Mutations in a central highly conserved non-DNA-binding region of OmpR, an Escherichia coli transcriptional activator, influence its DNA-binding ability. OmpR is a transcriptional activator for the expression of outer membrane porin genes ompF and ompC in Escherichia coli. Its C-terminal half has been identified as the DNA-binding domain (K. Tsung, R. Brissette, and M. Inouye, J. Biol. Chem. 264:10104-10109, 1989). Recent studies have indicated that the N-terminal non-DNA-binding domain of OmpR is involved in modulating OmpR function through interaction with the EnvZ protein, a kinase and phosphatase for OmpR. We isolated and characterized two mutations, G94D and E111K, in the N-terminal domain of OmpR and one mutation, R182C, in the DNA-binding domain of OmpR. All three mutations abolished the ability of OmpR to bind to the ompF and ompC promoters in vivo, thus giving an OmpF- OmpC- phenotype. The decreased DNA-binding ability of the mutant OmpRs was not due to diminished phosphorylation of their N termini, since all the mutant OmpRs were found to be normally phosphorylated by EnvZ in vitro. The mutant OmpRs produced from multicopy plasmids were also found to inhibit completely the production of OmpF and OmpC in wild-type cells, and the complete inhibition depended on the function of EnvZ which was produced in cis or in trans from plasmids. The relationship of the possible alterations in OmpR by the mutations with the observed diminished binding ability is discussed.


Nucleotide sequence of Streptococcus mutans superoxide dismutase gene and isolation of insertion mutants. A gene (sod) encoding superoxide dismutase (SOD) was cloned from Streptococcus mutans in Escherichia coli, and its nucleotide sequence was determined. The presumptive amino acid sequence of its product revealed that the SOD is basically of Mn type. Insertional inactivation of the sod gene resulted in the loss of SOD activity in crude extracts, indicating that the gene represents the only functional gene for SOD in S. mutans. Moreover, Southern blot analysis indicated that the S. mutans chromosome had no additional gene which was hybridizable with an oligonucleotide probe specific for an SOD motif. The SOD-deficient mutants were able to grow aerobically, albeit more slowly than the parent strains.


Isolation and characterization of a Tn551-autolysis mutant of Staphylococcus aureus. A Lyt- mutant with reduced autolytic activity was isolated after Tn551 mutagenesis of the methicillin-susceptible Staphylococcus aureus laboratory strain RN450. The Lyt- phenotype could be transferred back into the parent and into a variety of other S. aureus strains by transduction of the transposon marker. Southern analysis has located the Tn551 insert to a 3.2-kb HindIII DNA fragment on the SmaI B fragment of the staphylococcal chromosome. The Lyt- phenotype included reduced rates of cell wall turnover and autolysis induced by detergent or methicillin treatment; however, the rate of methicillin-induced killing was not affected. Peptidoglycans prepared from the parental and mutant cells showed identical muropeptide compositions, as resolved by a high-resolution high-pressure liquid chromatography technique. On the other hand, LiCl extracts of the mutant cells contained reduced amounts of total protein and lower specific cell wall-degrading activity compared with those of extracts of parental cells. The profile of bacteriolytic enzymes as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed multiple band differences between mutant and parental cells; a major lytic band with properties characteristic of the staphylococcal endo-beta-N-acetylglucosaminidase was completely absent from the Lyt- cells. The Lyt- phenotype transduced into a series of methicillin-resistant strains of both homogeneous and heterogeneous phenotypes caused only a modest decrease in the level of methicillin resistance, as determined by population analysis.


rec-2-dependent phage recombination in Haemophilus influenzae. The genetic transformation mutant Rd(DB117)rec- has a pleiotropic phenotype that includes reduced levels of phage recombination. Physical mapping experiments showed that this strain has a 78.5-kbp insertion in the rec-2 gene. The rec-2 dependence of phage recombination was reexamined to determine whether the defective phenotype in Rd(DB117)rec- was due to the simple disruption of the rec-2 gene or whether trans-acting factors from the inserted DNA were responsible. Analysis of strains with transposon insertions in the rec-2 gene showed that they were also defective for phage recombination. Therefore, the phage recombination defect was due solely to the disruption of the rec-2 gene. Strain KB6 is proficient for phage recombination but has a defect in genetic transformation resembling that of Rd(DB117)rec-. The transformation defect of KB6 could be complemented by the wild-type rec-2 gene, showing that the rec-2 contributions to genetic transformation and phage recombination were uncoupled in this strain. The rec-2-dependent phenotype of KB6 suggests that the rec-2 gene participates in genetic transformation and phage recombination in different ways.


IncN plasmid pKM101 and IncI1 plasmid ColIb-P9 encode homologous antirestriction proteins in their leading regions. The IncN plasmid pKM101 (a derivative of R46), like the IncI1 plasmid ColIb-P9, carries a gene (ardA, for alleviation of restriction of DNA) encoding an antirestriction function. ardA was located about 4 kb from the origin of transfer, in the region transferred early during bacterial conjugation. The nucleotide sequence of ardA was determined, and an appropriate polypeptide with the predicted molecular weight of about 19,500 was identified in maxicells of Escherichia coli. Comparison of the deduced amino acid sequences of the antirestriction proteins of the unrelated plasmids pKM101 and ColIb (ArdA and Ard, respectively) revealed that these proteins have about 60% identity. Like ColIb Ard, pKM101 ArdA specifically inhibits both the restriction and modification activities of five type I systems of E. coli tested and does not influence type III (EcoP1) restriction or the 5-methylcytosine-specific restriction systems McrA and McrB. However, in contrast to ColIb Ard, pKM101 ArdA is effective against the type II enzyme EcoRI. The Ard proteins are believed to overcome the host restriction barrier during bacterial conjugation. We have also identified two other genes of pKM101, ardR and ardK, which seem to control ardA activity and ardA-mediated lethality, respectively. Our findings suggest that ardR may serve as a genetic switch that determines whether the ardA-encoded antirestriction function is induced during mating.


Phosphorylation site of NtrC, a protein phosphatase whose covalent intermediate activates transcription. The NtrC transcription factor is a member of a family of homologous prokaryotic regulatory proteins that participate in the transduction of extracellular and nutritional signals. It has been demonstrated that the phosphate group from a histidine residue of the phosphorylated NtrB protein autokinase is transferred to the NtrC protein. Phosphorylation of the NtrC protein is transient and activates its transcriptional enhancement activity. We have investigated the site of phosphorylation of the Salmonella typhimurium NtrC protein and find that it is an aspartate residue (Asp-54) that is found within a sequence conserved in all of the members of the family of regulatory proteins. We propose that this phosphorylation is an NtrC protein histidine phosphatase catalytic intermediate. This conclusion suggests that the NtrC family should be viewed not as kinase substrates but as enzymes that can catalyze the hydrolysis of their activated forms in a concentration-independent fashion. They are similar in this sense to eukaryotic signal-transducing GTPases.


Cloning and sequencing of Escherichia coli mutR shows its identity to topB, encoding topoisomerase III. We have cloned and sequenced the mutR gene from Escherichia coli, which results in an increased frequency of spontaneous deletions, by using a strain carrying a Tn10 derivative inserted into mutR. The analysis of 1,286 bp of mutR sequence shows that this gene is identical to the topB gene, which encodes topoisomerase III. The increased deletion formation is the first reported phenotype for cells lacking topoisomerase III, and this suggests that topoisomerase III is involved in reactions that normally reduce the levels of spontaneous deletions.


Polypeptide translocation across the endoplasmic reticulum membrane. Many polypeptides have been postulated to play direct roles in secretory protein translocation based on genetic criteria, cross-linking, and antibody inhibition. Much of the excitement in the next few years will come from the resolution of current controversies. What is the nature of the ribosome receptor, and is it essential for translocation? Is BiP required for translocation in mammalian cells? Are all of the polypeptides of signal peptidase and oligosaccharyltransferase required for catalytic function, or do some of them mediate steps of protein translocation? One of the best ways to resolve these problems will be to determine the importance of each in reconstituted translocation reactions by fractionation or immunodepletion, or by analysis in a purified reaction. Another approach is to identify homologues of these molecules in S. cerevisiae and to assess their importance in in vivo translocation. Several mechanistic questions remain to be addressed as well. Does the protein translocation apparatus consist of protein, or lipid, or both? How are integral membrane proteins inserted? How is the translocon gated to admit only unfolded or partially folded secretory polypeptides and to exclude cytoplasmic molecules? The answers to these questions will illuminate a basic enigma in cell biology that has remained unanswered for many years.


Structure and function of the human platelet thrombin receptor. Studies using monoclonal antibodies directed against a defined domain within the receptor N terminus. Based upon its recently cloned nucleotide sequence, the human platelet thrombin receptor is thought to be formed by a single polypeptide chain with seven transmembrane domains and an extracellular N terminus that can be cleaved by thrombin. As yet, however, little is known from studies of the receptor protein itself. To obtain such information, we have prepared monoclonal antibodies against a peptide corresponding to receptor residues Ser42 through Phe55, the domain immediately distal to the site of cleavage by thrombin. By flow cytometry, all of the antibodies reacted with the thrombin-responsive megakaryoblastic CHRF-288 and HEL cell lines, but not with the T-lymphoid Sup-T1 cell line. Functionally, the antibodies inhibited platelet responses to alpha-thrombin, gamma-thrombin, and trypsin, but had no effect on platelet activation by ADP, epinephrine, or the thromboxane analog U46619. Radioiodinated antibody bound to approximately 1,800 sites/platelet, a value similar to the reported number of moderate affinity thrombin binding sites per platelet. On Western blots, the antibodies recognized a 66-kDa protein in platelet, HEL, and CHRF-288 membranes. The discrepancy between this apparent size and the predicted mass of the receptor suggests that, as with other G protein-coupled receptors, one or more of the potential sites for N-linked glycosylation have been utilized. Therefore, these results suggest that: 1) the cloned thrombin receptor is involved in a broad range of platelet responses to thrombin, as well as gamma-thrombin and trypsin; 2) as predicted, the N terminus of the receptor is accessible on the platelet surface; 3) the moderate affinity thrombin binding site noted in earlier studies may be the receptor; 4) potentially as much as one third of the mass of the receptor is carbohydrate.


Insulin receptor protein-tyrosine phosphatases. Leukocyte common antigen-related phosphatase rapidly deactivates the insulin receptor kinase by preferential dephosphorylation of the receptor regulatory domain. A number of protein-tyrosine phosphatase(s) (PTPases) have been shown to dephosphorylate the insulin receptor in vitro; however, it is not known whether any individual PTPase has specificity for certain phosphotyrosine residues of the receptor that regulate its intrinsic tyrosine kinase activity. We evaluated the deactivation of the insulin receptor kinase by three candidate enzymes that are expressed in insulin-sensitive rat tissues, including the receptor-like PTPases LAR and LRP, and the intracellular enzyme, PTPase1B. Purified insulin receptors were activated by insulin and receptor dephosphorylation, and kinase activity was quantitated after incubation with recombinant PTPases from an Escherichia coli expression system. When related to the level of overall receptor dephosphorylation, LAR deactivated the receptor kinase 3.1 and 2.1 times more rapidly than either PTPase1B or LRP, respectively (p less than 0.03). To assess whether these effects were associated with preferential dephosphorylation of the regulatory (Tyr-1150) domain of the receptor beta-subunit, we performed tryptic mapping of the insulin receptor beta-subunit after dephosphorylation by PTPases. Relative to the rate of initial loss of 32P from receptor C-terminal sites, LAR dephosphorylated the Tris-phosphorylated Tyr-1150 domain 3.5 and 3.7 times more rapidly than either PTPase1B or LRP, respectively (p less than 0.01). The accelerated deactivation of the insulin receptor kinase by LAR and its relative preference for regulatory phosphotyrosine residues further support a potential role for this transmembrane PTPase in the physiological regulation of insulin receptors in intact cells.


The importance of Asn47 for structure and reactivity of azurin from Alcaligenes denitrificans as studied by site-directed mutagenesis and spectroscopy. To study the importance of a rigid copper site for the structure and function of azurin, a mutant with a reduced number of internal hydrogen bonds around the copper has been prepared and characterized. To this purpose, the previously cloned azu gene from Alcaligenes denitrificans (Hoitink, C. W. G., Woudt, L. P., Turenhout, J. C. M., Van de Kamp, M., and Canters, G. W. (1990) Gene (Amst.) 90, 15-20) was expressed in Escherichia coli and an isolation and purification procedure for the azurin was developed. The azurin obtained after heterologous expression in E. coli appears spectroscopically indistinguishable from azurin derived from A. denitrificans. The hydrogen bonding network around the copper site was altered by replacing Asn47 by a leucine by means of site-directed mutagenesis. Asn47 is a conserved residue in all blue copper proteins of which the primary structure has been reported. Characterization of the mutant protein with UV-visible, electron spin resonance, and NMR spectroscopy, and comparison with the wild type azurin revealed that the structure of the copper site as well as the overall structure of the protein have been largely retained. The redox activity as measured by the electron self-exchange rate appears not to have changed either. However, the mutant differs from the wild type azurin with respect to stability and midpoint potential. Midpoint potentials of mutant and wild type azurin amount to 396 and 286 mV, respectively. The difference is due to sizable entropic and enthalpic contributions which to a large extent cancel. Possible explanations for the outcome of these experiments are discussed.


Detection of a higher oxidation state of manganese-prostaglandin endoperoxide synthase. Addition of arachidonic acid or 5-phenyl-4-pentenylhydroperoxide to manganese-prostaglandin endoperoxide synthase (Mn-PGH synthase) produced a species with an absorbance maximum at 418 nm. This maximum is distinct from those of resting enzyme (372 and 468 nm) or reduced enzyme (434 nm). The formation of the 418 nm-absorbing species was observed immediately after the addition of hydroperoxide to enzyme but only after a 10-s lag period following addition of arachidonate. Mn-PGH synthase exhibited a peroxidase activity that was 0.8% that of Fe-PGH synthase. Addition of peroxidase reducing substrates to the oxidized form of Mn-PGH synthase diminished the absorbance at 418 nm. In the case of N,N,N',N'-tetramethylphenylenediamine, reduction of the 418 nm-absorbing species was accompanied by an increase in absorbance at 610 nm due to the oxidized form of the amine. Thus, the spectral and chemical properties of the 418 nm-absorbing species are consistent with its existence as a higher oxidation state of Mn-PGH synthase. Kinetic analysis indicated that formation of the higher oxidation state preceded or was coincident with oxygenation of the fatty acid substrate, eicosa-11,14-dienoic acid. The cyclooxygenase activity of Mn-PGH synthase was inhibited by the combination of glutathione and human plasma glutathione peroxidase at a glutathione peroxidase concentration 227-fold lower than the concentration that inhibited Fe-PGH synthase. The results suggest that Mn-PGH synthase forms a higher oxidation state following reaction with hydroperoxides added exogenously or generated endogenously from polyunsaturated fatty acid substrates. This higher oxidation state functions in the peroxidase catalytic cycle of Mn-PGH synthase, and its formation appears to be essential for activation of the cyclooxygenase catalytic cycle.


Carboxyl-terminal sequences can influence the in vitro import and intraorganellar targeting of chloroplast protein precursors. The transit peptide of the lumenal 33-kDa oxygen-evolving polypeptide (OEE1) is capable of directing the import and targeting of the foreign protein dihydrofolate reductase (DHFR) to the thylakoid lumen. The import results from the first part of this study indicate that methotrexate cannot block the import or intraorganellar targeting of OEE1-DHFR in chloroplasts in contrast to that reported for the import of cytochrome oxidase subunit IV (COXIV)-DHFR in mitochondria. These results suggest that the fusion of the OEE1 transit sequence to DHFR affected the protein's methotrexate binding properties. We further examined and compared the transport characteristics of a number of carboxyl-terminal truncated native chloroplast precursors to determine whether carboxyl domains contribute to the import and intraorganellar targeting mechanism of these proteins. The plastid precursors chosen for this study are targeted to one of the following chloroplast compartments: the stroma, the thylakoid membrane, and the lumen. In most cases, removal of carboxyl domains had a dramatic effect on one or more stages of the translocation pathway, such as import, processing, and intraorganellar targeting. The effects of carboxyl deletions varied from precursor to precursor and were dependent on the extent of the deletion. These combined results suggest that carboxyl domains in the mature part of the proteins can influence the function of the transit peptide, and as a result play an important role in determining the import and targeting competence of chloroplast precursors.


Nerve growth factor (NGF) receptor on rat glial cell lines. Evidence for NGF internalization via p75NGFR. Two types of nerve growth factor (NGF) receptors have been described: high affinity (class I) and low affinity (class II). Biological responses to NGF are thought to be mediated by class I receptors, whereas the role of class II receptors is less clear. While some neuronal cells express both receptor types, only class II receptors have been detected on glial cells. Two glial cell lines, peripheral Schwannoma D6P2T and central 33B glioma cells, were employed to investigate the properties of class II receptors in the absence of class I receptors. These cell lines were found to express NGF receptors identified as class II by a low nanomolar dissociation constant, rapid dissociation kinetics at 4 degrees C, and trypsin sensitivity. The receptor was found to bind brain-derived neurotrophic factor with similar affinity as NGF. The responsible binding molecule appeared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a heterogeneously glycosylated protein of 60-80 kDa with a tendency to aggregate. All receptor bands affinity-labeled with radioiodinated NGF were immunoprecipitated with anti-p75NGFR antibody, but not with anti-p140prototrk antiserum. In these cells, which express p75NGFR as only NGF receptor, a time- and temperature-dependent appearance of a nondisplaceable, trypsin-resistant, acid wash-stable ligand fraction, followed by an increase of trichloroacetic acid-soluble radiolabel in the medium was observed. This sequestration resembled receptor-mediated internalization with subsequent degradation of NGF. Whether this ligand processing indicates a functional role of p75NGFR in glial cells remains to be shown.


Gelsolin binds to polymeric actin at a low rate. Association of gelsolin with actin filament subunits was investigated by the decrease of the fluorescence intensity of a 7-nitro-2-oxa-1,3-diazole (NBD) label covalently linked to gelsolin. The rate constant of this reaction was found to be 4 x 10(3) M-1 s-1. Binding of NBD-labeled gelsolin to monomeric actin proceeds at a similar low rate. The rate of association of gelsolin that was unmodified to actin filament subunits was estimated too. Unmodified gelsolin was added to a mixture of actin filaments and actin-DNase I complex. The fractions of gelsolin that bound to actin filament subunits or to actin-DNase I complex depended on the relative rates of these two competing reactions. In this way it was possible to estimate the rate constant of association of unmodified gelsolin with actin filament subunits (2 x 10(4) M-1 s-1). Thus, gelsolin associates with actin filament subunits at a rate that is considerably slower than diffusion-controlled and similar to the rate of binding of gelsolin to monomeric actin.


Deoxycytidine deaminase-resistant stereoisomer is the active form of (+/-)-2',3'-dideoxy-3'-thiacytidine in the inhibition of hepatitis B virus replication. 2',3'-Dideoxy-3'-thiacytidine (+/-)-SddC) was found to have potent activity against human hepatitis B virus as well as human immunodeficiency viruses in culture. The (-)form ((-)-SddC) which is resistant to deoxycytidine deaminase was found to be the more active antiviral stereoisomer than the (+)-form ((+)-SddC). The (+)-SddC is susceptible to deamination by deoxycytidine deaminase and is 25- and 12-fold more toxic than (-)-SddC in CEM cells in terms of anti-cell growth and anti-mitochondrial DNA synthesis, respectively. Similar results were obtained using a mixture of their 5-fluoro analogs ((+/-)-FSddC). Unlike 2',3'-dideoxycytidine, which is a potent inhibitor of mitochondrial DNA synthesis and results in such delayed toxicity as peripheral neuropathy with long term usage, (-)-SddC does not affect mitochondrial DNA synthesis. The (-)form is phosphorylated to (-)-SddCMP and is subsequently converted to (-)-SddCDP and (-)-SddCTP. One additional major metabolite which has been tentatively assigned the name "(-)-SddCMP sialate" was also identified. No significant difference in terms of the profiles of the metabolites was found between 4 and 24 h. There is an appreciable amount of (-)-SddCTP detectable 24 h after removal of the drug. (-)-SddCTP was also found to be approximately 3-fold more potent than (+)-SddCTP in inhibiting human hepatitis B virus DNA polymerase. This is the first nucleoside analog with the unnatural sugar configuration demonstrated to have antiviral activity.


Differential dephosphorylation of the insulin receptor and its 160-kDa substrate (pp160) in rat adipocytes. A permeabilized rat adipocyte model was developed which permitted an examination of: 1) insulin receptor autophosphorylation, 2) phosphorylation of a putative insulin receptor substrate of 160 kDa, pp160, and 3) the dephosphorylation reactions associated with each of these phosphoproteins. Rat adipocytes, preincubated with [32P]orthophosphate for 2 h, were exposed to insulin (10(-7) M) at the time of digitonin permeabilization. Phosphorylation of pp160 and autophosphorylation of the insulin receptor increased as a function of Mn2+ concentration in the media with near maximum responses at 10 mM. Maximum response was at least as large as the intact cell response to 10(-7) M insulin. In contrast, magnesium did not increase phosphorylation of pp160 although an increase in receptor autophosphorylation was observed. Autophosphorylation was preserved at digitonin concentrations of 20-100 micrograms/ml, but pp160 phosphorylation was negligible beyond 40 micrograms/ml. Our previous work demonstrated that the insulin receptor was associated with a phosphotyrosine phosphatase activity in permeabilized adipocytes (Mooney, R., and Anderson, D. (1989) J. Biol. Chem. 264, 6850-6857). The current permeabilized adipocyte model made possible an examination of the effects of phosphotyrosine phosphatase inhibitors, including several divalent metal cations (Zn2+, Co2+, and Ni2+), vanadate, and molybdate on both net phosphorylation of pp160 and autophosphorylation of the insulin receptor. Zn2+ at 100 microM, Ni2+ at 1 mM, and Co2+ at 1 or 5 mM increased insulin-dependent phosphorylation of pp160 at least 5-fold and autophosphorylation 2-fold. At higher concentrations of Zn2+ (1 mM) and Ni2+ (5 mM), however, no increase in phosphorylation of pp160 was observed and autophosphorylation was inhibited. Vanadate (1 mM) and molybdate (100 microM) increased insulin-dependent phosphorylation of pp160 by 3-fold when tested separately and 7-fold in combination. Insulin receptor autophosphorylation was increased 50% by each and 3-fold when the agents were combined. Dephosphorylation of pp160 and the insulin receptor was analyzed directly by permeabilizing prelabeled insulin-treated adipocytes in the presence of EDTA (10 mM). Dephosphorylation of pp160 was especially rapid with a t1/2 of approximately 10 s. The t1/2 for the insulin receptor was 37 s. Zn2+ at 1 mM (a concentration that inhibited the insulin receptor kinase) was a strong inhibitor of dephosphorylation, prolonging the rate of pp160 dephosphorylation more than 12-fold and insulin receptor dephosphorylation 3-fold.(ABSTRACT TRUNCATED AT 400 WORDS)


The Rieske FeS center from the gram-positive bacterium PS3 and its interaction with the menaquinone pool studied by EPR. The Rieske 2Fe2S center from Bacillus PS3, a Gram-positive thermophilic eubacterium, has been studied by EPR spectroscopy. Its redox midpoint potential at pH 7.0 was determined to be +165 +/- 10 mV and was found to decrease with an apparent slope of -80 mV/pH unit above pH 7.9. The Qo-site inhibitor stigmatellin induced spectral changes analogous to those reported for Rieske centers from mitochondria and chloroplasts. The redox midpoint potential of the PS3 Rieske cluster was not affected by stigmatellin. The orientation of the g tensor was similar to other Rieske centers (gz and gy are oriented parallel, gx is oriented perpendicular to the membrane plane). The shape of the EPR spectrum of the Rieske cluster from PS3 changed as a function of the redox state of the menaquinone (MK) pool. This permitted the redox midpoint potential of the MK pool to be determined in the membrane. Values of -60 +/- 20 mV at pH 7.0 and of -130 +/- 20 mV at pH 8.0 were obtained. The results are compared with already published data from other Rieske centers. It is proposed that all Rieske centers that function in electron transport chains using MK as pool quinone show common features that distinguish them from Rieske centers operating in ubiquinone- or plastoquinone-based electron transfer chains.


The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin. An initial step in the replication of simian virus (SV40) DNA is the ATP-dependent formation of a double hexamer of the SV40 large tumor (T) antigen at the SV40 DNA replication origin. In the absence of DNA, T antigen assembled into hexamers in the presence of magnesium and ATP. Hexameric T antigen was stable and could be isolated by glycerol gradient centrifugation. The ATPase activities of hexameric and monomeric T antigen isolated from parallel glycerol gradients were identical. However, while monomeric T antigen was active in the ATP-dependent binding, untwisting, unwinding, and replication of SV40 origin-containing DNA, hexameric T antigen was inactive in these reactions. Isolated hexamers incubated at 37 degrees C in the presence of ATP remained intact, but dissociated into monomers when incubated at 37 degrees C in the absence of ATP. This dissociation restored the activity of these preparations in the DNA replication reaction, indicating that hexameric T antigen is not permanently inactivated but merely assembled into a nonproductive structure. We propose that the two hexamers of T antigen at the SV40 origin assemble around the DNA from monomer T antigen in solution. This complex untwists the DNA at the origin, melting specific DNA sequences. The resulting single-stranded regions may be utilized by the T antigen helicase activity to initiate DNA unwinding bidirectionally from the origin.


Retinoic acid activation and thyroid hormone repression of the human alcohol dehydrogenase gene ADH3. Mammalian alcohol dehydrogenase (ADH) catalyzes the oxidation of retinol to retinaldehyde, the rate-limiting step in the synthesis of retinoic acid. There exists a family of ADH isozymes encoded by unique genes, and it is unclear which isozymes are most important for regulation of retinoic acid synthesis during differentiation or development. A region in the human ADH3 promoter from -328 to -272 base pairs was shown previously to function as a retinoic acid response element (RARE), prompting an hypothesis for a positive feedback mechanism controlling retinoic acid synthesis (Duester, G., Shean, M. L., McBride, M. S., and Stewart, M. J. (1991) Mol. Cell. Biol. 11, 1638-1646). The ADH3 RARE contains three direct AGGTCA repeats which constitute the critical nucleotides of RAREs present in other genes. We dissected the ADH3 RARE and determined that receptor binding as well as transactivation are dependent upon only the two downstream AGGTCA motifs separated by 5 base pairs, a structure noticed previously for a RARE in the promoter for the retinoic acid receptor beta (RAR beta) gene. ADH3 and RAR beta RAREs functioned similarly in transfection assays, suggesting that the feedback mechanisms controlling ADH3 and RAR beta utilize a common RARE. We also found that the normal functioning of the ADH3 RARE was abrogated by thyroid hormone receptor in the presence of thyroid hormone. A negative thyroid hormone response element in the human ADH3 promoter was found to colocalize with the RARE. Since ADH production in rat liver is known to be repressed by thyroid hormone, these findings suggest that human ADH production may also be subject to thyroid hormone repression and that the mechanism involves an interference with retinoic acid induction.


Structural requirements for the growth factor activity of the amino-terminal domain of urokinase. High molecular weight urokinase-type plasminogen activator (uPA) in which proteolytic activity was inactivated (diisopropyl fluorophosphate (DFP)-uPA), its amino-terminal fragment (ATF, amino acids (aa) 1-143), and fucosylated and defucosylated growth factor domains (GFD, aa 4-43) were tested for growth-promoting effects and binding in human SaOS-2 osteosarcoma cells and U-937 lymphoma cells. DFP-uPA, ATF, and both the fucosylated and defucosylated GFD were capable of competing with 125I-ATF for binding to both SaOS-2 and U-937 cells. DFP-uPA, ATF, and fucosylated GFD were also mitogenic in SaOS-2 cells and increased cell numbers. However, defucosylated GFD was nonmitogenic in SaOS-2 cells and did not stimulate cell proliferation, even though it bound to these cells in a manner equivalent to the fucosylated GFD. A nonglycosylated high molecular weight uPA expressed and purified from Escherichia coli inhibited 125I-ATF binding to SaOS-2 cells but was also nonmitogenic. No mitogenic activity was observed in U-937 cells treated with the uPA forms capable of eliciting a mitogenic response in SaOS-2 cells. Proteolytically prepared kringle domain (aa 47-135) and low molecular weight uPA (aa 144-411) did not compete for 125I-ATF binding and did not elicit any mitogenic response in either of the cell lines tested. In addition, tissue plasminogen activator (tPA), which has been shown to be homologous to uPA in its growth factor domain and is also fucosylated, did not inhibit 125I-ATF binding nor elicit any mitogenic response. These results demonstrate that the GFD, implicated in binding to the uPA receptor, is also responsible for growth factor like activity in SaOS-2 cells and that the fucosylation at Thr18 within this domain may serve as a molecular trigger in eliciting this response.


Protein ubiquitination is regulated by phosphorylation. An in vitro study. Protein ubiquitination has been implicated in ATP-dependent protein turnover and in a number of biological processes in eukaryotic cells. The ubiquitination activating enzyme, E1, and ubiquitin carrier protein, E2, are two essential enzymes in the protein ubiquitination machinery. Using purified E1 and E2 from rabbit reticulocytes and various protein kinases, which include cAMP-dependent protein kinase, protein kinase C, and protein tyrosine kinase, we demonstrated that E1 is phosphorylated by protein kinase C, with a stoichiometry of 0.65 mol of phosphate/mol of E1, and one of the E2 isoforms, E2(32kDa), is phosphorylated by protein tyrosine kinase to 2 eq of phosphate/mol of protein. Phosphorylation of E1 causes a 2-fold enhancement of its activity as monitored by ubiquitin-dependent ATP in equilibrium PPi exchange. When 1 eq of phosphate was incorporated into E2(32kDa), a 2.4-fold activation was also observed for its activity to catalyze the ubiquitination of histone H2A. The regulatory significance of this finding is discussed.


Adjacent nuclear factor-1 and activator protein binding sites in the enhancer of the neurotropic JC virus. A common characteristic of many brain-specific genes. JC virus is a neurotropic virus that causes the demyelinating disease progressive multifocal leukoencephalopathy in humans. In order to understand the neurotropic nature of this virus, we examined the binding of nuclear proteins to the viral regulatory region. A close association of nuclear factor-1 (NF-1) and Jun protein binding sites was found. These binding sites were either adjacent or overlapped each other. Depending on the order of binding, there was some interference of binding of the NF-1 protein by Jun even at a non-Jun binding site. This suggests that there may be a direct interaction between these proteins. Examination of the regulatory region of a number of genes expressed in the central and peripheral nervous systems revealed that many of these genes apparently have adjacent NF-1 and activator protein binding sites immediately upstream from the mRNA start site. Since it had been demonstrated that nuclear proteins from brain and non-brain cells could interact with these sites, it is probable that the NF-1- and Jun-related proteins which interact at these sites are involved in the basal activity of these genes. It appears that adjacent binding sites for NF-1 and Jun immediately upstream from the mRNA start site may be a characteristic of many genes expressed in the nervous system.


The fate of parental nucleosomes during SV40 DNA replication. The fate of parental nucleosomes during the replication of chromatin templates was studied using a modification of the cell-free SV40 DNA replication system. Plasmid DNA molecules containing the SV40 origin were assembled into chromatin with purified core histones and fractionated assembly factors derived from HeLa cells. When these templates were replicated in vitro, the resulting progeny retained a nucleosomal organization. To determine whether the nucleosomes associated with the progeny molecules resulted from displacement of parental histones during replication followed by reassembly, the replication reactions were performed in the presence of control templates. It was observed that the progeny genomes resulting from the replication of chromatin templates retained a nucleosomal structure, whereas the progeny of the control DNA molecules were not assembled into chromatin. Additional experiments, involving direct addition of histones to the replication reaction mixtures, confirmed that the control templates were not sequestered in some form which made them unavailable for nucleosome assembly. Thus, our data demonstrate that parental nucleosomes remain associated with the replicating molecules and are transferred to the progeny molecules without displacement into solution. We propose a simple model in which nucleosomes ahead of the fork are transferred intact to the newly synthesized daughter duplexes.


Purification and substrate specificity of an endopeptidase from the human oral spirochete Treponema denticola ATCC 35405, active on furylacryloyl-Leu-Gly-Pro-Ala and bradykinin. An endopeptidase was purified to homogeneity from the cell extracts of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised dialysis, anion exchange fast protein liquid chromatography (FPLC), hydroxylapatite FPLC, immobilized metal affinity FPLC, FPLC chromatofocusing, and two consecutive gel permeation FPLC steps. The enzyme is a 62-kDa protein with an isoelectric point of 6.5-7.0. Experiments with enzyme inhibitors suggest that this enzyme is a metallopeptidase and that its activity is not dependent on sulfhydryl or serine residues. The enzyme is active on furylacryloyl-Leu-Gly-Pro-Ala (FALGPA; pH optimum near 6.25), bradykinin (Bk), and several Bk-related peptides. In FALGPA, the cleavage site is the Leu-Gly bond. An imino acid is absolutely necessary in position P'2. The shortest hydrolyzed peptide was FALGPA, the hydrolysis of which is strongly and competitively inhibited by Bk (Ki = 5.0 microM). The pyrophosphate ion and phosphoramidon also inhibited the hydrolysis of FALGPA. The enzyme does not hydrolyze all typical synthetic collagenase substrates, Azocoll, Azocasein, or Type I and Type IV collagens, or any other proteins tested. In Bk-related peptides, the hydrolyzed bond was Phe5-Ser6. Since a Bk antagonist and a Bk-potentiating pentapeptide also were good substrates, it is possible that the enzyme hydrolyzes Bks and related peptides only because of the coincidental, specific amino acid sequence of those substrates. A proposal is made that since a substantial portion of the amino acid sequence of FALGPA is present in collagen (and additionally acknowledging that the furylacryloyl residue structurally resembles that of proline), the natural substrates of this enzyme may be small, soluble collagen fragments produced by other enzymes from periodontal connective tissue, and that such peptides are important for the nutrition and pathogenicity of T. denticola.


Cooperative interactions between replication origin-bound molecules of herpes simplex virus origin-binding protein are mediated via the amino terminus of the protein. The virally encoded origin binding protein (OBP) of herpes simplex virus (HSV) is required for viral DNA synthesis. OBP binds at the replication origin to initimultienzyme replication complex (Challberg, M. D., and Kelly, T. J. (1989) Annu Rev. Biochem. 58, 671-717), OBP binds to two sites at the replication origin. The sequence-specific interaction of OBP with each binding site is localized to the major groove, and in both HSV origins the two interaction surfaces are in phase, aligned on the same face of the helix (Hazuda, D. J., Perry, H. C., Naylor, A. M., and McClements, W. L. (1991) J. Biol. Chem. 261, 24621-24625). Using native gel electrophoresis, we now demonstrate that OBP binding to the origin is highly cooperative and that cooperativity requires the putative NH2-terminal leucine zipper. Neither the phase nor orientation of the binding sites affect cooperativity, suggesting that the interaction promotes wrapping of origin DNA around the OBP multimer. A comparison of OBP DNase I footprints with the DNase I footprints of a truncated protein defective in cooperativity demonstrates that the interaction between OBPs bound at sites I and II affects the conformation of the intervening DNA, particularly when the phase or orientation of the two sites is different from wild type. OBP may elicit a unique nucleoprotein structure which facilitates unwinding of the origin and/or assembly of the replication complex. We also demonstrate that OBP can exchange binding sites, forming interduplex complexes. This property may be important for reinitiation of DNA replication.


Luteinization-associated changes in protein stability of the regulatory subunit of the type I cAMP-dependent protein kinase. The purpose of this study was to determine how RI alpha, the R subunit of the type I cAMP-dependent protein kinase, is regulated in rabbit ovarian follicles in response to the preovulatory luteinizing hormone surge. When soluble extracts from rabbit preovulatory follicles and 7-day-old corpora lutea were photoaffinity-labeled with 8-N3-[32P]cAMP, 3-fold more RI alpha was detected in corpora lutea than in follicles. Based on DEAE-cellulose chromatography, both type I holoenzyme and free RI alpha increased during luteinization. Western blot analysis of soluble extracts obtained from follicles and corpora lutea at various time points after human chorionic gonadotropin (hCG) injection revealed a 6-10-fold increase in RI alpha protein by 5 h after hCG injection. However, based on Northern blot analysis and solution hybridization/RNase protection assays, this increase in RI alpha protein was not due to an increase in RI alpha mRNA. These results suggested that RI alpha subunit levels were post-transcriptionally regulated. Half-life determinations indicated a 2.1-fold increase in the stability of RI alpha when follicles were incubated in the presence of hCG. The effect of hCG on the stability of RI alpha could also be mimicked by forskolin, thus suggesting that a rise in cAMP levels in follicles during the luteinizing hormone surge plays a role in RI alpha subunit stability. We conclude that RI alpha protein is stabilized in follicles by hCG treatment and the consequent rise in follicular cAMP levels.


Crystal structure of a wheat germ agglutinin/glycophorin-sialoglycopeptide receptor complex. Structural basis for cooperative lectin-cell binding. The crystal structure of wheat germ agglutinin isolectin 1 (WGA1) complexed with a tryptic sialoglycopeptide fragment (T-5) from its erythrocyte receptor glycophorin A, which contains the O-linked tetrasaccharide NeuNAc-alpha 2,3-Gal-beta 1,3-(alpha 2,6-NeuNAc) Gal-NAc-alpha 1-O-Thr, has been determined by molecular replacement techniques and refined at 2.0-A resolution (R = 18.1%). The structure reveals that association between WGA1 dimers, composed of two identical four-domain (A-D) monomers, and T-5 is asymmetric and involves sialic acid binding at three nonequivalent aromatic residue-rich sites. Two independent binding modes are observed. In the dominant ("major") binding mode, the two highest affinity sites are utilized to cross-link neighboring crystallographically related WGA1 dimers. The branched tetrasaccharide has an extended rigid conformation, and its terminal alpha 2,6-NeuNAc and alpha 2,3-NeuNAc residues occupy specificity sites in domains B1 (monomer 1) and C2 (monomer 2) on opposing dimers, respectively. This asymmetric selection of binding sites leads to infinite open-ended arrays of interlinked lectin molecules. In the subsidiary "minor" binding mode, only the terminal alpha 2,6-NeuNAc, anchored to the aromatic residue-rich binding site in domain A2, is clearly visible. The remaining portion of T-5 is disordered. This structure presents the first evidence for NeuNAc binding in the aromatic residue-rich sites of domains A and C and suggests a preference of WGA for alpha 2,6-linked NeuNAc. Moreover, the unusual asymmetric WGA1-tetrasaccharide association, involving domain binding sites that differ in their binding affinities for NeuNAc, offers explanations for the widely observed cooperative cell binding behavior of WGA.


Isolation and characterization of a gene encoding rat nucleoside diphosphate kinase. Two overlapping genomic clones for a rat nucleoside diphosphate kinase (NDP kinase) have been isolated and characterized. Complete sequencing of the genomic segment including the whole coding region for the enzyme revealed that the gene consists of four exons spanning 5.5 kilobase pairs. Primer extension analyses and ribonuclease protection assays indicated that the transcription may start from multiple sites with the major initiation site at 3 base pairs upstream from the translation initiation site, Met-1. Neither CAAT-box nor TATA-box could be assigned for each transcription initiation site, whereas five putative Sp1-binding sites (GC-boxes) were present in the 5'-flanking region. These features of the NDP kinase gene represent those of housekeeping genes. In genomic Southern blotting using a full-length rat NDP kinase cDNA as a probe, many positively hybridized fragments were detected. In support of this, five possible processed pseudogenes were identified in different DNA segments although many other NDP kinase-related genomic fragments remained to be characterized. These results demonstrate that the NDP kinase gene may consist of a multiple gene family.


Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells. Two peaks of mitogen-activated protein (MAP) kinase activator activity are resolved upon ion exchange chromatography of cytosolic extracts from epidermal growth factor-stimulated A431 cells. Two forms of the activator (1 and 2) have been purified from these peaks, using chromatography on Q-Sepharose, heparin-agarose, hydroxylapatite, ATP-agarose, Sephacryl S-300, Mono S, and Mono Q. The two preparations each contained one major protein band with an apparent molecular mass of 46 or 45 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Evidence identifying the MAP kinase activators as the 46- and 45-kDa proteins is presented. Using inactive mutants of MAP kinase as potential substrates, it was found that each preparation of MAP kinase activator catalyzes phosphorylation of the regulatory residues, threonine 188 and tyrosine 190, of Xenopus MAP kinase. These results support the concept that the MAP kinase activators are protein kinases. These MAP kinase kinases demonstrate an apparent high degree of specificity toward the native conformation of MAP kinase, although slow autophosphorylation on serine, threonine, and tyrosine residues and phosphorylation of myelin basic protein on serine and threonine residues is detected as well.


Iodination of tyrosine 59 of ubiquitin selectively blocks ubiquitin's acceptor activity in diubiquitin synthesis catalyzed by E2(25K). Covalent ligation of multiubiquitin chains targets eukaryotic proteins for degradation. Ubiquitin-conjugating enzyme E2(25K) utilizes isolated ubiquitin as the substrate for synthesis of such chains, in which successive ubiquitin units are linked by isopeptide bonds involving the side chain of Lys-48 of one ubiquitin and the COOH group of Gly-76 of the next. During continuous synthesis of multiubiquitin chains in the presence of purified ubiquitin-activating enzyme and E2(25K), there was a slight discrimination against radioiodinated ubiquitin (2.3-fold reduction in specific radioactivity of diubiquitin relative to value expected for no discrimination). Single-turnover experiments employing stoichiometrically iodinated ubiquitin derivatives indicated that E2(25K) discriminates extremely strongly (greater than 20-fold reduction in kcat/Km for diubiquitin synthesis) against ubiquitin that is monoiodinated at Tyr-59. The modest overall selection effect observed in continuous reactions is in part due to the occurrence of discrimination only when iodotyrosylubiquitin is the acceptor (Lys-48 donor) in diubiquitin synthesis; iodotyrosylubiquitin is kinetically competent when it is the species being transferred to native ubiquitin. The competence as acceptor of a site-directed mutant form of ubiquitin bearing a Tyr to Phe substitution at position 59 indicated that discrimination against iodotyrosylubiquitin by E2(25K) is not due to loss of the hydrogen-bonding interactions of Tyr-59. Rather, iodotyrosylubiquitin may be unable to react with the ubiquitin adduct of E2(25K) for steric reasons. Discrimination against iodotyrosylubiquitin as acceptor is unique to E2(25K) among three enzymes surveyed: iodotyrosylubiquitin is a fully competent acceptor in diubiquitin synthesis catalyzed by E2(25K) and is also utilized for multiubiquitin chain synthesis by E2(14K) and ubiquitin-protein ligase. These findings should assist in the design of future studies concerning E2(25K) structure and function.


Mouse mammary tumor virus gene expression is suppressed by oligomeric ellagitannins, novel inhibitors of poly(ADP-ribose) glycohydrolase. Oligomeric ellagitannins (nobotanins B, E, and K) were found to be potent inhibitors of poly(ADP-ribose) glycohydrolase purified from mouse mammary tumor 34I cells. Kinetic analysis revealed that the inhibition of nobotanin B (dimer) was competitive with respect to the substrate poly(ADP-ribose), whereas nobotanin E (trimer) and nobotanin K (tetramer) exhibited mixed-type inhibition. These results suggest that the dimeric structure of ellagitannin may have a functional domain that competes with poly(ADP-ribose) on the poly(ADP-ribose) glycohydrolase molecule. To determine the inhibitory effects of oligomeric ellagitannins on poly(ADP-ribose) glycohydrolase in vivo, we examined their effects on de-poly(ADP-ribosyl)ation of some chromosomal proteins in intact 34I cells that was induced by glucocorticoid treatment. Nobotanin B caused concentration-dependent inhibition of glucocorticoid-induced de-poly(ADP-ribosyl)ation of HMG 14 and 17 and histone H1 in intact 34I cells. Interestingly, this inhibition was associated with suppression of the glucocorticoid-sensitive mouse mammary tumor virus (MMTV) mRNA synthesis. In contrast, nobotanin E and K had little inhibitory effect on either de-poly(ADP-ribosyl)ation of these proteins or induction of MMTV transcription after glucocorticoid treatment. Nobotanin B but not E and K was taken into 34I cells. These results may suggest that the suppression of glucocorticoid-sensitive MMTV transcription results from in vivo inhibition of poly(ADP-ribose) glycohydrolase by nobotanin B. These results also indicate the importance of de-poly(ADP-ribosyl)ation of HMG 14 and 17 and histone H1 in regulation of transcription of the glucocorticoid-sensitive MMTV gene.


Molecular cloning and promoter analysis of the rat liver farnesyl diphosphate synthase gene. The isolation and characterization of the rat genomic clone encoding the cholesterogenic enzyme farnesyl diphosphate (FPP) synthase is reported. The gene is localized on a 15-kilobase (kb) genomic fragment, spans approximately 12 kb and contains eight exons. Sequences containing from 3.9 kb to 132 base pairs (bp) of the putative promoter were joined to the coding region of the bacterial reporter gene chloramphenicol acetyltransferase (CAT). The CAT activities or CAT mRNA levels of the hybrid genes were determined following either transient transfections into human hepatoma HepG2 cells or stable transfections into Chinese hamster ovary cells. The transient transfections identified a 319-bp fragment that was required for a 4-fold induction in the absence of sterols. Sequence analysis of this region showed it contained five potential copies of the sterol regulatory element (SRE-1) (Smith, J.R., Osborne, T.F., Brown, M.S., Goldstein, J.L., and Gil, G. (1988) J. Biol. Chem. 263, 18480-18487) previously identified in the promoters of the 3-hydroxy-3-methyl-coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and low density lipoprotein receptor genes. Further mutational and deletion analysis of the FPP synthase promoter-CAT constructs followed by stable transfection and primer extension of the CAT mRNA levels indicated that these potential SRE-1 regulatory elements were not involved in the sterol-mediated transcriptional regulation of the gene. Our analyses have identified a 115-bp region that is required for the transcriptional induction of FPP synthase in the absence of sterols. These results suggest that the FPP synthase gene may be regulated at the transcriptional level by a different mechanism than other sterol regulated genes.


Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the beta 1, gamma 2S, and gamma 2L subunits of the gamma-aminobutyric acid type A receptor. Gamma-aminobutyric acid Type A (GABAA) receptors are the major sites of synaptic inhibition in the central nervous system. These receptors are thought to be pentameric complexes of homologous transmembrane glycoproteins. Molecular cloning has revealed a multiplicity of different GABAA receptor subunits divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology. Within the proposed major intracellular domain of these subunits, there are numerous potential consensus sites for protein phosphorylation by a variety of protein kinases. We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). The purified fusion protein of the intracellular domain of the beta 1 subunit was an excellent substrate for both PKA and PKC. PKA and PKC phosphorylated the beta 1 subunit fusion protein on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 409 in the intracellular domain of the beta 1 subunit to an alanine residue eliminated the phosphorylation of the beta 1 subunit fusion protein by both protein kinases. The purified fusion proteins of the major intracellular domain of the gamma 2S and gamma 2L subunits of the GABAA receptor were rapidly and stoichiometrically phosphorylated by PKC but not by PKA. The phosphorylation of the gamma 2S subunit occurred on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 327 of the gamma 2S subunit fusion protein to an alanine residue eliminated the phosphorylation of the gamma 2S fusion protein by PKC. The gamma 2L subunit is an alternatively spliced form of the gamma 2S subunit that differs by the insertion of 8 amino acids (LLRMFSFK) within the major intracellular domain of the gamma 2S subunit. The PKC phosphorylation of the gamma 2L subunit occurred on serine residues on two tryptic phosphopeptides. Site-specific mutagenesis of serine 343 within the 8-amino acid insert to an alanine residue eliminated the PKC phosphorylation of the novel site in the gamma 2L subunit. No phosphorylation of a purified fusion protein of the major intracellular loop of the alpha 1 subunit was observed with either PKA or PKC. These results identify the specific amino acid residues within GABAA receptor subunits that are phosphorylated by PKA and PKC and suggest that protein phosphorylation of these sites may be important in regulating GABAA receptor function.(ABSTRACT TRUNCATED AT 400 WORDS)


Rab geranylgeranyl transferase. A multisubunit enzyme that prenylates GTP-binding proteins terminating in Cys-X-Cys or Cys-Cys. Rab proteins are membrane-bound prenylated GTP-binding proteins required for the targeted movement of membrane vesicles from one organelle to another. In the current paper we have characterized and purified an enzyme that attaches geranylgeranyl residues to Rab proteins that bear the COOH-terminal sequence Cys-X-Cys (such as Rab3A) and Cys-Cys (such as Rab1A). This enzyme is designated Rab geranylgeranyl transferase (Rab GG transferase). At high salt concentrations, Rab GG transferase from rat brain cytosol separates into two components, designated A and B, both of which are required for activity. We purified Component B to apparent homogeneity and found that it contains two peptides of 60 and 38 kDa. The purified Rab GG transferase did not attach geranylgeranyl to p21H-ras-CVLL, which is prenylated by a GG transferase of the CAAX type that resembles the CAAX farnesyltransferase. Rab GG transferase was strongly inhibited by Zn2+, a cation that is absolutely required by farnesyltransferase. The Rab GG transferase was also inhibited by NaCl concentrations in excess of 100 mM. Together with previous data, the current findings indicate that mammalian cells possess at least three protein prenyltransferases (CAAX farnesyltransferase, CAAX GG transferase, and Rab GG transferase) that are specific for different classes of low molecular weight GTP-binding proteins and other proteins.


The phosphorylation state of the DegU response regulator acts as a molecular switch allowing either degradative enzyme synthesis or expression of genetic competence in Bacillus subtilis. Two classes of mutations were identified in the degS and degU regulatory genes of Bacillus subtilis, leading either to deficiency of degradative enzyme synthesis (degS or degU mutations) or to a pleiotropic phenotype which includes overproduction of degradative enzymes and the loss of genetic competence (degS(Hy) or degU(Hy) mutations). We have shown previously that the DegS protein kinase and the DegU response regulator form a signal transduction system in B. subtilis. We now demonstrate that the DegS protein kinase also acts as a DegU phosphatase. We present evidence that the DegU response regulator has two active conformations: a phosphorylated form which is necessary for degradative enzyme synthesis and a nonphosphorylated form required for expression of genetic competence. The degU146-encoded response regulator, allowing expression of genetic competence, has been purified and seems to be modified within the putative phosphorylation site (D56----N) since it is no longer phosphorylated by DegS. Both the degU146 mutation as well as the degS220 mutation, which essentially abolishes DegS protein kinase activity, lead to deficiency of degradative enzyme synthesis, indicating the requirement of phosphorylated DegU for the expression of this phenotype. We also purified the degU32(Hy)-encoded protein and showed that this response regulator is phosphorylated by the DegS protein kinase in vitro. In addition, the phosphorylated form of the degU32(Hy)-encoded protein presented a strongly increased stability as compared with the wild type DegU protein, thus leading to hyperproduction of degradative enzymes in vivo.


Chronotropic and inotropic responses to adrenoceptor agonists in vitro after chronic dobutamine treatment in the rabbit. 1. Effects of long-term administration of the inotropic agent dobutamine (beta-and alpha 1-adrenoceptor agonist) on cardiac adrenoceptor function were studied in the rabbit. 2. After 2 weeks' continuous i.v. infusion of dobutamine (Dobutrex, 20 micrograms kg-1 min-1), spontaneous atrial rates in vitro were similar to controls, as were maximal rates in response to isoprenaline (262 +/- 17 vs 268 +/- 21 b.p.m., P greater than 0.05) but the chronotropic potency of isoprenaline was reduced, as shown by a nine-fold increase in EC50. 3. Basal developed tension of papillary muscles was greater after chronic dobutamine treatment and increases in contraction force in response to beta-activation by isoprenaline and noradrenaline were significantly higher. Maximal developed tensions were 93 +/- 14% (P less than 0.02) and 94 +/- 25% (P less than 0.05) respectively greater than those of control muscles. 4. The inotropic potency of isoprenaline, but not noradrenaline, was reduced significantly after chronic dobutamine with a two-fold increase in EC50. 5. Responses to alpha 1-adrenoceptor activation by phenylephrine were unchanged after dobutamine treatment. 6. These changes are consistent with functional desensitization of the myocardium by the prolonged beta- but not alpha 1-agonist activity of dobutamine. In contrast, there was an enhanced effectiveness of beta-adrenoceptor activation.


Peripheral 2-hydroxy-saclofen-sensitive GABA-B receptors mediate both vagal-dependent and vagal-independent acid secretory responses in rats. 1. The present study investigates the effects of peripherally administered baclofen on gastric acid secretion from the perfused stomach of anaesthetized rats. 2. Intravenous (i.v.) baclofen caused a marked dose-dependent increase in acid secretion which was antagonized by i.v. 2-hydroxy-saclofen, whereas intracerebroventricular (i.c.v.) 2-hydroxy-saclofen and i.c.v. or i.v. phaclofen and bicuculline were ineffective. In addition, 2-hydroxy-saclofen did not affect acid hypersecretion stimulated by histamine. 3. The secretagogue action of baclofen was fully prevented by cimetidine, but only partially attenuated by atropine, proglumide or bilateral cervical vagotomy. Moreover, the vagotomy-resistant excitatory effect of baclofen was abolished by 2-hydroxy-saclofen or cimetidine, but not by atropine or proglumide. 4. In vagotomized rats whose gastric secretion was maximally increased by electrical stimulation of the left vagus nerve, i.v. injection of baclofen further potentiated acid output, this action being prevented by cimetidine. 5. Taken together, the present results provide evidence that peripheral GABA-B receptors mediate the gastric hypersecretory effect of parenterally administered baclofen to anaesthetized rats, and suggest that both vagal cholinergic and extravagal pathways are involved in the stimulant effect.


Control of electrolyte transport in the kidney through a dopamine- and cAMP-regulated phosphoprotein, DARPP-32. 1. DARPP-32 is a phosphoprotein regulated by dopamine and cAMP. In its phosphorylated form it acts as an inhibitor of protein phosphatase-1, thereby regulating the phosphorylation state of phosphoproteins in the basal ganglia. 2. In the kidney, DARPP-32 has been detected in the medullary thick ascending limb of Henle (mTAL) and, to a lesser degree, in the proximal convoluted tubule by means of immunohistochemistry and in situ hybridization. 3. In single microdissected tubules of rat kidney, Na+, K(+)-ATPase activity, measured as ouabain-sensitive ATP hydrolysis, has been shown to be inhibited to the same degree by the DA1 agonist fenoldopam, cAMP and a synthesized and phosphorylated DARPP-32 peptide, D32(8-38). 4. It is concluded that the DA1 receptor-mediated inhibition of Na+ transport in the mTAL by dopamine occurs via cAMP accumulation and the phosphoprotein, DARPP-32.


Disulfide bond formation during the folding of influenza virus hemagglutinin. To study the importance of individual sulfhydryl residues during the folding and assembly in vivo of influenza virus hemagglutinin (HA), we have constructed and expressed a series of mutant HA proteins in which cysteines involved in three disulfide bonds have been substituted by serine residues. Investigations of the structure and intracellular transport of the mutant proteins indicate that (a) cysteine residues in the ectodomain are essential both for efficient folding of HA and for stabilization of the folded molecule; (b) cysteine residues in the globular portion of the ectodomain are likely to form native disulfide bonds rapidly and directly, without involvement of intermediate, nonnative linkages; and (c) cysteine residues in the stalk portion of the ectodomain also appear not to form intermediate disulfide bonds, even though they have the opportunity to do so, being separated from their correct partners by hundreds of amino acids including two or more other sulfhydryl residues. We propose a role for the cellular protein BiP in shielding the cysteine residues of the stalk domain during the folding process, thus preventing them from forming intermediate, nonnative disulfide bonds.


Ubiquitin-activating enzyme, E1, is associated with maturation of autophagic vacuoles. The ubiquitin-activating enzyme, E1, is required for initiating a multi-step pathway for the covalent linkage of ubiquitin to target proteins. A CHO cell line containing a mutant thermolabile E1, ts20, has been shown to be defective in stress-induced degradation of proteins at restrictive temperature (Gropper et al., 1991. J. Biol. Chem. 266:3602-3610). Parental E36 cells responded to restrictive temperature by stimulating lysosome-mediated protein degradation twofold. Such a response was not observed in ts20 cells. The absence of accelerated degradation in these cells at 39.5 degrees C was accompanied by an accumulation of autolysosomes. The fractional volume of these degradative autophagic vacuoles was at least sixfold greater than that observed for either E36 cells at 30.5 degrees or 39.5 degrees C, or ts20 cells at 30.5 degrees C. These vacuoles were acidic and contained both acid phosphatase and cathepsin L, but, unlike the autolysosomes observed in E36 cells, ubiquitin-conjugated proteins were conspicuously absent. Combined, our results suggest that in ts20 cells, which are unable to generate ubiquitin-protein conjugates due to heat inactivation of E1, the formation and maturation of autophagosomes into autolysosomes is normal, but the conversion of autolysosomes into residual bodies is disrupted.


Membrane anchoring of the autoantigen GAD65 to microvesicles in pancreatic beta-cells by palmitoylation in the NH2-terminal domain. Pancreatic beta-cells and gamma-aminobutyric acid (GABA)-secreting neurons both express the enzyme glutamic acid decarboxylase (GAD) which is a major target of autoantibodies associated with beta-cell destruction and impairment of GABA-ergic neurotransmitter pathways. The predominant form of GAD in pancreatic beta-cells, GAD65, is synthesized as a soluble hydrophilic molecule, which is modified to become firmly membrane anchored. Here we show by immunogold electron microscopy that GAD65 is localized to the membrane of small vesicles which are identical in size to small synaptic-like microvesicles in pancreatic beta-cells. The NH2-terminal domain of GAD65 is the site of a two-step modification, the last of which results in a firm membrane anchoring that involves posttranslational hydroxylamine sensitive palmitoylation. GAD65 can be released from the membrane by an apparent enzyme activity in islets, suggesting that the membrane anchoring step is reversible and potentially regulated. The hydrophobic modifications and consequent membrane anchoring of GAD65 to microvesicles that store its product GABA may be of functional importance and, moreover, significant for its selective role as an autoantigen.


Schwann cells of the myelin-forming phenotype express neurofilament protein NF-M. Immature Schwann cells of the rat sciatic nerve can differentiate into myelin-forming or non-myelin-forming cells. The factors that influence this divergent development are unknown but certain markers such as galactocerebroside distinguish the two cell populations at an early stage of Schwann cell differentiation. Because myelination requires extensive changes in cell morphology, we have investigated the composition and structure of the Schwann cell cytoskeleton at a time when these cells become committed to myelination. Here we show that Schwann cells express a cytoskeletal protein of M(r) 145 before diverging into the myelin-forming path, i.e., before they acquire cell-surface galactocerobroside. The p145 protein has the characteristics of an intermediate filament (IF) protein and immunoelectron microscopy shows that it colocalizes with vimentin, which suggests that these two proteins can coassemble into IFs. Elevated intracellular cAMP levels, which can mimic some of the early effects of axons on Schwann cell differentiation, induced p145 synthesis, therefore, we conclude that myelin-forming Schwann cells express this protein at a very early stage in their development. Immunological comparisons with other IF proteins revealed a close similarity between p145 and the neurofilament protein NF-M; the identification of p145 as NF-M was confirmed by isolating and sequencing a full-length clone from a Schwann cell cDNA library. These data demonstrate that Schwann cells remodel their IFs by expressing NF-M before acquiring the myelin-forming phenotype and that IF proteins of the neurofilament-type are not restricted to neurons in the vertebrate nervous system.


Thrombin receptor activation causes rapid neural cell rounding and neurite retraction independent of classic second messengers. The protease thrombin is a potent activator of various cell types. Thrombin cleaves and thereby activates its own seven-transmembrane-domain receptor which couples to G proteins. Thrombin also can inhibit neuronal differentiation, supposedly by degrading components of the extracellular matrix. Here we report that active thrombin induces immediate cell rounding and neurite retraction in differentiating N1E-115 and NG108-15 neural cells in serum-free culture. Serum (0.5-5% vol/vol) evokes similar responses, but the cell-rounding and neurite-retracting activity of serum is not attributable to thrombin. Neural cell rounding is transient, subsiding after 10-15 min, and subject to homologous desensitization, whereas retracted neurites rapidly degenerate. Thrombin action is inhibited by cytochalasin, but not colchicine. A novel 14-amino acid peptide agonist of the thrombin receptor fully mimics thrombin's morphoregulatory activity, indicating that thrombin-induced shape changes are receptor-mediated and not secondary to extracellular matrix degradation. Although thrombin receptors couple to phosphoinositide hydrolysis and Ca2+ mobilization, thrombin-induced shape changes appear to depend neither on the Ca2+/protein kinase C- nor the cyclic nucleotide-mediated signal transduction pathways; however, the morphological response to thrombin is blocked by pervanadate, an inhibitor of tyrosine phosphatases, and by broad-specificity kinase inhibitors. Our results suggest that the thrombin receptor communicates to an as-yet-uncharacterized effector to reorganize the actin cytoskeleton and to reverse the differentiated phenotype of neural cells.


Diacylglycerol activation of protein kinase C results in a dual effect on Na+,K(+)-ATPase activity from intact renal proximal tubule cells. This study evaluated the effect of L-1-oleoyl-2-acetyl-sn-3-glycerol (OAG) on ouabain-sensitive Na,K-dependent oxygen consumption (Na,K-QO2) in intact renal proximal tubule cells (RPTC). Basal Na,K-QO2 (nmol O2/mg protein per min) was 20.0 +/- 1.0. Incubation with 10 nM of OAG induced a dual effect on Na,K-QO2, with an initial stimulation (maximal at 10 min, 37.1 +/- 5.0), followed by an inhibition (significant at 20 min, 16.3 +/- 1.0). No changes in ouabain-insensitive QO2 were observed in any of the protocols. The effects were abolished by sphingosine, a protein kinase C inhibitor. Stimulation was abolished by amiloride 0.1 mM. Amiloride had no effect on Na,K-QO2 at the concentration used. Stimulation was not potentiated by the sodium ionophore, amphotericin B, and the later inhibition was still observed in the presence of amphotericin B. The initial stimulation was attributed to an increase in sodium permeability, while the later inhibition was attributed to a direct effect on the Na,K-pump. Regulation of Na+,K(+)-ATPase activity by protein kinase C in intact RPTC can be accomplished by a direct effect on the protein or as a secondary effect consequent upon changes in intracellular sodium.


The heroic age of neurophysiology.

The heroic age of neurophysiology. Building on 19th- and early 20th-century conceptualizations--the synapse, the cellular nature of neuronal structure, the electrical basis of neural impulses--the heroic age, starting around World War I, set the stage for molecular definition of neuromuscular transmission. Without targeting specific diseases, the work took us to the verge of controlling some of humankind's cruelest dysfunctions.


Determination by high-performance liquid chromatography with electrochemical detection of free and conjugated N-acetyldopamine excretion in urine of children with neuroblastoma and nephroblastoma. A simple method for the determination of N-acetyldopamine (NADA) (both free and conjugated) in children's urine by high-performance liquid chromatography with electrochemical detection has been developed. Conjugated NADA was measured as the free compound after enzymatic hydrolysis and purification on alumina. The total analysis time is 25 min. The results show a linearity of the whole assay from 0.005 to 20 mumol/l NADA; the sensitivity is 0.2 pmol per 20 microliters injected sample. Mean recoveries of 96.7 and 86.6% were obtained for free and total NADA, respectively. Modifications of the retention times (between 2 and 50 min) induced by changes in the eluent were determined. Conjugated NADA accounted for about 90% of the total excretion of NADA. These results suggest that this compound could play an important part in neuroblastoma; its concentration is thirteen times higher in children with neuroblastoma than in normal subjects.


Detection and differentiation of chicken anemia virus isolates by using the polymerase chain reaction. Complementary oligonucleotide primers which flank a 675-bp DNA fragment encompassing part of the putative gene for the capsid protein of chicken anemia virus (CAV) were used for the enzymatic amplification of CAV DNA by the polymerase chain reaction (PCR). Application of a dot blot hybridization assay by using a 32P-labeled cloned CAV DNA probe allowed PCR product amplified from as little as 0.1 fg of the target DNA sequence to be detected. When it was used for PCR amplification, DNA extracted from thymus tissue by a guanidine isothiocyanate-based method proved to be more efficient than that extracted by methods involving phenol or boiling. DNAs specified by 14 CAV isolates originating in the United Kingdom, Ireland, Germany, Sweden, the United States, Japan, and Australia were amplified. Restriction endonuclease analysis of the PCR-amplified DNAs with the enzymes HaeIII, HinfI, and HpaII indicated that the 14 CAV isolates can be assigned to seven groups, with isolates from different countries usually exhibiting the greatest number of restriction site differences.


Development of a biotin-streptavidin-enhanced enzyme-linked immunosorbent assay which uses monoclonal antibodies for detection of group C rotaviruses. A biotin-streptavidin-enhanced enzyme-linked immunosorbent assay (ELISA) which uses monoclonal antibodies (MAbs) for the detection of group C rotaviruses was developed. An assay in which plates were coated with three pooled MAbs and biotinylated polyclonal immunoglobulin G (IgG) (polyclonal antibody [PAb]) was used as the detector (MAb capture-PAb detector) was found to be the most sensitive and specific of the assays when it was compared with assays in which plates were coated with polyclonal antiserum and detection was done with either biotinylated polyclonal antiserum (PAb capture-PAb detector) or biotinylated pooled MAbs (PAb capture-MAb detector). The MAb capture-PAb detector ELISA detected 83% of samples confirmed to be positive for group C rotaviruses, whereas the PAb capture-PAb detector assay detected 63% of positive samples and the PAb capture-MAb detector assay detected 65% of positive samples. All three procedures detected both of the bovine and the two human group C rotaviruses, but none of the three procedures detected fecal samples containing group A and B rotaviruses or fecal samples negative for group C rotaviruses used in this study. The sensitivity of the MAb capture-PAb detector ELISA was determined by serially diluting fecal group C rotaviruses; antigens were detected in maximal positive dilution ranges of 1:1,000 to 1:3,000 for the samples tested. On the basis of the cell culture immunofluorescence assay infectivity titer of semipurified cell culture-passaged Cowden group C rotavirus, the sensitivity of the MAb capture-PAb detection ELISA for detection of homologous group C rotavirus was 53 fluorescent focus units per ml. Epitope mapping by use of the biotinylated MAbs in competition assay suggested that our MAbs may bind to three different but overlapping epitopes. These results suggest that the MAb capture-PAb detector ELISA can be used to study the epidemiology of group C rotaviruses in humans and animals.


Role of coproantibody in clinical protection of children during reinfection with rotavirus. Rotavirus is the major cause of severe, dehydrating infantile gastroenteritis. Infection is limited to the gut, but the relative roles of serum and secretory copro-immunoglobulin A (IgA) in protection are unclear. Specific copro-IgA is predictive of duodenal antirotaviral IgA and correlates with virus-neutralizing coproantibody. Copro-IgA conversion is a more sensitive marker of rotavirus reinfection than seroconversion. We measured rotavirus reinfections by copro-IgA conversion prospectively in 35 children recruited at a time of severe rotavirus illness. The children were followed up longitudinally for 14 to 31 months to determine whether high coproantibody levels correlated with clinical protection against rotavirus disease. Ninety-four percent of the children experienced reinfection, and 38% developed persistent elevations in specific copro-IgA termed plateaus. Plateau children had a higher mean annual rate of rotavirus infection and a lower ratio of symptomatic to total number of rotavirus reinfections than did nonplateau children. The annual rates of rotavirus infection and disease were significantly higher outside the plateau than inside it in children experiencing antirotavirus copro-IgA plateaus. Frequent rotavirus infection of children appears to stimulate production of a specific copro-IgA plateau which correlates with protection against an excess of infection and symptomatic disease.


General primer polymerase chain reaction in combination with sequence analysis for identification of potentially novel human papillomavirus genotypes in cervical lesions. We recently described the detection of potentially novel human papillomaviruses (HPV) genotypes (HPV types X [HPV X]) in cervical smears (A. J. C. van den Brule, C. J. L. M. Meijer, V. Bakels, P. Kenemans, and J. M. M. Walboomers, J. Clin. Microbiol. 28:2739-2743, 1990) by using the general primer-mediated polymerase chain reaction method (GP-PCR). In this study, the HPV specificities of GP-PCR products were determined by sequence analyses. M13 bacteriophage clones of PCR products derived from cloned unsequenced HPV genotypes 13, 32, 35, 43, 44, 45, 51, and 56 were subjected to dideoxy sequencing. Analyses of the putative amino acid sequences of these HPV types in addition to published HPV sequence data revealed stretches of highly conserved amino acid residues present in all HPV types, resulting in an HPV amino acid consensus sequence. Subsequently, HPV X-specific PCR products found in premalignant cervical lesions (n = 3), carcinomas in situ (n = 6), and invasive cancer (n = 6) were analyzed for their nucleotide sequences. Comparison of these sequences with published HPV nucleotide sequences and data obtained in this study revealed three HPV type 35, two HPV type 45, one HPV type 51, two HPV type 56, and six unique HPV X sequences, of which three types were present in four cases of carcinomas (in situ). The nucleotide sequences determined appeared to be unique after a data bank search. Furthermore, the sequences of all HPV X isolates matched the HPV amino acid consensus sequence, thus confirming HPV specificity. This study illustrates the power of GP-PCR in combination with sequence analysis to determine HPV specificity and genotyping of PCR products derived from sequenced as well as unsequenced HPVs, including novel, not yet identified HPV types.


Detection of cytomegalovirus (CMV) in granulocytes by polymerase chain reaction compared with the CMV antigen test. To compare the sensitivity and suitability of detection of active cytomegalovirus (CMV) infection by using monoclonal antibodies against CMV antigen (antigen test to detect antigenemia) and the polymerase chain reaction (PCR; to detect viral DNA) in granulocytes, 19 heart and 2 lung transplant recipients were closely monitored by these tests for at least 3 months after transplantation. All patients were CMV seropositive or had a seropositive donor. In total, 201 samples were tested; 46 were positive by both tests, 9 samples showed only antigenemia, 54 samples were positive by PCR only, and 102 samples were negative by both tests. PCR was positive earlier after transplantation in eight patients, whereas antigenemia was positive earlier after transplantation in one patient. In another four patients, both tests were positive at the same time. PCR was, on average, positive for a longer period of time. Discordant results showing a positive antigen test and a negative PCR were partly due to sampling error; some were positive by PCR on retesting. Samples which were negative by the antigen test and positive by PCR were taken at the beginning or at the end of an active CMV infection. In two patients, no active CMV infection was detected by the antigen test, cultures of urine and saliva, or serology, although PCR was positive for a long period of time in the two patients.


Comparative evaluation of four commercially available monoclonal antibodies for culture confirmation of herpes simplex virus infection. Four fluorescein isothiocyanate-labelled monoclonal antibody typing reagents were compared for intensity of fluorescence and sensitivity in confirming herpes simplex virus (HSV) in cell culture. A total of 125 (50 HSV type 1 [HSV-1] and 75 HSV-2) specimens positive for HSV were concurrently stained with type-specific monoclonal antibodies from Bartels (Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash.), Kallestad (Pathfinder; Kallestad Diagnostics, Chaska, Minn.), Diagnostic Products Corp. (DPC) (PathoDx; DPC, Los Angeles, Calif.), and Syva (Microtrak; Syva Co., Palo Alto, Calif.). Fifty cultured specimens not displaying HSV-associated cytopathic effect were also stained. Each of the four reagents confirmed the 50 negative cultures and 125 positive cultures. One HSV-1-positive culture was missed by the Kallestad antibody. Ten HSV-1 isolates displayed dull fluorescence when stained with the Syva HSV-2 reagent. The other three reagents stained only the HSV-1 well with these 10 specimens. The Syva and Bartels stains were brighter, in general, than the Kallestad and DPC stains. However, the Kallestad and DPC stains were brilliant enough to allow interpretation without hesitation. The differences in intensity and background staining emphasize the need for laboratories to routinely perform quality checks on their reagents and to test new products which become commercially available.


Low predictive value of polymerase chain reaction for diagnosis of cytomegalovirus disease in liver transplant recipients. The polymerase chain reaction (PCR) and viral culture techniques were prospectively compared for the detection of cytomegalovirus (CMV) in blood samples from 24 liver transplant recipients. Nine patients had one or more episodes of viremia, seven of which were clinically symptomatic infections. All samples in which CMV was isolated by culture were positive by the PCR. However, the PCR result was also positive for one or more samples from 11 patients who never developed CMV-related symptoms. Although the PCR is a very sensitive technique for CMV detection in blood samples from liver transplant recipients, it is not useful as a marker of symptomatic CMV disease.


Development and distribution of noradrenergic and cholinergic neurons and their trophic phenotypes in the avian ceruleus complex and midbrain tegmentum. We investigated the development of noradrenergic and cholinergic neurons in the ceruleus complex and mesencephalic tegmentum in embryonic and posthatch chickens and compared the distribution of transmitter phenotypes with the expression of nerve growth factor receptor (NGFR) mRNA and fibroblast growth factor receptor (FGFR) mRNA. Noradrenergic and cholinergic neurons were visualized by using antibodies against dopamine-beta-hydroxylase (DBH) and choline acetyltransferase (ChAT), respectively. Expression of receptors for trophic factors was determined by using in situ hybridization techniques. Noradrenergic neurons concentrate in caudal parts of the locus ceruleus and nucleus subceruleus. Cholinergic ceruleus neurons are abundant in the nucleus mesencephalicus profundus, pars ventralis (MPv) as well as in the nucleus subceruleus and locus ceruleus. This cholinergic population resembles the cholinergic pontomesencephalotegmental complex of mammals. Both DBH and ChAT label is evident at and after six days of incubation (E6). The distribution and numbers of immunolabeled neurons are similar in the embryonic and posthatch chick. Initially, many tegmental and ceruleus neurons express substantial levels of NGFR mRNA (E7-E9). After E9, expression of NGFR mRNA decreases in most of these neurons, except for a distinct subpopulation of neurons in caudal parts of the ceruleus complex with increased levels of NGFR transcripts. These NGFR-positive neurons coincide in number and distribution with the noradrenergic subpopulation of the ceruleus complex (800-900 neurons). Expression of FGFR mRNA was first detected in ceruleus neurons at E13. Neurons with FGFR transcripts have the same number and distribution as the neurons with the cholinergic phenotype (2,000-2,300 neurons). Transmitter heterogeneity in the ceruleus complex is reflected by a heterogeneity of receptors for trophic factors, with NGFR expressed in the noradrenergic subpopulation, and FGFR expressed in the cholinergic subpopulation. These findings provide evidence for new chemoarchitectonic subdivisions of the avian ceruleus complex. The data showing onset of ChAT expression prior to the onset of FGFR expression argue against a role of FGFR in the determination of the cholinergic transmitter phenotype. Expression of NGFR in the noradrenergic ceruleus subpopulation reveals remarkable species differences as compared to mammals.


Humoral IgG antibodies to oral microbiota in a population at risk for root-surface caries. Mutans streptococci have been strongly implicated in the initiation of dental caries on coronal surfaces. Their role in development of root-surface caries is less clear. The etiologic agents of both types of dental caries are likely to elicit systemic immune responses. The objective of the present study, therefore, was to study the association of clinical variables of disease with humoral IgG antibodies to nine oral micro-organisms in 314 adult subjects, aged 45-65 years, who were at risk for root-surface caries. Antibody activity to Streptococcus mutans strain Ingbritt, S. mutans/S. sobrinus GTFs, S. faecalis strain 19433, Actinomyces viscosus strain WVU 626, Actinomyces naeslundii strain 12, Lactobacillus casei, Actinobacillus actinomycetemcomitans strain Y4, Porphyromonas gingivalis strain 381, Eikenella corrodens strain 1073, and Wolinella recta strain 371 was measured by ELISA. Pearson correlation coefficients among log10 antibody levels within subjects revealed marked positive correlations among subgingival bacteria, generally weak positive correlations among supragingival micro-organisms, and no correlations between elements of the supragingival battery with the subgingival battery. IgG antibody levels to mutans streptococcal antigens were significantly correlated with subject DMF scores (r = 0.23; p less than 0.0001). No significant correlation was seen between DMF scores and antibody to any other supragingival micro-organism tested. Further relationships between levels of S. mutans antibody and individual clinical variables were analyzed by step-wise multiple linear regression, resulting in a model that was highly significant (p = 0.0001), with an r2 = 0.14. Numbers of missing teeth, coronal caries, root-surface caries, and root-surface restorations were each positively associated with antibody levels to mutans streptococci.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of skin-chamber fluids from human allergic reactions on neutrophil activation. We have found activated neutrophils (PMNs) in skin chambers overlying the site of ongoing human allergic reactions. To determine whether this activation is due to component(s) released early in these IgE-mediated reactions, we investigated the in vitro effects of skin chamber fluids (CF) on resting blood PMNs. These skin CF had been previously obtained, at different time periods, at pollen-antigen-challenged or buffer-control-challenged sites in sensitive human subjects. PMNs incubated with CF obtained during the first hour of antigen challenge (first-hour CF) generated significantly more superoxide (O2-) than spontaneous production (p less than 0.001) and more than PMNs incubated with first-hour buffer-site CF (p less than 0.002). A pattern similar to O2- generation was observed in lactoferrin secretion during the incubation of the three cell aliquots described above (p less than 0.001). After these incubations, the subsequent responsiveness of the PMNs present in the cell buttons to opsonized zymosan, a PMN activator, was assessed. PMNs previously incubated with first-hour CF generated significantly more net O2- in response to opsonized zymosan than did PMNs previously incubated with first-hour buffer-site CF (p less than 0.001) or buffered saline (p less than 0.001). Again, a similar difference in the patterns of net lactoferrin secretion was observed (p less than 0.02). These events were not associated with PMN damage. Analogous studies with CF previously obtained during the second- to first-hour of antigen or buffer challenge stimulated much less than first-hour CF. We conclude that one or more components released early in IgE-mediated skin reactions can activate PMNs. The nature of the activating component(s) is unknown, with no evidence of activation by histamine, antigen, or other components found to date in first-hour CF.


Distinction of hemangiomas from hepatic tumors with delayed enhancement by incremental dynamic CT. To analyze the patterns of contrast enhancement and to evaluate clinical utility, we performed table incremental dynamic CT in 21 patients with 30 hepatic hemangiomas and in 12 patients with 26 malignant neoplasms, which showed delayed enhancement. On incremental CT, dense, spotty peripheral enhancement was present in 23 of the 30 (77%) hemangiomas. In contrast, a circumferential bead- or band-like peripheral enhancement was seen in 19 of 26 (73%) malignant neoplasms. The findings were characteristic. We conclude that incremental CT is useful in the differential diagnosis of hepatic hemangioma in routine examination.


Protective effect of nedocromil sodium on the interleukin-1-induced production of interleukin-8 in human bronchial epithelial cells. Cellular constituents of the bronchial mucosa may participate in the recruitment of polymorphonuclear leukocytes to the inflamed airways through the generation of the neutrophil chemotactic peptide, interleukin-8 (IL-8). One important aspect of this interaction could be represented by the ability of pulmonary monokines, particularly IL-1, to induce gene expression of IL-8 in bronchial epithelial cells. In this study, we have evaluated the constitutive and IL-1-induced production of IL-8 by cultured human bronchial epithelial cells and examined the ability of the anti-inflammatory drug, nedocromil sodium, to inhibit the cytokine synthesis and release. Northern blot analysis demonstrated that IL-1 beta-treated human bronchial epithelial cells expressed appreciable levels of IL-8 mRNA during a period of 8 hours. This finding was associated with an increased release of immunoreactive IL-8 to the culture media, as assessed by specific ELISA. The bronchial epithelial cell-derived IL-8 demonstrated specific biologic activity, since the supernatants of stimulated bronchial epithelial cells possessed neutrophil chemotactic activity that could be inhibited by an antibody against human IL-8. Nedocromil sodium reduced the IL-1-induced release of IL-8 in a dose-dependent manner, but concentrations of the compounds, up to 10(-5) mol/L, did not affect the constitutive production of this cytokine.


Perirenal metastases from lung cancer: CT diagnosis. Three patients with primary lung cancer had perirenal metastases detected with CT. In two cases a symptomatic perirenal mass was the first evidence of metastatic spread and CT-guided biopsy of the perirenal lesion confirmed the diagnosis of metastatic lung cancer. The perirenal space is an unusual but potentially significant site of metastasis from lung cancer as well as other tumors such as malignant melanoma. It is suggested that connections between perirenal and intrathoracic lymphatics are the most probable mechanism of this pattern of spread by lung cancers.


A comparison of vascularized and conventional sural nerve grafts. Long-term results of a randomized series of 27 vascularized and 22 conventional sural nerve grafts in patients with comparable upper extremity injuries are reported. Recovery speed and outcome depended on (1) whether or not there was an overlying skin defect, (2) how the defect was closed, and (3) which nerve was injured and at what level. Generally, a vascularized nerve graft is indicated when the nerve gap is more than 6 cm and is associated with a massive skin defect or the graft is performed after reimplantation. Otherwise, results achieved with a conventional graft are equally good.


The effects of acid secretagogues on protective agents of gastric cells from adult rabbits in vitro. Endogenous prostaglandin and mucus have been recognized as important protective factors in the gastric mucosa. However, the regulatory mechanisms of these agents have not been well studied. The aim of the present study was to investigate the effects of acid secretagogues on cyclic adenosine monophosphate (cAMP) formation, prostaglandin E2 (PGE2) production, and mucus secretion by isolated parietal cells and culture mucous cells from adult rabbits. Rabbit parietal cells were enriched by nonlinear Percoll gradients after the isolation of rabbit gastric mucosal cells with collagenase and ethylenediaminetetraacetic acid (EDTA). Rabbit gastric mucous cells were cultured in 10% fetal bovine serum added to Ham's F12 medium. As gastric acid secretagogues, histamine, carbachol, gastrin, and 2-chloroadenosine were tested. To evaluate the effects of the second messengers of cellular signal transduction on protective agents, A23187, which is a calcium ionophore, and cAMP were used. PGE2 and cAMP were measured by radioimmunoassay. The release of [3H]glucosamine from prelabeled cells was used as an indicator of mucus secretion. Histamine, carbachol, gastrin, and 2-chloroadenosine did not modulate PGE2 production by parietal cells. Exogenously administered cAMP did not affect PGE2 production by parietal cells, whereas it was significantly increased by A23187. 2-Chloroadenosine but not histamine or carbachol significantly increased cAMP formation by mucous cells. Histamine, carbachol, and gastrin did not have significant effects on PGE2 production by mucous cells. 2-Chloroadenosine, which increased cAMP, also did not modulate PGE2 production. A23187 but not cAMP increased PGE2 production by mucous cells. None of the acid secretagogues used in the present study modulated mucus secretion. A23187 but not cAMP significantly increased mucus secretion by cultured mucus cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Immunohistochemical localization of cells that synthesize leukotriene B4 in human gastric mucosa. We evaluated the cell location of leukotriene B4 (LTB4) in human gastric mucosa by immunohistochemistry using a novel fixation procedure with 0.5% glutaraldehyde-8% paraformaldehyde followed by periodate-lysine-2% paraformaldehyde. The location of LTA4 hydrolase, which synthesizes LTB4, was also investigated in human gastric mucosa. Staining for LTB4 was mostly in a granular pattern in the cytoplasm of parietal and surface epithelial cells. LTB4 was also stained in some vascular endothelial cells and a few polymorphonuclear cells in the lamina propria. LTA4 hydrolase was similarly stained in these cells. These results suggest that parietal, surface epithelial, and vascular endothelial cells are the cells that synthesize LTB4 in the human gastric mucosa. Polymorphonuclear cells might also synthesize a smaller amount of LTB4.


Candoxatril, a neutral endopeptidase inhibitor: efficacy and tolerability in essential hypertension. To examine the efficacy and tolerability of the neutral endopeptidase inhibitor, candoxatril (UK 79,300) as monotherapy in essential hypertension. Double-blind, placebo-controlled, parallel-group study of 28 days' duration. Three hospital outpatient departments participating in the Glasgow Blood Pressure Clinic (Glasgow, UK). Forty patients with essential hypertension with diastolic blood pressure 95-114 mmHg after a 2-4 week placebo run-in period. Twenty-eight days' treatment with candoxatril 200 mg twice daily or matching placebo capsules. Changes in supine and erect blood pressure, and volunteered side effects during double-blind treatment. When measured at the end of the dose interval, the fall in supine blood pressure following candoxatril was not significantly greater than that after placebo. Compared with placebo, a significant effect for candoxatril was seen only for systolic blood pressure in the erect posture; the fall in erect diastolic blood pressure attributable to candoxatril was insignificant. Median plasma atrial natriuretic peptide concentration increased in candoxatril-treated patients and decreased in the placebo group. No stimulation of the renin-aldosterone axis was seen. There was a non-significant trend towards greater urinary excretion of cyclic guanosine monophosphate after candoxatril. Mean plasma concentration of candoxatril at (UK 73,967--the active metabolite of candoxatril) reached a peak of 1010 +/- 437 ng/ml after acute dosing, and 1328 +/- 405 ng/ml after chronic dosing; time to maximum concentration was 2 h in each case. Candoxatril was well-tolerated; numbers of adverse events did not differ between active treatment and placebo. Although atrial natriuretic peptide levels were significantly increased, candoxatril 200 mg twice daily for 28 days did not produce a clinically relevant fall in blood pressure. Our results cast some doubt upon the role of neutral endopeptidase inhibition in the treatment of unselected hypertensive patients.