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PubMed1991

Penile detumescence: characterization of three phases.

In 22 dogs in which erection was induced by cavernous nerve stimulation, we analyzed the intracavernous pressure changes during detumescence without and with acute clamping of the aorta or electrostimulation of the lumbar sympathetic chains. Additionally, the degree of venous outflow obstruction was assessed by saline perfusion of the cavernous body during aortic occlusion. Detumescence had three distinct phases: an initial phase exhibiting a small pressure increase; a second phase showing a slow pressure decrease; and a third phase in which a fast decrease occurred. The first phase was abolished by aortic clamping, whereas the other phases were not significantly affected. Sympathetic stimulation abolished or prevented the second phase. Perfusion of the cavernous body during the second phase resulted in a pressure rise to off-scale values; however, when initiated during the terminal phase or in the nonstimulated penis, the pressure increase was slight. Our study indicates that the arterial flow rate influences the duration of the first phase of detumescence and that venous drainage is completely restored in the third phase. Furthermore, sympathetic stimulation causes an almost immediate full restoration of venous drainage, as cavernous perfusion initiated with an intracavernous pressure about twice as high as without sympathetic stimulation failed to increase pressure to off-scale values.

Animals↗

PubMed1988

Structural characterization of heparan sulfate proteoglycan subclasses isolated from bovine aortic endothelial cell cultures.

Labeled heparan sulfate proteoglycans (HSPG) were isolated from wounded and confluent cultures of bovine aortic endothelial cells by nondegradative extraction with 4 M guanidine hydrochloride and detergent. HSPG were separated from more highly charged chondroitin or dermatan sulfate proteoglycans by ion-exchange chromatography, and subclasses of different hydrodynamic size were isolated by gel filtration. Three major subclasses of HSPG were characterized structurally with respect to the presence and relative size of protein core, the presence and amount of nonsulfated oligosaccharide, and size and structure of heparan sulfate (HS) chains. The largest (600-800-kDa) HSPG subclass (I), isolated from cell layers and media of confluent cultures, bears 38-kDa HS chains on an apparently heterogeneous class of relatively large glycoprotein cores. HSPG II (150-200 kDa), isolated from cell layer or media, has 22-kDa HS chains and smaller core glycoproteins (less than 50 kDa). HSPG III, the subclass of smallest hydrodynamic size, has 13-kDa HS chains and a glycopeptide core of less than 15 kDa. All subclasses bear varying proportions of non-sulfated oligosaccharides of similar sizes. Comparisons of HS chain structure indicated that the different subclasses have similar proportions (49-55%) of N-sulfate, with both O-sulfate and highly N-sulfated blocks of disaccharide distributed similarly along HS chains. In addition, HS chains from subclasses II and III contain sequences that are insensitive to periodate oxidation or heparitinase digestion, suggesting that they contain increased proportions of iduronate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Electric charge effects on phospholipid headgroups. Phosphatidylcholine in mixtures with cationic and anionic amphiphiles.

The influence of electric surface charges on the polar headgroups and the hydrocarbon region of phospholipid membranes was studied by mixing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with charged amphiphiles. A positive surface charge was generated with dialkyldimethylammonium salts and a negative surface charge with dialkyl phosphates. The POPC:amphiphile ratio and hence the surface charge density could be varied over a large range since stable liquid-crystalline bilayers were obtained even for the pure amphiphiles in water. POPC was selectively deuterated at both methylene segments of the choline moiety and at the cis double bond of the oleic acyl chain. Additional experiments were carried out with 1,2-dipalmitoyl-rac-glycero-3-phosphocholine labeled at the C-2 position of the glycerol backbone. Deuterium, phosphorus, and nitrogen-14 nuclear magnetic resonance (NMR) spectra were recorded for liquid-crystalline bilayers with varying concentrations of amphiphiles. Although the hydrocarbon region and the glycerol backbone were not significantly influenced by the addition of amphiphiles, very large perturbations of the phosphocholine headgroup were observed. Qualitatively, these results were similar to those observed previously with other cationic and anionic molecules and suggest that the electric surface charge is the essential driving force in changing the phospholipid headgroup orientation and conformation. While the P-N dipole is approximately parallel to the membrane surface in the pure phospholipid membrane, the addition of a positively charged amphiphile or the binding of cationic molecules moves the N+ end of the dipole toward the water phase, changing the orientation of the phosphate segment by more than 30 degrees at the highest amphiphile concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Ampholyte Mixtures↗

PubMed1980

Inhibition of ion permeability control properties of acetylcholine receptor from Torpedo californica by long-chain fatty acids.

The characteristics of fatty acid inhibition of acetylcholine receptor function were examined in membrane vesicles prepared from Torpedo californica electroplax. Inhibition of the carbamylcholine-induced increase in sodium ion permeability was correlated with the bulk melting point of exogenously incorporated fatty acids. Above its melting temperature, a fatty acid could inhibit the large increase in cation permeability normally elicited by agonist binding to receptor. Below its melting temperature, a fatty acid was ineffective. None of the fatty acids altered any of the ligand binding properties of the receptor. Inhibitory fatty acids did not induce changes in membrane fluidity, as determined by electron paramagnetic resonance using spin-labeled fatty acids. The spin-labeled fatty acids also acted as inhibitors, and the extent of inhibition depended largely on the position of the nitroxide group along the fatty acid chain. Addition of noninhibitory fatty acid to the vesicle membranes did not protect the receptor from inhibition by spin-labeled fatty acids. The effects of free fatty acids on acetylcholine receptor function are attributed to the disruptions of protein-lipid interactions.

Acetylcholine↗

PubMed1989

Topography of glycosylation in yeast: characterization of GDPmannose transport and lumenal guanosine diphosphatase activities in Golgi-like vesicles.

"Outer-chain" addition of mannose residues to yeast glycoproteins occurs in the Golgi compartment of the cell. Essential steps in this process are thought to include transport of GDPmannose from the cytoplasm into the lumen of Golgi vesicles, transfer of mannose to glycoprotein acceptors, hydrolysis of the resulting GDP to GMP, and return of GMP and inorganic phosphate to the cytoplasm. We report detection and characterization of a GDPmannose transport activity and a GDPase by yeast vesicles. The active transport of GDPmannose as well as the GDPase and another presumed Golgi enzyme, alpha 1,2-mannosyltransferase, are concentrated in a subcellular fraction that can be partially separated, by velocity sucrose gradient centrifugation, from a fraction enriched in an endoplasmic reticulum marker enzyme.

Biological Transport↗

PubMed1990

Reconstitution of simian virus 40 DNA replication with purified proteins.

Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.

Antigens, Polyomavirus Transforming↗

PubMed1989

Molecular cloning of syndecan, an integral membrane proteoglycan.

We describe cDNA clones for a cell surface proteoglycan that bears both heparan sulfate and chondroitin sulfate and that links the cytoskeleton to the interstitial matrix. The cDNA encodes a unique core protein of 32,868 D that contains several structural features consistent with its role as a glycosamino-glycan-containing matrix anchor. The sequence shows discrete cytoplasmic, transmembrane, and NH2-terminal extracellular domains, indicating that the molecule is a type I integral membrane protein. The cytoplasmic domain is small and similar in size but not in sequence to that of the beta-chain of various integrins. The extracellular domain contains a single dibasic sequence adjacent to the extracellular face of the transmembrane domain, potentially serving as the protease-susceptible site involved in release of this domain from the cell surface. The extracellular domain contains two distinct types of putative glycosaminoglycan attachment sites; one type shows sequence characteristics of the sites previously described for chondroitin sulfate attachment (Bourdon, M. A., T. Krusius, S. Campbell, N. B. Schwartz, and E. Ruoslahti. 1987. Proc. Natl. Acad. Sci. USA. 84:3194-3198), but the other type has newly identified sequence characteristics that potentially correspond to heparan sulfate attachment sites. The single N-linked sugar recognition sequence is within the putative chondroitin sulfate attachment sequence, suggesting asparagine glycosylation as a mechanism for regulating chondroitin sulfate chain addition. Both 5' and 3' regions of this cDNA have sequences substantially identical to analogous regions of the human insulin receptor cDNA: a 99-bp region spanning the 5' untranslated and initial coding sequences is 67% identical and a 35-bp region in the 3' untranslated region is 81% identical in sequence. mRNA expression is tissue specific; various epithelial tissues show the same two sizes of mRNA (2.6 and 3.4 kb); in the same relative abundance (3:1), the cerebrum shows a single 4.5-kb mRNA. This core protein cDNA describes a new class of molecule, an integral membrane proteoglycan, that we propose to name syndecan (from the Greek syndein, to bind together).

Amino Acid Sequence↗

PubMed1981

Immunoglobulin expression in human lymphoblastoid cell lines with early B cell features.

Immunoglobulin expression was studied by direct immunofluorescence and by biosynthesis experiments in two human cell lines Raji and T 5.1. The basic phenotype of these cells was close to that of pre-B cells: large cells with intracytoplasmic IgM with a predominance of mu chains over light chains and no detectable surface immunoglobulins. The apparent molecular weight of heavy and light chains was abnormally large. Immunoglobulins were secreted at a low rate as pentameric IgM in the T 5.1 line and as subunits and free light chains in the Raji line. Spontaneous variations of this phenotype were observed: the cultured cells acquired mu and lambda chains, then additionally delta chains while they progressively lost detectable cytoplasmic mu chains, thus leading to a mature B cell phenotype. Subsequently, the cells had no detectable surface and cytoplasmic immunoglobulins and then they displayed a pre-B cell phenotype again. Attempts to induce further maturation using various potential inducers were unsuccessful.

B-Lymphocytes↗

PubMed1979

Yeast tRNA Leu UAG. Purification, properties and determination of the nucleotide sequence by radioactive derivative methods.

A second major species of leucine tRNA, tRNA Leu UAG (formerly designated tRNA Leu CUA) was purified from baker's yeast in a three-step procedure entailing BD-cellulose chromatography in the presence and absence of Mg2+ and Sephadex G-100 gel filtration. Results of aminoacylation and partial RNase T1 digestion experiments showed that this tRNA retains a native conformation under conditions that denature yeast tRNA Leu m5CAA (tRNA3 Leu). The primary structure of baker's yeast tRNA Leu UAG was elucidated by application of sensitive radioactive isotope derivative ("postlabeling") methods. Complete RNase T1 and A and partial RNase U2 fragments, prepared from non-radioactive tRNA and 5'-half and 3'-half molecules, were separated by two-dimensional polyethyleneimine-cellulose anion-exchange thin-layer chromatography and isolated by a novel micropreparative procedure affording high yields of these compounds in sufficient purity for subsequent tritium derivative analysis. Base composition and sequence of oligonucleotides were analyzed by tritium derivative methods. Molar ratios of the fragments were determined from the radioactivity of 3H-labeled nucleoside trialcohols in combination with base analysis. 2'-O-Methylated guanosine was characterized using the [gamma-32P]ATP/polynucleotide kinase reaction. The analysis of classical complete and partial RNase digests by the tritium derivative methods yielded the complete nucleotide sequence of the tRNA. A total of about 20 A260 units of the RNA was used for analysis, i.e. considerably less material than required for conventional spectrophotometric analysis. A different sequencing approach, consisting of a combination of "readout sequencing" with tritium sequencing of complete RNase T1 and A fragments, was applied to the 3'-half molecule. The 3'-half molecule was labeled with 32P at its 5' terminus, partially degraded with RNase T1, U2, and Phy1 and with alkali, and subjected to polyacrylamide gel electrophoresis. The sequence was read off the gel on the basis of cleavage patterns and size of the fragments. While the readout procedure provided only the positions of A, U, C, and G residues in the chain, additional information from tritium derivative analysis was utilized to define the positions of the modified nucleosides. The readout sequencing procedure was found to require less than 0.01 A260 unit of RNA and the analysis of the complete fragments about 6 A260 units. Interesting structural features of tRNA Leu UAG are (a) the location of unique, leucine tRNA iso-acceptor-specific sequences next to U-8, a constant nucleotide participating in synthetase recognition, (b) the occurrence of 1-methyladenosine in the T loop, a modification not present in the structurally related tRNA Leu m5CAA, and (c) the unusual presence of an unmodified uridine in the first position of the anticodon, which may be related to the unusual coding properties reported for this tRNA.

Adenine↗

PubMedDate not supplied

MTD calculations on quantitative structure-activity relationships of steroids binding to the progesterone receptor.

The minimal topological difference (MTD) method is used to describe quantitative structure-activity relationships (QSAR) for the progesterone-receptor binding affinity including 59 progestational steroids. Multiple correlation coefficients of r = 0.962 and r = 0.955 are obtained by use of the MTD variable and a measure of hydrophobicity for the series of progesterone and ethisterone derivatives, respectively. Hydrophobic effects are found to strongly influence receptor binding. In accordance with the hydrogen bonding concept, the optimized MTD receptor maps indicate cavity vertices in the regions of oxygen functions at C3 and in the 17 beta position. Receptor wall vertices are attributed in the areas of 4, 10 beta, and 13 beta substituents of 4-en-3-one steroids while 17 alpha side chains additionally contain receptor cavity vertices. A comparison of corresponding receptor maps suggests in accord with X-ray crystal structure data that progesterone and ethisterone derivatives are bound in somewhat different orientations relative to the receptor surface.

Animals↗

PubMed1980

Effects of propylthiouracil and methylmercaptoimidazole on thyroglobulin synthesis.

The effect of 6-propyl-2-thiouracil (PTU), and 1-methyl-2-mercaptoimidazole (MMI) on thyroglobulin (Tg) biosynthesis has been studied in vivo and in vitro. In vivo experiments were performed in rats treated for 20 days with PTU or MMI, analyzing soluble and particulate, cold and 125I-labelled, Tg. Thyroglobulin biosynthesis was also investigated by in vitro experiments, incubating thyroid tissue with labelled amino acid and carbohydrate in the presence of antithyroid compounds. It has been found that in vivo antithyroid agents decrease the amount of soluble Tg and increase the proportion of particulate Tg. Tg from treated animals is poorly iodinated being mainly represented by its 12S subunit. In vitro studies demonstrate that PTU and MMI inhibit Tg biosynthesis which is impaired in the polypeptide synthesis as well as in carbohydrate chains addition. Thus the inhibition of the hormonogenetic processes induced by antithyroid treatment leading to a depressed iodinating activity also appears to be related to a significant impairment of the production of the Tg molecule, the specific iodine acceptor.

Amino Acids↗

PubMed1990

Active site labeling of Escherichia coli transcription elongation complexes with 5-[4-azidophenacyl)thio)uridine 5'-triphosphate.

Escherichia coli RNA polymerase transcription elongation complexes have been prepared that contain a photo-cross-linking uridine analog at only the 3' end, or one or two nucleotides removed from the 3' end, in the nascent RNA chain. Additionally, complexes have been isolated in which the analog has been substituted for every UMP residue, at positions ranging from 20 to 140 nucleotides from the 3' end. The RNA has been photochemically cross-linked to the RNA polymerase to identify the subunits that form the binding site(s) for these regions in the nascent RNA. The photo-cross-linking nucleotide analog used for these studies was 5-[4-azidophenacyl)thio)uridine-5'-triphosphate (5-APAS-UTP), which acts as a 10-15 A probe. With 5-APAS-UMP positioned only at the 3' end of the RNA, or one or two nucleotides from the 3' end, only the beta subunit appeared to be contacted. When the analog was positioned throughout the RNA, both the beta and beta' subunits were contacted. No labeling of the sigma or alpha subunits was observed with any RNA length. In addition to placing this analog at specific positions in short RNAs, we have carried out transcription studies with 5-APAS-UTP to determine the optimal UTP to analog ratio for production of full length, photoreactive transcripts. Surprisingly, we found that when transcription complexes were stalled shortly after initiation, by deletion of one ribonucleoside triphosphate to synchronize transcription, changes in transcriptional pausing occurred downstream. These results suggest that events that occur early in transcription can affect the elongation and/or termination events that occur far downstream from the promoter. This effect occurred even with UTP but was greatly enhanced by replacement of UTP with either this analog or 4-thio-UTP. By enhancing the normal transcriptional pausing event, these analogs can serve as probes of the conformational changes that may exist in paused transcription complexes.

Affinity Labels↗

PubMed1991

Differential expression of small chondroitin/dermatan sulfate proteoglycans, PG-I/biglycan and PG-II/decorin, by vascular smooth muscle and endothelial cells in culture.

Cultured bovine aortic smooth muscle (BASM) and endothelial (BAE) cells, like articular chondrocytes, synthesize two populations of small chondroitin/dermatan sulfate (CS/DS) proteoglycans (PGs) of similar size as PG-I/biglycan and PG-II/decorin. However, Northern blot analyses demonstrated that BAE cells express detectable amounts of mRNA transcripts only for PG-I/biglycan, whereas BASM cells and articular chondrocytes express mRNA transcripts for both PG-I/biglycan and PG-II/decorin. Endothelial cells from human umbilical vein also expressed detectable amounts of mRNA transcripts only for PG-I/biglycan, and not PG-II/decorin. Antiserum raised against bovine PG-II/decorin immunoprecipitated an apparent single PG species with relative molecular mass (Mr) of approximately 120,000-180,000 from BASM cell and articular chondrocyte cultures but failed to immunoprecipitate an equivalent PG species from BAE cell cultures, consistent with the results from Northern blot analysis. In contrast, immunoprecipitations by antisera to PG-I/biglycan indicated that cultured endothelial cells synthesize two forms of PG-I/biglycan with Mr values slightly larger than 200,000 and 120,000-140,000. It is likely, based on the magnitude of the size difference, that these two forms of PG-I/biglycan differ in the number of glycosaminoglycan chains. Additionally, BASM but not BAE cells were found to express detectable amounts of mRNA transcripts for type I collagen. The above results indicate that the two main cell types of the vascular wall, endothelial and smooth muscle cells, express different sets of small interstitial CS/DS PGs and that the synthesis of PG-II/decorin by these cells correlates with the expression of type I collagen, a collagen known to interact specifically with this PG. These differences in the expression of extracellular matrix molecules may be important in regulating the cell type-specific functions of endothelial and smooth muscle cells within the vascular tissue.

Animals↗

PubMed1991

Purification and partial characterization of fibrillin, a cysteine-rich structural component of connective tissue microfibrils.

Fibrillin, a connective tissue macromolecule (Mr = 350,000) which is normally insoluble in its tissue form, has been purified from the medium of human skin fibroblast and ligament cells in culture. Analysis of the amino acid composition indicates that fibrillin contains approximately 14% cysteine, of which one-third appears to be in the free reactive sulfhydryl form. Electron microscopic images of fibrillin reveal an extended, flexible molecule approximately 148 nm long and 2.2 nm wide. These length measurements are consistent with shape calculations based upon velocity sedimentation data. It is likely that the material we have purified from cell culture medium represents monomeric fibrillin consisting of a single polypeptide chain. Additional ultrastructural immunohistochemical data presented here suggest a model for the parallel, head-to-tail alignment of fibrillin molecules in microfibrils.

Amino Acids↗

PubMedDate not supplied

[Conformational aspects of glycosylation].

The stereochemical conditions of the enzymatic process of carbohydrate chain addition to the amino acid sequence are reviewed. A particular attention is given to the process of N-glycosylation, which was studied by the method of theoretical conformational analysis.

Amino Acid Sequence↗

PubMed1987

Characterization of specific HLA-DQ alpha allospecificities by genomic, biochemical, and serologic analysis.

Bgl II restriction endonuclease digestion of genomic DNA from lymphoblastoid cell lines homozygous for HLA DR and DQ serological specificities, followed by hybridization with a DQ alpha cDNA probe, identified a genomic polymorphism characterized by two reciprocal patterns, one associated with DR 3, 5 and 8 and the other with DR 1, 2, 4, 7, and 9. The former pattern corresponded precisely to the reactivity of monoclonal antibody SFR20-DQ alpha 5, shown by Western blotting to react with isolated alpha-chains, but not with beta-chains. Additional variants of the DQ alpha genes were identified by using a locus-specific oligonucleotide probe for the DQ alpha gene, indicating differences among the DQ alpha 5-negative set of alleles. This analysis defines a set of DQ alpha allelic markers that are distinct from the well-established DQ serologic specificities DQw1, 2, 3 or "blank." Although most DQ alpha 5+ cells carry the DRw52 specificity associated with the DR beta 2 gene, analysis of DQ alpha polymorphisms on DR5, DQw1; DR8, DQw1; and DRw13, DQw1 cells verified that this DQ alpha family of alleles was not invariably linked to the DR beta 2 locus.

Epitopes↗

PubMed1977

Specific and non-specific interactions of two carcinogenic mycotoxins, luteoskyrin and rugulosin with nucleic acids.

(-) Luteoskyrin and (+) Rugulosin are 2 hepatotoxic and carcinogenic anthraquinone-like pigments from Penicillia which contaminate rice and other foodstuffs. They inhibit in vivo and in vitro replication, transcription and DNA repair of Protocaryots and Eucaryots presumably by forming chelate-complexes with nucleic acids. In the present work, characterization, specificity and structure of binary polychelates (pigment-metal)n and ternary complexes (pigment-metal-nucleic acids) are successively reported: whereas, unspecific complex II with double-stranded polynucleotides is shown to result from simple adsorption of polychelate onto the double helix, complex I is specific for single-stranded DNA (or polypurine nucleotides) and results from electrostatic association of separate pigment-metal units with the polyphosphate chain. Additional stabilization is obtained by hydrophobic interaction between stacked aromatic moieties of pigment and purine units. A model of interaction is proposed for both complexes I and II.

Carcinogens↗

PubMed1982

An analogue of ubiquinone which inhibits respiration by binding to the iron-sulfur protein of the cytochrome b-c1 segment of the mitochondrial respiratory chain.

A synthetic quinone, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), inhibits electron transfer reactions in the cytochrome b-c1 segment of the mitochondrial respiratory chain. Addition of UHDBT to isolated succinate-cytochrome c reductase complex has effects on reduction of the cytochromes b and c1 by succinate similar to those which result from removal of the iron-sulfur protein from the b-c1 complex. Thus, UHDBT inhibits reduction of cytochrome c1 by succinate and, if antimycin is added before succinate, UHDBT inhibits reduction of cytochrome b in addition to c1. UHDBT increases the midpoint potential of the iron-sulfur protein of the b-c1 complex from +280 to +350 mV at pH 7.2. The inhibitor also shifts the gx peak in the EPR spectrum of the iron-sulfur protein from g = 1.80 to 1.76 and shifts the gz peak from g = 2.02 to 2.03. It causes only a slight shift in the central gy = 1.90 signal. The efficacy of inhibition of cytochrome c reductase activity of isolated reductase complex by UHDBT appears to depend on the oxidation-reduction poise of some component(s) in the b-c1 complex. Inhibition is decreased and there is an extensive lag in the onset of inhibition under conditions favoring oxidation of the b-c1 complex; inhibition increases and the lag is eliminated under conditions favoring reduction of the b-c1 complex. The titer for inhibition of cytochrome c reductase activity of isolated reductase complex is one UHDBT per b-c1 complex. With reductase complex from which the iron-sulfur protein of the b-c1 complex is reversibly resolved, the titer for inhibition is proportional to the amount of iron-sulfur protein reconstituted to the complex. These results suggest that UHDBT inhibits mitochondrial respiration by binding to the iron-sulfur protein of the b-c1 complex, possibly at a site which is otherwise involved in binding ubiquinone, and that this binding is enhanced when the iron-sulfur protein is reduced.

Animals↗