subunits of the Na+/K(+)-ATPase, revealed that skeletal muscle PM contains the alpha 1 and alpha 2 catalytic subunits and the beta 1 and beta 2 subunits of the Na+ pump. Skeletal muscle IM were enriched in alpha 2, beta 1, and beta 2; alpha 1 was barely detectable in this fraction. After insulin treatment, alpha 2 content in the PM increased, with a parallel decrease in its abundance in the IM pool; insulin did not have any effect on alpha 1 isoform amount or subcellular distribution. The beta 1 subunit, but not beta 2, was also elevated in the PM after insulin treatment, but this increase originated from a sucrose gradient fraction different from that of the alpha 2 subunit. Our findings suggest that insulin induces an isoform-specific translocation of Na+ pump subunits from different intracellular sources to the PM and that the hormone-responsive enzyme in rat skeletal muscle is an alpha 2:beta 1 dimer.


Ceramide stimulates a cytosolic protein phosphatase. A sphingomyelin cycle has been identified whereby the action of certain extracellular agents results in reversible sphingomyelin hydrolysis and the concomitant generation of ceramide. Moreover, a cell-permeable ceramide, C2-ceramide (N-acetylsphingosine), is a potent modulator of cell proliferation and differentiation. We report herein that C2-ceramide, C6-ceramide, and natural ceramides activate a cytosolic serine/threonine protein phosphatase in a dose-dependent manner. Initial activation is observed at concentrations of ceramide as low as 0.1 microM with peak response occurring at 5-10 microM. However, other closely related sphingolipids, sphingosine and sphingomyelin, were largely inactive. Ceramide-stimulated phosphatase was inhibited by okadaic acid, an inhibitor of protein phosphatases, with an IC50 of 0.1-1 nM, depending on the concentration of ceramide. Ceramide-stimulated phosphatase was insensitive to Mg2+ and Mn2+ cations. Using sequential anion exchange chromatography, ceramide-stimulated phosphatase activity could be resolved from ceramide-nonresponsive phosphatases. The activity of partially purified enzyme was stimulated 3.5-fold by ceramide. The identification of a phosphatase as a molecular target for the action of ceramide defines a novel intracellular signaling pathway with potential roles in the regulation of cell proliferation and differentiation.


Regulation of neutrophil superoxide by antichymotrypsin-chymotrypsin complexes. The ability of neutrophils to generate free radicals is a crucial component of host defense (Babior, B. M. (1978) N. Engl. J. Med. 298, 659-668, 721-725. Neutrophil oxidants, however, can cause significant host tissue destruction (Weiss, S. J. (1989) N. Engl. J. Med. 320, 365-376), and the regulation of free radical production is not well understood. We have previously shown that recombinant antichymotrypsin (rACT), a serine protease inhibitor, inhibits superoxide production in intact neutrophils (Kilpatrick, L., Johnson, J. L., Nickbarg, E. B., Wang, Z., Clifford, T. F., Banach, M., Cooperman, B. S., Douglas, S. D., and Rubin, H. (1991) J. Immunol. 146, 2388-2393). Using a cell-free NADPH oxidase preparation, we now demonstrate that rACT alone has no effect on superoxide production and that antichymotrypsin-chymotrypsin (rACT.CT) complexes are required to inhibit superoxide, suggesting that neutrophil chymotrypsin-like proteases produce conformational changes in ACT, allowing it to become active in regulating superoxide production. Additionally, we have identified NADPH oxidase itself as the target for rACT.CT and have demonstrated that rACT.CT interferes specifically with activation of the NADPH oxidase without changing the Km for NADPH or the rate constant describing the rate-limiting step in activation. These observations suggest an important role for antichymotrypsin in the regulation of NADPH-oxidase activation, which is a prerequisite for neutrophil superoxide production, and predict possible therapeutic uses for rACT in conditions where unregulated neutrophil-free radical production has been implicated in the mechanism of tissue destruction.


A differential location of phosphoinositide kinases, diacylglycerol kinase, and phospholipase C in the nuclear matrix. Several enzymes involved in the phosphoinositide metabolism have been shown to be present in nuclei of rat liver and Friend cells. In this paper we demonstrate that nuclear matrices of mouse NIH 3T3-fibroblasts and rat liver cells, isolated by nuclease treatment and high salt extraction, contain phosphatidylinositol 4-kinase (PdtIns 4-kinase), phosphatidylinositol 4-phosphate 5-kinase (PtdIns(4)P 5-kinase), diacylglycerol kinase, and phospholipase C. By a selective extraction the nucleus can be dissected in the peripheral matrix (lamina-pore complex) and the internal matrix as shown by using marker antibodies. Surprisingly, PtdIns 4-kinase was found exclusively in the peripheral nuclear matrix, whereas PtdIns(4)P 5-kinase was found to be associated to internal matrix structures. Diacylglycerol kinase and phospholipase C activities were also preferentially detected in the internal matrix. These data demonstrate a differential localization of the phosphoinositide kinases in the nucleus and suggest that the phosphoinositide metabolism may play a specific role in the nucleus.


The 38-amino acid form of pituitary adenylate cyclase-activating polypeptide stimulates dual signaling cascades in PC12 cells and promotes neurite outgrowth. Vasoactive intestinal peptide (VIP) activates adenylylcyclase in sympathoadrenal cells at concentrations greater than 10(-6) M. We demonstrate here that two forms of a newly discovered peptide with homology to VIP named pituitary adenylate cyclase-activating polypeptide (PACAP) are much more potent activators of signal transduction in PC12 cells. Both the 27- and 38-amino acid forms of PACAP elevate cAMP levels in PC12 cells and stimulate adenylylcyclase in PC12 membranes, with an EC50 near 10(-9) M. PACAP38 additionally is a potent activator of the inositol lipid cascade in PC12 cells, elevating the content of inositol phosphates by 8-fold at 10(-8) M (EC50 = 7 x 10(-9) M). PACAP38 and PACAP27 have been thought to have essentially identical actions, but PACAP27 is 2-3 logs less potent in increasing inositol lipid levels. Moreover, PACAP38 at 10(-8) M is an effective inducer of neuronal morphology in PC12 cells, whereas PACAP27 is much less active in promoting neurite outgrowth. In contrast to the PACAP-preferring receptors on PC12 cells, another class of PACAP-binding sites with equal high affinities for VIP, PACAP38, and PACAP27 has been identified on several other cell types. We find that the cAMP content of rat CH3 pituitary cells, known to have high affinity VIP receptors, is in fact potently elevated by PACAP27 and PACAP38 as well as by VIP. However, PACAP38, even at 10(-6) M, is not capable of significant activation of inositol lipid turnover via these VIP/PACAP nondiscriminating sites.


Delivery of ion pumps from exogenous membrane-rich sources into mammalian red blood cells. Using polyethylene glycol-mediated fusion of ATP-ase-enriched (native) microsomes with red blood cells, we have delivered sarcoplasmic reticulum (SR) Ca-ATPase and kidney Na,K-ATPase into the mammalian erythrocyte membrane. Experiments involving delivery of the SR Ca-ATPase into human red cells were first carried out to assess the feasibility of the fusion protocol. Whereas there was little detectable 45Ca2+ uptake into control cells in either the absence or presence of extracellular ATP, a marked time-dependent uptake of 45Ca2+ was observed in the presence of ATP in cells fused with SR Ca-ATPase. Comparison of the kinetics of uptake into microsome-fused cells versus native SR vesicles supports the conclusion of true delivery of pumps into the red cell membrane. Thus, the time to reach steady state was more than two orders of magnitude longer in the (large) cells versus the native SR vesicles. Na,K-ATPase from dog and rat kidney microsomes were fused with red cells of humans, sheep, and dogs. Using dog kidney microsomes fused with dog red cells which are practically devoid of Na,K-ATPase, functional incorporation of sodium pumps was evidenced in ouabain-sensitive Rb+ uptake and Na+ efflux energized by intracellular ATP, as well as in ATP-stimulated Na+ influx and Rb+ efflux from inside-out membrane vesicles prepared from the fusion-treated cells. From analysis of the biphasic kinetics of ouabain-sensitive Na+ efflux under conditions of limited intracellular Na+ concentration, it is concluded that the kidney pumps are incorporated into a relatively small fraction (approximately 15%) of the red cells. This system provides a uniquely useful system for studying the behavior of native sodium pumps in a compartment (red cell) of small surface/volume ratio. The newly incorporated native kidney pumps, while of the same isoform as the endogenous red cell pump, behave differently from the endogenous red cell sodium pump with respect to their very low "uncoupled" Na+/O flux activity.