Two classes of DNA end-joining reactions catalyzed by vaccinia topoisomerase I. The ability of a eukaryotic DNA topoisomerase I to catalyze DNA rearrangements was examined in vitro using defined substrates and purified enzyme. Site-specific DNA strand cleavage by vaccinia topoisomerase I across from a nick generated double-strand breaks that could be religated to a heterologous blunt-ended duplex DNA regardless of the sequence of the acceptor molecule. Topoisomerase bound covalently at internal positions could religate the bound strand to an incoming acceptor provided that DNA molecule had sequence homology to the region 3' of the scissile bond. These end-joining reactions suggest two potential modes of topoisomerase-mediated recombination that differ in their requirements for DNA homology.


Interleukin-6 signal transducer gp130 mediates oncostatin M signaling. Oncostatin M (OM) is a multifunctional cytokine that is structurally and functionally related to interleukin 6 (IL-6) and leukemia inhibitory factor (LIF). The specific receptor for OM has been demonstrated (by chemical cross-linking) to be a 150-kDa protein in a number of cell lines. The IL-6 signal transducer, gp130, is also an affinity converter for the LIF receptor. It does not bind to either IL-6 or LIF, but associates with the alpha subunits of the receptors and transduces the signals. We examined the possible involvement of gp130 in OM binding and signaling. We demonstrate that: (a) anti-gp130 monoclonal antibodies (mAbs) block the inhibitory effect of OM on A375 cell growth, (b) the binding and cross-linking of 125I-OM to H2981 cells are completely abolished by anti-gp130 mAbs, (c) the cross-linked OM-receptor complex is immunoprecipitated by anti-gp130 mAbs, and (d) COS-7 cells transfected with the full-length cDNA encoding gp130 exhibit increased OM binding and cross-linking, which are also blocked by anti-gp130 mAbs. Therefore, we conclude that the 150-kDa OM binding protein previously characterized in a variety of cell lines is gp130. OM is the natural ligand for gp130 and gp130 mediates the biological responses of OM.


Structural investigations by extended X-ray absorption fine structure spectroscopy of the iron center of mitochondrial aconitase in higher plant cells. We have obtained iron K-edge extended x-ray absorption fine structure spectra of the plant mitochondrial aconitase in its active state, in the presence (aconitase (+)) and absence (aconitase (-)) of the substrate citrate. Analysis of the data indicates that oxygens are present in the first coordination shell, at an average Fe-O distance of 1.96/1.98 A (aconitase (+)/aconitase(-)). Part of these oxygens is provided by the citrate, which binds at 1.99 A from the iron in aconitase (+). The second shell (sulfur) contribution is split and is consistent with Fe-S distances of 2.30/2.29 and 2.56/2.59 A, and the third shell (iron) is consistent with an Fe-Fe distance of 2.83/2.84 A. Both Fe-S and Fe-Fe distances are longer than similar distances found in most Fe-S centers. A strong scattering at approximately 5 A has been identified as originating from an iron atom which is near to, but not part of, the Fe-S cluster. These data indicate that active plant mitochondrial aconitase contains a novel type of iron center.


Selective immunoneutralization of the multiple activities of Escherichia coli DNA polymerase I supports the model for separate active sites and indicates a complex 5' to 3' exonuclease. DNA polymerase I (pol I) from Escherichia coli has three well-defined activities: DNA polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. We have raised monoclonal antibodies to pol I which selectively neutralize each of these three activities, thus supporting the model of separate active sites for each activity, heretofore exclusively demonstrated with proteolytic fragments of pol I. Antibodies from each class could bind pol I in the presence of antibodies of another class, indicating the existence of significant spatial separation between each of the three sites. In addition, several of the neutralizing antibodies were able to distinguish particular activities of the 5'-3' exonuclease. One of them, for example, inhibited the RNase H activity but not the DNase activity. Two other antibodies could, in addition to inhibiting the polymerase and the 3'-5' exonuclease, either stimulate or inhibit the 5'-3' exonuclease depending upon the assay conditions, particularly the ionic strength.


Site-directed mutagenesis of tetraheme cytochrome c3. Modification of oxidoreduction potentials after heme axial ligand replacement. The nature of the axial ligands of a heme group is an important factor in maintaining the oxidation-reduction potential of a c-type cytochrome. Cytochrome c3 from Desulfovibrio vulgaris Hildenborough contains four bis-histidinyl coordinated hemes with low oxidation-reduction potentials. Site-directed mutagenesis was used to generate a mutant in which histidine 70, the sixth axial ligand of heme 4, has been replaced by a methionine. The mutant protein was expressed in Desulfovibrio desulfuricans G200 at a level similar to the wild type cytochrome. A model for the three-dimensional structure of D. vulgaris Hildenborough cytochrome c3 was generated on the basis of the crystal structure of D. vulgaris Miyazaki cytochrome c3 in order to investigate the effects of the H70M mutation. The model, together with NMR data, suggested that methionine 70 has effectively replaced histidine 70 as the sixth axial ligand of heme 4 without significant alteration of the structure. A large increase of at least 200 mV of one of the four oxidation-reduction potentials was observed by electrochemistry and is interpreted in terms of structure/potential relationships.


Differential regulation of glycogen synthase kinase-3 beta by protein kinase C isotypes. In cells, stimulation of protein kinase C (PKC) results in the dephosphorylation of specific residues proximal to the DNA binding domain of c-Jun, a major component of the AP-1 transcription factor. Since phosphorylation of this region of c-Jun inhibits interaction with DNA, this pathway may contribute to PKC activation of AP-1. To determine the mechanism(s) underlying this pathway, possible interactions between PKC and proteins implicated in c-Jun regulation are being investigated. Here it is shown that glycogen synthase kinase-3 beta (GSK-3 beta), a serine/threonine kinase that specifically targets the inhibitory c-Jun phosphorylation sites, is phosphorylated in vitro by particular forms of PKC (alpha, beta 1, gamma greater than beta 2; not epsilon). By contrast, the related GSK-3 alpha is not a substrate for any of these PKC isotypes. Phosphorylation of GSK-3 beta by PKC results in its specific inactivation. These results are consistent with a model in which activation of PKC stimulates c-Jun DNA binding by inhibiting its phosphorylation by GSK-3 beta.


NMR and isotopic exchange studies of the site of bond cleavage in the MutT reaction. The MutT protein, which prevents AT----CG transversions during DNA replication, hydrolyzes nucleoside triphosphates to yield nucleoside monophosphates and pyrophosphate. The hydrolysis of dGTP by the MutT protein in H(2)18O-enriched water, when monitored by high resolution 31P NMR spectroscopy at 242.9 MHz, showed 18O labeling of the pyrophosphate product, as manifested by a 0.010 +/- 0.002 ppm upfield shift of the pyrophosphate resonance, and no labeling of the dGMP product. This establishes that the reaction proceeds via a nucleophilic substitution at the beta-phosphorus of dGTP with displacement of dGMP as the leaving group. No exchange of 32P-labeled dGMP into dGTP was detected, indicating that water attacks dGTP directly or, less likely, an irreversibly formed pyrophosphoryl-enzyme intermediate. No exchange of 32P-labeled pyrophosphate into dGTP was observed, consistent with nucleophilic substitution at the beta-phosphorus of dGTP. Only six enzymes, all synthetases, have previously been shown to catalyze nucleophilic substitution at the beta-phosphorus of nucleoside triphosphate substrates. The MutT protein is the first hydrolase shown to do so.


A possible regulatory role for conserved promoter motifs in an adult-specific muscle myosin gene from mouse. The mouse gene encoding a myosin heavy chain (MHC) protein expressed in type IIB fibers of adult skeletal muscle has been cloned and its promoter isolated. A number of DNA sequence motifs are found within the first 2.5 kilobases of the promoter which are similar to motifs present in the promoters of other muscle-specific genes. One sequence located at approximately -940 base pairs corresponds to the motif called MEF1 which has been shown in other muscle genes to bind the myogenic regulatory factors of which MyoD is one example. The MEF1 site of this adult IIB MHC promoter does indeed bind MyoD although this factor is normally thought to be involved in early muscle cell differentiation. The IIB MHC promoter also has several motifs located in the first 200 base pairs which are strikingly conserved between this mouse gene and several chicken skeletal MHC genes. Of these evolutionarily conserved sequences, two motifs rich in A and T residues appear to be major contributors to the muscle-specific transcriptional activity of the mouse IIB MHC promoter when transfected into quail myogenic and non-myogenic cells. These observations suggest an important functional role for these AT-rich sequence motifs in the regulation of genes of the MHC family.


Molecular cloning of the rat vascular smooth muscle thrombin receptor. Evidence for in vitro regulation by basic fibroblast growth factor. To study thrombin's receptor-mediated effects on vascular cells, we cloned and characterized a cDNA encoding a rat smooth muscle cell thrombin receptor. A rat aortic smooth muscle (RASM) cell cDNA library was screened with a 500-base pair (bp) sequence from the human thrombin receptor, obtained by polymerase chain reaction (PCR) amplification of cDNA synthesized from human erythropoietic leukemia (HEL) cell mRNA with PCR primers based on the published human thrombin receptor sequence. Clone pRTHR17 contains a 3418-bp insert that includes 50 bp of the 5'-untranslated region and the entire coding and 3'-untranslated regions of the RASM cell thrombin receptor. The sequence of pRTHR17 is 85% similar, at the nucleotide level, and 78% similar, at the deduced amino acid level, to the human thrombin receptor. Although the putative thrombin cleavage and binding sites are present, there are significant differences between the rat and human receptors in their amino-terminal sequences. Detectable signals (consisting of a single band of 3.45 kb) are present by Northern analysis of mRNA from RASM cells, and rat lung, kidney, and testes, but not in aorta or other tissues probed. The results of Southern analysis of rat genomic DNA are consistent with the existence of a single copy of the gene encoding this receptor. The steady state thrombin receptor mRNA level is low in cultured growth-arrested RASM cells and not detectable in rat aorta. To determine whether regulation of the RASM cell thrombin receptor occurs under growth-stimulating conditions, growth-arrested RASM cells were treated with basic fibroblast growth factor (bFGF, recently proposed to be a major mitogen controlling vascular smooth muscle cell growth following injury (Lindner, V., and Reidy, M. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3739-3743)). There was a significant increase in thrombin receptor mRNA following the addition of bFGF. These data demonstrate that: 1) mRNA for a thrombin receptor similar to that reported from human megakaryocyte and hamster fibroblast cell lines is present in proliferating primary culture rat smooth muscle cells, 2) the most significant sequence differences are present in the amino-terminal tail of the thrombin receptor, and 3) the mRNA level for this receptor is regulated under growth-stimulating conditions in vitro.


Inhibition of the Na,K-ATPase by fluoride. Parallels with its inhibition of the sarcoplasmic reticulum CaATPase. Addition of lithium fluoride to a suspension of Na,K-ATPase undergoing turnover produced a slow (minutes) complete loss of ouabain-sensitive ATPase activity. Persistence of the effect in the presence of deferoxamine showed that fluoride inhibits independent of aluminum. The time course of onset of inhibition was adequately fit by a function corresponding to a monophasic transformation with a pseudo first-order rate constant (k(obs)). This constant varied hyperbolically with [Mg2+] (half-maximal effect at 9 mM Mg2+), whereas it increased with no sign of approaching saturation as the square of [F-], implying that inhibition requires binding of two fluorides/ATPase. The value of k(obs) was found to be increased by greater than 10-fold in the presence of potassium ([K+]1/2 = 0.6 mM) or ouabain. Sodium, ATP, and ADP, which favor the E1 form of the enzyme, had a protective effect. These results implicate the potassium-occluded MgE2(K2) complex as the main fluoride-susceptible form. Protection by Pi and orthovanadate suggests that fluoride exerts its effect at the phosphorylation site. Inhibition was reversible, although slowly, with t1/2 = 7 h at 37 degrees C. Sodium greatly accelerated reversal (t1/2 = 3 min with 150 mM Na+ present), and potassium antagonized this acceleration. The value of k(obs) for reactivation increased steeply with [Na+], with the sodium dependence being about the same at pH 8.0 as at pH 7.4. All of these effects have parallels to effects of fluoride on the sarcoplasmic reticulum CaATPase (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 5229-5235).


Affinity-purified c-Jun amino-terminal protein kinase requires serine/threonine phosphorylation for activity. The addition of phorbol esters to U937 leukemic cells stimulates the phosphorylation of c-Jun on serines 63 and 73. To isolate the protein kinase which stimulates this phosphorylation, we have used heparin-Sepharose chromatography followed by affinity chromatography over glutathione-Sepharose beads bound with a fusion protein of glutathione S-transferase and amino acids 5-89 of c-Jun (GST-c-Jun). Using this procedure we purify a 67-kDa protein which is capable of phosphorylating GST-c-Jun as well as the complete c-Jun protein. By making mutations in serines 63 and 73 and then creating a fusion protein with GST (GST-c-Jun mut), we demonstrate that this protein kinase specifically phosphorylates these sites in the c-Jun amino terminus. Treatment of purified c-Jun amino-terminal protein kinase (cJAT-PK) with phosphatase 2A inhibits its ability to phosphorylate GST-c-Jun. This inactivated enzyme can be reactivated by phosphorylation with protein kinase C (PKC), although PKC is not capable of phosphorylating the GST-c-Jun substrate. Because v-Jun cannot be phosphorylated in vivo, we compared the ability of cJAT-PK to bind to GST-v-Jun or GST-c-Jun mut. The cJAT-PK bound 50-fold better to GST-c-Jun mut than GST-v-Jun suggesting that the delta domain which is missing in v-Jun plays a role in binding the cJAT-PK. These results suggest that there is a protein kinase cascade mediated by protein phosphatases and PKC which regulates c-Jun phosphorylation.


Interleukin 3, granulocyte-macrophage colony-stimulating factor, and transfected erythropoietin receptors mediate tyrosine phosphorylation of a common cytosolic protein (pp100) in FDC-ER cells. Receptors for the hematopoietic growth factors erythropoietin, interleukin 3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are members of a structurally related receptor superfamily. Interestingly, while none of these receptors encode tyrosine kinase activities, induced tyrosine phosphorylation has been observed in various responsive cells stimulated with each factor. Toward defining possible common transduction pathways which are activated by these three cytokines, we have studied induced protein phosphorylation in murine myeloid FDC-P1 cells stably transfected with an erythropoietin receptor cDNA (FDC-ER cells). FDC-ER cells proliferate in response to erythropoietin (Quelle, D. E., and Wojchowski, D. M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4801-4805), and presently are shown to rapidly phosphorylate a M(r) 100,000 cytosolic protein (pp100) at tyrosine residues in response to this factor. Phosphorylation of pp100 also is induced in FDC-P1 and FDC-ER cells in response to IL-3 or GM-CSF. Importantly, quantitative analyses showed identical concentration dependencies for factor-induced pp100 phosphorylation and induced cell proliferation. Moreover, a selective loss of proliferative responsiveness to GM-CSF in FDC-ER cells was associated with a reduced capacity of GM-CSF to induce pp100 phosphorylation. Finally, limited differences in tryptic phosphopeptide maps of pp100 as isolated following exposure to erythropoietin, IL-3, or GM-CSF were observed, suggesting that these factors also may preferentially induce phosphorylation of pp100 at distinct sites. These findings are consistent with a role for pp100 as a common cytosolic transducer in the apparently convergent pathways of erythropoietin-, IL-3-, and GM-CSF-induced proliferation of myeloid progenitor cells.


Estrogen modulates Ca(2+)-independent lipid-stimulated kinase in the rabbit corpus luteum of pseudopregnancy. Identification of luteal estrogen-modulated lipid-stimulated kinase as protein kinase C delta. Rabbit corpora lutea were tested for the presence of phosphorylative responses sensitive to estrogen. Luteal Ca(2+)-independent lipid-stimulated kinase activity was detected by phosphorylation of the endogenous substrate, p76. Estrogen treatment, by way of estradiol-17 beta implant, increased levels of the lipid-stimulated phosphoprotein 2-3-fold throughout pseudopregnancy. Midpseudopregnant rabbit luteal extracts were further evaluated to determine the identity of the lipid-stimulated kinase. Results of low pH-activated phosphorylation were consistent with the identification of p76 as an autophosphorylated member of the protein kinase C (PKC) family. Partial purification of the luteal lipid-stimulated kinase was performed using sequential DEAE-cellulose/hydroxylapatite chromatographies and using gel filtration. Western immunoblot with type-specific anti-PKC delta antiserum showed coelution of kinase p76 activity with immunoreactive PKC delta. Immunoblot analysis confirmed that luteal levels of PKC delta were increased by estrogen treatment.


Two thrombin-activated Ca2+ channels in human platelets. The regulation of extracellular Ca2+ entry into fura-2-loaded human platelets was examined following stimulation with thrombin. In the presence of external Ca2+, stimulation of platelets with thrombin resulted in a rapid increase, followed by a plateau, in intracellular Ca2+ concentration ([Ca2+]i). Pretreatment with wortmannin, a specific inhibitor of myosin light chain kinase, suppressed only the plateau phase and had no effect on the initial rapid increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thrombin induced a transient and relatively small increase in [Ca2+]i caused by Ca2+ release from internal stores. When Ca2+ was added subsequently to the Ca(2+)-free medium within 10 min after thrombin activation, a marked increase in [Ca2+]i was seen, reflecting thrombin-stimulated external Ca2+ entry. With the Ca(2+)-free medium, wortmannin did not affect either the Ca2+ mobilization from the internal stores or the rapid external Ca2+ entry at early time points (within 5 s) after thrombin stimulation, whereas it significantly inhibited Ca2+ entry when Ca2+ was added later (at 3 min). Wortmannin inhibition of this late Ca2+ entry and that of 20-kDa myosin light chain phosphorylation after thrombin stimulation were dose- and preincubation time-dependent and correlated well with each other. These results suggest that two different channels are responsible for Ca2+ entry in human platelets at the early and late phases of thrombin stimulation and that the channel responsible for the late phase of Ca2+ entry may be activated by a mechanism involving myosin light chain kinase.


A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function. The determinants on the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) required for lysosomal enzyme sorting have been analyzed. Mouse L cells deficient in the mannose 6-phosphate/insulin-like growth factor-II receptor were transfected with normal bovine CD-MPR cDNA or cDNAs containing mutations in the 67-amino acid cytoplasmic tail and assayed for their ability to target the lysosomal enzyme cathepsin D to lysosomes. Cells expressing the wild-type bovine CD-MPR sorted 67 +/- 2% of newly synthesized cathepsin D compared with the base-line value of 47 +/- 1%. The presence of mannose 6-phosphate in the medium did not affect the efficiency of cathepsin D sorting, indicating that the routing of the ligand-receptor complex is completely intracellular. Mutant receptors with the carboxyl-terminal His-Leu-Leu-Pro-Met67 residues deleted or replaced with alanines sorted cathepsin D below the base-line value. A mutant receptor with the outermost Pro-Met residues replaced with alanines sorted cathepsin D better than the wild-type receptor, indicating that the essential residues for sorting are the His-Leu-Leu sequence. Disruption of a putative casein kinase II phosphorylation site at Ser57 had no detectable effect on sorting. The mutant receptor with the five-amino acid deletion was able to bind to a phosphopentamannose affinity column, proving that its ligand binding site was grossly intact. Resialylation experiments showed that this mutant receptor recycled from the cell surface to the Golgi at a rate similar to the normal CD-MPR, indicating that the defect in sorting is at the level of the Golgi.


Nucleotide binding by the poliovirus RNA polymerase. Cross-linking of ribonucleoside triphosphates (NTPs) to specific binding sites on the poliovirus RNA-dependent RNA polymerase has been performed by ultraviolet irradiation and by reduction of oxidized nucleotide-protein complexes. The latter method approached a cross-linking efficiency of 1 NTP/molecule of enzyme. Nucleotide competition experiments suggested that the same binding site is occupied by all NTPs. Analysis of peptides produced by proteinase Glu-C and trypsin digestion and labeled with [32P]GTP indicated that a lysine residue between Met-189 and Lys-228 in the polymerase was cross-linked to NTP. Nucleotide binding was exploited for rapid purification of the enzyme by GTP-agarose affinity chromatography. In addition, a set of cloned, modified polymerase molecules with reduced or absent polymerization activity was analyzed for binding efficiency to a GTP-agarose column. Some mutations eliminated GTP binding, whereas others generated proteins with varying affinities for GTP. Incubation of the poliovirus polymerase with high concentrations of NTP, particularly GTP, resulted in a dramatic protection against heat denaturation and activity loss. These data suggest that nucleotide binding results in an alteration of the enzyme conformation or the stabilization of an ordered conformation.


Identification of the yeast TOP3 gene product as a single strand-specific DNA topoisomerase. The TOP3 gene of the yeast Saccharomyces cerevisiae was postulated to encode a DNA topoisomerase, based on its sequence homology to Escherichia coli DNA topoisomerase I and the suppression of the poor growth phenotype of top3 mutants by the expression of the E. coli enzyme (Wallis, J.W., Chrebet, G., Brodsky, G., Golfe, M., and Rothstein, R. (1989) Cell 58, 409-419). We have purified the yeast TOP3 gene product to near homogeneity as a 74-kDA protein from yeast cells lacking DNA topoisomerase I and overexpressing a plasmid-borne TOP3 gene linked to a phosphate-regulated yeast PHO5 gene promoter. The purified protein possesses a distinct DNA topoisomerase activity: similar to E. coli DNA topoisomerases I and III, it partially relaxes negatively but not positively supercoiled DNA. Several experiments, including the use of a negatively supercoiled heteroduplex DNA containing a 29-nucleotide single-stranded loop, indicate that the activity has a strong preference for single-stranded DNA. A protein-DNA covalent complex in which the 74-kDa protein is linked to a 5' DNA phosphoryl group has been identified, and the nucleotide sequences of 30 sites of DNA-protein covalent complex formation have been determined. These sequences differ from those recognized by E. coli DNA topoisomerase I but resemble those recognized by E. coli DNA topoisomerase III. Based on these results, the yeast TOP3 gene product can formally be termed S. cerevisiae DNA topoisomerase III. Analysis of supercoiling of intracellular yeast plasmids in various DNA topoisomerase mutants indicates that yeast DNA topoisomerase III has at most a weak activity in relaxing negatively supercoiled double-stranded DNA in vivo, in accordance with the characteristics of the purified enzyme.


Inhibitory autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase analyzed by site-directed mutagenesis. Initial autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) occurs at Thr286 (the "autonomy" site) and converts the kinase from a Ca(2+)-dependent to a partially Ca(2+)-independent or autonomous enzyme. After removal of Ca2+/calmodulin, the autonomous kinase undergoes a "burst" of inhibitory autophosphorylation at sites distinct from the autonomy site which may be masked in the presence of bound calmodulin. This burst of Ca(2+)-independent autophosphorylation blocks the ability of calmodulin to activate the kinase. We have used site-directed mutagenesis to replace putative inhibitory autophosphorylation sites within the calmodulin binding domain of recombinant alpha-CaM kinase with nonphosphorylatable alanines and examined the effects on autophosphorylation, kinase activity, and calmodulin binding. Although prominent Ca(2+)-independent autophosphorylation occurs within the calmodulin binding domain at Thr305, Thr306, and Ser314 in wild-type alpha-CaM kinase, the inhibitory effect on kinase activity and calmodulin binding is retained in mutants lacking any one of these three sites. However, when both Thr305 and Thr306 are converted to alanines the kinase does not display inhibition of either activity or calmodulin binding. Autophosphorylation at either Thr305 or Thr306 is therefore sufficient to block both binding and activation of the kinase by Ca2+/calmodulin. Thr306 is also slowly autophosphorylated in a basal reaction in the continuous absence of Ca2+/calmodulin. Autophosphorylation of Thr306 by the kinase in either its basal or autonomous state suggests that in the absence of bound calmodulin, the region of the autoregulatory domain surrounding Thr306, rather than the region near the autonomy site, lies nearest the peptide substrate binding site of the kinase.


M-CAT binding factor is related to the SV40 enhancer binding factor, TEF-1. M-CAT binding factor (MCBF) governs the activity of the cardiac troponin T gene promoter. M-CAT motifs have also been implicated recently in the regulation of other contractile protein genes which like cardiac troponin T, do not require direct interaction with MyoD1 or related factors for activity. Mutational analysis of the M-CAT motif revealed that it can be functionally replaced by a regulatory motif of the SV40 enhancer which binds human transcription factor TEF-1. Biochemical analyses show that MCBF from muscle nuclei is indistinguishable from TEF-1 in terms of specificity of binding site recognition, fractionation on DNA-agarose, and apparent molecular weight. In addition, antibodies raised against amino- and carboxyl-terminal regions of human TEF-1 also bind to MCBF from chicken muscle. We conclude, therefore, that MCBF is closely related to TEF-1. Finally, MCBF/TEF-1 is highly enriched in the nuclei of striated muscle, as compared with other tissues, consistent with a role in muscle-specific promoter regulation.


Ouabain-resistant transfectants of the murine ouabain resistance gene contain mutations in the alpha-subunit of the Na,K-ATPase. A 6.5-kilobase murine genomic DNA fragment isolated by Levenson et al. (Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1489-1493) (called the ouabain resistance gene) has been shown to produce ouabain resistance in primate cells. Preliminary sequence information has revealed no homology with the coding sequence of the Na,K-ATPase. We have introduced this murine sequence into monkey and murine cells in an attempt to characterize its mechanism of action. In our experiments, transfection of this DNA fragment is associated with the low frequency (1 in 8 x 10(5) cells) appearance of ouabain-resistant clones of CV1, COS, and NIH 3T3 cells, an event not seen in control transfections. Characterization of a new clone of ouabain-resistant CV1 cells (called OR8 cells) revealed a 5-fold increase in the IC50 for ouabain inhibition of rubidium uptake and a 10-fold increase in cell survival on ouabain. Although the murine sequence was detectable in Southern blots of ouabain-resistant cells soon after transfection, this exogenous DNA was rapidly lost despite continued exposure to ouabain. Furthermore, we were unable to detect message expression by this genomic sequence in any of the three cell types tested. Instead, we found that all three ouabain-resistant cell lines exhibited point mutations in a domain of the alpha-subunit that has been implicated in ouabain sensitivity (H1-H2). One of these mutations (Asp121-Asn121 in OR8 cells) has been previously reported to cause ouabain resistance (Price, E.M., Rice, D.A., and Lingrel, J.B. (1989) J. Biol. Chem. 264, 21902-21906). Other novel mutations in the H2 transmembrane domain were also detected. We postulate that the "ouabain resistance gene" is important in the early selection process on ouabain but that the permanent ouabain-resistant phenotype is due to a stable mutation in one allele of the alpha-subunit of the Na,K-ATPase.


TraK protein of conjugative plasmid RP4 forms a specialized nucleoprotein complex with the transfer origin. Conjugative transfer of the self-transmissible IncP plasmid RP4 requires the product of the RP4 traK gene. By using the phage T7 expression system, the traK gene product was efficiently overproduced and purified to near homogeneity. traK encodes a basic protein (pI = 10.7) of 14.6 kDa that, as shown by DNA fragment retention assay, interacts exclusively with its cognate transfer origin. The apparent equilibrium constant K(app) for the complex of TraK and oriT-DNA was estimated to be 4 nM. Footprinting experiments using DNase I or hydroxyl radicals indicate that several TraK molecules interact specifically with an intrinsically bent region of oriT, covering a range of almost 200 base pairs. The TraK target sequence maps in the leading region adjacent to the relaxation nick site and recognition sequences involved in relaxosome formation but does not overlap them. Specific interactions between TraK and the DNA occur only on one side of the double helix. Electron microscopy of TraK-oriT complexes demonstrates that binding of TraK to its recognition region apparently shrinks the length of the target DNA, suggesting that the nucleic acid becomes wrapped around a core of TraK molecules. Formation of this structure could be favored by the presence of the sequence-directed bend in the TraK recognition region.


Regulation by thyrotropin-releasing hormone (TRH) of TRH receptor mRNA degradation in rat pituitary GH3 cells. In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) down-regulates TRH receptor (TRH-R) mRNA (Fujimoto, J., Straub, R.E., and Gershengorn, M.C. (1991) Mol. Endocrinol. 5, 1527-1532), at least in part, by stimulating its degradation (Fujimoto, J., Narayanan, C.S., Benjamin, J.E., Heinflink, M., and Gershengorn, M.C. (1992) Endocrinology 130, 1879-1884). Here we show that TRH regulates RNase activity in GH3 cells and that specific mRNA sequences are needed for in vivo regulation of TRH-R mRNA by TRH. TRH affected RNase activity in a biphasic manner with rapid stimulation (by 10 min) followed by a decrease to a rate slower than in control lysates within 6 h. This time course paralleled the effects of TRH on degradation of TRH-R mRNA in vivo. The regulated RNase activity was in a polysome-free fraction of the lysates and was not specific for TRH-R RNA. A truncated form of TRH-R RNA that was missing the entire 3'-untranslated region (TRHR-R5) was more stable than full-length TRH-R RNA (TRHR-WT). In contrast to TRHR-WT mRNA, TRHR-R5 mRNA and TRHR-D9 mRNA, which was missing the 143 nucleotides 5' of the poly(A) tail, were not down-regulated by TRH in stably transfected GH3 cells as their rates of degradation were not increased. These data show that TRH regulates RNase activity in GH3 cells, that the 3'-untranslated region bestows decreased stability on TRH-R mRNA and that the 3' end of the mRNA is necessary for regulation by TRH of TRH-R mRNA degradation. We present an hypothesis that explains specific regulation of TRH-R mRNA degradation by TRH in GH3 pituitary cells.


Transcriptional regulatory elements for basal expression of cytochrome P450IIC genes. To analyze the transcriptional regulatory elements in rabbit cytochrome P450IIC genes, varying lengths of the 5'-flanking regions of CYP2C1 and CYP2C2 were fused to a luciferase reporter gene. Promoter activity was assayed by transfection into HepG2 cells, a hepatic cell line, and monkey kidney COS-1 cells, a nonhepatic cell line. Activity of the CYP2C1 promoter in HepG2 cells increased slightly with progressive 5' deletions of the 5'-flanking region from nucleotide -3600 to -1500 relative to the transcription start site. Additional deletions to -900, -358, and -116 each reduced activity by about 50%, and deletion of the sequence from -116 to -67 reduced activity by a factor of 12. Activity of the CYP2C2 promoter increased about 3-fold with progressive 5' deletions of sequence from nucleotide -3500 to -410. In contrast, deletions of sequences from -251 to -193 and from -133 to -64 reduced promoter activity by factors of 2 and 8, respectively. In COS-1 cells, the maximum activities of the CYP2C1 and CYP2C2 promoters normalized to a Rous sarcoma viral promoter were about 10-20% of that in the HepG2 cells. The changes in activity between different constructions in COS-1 cells largely paralleled those in the HepG2 cells except for deletions of the sequences -133 to -64 and -116 to -67 for CYP2C1 and CYP2C2, respectively, which produced the largest reduction of promoter activity in HepG2 cells but had little effect in COS-1 cells. These results show that HepG2-specific regulatory elements are present in the regions between -120 and -65 in both genes. Nuclear proteins from HepG2 cells, but not from COS-1 cells, bound to sequences within these regions, and the binding was inhibited by an oligonucleotide containing a sequence conserved in rabbit P450IIC genes which has been designated the HepG2-specific P450 2C factor-1 (HPF1) motif. Mutation of this sequence eliminated the binding of nuclear proteins and reduced transcriptional activity 25-fold. The HPF1 binding sequence is conserved in CYP2A, CYP2C, and CYP2D genes and resembles the binding motif for hepatic nuclear factor-4. These results demonstrate that CYP2C1 and CYP2C2 contain several potential regulatory elements for basal expression, including one HepG2-specific sequence that may be important for liver expression.


Role of protein phosphatases in insulin-like growth factor II (IGF II)-stimulated mannose 6-phosphate/IGF II receptor redistribution. The regulated expression of mannose 6-phosphate/insulin-like growth factor II (M6P/IGF II) receptors in plasma membranes has previously been shown to be accompanied by marked changes in the phosphorylation state of the receptors (Corvera, S., Folander, K., Clairmont, K. B., and Czech, M. P. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7567-7571). In the present study we show that protein phosphatase 2A dephosphorylates the human M6P/IGF II receptor in vitro. Incubation of human fibroblasts with okadaic acid, a specific inhibitor of this phosphatase, resulted in a depletion of M6P/IGF II receptors at the cell surface without affecting their internalization kinetics. The phosphorylation state of the remaining cell surface receptors was 3-fold increased. Thus, the endocytosis rate of M6P/IGF II receptors appears to be unaltered by increased phosphorylation. While the decreased cell surface expression of receptors was reversible upon removal of okadaic acid the IGF II-induced redistribution of M6P/IGF II receptors to the plasma membrane (Braulke, T., Tippmer, S., Neher, E., and von Figura, K. (1989) EMBO J. 8, 681-686) was irreversibly inhibited by the phosphatase inhibitor. Receptor redistribution in response to protein kinase C activation was not affected by okadaic acid. These results suggest that the cell surface expression of M6P/IGF II receptor can be regulated by phosphatase-dependent and -independent pathways. In addition, the phosphorylation state and the steady-state cell surface number of transferrin receptors were not affected by okadaic acid, whereas it impaired the IGF II-stimulated receptor redistribution similarly as for M6P/IGF II receptors. The data indicate that okadaic acid-sensitive protein phosphatases may play a general role in terms of IGF II-modulated receptor recycling.


Nerve growth factor-induced phosphorylation cascade in PC12 pheochromocytoma cells. Association of S6 kinase II with the microtubule-associated protein kinase, ERK1. Microtubule-associated protein (MAP) kinases form a group of serine/threonine kinases stimulated by various growth factors such as nerve growth factor (NGF) and hormones such as insulin. Interestingly, MAP kinases are thought to participate in a protein kinase cascade leading to cell growth as they have been shown to phosphorylate and activate ribosomal protein S6 kinase. To further evaluate the interactions between the different components of this cascade, we looked at the possible coprecipitation of MAP kinase activator(s) or MAP kinase substrate(s) with MAP kinase. Using antipeptides to the C terminus of the M(r) 44,000 MAP kinase, ERK1, and cell extracts from unstimulated or NGF-treated PC12 cells, we obtained in addition to MAP kinase itself coprecipitation of a protein with a M(r) in the 90,000 range. We further show that this protein is a protein kinase since it becomes phosphorylated on serine residues, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to a polyvinylidene difluoride membrane. In vitro phosphorylation performed before sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates NGF-sensitive phosphorylation of this 90-kDa protein on both serine and threonine; the serine phosphorylation is likely to be due to autophosphorylation, and the threonine phosphorylation due to phosphorylation by the copurifying MAP kinase. Furthermore, immunoprecipitation of this 90-kDa protein was obtained with antibodies to S6 kinase II. Finally, using in situ chemical cross-linking, we were able to demonstrate in intact cells the occurrence of an anti-ERK1 immunoreactive species with a molecular mass of approximately 125,000 compatible with a complex between ERK1 and a 90-kDa S6 kinase. Taken together, our observations demonstrate that the 44-kDa MAP kinase is associated, in intact PC12 cells, with a protein kinase which is very likely to be S6 kinase II. In conclusion, our data represent strong evidence for a physiological role of the MAP kinase-S6 kinase cascade in PC12 cells. Finally, our antipeptides provide us with a powerful tool to search for additional physiologically relevant substrates for MAP kinase, a key integrator enzyme for growth factors and hormones.


Transcriptional regulation of the human insulin receptor promoter. The ability of the human insulin receptor promoter to direct expression of a linked chloramphenicol acetyltransferase gene was assessed in transient transfections into HepG2 and Hela cells. A 5'-deletional analysis of the promoter showed that regions between -646 and -489 were important for the activity of the proximal promoter. In addition, a possible negative regulatory element was identified between -1311 and -877 and a positive element between -1823 and -1311. DNase I footprint and gel retardation analysis showed that multiple factors bind to the human insulin receptor promoter. In particular, DNase I protection patterns were observed over the Sp1 sites at -620 to -599 and -438 to -392, a TC box at -533, four homopyrimidine/homopurine sites clustered around -1150, and a site at -1420 that contains the motif TGGCCC which has been shown to bind the liver-specific transcription factor LF-A1.


Atrial natriuretic peptide inhibits the production and secretion of endothelin from cultured endothelial cells. Mediation through the C receptor. Atrial natriuretic peptide (ANP) and endothelin-1 (ET-1) are vasoactive peptides produced in cells of the cardiovascular system. We examined the effects of ANP on ET-1 transcription, production (translation), and secretion in cultured bovine aortic endothelial cells (BAEC). ANP and C-ANP 4-23 (a specific ligand for the C or non-guanylate cyclase receptor) equipotently inhibited the synthesis of prepro-ET-1 and ET-1 proteins in BAEC by at least 50%. Both of these forms of ANP and another C receptor specific ligand, nanopiperazine ANP (11-15)-NH2, inhibited ET-1 secretion by as much as 55%. LY 83583, an inhibitor of ANP-induced cGMP generation, failed to reverse the ANP-induced inhibition of ET-1 secretion. This further indicated that the guanylate cyclase-linked B receptor is not involved. The decreased ET-1 secretion caused by C-ANP 4-23 was reversed by 8-bromo-cAMP or amiloride, which prevents ANP-induced inhibition of cAMP. We also found that ANP and C-ANP 4-23 augmented ET-1 mRNA levels in BAEC by prolonging the mRNA half-life. ANP or cycloheximide comparably inhibited ET-1 translation while increasing ET-1 mRNA levels, suggesting that the two events are related. These results indicate that ANP inhibits ET-1 protein production and secretion while stabilizing the ET-1 mRNA. The effects of ANP are mediated through the C receptor and are probably the result of ANP inhibiting the generation of cAMP. These findings suggest a potentially important new function for this receptor to mediate, in part, the interactions of ANP and ET in the vasculature.


N-linked oligosaccharide chains of the insulin receptor beta subunit are essential for transmembrane signaling. Insulin receptor (IR) is a glycoprotein possessing N-linked oligosaccharide side chains on both alpha and beta subunits. The present study focuses for the first time on the potential contribution of N-linked oligosaccharides of the beta subunit in the processing, structure, and function of the insulin receptor. To investigate this point, a receptor mutant (IR beta N1234) was obtained by stable transfection into Chinese hamster ovary cells of an IR cDNA modified by site-directed mutagenesis on the four potential N-glycosylation sites (Asn-X-Ser/Thr) of the beta subunit. The mutated receptor presents an alpha subunit of 135 kDa, indistinguishable from the wild type alpha subunit, but the beta subunit has a reduced molecular mass (80 kDa instead of 95 kDa) most likely due to the absence of N-glycosylation. Metabolic labeling experiments indicate a normal processing and maturation of this mutated receptor which is normally expressed at the surface of the cells as demonstrated by indirect immunofluorescence. The affinity of the mutant for insulin (Kd = 0.12 nM) is similar to that of the wild type receptor (Kd = 0.12 nM). However, a major defect of the mutated IR tyrosine kinase was assessed both in vitro and in vivo by (i) the absence of insulin-stimulated phosphorylation of the poly(Glu-Tyr) substrate in vitro; (ii) the reduction of the insulin maximal stimulation of the mutated IR autophosphorylation in vitro (2-fold stimulation for the mutant receptor as compared to a 7-fold stimulation for the wild type); and (iii) a more complex alteration of the mutated receptor tyrosine autophosphorylation in vivo (3-fold increase of the basal phosphorylation and a 4-fold simulation of this phosphorylation as compared to the wild type receptor, the phosphorylation of which is stimulated 14-fold by insulin). The physiological consequences of this defect were tested on three classical insulin cellular actions; in Chinese hamster ovary IR beta N1234, glucose transport, glycogen synthesis, and DNA synthesis were all unable to be stimulated by insulin indicating the absence of insulin transduction through this mutated receptor. These data provide the first direct evidence for a critical role of oligosaccharide side chains of the beta subunit in the molecular events responsible for the IR enzymatic activation and signal transduction.


Structural elements required for the cooperative binding of the herpes simplex virus origin binding protein to oriS reside in the N-terminal part of the protein. The origin binding protein (OBP) of herpes simplex virus type 1 is required to activate a viral origin of replication in vivo. We have used intact OBP as well as a truncated form of the protein expressed in Escherichia coli to investigate the protein-protein interactions, as well as the protein-DNA interactions involved in the formation of a nucleoprotein complex at a viral origin of replication (oriS) in vitro. The salient findings demonstrate that the N-terminal part of OBP is required for the cooperative binding of OBP to three sites (boxes I, II, and III) within oriS. A detailed model for the interaction of OBP with the viral origins of replication oriS and oriL is presented.


Glucose-induced degradation of the MDH2 isozyme of malate dehydrogenase in yeast. MDH2, the nonmitochondrial isozyme of malate dehydrogenase in Saccharomyces cerevisiae, was determined to be a target of glucose-induced proteolytic degradation. Shifting a yeast culture growing with acetate to medium containing glucose as a carbon source resulted in a 25-fold increase in turnover of MDH2. A truncated form of MDH2 lacking amino acid residues 1-12 was constructed by mutagenesis of the MDH2 gene and expressed in a haploid yeast strain containing a deletion disruption of the corresponding chromosomal gene. Measurements of malate dehydrogenase specific activity and determination of growth rates with diagnostic carbon sources indicated that the truncated form of MDH2 was expressed at authentic MDH2 levels and was fully active. However, the truncated enzyme proved to be less susceptible to glucose-induced proteolysis, exhibiting a 3.75-fold reduction in turnover rate following a shift to glucose medium. Rates of loss of activity for other cellular enzymes known to be subject to glucose inactivation were similarly reduced. An extended lag in attaining wild type rates of growth on glucose measured for strains expressing the truncated MDH2 enzyme represents the first evidence of a selective advantage for the phenomenon of glucose-induced proteolysis in yeast.


Calcium depletion blocks proteolytic cleavages of plasma protein precursors which occur at the Golgi and/or trans-Golgi network. Possible involvement of Ca(2+)-dependent Golgi endoproteases. The effects of calcium depletion on the proteolytic cleavage and secretion of plasma protein precursors were investigated in primary cultured rat hepatocytes and HepG2 cells. When the cells were incubated with A23187, the calcium-specific ionophore, in a medium lacking CaCl2, precursors of serum albumin and the third and fourth components of complement, C3 and C4, respectively, were found to be released into the medium. The addition of ionomycin or EGTA to the medium inhibited the processing of pro-C3 as well. Blocking the secretory pathway either at the mixed endoplasmic reticulum/Golgi in the presence of brefeldin A or at the endoplasmic reticulum/tubular-vesicular structure at a reduced temperature caused accumulation of pro-C3 within hepatocytes or HepG2 cells, indicating that the cleavage of the precursor occurs at a later stage of the secretory pathway. Once the blockade was released by incubating the cells either in the brefeldin A-free medium or at 37 degrees C, the secretion of plasma proteins resumed, irrespective of the presence of A23187. However, the processing of pro-C3 was almost completely inhibited in the presence of A23187, with only the precursor being released into the medium, implying that a decline in Ca2+ levels within the cell modulates the activity of a Golgi endoprotease responsible for the cleavage of pro-C3. When incubated with isolated Golgi membranes, pro-C3 secreted from Ca(2+)-depleted cells was cleaved in vitro into their subunits in the presence of Ca2+ but not in its absence, pointing to the involvement of a Ca(2+)-dependent Golgi endoprotease in the processing of pro-C3. These results collectively suggest that calcium depletion blocks the proteolytic cleavages of plasma protein precursors presumably by exhausting a Ca2+ pool available to the Ca(2+)-dependent processing enzyme(s) located at the Golgi and/or trans-Golgi network.


Transient elevation of cardiac beta-adrenoceptor responsiveness and receptor number in the streptozotocin-diabetic rat. 1. The effects on cardiac responsiveness of diabetes of up to 12 weeks duration has been examined in streptozotocin-pretreated rats. 2. Two weeks of diabetes resulted in a supersensitivity of isolated left atria and papillary muscles to isoprenaline, which was associated with an increase in the density of ventricular [3H]-dihydroalprenolol binding sites. 3. The beta-adrenoceptor supersensitivity was still evident in both tissues after 4 weeks of diabetes, but in left atria the supersensitivity was reduced compared with that observed at 2 weeks, while for papillary muscles it was greater than at 2 weeks. 4. Following 12 weeks of diabetes, responses of papillary muscles to isoprenaline were similar to controls, while beta-mediated responses of left atria were significantly depressed. 5. The alpha-adrenoceptor-mediated responses of cardiac tissues to phenylephrine were similar to controls following diabetes of 2 or 4 weeks duration. At 12 weeks, however, papillary muscle alpha-adrenoceptor-mediated responses were enhanced. The change in ventricular responsiveness to phenylephrine was not accompanied by any change in [3H]-prazosin binding to ventricular membranes. 6. The results demonstrate a transient elevation of cardiac beta-adrenoceptor sensitivity and receptor density during acute diabetes and illustrate the time- and tissue-dependence of diabetes-induced changes in cardiac adrenoceptor sensitivity.


Effect of [D-Phe6] bombesin (6-13) methylester, a bombesin receptor antagonist, towards bombesin-induced contractions in the guinea-pig and rat isolated urinary bladder. 1. The effect of [D-Phe6] bombesin (6-13) methylester (OMe), a newly developed potent antagonist of bombesin receptors, has been investigated against bombesin-induced contractions of the guinea-pig and rat isolated urinary bladder. 2. Bombesin (0.1 nM-10 microM) produced a concentration-dependent contraction of the guinea-pig isolated bladder which approached the same maximum response as KCl (80 mM). The response to bombesin was antagonized in a competitive manner (rightward shift of the concentration-response curve without depression of the maximal response) by [D-Phe6] bombesin (6-13) OMe (0.3-10 microM). Degree of antagonism was concentration-dependent between 0.3 and 3 microM (dose ratios = 2.4, 9 and 39 in the presence of 0.3, 1, 3 microM of the antagonist). However, a larger concentration (10 microM) of the antagonist was not more effective (dose ratio = 36) than 3 microM. 3. Neither the action of bombesin nor the activity of the antagonist was influenced by peptidase inhibitors (bestatin, captopril and thiorphan 3 microM each) or by atropine, indomethacin, chlorpheniramine and desensitization of P2x purinoceptors by alpha, beta methylene ATP. 4. The bombesin antagonist was ineffective against contraction of the guinea-pig urinary bladder produced by the NK-1 tachykinin receptor-selective agonist, [Sar9] substance P sulphone. The action of the NK-1 receptor agonist was antagonized by L 668, 169 (3 microM), a cyclic peptide tachykinin antagonist. L 668, 169 had no effect toward bombesin-induced contraction. 5. The bombesin antagonist (1-10 microM) had no effect against the non-adrenergic non-cholinergic response of the guinea-pig isolated urinary bladder to electrical field stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)


Preparation of dense hydroxylapatite or rhenanite containing bioactive glass composites. The effect of time at 600 degrees C and of small additions of Al2O3 and B2O3 on the sintering of two composite materials of (1) hydroxylapatite (Ca10(PO4)6(OH)2) and bioactive glass (SiO2-CaO-P2O5-Na2O) or (2) rhenanite (CaNaPO4) and bioactive glass were studied. Scanning microscopy, quantitative EDX, x-ray diffraction, helium gas density measurements, and diametral measurements were performed on the resulting composites. No reactions were observed with the SEM or XRD between the hydroxylapatite particles and the glass matrix within sufficient sintering times to achieve maximum density. The rheunanite-containing composites were observed to form Na2O2CaO3SiO2 crystals by x-ray diffraction, probably as a result of dissolution of the rhenanite particle surfaces into the glass phase, the crystals formed in the glass or at the interface of the glass, and the ceramic particles. However, within the short sintering times needed to achieve maximum density the rhenanite particles remained mostly intact. The rhenanite-containing materials gave better results than the hydroxylapatite-containing materials. The glass composition had a great effect on the densification process.


Intracellular retention of membrane-anchored v-sis protein abrogates autocrine signal transduction. An important question regarding autocrine transformation by v-sis is whether intracellularly activated PDGF receptors are sufficient to transform cells or whether activated receptor-ligand complexes are required at the cell surface. We have addressed this question by inhibiting cell surface transport of a membrane-anchored v-sis protein utilizing the ER retention signal of the adenoviral transmembrane protein E3/19K. A v-sis fusion protein containing this signal was retained within the cell and not transported to the cell surface as confirmed by immunofluorescent localization experiments. Also, proteolytic maturation of this protein was suppressed, indicating inefficient transport to post-Golgi compartments of the secretory pathway. When compared with v-sis proteins lacking a functional retention signal, the ER-retained protein showed a diminished ability to transform NIH 3T3 cells, as measured by the number and size of foci formed. In newly established cell lines, the ER-retained protein did not down-regulate PDGF receptors. However, continued passage of these cells selected for a fully transformed phenotype exhibiting downregulated PDGF receptors and proteolytically processed v-sis protein. These results indicate that productive autocrine interactions occur in a post-ER compartment of the secretory pathway. Transport of v-sis protein beyond the Golgi correlated with acquisition of the transformed phenotype. Furthermore, suramin treatment reversed transformation and upregulated the expression of cell surface PDGF receptors, suggesting an important role for receptor-ligand complexes localized to the cell surface.


Portosystemic collateral shunts originating from the left portal veins in portal hypertension: demonstration by color Doppler flow imaging. Color Doppler flow imaging was performed in 121 patients with portal hypertension. Portosystemic collateral shunts originating from the left portal veins were seen in 41 of the patients. A single collateral shunt was seen in 27 of these, and multiple collateral shunts were seen in the other 14. Collateral shunts running in the ligamentum teres were seen in 26 of the 41 patients; the veins ran through the liver parenchyma in 25 of these. B-mode ultrasound imaging could not clearly demonstrate vascular structures in 55% of the collateral shunts. Color Doppler flow imaging provided a clear picture of the course of the portosystemic collateral shunts originating from the left portal vein.


Transcriptional downregulation of gap-junction proteins blocks junctional communication in human mammary tumor cell lines. Subtractive hybridization, selecting for mRNAs expressed in normal human mammary epithelial cells (NMECs) but not in mammary tumor cell lines (TMECs), led to the cloning of the human gap junction gene connexin 26 (Cx26), identified by its sequence similarity to the rat gene. Two Cx26 transcripts derived from a single gene are expressed in NMECs but neither is expressed in a series of TMECs. Northern analysis using rat Cx probes showed that Cx43 mRNA is also expressed in the normal cells, but not in the tumor lines examined. Connexin genes Cx31.1, Cx32, Cx33, Cx37, and Cx40 are not expressed in either normal cells or the tumor lines examined. In cell-cell communication studies, the normal cells transferred Lucifer yellow, while tumor cells failed to show dye transfer. Both Cx26 and Cx43 proteins were immunolocalized to membrane sites in normal cells but were not found in tumor cells. Further analysis demonstrated that Cx26 is a cell-cycle regulated gene expressed at a moderate level during G1 and S, and strongly up-regulated in late S and G2, as shown with lovastatin-synchronized NMECs. Cx43, on the contrary is constitutively expressed at a uniform low level throughout the cell cycle. Treatment of normal and tumor cells with a series of drugs: 5dB-cAMP, retinoic acid, okadaic acid, estradiol, or TGFb had no connexin-inducing effect in tumor cells. However, PMA induced re-expression of the two Cx26 transcripts but not of Cx43 in several TMECs. Thus Cx26 and Cx43 are both downregulated in tumor cells but respond differentially to some signals. Modulation of gap-junctional activity by drug therapy may have useful clinical applications in cancer.


Color Doppler imaging of the cystic artery. Color Doppler ultrasonography was used to examine the gallbladder vascular supply in 30 volunteers and in 30 biliary lithotripsy patients pre- and 2 hours posttreatment. A preliminary study of 10 cases of acute cholecystitis was also performed. A cystic artery waveform was obtained in 24 of the volunteers, 18 pre- and postlithotripsy patients, and in 1 of 10 patients with acute cholecystitis. The mean pulsatility indices obtained were 1.04 in the volunteers, 0.98 pre- and 1.02 postlithotripsy (p greater than 0.1). This study shows that the normal gallbladder blood supply can be consistently identified using color Doppler flow imaging and that there is no appreciable difference in the cystic artery pulsatility index following medium-energy shock-wave extracorporeal lithotripsy. Preliminary results in acute cholecystitis suggest that flow is decreased below the sensitivity of the color Doppler imaging system used in this investigation.


Diagnosis of portal vein tumor thrombus by pulsed Doppler ultrasonography. The blood flow of portal vein thrombus was studied by pulsed Doppler ultrasonography in 21 patients with tumor thrombus and 14 with blood clot thrombus. Pulsatile waves were observed in 19 of the 21 with tumor thrombus, with backward continuous waves in 3, and forward/backward continuous waves in 2. In contrast, forward continuous waves were observed in 2 of the 14 with blood clot thrombus. By color imaging, the blood flow could be observed in 9 of 14 with tumor thrombus, but in 1 of 7 with blood clot thrombus. Pulsatile and backward continuous waves were characteristic of tumor thrombus.


Continuous recording of changes in intracranial pressure using interference echoes of ultrasonic wave: a preliminary report of practicality and clinical evaluation. We attempted to examine intracranial pressure (ICP) noninvasively using probes receiving interference echoes of ultrasound from thin layers between the skull and the surface of the cerebrum. The reception of echo through bone flaps less than 5-mm thick was good. Normal adults (4) and patients (4) were examined by attaching ultrasound probes to the frontotemporal scalp. Recordings were performed during rest, Valsalva maneuver, hyperventilation, and pumping of a shunt-flushing device. Ultrasound recordings of ICP were compared with those of the scalp using two different time windows. The effect of scalp vessels on the ultrasound recordings of ICP was evaluated. The waves from a pressure transducer and the ultrasound probe were similar. This method was found to be useful for ICP recording.


Measurements of uterine volume: a comparison between measurements by ultrasonography and by water displacement. The uterine volume was measured in 30 patients 24 hours before hysterectomy by ultrasonography using the prolate ellipsoid formula. At hysterectomy the volumes of the same uteri were measured again by water displacement. A comparison is made between the two methods of measurements. Although the correlation between the methods is high (r = 0.964) and the mean percentage bias very small (+2.0%), the agreement between the methods for 95% of subjects is between -33.6, 8% and +37.6%. This is a potentially large disagreement relative to the typical size of the uterus, indicating that ultrasonic uterine volume measurement by the prolate ellipsoid method is not adequately precise for clinical management of the individual patient.


Tuberculous pleural effusions: ultrasonic diagnosis. Twenty patients with tuberculous pleural effusions were studied with ultrasonography. In 18 patients, ultrasonography demonstrated regular pleural thickening which was less than 1 cm except in 1 case. In 4 cases there were a few pleural nodules, whereas in 2 cases the pleural surface showed small nodularity. The latter finding may be diagnostic for a tuberculous etiology. Eighteen patients had multiple, delicate, mobile septations in the effusions, and a lattice-like appearance had formed in 6 cases. Computed tomography was obtained in 7 cases, and pleural thickening was demonstrated in 6 of them. Ultrasonography is a useful imaging modality in the diagnosis of tuberculous pleurisy.


A statistical study of cochlear nerve discharge patterns in response to complex speech stimuli. Cochlear nerve discharge patterns in response to the synthesized consonant-vowel stimulus /da/ were collected from a population of 223 auditory-nerve fibers from a single cat. For each nerve fiber, discharges were measured from multiple, independent stimulus presentations, with the means and variances of the post-stimulus time histograms and Fourier transforms of response generated from the ensemble of stimulus presentations. The statistics were not consistent with those predicted via an inhomogeneous Poisson counting process model. Specifically, the synchronized components as measured by the Fourier transforms of post-stimulus time histogram responses have variances that are as much as a factor of 3 times lower than the predicted by the Poisson model. To account for the non-Poisson nature of the statistics, the Markov process model of Siebert/Gaumond was adopted. Using the maximum-likelihood and minimum description length algorithms, introduced by Miller [J. Acoust. Soc. Am. 77, 1452-1464 (1985)] and Mark and Miller [J. Acoust. Soc. Am. 91, 989-1002 (1992)], estimates of the stimulus and recovery functions were computed for each nerve fiber. Then, Markov point processes were simulated with the stimulus and recovery functions generated from these nerve fibers. The statistics of the simulated Markov processes are shown to have almost identical first- and second-order statistics as those measured for the population of auditory-nerve fibers, and demonstrates the effectiveness of the Markov point process model in accounting for the correlation effects associated with the discharge history-dependent refractory properties of auditory nerve response.


Uncoupling of atrial natriuretic peptide extraction and cyclic guanosine monophosphate production in the pulmonary circulation in patients with severe heart failure. This study was designed to evaluate the role of endogenous atrial natriuretic peptide in the pulmonary circulation in patients with chronic heart failure. Plasma atrial natriuretic peptide concentrations in patients with heart failure have been reported to be higher than those in normal subjects and to increase as the severity of heart failure progresses. Although endogenous atrial natriuretic peptide is thought to improve the condition of patients with heart failure by reducing preload and afterload, recent findings have indicated that a high plasma atrial natriuretic peptide level is a prognostic predictor in patients with heart failure. To evaluate the pathophysiologic role of endogenous atrial natriuretic peptide in the pulmonary circulation, plasma atrial natriuretic peptide and cyclic guanosine monophosphate (cGMP) levels were determined in the main pulmonary artery and pulmonary capillary wedge region in 80 patients with chronic congestive heart failure (New York Heart Association functional classes II to IV). The plasma atrial natriuretic peptide level decreased significantly from the main pulmonary artery to the pulmonary capillary wedge region, whereas the plasma cGMP level increased significantly from the main pulmonary artery to the pulmonary capillary wedge region. In patients with mild chronic heart failure (n = 50), the plasma atrial natriuretic peptide level correlated with the cGMP level in the main pulmonary artery (gamma = 0.71, p less than 0.001). The atrial natriuretic peptide extraction level, calculated as (Atrial natriuretic peptide in the main pulmonary artery--Atrial natriuretic peptide in the pulmonary capillary wedge region) x Cardiac output x (1-hematocrit/100) (ng/min), also correlated with the cyclic guanosine monophosphate production level, calculated as (cGMP in the pulmonary capillary wedge region--cGMP in the main pulmonary artery) x Cardiac output x (1-hematocrit/100) (nmol/min) (gamma = 0.78, p less than 0.001). In contrast, such correlations were not found in patients with severe chronic heart failure (n = 30). In these patients, the atrial natriuretic peptide extraction level was significantly higher but there was no significant difference in the cGMP production level between the two groups (mild and severe chronic heart failure). Therefore, the molar ratio of cGMP production to atrial natriuretic peptide extraction in the pulmonary circulation was significantly lower in patients with severe chronic heart failure (88 +/- 16 vs. 480 +/- 41, p less than 0.001). These results indicate that down-regulation of atrial natriuretic peptide receptors coupled to guanylate cyclase may occur in the pulmonary vascular beds of patients with severe chronic heart failure.


Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180. Bullous pemphigoid (BP) is an autoimmune skin disease that is characterized by the presence of subepidermal blisters resulting from a disruption of the adhesive interactions between basal keratinocytes and the cutaneous basement membrane. Autoantibodies from patients suffering from this disorder recognize two epidermal antigens, BP180 and BP230, both of which have been localized to the hemidesmosome, a transmembrane structure of stratified, squamous epithelia that functions in cell-matrix adhesion. In the present study we report the primary structural analysis of BP180 based on the sequence of a series of overlapping cDNA clones encompassing 4,669 bases of the BP180 transcript. A polymerase chain reaction-based protocol was used to confirm the contiguity of the cDNA segments. This cloned portion of the BP180 transcript was found to contain one long open reading frame (ORF) 4.596 bases in length. This ORF encodes a polypeptide of 155,000 Daltons with an isoelectric point of 9.7. The carboxy-terminal half of BP180, a stretch of 916 amino acids, consists of 15 collagen domains of variable length (15 to 242 amino acids) that are separated from one another by short stretches of non-collagen sequences. Located 76 amino acids upstream of the collagenous region is a putative transmembrane domain, a structural feature that distinguishes BP180 from all of the well-characterized members of the collagen family. This membrane-spanning domain is predicted to function as a signal-anchor sequence, directing the C-terminal collagenous segment of this protein to the exterior of the cell. The putative intracellular domain is highly basic with an isoelectric point of 10.37. This molecular analysis predicts that the BP180 antigen is an integral membrane protein of the hemidesmosome that contains a long extracellular collagenous tail. This combination of structural features suggests that BP180 may function as a cell-matrix adhesion molecule, with the collagenous region acting as a potential site of interaction with basement membrane components. Autoantibody-mediated disruption of such an adhesive interaction may play a critical role in the development of sub-epidermal blisters in BP patients.


Expression of growth hormone receptor, insulin-like growth factor 1 (IGF-1) and IGF-1 receptor mRNA and proteins in human skin. A cDNA corresponding to the membrane receptor for growth hormone (GH) was amplified by polymerase chain reaction (PCR) directly from human skin. The cDNA was cloned and found to have complete sequence homology to the extracellular domain of human liver GH receptor (GH-R). Northern analysis, using the cloned GH-R as probe, revealed relatively higher levels of GH-R transcripts in cultured human dermal fibroblasts compared to cultured keratinocytes or keratome biopsies. Semi-quantitative PCR analysis indicated that the level of GH-R mRNA in cultured melanocytes was similar to that in fibroblasts. The receptor protein encoded by GH-R mRNA in fibroblasts was shown by affinity cross-linking to have an apparent M(r) of 115-120 kDa, similar to that of 3T3-F442A fibroblasts used as a control. mRNA transcripts for the major mediator of GH actions, insulin-like growth factor 1 (IGF-1), were detected by PCR in fibroblasts, melanocytes, and keratome biopsies, but not in keratinocytes. In contrast, IGF-1 receptor mRNA were abundant in cultured keratinocytes and skin biopsies, as determined by Northern analysis. IGF-1 but not GH (5-50 ng/ml) promoted clonal proliferation of cultured keratinocytes. In contrast, GH (10 ng/ml) after 5 d markedly increased fibroblast cell numbers (70%, p less than 0.009) over 0.2% serum control. These data indicate that human skin cells possess the molecular elements necessary to respond to GH and raise the possibility that GH may influence skin growth in vivo.


Role of liver endothelial and Kupffer cells in clearing low density lipoprotein from blood in hypercholesterolemic rabbits. The role of liver endothelial and Kupffer cells in the hepatic uptake of cholesterol-rich low density lipoprotein (LDL) was studied in rabbits fed a diet containing 2% (w/w) cholesterol for 3 weeks. 125I-labeled tyramine cellobiose-labeled cholesterol-rich LDL was injected intravenously into rabbits, and parenchymal and nonparenchymal liver cells were isolated 24 h after injection. The hepatic uptake was 9 +/- 3% of injected dose in cholesterol-fed rabbits 24 h after injection, as compared to 36 +/- 9% in control-fed rabbits (n = 6 in each group; significant difference, P less than 0.005). Endothelial and Kupffer cells took up 2.7 +/- 0.5% and 1.2 +/- 0.8% of injected dose in the hypercholesterolemic rabbits, as compared to 1.9 +/- 0.8% and 0.8 +/- 0.3% in control animals. The amount accounted for by the parenchymal cells was markedly reduced in the cholesterol-fed rabbits to 7.3 +/- 2.7% of injected dose, as compared to 32.8 +/- 7.6% in controls (P less than 0.02). On a per cell basis, the nonparenchymal cells of cholesterol-fed rabbits took up as much LDL as the parenchymal cells (0.6 +/- 0.2, 0.7 +/- 0.1, and 0.6 +/- 0.4% of injected dose per 10(9) parenchymal, endothelial, and Kupffer cells, respectively). This is in marked contrast to the control animals, in which parenchymal cells took up about 6 times more LDL per cell than endothelial and Kupffer cells (3.2 +/- 0.9, 0.7 +/- 0.3, and 0.5 +/- 0.1% of injected dose per 10(9) cells). Thus, 30% of the hepatic uptake of LDL in the cholesterol-fed rabbits took place in nonparenchymal cells, as compared to 6% in controls. Consistent with these data, the concentrations of cholesteryl ester in endothelial and Kupffer cells in rabbits fed the high cholesterol diet were about twofold higher than in parenchymal cells (428 +/- 74 and 508 +/- 125 micrograms/mg protein, respectively, vs. 221 +/- 24 micrograms/mg protein in parenchymal cells). In contrast to cells from normal rabbits, Kupffer and endothelial cells from cholesterol-fed rabbits accumulated significant amounts of Oil Red O-positive material (neutral lipids). Electron microscopic examination of these cells in situ as well as in culture revealed numerous intracellular lipid droplets. Slot blot hybridization of RNA from liver parenchymal, endothelial, and Kupffer cells showed that cholesterol feeding reduced the level of mRNA specific for the apoB,E receptor to a small and insignificant extent in all three cell types (to 70-80% of that observed in control animals).(ABSTRACT TRUNCATED AT 400 WORDS)


Hypoinsulinemia in a patient with isolated ACTH deficiency. We report on a patient with isolated ACTH deficiency accompanying deficient insulin response to glucose. The subject is a 59 year-old male. He was admitted to our hospital because of a hypoglycemic attack. Urinary excretion of 17-hydroxycorticosteroid (17-OHCS) and 17-ketosteroid (17-KS) was low. Plasma ACTH levels decreased and serum cortisol levels were undetectable. ACTH did not respond to CRF stimulation for three consecutive days. Antibodies to At-T 20 cells (anterior pituitary cell-derived cell line) were positive in the serum of this patient. Serum immunoreactive insulin (IRI) levels were not detected throughout the day. The urinary excretion of C-peptide decreased to 9.8 micrograms/day. IRI responses on 75 g oral glucose tolerance test (OGTT) were examined at one and two months after the start of cortisol supplementation. No remarkable IRI response was observed, although blood glucose levels were maintained within the normal range. Eight months after the start of cortisol supplementation, the insulin response to 75 g OGTT appeared. The present study suggests that the recovery from hypo-insulinemia may be delayed even after normalization of serum cortisol levels in ACTH deficiency. This may be possibly due to the salutary effects of cortisol in maintaining plasma insulin levels. The time lag may be due to other factors also.


Monoclonal antiprothrombinase (3D4.3) prevents mortality from murine hepatitis virus (MHV-3) infection. The induction of monocyte/macrophage procoagulant activity (PCA) has been implicated in the pathogenesis of murine hepatitis virus strain 3 (MHV-3) infection and disease. Previously, we have shown that induction of PCA by MHV-3 correlated with resistance/susceptibility to infection in different mouse strains. In this study, all BALB/cJ mice that were infected with 10(3) plaque-forming units of MHV-3 developed severe liver disease and died within 96-120 h. Examination of the livers of these animals showed marked hepatic necrosis, deposition of fibrin, and cellular expression of PCA by direct immunofluorescence staining in areas of necrosis as well as in hepatic sinusoids. Splenic mononuclear cells recovered from these mice expressed high concentrations of PCA with time after infection. Infusion into mice of a high-titered monoclonal antibody that neutralized PCA (3D4.3) attenuated the development of hepatic necrosis and enhanced survival in a dose-dependent manner. All of the animals receiving 100 micrograms, and 44% and 22% of the animals that received 50 and 25 micrograms per day, respectively, survived for 10 d and made a full recovery. Administration of the antibody resulted in a dose-dependent reduction in fibrin deposition, PCA expression as detected by direct immunofluorescence staining and by a functional assay. In animals treated with high concentrations of antibody, titers of antibody to PCA fell from 87 +/- 15 micrograms/ml to 100 +/- 7 ng/ml during the active phase of the disease, consistent with sequestration due to binding of the immunoglobulin to cells expressing PCA. Surviving animals, when rechallenged with MHV-3, had a 40% mortality, consistent with the known rates of metabolism of immunoglobulin. This further suggested that protection was by a passive mechanism. The results reported here demonstrate that a neutralizing antibody to PCA protects animals from fulminant hepatitis and death associated with MHV-3 infection, and supports the notion that PCA is a potent inflammatory mediator that plays a pivotal role in the pathogenesis of liver injury resulting from MHV-3 infection.


Cytomegalovirus prevents antigen presentation by blocking the transport of peptide-loaded major histocompatibility complex class I molecules into the medial-Golgi compartment. Selective expression of murine cytomegalovirus (MCMV) immediate-early (IE) genes leads to the presentation by the major histocompatibility complex (MHC) class I molecule Ld of a peptide derived from MCMV IE protein pp89 (Reddehase, M.J., J. B. Rothbard, and U.H. Koszinowski. 1989. Nature (Lond.). 337:651). Characterization of endogenous antigenic peptides identified the pp89 peptide as the nonapeptide 168YPHFMPTNL176 (del Val, M., H.-J. Schlicht, T. Ruppert, M.J. Reddehase, and U.H. Koszinowski. 1991. Cell. 66:1145). Subsequent expression of MCMV early genes prevents presentation of pp89 (del Val, M., K. Münch, M.J. Reddehase, and U.H. Koszinowski. 1989. Cell. 58:305). We report on the mechanism by which MCMV early genes interfere with antigen presentation. Expression of the IE promoter-driven bacterial gene lacZ by recombinant MCMV subjected antigen presentation of beta-galactosidase to the same control and excluded antigen specificity. The Ld-dependent presence of naturally processed antigenic peptides also in nonpresenting cells located the inhibitory function subsequent to the step of antigen processing. The finding that during the E phase of MCMV gene expression the MHC class I heavy chain glycosylation remained in an Endo H-sensitive form suggested a block within the endoplasmic reticulum/cis-Golgi compartment. The failure to present antigenic peptides was explained by a general retention of nascent assembled trimolecular MHC class I complexes. Accordingly, at later stages of infection a significant decrease of surface MHC class I expression was seen, whereas other membrane glycoproteins remained unaffected. Thus, MCMV E genes endow this virus with an effective immune evasion potential. These results also indicate that the formation of the trimolecular complex of MHC class I heavy chain, beta 2-microglobulin, and the finally trimmed peptide is completed before entering the medial-Golgi compartment.


Stimulation of tumor necrosis factor alpha production in human monocytes by inhibitors of protein phosphatase 1 and 2A. The protein phosphatase 1 and 2A inhibitor, okadaic acid, has been shown to stimulate many cellular functions by increasing the phosphorylation state of phosphoproteins. In human monocytes, okadaic acid by itself stimulates tumor necrosis factor alpha (TNF-alpha) mRNA accumulation and TNF-alpha synthesis. Calyculin A, a more potent inhibitor of phosphatase 1, has similar effects. TNF-alpha mRNA accumulation in okadaic acid-treated monocytes is due to increased TNF-alpha mRNA stability and transcription rate. The increase in TNF-alpha mRNA stability is more remarkable in okadaic acid-treated monocytes than the mRNA stability of other cytokines, such as interleukin 1 alpha (IL-1 alpha), IL-1 beta, and IL-6. Gel retardation studies show the stimulation of AP-1, AP-2, and NF-kappa B binding activities in okadaic acid-stimulated monocytes. This increase may correlate with the increase in TNF-alpha mRNA transcription rate. In addition to the stimulation of TNF-alpha secretion by monocytes, okadaic acid appears to modulate TNF-alpha precursor processing, as indicated by a marked increase in the cell-associated 26-kD precursor. These results suggest that active basal phosphorylation/dephosphorylation occurs in monocytes, and that protein phosphatase 1 or 2A is important in regulating TNF-alpha gene transcription, translation, and posttranslational modification.


Properties of cyclic nucleotide-gated channels mediating olfactory transduction. Activation, selectivity, and blockage. Cyclic nucleotide-gated channels (cng channels) in the sensory membrane of olfactory receptor cells, activated after the odorant-induced increase of cytosolic cAMP concentration, conduct the receptor current that elicits electrical excitation of the receptor neurons. We investigated properties of cng channels from frog and rat using inside-out and outside-out membrane patches excised from isolated olfactory receptor cells. Channels were activated by cAMP and cGMP with activation constants of 2.5-4.0 microM for cAMP and 1.0-1.8 for cGMP. Hill coefficients of dose-response curves were 1.4-1.8, indicating cooperativity of ligand binding. Selectivity for monovalent alkali cations and the Na/Li mole-fraction behavior identified the channel as a nonselective cation channel, having a cation-binding site of high field strength in the pore. Cytosolic pH effects suggest the presence of an additional titratable group which, when protonated, inhibits the cAMP-induced current with an apparent pK of 5.0-5.2. The pH effects were not voltage dependent. Several blockers of Ca2+ channels also blocked olfactory cng channels. Amiloride, D 600, and diltiazem inhibited the cAMP-induced current from the cytosolic side. Inhibition constants were voltage dependent with values of, respectively, 0.1, 0.3, and 1 mM at -60 mV, and 0.03, 0.02, and 0.2 mM at +60 mV. Our results suggest functional similarity between frog and rat cng channels, as well as marked differences to cng channels from photoreceptors and other tissues.


Is the mammalian cell plasma membrane a barrier to oxygen transport? Oxygen transport in the Chinese hamster ovary (CHO) plasma membrane has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed in the lipid bilayer portion of the membrane at various distances from the membrane surface using the long-pulse saturation-recovery electron spin resonance (ESR) technique. The collision rate was estimated for 5-, 12-, and 16-doxylstearic acids from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameters across the membrane were obtained showing that the oxygen diffusion-concentration product is lower than in water for all locations at 37 degrees C. From oxygen transport parameter profiles, the membrane oxygen permeability coefficients were estimated according to the procedure developed earlier by Subczynski et al. (Subczynski, W. K., J. S. Hyde, and A. Kusumi. 1989. Proceedings of the National Academy of Sciences, USA. 86:4474-4478). At 37 degrees C, the oxygen permeability coefficient for the plasma membrane was found to be 42 cm/s, about two times lower than for a water layer of the same thickness as the membrane. The oxygen concentration difference across the CHO plasma membrane at physiological conditions is in the nanomolar range. It is concluded that oxygen permeation across the cell plasma membrane cannot be a rate-limiting step for cellular respiration. Correlations of the form PM = cKs between membrane permeabilities PM of small nonelectrolyte solutes of mol wt less than 50, including oxygen, and their partition coefficients K into hexadecane and olive oil are reported. Hexadecane: c = 26 cm/s, s = 0.95; olive oil: c = 23 cm/s, s = 1.56. These values of c and s differ from those reported in the literature for solutes of 50 less than mol wt less than 300 (Walter, A., and J. Gutknecht. 1986. Journal of Membrane Biology. 90:207-217). It is concluded that oxygen permeability through membranes can be reliably predicted from measurement of partition coefficients.


Stability, frequency and multiplicity of transposon insertions in the pyoverdine region in the chromosomes of different fluorescent pseudomonads. Tn5 mutagenesis of different fluorescent pseudomonads was achieved by conjugational transfer of the suicide vector pSUP 10141. Pyoverdine negative (Pvd-) mutants were detected by the absence of fluorescence on King's B medium and by their inability to grow in the presence of the iron chelator EDDHA [ethylenediamine di(o-hydroxyphenylacetic acid)]. In P. fluorescens ATCC 17400 and three rhizosphere isolates (one P. putida and two P. fluorescens), the percentage of Pvd- mutants ranged between 0 and 0.54%. In a P. chlororaphis rhizosphere isolate, this percentage was higher (4%). In these mutants both of the Tn5 antibiotic resistances (Km and Tc) were stable and the transposon could be detected by hybridization. In Pvd- mutants of P. fluorescens ATCC 17400, the transposon was found to be inserted twice in the chromosome while single insertions were detected in the DNA of other, randomly tested mutants. In P. aeruginosa PAO1, where 13.1% of the mutants were Pvd-, both antibiotic resistances were rapidly lost and accordingly no transposon insertion could be detected by hybridization. However, the Pvd- phenotype was generally stable in these mutants. The plasmid pNK862 containing a mini-Tn10 transposon was introduced by electroporation into P. aeruginosa PAO1 and Kmr mutants were recovered, 89% of which were Pvd- and confirmed to be P. aeruginosa by PCR amplification of the P. aeruginosa lipoprotein gene. The mini-Tn10 insertions were also found to be unstable in PAO1.


Inhibition of lipopolysaccharide synthesis in Agrobacterium tumefaciens and Aeromonas salmonicida. Lipopolysaccharide (LPS) synthesis was inhibited, new lipid A metabolites accumulated, and growth ceased, when the plant pathogen Agrobacterium tumefaciens and the fish pathogen Aeromonas salmonicida were treated with an antibacterial agent which specifically inhibits CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthase). The new lipid A metabolites were purified by chromatography on DEAE-cellulose and chemically analysed. Metabolites isolated from both bacterial species contained glucosamine and phosphate in a 1:1 molar ratio, and 3-OH-C14:0 was the major fatty acid present (1 mol and 1.4 mol per mol glucosamine for A. tumefaciens and A. salmonicida, respectively). Inhibition of LPS synthesis by CMP-KDO synthase inhibitor had no effect on the initial kinetics of A. tumefaciens attachment to cultured carrot cells, but did inhibit cell aggregation normally induced by bacterial cellulose synthesis. Bacteria treated with inhibitor remained viable and able to synthesize protein at 15% the rate of control cells, indicating that the lack of cellulose-induced aggregation was not due to the inability of bacteria to make protein, but rather the inability to respond normally to the bacterial-plant cell interaction.


The inhibitory control of the substantia nigra over generalized non-convulsive seizures in the rat. A system exerting inhibitory control over generalized epilepsies and involving neurons from the substantia nigra has been described by several authors in experimental models of convulsive seizures. In the present study, the existence of such a control system governing absence epilepsy was investigated using models of non-convulsive seizures in the rat. Activation of the GABAergic neurotransmission within the substantia nigra by local injection of GABA agonists (muscimol, THIP) or an inhibitor of GABA degradation (gamma-vinyl GABA) suppresses generalized non convulsive seizures, whether they are genetically determined or induced by systemic injections of gamma-butyrolactone (100 and 200 mg/kg), pentylenetetrazole (20 mg/kg) or THIP (7.5 mg/kg). The ascending dopaminergic nigral output or the GABAergic fibres to the ventromedial thalamus are not critically involved in this control system. By contrast, the GABAergic nigro-collicular pathway appears crucial: bilateral lesion of the superior colliculus abolishes the anti-epileptic effects of intranigral injection of muscimol and blockade of the GABAergic transmission within the superior colliculus results in a suppression of generalized non-convulsive seizures. Finally, activation of collicular cell bodies by low doses of kainic acid significantly suppresses absence seizures. These results suggest the existence of a control system inhibiting generalized non-convulsive seizures which is activated by the release of the tonic inhibition exerted by the nigral GABAergic fibres on collicular neurons. The similarities between this system and the control system described for convulsive seizures are discussed.


Experimental absence seizures: potential role of gamma-hydroxybutyric acid and GABAB receptors. We have investigated whether the pathogenesis of spontaneous generalized non-convulsive seizures in rats with genetic absence epilepsy is due to an increase in the brain levels of gamma-hydroxybutyric acid (GHB) or in the rate of its synthesis. Concentrations of GHB or of its precursor gamma-butyrolactone (GBL) were measured with a new GC/MS technique which allows the simultaneous assessment of GHB and GBL. The rate of GHB synthesis was estimated from the increase in GHB levels after inhibition of its catabolism with valproate. The results of this study do not indicate significant differences in GHB or GBL levels, or in their rates of synthesis in rats showing spike-and-wave discharges (SWD) as compared to rats without SWD. Binding data indicate that GHB, but not GBL, has a selective, although weak affinity for GABAB receptors (IC50 = 150 microM). Similar IC50 values were observed in membranes prepared from rats showing SWD and from control rats. The average GHB brain levels of 2.12 +/- 0.23 nmol/g measured in the cortex and of 4.28 +/- 0.90 nmol/g in the thalamus are much lower than the concentrations necessary to occupy a major part of the GABAB receptors. It is unlikely that local accumulations of GHB reach concentrations 30-70-fold higher than the average brain levels. After injection of 3.5 mmol/kg GBL, a dose sufficient to induce SWD, brain concentrations reach 240 +/- 31 nmol/g (Snead, 1991) and GHB could thus stimulate the GABAB receptor. Like the selective and potent GABAB receptor agonist R(-)-baclofen, GHB causes a dose-related decrease in cerebellar cGMP. This decrease and the increase in SWD caused by R(-)-baclofen were completely blocked by the selective and potent GABAB receptor antagonist CGP 35348, whereas only the increase in the duration of SWD induced by GHB was totally antagonized by CGP 35348. The decrease in cerebellar cGMP levels elicited by GHB was only partially antagonized by CGP 35348. These findings suggest that all effects of R(-)-baclofen are mediated by the GABAB receptor, whereas only the induction of SWD by GHB is dependent on GABAB receptor mediation, the decrease in cGMP being only partially so. Taken together with the observations of Marescaux et al. (1992), these results indicate that GABAB receptors are of primary importance in experimental absence epilepsy and that GABAB receptor antagonists may represent a new class of anti-absence drugs.


GABAB receptor antagonists: potential new anti-absence drugs. The availability of new antagonists of the GABAB receptor which readily cross the blood-brain barrier has made it possible to investigate the role of GABAB-receptor-mediated transmission in the control of spike-and-wave discharges (SWD) in a strain of rats (GAERS) with genetic absence epilepsy. Systemic administration of R-Baclofen, a GABAB agonist, increased the duration of SWD, or elicited SWD-like oscillations in the cortical EEG of non-epileptic control rats. Conversely, administration of CGP 35348, a GABAB antagonist, either i.p. or p.o., dose-dependently suppressed the spontaneous SWD, as well as the SWD aggravated by concomitant injection of various GABAmimetic drugs, GHB, or anti-convulsants known to exacerbate absence seizures. These results demonstrate the involvement of GABAB-mediated neurotransmission in the development of SWD in generalized non-convulsive epilepsy. GABAB antagonists may thus be considered to be potentially specific anti-absence drugs.


GABA receptors in rats with spontaneous generalized nonconvulsive epilepsy. We have used the technique of autoradiography to study the binding of [3H]-GABA to GABAA and GABAB receptors in brains taken from rats that are genetically predisposed to petit mal type seizures. A range of concentrations of [3H]-GABA were employed to test the hypothesis that this predisposition was due to regional changes in either the number of GABAA or GABAB receptors, or affinity of GABA for these receptors. We found no statistical difference in the levels of radioligand binding to GABAA and GABAB receptors in animals susceptible to seizures compared to control animals in any of the brain regions studied over the concentration range 25 nM to 400 nM. This indicated that there was no change in either the Kd (affinity) or Bmax (receptor number) in these animals and that the pharmacological basis for the efficacy of GABAB antagonists in this seizure condition probably lies elsewhere.


A strategy for identifying novel, mechanistically unique inhibitors of topoisomerase I. While the design of molecules that inhibit or antagonize the functions of specific macromolecules is now well precedented, in many cases the structural information requisite to the design process is lacking. The tools of molecular biology can now furnish the target macromolecules for use in mechanism-based exploration; highly defined assays can be devised based upon the known biochemistry of these macromolecules to permit the discovery of novel inhibitors or antagonists present in chemical collections. Presently, we describe a set of assays directed toward the discovery of novel inhibitors of eukaryotic topoisomerase I, an enzyme critical to maintenance of chromosomal DNA topology and therefore essential for normal replication and transcription. The identification of chebulagic acid as an extraordinarily potent and mechanically novel inhibitor of topoisomerase I illustrates the potential of this approach.


Fistularin 3 and 11-ketofistularin 3. Feline leukemia virus active bromotyrosine metabolites from the marine sponge Aplysina archeri. Two brominated tyrosine metabolites, fistularin 3 [1] and a new compound 11-ketofistularin 3 [2] have been isolated from a marine sponge, Aplysina archeri. Their structures were determined on the basis of spectral data. Both compounds inhibited the growth of feline leukemia virus.


Inhibitory effect of tannic acid on gastric H+,K(+)-ATPase. The effect of tannic acid on gastric H+,K(+)-ATPase was studied. Tannic acid dose-dependently inhibited pig gastric H+,K(+)-ATPase activity with an IC50 value of 2.9 x 10(-8) M. Tannic acid also inhibited K(+)-stimulated p-nitrophenyl phosphatase (K(+)-pNPPase) activity, which is found in gastric H+,K(+)-ATPase preparations, as well as H+,K(+)-ATPase activity, with an IC50 value of 4.1 x 10(-7) M. Kinetic studies showed that the inhibition of H+,K(+)-ATPase activity by tannic acid was competitive with respect to ATP and noncompetitive with respect to K+. These results show that tannic acid is a potent inhibitor of gastric H+,K(+)-ATPase; this may be related to its anti-secretory and anti-ulcerogenic effects.


Peripheral nerve involvement in Churg-Strauss syndrome. Peripheral neuropathy associated with bronchial asthma, multisystem organ dysfunction and idiopathic hypereosinophilia may be found in Churg-Strauss syndrome, hypereosinophilic syndrome and polyarteritis nodosa. Some authors have diagnosed their patients according to the presence in tissue biopsies of the three histological criteria of Churg and Strauss (necrotizing vasculitis, tissue eosinophilic infiltration, extravascular granulomas). We have observed three patients with a common history of a prodromal phase of allergic diseases (bronchial asthma and rhinitis) followed by a vasculitic phase with mononeuritis multiplex, purpura and arthritis, associated with hypereosinophilia of more than 1500 cells/mm3. All responded well to steroid treatment. Sural nerve biopsy revealed true vasculitis in two of these cases and a mild perivascular inflammatory infiltration in the other. On the basis of their characteristic clinical pattern, we think that our cases best fit the diagnosis of Churg-Strauss syndrome even though the typical histological features were not found in the sural nerves examined.


Baclofen-assisted detoxification from opiates. A pilot study. In an open label pilot study, five opiate-dependent patients underwent baclofen-assisted opiate detoxification after abrupt discontinuation of methadone. Patients received baclofen in oral doses up to 80 mg/day, and all patients subjectively reported some reduction in discomfort. However, 3 of 5 (60%) patients could not complete detoxification with baclofen, primarily because of insufficient suppression of vomiting, myalgias, and headache. These patients successfully completed their detoxification with clonidine. These findings suggest that, in the dose range studied, baclofen is of limited use as a primary treatment for opiate dependence, although adjunctive roles for this medication in detoxification should be explored.