Sequence-specific 1H and 15N resonance assignments for both equilibrium forms of the soluble heme binding domain of rat ferrocytochrome b5. 15N and 1H resonance assignments for backbone and side-chain resonances of both equilibrium forms of rat ferrocytochrome b5 have been obtained, using 15N-1H heteronuclear correlation methods employing globally 15N-labeled protein. Unlike other cytochrome b5 species assigned to date (Guiles et al., 1990) the rat cytochrome exists as an equilibrium distribution of conformers in nearly equal abundance (Lee et al., 1990). The ratio of conformers present in all other species variants is approximately 1:9. More than 40% of all residues of the rat protein exhibit NMR-detectable heterogeneity due to the 180 degrees rotation of the heme about the alpha, gamma-meso axis. NOESY and HOHAHA relayed 15N-1H double-DEPT heteronuclear correlation methods were an indispensible tool for the deconvolution of a system with this level of heterogeneity. Differences in the resonance assignments between the two equilibrium conformers were found to be as great as differences between species variants we have previously reported. On the basis of the magnitude and extent of the observed chemical shift differences and specific NOESY connectivities observed in the two isomers, we believe the two equilibrium conformers differ not only by a simple back-to-front flip of the heme but also by an additional rotation about an axis normal to the heme plane as has been previously suggested by Pochapsky et al. (1990). A short segment of the protein at the N-terminus could not be assigned, presumably due to rapid exchange of solvent-accessible amide protons in this disordered segment of the protein. Assignments for 93 of the 98 residues of this 12-kDa protein have been obtained.


Hydrogen exchange in native and alcohol forms of ubiquitin. Ubiquitin adopts a non-native folded structure in 60% methanol solution at low pH. Two-dimensional nuclear magnetic resonance (2D NMR) was used to measure the hydrogen-exchange rates of backbone amide protons of ubiquitin in both native and methanol forms, and to characterize the structure of ubiquitin in the methanol state. Protection factors (the ratios of experimentally determined exchange rates to the rates calculated for an unfolded polypeptide) for protons in the native form of ubiquitin range from less than 10 to greater than 10(5). Most of the protons that are protected from exchange are located in regions of hydrogen-bonded secondary structure. The most strongly protected backbone amide protons are those of residues comprising the hydrophobic core. Hydrogen exchange from ubiquitin in methanol solution was too rapid to measure directly by 2D NMR, so a labeling scheme was employed, in which exchange with solvent occurred while the protein was in methanol solution. Exchange was quenched by dilution with aqueous buffer after the desired labeling time, and proton occupancies were measured by 1H NMR of the native form of the protein. Protection factors for protons in the methanol form of ubiquitin range from 2.6 to 42, with all protected protons located in hydrogen-bonded structure in the native form. Again, the most strongly protected protons are those of residues in the hydrophobic core. Comparison of the patterns of the hydrogen-exchange rates in the native and methanol forms indicates that almost all of the native secondary structure persists in the methanol form, but that it is almost uniformly destabilized by 4-6 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)


Resolution of NADH:ubiquinone oxidoreductase from bovine heart mitochondria into two subcomplexes, one of which contains the redox centers of the enzyme. NADH:ubiquinone oxidoreductase (complex I) was purified from bovine heart mitochondria by solubilization with n-dodecyl beta-D-maltoside (lauryl maltoside), ammonium sulfate fractionation, and chromatography on Mono Q in the presence of the detergent. Its subunit composition was very similar to complex I purified by conventional means. Complex I was dissociated in the presence of N,N-dimethyldodecylamine N-oxide and beta-mercaptoethanol, and two subcomplexes, I alpha and I beta, were isolated by chromatography. Subcomplex I alpha catalyzes electron transfer from NADH to ubiquinone-1. It is composed of about 22 different and mostly hydrophilic subunits and contains 2.0 nmol of FMN/mg of protein. Among its subunits is the 51-kDa subunit, which binds FMN and NADH and probably contains a [4Fe-4S] cluster also. Three other potential Fe-S proteins, the 75- and 24-kDa subunits and a 23-kDa subunit (N-terminal sequence TYKY), are also present. All of the Fe-S clusters detectable by EPR in complex I, including cluster 2, are found in subcomplex I alpha. The line shapes of the EPR spectra of the Fe-S clusters are slightly broadened relative to spectra measured on complex I purified by conventional means, and the quinone reductase activity is insensitive to rotenone. Similar changes were found in samples of the intact chromatographically purified complex I, or in complex I prepared by the conventional method and then subjected to chromatography in the presence of lauryl maltoside. Subcomplex I beta contains about 15 different subunits. The sequences of many of them contain hydrophobic segments that could be membrane spanning, including at least two mitochondrial gene products, ND4 and ND5. The role of subcomplex I beta in the intact complex remains to be elucidated.


Demonstration by FTIR that the bo-type ubiquinol oxidase of Escherichia coli contains a heme-copper binuclear center similar to that in cytochrome c oxidase and that proper assembly of the binuclear center requires the cyoE gene product. Amino acid sequence data have revealed that the bo-type ubiquinol oxidase from Escherichia coli is closely related to the eukaryotic aa3-type cytochrome c oxidases. In the cytochrome c oxidases, the reduction of oxygen to water occurs at a binuclear center comprised of heme a3 and Cu(B). In this paper, Fourier transform infrared (FTIR) spectroscopy of CO bound to the enzyme is used to directly demonstrate that the E. coli bo-type ubiquinol oxidase also contains a heme-copper binuclear center. Photolysis of CO ligated to heme o at low temperatures (e.g., 30 K) results in formation of a CO-Cu complex, showing that there is a heme-Cu(B) binuclear center similar to that formed by heme a3 and Cu(B) in the eukaryotic oxidase. It is further demonstrated that the cyoE gene product is required for the correct assembly of this binuclear center, although this polypeptide is not required as a component of the active enzyme in vitro. The cyoE gene product is homologous to COX10, a nuclear gene product from Saccharomyces cerevisiae, which is required for the assembly of yeast cytochrome c oxidase. Deletion of the cyoE gene results in an inactive quinol oxidase that is, however, assembled in the membrane. FTIR analysis of bound CO shows that Cu(B) is present in this mutant but that the heme-Cu(B) binuclear center is abnormal. Analysis of the heme content of the membrane suggests that the cyoE deletion results in the insertion of heme B (protoheme IX) in the binuclear center, rather than heme O.(ABSTRACT TRUNCATED AT 250 WORDS)


Reevaluation of the stoichiometry of cytochrome b559 in photosystem II and thylakoid membranes. The stoichiometry of cytochrome b559 (one or two copies) per reaction center of photosystem II (PSII) has been the subject of considerable debate. The molar ratio of cytochrome b559 has a number of significant implications on our understanding of the functional role of cytochrome b559, the mechanism of electron donation in PSII, and the stoichiometry of the other redox-active, reaction center components. We have reinvestigated the stoichiometry of cytochrome b559 in PSII-enriched and thylakoid membranes, using differential absorbance and electron paramagnetic resonance spectroscopies. The data from both quantitation procedures strongly indicate only one copy of cytochrome b559 per reaction center in PSII-enriched membranes and also suggest one copy of cytochrome b559 per reaction center in thylakoid membranes.


Molecular changes following oxidoreduction of cytochrome b559 characterized by Fourier transform infrared difference spectroscopy and electron paramagnetic resonance: photooxidation in photosystem II and electrochemistry of isolated cytochrome b559 and iron protoporphyrin IX-bisimidazole model compounds. The vibrational infrared absorption changes associated with the oxidation of cytochrome b559 (Cyt b559) have been characterized. In photosystem II (PS II) enriched membranes, low-potential (LP) and high-potential (HP) Cyt b559 were investigated by light-induced FTIR difference spectroscopy. The redox transition of isolated Cyt b559 is characterized by protein electrochemistry. On the basis of a model of the assembly of Cyt b559 with the two axial Fe ligands being histidine residues of two distinct polypeptides, each forming a transmembrane alpha-helix [Cramer, W.A., Theg, S.M., & Widger, W.R. (1986) Photosynth. Res. 10, 393-403], the bisimidazole and bismethylimidazole complexes of Fe protoporphyrin IX were electrochemically oxidized and reduced to detect the IR oxidation markers of the heme and its two axial ligands. Major bands at 1674/1553, 1535, and 1240 cm-1 are tentatively assigned to nu 37 (CaCm), nu 38-(CbCb) and delta (CmH) modes, respectively; other bands at 1626, 1613, 1455, 1415, and 1337 cm-1 are assigned to porphyrin skeletal and vinyl modes. Modes at 1103 and 1075/1066 cm-1 are assigned to the 4-methylimidazole and imidazole ligands, respectively. For the isolated Cyt b559, it is shown that both the heme (at 1556-1535, 1337, and 1239 cm-1), the histidine ligands at 1104 cm-1 and the protein (between 1600 and 1700 cm-1 and at 1545 cm-1) are affected by the charge stabilization. The excellent agreement between model compounds and isolated Cyt b559 reinforces the validity of the model of a heme iron coordinated to two histidine residues for Cyt b559. A differential signal at 1656/1641 cm-1 is assigned to peptide C = O mode(s). We speculate that this signal reflects the change in strength of a hydrogen bond formed between the histidine ligand(s) and the polypeptide backbone upon oxidoreduction of the cytochrome. In PS II membranes, the signals characteristic of Cyt b559 photooxidation are found at 1660/1652 and 1625 cm-1, for both the high- and low-potential forms. The differences observed in the amplitude of the 1660/1652-cm-1 band, at 1700 and 1530-1510 cm-1 in the light-induced FTIR difference spectra of Cyt b559 HP and LP, show that the mechanisms of heme oxidation in vivo imply different molecular processes for the two forms Cyt b559 HP and LP.


Selective removal of subunit VIb increases the activity of cytochrome c oxidase. Bovine heart cytochrome c oxidase was gel-filtered on Sephacryl S-300 in 0.05% dodecyl maltoside and in the presence or absence of 1 M KCl. The presence of KCl selectively removed subunit VIb from the enzyme complex, resulting in about doubling of enzymatic activity and an increase of the Km for ferrocytochrome c. In contrast, the proton pumping activity of the enzyme was unchanged. The increase of activity is due to removal of subunit VIb and not of lipids, because titration with asolectin or dodecyl maltoside could not abolish the difference in activity between the 12- and 13-subunit enzyme. Attempts to reconstitute cytochrome c oxidase from its separated components were unsuccessful. It is concluded that subunit VIb suppresses the activity of the mammalian enzyme complex by interaction with the active center.


Effect of Asp-235-->Asn substitution on the absorption spectrum and hydrogen peroxide reactivity of cytochrome c peroxidase. The spectroscopic properties of a mutant cytochrome c peroxidase, in which Asp-235 has been replaced by an asparagine residue, were examined in both nitrate and phosphate buffers between pH 4 and 10.5. The spin state of the enzyme is pH dependent, and four distinct spectroscopic species are observed in each buffer system: a predominantly high-spin Fe(III) species at pH 4, two distinct low-spin forms between pH 5 and 9, and the denatured enzyme above pH 9.3. The spectrum of the mutant enzyme at pH 4 is dependent upon specific ion effects. Increasing the pH above 5 converts the mutant enzyme to a predominantly low-spin hydroxy complex. Subsequent conversion to a second low-spin form is essentially complete at pH 7.5. The second low-spin form has the distal histidine, His-52, coordinated to the heme iron. To evaluate the effect of the changes in coordination state upon the reactivity of the enzyme, the reaction between hydrogen peroxide and the mutant enzyme was also examined as a function of pH. The reaction of CcP(MI,D235N) with peroxide is biphasic. At pH 6, the rapid phase of the reaction can be attributed to the bimolecular reaction between hydrogen peroxide and the hydroxy-ligated form of the mutant enzyme. Despite the hexacoordination of the heme iron in this form, the bimolecular rate constant is approximately 22% that of pentacoordinate wild-type yeast cytochrome c peroxidase. The bimolecular reaction of the mutant enzyme with peroxide exhibits the same pH dependence in nitrate-containing buffers that has been described for the wild-type enzyme, indicating a loss of reactivity with the protonation of a group with an apparent pKa of 5.4. This observation eliminates Asp-235 as the source for this heme-linked ionization and strengthens the hypothesis that the pKa of 5.4 is associated with His-52. The slower phase of the reaction between peroxide and the mutant enzyme saturates at high peroxide concentration and is attributed to conversion of unreactive to reactive forms of the enzyme. The fraction of enzyme which reacts via the slow phase is dependent upon both pH and specific ion effects.


Solution structure of a platelet receptor peptide bound to bovine alpha-thrombin. NMR experiments were carried out to study the interaction of thrombin with a synthetic peptide, ESKATNATLDPR, derived from the newly-identified platelet receptor for thrombin [Vu, T.-K. H., Hung, D. T., Wheaton, V. I., & Coughlin, S. R. (1991) Cell 64, 1057-1068]. On the basis of the observation of the thrombin-induced line broadening and transferred NOEs, binding of the peptide was found to be located exclusively within residues LDPR of the proteolytic cleavage site LDPR/S essential for receptor activation by thrombin. Measurement of transferred NOEs and molecular modeling indicate that the side chain of the Asp(P3) residue may form a hydrogen bond with thrombin and, by doing so, it is brought near a positively-charged thrombin residue Arg(221A), thereby partially neutralizing the negative charge of an Asp residue at this site of protein substrates. The hydrophobic side chains of residues Leu(P4) and Pro(P2) reside on the same side of the peptide backbone as indicated by transferred NOEs and were found by modeling to fit into a hydrophobic cage around the thrombin active site. These results suggest that the interaction of thrombin with protein substrates such as prothrombin, protein C, protein S, the platelet receptor, and the A alpha- and B beta-chains of fibrinogen all follow the same canonical binding mode in that the substrate forms an antiparallel beta-strand with thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)


Locations of contacts between individual zinc fingers of Xenopus laevis transcription factor IIIA and the internal control region of a 5S RNA gene. A set of mutants of transcription factor IIIA (TFIIIA) have been prepared in which successive zinc-finger domains have been deleted from the carboxyl terminus of the protein. These have been analyzed by hydroxyl radical footprinting to map the location of contacts to DNA by individual zinc-finger domains of TFIIIA. The results suggest that the nine zinc fingers of TFIIIA are organized into three DNA-binding domains of three fingers each. The spatial relationship between zinc-finger contacts to 5S DNA suggests that the two domains which interact with either end of the DNA-binding site of TFIIIA (fingers 1-3 and 7-9) have a compact conformation, similar to that exemplified by the zif268 cocrystal structure [Pavletich, N.P., & Pabo, C.O. (1991) Science 252, 809-817]. However, the central domain (fingers 4-6) has a much more extended conformation, following a path nearly parallel to the helix axis and contacting over 20 base pairs of DNA in the center of the binding site of TFIIIA. These results strongly support two recently proposed and radically new models for the TFIIIA/5S DNA complex [Hayes, J.J., & Tullius, T.D. (1992) J. Mol. Biol. 227, 407-417; Clemens, K.R., Liao, X., Wolf, V., Wright, P.E., & Gottesfeld, J.M. (1992) Proc. Natl. Acad. Sci. U.S.A. (in press)].


Manganese-mediated oxidative damage of cellular and isolated DNA by isoniazid and related hydrazines: non-Fenton-type hydroxyl radical formation. The mechanism by which hydrazines induce damage to cellular and isolated DNA in the presence of metal ions has been investigated by pulsed-field gel electrophoresis (PFGE), DNA sequencing methods, and the ESR spin-trapping technique. For the detection of single-strand breaks by PFGE, an experimental procedure with alkali treatment has been designed. Isoniazid, hydrazine, and phenylhydrazine induced DNA single- and double-strand breaks in cells pretreated with Mn(II), whereas iproniazid did not. With isolated 32P-DNA, isoniazid produced DNA damage in the presence of Cu(II), Mn(II), or Mn(III). Iproniazid damage isolated DNA only in the presence of Cu(II). The Cu(II)-mediated DNA damage by isoniazid or iproniazid is due to active oxygen species other than hydroxyl free radical (.OH), presumably the Cu(I)-peroxide complex. Cleavage of isolated DNA by isoniazid plus Mn(II) occurred without marked site specificity. The DNA damage was inhibited by .OH scavengers and superoxide dismutase (SOD) but not by catalase, suggesting the involvement of .OH formed via O2- but not via H2O2. Consistently, in ESR experiments .OH formation was observed during Mn(II)-catalyzed autoxidation of isoniazid, and the .OH formation was inhibited by SOD, but not by catalase. Iproniazid plus Mn(II) produced no or little .OH. We propose a reaction mechanism for the .OH formation without a H2O2 intermediate during manganese-catalyzed autoxidation of hydrazine. The present and previous data raise the possibility that hydrazines plus Mn(II)-induced cellular DNA damage may occur, at least in part, through the non-Fenton-type reaction.


Characterization of the phosphorylated enzyme intermediate formed in the adenosine 5'-phosphosulfate kinase reaction. Adenosine 5'-phosphosulfate (APS) kinase (ATP:APS 3'-phosphotransferase) catalyzes the ultimate step in the biosynthesis of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the primary biological sulfuryl donor. APS kinase from Escherichia coli is phosphorylated upon incubation with ATP, yielding a protein that can complete the overall reaction through phosphorylation of APS. Rapid-quench kinetic experiments show that, in the absence of APS, ATP phosphorylates the enzyme with a rate constant of 46 s-1, which is equivalent to the Vmax for the overall APS kinase reaction. Similar pre-steady-state kinetic measurements show that the rate constant for transfer of the phosphoryl group from E-P to APS is 91 s-1. Thus, the phosphorylated enzyme is kinetically competent to be on the reaction path. In order to elucidate which amino acid residue is phosphorylated, and thus to define the active site region of APS kinase, we have determined the complete sequence of cysC, the structural gene for this enzyme in E. coli. The coding region contains 603 nucleotides and encodes a protein of 22,321 Da. Near the amino terminus is the sequence 35GLSGSGKS, which exemplifies a motif known to interact with the beta-phosphoryl group of purine nucleotides. The residue that is phosphorylated upon incubation with ATP has been identified as serine-109 on the basis of the amino acid composition of a radiolabeled peptide purified from a proteolytic digest of 32P-labeled enzyme. We have identified a sequence beginning at residue 147 which may reflect a PAPS binding site. This sequence was identified in the carboxy terminal region of 10 reported sequences of proteins of PAPS metabolism.


The cationic locus on the recombinant kringle 2 domain of tissue-type plasminogen activator that stabilizes its interaction with omega-amino acids. The properties of the cationic locus within the recombinant (r) kringle 2 domain (residues 180-261) of tissue-type plasminogen activator ([K2tPA]) that are responsible for stabilization of its interaction with the carboxylate moiety of omega-amino acid ligands have been assessed by determination of the binding constants of several such ligands to a variety of r-[K2tPA] mutants obtained by oligonucleotide-directed mutagenesis. We have generated, expressed in Escherichia coli, and purified alanyl mutants of individual histidyl,lysyl, and arginyl residues of r-[K2tPA] and determined the dissociation constants of several omega-amino acids, viz., 6-aminohexanoic acid (6-AHxA), 7-aminoheptanoic acid (7-AHpA), L-lysine (L-Lys), and trans-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA), to each of the r-[K2tPA] variants. We find that K33 plays the most significant role as a cationic partner of the complementary carboxylate group of these ligands. When K33 is altered to a variety of other amino acids, the K33R mutant best stabilizes binding of all of these ligands. However, the r-K33L and r-K33F variants selectively interact with 7-AHpA almost as strongly (ca. 2-fold reduction in binding strength) as wild-type r-[K2tPA]. Increased polarity (K33Q) or a negative charge (K33E) at this sequence position significantly destabilizes binding of omega-amino acids to the muteins. We also found that the r-K33E mutant and, to a lesser extent, the r-K33Q variant selectively interact with a new ligand, 1,6-diaminohexane. These observations show that the omega-amino acid binding site of wtr-[K2tPA] could be redesigned to provide a new binding specificity.(ABSTRACT TRUNCATED AT 250 WORDS)


Interaction of porcine uterine fluid purple acid phosphatase with vanadate and vanadyl cation. Uteroferrin, the purple acid phosphatase from porcine uterine fluid, is noncompetitively inhibited by vanadate in a time-dependent manner under both aerobic and anaerobic conditions. This time-dependent inhibition is observed only with the diiron enzyme and is absent when the FeZn enzyme is used. The observations are attributed to the sequential formation of two uteroferrin-vanadium complexes. The first complex forms rapidly and reversibly, while the second complex forms slowly and results in the production of catalytically inactive oxidized uteroferrin and V(IV), which is observed by EPR. The redox reaction can be reversed by treatment of the oxidized enzyme first with (V(IV)) and then EDTA to generate a catalytically active uteroferrin. Multiple inhibition kinetics suggests that vanadate is mutually exclusive with molybdate, tungstate, and vanadyl cation. The binding site for each of these anions is distinct from the site to which the competitive inhibitors phosphate and arsenate bind. The time-dependent inhibition by vanadate of uteroferrin containing the diiron core represents a new type of mechanism by which vanadium can interact with proteins and gives additional insight into the binding of anions to uteroferrin.


Redox properties of electron-transfer flavoprotein ubiquinone oxidoreductase as determined by EPR-spectroelectrochemistry. We have determined the formal potential values for each electron transfer to electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO), in order to further characterize the thermodynamics of electron transport from various acyl-CoA thioesters to the mitochondrial ubiquinone pool. ETF-QO contains one [4Fe-4S]2+,1+ cluster and one FAD prosthetic group. A preliminary visible-spectroelectrochemical titration showed that the two redox centers were reduced almost simultaneously. Since the visible spectra of the chromophores overlap, it was not possible to resolve the formal potential value for each electron transfer to the protein using this method. Accordingly, an EPR-spectroelectrochemical cell was designed so that each formal potential value could be resolved by EPR quantitation of the flavin semiquinone and the reduced iron-sulfur cluster during the titration. The formal potential values for electron transfer to ETF-ubiquinone oxidoreductase at pH 7.5 and 4 degrees C were E1 degrees' = +0.028 V and E2 degrees' = -0.006 V for the first and second electron transfers, respectively, to the FAD and E degrees' = +0.047 V for the iron-sulfur cluster. The thermodynamics of electron transport from the acyl-CoA substrates of beta-oxidation to the mitochondrial electron transport chain have been fully resolved with completion of this work. The results are discussed in terms of their significance to the overall electron transport process from beta-oxidation.


Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane Ca2+ pump. The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree. The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b. In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site. The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site. The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2. A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency. Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms.


Bis(benzylisoquinoline) analogs of tetrandrine block L-type calcium channels: evidence for interaction at the diltiazem-binding site. Bis(benzylisoquinoline) alkaloids block Ca2+ uptake through the L-type Ca2+ channel and modulate binding of ligands to four distinct sites (dihydropyridine, benzothiazepine, aralkylamine, and (diphenylbutyl)piperidine) in the Ca2+ entry blocker receptor complex of the channel. These alkaloids are structural analogs of tetrandrine, which has previously been demonstrated to block the L-type Ca2+ channel through interaction at the benzothiazepine (diltiazem) site (King et al., 1988). Different alkaloid conformational classes display either alpha-beta, beta-alpha, alpha-alpha, or beta-beta stereochemistry at the two chiral isoquinoline carbons. Compounds from all four classes were tested for their ability to interact with Ca2+ entry blocker ligands. All analogs completely inhibit diltiazem binding, but many only partially inhibit D-600 and fluspirilene binding. For dihydropyridine binding, the compounds show either stimulation or inhibition or exhibit no effect. This profile is quite different from the interaction displayed by diltiazem or tetrandrine. Scatchard analyses show effects predominantly on Kd for diltiazem, D-600, and PN200-110 binding. Representative conformers do not effect diltiazem dissociation rates but alter dissociation kinetics of ligands which bind to the other three sites. A correlation of the ability of these compounds to inhibit Ca2+ uptake through the L-type Ca2+ channel in GH3 cells exists only with their inhibition of diltiazem binding but not with inhibition of binding of ligands representing other classes of Ca2+ entry blockers. These data, taken together, indicate that a variety of bis(benzylisoquinoline) congeners act to block the L-type Ca2+ channel by binding to the benzothiazepine site on the channel.(ABSTRACT TRUNCATED AT 250 WORDS)


Structural consequences of a carcinogenic alkylation lesion on DNA: effect of O6-ethylguanine on the molecular structure of the d(CGC[e6G]AATTCGCG)-netropsin complex. Exposure of cells to alkylating agents produces DNA lesions, most of which are repaired. However some alkyl lesions persist and play a role in inducing point mutations and the subsequent carcinogenic conversion. O6-Ethylguanine (e6G) is a relatively persistent alkylation lesion caused by the exposure of DNA to N-ethyl-N-nitrosourea. We study the consequence of the e6G incorporation in DNA by X-ray crystallography. We have obtained crystals of the modified DNA dodecamer d(CGC[e6G]AATTCGCG) and the unmodified d(CGCGAATTCGCG), complexed to the minor groove binding drug netropsin. The space group of both crystals is P2(1)2(1)2(1), isomorphous to other related dodecamer DNA crystals. The structures have been solved by the molecular replacement method and refined by the constrained least-squares procedure to R-factors of approximately 16% at resolution of approximately 2.5 A. The two independent e6G-C base pairs in the DNA duplex adopt different base-pairing schemes. The e6G4-C21 base pair has a configuration similar to a normal Watson-Crick base pair, except with one three-centered hydrogen bond pair and one direct hydrogen bond between e6G4 and C21. In contrast, the e6G16-C9 base pair adopts a wobble configuration. The ethyl group is in the proximal orientation (to N7) in both base pairs. These observations enrich and support those found in the crystal structure of d(CGC[e6G]AATTCGCG), complexed to minor groove binding drugs Hoechst 33258 and Hoechst 33342 [Sriram et al. (1992) EMBO J. 11, 225-232]. We suggest that a dynamic equilibrium between these two configurations for the e6G-C base pair is likely and would present an ambiguous signal to the cellular transcription, replication, or repair mechanisms. In contrast, thymine can pair with e6G in only one way, albeit imperfect, mimicking a Watson-Crick base pair. This may be a plausible explanation of why thymine is found preferentially incorporated across the e6G during replication. In addition, we analyze the influence of the alkylation lesion on DNA and the molecular details of netropsin-DNA interaction. In the present two new netropsin complexes, the netropsin spans across five base pairs (starting halfway between C3-G22 and e6G4-C21 base pairs and ending at T8-A17 base pair) in the narrow minor groove. This is in contrast to the earlier crystal structure of netropsin complexed with another DNA dodecamer having the same AATT central core sequence, d(CGCGAATT[br5C]GCG) [Kopka et al. (1985) J. Mol. Biol. 272, 390-395]. In the latter structure, the netropsin lies between G4-br5C21 and br5C9-G16 base pairs.(ABSTRACT TRUNCATED AT 400 WORDS)


Proton transfer during the reaction between fully reduced cytochrome c oxidase and dioxygen: pH and deuterium isotope effects. The pH dependence of proton uptake and electron transfers during the reaction between fully reduced cytochrome c oxidase and oxygen has been studied using the flow-flash method. Proton uptake was monitored using different pH indicators. We have also investigated the effect of D2O on the electron-transfer reactions. Proton uptake was biphasic throughout the pH range studied (6.3-9.3), and the decrease of the observed rate constants at increasing pH could be described by titration curves with pKa values of 8-8.5. Of the four phases resolved in the redox reaction, the rate constants for the first two were independent of pH, whereas that of the third decreased at increasing pH with a pKa of 7.9. All phases except the first were slower in D2O than in H2O. The values obtained for kH/kD were 1.0 for the first phase, 1.4 for the second and third phases, and 2.5 for the fourth phase. We suggest from these results that the fast phase of proton uptake is initiated by the second phase of the redox reaction and that this step includes a partially rate-limiting internal proton transfer. The third and fourth phases of the redox reaction are suggested to be rate limited by proton uptake from the medium. The pH dependencies of the proton uptake reactions are consistent with the participation of a titrable group in the protein in proton transfer from the medium to the oxygen-binding site.


Intramolecular electron transfer in cytochrome c oxidase: a cascade of equilibria. Intramolecular electron redistribution in cytochrome c oxidase after photolysis of the partially reduced CO-bound enzyme was followed at a number of different wavelengths by absorption spectroscopy. Spectra were constructed for the first two phases of this process. The first phase (tau = 3 microseconds) has a spectrum essentially identical to the difference between the Fea and Fea3 reduced-minus-oxidized spectra, indicating a 1:1 stoichiometry between the amount of Fea3 oxidized and Fea reduced. It is not necessary to invoke reduction or oxidation of other redox carriers in this phase. The second phase (tau = 35 microseconds) spectrum appears to be a linear combination of the Fea3 and Fea reduced-minus-oxidized difference spectra, reflecting the oxidation of four parts of Fea3 for every part of Fea oxidized. This process can be described in terms of transfer to CuA of electrons from the Fea3<==>Fea equilibrium system established in the first phase. The relative contributions of Fea3 and Fea in the second phase allow us to calculate the equilibrium constant for Fea3<==>Fea electron exchange, which yields a delta Em of 36 mV for the two centers (Fea3 more positive). Together with the apparent rate constant for the fast phase, this equilibrium constant yields, in turn, the forward (kf) and reverse (kr) rates for electron transfer from Fea to Fea3 as follows: kf = 2.4 x 10(5) s-1 and kr = 6 x 10(4) s-1. kf is much faster than any observed step in the reaction of the reduced enzyme with O2. Thus, the catalytic mechanism of O2 reduction to water is not rate-limited by electron transfer from Fea to the binuclear Fea3/Cu(B) site.


Mutagenesis of methionine-183 drastically affects the physicochemical properties of cytochrome c1 of the bc1 complex of Rhodobacter capsulatus. Site-directed mutagenesis was used to investigate which of the highly conserved methionine residues (M183 and M205) provides the sixth axial ligand to the heme Fe in the cyt c1 subunit of the bc1 complex from the bacterium Rhodobacter capsulatus. These residues were changed to leucine (cM183L) and valine (cM205V). Two additional mutants were constructed, 1 in which a stop codon was inserted at M205 (cM205*) and the second in which 127 amino acids were deleted between the signal sequence and the putative C-terminal transmembrane alpha-helix (c delta SfuI). Only cM205V grew photosynthetically, and membranes isolated from this strain catalyzed quinol-dependent reduction of cyt c in amounts similar to that in a wild-type strain. Even though cM183L could not grow photosynthetically, it contained all the appropriate polypeptides and cofactors of the bc1 complex, as shown by SDS-PAGE and optical difference spectroscopy of intact membrane particles. Neither of the two deletion mutants contained a stable complex. Flash absorption spectroscopy using chromatophores showed no cytochrome c rereduction after oxidation by the reaction center in cM183L. The bc1 complex from each strain was isolated and characterized. Oxidation reduction midpoint potential titrations revealed that cyt c1 from cM183L had a dramatically shifted Em value (delta Em = -390 mV) compared with wild type and cM205V. While the optical absorption spectrum of cyt c1 from cM183L suggested that the c-type heme was low-spin, nonetheless it was able to react with the exogenous ligand carbon monoxide. The overall data support that M183, and not M205, is the sixth ligand to the heme Fe of cyt c1 of the bc1 complex.


Protein-tyrosyl radical interactions in photosystem II studied by electron spin resonance and electron nuclear double resonance spectroscopy: comparison with ribonucleotide reductase and in vitro tyrosine. The stable tyrosine radical in photosystem II, YD*, has been studied by ESR and ENDOR spectroscopies to obtain proton hyperfine coupling constants from which the electron spin density distribution can be deduced. Simulations of six previously published ESR spectra of PSII (one at Q band; five at X band, of which two were after specific deuteration and two others were of oriented membranes) can be achieved by using a single set of magnetic parameters that includes anisotropic proton hyperfine tensors, an anisotropic g tensor, and noncoincident axis systems for the g and A tensors. From the spectral simulation of the oriented samples, the orientation of the phenol head group of YD* with respect to the membrane plane has been determined. A similar orientation for YZ*, the redox-active tyrosine in PSII that mediates electron transfer between P680 and the oxygen-evolving complex, is expected. ENDOR spectra of YD* in PSII preparations from spinach and Synechocystis support the set of hyperfine coupling constants but indicate that small differences between the two species exist. Comparison with the results of spectral simulations for tyrosyl radicals in ribonucleotide reductase from prokaryotes or eukaryotes and with in vitro radicals indicates that the spin density distribution remains that of an odd-alternant radical but that interactions with the protein can shift spin density within this basic pattern. The largest changes in spin density occur at the tyrosine phenol oxygen and at the ring carbon para to the oxygen, which indicates that mechanisms exist in the protein environment for fine-tuning the chemical and redox properties of the radical species.


Site-specific dephosphorylation of smooth muscle myosin light chain kinase by protein phosphatases 1 and 2A. Smooth muscle myosin light chain kinase is phosphorylated at two sites (A and B) by different protein kinases. Phosphorylation at site A increases the concentration of Ca2+/calmodulin required for kinase activation. Diphosphorylated myosin light chain kinase was used to determine the site-specificity of several forms of protein serine/threonine phosphatase. These phosphatases readily dephosphorylated myosin light chain kinase in vitro and displayed differing specificities for the two phosphorylation sites. Type 2A protein phosphatase specifically dephosphorylated site A, and binding of Ca2+/calmodulin to the kinase had no effect on dephosphorylation. The purified catalytic subunit of type 1 protein phosphatase dephosphorylated both sites in the absence of Ca2+/calmodulin but only dephosphorylated site A in the presence of Ca2+/calmodulin. A protein phosphatase fraction was prepared from smooth muscle actomyosin by extraction with 80 mM MgCl2. On the basis of sensitivity to okadaic acid and inhibitor 2, this activity was composed of multiple protein phosphatases including type 1 activity. This phosphatase fraction dephosphorylated both sites in the absence of Ca2+/calmodulin. However, dephosphorylation of both sites A and B was completely blocked in the presence of Ca2+/calmodulin. These results indicate that two phosphorylation sites of myosin light chain kinase are dephosphorylated by multiple protein serine/threonine phosphatases with unique catalytic specificities.


Monoclonal antibody AG-1 initiates platelet activation by a pathway dependent on glycoprotein IIb-IIIa and extracellular calcium. The biochemical responses of intact human platelets to the monoclonal antibody (mAb) AG-1 were investigated. AG-1 is a murine IgG mAb that recognizes a series of platelet membrane glycoproteins (Gp) from M(r) 21,000 to 29,000, one of which is the M(r) 24,000 (p24) receptor for anti-CD9 mAbs. AG-1 causes platelet aggregation and secretion. Platelets binding AG-1 demonstrate a dose- and time-dependent breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), production of diacylglycerol, and generation of phosphatidic acid (PA). These events are associated with the activation of protein kinase C (PKC), an increase in intracellular calcium, and fibrinogen binding. Platelet PA generation and PKC activation in response to AG-1 are inhibited by mAbs to platelet GpIIb-IIIa or by extracellular EGTA, but not by a mAb to platelet GpIb or by inhibiting platelet Na+/H+ exchange with 5-(N-ethyl-N-isopropyl)amiloride. Platelet cytoplasmic free calcium ([Ca2+]i) is elevated in response to AG-1, and this elevation is inhibited by mAbs to GpIIb-IIIa, an RGDS peptide or by chelating extracellular calcium. These results suggest that AG-1 binding to a unique platelet-surface glycoprotein initiates platelet responses through the activation of PIP2-specific phospholipase C, and that this occurs through a signal pathway that is dependent on GpIIb-IIIa and extracellular calcium.


Reduction kinetics of the four hemes of cytochrome c3 from Desulfovibrio vulgaris by flash photolysis. The reduction of the tetraheme cytochrome c3 (from Desulfovibrio vulgaris, strains Miyazaki F and Hildenbourough) by flavin semiquinone and reduced methyl viologen follows a monophasic kinetic profile, even though the four hemes do not have equivalent reduction potentials. Rate constants for reduction of the individual hemes are obtained subsequent to incrementally reducing the cytochrome by phototitration. The dependence of each rate constant on the reduction potential difference between the heme and the reductant can be described by outer sphere electron transfer theroy. Thus, the very low reduction potentials of the cytochrome c3 hemes compensate for the very large solvent accessibility of the hemes. The relative rate constants for electron transfer to the four hemes of cytochrome c3 are consistent with the assignments of reduction potential to hemes previously made by Park et al. (Park, J.-S., Kano, K., Niki, S. and Akutsu, H. (1991) FEBS Lett. 285, 149-151) using NMR techniques. The ionic strength dependence of the observed rate constant for reduction by the methyl viologen radical cation indicates that ionic strength substantially alters the structure and/or the heme reduction potentials of the cytochrome. This result is confirmed by reduction with a neutral flavin species (5-deazariboflavin semiquinone) in which the reactivity of the highest potential heme decreases and the reactivity of the lowest potential heme increases at high (500 mM) ionic strength, and by the sensitivity of heme methyl resonances to ionic strength as observed by 1H-NMR. These unusual ionic strength-dependent effects may be due to a combination of structural changes in the cytochrome and alterations of the electrostatic fields at elevated ionic strengths.


Identification, purification and reconstitution of thiamin metabolizing enzymes in human red blood cells. Thiamin and its mono- (TMP), di- (TDP) and triphosphate (TTP) were assayed in adult human whole blood using high-performance liquid chromatography (HPLC). TDP and TTP were detected in red blood cells (RBC), but not in plasma. After incubation with 20 microM thiamin and 5 mM glucose for 2 h, the TDP and TTP contents of RBC increased from 111 to 222 and 0.6 to 2.2 nmol/l of packed RBC, respectively, suggesting enzymatic conversion of thiamin to TDP and then to TTP. Thiamin pyrophosphokinase (TPK, EC 2.7.6.2) had not been isolated before from human materials, nor had cytosolic adenylate kinase (AK1, EC 2.7.4.3) in human RBC been demonstrated to catalyze the phosphorylation of TDP to TTP, although AK1 from pig and chicken skeletal muscle possess TTP-synthesizing activity. TPK and AK1 in a human RBC lysate were therefore purified by a series of the conventional techniques. The specific activity of the purified TPK, which was obtained as a single protein, was 720 nmol TDP formed/mg protein per h at 37 degrees C. A partially purified AK1 preparation catalyzed the formation of TTP from TDP (specific activity, 170 nmol/mg protein per h at 37 degrees C) in addition to its proper reaction to form ATP from ADP. After incubation of the purified TPK and AK1 with 20 microM thiamin in the presence of ATP, ADP and Mg2+ at 37 degrees C for 48 h, the amounts of TDP and TTP synthesized were 465 and 54.0 pmol/250 microliters reaction mixture, respectively. Neither TDP nor TTP was formed when TPK was omitted from the reaction mixture and an omission of AK1 resulted in the formation of TDP alone. These results indicate that thiamin is converted to TDP by TPK and, subsequently, to TTP by AK1 in human RBC.


Role of the reactive cysteine residue in restriction endonuclease Cfr9I. Chemical modification studies were performed to elucidate the role of Cys-residues in the catalysis/binding of restriction endonuclease Cfr9I. Incubation of restriction endonuclease Cfr9I with N-ethylmaleimide (NEM), iodoacetate, 5,5'-dithiobis (2-nitrobenzoic acid) at pH 7.5 led to a complete loss of the catalytic activity. However, no enzyme inactivation was detectable after modification of the enzyme with iodoacetamide and methyl methanethiosulfonate. Complete protection of the enzyme against inactivation by NEM was observed in the presence of substrate implying that Cys-residues may be located at or in the vicinity of the active site of enzyme. Direct substrate-binding studies of native and modified restriction endonuclease Cfr9I using a gel-mobility shift assay indicated that the modification of the enzyme by NEM was hindered by substrate binding. A single Cys-residue was modified during the titration of the enzyme with DTNB with concomitant loss of the catalytic activity. The pH-dependence of inactivation of Cfr9I by NEM revealed the modification of the residue with the pKa value of 8.9 +/- 0.2. The dependence of the reaction rate of substrate hydrolysis by Cfr9I versus pH revealed two essential residues with pKa values of 6.3 +/- 0.15 and 8.7 +/- 0.15, respectively. The evidence presented suggests that the restriction endonuclease Cfr9I contains a reactive sulfhydryl residue which is non-essential for catalysis, but is located at or near the substrate binding site.


Beta-adrenergic receptor binding in human and rat hypothalamus. Quantitative autoradiographic analysis of beta-adrenergic binding sites was conducted in human postmortem hypothalamus using the radioligand 125I-pindolol. The focus was on the hypothalamic nuclei most clearly involved in corticotropin-releasing hormone (CRH) release, the PVN and SON. For comparison, the distribution of hypothalamic beta-adrenergic receptors was evaluated in the rat. A high level of beta-adrenergic receptor binding was found in the human paraventricular nucleus (PVN) and supraoptic nucleus (SON), but not in the rat. The majority of the beta-adrenergic receptors found in the human hypothalamus were of the beta 2-subtype. In contrast, in the rat hypothalamus, the majority of receptors were of the beta 1-subtype. These results show that the anatomical loci exist for direct beta-adrenergic influence on hypothalamic neuroendocrine function in the human and that the topography of beta-adrenergic receptors is markedly different in the rat and human hypothalamus.


Tricyclic heteroaromatic systems. Synthesis and benzodiazepine binding activity of 1-substituted-3-methyl- and 3-phenylpyrazolo[4,5-c]quinolin-4-ones. The synthesis and the benzodiazepine binding activity of some 3-methyl- and 3-phenylpyrazolo[4,5-c]quinolin-4-ones bearing a heterocyclic or a substituent which is different from an aryl moiety at position-1 are reported. Molecular modelling is used to correlate the binding affinity to the chemical features and to justify the reduced receptor affinities of the reported compounds with respect to that of CGS 8216 which is taken as the lead compound.


[Membrane mechanisms of testosterone action]. The experiments on adult Wistar rats have shown that testosterone administration provoke hyperpolarization of hepatocytes and adrenocorticocytes plasmatic membranes. It was discovered that this hyperpolarization was caused by cell Na, K-ATPase activation. Inhibitors of protein biosynthesis prevent both testosterone-induced hyperpolarization and Na, K-ATPase activation. It is shown that some hyperpolarization factor appeared in the cytosol and blood serum under testosterone influence. It was suggested that this mechanism is mediated by cell genome.


[Molecular mechanisms of the development of cardiovascular pathology]. Changes of a number of functions of beta-receptor complex mechanism have been analyzed by means of the microwave-dielectrometry method on native erythrocytes at various stages of cardiovascular pathology. The control scheme of beta-receptor complex has been analyzed in the adenylate cyclase system and the regions have been extracted which are subjected to an attack of pathological factors of the effect.


[Effects of nitroxyl radicals on pathogenic properties of herpesvirus (the spin-label technique in the study of virus-macrophage interactions)]. The effect of various concentrations of both methyl ether of 5-doxyl-stearic acid (M5DS) and 4-maleimido-TEMPO (4MT) on the pathogenicity of herpes simplex virus (HSV-1) was studied. It is known that the reagents modify the lipid matrix and the proteins of virion envelope. The decrease of the HSV-1 pathogenicity was shown when using the concentration of reagents more 5 x 10(-5) M. HSV-1 having high pathogenicity and cytotoxicity was obtained when the concentrations of the reagents were less 5 x 10(-5) M.


[Radiopaque magnetic medium oleoferrotrast, its x-ray specific properties and toxicity]. The paper reviews results of physicochemical and x-ray specific properties of a new magnetic radiopaque medium oleoferrotrast. The latter proved nontoxic, has radiopaque and magnetic adequacy for special x-ray examinations.


In vitro activation of lipophilic tributyltins by superoxide produces tributylstannyl superoxo radicals, proposed initiators of lipid peroxidation: an EPR model study. Tri-n-butyltin(TBT) compounds having the chemical formula (C4H9)3SnX are broad-spectrum biocidal agents whose toxic effect is primarily at the membrane level. Red blood cells (RBC) exposed to micromolar concentrations of tributyltin compounds (TBTX) undergo morphological changes and hemolysis. Determination of the mechanism of action whereby TBT elicits membrane damage continues to be a challenging endeavor. Because xenobiotic TBT+ and endogenous O2.- have been found to penetrate and alter RBC membrane function, it is hypothesized that they may combine chemically within the RBC hydrophobic lipid bilayer during TBT insult to initiate lipid peroxidative processes. The present study has been designed (1) to determine if TBT+ and O2.- combine chemically in aprotic media and, if so, (2) to characterize any free-radical complex(es) generated. The reactions of the membrane-active TBTX compounds (X = OCH3, Cl, Br, or I) with O2.- have been investigated in the aprotic solvent system cis-dicyclohexano-18-crown-6 ether/DMSO using EPR techniques. When incremental amounts of each TBT halide were added to O2.- solutions at room temperature, the EPR signal characteristic of O2.- diminished in intensity and disappeared when a 1:1 O2.-/TBT+ mole ratio was attained. Although the same phenomenon was observed for all TBT halides used, only the KO2/TBTI reaction produced detectable amounts of a new oxygen-centered free-radical complex. The EPR spectral parameters calculated from the product anisotropic frozen glass spectra were gx = 2.054, a(x) = 31.7 G, gy = 2.021, gz = 2.002, gav = 2.026.(ABSTRACT TRUNCATED AT 250 WORDS)


Intraarterial chemotherapy with limb-sparing resection of large soft-tissue sarcomas of the extremities. Fifteen patients with large (average, 15-cm), high-grade soft-tissue sarcomas of the extremities received prolonged selective intraarterial infusions of chemotherapeutic agents in an attempt to permit limb-sparing resection of these tumors, which would otherwise have required amputation. There were seven malignant fibrous histiocytomas, four liposarcomas, two fibrosarcomas, one leiomyosarcoma, and one rhabdomyosarcoma; 73% were grade III. Seven patients underwent two catheterizations, for a total of 22 infusions, which averaged 11.3 days each. There were four catheterization-related complications, including catheter occlusion or dislodgement in one patient each and two cases of arterial thromboembolism in patients in whom anticoagulant dose was not adequate. Both of the latter patients required thrombectomy; one developed gangrene, which precluded limb-sparing surgery. Thirteen of the 15 patients underwent limb-sparing resections, and two underwent amputations. No wound complications occurred. With a median follow-up of 36 months (mean, 34 months), life-table analysis indicates overall and disease-free survivals of 72% and 59%, respectively, at 2 years and 64% and 59% at 3 years. In comparison to other reported therapies, this technique permits limb salvage in most patients without the high wound complication rate associated with preoperative radiation therapy, with equivalent local disease control and survival.


SK&F 104353, a selective leukotriene receptor antagonist, inhibits leukotriene D4- and antigen-induced bronchoconstriction in cynomolgus monkeys. The ability of SK&F 104353 to prevent and reverse leukotriene (LT) D4- and antigen (Ag)-induced bronchoconstriction was examined in anesthetized, spontaneously breathing cynomolgus monkeys. Aerosol administration of LTD4 (10 micrograms/ml; 20 breaths) produced a sustained increase in pulmonary resistance and decrease in dynamic lung compliance. Aerosolized SK&F 104353 (150 breaths, 0.3 or 4.4 mg/ml) administered 15 min prior to LTD4 challenge antagonized these changes in a dose-dependent manner. When given intravenously 6 min after LTD4, SK&F 104353 (5 mg/kg) rapidly and completely reversed the ongoing bronchoconstriction. In mepyramine-pretreated (2 mg/kg i.v.) monkeys sensitive to aerosolized Ascaris suum Ag, intravenously administered SK&F 104353 (5 mg/kg) substantially reversed, but did not abolish, Ag-induced bronchoconstriction when administered 12 min after the Ag challenge. In contrast, SK&F 104353 (5 mg/kg i.v.) did not reverse Ag-induced bronchoconstriction in animals that had not been pretreated with mepyramine. Similar results were obtained when SK&F 104353 (20 mg/kg i.v.) was administered (as a pretreatment) 5 min prior to Ag under these conditions. Thus, SK&F 104353 reduced Ag-induced bronchoconstriction in mepyramine-pretreated monkeys, but had little effect in the absence of mepyramine. The data suggest that LTs, in addition to histamine, play a role in allergic bronchoconstriction in cynomolgus monkeys.


Effect of cis-diaminedichloroplatinum (II) (cis-DDP) on the intracellular Na+/K(+)-ratio in K 562 leukemia cells as revealed by X-ray microanalysis. Changes of intracellular ionic homeostasis are believed to play a role in the cytostatic action of cis-DDP. It has been observed by means of X-ray microanalysis that cis-DDP did not alter the intracellular Na+/K(+)-ratio of K 562 leukemia cells during incubation periods which lasted shorter than the average doubling time of the cells of nearly 15 h. After 24 h the treated cells displayed at least two main populations in the distribution histogram of the Na+/K(+)-ratio. The results indicated that the passage of cis-DDP through the plasma membrane by itself did not change the monovalent electrolyte balance at the early stage of its action in K 562 cells.


Nuclear localization signal(s) required for nuclear targeting of the maize regulatory protein Opaque-2. The maize regulatory protein Opaque-2 (O2) localizes to the nucleus in both maize and tobacco cells. Here we show that in-frame carboxy- and amino-terminal fusions of O2 to reporter protein beta-glucuronidase (GUS) were sufficient to direct GUS to the nucleus in transgenic tobacco plants and in transiently transformed onion cells. Two independent regions of O2 containing 135 and 149 amino acids were identified that were able to redirect GUS to the nucleus in both systems. A quantitative biochemical analysis of GUS in nuclei isolated from transgenic tobacco plants revealed that the second region was more efficient than the first one. The precise location of nuclear localization signals (NLSs) was determined using an onion transformation system. The first NLS was located between residues 101 and 135 and had the structure of a simian virus 40 NLS. The second NLS was located in the basic, DNA binding domain (between residues 223 and 254) and had a bipartite structure. The presence of one of the O2 NLSs in the basic domain is in complete agreement with similar findings of NLSs in the basic domain of three other basic/leucine zipper proteins, suggesting that this domain may be bifunctional. The effect of amino- versus carboxy-terminal GUS fusions is discussed.


The TMK1 gene from Arabidopsis codes for a protein with structural and biochemical characteristics of a receptor protein kinase. Genomic and cDNA clones that code for a protein with structural and biochemical properties similar to the receptor protein kinases from animals were obtained from Arabidopsis. Structural features of the predicted polypeptide include an amino-terminal membrane targeting signal sequence, a region containing blocks of leucine-rich repeat elements, a single putative membrane spanning domain, and a characteristic serine/threonine-specific protein kinase domain. The gene coding for this receptor-like transmembrane kinase was designated TMK1. Portions of the TMK1 gene were expressed in Escherichia coli, and antibodies were raised against the recombinant polypeptides. These antibodies immunodecorated a 120-kD polypeptide present in crude extracts and membrane preparations. The immunodetectable band was present in extracts from leaf, stem, root, and floral tissues. The kinase domain of TMK1 was expressed as a fusion protein in E. coli, and the purified fusion protein was found capable of autophosphorylation on serine and threonine residues. The possible role of the TMK1 gene product in transmembrane signaling is discussed.


The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase. An S-receptor kinase (SRK) cDNA, SRK-910, from the active S-locus in a self-incompatible Brassica napus W1 line has been isolated and characterized. The SRK-910 gene is predominantly expressed in pistils and segregates with the W1 self-incompatibility phenotype in an F2 population derived from a cross between the self-incompatible W1 line and a self-compatible Westar line. Analysis of the predicted amino acid sequence demonstrated that the extracellular receptor domain is highly homologous to S-locus glycoproteins, whereas the cytoplasmic kinase domain contains conserved amino acids present in serine/threonine kinases. An SRK-910 kinase protein fusion was produced in Escherichia coli and found to contain kinase activity. Phosphoamino acid analysis confirmed that only serine and threonine residues were phosphorylated. Thus, the SRK-910 gene encodes a functional serine/threonine receptor kinase.


Tourist: a large family of small inverted repeat elements frequently associated with maize genes. The wx-B2 mutation results from a 128-bp transposable element-like insertion in exon 11 of the maize Waxy gene. Surprisingly, 11 maize genes and one barley gene in the GenBank and EMBL data bases were found to contain similar elements in flanking or intron sequences. Members of this previously undescribed family of elements, designated Tourist, are short (133 bp on average), have conserved terminal inverted repeats, are flanked by a 3-bp direct repeat, and display target site specificity. Based on estimates of repetitiveness of three Tourist elements in maize genomic DNA, the copy number of the Tourist element family may exceed that of all previously reported eukaryotic inverted repeat elements. Taken together, our data suggest that Tourist may be the maize equivalent of the human Alu family of elements with respect to copy number, genomic dispersion, and the high frequency of association with genes.


Operation for polysyndactyly of the fifth toe using Z-plasty. A new operative procedure was devised to treat polysyndactyly of the fifth toe. This procedure, which consists of removal of the fifth toe, correction of the alignment of the preserved sixth toe by arthroplasty and construction of an interdigital space by Z-plasty, is technically simple and produces good results, both functionally and aesthetically.


Characterization of opioid receptor-mediated regulation of incertohypothalamic dopamine neurons: lack of evidence for a role of 5-hydroxytryptaminergic neurons in mediating the stimulatory effects of morphine. The purpose of the present study was to characterize opioid receptor-mediated regulation of incertohypothalamic dopaminergic (DA) neurons in the rat brain by examining the acute effects of selective mu or kappa opioid receptor agonists and antagonists on concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) in the medial zona incerta (MZI) and the dorsomedial hypothalamic nucleus (DMN) which contain cell bodies and terminals, respectively, of these neurons. Morphine caused a dose- and time-related increase in concentrations of DOPAC in MZI and DMN; this stimulatory effect was blocked by the mu opioid receptor antagonist naltrexone. In contrast, activation or blockade of kappa opioid receptors following administration of U-50,488 or nor-binaltorphimine, respectively, had no effect on DOPAC concentrations in either the MZI or DMN. The basal activity of incertohypothalamic DA neurons and their response to morphine was similar in male and female rats. Morphine also increased the concentrations of 5-hydroxyindoleacetic acid in MZI and DMN, indicating that morphine increases the activity of 5-hydroxytryptamine (5HT) neurons projecting to these regions. This might suggest that morphine-induced activation of incertohypothalamic DA neurons is mediated by 5HT neurons; but 5,7-dihydroxytryptamine-induced lesions of 5HT neurons did not alter the ability of morphine to increase DOPAC concentrations in MZI and DMN. These results indicate that the stimulatory effects of mu opioid receptor activation on incertohypothalamic DA neurons is not dependent upon the presence of 5HT neurons.


Two types of chloride channel in the apical membrane of rat choroid plexus epithelial cells. The patch clamp technique has been used to study ion channel activity in the apical (ventricular) membrane of epithelial cells from the rat choroid plexus. Two different classes of Cl(-)-selective channel were identified. A low conductance (26 pS) channel which was the predominant feature in cell-attached and inside-out patches. The occurrence of this channel appeared to increase in tissue bathed in forskolin. It was activated in inside-out patches by increasing the Ca2+ concentration at the intracellular face of the membrane and by depolarising potentials. The second class of channel was observed infrequently (2% of patches) and appeared to be similar to 'maxi'-Cl- channels which have been described in many other cell types. It had a conductance of 320 pS, opened to sub-conductance levels and displayed a marked voltage dependence in inside-out patches. The possible contribution of these channels to Cl- transport during the production of cerebrospinal fluid (CSF) is discussed.


Different types of norepinephrinergic receptors are involved in preoptic area mediated independent modulation of sleep-wakefulness and body temperature. The preoptic area is known to regulate sleep-wakefulness and body temperature. It was suggested earlier that though sleep-wakefulness and body temperature may affect each other, the preoptic area mediated influence on those two physiological phenomena is likely to be independent of alteration in each other. Since intrapreoptic area norepinephrine could modulate both those functions, study of that system was undertaken. It was hypothesized that since the preoptic area has different types of norepinephrinergic receptors (viz. alpha 1, alpha 2 and beta), independent modulation of those two functions was probably due to activation or inactivation of separate receptors. Hence, the effects of different agonist and antagonist of those receptors individually as well as in combination into the preoptic area were studied on those two functions in freely moving rats. The results suggest that norepinephrine induced preoptic area mediated influence on the body temperature is primarily regulated by the alpha 1 receptors while the sleep and wakefulness are regulated by alpha 2 and beta receptors, respectively. The finding should help in explaining several poorly understood observations reported earlier and it suggests that similar phenomena may possibly exist in other system involving other neurotransmitters as well.


Autoradiographic characterization of [3H]imipramine and [3H]citalopram binding in rat and human brain: species differences and relationships to serotonin innervation patterns. The neuroanatomical distribution of binding sites for [3H]imipramine and [3H]citalopram was assessed by in vitro autoradiography in select regions of the rat and human forebrain. To determine involvement of serotonin-containing terminals in the binding of [3H]imipramine and [3H]citalopram, binding of these compounds was measured in rats after destroying serotonin-containing neurons with 5,7-dihydroxytryptamine (5,7-DHT). Treatment with this neurotoxin decreased serotonin content by 90% and reduced [3H]citalopram binding to a similar extent. These results demonstrate that [3H]citalopram binding is a reliable marker for serotonin-containing terminals. Binding of [3H]imipramine was reduced by only 15-35% after 5,7-DHT treatment. These latter results suggest that only a small fraction of [3H]imipramine binding to brain sections is associated with serotonergic terminals under standard conditions used in autoradiographic studies with the ligand. Dose-response effects of fluoxetine and desipramine on displacement of [3H]imipramine binding in forebrain regions indicate that the ligand labels predominantly high capacity, low affinity binding sites. To determine the utility of the rat brain as a model for [3H]imipramine and [3H]citalopram binding in the human brain, binding of the ligands was compared in human and rat hypothalamus, amygdala, and hippocampus. The pharmacological characteristics of [3H]imipramine and [3H]citalopram binding were similar in the rat and human brain. However, substantial species differences were observed in topographic patterns of [3H]imipramine binding within the hippocampus and hypothalamus. The distribution of [3H]citalopram binding sites within the amygdala and hypothalamus were also strikingly different in rats compared to humans. This work provides the first demonstration that marked species differences exist in the topography of serotonergic innervation and in the distribution of [3H]imipramine binding sites within the rat and human brain regions examined.


Changes in [125I]hCGRP binding in rat spinal cord in an experimental model of acute, peripheral inflammation. [125I]Human calcitonin gene-related peptide ([125I]hCGRP) binding in the dorsal horn of the spinal cord exhibited differential changes among laminae over time in response to unilateral adjuvant-induced inflammation. In laminae I/II, 4 days after induction of inflammation, the binding decreased 36% on the side of the spinal cord ipsilateral to the inflammation, while there was no change on the contralateral side. The decrease ipsilateral to inflammation was due primarily to a decrease in the Bmax of the high affinity binding site for CGRP. In lamina V, the binding increased 18% on both sides of the spinal cord at the same time point. In lamina X, the binding increased 16% on both sides of the spinal cord at 2 days after induction of inflammation and remained increased at 8 days. The increases in [125]hCGRP binding in laminae V and X were primarily due to a decrease in the Kd of the low affinity binding site for CGRP. the accompanying hyperalgesia was first measured at 2 days after induction of inflammation and persisted at 8 days. Because the changes in [125I]hCGRP binding did not parallel the hyperalgesia accompanying the unilateral adjuvant-induced inflammation, we believe that CGRP receptors are not directly involved with the hyperalgesia but may be involved with other plastic changes observed in the spinal cord during unilateral adjuvant-induced inflammation.


Insulin-like growth factor I modulates voltage-dependent Ca2+ channels in neuronal cells. Insulin and insulin-like growth factors are neuroactive peptides. We investigated the effect of insulin-like growth factor I (IGF-I) on Ca2+ channel currents in 108CC15 neuroblastoma x glioma (N x G) cells and a possible role of protein kinase C (PKC). Whereas the native IGF-I enhanced the Ca2+ channel current density in N x G cells, the boiled IGF-I had no effect. The effect of IGF-I occurred after 1-2 h incubation and reversed within 24 h. Ca2+ channel currents recorded in control cells were mainly of a low-threshold fast inactivating type and showed a mean density of 5.9 +/- 0.3 pA/pF. Current density in cells incubated with IGF-I (0.2 micrograms/ml) for 2 h increased to 9.2 +/- 0.8 pA/pF. Ca2+ channel currents in cells treated with IGF-I showed an enhanced amount of a high-threshold slowly inactivating Ca2+ current type sensitive to the dihydropyridine isradipine and the snail toxin omega-conotoxin. The effect of IGF-I was suppressed by coincubation with the PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and staurosporin which were both without effect on current density in control cells. Whereas the inactive phorbol ester phorbol 12-myristate 13-acetate (PMA) failed to modulate Ca2+ channels in N x G cells, stimulation of PKC by the active phorbol ester PMA mimicked the effect of IGF-I. The effects of IGF-I and phorbol ester were not additive. Our data suggest an intracellular mechanism dependent on PKC and we propose a physiological relevance of the observed Ca2+ channel modulation by IGF-I in the neuroactivity of the peptide.


Uncoupling of GABA-benzodiazepine receptors in chick cerebral cortical neurons requires co-activation of both receptor sites. Primary cultures of chick cerebral cortical neurons were exposed to 1 microM flurazepam in vitro, and the effect of flurazepam on GABA-activated membrane current (IGABA) was examined using whole-cell voltage-clamp recording. Exposure of chick cerebral cortical neurons to flurazepam alone for 3-10 days resulted in a significant decrease in the degree of potentiation of IGABA elicited by 0.5 microM flurazepam. When GABA or nipecotic acid (a GABA uptake blocker) were added with flurazepam during chronic drug exposure, neuronal responses to GABA were significantly less sensitive to modulation by 0.5 microM flurazepam compared to flurazepam treatment alone. Furthermore, this effect was significantly reduced by co-administration of the GABAA receptor antagonist bicuculline. These results suggest that tolerance to benzodiazepines in vitro requires activation of both the GABA and the benzodiazepine binding sites on the GABAA receptor-channel complex.


Modulation of ionic currents in isolated type I cells of the neonatal rat carotid body by p-chloromercuribenzenesulfonic acid. The sulphydryl reagent, p-chloromercuribenzenesulfonic acid (PCMBS), irreversibly excites the carotid body in vivo. We tested the effects of PCMBS on ionic currents in isolated type I cells, using the whole-cell patch-clamp technique. PCMBS selectively and irreversibly inhibited the Ca(2+)-activated K+ current (IKCa) in a dose-dependent manner (0.01-1 mM). The same concentrations of PCMBS did not affect the Ca(2+)-independent K+ current (IKv), but caused a transient enhancement of the Ca2+ current (ICa). The inhibition of IKCa by PCMBS is similar to the previously reported effects of hypoxia, and suggests a central role for the channels underlying this current in chemotransduction.


[Immune status of the spleen and surgical treatment in patients with advanced gastric cancer]. The immune status of spleen and the effect of surgical treatment in advanced gastric cancer (AGC) patients were evaluated by means of testing NK cell activity, T-lymphocyte subsets and circulating immune complex (CIC). The results showed (1) the significant impairment of NK cell activity and T-lymphocyte subsets, were decreased in CD3+, CD4+ cells and increased in CD6+ cells resulting in CD4+/CD8+ cell ratio decrease in peripheral blood lymphocytes (PBL), splenic venous blood lymphocytes (SVL) and spleen cells (SC) of AGC, as compared with PBL of normal population; (2) NK cell activity or CD4+/CD8+ cell ratio of SVL and SC were significantly lower than those of PBL in AGC patients, mainly caused by marked decrease of CD4+ cells in SC; (3) NK cell activity, CD4+ cells and CD4+/CD8+ cell ratio were significantly elevated in most of AGC cases receiving either radical gastrectomy (R2+) or extensive radical gastrectomy (R3). A striking declination in CD8+ cells were found only after R3 operation; (4) after a short period (10-14 days) or a long period (2-4 years) of radical gastrectomy, NK cell activity and T-lymphocyte subsets showed no significant differences between R2+ and R3 operation. As a rule, AGC would weaken immune function of patients, and along with the development of the tumor, the immunosuppression in the spleen would be generated gradually. For these reasons, a complete tumor resection would be necessary to improve the immunocompetence, and the combined splenectomy might be advisable if indicated.


[Effects of the spleen on immune state in patients with gastric cancer]. For analysing the effects of the spleen on immune state of gastric cancer patients, T-lymphocyte subsets and serum immunosuppressive factors of splenic venous and peripheral blood were assayed. The results showed that the proportion of CD4+ cells in peripheral blood, especially in splenic venous blood of early cancer patients was higher than that in advanced cancer patients. The proportion of CD8+ cells in splenic venous blood of the advanced cancer patients was higher not only than that in peripheral blood of advanced cancer patients, but also than that in splenic venous blood of early cancer patients. The ratio of CD4+/CD8+ cells in peripheral blood, especially in splenic venous blood showed a decrease with the increase of cancer stage. The immunosuppressive activity of sera of peripheral blood, especially of splenic venous blood was increased significantly in keeping with the cancer stage. It seems that the spleen takes part in regulation of tumor immunity. In early cancer patients it shows some anticancer effects on forming and releasing supplementary T cells (CD4+). However, in advanced cancer patients it effects on the development of immunosuppression through forming and releasing suppressive T cells and serum immunosuppressive factors.


[Effects of spleen on inducing portal hypertension and liver cirrhosis in rats]. The role of spleen on inducing the portal hypertension and cirrhotic rat by carbon tetrachloride (CCl4) and ethyl alcohol was studied. Totally 412 male rats were divided randomly into four groups: group A (n = 42) underwent splenectomy before induction by CCl4; group B (n = 42) underwent splenectomy after induction by CCl4 and alcohol of 4 weeks; group C (n = 42) received sham-splenectomy and group D (n = 14) served as a control group. Free portal pressure (FPP), function of liver, the index of spleen (weight of spleen/weight of body) as well as the spleen and liver biopsy were evaluated at different time. The results showed that, in group C, the index of spleen and the area of splenic white pulp had significant increase. The degree of hepatic damage, the number of the infiltrating cells and the degree of hepatic fibrosis were significantly greater in sham-operated animals than in others (group A and B). These results suggested that the spleen should be an immunomodulatic organ in playing an exacerbation role of hepatic cirrhosis, and the effect of splenectomy be a preventive role against the induced rat liver cirrhosis, at least, at certain stage.


[Pathological classification of splenomegaly in portal hypertension]. The morphometric analysis was carried out for 150 cases splenomegaly pathological sections (1010 sections) after being stained with histochemical, immunohistochemical, hematoxylin and eosin methods, summarizing that the proliferation of three types of fibers, the number and distribution of T, B lymphocytes and macrophages, the changes of sections stained with HE, the ultrastructure of splenomegaly under electron microscope would be the criteria for partially preserving splenomegaly. The data of morphometry was treated by IBM/PC computer. The research was aimed to establish a pathological indication for preserving partial splenomegaly clinically. The study demonstrated that the pathological classification, based on the fibrosis degree of splenomegaly, may objectively reflect the changes of immunological function of splenomegaly, by which it was also revealed that the immunological function in pathological class II and III was markedly lowered. Because the proportion of class I splenomegaly remained only in 31% of good function, there are less splenomegaly worth preserving.


Pharmacological and functional characterization of excitatory amino acid mediated cytotoxicity in cerebral cortical neurons. The cytotoxic action of the excitatory amino acids (EAAs) glutamate, N-methyl-D-aspartate (NMDA), quisqualate (QA), kainate (KA) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA) was studied in cerebral cortical neurons in culture. The pharmacological profile of these actions was characterized using the NMDA selective antagonist D-(-)-2-amino-5-phosphonopentanoate (APV) and the non-NMDA selective antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX), 2-amino-3[3-(carboxymethoxy)-5-methylisoxazol-4-yl]-propionate (AMOA) and 2-amino-3-[2-(3-hydroxy-5-methylisoxazol-4-yl)methyl-5-methyl-3- oxoisoxazolin-4-yl]propionate (AMNH). The role of intracellular Ca++ homeostasis and cGMP production for development of EAA mediated cytotoxicity was assessed by measurements of changes in [Ca++]i using the fluorescent Ca++ chelator Fluo-3 and in cGMP concentrations using a conventional radioimmune assay. It was found that glutamate toxicity involves both NMDA and non-NMDA receptor activation and that aberrations in Ca++ homeostasis brought about by Ca++ influx and/or liberation of Ca++ from internal stores are important for development of toxicity. The drug dantrolene which prevents release of Ca++ from such stores can prevent toxicity induced by glutamate, NMDA and QA completely but has no effect on KA and AMPA toxicity. Changes in cGMP levels appear to play a role for development of glutamate, NMDA and KA toxicity but does not seem to be involved in that triggered by QA and AMPA.


[Effects of Ligustricum Wallichii on acute nephrotoxicity induced by cyclosporine A in rats]. We investigated the influence of cyclosporine A (CsA) on renal function, renin-angiotensin system (RAS) and platelet aggregation in addition to the effect of Ligusticum Wallichii (LW) on CsA actions in SD rats. Infusion of CsA (50 mg/kg, iv) resulted in a significant fall in glomerular filtration rate (GFR) and renal plasma flow (RPF) and a significant increase of plasma renin activity (PRA), angiotensin II (A II) level and percentage platelet aggregation. At the same time, treatment with 20% LW (8 ml/kg, iv) before CsA infusion significantly prevented the decline of GFR and RPF as well as the enhancement of platelet aggregation, but had no influence on CsA-mediated RAS activation. These results suggested that LW may be beneficial to the acute nephrotoxicity induced by CsA.


[Clinical study on the prevention of kernicterus caused by hereditary glucose-6-phosphate dehydrogenase deficiency]. A combined preventive scheme was conducted in four hospitals in Guangzhou to lower the rate of kernicterus and mental retardation caused by related neonatal jaundice due to G6PD deficiency. Observation was focused on 330 G6PD deficient infants, and the effects were measured according to the incidence of hyperbilirubinemia and kernicterus. The results, as compared to those of a retrospective study, showed that the incidence of hyperbilirubinemia was significantly decreased (51.4% to 21.2%), and neither kernicterus nor mental retardation infant was found in this series (12.5% in the control group). The authors conclude that this combined scheme is extremely effective and can be used in any large population area in which there is a high gene frequency of G6PD deficiency.


[A case-control study on the relationship between smoking and genetic background and lung cancer]. A case-control study on the relationship between smoking and genetic background and lung cancer was carried out. The cases (probund) were death cases of lung cancer diagnosed between 1984 and 1986 in Quanshan County, and the controls were healthy people at the same period. Cases and controls were 1:1 in match with sex, age and residence. 196 pairs were so matched. The results were adjusted for smoking habit and family history of lung cancer using conditionally logistic regression model. There were significant differences shown in quantity of smoking (packet/year) (beta = 0.070 9) and age to start (years) (beta = -0.055 6) between cases and controls. There were also significant differences in history of lung cancer of first and second relatives (living together or not) between cases and controls. The positive interaction between smoking habit and genetic background of lung cancer was also found.


[Total hip replacement for congenital dislocation of the hip in elderly patients]. From 1986 to 1989, total hip replacement was performed on ten patients (twelve hips) with congenital dislocation of the hip. The average age of the patients was fifty-three years (ranging from forty-eight to sixty years), and the mean follow-up period was twenty-eight months (ranging from six to forty-eight months). Of the ten patients, eight had unilateral replacement and two had bilateral replacement, for a total of twelve arthroplasties. Eight arthroplasties were performed on the right and four on the left. Most patients left the hospital two to three weeks after operation. All the patients obtained satisfactory results. Only one patient developed late infection two years postoperatively. This patient occasionally used a cane for walking. The other patients had no hip pain, the motion of the hips was approximate to a normal person. No patient required a shoe-lift for limb-length discrepancy.


[Diagnostic imaging of chondroblastoma: a radiological, pathological and postoperative analysis of 58 cases]. Imageological data of 58 cases with chondroblastoma including plain radiographs in all cases, CT scan in 10 and scintigraphy of bone in 6 proved pathologically were reviewed and analysed. 33 cases were followed-up for 1 to 20 years after operation. The authors hold that plain radiograph is still the primary approach to assessment of this disorder. Position of the disease, calcification inside the focus, scalloping sclerotic rim and periosteal new bone formation frequently seen in metaphysis near the lesion are essential X-ray appearance. CT scan has some advantages in showing fine pathological fracture of cortex, subtel calcification, scope of involvement and recurrent signs in earlier stage, whereas bone scintigraphy is non-specific. The main factors of postoperative recurrence are incomplete curettage and more active biological behaviour of the tumor itself in part of cases. And some points for diagnosing chondroblastoma with atypical X-ray manifestations were also presented.


[Chondronecrosis induced in rhesus monkeys fed with grains and water of Kaschin-Beck's disease endemic area]. Immature Rhesus monkeys were fed with grains and/or water of Kashin-Beck disease (KBD) endemic area for 6 or 18 months. Multifocal and zonal chondronecrosis and a series of secondary reactions following necrosis were found in articular cartilage and growth plate in most of the monkeys. These changes are very similar to the characteristics of human KBD. The results suggest that growing Rhesus monkey should be the susceptible animal for reproduction of chondronecrosis model of KBD and the articular cartilage of greater trochanter proximal to femur is a portion predisposing to cartilage lesions. The experiment also indicated that pathogenic factor resulting in chondronecrosis is still remaining in water and grains of endemic area, though the area is no longer actively endemic for KBD.


[Ultrastructural examination and stereological study of bone marrow megakaryocytes in children with idiopathic thrombocytopenic purpura]. The ultrastructure and surface architecture of megakaryocytes (MK) were examined in 19 children with idiopathic thrombocytopenic purpura (ITP), the stereological parameters of alpha granules were computed. Transmission electron microscopy revealed vacuolization of cytoplasma of MKs, decrease in the number of alpha granules and degeneration of mitochondria. The granular MKs of chronic ITP appeared to be focal distribution of demarcation membrane system (DMS) in two cases with formation of large membrane complex. Staining with tannic acid indicated vesicular, tubular and cisternal dilation of DMA. Rough endoplasmic reticulum and Golgi complex were much more abundant than in normal MKs. In 5 cases of ITP, we found emperipolesis of hematopoietic cells within MKs. Under scanning electron microscopy many MKs showed complex surface processes including crowded blebs, villi and giant irregular projections. Occasionally perforations on the surface membrane could be seen. Stereological analysis showed that the Nv, Vv, and Sv of alpha granules of both acute and chronic ITP were markedly decreased as compared with normal control (P < 0.001), and the Nv of acute ITP was notably less than that of chronic ITP (P < 0.05). However the S/V, M.DIAM. and M. VOL. were no significant difference among normal control, acute and chronic ITP (P > 0.05).


[Pathologic-ultrasonographic comparative study of carcinomatous lesions of experimental hepatic carcinoma in rats]. 3'-Me-DAB was used to induce hepatic carcinoma in male Wistar rats. The pathologic-ultrasonographic comparative observation of forty-three carcinoma nodes of twenty-five rats showed that the echoic patterns of the nodes were determined not by the diameter, but by the pathologic types, the extent of hemorrhage or necrosis of lesions and the proliferative degree of the lattice fibers. The irregular vascularization, the dilatation of vessels and the tumoral cell fatty degeneration are related to the echoic patterns as well. This article also reports a preliminary study of the possibility that different structures and compositions of the carcinomatous nodes will produce different echoic levels.


[Changes in beta-endorphin receptor on T-lymphocytes in burned patients and their significance]. The changes in beta-endorphin receptor on the peripheral T lymphocytes were assayed in 30 burned patients (average burn area 38.1 +/- 30.0% of TBSA) and 20 healthy volunteers with radioactive ligand I-beta-endorphin. Results showed that the binding sites of beta-endorphin receptor on T lymphocytes were significantly decreased in various degrees, while kd values were increased, in patients major burns. In patients with smaller burns, the above parameters showed no significant difference from the normal. There were also decreased binding sites when complications such as infection occurred.


Syntheses of specifically deoxygenated methyl alpha-isomaltotriosides. Specifically deoxygenated methyl alpha-isomaltotriosides were synthesized by the silver perchlorate-mediated condensation of a protected alpha-isomaltosyl chloride with suitably blocked derivatives of methyl alpha-D-glucopyranoside deoxygenated respectively at positions 2, 3, and 4, followed by deprotection.


Oxygen free radical injury and Gs mediated signal transduction in the stunned porcine myocardium. The aim was to investigate involvement of oxygen free radicals and any changes in the Gs mediated beta adrenergic signalling system of stunned porcine myocardium. Myocardial stunning was induced in eight pentobarbitone anaesthetised pigs by brief occlusions of the distal left anterior descending coronary artery for periods of up to 10 min. Segment length function was measured in the ischaemic region and in a control region supplied by the circumflex artery. Left ventricular biopsies were obtained from the two regions 1 h after the last occlusion for ultrastructural and biochemical studies. Timolol has been used to prevent arrhythmia during ischaemia. At the time when biopsies were obtained, percent systolic shortening was reduced to 58% in the region subjected to ischaemia and was only minimally reduced in the control region. In the biopsies from the stunned region: (1) electron microscopy showed mild and reversible intracellular changes in the stunned myocardium; (2) the activities of superoxide dismutase and glutathione peroxidase were decreased by 66% and 52%, respectively; (3) the content of malondialdehyde was increased by 49%; (4) neither density nor affinity of beta adrenoceptors showed any changes; (5) there were no alterations in messenger RNA encoding for the alpha subunit of the stimulatory guanine nucleotide binding protein (Gs), demonstrated by northern and dot-blot hybridisations; (6) ELISA technique utilising a specific antipeptide antibody showed no quantitative change in Gs; (7) the activity of adenyl cyclase was unchanged. Even though the stunned porcine myocardium showed substantial evidence of free radical injury, the beta adrenergic signalling system was intact.


Combined class I antiarrhythmic agents have differential effects on tonic and use dependent block of maximum rate of depolarisation of action potentials in guinea pig cardiac muscle. The aim was to study the difference between tonic and use dependent block of the cardiac sodium channel produced by the combined application of the same subclass of antiarrhythmic agents (class Ia or Ib). Conventional glass microelectrode technique was used to record the maximum rate of depolarisation (dV/dtmax) of action potentials reflecting sodium channel availability, before and after the combined application of quinidine plus disopyramide, aprindine plus lignocaine, aprindine plus mexiletine, and lignocaine plus mexiletine. Guinea pig papillary muscles (n = 4-8 per experiment) were used for the study. All combinations increased tonic block additively compared to use of a single drug. On the other hand, use dependent block was increased by the combination of quinidine 10 microM plus disopyramide 30 microM compared to a single drug, and was not changed by lignocaine 50 microM plus mexiletine 20 microM, whereas it was decreased by aprindine 3 microM plus lignocaine 50 microM or mexiletine 20 microM. When concentrations of mexiletine and lignocaine were increased, both tonic and use dependent block in a single drug were increased dose dependently, whereas the combination produced an additive increase in tonic block but no change in use dependent block compared to a single drug. The results suggested that the binding and unbinding process of the drug to produce tonic block was different from that to produce use dependent block, and that combination of different drugs produced diverse effects on use dependent block even though state dependent affinity of individual drugs seemed similar. These two factors must be born in mind in evaluating the combination therapy.


Augmentation of beta adrenergic receptors in cardiomyopathic hamsters (BIO 14.6) with heart failure. The aim was to characterise the transmural distribution of beta adrenergic receptors in failing myocardium in cardiomyopathy. Using a quantitative autoradiographic technique with 125I-cyanopindolol (ICYP), the density and transmural distribution of beta adrenergic receptors were compared between eight cardiomyopathic BIO 14.6 Syrian hamsters with heart failure and six normal age matched controls (BIO 14.6HAM). Binding of ICYP to transmural slices of hamster myocardium was rapid, saturable, stereoselective, and displaceable by antagonists. The binding isotherm showed a significant increase in the total tissue content of beta adrenergic receptors in the failing myocardium of cardiomyopathic hamsters: 15.3(SEM 1.6) fmol.mg-1 protein v 9.4(1.2) fmol.mg-1 protein in normal myocardium of control hamsters (p < 0.05). There was no difference in receptor affinity. Quantitative autoradiography showed regional heterogeneity of beta adrenergic receptors in cardiomyopathic hamsters, with an increase of beta adrenergic receptor density in the septal and subendocardial regions. In addition, the regions with increased interstitial fibrosis corresponded to the sites of increased beta adrenergic receptor density. The transmural distribution of beta adrenergic receptor is heterogeneous in the failing myocardium of cardiomyopathic hamsters and an increased beta adrenergic receptor density may be associated with the development of cardiomyopathy.


Effect of brain lesions on [3H]ohmefentanyl binding site densities in the rat striatum and substantia nigra. We have recently demonstrated that [3H]ohmefentanyl, a non-peptidergic opioid ligand which was suggested to cross the blood brain barrier in contrast to other peptidergic opioid ligands, bound not only to mu opioid receptor sites but also to sigma sites. In order to examine whether [3H]ohmefentanyl can be used as a marker for mu sites, we investigated the effects of brain lesions on [3H]ohmefentanyl binding site densities, as compared with [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin ([3H]DAGO), a selective mu ligand. These binding site densities were measured by quantitative autoradiography in the rat striatum and substantia nigra, two brain structures known to contain a high density of mu receptors, following lesions of the nigro-striatal dopaminergic pathway and striatal intrinsic neurons. Following unilateral nigral lesion with 6-hydroxydopamine, [3H]ohmefentanyl binding site densities were decreased in the patches (-35%) and matrix (-20%) of the ipsilateral striatum and in the lesioned substantia nigra pars compacta (-49%). Unilateral striatal lesion with quinolinic acid induced 72%, 61% and 50% decreases in [3H]ohmefentanyl binding in the patches and matrix of the lesioned striatum and in the ipsilateral substantia nigra pars reticulata, respectively. Similar results were obtained in the binding of [3H]DAGO. Indeed, a significant linear correlation was observed between [3H]ohmefentanyl and [3H]DAGO binding site densities. Therefore, mu opioid receptors may be mainly located on intrinsic neurons in the striatum, dopaminergic cell bodies in the substantia nigra pars compacta and nerve terminals of striatal efferents in the substantia nigra pars reticulata.(ABSTRACT TRUNCATED AT 250 WORDS)


Photoaffinity labeling of the electroplax sodium channel with a photoreactive mu-conotoxin carrying a radioactive and chromogenic diazirine. Photoreactive derivatives of mu-conotoxin GIIIA carrying a radioactive and chromogenic diazirine have been prepared as a photoaffinity labeling reagent for muscle-type sodium channels. When the reagent was photolyzed in the presence of the eel sodium channel, radioactivity was specifically incorporated into the channel protein.


HPV genital infections and contraception. Human Papilloma Virus genital infections are, especially during fertile age, a very common finding in performing PAP smear and colposcopy. The interest in these infections arises from their frequent association with dysplasia and their possible role in the etiology of carcinoma of the lower genital tract. The natural history of HPV infections is not very well known and foreseen. Epidemiological studies indicate that the progression, regression or stability of induced HPV lesions are correlated with cofactors which are considered risk factors in carcinoma of the lower genital tract. The aim of this study is to evaluate the influence of estroprogestinic contraception and other methods on the evolution of the HPV genital infections.


Immune aspects of pathology of the placental bed contributing to pregnancy pathology. Interest has recently focused on the role of the placental bed in the pathogenesis of a variety of pregnancy disorders. Considerable advances have been made in the understanding of the complex relationships between maternal and fetal trophoblast in the placental bed in normal pregnancy. Invasion of uterine spiral arteries by extravillous trophoblast effects the physiological changes required to accommodate increased blood flow to the fetoplacental unit. Control of trophoblast invasion may depend on intrinsic properties, such as production of proteolytic enzymes and expression of a non-classical class I MHC antigen, but maternal cells within decidua may also play a role. Leukocytes form a major component of human decidualized endometrium and in the first trimester consist of granulated lymphocytes, macrophages and T lymphocytes. Suggested roles for decidualized leukocytes include natural killer cell activity, cytokine secretion, antigen presentation and immunosuppression. Several pregnancy disorders, including pre-eclampsia and intrauterine growth retardation, may be due to abnormal maternofetal cellular relationships within the placental bed causing inadequate invasion of spiral arteries and acute atherosis. However, the role of immunological factors in the pathogenesis of these disorders is uncertain since deposition of immunoglobulins and complement has also been detected in spiral arteries in normal pregnancy. Placenta accreta may reflect undue invasiveness of trophoblast and immunohistochemical studies of subinvolution of uteroplacental arteries also suggest an abnormal maternofetal relationship in the placental bed. Although the in vivo role of decidual leukocytes is not known, studies of infertile endometrium have reported a deficiency of granulated lymphocytes, suggesting a possible role in early implantation and placentation. Granulated lymphocytes may also play a role in pregnancy loss. There have been considerable advances in understanding of the abnormal maternofetal relationships in the placental bed which can lead to pregnancy disorders. However, the aetiology and pathogenesis of the various clinical conditions is unlikely to be fully established until regulatory mechanisms in normal pregnancy are elucidated.