Binding of two Sr2+ ions changes the chemical specificities for phosphorylation of the sarcoplasmic reticulum calcium ATPase through a stepwise mechanism.

The sequential binding of Sr2+ and Ca2+ to the cytoplasmic transport sites of the sarcoplasmic reticulum calcium ATPase allows the formation of two different mixed complexes: cE.Sr.Ca, with Sr2+ bound to the "inner" site and Ca2+ bound to the "outer" site, and cE. Ca.Sr, with Ca2+ bound to the inner site and Sr2+ bound to the outer site (pH 7.0, 25 degrees C, 10 mM MgCl2, 100 mM KCl). Both cE.Sr.45Ca and cE.45Ca.Sr react with ATP to internalize one 45Ca/phosphoenzyme. The value of K0.5 = 83 microM Sr2+ for activation of the enzyme for phosphorylation by ATP is much larger than K0.5 = 28 microM Sr2+ for inhibition of phosphoenzyme formation from inorganic phosphate (eta H = 1.0-1.3). These results are consistent with the sequential binding of two strontium ions with negative cooperativity and dissociation constants of KSr1 = 35 microM and KSr2 = 55 microM. The species cE.Sr2 and cE.Ca2 react rapidly with ATP but not inorganic phosphate. However, enzyme with one strontium bound, cE.Sr, does not react with either inorganic phosphate or ATP. Therefore, the conformational changes in the enzyme that alter the chemical specificity for phosphorylation by ATP and by inorganic phosphate are different. This requires the existence of at least three forms of the unphosphorylated enzyme with three different chemical specificities for catalysis.


Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase. Novel fragments produced by trifluoroacetic acid.

Inositol glycans were prepared from reductively radiomethylated human erythrocyte acetylcholinesterase by sequential treatment with Proteinase K, methanolic KOH, and phosphatidylinositol-specific phospholipase C. Four glycans denoted alpha-delta were resolved by anion exchange high performance liquid chromatography (HPLC). Each glycan was subjected to hydrolysis in 4 M trifluoroacetic acid, and their hexose and hexose phosphate compositions were determined by anion exchange HPLC. The predominant glycan alpha showed a relative stoichiometry of 2 mannoses, 1 mannose 6-phosphate, 1 radiomethylated glucosamine, 1 radiomethylated ethanolamine, and 1 inositol. In contrast, the stoichiometry of glycan beta was 1 mannose, 2 mannose 6-phosphates, 1 radiomethylated glucosamine, 2 radiomethylated ethanolamines, and 1 inositol. Glycans alpha and beta were analyzed by electrospray ionization-mass spectrometry, and respective parent ions of m/z 1266 and 1417 were observed. The fragmentation pattern produced by collision-induced dissociation mass spectrometry of these parent ions was consistent with a common linear core glycan sequence prior to radiomethylation of ethanolamine-phosphate-mannose - mannose - mannose - glucosamine - inositol. Glycan alpha contained a single additional radiomethylated phosphoethanolamine branching from the mannose adjacent to glucosamine, whereas glycan beta contained two additional radiomethylated phosphoethanolamines, one branching from each of the mannoses nearest to glucosamine. Trifluoroacetic acid hydrolysis did not cleave within the N,N-dimethylglucosamine-inositol-phosphate moiety in these glycans, and this component was resolved by anion exchange HPLC and structurally confirmed by mass spectrometry. Dephosphorylation of this component by treatment with 50% HF produced N,N-dimethylglucosamine-inositol, and this conjugate was shown to have a characteristic elution time on cation exchange chromatography in an amino acid analyzer. Both of these fragments involving an intact radiomethylated glucosamine-inositol bond are proposed as new diagnostic indicators in the search for minor glycoinositol phospholipids in cells and tissues.


Location of the two catalytic sites in intestinal lactase-phlorizin hydrolase. Comparison with sucrase-isomaltase and with other glycosidases, the membrane anchor of lactase-phlorizin hydrolase.

Lactase-phlorizin hydrolase was isolated by immunoadsorption chromatography from rabbit brush-border membrane vesicles. Inactivation of the enzyme with [3H]conduritol-B-epoxide, a covalent active site-directed inhibitor, labeled glutamates at positions 1271 and 1747. Glu1271 was assigned to lactase, Glu1747 to phlorizin hydrolase activity. In contrast, the nucleophiles in the active sites of sucrase-isomaltase are aspartates (Asp505 and Asp1394). Asp505 is a part of the isomaltase active site and is localized on the larger subunit, which carries the membrane anchor also, while Asp1394 is a part of the active of sucrase. Alignment of these 2 nucleophilic Glu residues in lactase-phlorizin hydrolase and of their flanking regions with published sequences of several other beta-glycosidases allows the classification of the configuration retaining glycosidases into two major families: the "Asp" and the "Glu" glycosidases, depending on the carboxylate presumed to interact with the putative oxocarbonium ion in the transition state. We offer some predictions as to the Glu acting as the nucleophile in the active site of some glycosidases. By hydrophobic photolabeling, the membrane-spanning domain of lactase-phlorizin hydrolase was directly localized in the carboxyl-terminal region thus confirming this enzyme as a monotopic type I protein (i.e. with Nout-Cin orientation) of the brush-border membranes. A simplified version of the Me2+ precipitation method to efficiently and simply prepare brush-border membrane vesicles is also reported.


Structure of the vacuolar ATPase from Neurospora crassa as determined by electron microscopy.

We have examined the structure of the vacuolar ATPase of Neurospora crassa using negatively stained preparations of vacuolar membranes and of detergent-solubilized and gradient-purified ATPase complexes. We also examined the peripheral sector (V1) of the enzyme after it had been removed and purified. Using different stains, vacuolar membranes displayed ball-and-stalk structures similar to those of the intact mitochondrial ATPase. However, the vacuolar ATPase was clearly different from the mitochondrial ATPase in both size and structural features. The vacuolar enzyme had a much larger head domain with a distinct cleft down the middle of the complex. This domain was held above the membrane by a prominent stalk. Most intriguing was the presence of basal components. These structures appeared to project from the vacuolar membrane near the base of the stalks. Detergent-solubilized, gradient-purified ATPases displayed the same head, stalk, and basal features as those found with the intact enzyme on vacuolar membranes. The mitochondrial ATPase was significantly smaller, and no clefted head domains or basal components were observed. When V1 and F1 particles were directly compared, a significant difference in size and shape between these two soluble ATPase sectors was apparent. V1 retained all of the features seen in the globular head of the intact complex: V-shaped, triangular, and square forms around a stain-filled core.


A novel ganglioside expressed by mouse hematopoietic cell lines.

Mouse progenitor T cell-derived cell lines were established by fusion of cells of hematopoietic organs such as bone marrow and fetal liver with T lymphoma (BW5147) to determine their characteristic cell-surface components. The hybridomas with the phenotype of Thy-1+, CD3-, CD4-, CD8- and expression of T cell receptor gene mRNA (BM216 and FL339) were selected for progenitor T cell-derived cell lines, and their ganglioside compositions were studied. A ganglioside component with a mobility slightly faster than that of bovine brain GD1a on high-performance thin-layer chromatography was found in the cell extracts of these cell lines as one of the most abundant components and was absent in the extract of the parental cell line (BW5147). The structure of the ganglioside was determined to be: NeuAc alpha-Gal beta-Gal beta-Gal alpha-Gal beta-Glc beta-ceramide. Gangliosides with such a sequence have never been found before, suggesting the possibility that the ganglioside is expressed as a surface marker of the cells in hematopoietic organs committed to a specific cell lineage, presumably to T cell lineage. cells in hematopoietic organs committed to a specific


Determination of the 1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide- induced cross-link between the beta and epsilon subunits of Escherichia coli F1-ATPase.

The zero-length cross-link between the inhibitory epsilon subunit and one of three catalytic beta subunits of Escherichia coli F1-ATPase (alpha 3 beta 3 gamma delta epsilon), induced by a water-soluble carbodiimide, 1-ethyl-3-[(3-dimethylamino) propyl]-carbodiimide (EDC), has been determined at the amino acid level. Lability of cross-linked beta-epsilon to base suggested an ester cross-link rather than the expected amide. A 10-kDa cross-linked CNBr fragment derived from beta-epsilon was identified by electrophoresis on high percentage polyacrylamide gels. Sequence analysis of this peptide revealed the constituent peptides to be Asp-380 to Met-431 of beta and Glu-96 to Met-138 of epsilon. Glu-381 of beta was absent from cycle 2 indicating that it was one of the cross-linked residues, but no potential cross-linked residue in epsilon was identified in this analysis. A form of epsilon containing a methionine residue in place of Val-112 (epsilon V112M) was produced by site-directed mutagenesis. epsilon V112M was incorporated into F1-ATPase which was then cross-linked with EDC. An 8-kDa cross-linked CNBr fragment of beta-epsilon V112M was shown to contain the peptide of epsilon between residues Glu-96 and Met-112 and the peptide of beta between residues Asp-380 and Met-431. Again residue Glu-381 of beta was notably reduced and no missing residue from the epsilon peptide could be identified, but the peptide sequence limited the possible choices to Ser-106, Ser-107, or Ser-108. Furthermore, an epsilon mutant in which Ser-108 was replaced by cysteine could no longer be cross-linked to a beta subunit in F1-ATPase by EDC. Both mutant forms of epsilon supported growth of an uncC-deficient E. coli strain and inhibited F1-ATPase. These results indicate that the EDC-induced cross-link between the beta and epsilon subunits of F1-ATPase is an ester linkage between beta-Glu-381 and, likely, epsilon-Ser-108. As these residues must be located immediately adjacent to one another in F1-ATPase, our results define a site of subunit-subunit contact between beta and epsilon.


Ankyrin regulation: an alternatively spliced segment of the regulatory domain functions as an intramolecular modulator.

This study of two forms of ankyrin (protein 2.1 and 2.2) from human erythrocytes has revealed a role for alternate exon usage at the level of regulation of protein interactions. The smaller form of ankyrin (protein 2.2), which lacks a portion of the regulatory domain due to alternative splicing of pre-mRNA, exhibits increased affinity for the cytoplasmic domain of the anion exchanger, spectrin, and tubulin. Direct evidence that at least one of these associations is modulated by the alternatively spliced segment of the regulatory domain is provided by experiments utilizing a polypeptide that is comprised of residues 1513-1674 corresponding to the portion of the regulatory domain missing from protein 2.2. Addition of this regulatory domain polypeptide to binding assays reversed the increase in affinity of protein 2.2 for the anion exchanger. The inhibitory activity of the regulatory domain polypeptide in these assays is accompanied by a direct interaction with a site that is available on the smaller form of ankyrin and is distinct from the binding site for the anion exchanger. These results support the idea that the alternatively spliced segment within the regulatory domain of erythrocyte ankyrin performs a repressor function and acts through an allosteric mechanism involving interaction(s) at a site separate from the binding site for the anion exchanger.


Kinetic characterization of the substrate reaction between a complex of antithrombin with a synthetic reactive-bond loop tetradecapeptide and four target proteinases of the inhibitor.

A tetradecapeptide corresponding to the P1 to P14 region of the reactive-bond loop of antithrombin (AT) binds to the inhibitor, presumably as a middle strand of the A beta-sheet, thereby converting AT from an inhibitor to a substrate of thrombin (Björk, I., Ylinenjärvi, K., Olson, S.T., and Bock, P. E. (1992) J. Biol. Chem. 267, 1976-1982). The kinetics of cleavage of the AT reactive bond in the AT-peptide complex by four target proteinases were quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. The kcat/Km values for thrombin and factor IXa were indistinguishable from the second-order rate constants for AT inhibition of these enzymes, whereas the values for factor Xa and plasmin were 10-17-fold higher than the inhibition rate constants. Heparin with high affinity for AT accelerated the substrate reaction with thrombin to an extent consistent with the reduced heparin affinity of the AT-peptide complex. These data show that blocking by the peptide of the putative intramolecular association of the P1 to P14 region of the AT reactive-bond loop with the A beta-sheet leads to AT functioning as a substrate of its target enzymes with an efficiency that equals or exceeds the action of uncomplexed AT as an inhibitor and with the expected heparin activation. The results thus suggest that a substrate-like attack of the proteinase on the inhibitor reactive bond in an exposed loop initiates the inhibition reaction. This attack presumably induces the subsequent trapping of the enzyme by the insertion of the reactive-bond loop into the A beta-sheet.


Effects of decreased cytosine content on rho interaction with the rho-dependent terminator trp t' in Escherichia coli.

We have introduced multiple cytosine-to-uracil mutations in the rho-dependent transcription terminator trp t' and have characterized a subset of the resulting mutant derivatives in vitro for termination efficiency, affinity for rho, and stimulation of rho-ATPase activity. No specific cytosine residue appears to be required for termination, and at least 13 of the 28 cytosine residues in the 104 nucleotide trp t' region can be mutated with little effect on termination efficiency. One derivative with 11 mutations is significantly less efficient than other derivatives with a similar number of mutations, implying that the pattern of alterations, as well as the number, can affect termination efficiency. Derivatives with 20 and 28 cytosines mutated are non-functional in termination, consistent with the idea that cytosines are required for rho-dependent termination. Our results are inconsistent, however, with the hypothesis that the termination efficiency is directly related to the cytosine/guanine ratio in the nascent transcript. The results support the idea that neither RNA binding affinity nor ATPase activation per se are accurate predictors of rho-dependent termination efficiency.


Pyruvic acid is attached through its central carbon atom to the amino terminus of the recombinant DNA-derived DNA-binding protein Ner of bacteriophage Mu.

Ner protein of bacteriophage Mu, produced by recombinant DNA techniques in Escherichia coli, has been found to possess a molecule of pyruvic acid attached covalently through carbon-2 to the amino-terminal cysteine residue. The intact protein and the amino-terminal chymotryptic peptide were found by mass spectrometry to be 70 mass units heavier than expected. The modified peptide was unstable under mildly acid or mildly basic conditions. Two-dimensional nuclear magnetic resonance spectroscopy of the modified and unmodified forms of the amino-terminal chymotryptic peptide was consistent with the presence of pyruvate linked through carbon-2 to the amino-terminal Cys residue. Treatment of the modified form with 2,4-dinitrophenylhydrazine in acid medium led to the expected hydrazone of pyruvic acid, which was identified by high pressure liquid chromatography. Of the two proteins known to be modified by pyruvate through its central carbon (the other being human adult hemoglobin, in which the modified form represents only a very minor fraction), Ner is the first protein found to be modified quantitatively. Given the instability of the modification, it may be more prevalent than recognized hitherto. Incubation with 2,4-dinitrophenylhydrazine may offer a useful means of detecting the presence of pyruvate linked to proteins in this way.


Identification and characterization of columbid annexin Icp37. Insights into the evolution of annexin I phosphorylation sites.

Annexin I (AnxI) contains phosphorylation sites in its "hinge region" that have been implicated in the regulation of cell growth and/or differentiation. A pigeon (Columba livia) isoform of this protein, annexin Icp35 (cp35), has a very similar amino acid sequence overall but an unrelated sequence that lacks phosphorylation sites in the hinge region. We now report the identification and characterization of annexin Icp37 (cp37) from pigeon. Genomic cloning and Southern blot analysis demonstrated that cp37 and cp35 were encoded by separated genes. Prolactin induced the expression of cp35 mRNA but not cp37. The amino acid sequence of cp37 was deduced from a cDNA clone and found to share 93 and 75% sequence identity with cp35 and human AnxI, respectively. The amino acid sequence of cp37 bore similarities to both AnxI and cp35 in the critical hinge region. Like AnxI, cp37 contained consensus phosphorylation sites in its amino acid sequence and was phosphorylated on tyrosine by the EGF receptor/kinase and on serine by protein kinase C in vitro. Despite the functional similarities between cp37 and AnxI, the nucleotide sequence that encoded the hinge region of cp37 was very similar to the analogous region of cp35, but different from that of AnxI. We propose that certain features shared by cp37 and AnxI are the products of convergent evolution. The fact that evolution independently selected for two annexin I-like genes (cp37 and anxI) encoding analogous phosphorylation sites is strong evidence that phosphorylation is important for the regulation of the biological activity of these proteins.


The asparagine-linked oligosaccharides on tissue factor pathway inhibitor terminate with SO4-4GalNAc beta 1, 4GlcNAc beta 1,2 Mana alpha.

Tissue factor pathway inhibitor (TFPI) produced by endothelial cells contains sulfated Asn-linked oligosaccharides. We have determined that greater than 70% of the oligosaccharides on recombinant TFPI expressed in 293 cells terminate with the sequence SO4-4GalNAc beta 1, 4GlcNAc beta 1, 2Man alpha. Oligosaccharides terminating with this sequence have previously been described on lutropin, thyrotropin, and pro-opiomelanocortin: glycoproteins synthesized in the anterior pituitary. A GalNAc-transferase that recognizes the tripeptide motif Pro-Xaa-Arg/Lys 6-9 residues N-terminal to Asn glycosylation sites accounts for the specific addition of GalNAc to the oligosaccharide acceptor on these glycoproteins, whereas a GalNAc beta 1,4GlcNAc beta 1, 2Man alpha-4-sulfotransferase accounts for the addition of sulfate. The sulfated oligosaccharides present on these hormones are responsible for their rapid clearance from plasma by a receptor in hepatic reticuloendothelial cells. GalNAc- and sulfotransferase activities with the same properties as those expressed in the pituitary are detected at high levels in 293 cells and at lower levels in endothelial cells. Chinese hamster ovary (CHO) cells do not contain detectable levels of either transferase and rTFPI expressed in CHO cells does not contain sulfated Asn-linked oligosaccharides. TFPI contains the sequence Pro-Phe-Lys, 9 residues N-terminal to the glycosylation site at position 228; this tripeptide may act as the recognition sequence for the GalNAc-transferase. rTFPI produced by 293 cells, but not that produced by CHO cells, is bound by the receptor on hepatic reticuloendothelial cells suggesting the sulfated structures play a role in the biologic behavior of TFPI.


Mutagenesis of the C-terminal nucleotide-binding site of an anion-translocating ATPase.

An oxyanion-translocating ATPase encoded by a bacterial plasmid confers resistance to antiomonials and arsenicals in Escherichia coli by extrusion of the toxic oxyanions from the cytosol. The anion pump is composed of two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein is an oxyanion-stimulated ATPase with two nucleotide-binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein. The ArsA protein can be labeled with [alpha-32P]ATP by a UV-catalyzed reaction. Previously reported mutations in the N-terminal site abolish photoadduct formation. Using site-directed mutagenesis the glycine-rich region of the C-terminal putative nucleotide-binding sequence was altered. Three C-terminal site mutant proteins (GR337, KE340, KN340) were analyzed, as well as one additional N-terminal mutant protein (KE21). Strains bearing the mutated plasmids were arsenite sensitive to varying degrees. The purified ArsA protein from mutant KE340 retained approximately 20% of the wild type oxyanion-stimulated ATPase activity, while the purified proteins from the other mutants were catalytically inactive. The KE21 mutation in the N-terminal nucleotide-binding site eliminated photoadduct formation with [alpha-32P] ATP, while the purified proteins with mutations in the C-terminal site retained the ability to form a photoadduct. Each mutant protein was capable of forming a membrane-bound complex in arsB expressing strains. These results suggest first that both sites are required for resistance and ATPase activity, and second that the conserved lysyl residue in the glycine-rich loop of the C-terminal nucleotide-binding site is not essential for catalytic activity.


Plasmin and the regulation of tissue-type plasminogen activator biosynthesis in human endothelial cells.

Plasmin inhibited the biosynthesis of tissue-type plasminogen activator (tPA) antigen by human umbilical vein endothelial cells (HUVEC) in a dose-dependent manner. The amount of tPA antigen found in the 24-h conditioned medium of cells treated with 100 nM plasmin for 1 h was 20-30% of that in the control group. However, in contrast to tPA, such treatment led to a 3-fold increase in plasminogen activator inhibitor (PAI) activity, whereas the amount of PAI type 1 antigen was unchanged. The effects of plasmin on HUVEC were binding- and catalytic activity-dependent and were specifically blocked by epsilon-aminocaproic acid. Microplasmin, which has no kringle domains, was less effective in reducing tPA antigen biosynthesis or enhancing PAI activity in HUVEC. Kringle domains of plasmin affected neither tPA antigen nor PAI activity of the cells. Other proteases including chymotrypsin, trypsin, and collagenase at comparable concentrations did not have a significant effect on the biosynthesis of tPA antigen or PAI activity of HUVEC. Thrombin stimulated the biosynthesis of tPA and PAI-1 antigens by HUVEC. Thrombin also stimulated an increase in the protein kinase activity in HUVEC, whereas plasmin inhibited the protein kinase activity of the cells. It is possible that plasmin regulates the biosynthesis of tPA in HUVEC through the signal transduction pathway involving protein kinase.


Functional consequences of alterations to Glu309, Glu771, and Asp800 in the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis was used to replace Glu309, Glu771, and Asp800 in the Ca(2+)-ATPase of rabbit fast twitch muscle sarcoplasmic reticulum with their corresponding amides. These residues are predicted to lie in the transmembrane domain and have been suggested as oxygen ligands for Ca2+ binding at high affinity sites (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). The Glu309----Gln and Asp800----Asn mutants were unable to form a phosphoenzyme from ATP at the Ca2+ concentrations examined (up to 12.5 mM), whereas the Glu771----Gln mutant phosphorylated from ATP at 2.5 mM Ca2+. In all three mutants, Ca2+ at concentrations well below 12.5 mM prevented or inhibited phosphorylation with Pi, suggesting that at least one calcium-binding site was functioning in each mutant. In the mutants Glu309----Gln and Glu771----Gln, the ADP-insensitive phosphoenzyme intermediate was unusually stable, as indicated by a very low rate of dephosphorylation observed in kinetic experiments and by an increased apparent affinity for Pi determined in equilibrium phosphorylation experiments. These data indicate a central role of Glu309 and Glu771 in the energy-transducing conformational changes and/or in the activation of phosphoenzyme hydrolysis.


Host factor requirements for processive antitermination of transcription and suppression of pausing by the N protein of bacteriophage lambda.

The N protein of phage lambda prevents termination of transcription by Escherichia coli RNA polymerase at Rho-dependent and -independent terminators in the lambda early operons. The modification of RNA polymerase by N requires an N-utilization (nut) site, present in each lambda early operon, and involves the E. coli factors NusA, NusB, NusG, and ribosomal protein S10. We show that, in the presence of NusA, N inhibits pausing by RNA polymerase and Rho-dependent termination in vitro at three sites in the lambda terminator tR1 which are located less than 100 base pairs downstream from nutR. NusA is also sufficient for partial antitermination at sites located farther downstream from nutL and nutR if there is a high concentration of N in the reaction. At low concentrations of N, the additional factors NusB, S10, and NusG are essential for antitermination at distal sites. In these conditions, the presence of NusA, NusB, S10, and NusG in the reaction enables N-modified RNA polymerase to elongate efficiently and processively through Rho-dependent and -independent terminators over distances as great as 7 kilobases downstream from the lambda nut sites. This substantial processivity of antitermination in vitro also occurs in vivo and probably reflects the stable association of N, NusA, NusB, S10, and NusG with RNA polymerase and nut site RNA in elongation complexes transcribing the lambda chromosome.


Structure of the rat plasma membrane Ca(2+)-ATPase isoform 3 gene and characterization of alternative splicing and transcription products. Skeletal muscle-specific splicing results in a plasma membrane Ca(2+)-ATPase with a novel calmodulin-binding domain.

We have isolated the rat gene encoding isoform 3 of the plasma membrane Ca(2+)-ATPase (PMCA3) and have determined its exon/intron organization. The PMCA3 gene contains 24 exons and spans approximately 70 kilobases. In addition, we have analyzed the splicing and polyadenylation patterns leading to the production of an alternative 4.5-kilobase (PMCA3) skeletal muscle mRNA that differs from the previously characterized 7.5-kilobase brain mRNA (Greeb, J., and Shull, G. E. (1989) J. Biol. Chem. 264, 18569-18576). cDNA cloning, Northern blot hybridization, and polymerase chain reaction analyses of the 4.5-kilobase mRNA demonstrate (i) the inclusion of a novel 68-nucleotide exon (exon 22) that is specific for skeletal muscle and significantly alters the calmodulin-binding domain and (ii) the utilization of an alternative polyadenylation site following exon 23 which eliminates the last coding exon (exon 24) and 3'-untranslated sequence of the 7.5-kilobase mRNA. We have also identified a 42-nucleotide exon (exon 8) that is included in the skeletal muscle PMCA3 mRNAs, but may be either included or excluded in the brain mRNAs. Exon 8 is inserted immediately before the sequence encoding a putative phospholipid binding domain and thus may alter regulatory interactions of the enzyme with acidic phospholipids.


Lack of scientific evidence for the treatment of lateral epicondylitis of the elbow. An attempted meta-analysis.

We have reviewed 185 articles published since 1966 to assess the scientific evidence for methods of treatment for lateral epicondylitis of the elbow. Of the 185 articles, 78 discussed treatment, but since the natural history of the syndrome is uncertain we considered only those series with concurrent control groups. Only 18 of these were randomised and controlled studies. We then graded these papers for scientific validity, using the methods of Chalmers et al (1981). The mean score of the 18 articles was only 33%, with a range from 6% to 73%. A minimum of 70% is required for a valid clinical trial, and we therefore concluded that there was insufficient scientific evidence to support any of the current methods of treatment. There were too many methodological differences to allow a quantitative meta-analysis, but our qualitative review established the importance of the natural evolution of the syndrome and of the placebo effect of all treatments. Properly designed, controlled trials are needed.


Annular tears and disc degeneration in the lumbar spine. A post-mortem study of 135 discs.

We studied 135 lumbar discs from 27 spines removed post-mortem from subjects of an average age of 31.5 years. Defects of the annulus fibrosus were classified as peripheral, circumferential or radiating; the nucleus pulposus as normal, moderately or severely degenerate. Peripheral tears were more frequent in the anterior annulus, except in the L5-S1 disc. Circumferential tears were equally distributed between the anterior and the posterior annulus. Almost all the radiating tears were in the posterior annulus, and closely related to the presence of severe nuclear degeneration. Histology suggested that peripheral tears were due to trauma rather than biochemical degradation, and that they developed independently of nuclear degeneration. The association of peripheral annular lesions with low back pain is uncertain but our study suggests that they may have a role in the pathogenesis of discogenic pain.


Apical endosomes isolated from kidney collecting duct principal cells lack subunits of the proton pumping ATPase.

Endocytic vesicles that are involved in the vasopressin-stimulated recycling of water channels to and from the apical membrane of kidney collecting duct principal cells were isolated from rat renal papilla by differential and Percoll density gradient centrifugation. Fluorescence quenching measurements showed that the isolated vesicles maintained a high, HgCl2-sensitive water permeability, consistent with the presence of vasopressin-sensitive water channels. They did not, however, exhibit ATP-dependent luminal acidification, nor any N-ethylmaleimide-sensitive ATPase activity, properties that are characteristic of most acidic endosomal compartments. Western blotting with specific antibodies showed that the 31- and 70-kD cytoplasmically oriented subunits of the vacuolar proton pump were not detectable in these apical endosomes from the papilla, whereas they were present in endosomes prepared in parallel from the cortex. In contrast, the 56-kD subunit of the proton pump was abundant in papillary endosomes, and was localized at the apical pole of principal cells by immunocytochemistry. Finally, an antibody that recognizes the 16-kD transmembrane subunit of oat tonoplast ATPase cross-reacted with a distinct 16-kD band in cortical endosomes, but no 16-kD band was detectable in endosomes from the papilla. This antibody also recognized a 16-kD band in affinity-purified H+ ATPase preparations from bovine kidney medulla. Therefore, early endosomes derived from the apical plasma membrane of collecting duct principal cells fail to acidify because they lack functionally important subunits of a vacuolar-type proton pumping ATPase, including the 16-kD transmembrane domain that serves as the proton-conducting channel, and the 70-kD cytoplasmic subunit that contains the ATPase catalytic site. This specialized, non-acidic early endosomal compartment appears to be involved primarily in the hormonally induced recycling of water channels to and from the apical plasma membrane of vasopressin-sensitive cells in the kidney collecting duct.


Tripeptide RGD-dependent adhesion of articular chondrocytes to synovial fibroblasts.

Cell-cell interactions play an important role in the development of cartilage. Heterologous and homologous cell-cell interactions are critical for chondrogenic differentiation during development. Cell-cell interactions in the formation of fracture callus and cartilage neoplasia also invoke the process of cartilage differentiation. We have investigated cell-cell interactions between articular chondrocytes and synovial fibroblasts and show that there was enhanced binding between these two cell types compared to background binding of the labelled cells to the tissue culture plastic surface. The binding of chondrocytes to fibroblasts was temperature- and calcium-dependent, suggesting ligand-integrin involvement. The peptide, GRGDSP, which competes with the ligand-integrin through the tripeptide RGD (arginine-glycine-aspartic acid), almost completely inhibited chondrocyte attachment to synovial fibroblasts. The control peptide, GRGESP, had no inhibitory effect on binding. Antibodies to fibronectin (Fn) inhibited chondrocyte attachment by about 50%. Monoclonal antibodies to the alpha and beta chains of the fibronectin receptor (FnR) interfered with the attachment of chondrocytes to synovial fibroblasts. A combination of antibodies to Fn and to FnR did not completely abrogate chondrocyte binding, suggesting that other ligand-receptors were involved in the adhesion process. Chondrocytes and fibroblasts were shown to express membrane-associated Fn and FnR, by immunofluorescence. The alpha and beta chains of FnR, migrating at 110 and 140 kDa, respectively, could be immunoprecipitated from [35S]methionine-labelled synovial fibroblasts and chondrocytes. Northern blots showed the presence of mRNA for the alpha and beta chains of fibronectin receptors in fibroblasts and chondrocytes. Changes in cell shape were observed in chondrocytes on attachment to fibroblasts, i.e. the chondrocytes appeared fibroblast-like, suggesting that the chondrocytes had dedifferentiated. These studies suggest that chondrocytes specifically bind to synovial fibroblasts through RGD-dependent receptors. beta 1 Integrins are involved in this adhesion process and these heterlogous cell interactions appear to have a negative influence on chondrogenic differentiation.


Lymphocyte adhesion to high endothelium is mediated by two beta 1 integrin receptors for fibronectin, alpha 4 beta 1 and alpha 5 beta 1.

Using a rat model we have previously proposed a role for fibronectin as an adhesive ligand on high endothelial cells (HEC) for recirculating lymphocytes. Lymphocyte adhesion to high endothelial cells was blocked by CS1 peptide (from the type III connecting segment of fibronectin) and RGD-containing peptides using two different in vitro assays of lymphocyte-HEC recognition, the frozen section assay and cultured HEC. In order to study the receptors utilised by lymphocytes to bind to HEC we have developed a xenogeneic model in which the adhesion of human lymphocytes to HEC cultured from rat lymph nodes is measured. The basic properties of lymphocyte-HEC interaction were retained using human lymphocytes. CS1 peptide and RGD-containing peptides gave similar profiles of inhibition of lymphocyte adhesion as found previously using rat cells. FACS analysis showed that the majority of peripheral blood lymphocytes expressed two beta 1 integrin receptors, alpha 4 beta 1 and alpha 5 beta 1, which are known to recognise distinct adhesion domains in fibronectin. A subpopulation of lymphocytes also expressed alpha 3 beta 1, which, like alpha 5 beta 1, has been reported to be an RGD-dependent adhesion receptor for the central cell binding domain of fibronectin. Anti-alpha 4 and anti-alpha 5 subunit monoclonal antibodies maximally inhibited adhesion to HEC by 60% and 65%, respectively. Monoclonal antibodies to the common beta 1 subunit gave slightly higher inhibition at 70%. These results suggest that lymphocytes employ one or both of two different beta 1 integrin fibronectin receptors to bind to HEC. The simultaneous or alternate engagement of two fibronectin receptors on the lymphocyte surface by immobilised fibronectin in the endothelial layer may contribute to the stabilisation of adhesive contacts or to the subsequent transendothelial migration of lymphocytes. In contrast to lymphocytes, peripheral blood neutrophils did not express any members of the beta 1 integrin family. The selective expression of beta 1 integrins by lymphocytes and not neutrophils contrasted with the widespread distribution of the other homing-associated adhesion molecules, LECAM-1, CD44 and LFA-1, on these two cell types. It is thus possible that the selective expression of beta 1 integrins regulates the constitutive migration of lymphocytes but not neutrophils into organised lymphoid tissues.


Albumin impregnated vascular grafts: albumin resorption and tissue reactions.

This study aimed to determine the kinetics of albumin resorption from and the healing of two types of albumin impregnated Vasculour II (Bard Cardiovascular) Dacron grafts (ACG-A and ACG-B) using whole blood preclotted Vasculour II Dacron grafts (without albumin) as controls (PCC). Prostheses measuring 4 mm ID x 50 mm length were implanted in the aortoiliac position in 24 dogs (ACG-A n = 12, ACG-B n = 24, PCC n = 12) and explanted after 1, 2 4, and 6 months. Platelet count, platelet aggregometry to 10(-5) M ADP, prothrombin time (PT), and partial thromboplastin time (PTT) were determined preoperatively and at explantation. Sections of the explanted grafts were assayed for human albumin by immunohistochemical techniques utilizing a rabbit polyclonal mono-specific antibody for human albumin followed by the addition of a biotinylated goat anti-rabbit IgG. Immunoperoxidase staining was then performed using Avidin D horse-radish peroxidase. Histology of the grafts (light microscopy, scanning electron microscopy, and transmission electron microscopy) as well as percent thrombus free surface area (TFSA) by computerized planimetry were also determined. Seven of 48 grafts were occluded (85.4% patency) with no difference among the three groups. Platelet aggregometry was not predictive of graft patency. No change in PT or PTT occurred nor was there any difference among the three groups. Retained albumin was detected in every one-month explant but not beyond that time, with the sensitivity for detecting human albumin in this assay being 20 mg albumin per gram of Dacron. All ACG explants at one month revealed inner capsular fibrin coagula not present in PCC specimens.(ABSTRACT TRUNCATED AT 250 WORDS)


The influence of competitive flow on graft patency.

To evaluate the effects of competitive flow, bilateral aorto-iliac ePTFE grafts, 10 cm in length and with an internal diameter of 6 mm, were placed in ten mongrel dogs. On one side the external iliac artery was ligated (the control side); on the opposite side (the experimental side) flow continued through the native aorto-iliac system, as well as the graft, to allow a situation of competitive flow. Flow measurements showed the experimental graft carried 38.6% of the total blood flow going to the ipsilateral external iliac artery. After 30 days the grafts were exposed and patency and flow were evaluated by Doppler ultrasound. The control side showed 60% patency; all of the grafts on the experimental side were occluded. In addition, complete endothelial healing was observed at the occluded ostia of all experimental grafts. We conclude that competitive flow does influence graft patency and that graft thrombosis appears to be a relatively early phenomenon, as evidenced by the endothelial healing which occurred.


Therapeutic dissection after successful coronary balloon angioplasty: no influence on restenosis or on clinical outcome in 693 patients. The MERCATOR Study Group (Multicenter European Research Trial with Cilazapril after Angioplasty to prevent Transluminal Coronary Obstruction and Restenosis).

The objective of this study was to examine the relation between an angiographically visible coronary dissection immediately after successful coronary balloon angioplasty and a subsequent restenosis and long-term clinical outcome. The study population comprised all 693 patients who participated in the MERCATOR trial (randomized, double-blind, placebo-controlled restenosis prevention trial of cilazapril, 5 mg two times a day). Cineangiographic films were processed and analyzed at a central angiographic core laboratory, without knowledge of clinical data, with use of an automated interpolated edge detection technique. Dissection was judged according to the National Heart, Lung, and Blood Institute classification. Angiographic follow-up was obtained in 94% of patients with 778 lesions. Two approaches were used to assess the restenosis phenomenon: 1) categoric, using the traditional cutoff criterion of greater than 50% diameter stenosis at follow-up, and 2) continuous, defined as absolute change in minimal lumen diameter (mm) between the postcoronary angioplasty and follow-up, adjusted for the vessel size (relative loss). Clinical outcome was ranked according to the most serious adverse clinical event per patient during the 6-month follow-up period, ranging from death, nonfatal myocardial infarction, coronary revascularization and recurrent angina requiring medical therapy to none of these. Dissection was present in 247 (32%) of the 778 dilated lesions. The restenosis rate was 29% in lesions with and 30% in lesions without dissection (relative risk 0.97; 95% confidence interval 0.77 to 1.23). The relative loss in both groups was 0.10 (mean difference 0; 95% confidence interval -0.03 to 0.03). Clinical outcome ranged from death in 4 patients (0.9%) without dissection and 1 patient (0.4%) with dissection; nonfatal myocardial infarction in 4 (0.9%) without and 8 (3.2%) with dissection; coronary revascularization in 73 (16.6%) without and 32 (12.7%) with dissection; recurrent angina requiring medical therapy in 88 (20%) without and 47 (18.7%) with dissection to no serious adverse event in 272 (61.7%) without and 114 (65.1%) with dissection. These data indicate that a successfully dilated coronary lesion with an angiographically visible dissection is no more likely to develop restenosis, and is not associated with a worse clinical outcome, at 6-month follow-up than is a dilated lesion without visible dissection on the post-balloon angioplasty angiogram.


Hemodynamic and volume correlates of left ventricular diastolic relaxation and filling in patients with aortic stenosis.

The aim of the present study was to evaluate the hemodynamic and volume correlates of early diastolic filling and isovolumetric relaxation in patients with aortic stenosis. Left ventricular diastolic relaxation and filling have been found to be heterogeneous in patients with aortic stenosis. Potential mechanisms underlying this heterogeneity include individual differences in the severity of muscle hypertrophy or systolic dysfunction, or both, in the presence and severity of mitral regurgitation and in the level of left atrial pressure. Right (fluid-filled) and left (high fidelity micromanometer) ventricular pressures, left ventricular volumes (contrast angiography) and transmitral inflow dynamics (Doppler echocardiography) were measured in 17 patients with isolated severe aortic stenosis (valve area less than 0.75 cm2). Measurements included left ventricular end-diastolic and end-systolic volumes, left ventricular ejection fraction, peak positive and negative first derivative of left ventricular pressure (dP/dt), the time constant of isovolumetric relaxation (tau), left ventricular end-diastolic pressure, left ventricular mass, left ventricular end-systolic stress, mean capillary wedge pressure and peak early (E) and late (A) transmitral filling velocities. Patients were subclassified according to left ventricular ejection performance at rest and mean capillary wedge pressure. Patients with normal ejection performance and normal mean capillary wedge pressure had a normal rate of isovolumetric left ventricular pressure decay and an abnormal diastolic filling pattern, with diastolic filling occurring primarily during atrial systole. In contrast, in patients with systolic dysfunction and elevated mean capillary wedge pressure, isovolumetric pressure decay was prolonged and diastolic filling occurred essentially during the rapid filling period, with reduced atrial contribution to left ventricular filling and a short isovolumetric relaxation period. Stepwise multiple linear regression analysis identified two variables as independent predictors of transmitral velocity profile and three variables independently predictive of the rate of left ventricular pressure decay. The single most important predictor of transmitral filling pattern was the pulmonary capillary wedge pressure (p less than 0.0001), followed by the left ventricular peak negative dP/dt (p = 0.002). The single most powerful predictor of the rate of reduction in left ventricular pressure was left ventricular mass index (p less than 0.0001), followed by end-systolic volume index (p = 0.0002) and left ventricular peak negative dP/dt (p = 0.0029). In patients with aortic stenosis, left ventricular filling is essentially determined by left atrial pressure, whereas isovolumetric relaxation more closely depends on the severity of muscle hypertrophy and chamber dilation.


Left ventricular volume determined echocardiographically by assuming a constant left ventricular epicardial long-axis/short-axis dimension ratio throughout the cardiac cycle.

The purpose of this study was to develop and test a simplified echocardiographic method to calculate left ventricular volume. This method was based on the assumption that the ratio of the left ventricular epicardial long-axis dimension to the epicardial short-axis dimension was constant throughout the cardiac cycle. With use of this constant ratio, the method developed to calculate left ventricular volume at a given point in the cardiac cycle required the left ventricular endocardial long-axis dimension to be measured at only one point in the cardiac cycle. Studies were performed in 13 normal dogs, 8 normal puppies, 9 normal pigs, 12 dogs with aortic stenosis, 13 dogs with acute mitral regurgitation, 12 dogs with chronic mitral regurgitation, 7 dogs that had undergone mitral valve replacement and 6 pigs that had had chronic supraventricular tachycardia. Animals with aortic stenosis developed left ventricular pressure overload hypertrophy with a 60% increase in left ventricular mass; chronic mitral regurgitation caused left ventricular volume overload hypertrophy with a 46% increase in left ventricular volume; supraventricular tachycardia caused a dilated cardiomyopathy with a 55% decrease in left ventricular ejection fraction. The left ventricular epicardial long-axis/short-axis dimension ratio remained constant throughout the cardiac cycle in each animal group. End-diastolic and end-systolic volumes calculated with the simplified echocardiographic method correlated closely with angiographically measured volumes; for end-diastolic volume, echocardiographic end-diastolic volume = 1.0 (angiographic end-diastolic volume) -1.8 ml, r = 0.96; for end-systolic volume, echocardiographic end-systolic volume = 0.98 (angiographic end-systolic volume) -0.7 ml, r = 0.95. Thus the left ventricular epicardial long-axis/short-axis dimension ratio was constant throughout the cardiac cycle in a variety of animal species and age groups and in the presence of cardiac diseases that significantly altered left ventricular geometry and function. The simplified echocardiographic method examined provided an accurate determination of left ventricular volumes.


Segmental systolic responses to brief ischemia and reperfusion in the hypertrophied canine left ventricle.

Left ventricular hypertrophy is associated with increased mortality, increased myocardial infarct size and an increased incidence of sudden death. Although reperfusion after ischemia has been shown to result in decreased infarct size and recovery of systolic thickening, it is unknown how left ventricular hypertrophy might influence recovery of regional systolic thickening after ischemia and reperfusion. We hypothesized that left ventricular hypertrophy might attenuate or abolish the functional response to reperfusion. Three groups of chronically instrumented, conscious dogs (dogs with left ventricular hypertrophy and hypertension; dogs with left ventricular hypertrophy and reduced blood pressure and a control group without hypertrophy and with normal blood pressure) underwent 15 min of ischemia and 24 h of reperfusion. Segmental systolic thickening was measured by sonomicrometers and myocardial segments were grouped by percent of control segmental systolic thickening retained at 15 min of ischemia (class 1 greater than or equal to 67%, class 2 from 0% to 66%, class 3 less than 0% control systolic thickening). The recovery of each class of segment was measured serially during reperfusion. Hemodynamic variables and regional myocardial blood flow were also measured. There were no differences among groups in recovery of segmental systolic thickening for class 1 segments. Systolic thickening in class 2 (hypokinetic) segments was significantly depressed (p less than 0.05 compared with control value) in the group with left ventricular hypertrophy and reduced blood pressure (but not in the group with hypertrophy and hypertension) during early reperfusion; systolic thickening in class 3 (dyskinetic) segments showed a similar trend in the group with hypertrophy and reduced pressure. Although left ventricular hypertrophy with hypertension did not attenuate the contractile response to reperfusion, hypertrophy with reduced blood pressure was associated with significantly greater depression of segmental systolic thickening early during reperfusion.


Prevalence of latex allergy in operating room nurses.

The twofold purpose of this study was to assess the prevalence of latex sensitivity in a large group of operating room nurses and to evaluate the relationship between questionnaire responses and skin tests. Of the total target population of 268 operating room nurses, 248 (93%) answered the questionnaire and 197 had skin prick tests to latex (1/10 wt/vol solution). Symptoms associated with glove wearing were acknowledged by 41.1% of nurses. Skin tests to latex were positive in 21 nurses (10.7%), 4.4 times more often in atopic nurses. Among nurses complaining of local symptoms, only 18.6% had positive skin tests. Itching of the hands during glove wearing correlated poorly with latex sensitivity, but correlation with local urticaria was better. Atopic nurses complaining of urticaria had latex allergy in 70% of cases. Thus latex allergy is common in nurses, especially atopic nurses. A questionnaire is unreliable in predicting latex sensitivity and must be supported by latex skin test. More data will be needed to assess the risk of anaphylactic perioperative reactions in operating room nurses.


Origin and fate of IgE-bearing lymphocytes. II. Gut-associated lymphoid tissue as sites of first appearance of IgE-bearing B lymphocytes and hapten-specific IgE antibody-forming cells in mice immunized with benzylpenicilloyl-keyhole limpet hemocyanin by various routes: relation to asialo GM1 ganglioside+ cells and IgE/CD23 immune complexes.

The organs in which B cells bearing membrane-bound IgE (sIgE+) and benzylpenicilloyl (BPO)-specific IgE antibody-forming cells (AFC) first appeared were determined in BALB/c mice given BPO-keyhole limpet hemocyanin (10 micrograms) in aluminum hydroxide by various routes (i.p, gavage, s.c., i.v., or i.m.). In mice immunized by the i.p. route, the numbers and location of sIgE+ B cells and asialo GM1 ganglioside (AsGm1+) cells, the location of IgE/CD23 immune complexes, and the numbers of BPO-specific IgE AFC in lymphoid organs were determined. With all routes of immunization, no sIgE+ B cells or BPO-specific IgE AFC were ever detected in any organ before day 8. On day 8, with the s.c., i.v., or i.m. routes, sIgE+ B cells and IgE AFC appeared exclusively in Peyer's patches (PP); with the i.p. or gavage routes, sIgE+ B cells simultaneously appeared in both PP and mesenteric lymph nodes, whereas IgE AFC appeared only in PP. In mice immunized by the i.p. route, IgE/CD23 immune complexes and strikingly increased numbers of AsGm1+ cells transiently appeared only in PP after the appearance and preceding the "disappearance" of the sIgE+ B cells and IgE AFC. The data suggest that specific IgE responses originate in gut-associated lymphoid tissue and appear later in spleen. The data also associate the appearance of IgE/CD23 immune complexes and AsGm1+ cells with the "disappearance" of sIgE+ B cells and IgE AFC from PP.


Microfilament assembly modulates phospholipase C-mediated signal transduction by the TCR/CD3 in murine T helper lymphocytes.

Cytoskeletal involvement in the response to TCR/CD3 ligation and in signal transduction was investigated in a murine Th cell type 2 clone. Cells coated with the hamster anti-CD3 mAb, 145-2C11 (2C11 mAb), and exposed to goat anti-hamster demonstrated an increase in polymerized actin as well as an increase in inositol phospholipid hydrolysis mediated by activation of phospholipase C. Pretreatment with cytochalasins (Cyt) (D or B), drugs that interact with cellular actin, prevented actin polymerization, and augmented the initial rate and total amount of inositol phosphates produced. Drugs modifying microtubule function were ineffective. The intracellular Ca2+ rise attributed to InsP3 and InsP4 generated in response to CD3 perturbation was augmented by CytD. CytD treatment did not affect inositol phosphate generation resulting from the stimulation of guanine nucleotide-binding proteins with aluminium tetrafluoride, indicating that the action of CytD was specific for receptor-mediated inositol phospholipids. CytD decreased the rate of anti-CD3-induced receptor internalization. These data suggest that the assembly of microfilaments plays a role in CD3 internalization and that a CytD-sensitive mechanism uncouples the TCR/CD3 complex from phospholipase C-mediated signaling.


CD45 expression by B cells. Expression of different CD45 isoforms by subpopulations of activated B cells.

To determine the effect of distinct activation stimuli on CD45 expression by B cells, we have examined the expression of CD45 molecules on murine B cells stimulated with LPS or the Th cell cytokine IL-5. Analysis of CD45 by flow cytometry revealed that unstimulated and stimulated B cells expressed homogeneous amounts of total CD45 but that stimulation with IL-5 resulted in a CD44hi, hyaluronate-adherent subpopulation of activated B cells that expressed a markedly altered pattern of expression of exon-specific CD45R or B220 determinants. The predominant CD45 immunoprecipitated from either unstimulated or LPS-stimulated B cells was of the high molecular mass form (approximately 220 kDa) usually associated with B cells. In contrast, the CD45 proteins immunoprecipitated from the hyaluronate-adherent subpopulation of IL-5-activated B cells were predominantly lower m.w. forms. PCR analysis of amplified CD45 cDNA also showed distinct expression profiles characteristic of each B cell population. The highest molecular size PCR product, corresponding to expression of all three variably expressed CD45 exons (A, B, and C) was prominent in resting B cells and in LPS-activated B cells but was selectively reduced in hyaluronate-adherent IL-5-activated B cells, where lower molecular size PCR products predominated, corresponding to expression of one or two of the variable exons. In contrast, LPS-activated B cells expressed reduced levels of these one- or two-exon forms. In addition, all B cell populations expressed a lower m.w. PCR product corresponding in size to the product expected when exons A, B, and C are spliced out of CD45 mRNA. Thus, analysis of alternative splicing of CD45 mRNA, as well as cell surface expression of CD45 provides a novel parameter for analysis of B cell activation by different stimuli.


Activation requirements for CD4+ T cells differing in CD45R expression.

Murine CD4+ T cells can be subdivided into naive and memory T cells based on surface phenotype, on recall response to Ag, and on differences in activation requirements. Furthermore, several studies have shown that two signals are required for CD4+ T cell activation; one signal is provided by occupancy of the TCR and the other signal is provided by the APC. In this report, analysis of naive and memory CD4 T cells, separated on the basis of CD45 isoform expression, has shown that their requirements for two signals differ. Activation of memory CD4 T cells to proliferate and secrete IL-2/IL-4 only required occupancy of the TCR complex, whereas activation of naive CD4 T cells required an APC-derived signal as well. Moreover, the signal induced by anti-CD3 antibodies differs from the signal provided by anti-V beta cross-linking of the TCR because both antibodies activate memory CD4 T cells but only anti-CD3 activates naive CD4 T cells. Together these data suggest that the consequence of stimulation through the TCR/CD3 signal complex differs between memory and naive CD4 T cells.


Activation requirements of newborn thymic gamma delta T cells.

We have analyzed the requirements for the induction of proliferative responses by thymic CD4-CD8- gamma delta T cells. Enriched populations of CD4-CD8- thymocytes from newborn mice, purified by negative selection with anti-CD4, anti-CD8, and anti-TCR alpha beta mAbs were found to contain approximately 20% gamma delta T cells that were p55IL-2R-. When these cells were cultured with a panel of lymphokines (IL-1, -2, -4, and -7), a small response was observed to some of the cytokines tested individually; however, combinations of certain lymphokines (IL-1 + 2, IL-1 + 7, and IL-2 + 7) were found to induce significant proliferation and the selective outgrowth (75-90%) of gamma delta T cells. These cells were IL-2R+, remained CD4-, yet expressed variable levels of CD8. A limited analysis with specific anti-V gamma and V delta mAb suggested that there had not been a selective expansion of preexisting V gamma 2, V gamma 3, or V delta 4 populations in response to the stimulatory lymphokine combinations. Thymic CD4-CD8- gamma delta T cells were unresponsive to stimulation with immobilized anti-pan gamma delta mAb alone. However, in the presence of immobilized anti-pan gamma delta mAb and IL-1, IL-2, or IL-7, but not IL-4, a vigorous proliferative response was observed. Phenotypic analysis showed that 80 to 95% of the proliferating cells were polyclonally expanded gamma delta T cells, expressed the p55IL-2R, and the majority remained CD4-CD8-. Blocking studies with anti-IL-2R mAb showed that stimulation with anti-pan gamma delta + IL-1, but not anti-pan gamma delta + IL-7 was dependent on endogenously produced IL-2. Collectively, these studies suggest that the activation requirements of newborn thymic gamma delta T cells differ markedly from alpha beta T cells in that gamma delta T cells 1) respond to combinations of cytokines in the absence of TCR cross-linking, 2) can respond to TCR cross-linking in the presence of exogenous cytokines, 3) but are unable to activate endogenous cytokine production solely in the presence of TCR cross-linking.


Functional comparison of Fc epsilon RI, Fc gamma RII, and Fc gamma RIII in mast cells.

The cellular responses initiated by cross-linking rodent Fc gamma RII-b1, Fc gamma RII-b2, Fc gamma RIII, and Fc epsilon RI in mast cells were compared. Individual murine Fc gamma R isoforms were transfected into rat basophilic leukemia cells and after cross-linking the FcR, changes in the phosphorylation of protein tyrosines, in the level of intracellular Ca2+, in the hydrolysis of phosphoinositides, and in the release of arachidonic acid metabolites and hexosaminidase were monitored. Cross-linking of Fc gamma RIII initiated all of these early and late biochemical functions, and although they were quantitatively somewhat smaller, the responses were qualitatively indistinguishable from those stimulated by the endogenous Fc epsilon RI. However, despite ample expression, neither Fc gamma RII-b1 nor Fc gamma RII-b2 stimulated these functions when cross-linked. The functional differences between Fc gamma RII and Fc gamma RIII were studied further by assessing the responses to cross-linking of the endogenous Fc gamma R (Fc gamma RII-b1, Fc gamma RII-b2, and Fc gamma RIII) on P815 mouse mastocytoma cells that had been transfected with normal or functionally defective Fc epsilon RI. Two types of mutant subunits had previously been observed to impair the activity of Fc epsilon RI: gamma-chains missing the cytoplasmic domain, and beta-chains missing the COOH-terminal cytoplasmic domain. In both types of transfectants the functional inhibition of the endogenous Fc gamma R paralleled that of the transfected Fc epsilon RI. These results are consistent with the gamma subunit being associated with the functions of Fc gamma RIII as well as of Fc epsilon RI. The functional results also complement the recently reported evidence that Fc gamma RIII can interact with Fc epsilon RI beta-subunits (J. Exp. Med. 175:447, 1992).


Complex inositol polyphosphate response induced by co-cross-linking of CD4 and Fc gamma receptors in the human monocytoid cell line U937.

The cell-surface Ag CD4, which is characteristic for Th lymphocytes, can also be found with a lower density on monocytes/macrophages. Co-cross-linking of CD4 and Fc gamma R by an anti-CD4 mAb (MAX.16H5) and by excess of goat anti-mouse Ig induced a biphasic increase of the free cytosolic Ca(2+)-concentration ([Ca2+]i) in the human monocytoid cell line U937 as measured by FURA-2 fluorescence. A rapid rise from 100 to 150 nM [Ca2+]i to 750 to 900 nM within 1 min was followed by a decline to about 200 to 300 nM within the next 2 to 3 min. This kinetic is characteristic also for blood monocytes and differs significantly from CD4-mediated Ca(2+)-mobilization in T lymphocytes. The rise in [Ca2+]i in U937 cells was not observed when F(ab)2 fragments of MAX.16H5 and F(ab)2 fragments of the cross-linker were used indicating the involvement of Fc gamma R. Time course analysis using HPLC and a recently developed post-column dye system for mass analysis revealed a complex inositol polyphosphate response with rapid increases not only in inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, but also in D/L-inositol 1,4,5,6-tetrakisphosphate, inositol 1,3,4,5,6-pentakisphosphate, and inositol hexakisphosphate after co-cross-linking of CD4 and Fc gamma R. In conclusion, co-cross-linking of CD4 and Fc gamma R, which may occur in vivo during HIV infection or treatment with therapeutic anti-CD4 antibodies, appears to be a strong activation mechanism for the inositol polyphosphate/Ca2+ signal transduction pathway in U937 cells.


Heterogeneous mechanisms of human cytotoxic T lymphocyte generation. I. Differential helper cell requirement for the generation of cytotoxic effector cells from CD8+ precursor subpopulations.

The subpopulations of CD8+ T cells defined by CD45RA Ag expression have been hypothesized to represent cells varying in their relative maturation along a common, activation-dependent differentiation pathway. Previous studies have shown that both the CD8+CD45RA+ and CD8+CD45RA- subsets contain precursor cells capable of developing into alloreactive CTL. In the current study, we have examined the mechanisms involved in the generation of CTL effector cells from these two CD8+ subsets. Purified CD8+CD45RA+ or CD8+CD45RA- cells were stimulated with allogeneic non-T cells, either alone or in the presence of CD4+ Th cells. Although the generation of CTL from CD8+CD45RA- precursor cells consistently required the presence of CD4+ Th cells, cytotoxic effector cells could be generated from CD8+CD45RA+ precursor cells in the absence of CD4+ cells. Several lines of evidence indicated that the helper cell-independent generation of cytotoxic effector cells from CD8+CD45RA+ precursors resulted from the unique ability of this subset to produce and use IL-2 in an autocrine fashion: 1) exogenous IL-2 could replace the effects of CD4+ helper cells for either CD8+ subset; 2) the helper cell-independent functional maturation of CD8+CD45RA+ cells could be blocked by anti-CD25 or anti-IL-2 antibodies; and 3) CD8+CD45RA+ cells produced IL-2 after activation with allogeneic cells. The finding that precursors for helper cell-independent CTL generation are restricted to the CD8+CD45RA+ subset suggests that this capability may vary as a function of the maturation of CD8+ cells.


Tumor necrosis factor-alpha and IFN-gamma expression in human thymus. Localization and overexpression in Down syndrome (trisomy 21).

In vitro studies suggest that TNF-alpha and IFN-gamma regulate thymocyte proliferation, but little evidence exists for the constitutive production of these cytokines in normal human thymus. In paired experiments, we examined frozen sections of postnatal human thymus from four control children and four age-matched children with Down syndrome (DS) (trisomy 21) for TNF-alpha and IFN-gamma mRNA expression using in situ hybridization. We studied thymuses from children with DS because this aneuploid condition is associated with a greatly increased incidence of infection and has abnormal thymic anatomy and patterns of thymocyte maturation. We found cells expressing constitutive levels of TNF-alpha mRNA in the trabeculae, corticomedullary junctions, and medulla of both control and DS thymuses and the number of these cells was an average of 3.9-fold higher in DS thymuses than in age-matched control thymuses. DS thymuses also contained an average of 3 fold higher numbers of cells with mast cell morphology, identified by toluidine blue histologic staining and electron microscopy. In both DS and control thymuses the mast cells colocalized with TNF-alpha mRNA-expressing cells. In addition, TNF-alpha protein- expressing cells, identified by immunohistochemistry, displayed a granular pattern of staining that is characteristic of mast cells. These results suggest that mast cells may be one source of TNF-alpha in human postnatal thymus. Discrete cells expressing IFN-gamma mRNA were distinctly localized to the cortical region of both DS and control thymuses and were 2.4-fold more abundant in DS thymuses than in the controls. Our results demonstrate, for the first time, the constitutive production and location of TNF-alpha and IFN-gamma in postnatal human thymus. The overexpression of both of these cytokines in DS thymuses suggests a dysregulation in cytokine production in DS and may provide an explanation for the abnormal thymic anatomy and thymocyte maturation associated with this syndrome.


Reconstitution of long-term T helper cell function after zidovudine therapy in human immunodeficiency virus-infected patients.

Peripheral blood mononuclear cells from 12 asymptomatic patients infected with immunodeficiency virus (HIV) and 4 patients with AIDS were analyzed before and during therapy with zidovudine for T helper cell (Th) function. Th function improved by more than fourfold to one or more of three stimuli tested in 9 (75%) of 12 asymptomatic patients on zidovudine therapy and in 3 of 4 patients with AIDS. Only 6 (7.4%) of 80 untreated HIV-infected control patients showed spontaneous improvement in Th function (P less than 10(-6)). Improved Th function was detected as early as 5 weeks into therapy in 6 patients and continued to be evident for greater than 1 year after start of therapy in 6 patients and for greater than 2 years in 2 patients. No correlation was observed between improved Th function and changes in CD4+ or CD8+ cell numbers or in levels of serum HIV p24 antigen or beta 2-microglobulin. These results suggest inclusion of in vitro Th function as a useful marker in determining the efficacy of antiretroviral drug therapy of HIV-infected patients.


The Gambia Hepatitis Intervention Study: follow-up of a cohort of children vaccinated against hepatitis B.

Hepatitis B vaccine has been introduced into The Gambia's national Expanded Programme on Immunization, with the aim of evaluating the impact on chronic liver diseases and in particular on hepatocellular carcinoma. As part of the project, a cohort of 1000 children was recruited to assess the long-term protection induced by the vaccine. The first 3 years of follow-up are described. By the age of 3 years, 95% of the children had protective antibody levels; 4 (0.6%) are known to be carriers in contrast to the 90-140 that would be expected in the absence of vaccination. The protective effectiveness of vaccine in preventing chronic carriage in the first 3 years of life is thus estimated as 95%. Since the risk of becoming a carrier is highest in those infected early in life, these results are encouraging.


Modulation of plasma triglyceride levels by apoE phenotype: a meta-analysis.

The relationship between apoE phenotype and plasma lipid levels was analyzed in the combined data of published studies. Accordingly, 45 population samples from 17 different countries were included in the analysis. The mean plasma values of cholesterol (CH), triglyceride (TG), and high density lipoprotein (HDL)-CH of the apoE 2/2, 3/2, 4/3, 4/4, and 4/2 groups were compared with the same parameters of the E 3/3 subset. The standardized difference between the plasma lipid concentrations of the apoE subgroups and of their respective apoE 3/3 control (Z-score), as well as their mean weighted value, were calculated for each study and in each subgroup. The analysis confirmed that subjects carrying the epsilon 2 and epsilon 4 alleles had, respectively, lower (Z2/2 = -0.39, Z3/2 = -0.34) and higher (Z4/3 = 0.15, Z4/4 = 0.29) plasma cholesterol values than subjects carrying the epsilon 3/epsilon 3 genotype. In addition, results indicated a consistent relationship between plasma TG levels and apoE phenotype among different populations. TG concentrations were significantly higher in apoE 2/2, 3/2, 4/3 and E 4/2 than in E 3/3 subsets (Z2/2 = 0.42, Z3/2 = 0.14, Z4/3 = 0.13, Z4/2 = 0.19). Further, this trend was found in samples of normolipidemic adults and children, in diabetic and obese individuals, as well as in hyperlipidemic subjects indicating an ubiquitous relationship. Concurrently, HDL-CH was significantly lower in the apoE 4/3 (Z4/3 = -0.09) than in the E 3/3 subset.(ABSTRACT TRUNCATED AT 250 WORDS)


Huntington's disease in Chinese: a hypothesis of its origin.

The period prevalence (1984-91) of Huntington's disease (HD) in Hong Kong Chinese was 3.7 per million population. HD patients in Mainland China and Hong Kong showed similar hereditary pattern, clinical and pathological features as in the West. Chinese HD patients were male predominant with a younger age of onset and death. Their ancestral origin could be traced mostly to the coastal provinces of China. It is proposed that Chinese HD patients may have a European origin and share the same gene pool as their white counterparts.


Age-dependence of effects of A1 adenosine receptor antagonism in rat hippocampal slices.

1. Population excitatory postsynaptic potentials (EPSPs) and population spikes evoked in area CA1 of hippocampal slices from aged Fischer 344 rats were significantly smaller in amplitude than responses obtained in slices from young Fischer 344 rats. 2. The A1 adenosine receptor antagonist 8-cyclopentyltheophylline (8-CPT) produced a concentration-dependent increase in synaptic potentials in slices from both young and aged rats. Low concentrations (1 nM) of 8-CPT were effective in producing increases in both population spike amplitudes and population EPSP slopes in young and aged rat slices. Response increases were maximized by 100 nM 8-CPT in slices from rats of both age groups. 3. Adenosine antagonism produced greater average increases in synaptic responses in hippocampal slices from aged rats at all concentrations tested (1.0 nM-1.0 microM). A qualitative age-related difference in the response to 8-CPT was also observed; 8-CPT produced a late component, consisting of multiple population spikes, in evoked responses in slices obtained from aged but not young rats. 4. Adenosine antagonism significantly increased the maximum evocable response (both spike amplitude and EPSP slope) in slices from aged rats, relative to increases observed in slices from young rats. This suggested that smaller synaptic potentials seen in slices from aged rats were in part due to greater levels of "tonic" adenosinergic inhibition. 5. Slices from young and aged rats were incubated in the adenosine reuptake inhibitor soluflazine (R64719; 1.0, 10, and 100 microM) and the inhibition of population EPSPs was observed for 60 min. No difference was observed in the rate of inhibition or the maximal level of inhibition produced by soluflazine, in slices from rats of either age group. 6. Application of (+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclo-hepten- 5,10-imine hydrogen maleate (MK-801) and 2-amino-5-phosphonopentanoic acid (2-AP5), antagonists of N-methyl-D-aspartate (NMDA) excitatory amino acid (EAA) receptors, reduced the late multiple population spike component in slices from aged rats incubated in 8-CPT. A smaller direct effect of the NMDA antagonists was observed in slices from aged rats in the absence of 8-CPT treatment at maximal response levels. No effect of NMDA receptor antagonism was observed in slices from young rats under either condition. 7. Hippocampal tissue, from young and old rats utilized in the electrophysiological experiments, was assayed for A1 adenosine binding site density with a saturating concentration of radiolabeled agonist and antagonist. Guanine nucleotide modulation of agonist binding was also measured.(ABSTRACT TRUNCATED AT 400 WORDS)


Electrophysiology of glutamate neurotoxicity in vitro: induction of a calcium-dependent extended neuronal depolarization.

1. Physiological responses of hippocampal pyramidal neurons in primary culture to prolonged glutamate (GLU) exposure (500 microM in all experiments) were studied with the use of patch electrodes and whole-cell current-clamp recording techniques. In some experiments, perforated patch recordings were employed with electrodes containing the pore-forming antibiotic nystatin. 2. After washout of GLU after a 10-min exposure, pyramidal neurons remained depolarized by greater than or equal to 20 mV from rest for the duration of the recording (30 min to less than 4 h). This depolarization was accompanied by a 57.8% increase in membrane conductance and was termed an extended neuronal depolarization (END). The percentage of neurons in which END was induced varied with the duration of GLU exposure, with a 4-, 6-, 8-, 10-, and 20-min GLU exposure eliciting END in 12.5, 41.7, 81.8, 100, and 100% of neurons. END induction appeared to be an all-or-none phenomenon, because END levels did not differ when compared across GLU exposure times. 3. During the END, cells retained both the ability to fire action potentials and the ability to respond to GLU, appeared viable when examined anatomically, and still excluded vital dyes. This supports the conclusion that END is not a nonspecific consequence of cell death. Rather, END is a discrete physiological process triggered by prolonged GLU exposure. The results raise questions concerning the reversibility of END induction, i.e., can neurons be "rescued" once END is induced, or will these cells inevitably go on to die? 4. END induction was dependent on a rise in intracellular free calcium ([Ca]i). END was prevented by strong buffering of [Ca]i or by substitution of external Ba2+ for Ca2+. However, substitution of Mn2+ for Ca2+ still permitted END induction. In cells recorded with the perforated-patch technique, maintaining normal [Ca]i levels, END could be induced, but less readily than under unbuffered [Ca]i conditions. 5. END could not be induced by a 10- to 20-min current-clamp depolarization to 0 mV, nor by 10-min GLU application while the membrane potential was voltage clamped at rest in a solution containing 1 mM Mg2+. In addition, END induction by GLU could be blocked by application of MK-801 (10-30 microM) but not 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX (100-200 microM)]. 6. The dependence of both delayed neuronal cell death and END induction on GLU exposure duration were similar.(ABSTRACT TRUNCATED AT 400 WORDS)


Secretion of vitamin A and retinol-binding protein into plasma is depressed in rats by N-(4-hydroxyphenyl)retinamide (fenretinide).

In clinical trials the cancer preventive drug N-(4-hydroxyphenyl)retinamide (HPR) markedly lowers plasma concentrations of retinol and retinol-binding protein (RBP). Five hours after injection of HPR (51 mumol/kg), serum concentrations of retinol and RBP were 33 and 42% lower, respectively, than control values in rats. Because the mean transit time for retinol disappearance from serum of HPR-treated rats (1.9 h) was similar to that for radiolabeled retinol in control rats in previous studies, plasma retinol turnover is apparently not accelerated by HPR treatment. To study the effects of HPR on the secretion of the retinol-RBP complex from liver, control or HPR-treated rats were injected with chylomicrons containing [3H]vitamin A and [14C]triglycerides. Both labels were rapidly cleared from plasma in the two groups. In control rats [3H]retinol concentrations began to increase in plasma after 30 min due to liver secretion of retinol bound to RBP. In HPR-treated rats, secretion was apparently inhibited because the amount of [3H]retinol bound to RBP at 4.66 h was only 2.6% of the control level. We conclude that HPR partially blocks the secretion of the retinol-RBP complex from liver and other tissues, and thus depresses plasma concentrations of vitamin A and RBP.


Enhanced sensitivity to the behavioral effects of cocaine after chronic administration of D2-selective dopamine antagonists in the squirrel monkey.

The behavioral effects of cocaine (0.03-3.0 mg/kg i.v.) were determined in squirrel monkeys (Saimiri sciureus) trained to respond under a fixed-interval 300-sec schedule of stimulus termination. A session consisted of 13 consecutive fixed-interval components, each followed by a 60-sec timeout. Graded doses of cocaine were injected during selected timeout periods using a cumulative-dosing procedure. Subsequently, two dopamine D2-selective antagonists, spiperone and raclopride, and a D1-selective antagonist, SCH 23390, were administered chronically for a 2-week period. Due to pronounced time course differences, raclopride and SCH 23390 were infused continuously via osmotic minipump, and spiperone was administered i.m. twice per week. Spiperone and raclopride markedly suppressed responding during the 2-week period. When the effects of cocaine were redetermined 3 days after spiperone or 1 day after raclopride administration was terminated, there was a parallel leftward shift in the dose-effect curve, indicating enhanced sensitivity to cocaine. Three days later, sensitivity to cocaine had changed and was similar to that obtained before chronic drug administration. In contrast, SCH 23390 did not alter sensitivity to cocaine after chronic administration was terminated, even though it did attenuate the behavioral effects of cocaine as effectively as spiperone and raclopride. Chronic administration of spiperone did not alter sensitivity to nisoxetine, a norepinephrine uptake inhibitor, or quipazine, a serotonin agonist. The acute administration of spiperone in combination with cocaine also differed markedly from nisoxetine and quipazine. The pronounced rate-decreasing effect of spiperone was attenuated by cocaine in a dose-dependent manner, but not by nisoxetine or quipazine.(ABSTRACT TRUNCATED AT 250 WORDS)


Potent indole- and quinoline-containing N-methyl-D-aspartate antagonists acting at the strychnine-insensitive glycine binding site.

The N-methyl-D-aspartate (NMDA)-preferring glutamate receptor subtype possesses, in addition to the recognition site for glutamate, a binding site for glycine. We report here on the pharmacological properties of 3-(4,6-dichloro-2-carboxyindol-3-yl)-propionic acid (MDL 29,951) and 4-carboxymethylamino-5,7-dichloroquinoline-2-carboxylic acid (MDL 100,748), two novel glycine antagonists of NMDA receptor activation in vitro and in vivo. We have measured in parallel the effects of two previously described glycine antagonists, 7-chlorokynurenic acid and 5,7-dichlorokynurenic acid. All were potent inhibitors of [3H]glycine binding. Ki values (microM) were 0.36 (7-chlorokynurenic acid), 0.08 (5,7-dichlorokynurenic acid), 0.07 (MDL 100,748) and 0.14 (MDL 29,951). MDL 100,748 and MDL 29,951 were approximately 2000-fold selective for the glycine binding site relative to the glutamate recognition sites. All four compounds completely inhibited the use-dependent binding of [3H]N-[1-(2-thienyl) cyclohexyl]-piperidine and were noncompetitive, glycine-reversible inhibitors of both NMDA-induced biochemical and electrophysiological responses in brain slice preparations. A competitive interaction with the glycine binding site was also evident in that MDL 29,951 and MDL 100,748 produced parallel rightward shifts in the glycine requirement for demonstration of NMDA-stimulated elevations in cytosolic calcium in cultured neuronal preparations. The glycine antagonists were potent anticonvulsants after their i.c.v. administration to audiogenic seizure-susceptible DBA/2J mice. Because the compounds chosen encompass a variety of chemical structures, the results indicate that glycine is required for NMDA receptor activation and that bioavailable glycine antagonists may form the basis of a novel therapy for epilepsy.


Determination of the absolute configuration of the active amlodipine enantiomer as (-)-S: a correction.

The active (-) enantiomer of amlodipine was originally reported to have R configuration. This does not concur with other 1,4-dihydropyridines with known absolute configuration. This configuration has now been determined by X-ray structural analysis using (1S)-camphanic acid and (S)-2-methoxy-2-phenylethanol as chiral probes. Both determinations gave the S configuration for the amlodipine (-) enantiomer with the greater Ca-antagonistic activity.


Non-prostanoid thromboxane A2 receptor antagonists with a dibenzoxepin ring system. 1.

A series of 11-[[2-[(arylsulfonyl)amino]ethyl]thio]-6,11- dihydrodibenz[b,e]oxepin-2-carboxylic acids and related derivatives were synthesized. The compounds were tested for their antagonizing effects on guinea pig platelet TXA2/PGH2 receptors. Structure-activity relationships are discussed. (+/-)-11-[[2-[(Styrylsulfonyl)amino]ethyl]-thio]-6,11- dihydrodibenz[b,e]oxepin-2-carboxylic acid (41) and (+/-)-11-[[2-[(phenylsulfonyl)amino]ethyl]thio]-6,11- dihydrodibenz[b,e]thiepin-2-carboxylic acid (4af) were the most promising compounds with K(i) values of 6.5 +/- 0.29 and 3.7 +/- 0.31 nM, respectively, for the TXA2/PGH2 receptor. These compounds also significantly inhibited U-46619-induced guinea pig platelet aggregation ex vivo (10 mg/kg po). Compound 41 was resolved into its optically active form. The (-)-isomer was 60-fold more potent than the (+)-isomer in the TXA2/PGH2 receptor binding assay. Some compounds tested in this study showed both TXA2/PGH2 receptor antagonizing and TXA2 synthase inhibitory effects.


Non-prostanoid thromboxane A2 receptor antagonists with a dibenzoxepin ring system. 2.

A series of 11-[2-(1-benzimidazolyl)ethylidene]-6,11-dihydrodibenz[b,e]oxep in-2- carboxylic acid derivatives and related compounds were synthesized and found to be potent TXA2/PGH2 receptor antagonists. Each compound synthesized was tested for its ability to displace [3H]U-46619 binding from guinea pig platelet TXA2/PGH2 receptors. Structure-activity relationship studies revealed that the following key elements were required for enhanced activities: (1) an (E)-2-(1-benzimidazolyl)ethylidene side chain in the 11-position of the dibenzoxepin ring system and (2) a carboxyl group in the 2-position of the dibenzoxepin ring system. The studies also indicated that the TXA2/PGH2 receptor binding affinities of this series of compounds in guinea pig platelet were poorly correlated with those in human platelet. Introduction of substituent(s) to the benzimidazole moiety was effective and sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)ethylidene]- 6,11-dihydrodibenz[b,e]oxepin-2-carboxylate monohydrate (57) recorded the highest affinity for human platelet TXA2/PGH2 receptor with a K(i) value of 1.2 +/- 0.14 nM. It demonstrated potent inhibitory effects on U-46619-induced guinea pig platelet aggregation (in vitro and ex vivo) and human platelet aggregation (in vitro). Compound 57, now designated as KW-3635, is a novel, orally active, and specific TXA2/PGH2 receptor antagonist with neither TXA2/PGH2 receptor agonistic nor TXA2 synthase inhibitory effects. It is now under clinical evaluation.


Terminal regions of the NS-1 protein of the parvovirus minute virus of mice are involved in cytotoxicity and promoter trans inhibition.

The nonstructural (NS) transcription unit of minute virus of mice (MVMp) encodes proteins that are involved in viral DNA replication and in the regulation of homologous and heterologous promoters. Moreover, it has been shown that NS-protein accumulation is toxic for transformed cells. With the aim of identifying the NS-protein function(s) responsible for cytotoxicity, point mutations and deletions were introduced in the NS-protein-coding sequence of MVMp. This strategy indicated that in transformed human NBE cells, the NS-1 protein is indispensable for MVMp DNA replication, trans activation of the late parvoviral promoter P38, trans inhibition of the long terminal repeat promoter of the Rous sarcoma virus, and cytotoxicity. Moreover, some mutations led to the dissociation of the replicative and regulatory functions of the NS-1 protein and showed that cytotoxicity correlated with the latter, more particularly with the capacity to trans inhibit the heterologous promoter. The NS-1 sequences required for cytotoxicity were found to be restricted to the amino- and carboxy-terminal portions of the protein. Although the cytotoxicities of NS-1 extremities were weak when the extremities were tested separately, the cytotoxicities were comparable to that of the full protein when the extremities were fused. Interestingly, an overall negative charge can be predicted from the NS-1 sequence over about 100 amino acids at both ends. The conservation of this charge distribution among the NS proteins of different parvoviruses suggests that NS-1 may bear some similarities to acidic transcriptional activators.


Effects of reperfusion on left ventricular ejection fraction and volume after acute myocardial infarction.

The effects of reperfusion on left ventricular (LV) function and volume were studied in patients with evolving acute myocardial infarction (AMI). We analyzed the LV ejection fraction and volume in patients who had been admitted within 24 h of the onset of their first AMI with culprit lesion of #6, #7 and #1 (American Heart Association classification). Sixty-five patients (Re group) received successful reperfusion therapy within 6 h after the AMI. The other 60 patients (Oc group), who were admitted from 6 to 24 h after the AMI, received conservative therapy. Patients with re-obstruction of the culprit lesion after reperfusion therapy were excluded from the Re group. Patients with spontaneous recanalization following conservative therapy were excluded from the Oc group. The LV ejection fraction (LVEF), LV end-systolic volume index (LVESVI), and LV end-diastolic volume index (LVEDVI) were measured using a modified Dodge's formula by left ventriculography performed 4 weeks after the AMI. LVEF in the Re group was significantly greater than in the Oc group (57 +/- 12 vs 49 +/- 11%) (mean +/- SD, p less than 0.01). LVESVI in the Re group was significantly smaller than in the Oc group (30 +/- 13 vs 38 +/- 16 ml/m2, p less than 0.01). Although LVEDVI was not significantly different between the 2 groups, in patients with a responsible coronary lesion of segment #6, LVEDVI in the Re group was significantly smaller than in the Oc group (67 +/- 14 vs 77 +/- 18 ml/m2, p less than 0.05). Although LVEF and LV volume correlated in both groups, the correlation was weak (r = 0.40-0.42), suggesting that LV volume was not dependent solely on LV functional recovery. The incidence of ventricular aneurysm in the Re group was significantly lower than in the Oc group (15.4 vs 45.0%, p less than 0.01). Multivariate analysis selected reperfusion of the responsible coronary artery as one of the factors significantly associated with a reduction of LVEDVI, LVESVI, an improvement of LVEF, and a decrease in the rate of aneurysm formation. In summary, our results indicated that reperfusion improved EF, reduced LV volume, and decreased the rate of aneurysm formation as compared to non-reperfusion, which suggests that reperfusion therapy is beneficial for both functional recovery and ventricular remodeling.


Map of cis-acting sequences that determine alternative pre-mRNA processing in the E3 complex transcription unit of adenovirus.

The E3 complex transcription unit of adenovirus encodes four major mRNAs (a, c, f, and h) and two minor (d and e) mRNAs with overlapping exons, alternative splice sites, and two polyadenylation sites, termed E3A (upstream) and E3B (downstream). mRNAs a and d use the E3A polyadenylation site, and mRNAs c, e, f, and h use the E3B site. We have analyzed virus mutants with deletions throughout the E3 region in order to identify cis-acting sequences that function in E3 pre-mRNA processing. The results presented in this report as well as previous results are summarized as follows. (i) Deletions in the first (5') intron at nucleotides (nt) 372 to 768 in E3 had no effect unless they removed the consensus sequence for the nt 372 5' splice site; however, the overall pattern of E3 mRNAs did not change significantly. (ii) Deletions in region I (nt 1441 to 2044) eliminated mRNAs a and c and resulted in corresponding increases in mRNAs f and h; we propose that region I contains sequences that suppress splicing. (iii) Mutations in region II (nt 2161 to 2243) resulted in nearly exclusive synthesis of mRNA f; this phenotype is understood and is discussed. (iv) Changing the AUUAAA component of the E3A poly(A) addition signal to AAUAAA resulted in increased mRNA a levels, suggesting that the E3A poly(A) addition signal is intrinsically inefficient. (v) Deletions in region III (nt 2488 to 3002) decreased mRNA a levels about two- to threefold and specifically increased mRNA f levels; we suggest that region III facilitates use of the E3A polyadenylation site. (vi) Deletions in region IV (nt 2904 to 3251) increased mRNA a levels about two- to threefold; we suggest that region IV may contain sequences that facilitate use of the E3B polyadenylation site. A map of sequences that determine alternative pre-mRNA processing in region E3 is now nearly complete.


[Effect of antihypertensive treatment in elderly hypertensive patients with cardiac hypertrophy].

In elderly hypertensive patients effect of antihypertensive treatment with Ca antagonist or ACE inhibitor on the heart were examined. Twenty-four elderly hypertensive patients with cardiac hypertrophy, aged 65-79 years old (mean +/- SEM, 71 +/- 1) were treated with Ca antagonist (nifedipine or nicardipine) or ACE inhibitor (captopril or enalapril) for 3 months. Thirteen patients had essential hypertension (EH: SBP greater than or equal to 160 mmHg and DBP greater than or equal to 95 mmHg, 70 +/- 1 years) and 11 had isolated systolic hypertension (ISH: SBP greater than or equal to 160 mmHg and DBP less than 95 mmHg, 74 +/- 2 years). Blood pressure (BP) and heart rate were measured every two weeks. In all patients, M-mode echocardiography was performed to measure left ventricular mass index (LVMI) and ejection fraction (EF), and the sympathetic nervous (plasma norepinephrine and epinephrine) and the renin-angiotensin system (plasma renin activity and aldosterone concentration), were assessed before and after 3 months of treatment. BP significantly decreased from 174 +/- 3/97 +/- 1 to 149 +/- 4/84 +/- 2 mmHg in EH and from 167 +/- 3/82 +/- 2 to 144 +/- 4/74 +/- 2 mmHg in ISH. LVMI was significantly reduced from 204 +/- 14 to 174 +/- 16 g/m2 in EH and from 179 +/- 14 to 156 +/- 12 g/m2 in ISH. EF showed no significant changes in either group. In ISH, the change in LVMI was significantly correlated with the change in systolic BP (r = 0.74, p less than 0.05). In EH, there was no significant relation between BP and LVMI changes.(ABSTRACT TRUNCATED AT 250 WORDS)


[Calcium metabolism in various forms of hypertrophy of the "hypertensive" heart].

To study the changes occurred in integral calcium metabolic parameters in evolving "hypertensive" heart, a total of 100 patients with Stage I-II (WHO Classification) were examined. Myocardial structure and function were echocardiographically examined. Plasma concentrations of ionized calcium were measured by direct potentiometry. Plasma levels of calcitonin and parathyroid hormone were measured by radioimmunoassay. As the "hypertensive" heart progressed, the ionization of serum calcium was found to enhance along with an increase in calcitoninemia. In early formation of the "hypertensive" heart, parathyroid hormone remained unchanged, but became elevated only in severe left ventricular hypertrophy. The findings may explain the features of the clinical aspects of variant left ventricular hypertrophies in the "hypertensive" heart.


[The treatment of acute appendicitis].

The article deals with the experience in the treatment of 3,368 patients with acute appendicitis from 1979 to 1989. Among them were 2,398 adults and 976 children. Complications developed in 190 (5.6%) patients, 5 (0.15%) patients died. The mortality rate among children was 0.3%. Complications and deaths were connected with neglected forms of the disease. 695 (20.6%) patients were admitted later than 24 hours after the onset of the disease. The patients diagnosed the disease themselves and resorted to self-treatment. Health education measures were poorly effective; it is suggested that this work should be started from early childhood. A permanently functioning seminar on acute surgical diseases was organized at the institution for doctors not practicing in surgery, but diagnostic errors were made at the prehospital stage in 117 cases by district therapists, pediatricians, and the staff of emergency aid teams. There were also cases in which operation was undertaken late after admission to the hospital and cases of hyperdiagnosis. The late-term results were studied in 109 patients without morphological changes in the removed vermiform processes, in 51.4% of them other diseases were encountered in the long-term period.


[The ultrasonic diagnosis of acute appendicitis and its complications].

The article discusses the possibility of ultrasonic examination (USE) in the diagnosis of acute appendicitis and its complications. Forty-five patients were examined, 15 of them had a doubtful clinical picture of acute appendicitis and in 30 destructive acute appendicitis was suspected. During USE the vermiform process was not detected in 5 patients, uncomplicated acute appendicitis was diagnosed in 10 and complicated acute appendicitis (appendicular infiltrate, abscess) in 30 patients. Eight patients underwent laparoscopy, 29 patients were subjected to an operative intervention, 15 patients received a course of nonoperative treatment followed by appendectomy 3-6 months later. As the result of the study the USE signs of acute appendicitis and its complications were specified. USE may be of significant aid to the surgeon in the diagnosis of this disease.


[Laparoscopy in the diagnosis of acute appendicitis in pregnant women].

Diagnostic laparoscopy in 96 pregnant women with suspected acute appendicitis revealed inflammation of the vermiform process only in two (2.1%) of them. In 81 cases acute surgical diseases of the abdominal organs were not found, extrauterine pregnancy was revealed in 9 and other diseases of the abdominal organs were detected in 4 cases. The threat of abortion was mistaken for acute appendicitis most frequently. Unjustified operation in such cases intensifies still more the signs of the threat of abortion. Analysis allows the assertion that the laparoscopic method should be decisive in the examination of pregnant women with a doubtful clinical diagnosis in suspected acute appendicitis. Laparoscopy is indicated when the reserves of the generally accepted clinical methods and noninvasive instrumental examination do not allow the diagnosis of acute appendicitis to be established or excluded; it is also recommended in establishing the exact diagnosis and determining the approach and volume of an operative intervention when there is a clinical picture of an acute surgical disease of the abdominal cavity.


[Plastic operations in rectal fistulae].

Experiments were conducted on 48 dogs to study the terms and degree of resolution of various plastic materials--areas of fascia lata of the animal's thigh, as well as explants (medical glue compositions, biological absorbable lavsan-armored Soviet medical films) in the pararectal tissues and in artificial formation of rectal fistulas. It is shown that auto- and explantation may be performed in the treatment of rectal fistulas in patients. The suggested method was applied in the clinic in 136 operations for complex trans- and extrasphincteric pararectal fistulas. The results were followed-up for 12 months to 5 years and proved to be good in 126 patients.


[Torsion of the colonic epiploic appendages].

Operations were carried out on 21 patients with torsion of epiploic appendages of the colon (sigmoid 19, ascending 1, hepatic flexure 1). This pathologic condition has no specific manifestations and occurs under the mask of other emergencies. The suggested symptom of "pain displacement" in combination with other signs allowed the correct diagnosis to be established before the operation in two thirds of patients. Patients with torsion of epiploic appendages must be subjected to an emergency operation for removal of the necrotic appendage by ligation of its vascular pedicle and its peritonization with seromuscular sutures.