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PubMed2000

Time to pregnancy among female greenhouse workers.

OBJECTIVES: This study examined the possibility that work in greenhouses with potential exposure to pesticides entails a risk for reduced fecundity in terms of increased time to pregnancy. METHODS: Among 1767 female members of the Danish Gardeners Trade Union, telephone interview data were obtained on the 492 most recent pregnancies of women employed when they stopped contraception to get a child (the starting time). The pregnancies were classified according to job characteristics at the starting time. The ratio between the likelihood of pregnancy during a month for the exposed persons versus the referents (the fecundability ratio) was estimated by discrete proportional hazards regression. RESULTS: The adjusted fecundability ratio for workers in flower greenhouses versus other union members was 1.11 [95% confidence interval (95% CI) 0.90-1.36]. Among workers in flower greenhouses the handling of cultures many hours per week, the spraying of pesticides, and the nonuse of gloves was related to reduced fecundability [adjusted fecundability ratio 0.69 (95% CI 0.47-1.03), 0.78 (95% CI 0.59-1.06), and 0.67 (95% CI 0.46-0.98), respectively]. CONCLUSIONS: The findings suggest that female workers in flower greenhouses may have reduced fecundability and that exposure to pesticides may be part of the causal chain. Additional studies of fertility among women working in greenhouses are highly warranted.

Adult↗

PubMed1999

Chemical cleavage of the overexpressed mitochondrial F1beta precursor with CNBr: a new strategy to construct an import-competent preprotein.

We have isolated a soluble import-competent 15 kDa N-terminal fragment of the overexpressed Nicotiana plumbaginifolia F1beta precursor of the ATP synthase (N15pF1beta). The isolation was achieved after chemical cleavage, with CNBr, of the insoluble precursor collected in inclusion bodies, followed by purification of the fragment using ion-exchange chromatography. The purity of the final product was estimated to be more than 99%. N15pF1beta contained a presequence of 54 amino acid residues (except for the N-terminal methionine residue) and 82 N-terminal residues of the mature protein. N15pF1beta was shown to be imported into isolated potato tuber mitochondria and to be processed by the isolated mitochondrial processing peptidase (MPP) integrated into the cytochrome bc1 complex of the respiratory chain. Addition of N15pF1beta at micromolar concentrations resulted in the inhibition of import of F1beta precursor and alternative oxidase precursor, synthesized in vitro, into isolated mitochondria as well as the processing of these precursors catalysed by the isolated MPP-bc1 complex. N15pF1beta conjugated via a biotin link to avidin blocked import sites even after the reisolation of mitochondria and inhibited the import of the mitochondrial precursors, indicating that it can be used as a substrate for the generation of a stable translocation intermediate. Our results present a novel procedure for the production of an N-terminal fragment of the F1beta precursor that contains all information necessary for mitochondrial targeting and processing and that can be used for structural and functional studies of the mitochondrial protein import system. This procedure has a general value because it can be used for the production of chemical quantities of any mitochondrial import substrate and presequence peptide.

Avidin↗

PubMed2000

The penultimate rotamer library.

All published rotamer libraries contain some rotamers that exhibit impossible internal atomic overlaps if built in ideal geometry with all hydrogen atoms. Removal of uncertain residues (mainly those with B-factors >/=40 or van der Waals overlaps >/=0.4 A) greatly improves the clustering of rotamer populations. Asn, Gln, or His side chains additionally benefit from flipping of their planar terminal groups when required by atomic overlaps or H-bonding. Sensitivity to skew and to the boundaries of chi angle bins is avoided by using modes rather than traditional mean values. Rotamer definitions are listed both as the modal values and in a preferred version that maximizes common atoms between related rotamers. The resulting library shows significant differences from previous ones, differences validated by considering the likelihood of systematic misfitting of models to electron density maps and by plotting changes in rotamer frequency with B-factor. Few rotamers now show atomic overlaps in ideal geometry; those overlaps are relatively small and can be understood in terms of bond angle distortions compensated by favorable interactions. The new library covers 94.5% of examples in the highest quality protein data with 153 rotamers and can make a significant contribution to improving the accuracy of new structures. Proteins 2000;40:389-408.

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↗

PubMed1981

Post-translational glycosylation-induced activation of aglycoinsulin receptor accumulated during tunicamycin treatment.

Tunicamycin, which inhibits N-linked oligosaccharide chain addition to nascent polypeptides, interrupts glycosylation of the insulin receptor in 3T3-L1 adipocytes giving rise to inactive receptors. Chronic exposure of cells to low levels (100 ng/ml) of high performance liquid chromatography-purified tunicamycin causes a greater than or equal to 90% depletion of insulin binding to cell surface and Triton X-100-extractable receptors and a 93% inhibition of [3H]glucosamine incorporation into protein in alkali-stable form. Under identical conditions, protein synthesis was inhibited less than 10%. Recovery of insulin binding activity after the removal of tunicamycin achieves 70-80% of control activity within 36 h. Concomitant with the withdrawal of tunicamycin, cells were shifted to medium containing heavy (greater than 95% 15N, 13C, 2H) amino acids after which Triton X-100-solubilized "light" and "heavy" insulin receptors were separated isopycnically on CsCl density gradients. A kinetic analysis of the recovery of functional receptors revealed that the initial appearance of previously synthesized light receptor was followed, after a short lag, by newly synthesized heavy receptor. Similar levels of light receptor, but no new heavy receptor, accrue in the presence of cycloheximide. This strongly suggests that inactive aglycoinsulin receptor accumulated post-translationally during chronic treatment with tunicamycin and then re-entered the glycosylation pathway when the inhibitor was removed giving rise to a functional insulin receptor.

Animals↗

PubMed1993

Effects of long-chain fatty acids on the channel activity of the nicotinic acetylcholine receptor.

We have analyzed the effect of free fatty acids on the function of the acetylcholine receptor (AChR) at the single-channel level, using the patch-clamp technique. Long-chain fatty acids, in the presence of albumin as a carrier, were applied to intact cells or to the cytoplasmic surface of excised membrane patches. In the latter case, AChR channels underwent immediate changes in their behaviour and only very brief opening events were apparent. This could be accounted for by a four-fold reduction in the channel mean open time, with no significant changes occurring in the conductance. An increase in the duration of intermediate closed intervals and a decrease in the burst duration were also observed. The modification appeared not to be critically dependent on the degree of saturation of fatty acyl chains. Addition of free fatty acids in the absence of albumin, as well as treatment of the excised membrane patches with phospholipase A2, resulted in complete inhibition of AChR channel activity. In intact cells, fatty acids could reach and affect AChR channels in the plasmalemma under the patch pipette when added from outside the patch-clamped area. The fatty acid-modified AChR was still able to undergo desensitization. The open channel probability was higher than 0.8 for 100 microM agonist, decreasing to 0.4 in the fatty acid-modified receptors. The results are discussed within the framework of the hypothesis that some lipophilic compounds exert their action on the AChR-lipid interface (the "annulus") (see Barantes, 1993).

Acetylcholine↗

PubMed1993

A monoclonal antibody to recombinant human IFN-alpha receptor inhibits biologic activity of several species of human IFN-alpha, IFN-beta, and IFN-omega. Detection of heterogeneity of the cellular type I IFN receptor.

A cDNA encoding a 63-kDa human IFN-alpha R (hIFN-alpha R) has recently been cloned. Mouse cells transfected with this cDNA failed to show any signal transmission for IFN-beta, suggesting the involvement of additional chains in the receptor complex. We have expressed in Escherichia coli and COS 7 cells a soluble recombinant protein (sIFN-alpha R) comprising the extracellular domain of the hIFN-alpha R fused at the carboxyl terminus to a sequence of five histidine residues. The sIFN-alpha R was affinity purified and used to generate mAb. One mAb, 64G12, which recognized the cellular receptor as well as the recombinant proteins was found to neutralize the biologic activity of IFN-alpha, -beta, and -omega and natural type I IFN, but not IFN-gamma. To our knowledge, this is the first report of a neutralizing mAb against the human IFN-alpha R. Interestingly, we found that the affinity of this antibody for the cellular receptor depended on the cell line used. These results provide strong evidence that the cloned hIFN-alpha R chain is required for binding and signal transmission of all subspecies of type I IFN and suggest a structural heterogeneity of the receptor at the cell surface.

Animals↗

PubMedDate not supplied

Immune complexes from sera of patients with juvenile rheumatoid arthritis reveal novel 40 and 60 kd bands.

OBJECTIVE: Immune complexes (IC) have been detected in the majority of sera of patients with juvenile rheumatoid arthritis (JRA). Our aim was to determine the possible constituents of these IC. METHODS: One hundred and four sera from patients with JRA were separated on a Sepharose 4B column to which were bound F(ab')2 fragments of goat anti-human IgM antibody to separate IgM-containing IC. The column was sequentially eluted with 1 M ammonia and 0.1 M glycine-HCl buffer, pH 3. The eluted material was treated with sodium dodecyl sulfate (SDS) and simultaneously reduced with 2-mercaptoethanol. Individual components were then separated by SDS-gradient polyaclylamide gel electrophoresis and were transferred to nitrocellulose by Western blotting. The nitrocellulose strips were overlaid with the patient's own serum and developed with specific antiserum to human IgM and IgG. RESULTS: Four bands were noted in the majority of JRA patients, including a band in the 70-80 kD area to the IgM heavy chain and in the 50 kD area to the IgG heavy chain. Additional bands specific for JRA patients' sera were noted in the 40 kD and 60 kD areas. Overall, 61 of 104 sera demonstrated all four bands; 78 showed the 40 kD band and 73 the 60 kD band. CONCLUSION: These IC were previously shown to contain IgM rheumatoid factor (RF), IgG-RF, and IgG in addition to the 40 and/or 60 kD constituents present in the isolates. Preliminary studies on these constituents suggest the possibility of the 60 kD band being the human 60 kD mitochondrial heat shock protein. Further investigations of these immune complex components may aid in the understanding of the pathophysiology of JRA.

Adolescent↗

PubMedDate not supplied

[Effect of chain length on formation of a double-layer structure of Z-(Gly-Pro-Gly)n-OMe oligotripeptides].

Effect of chain length on the formation of the double-layered sheet in synthetic oligotripeptides Z-(Gly-Pro-Gly)n-OMe (n = 1,2,3,4) in the solid state has been studied by the IR-spectroscopy method. The major spectral features characteristic for the polymer structure was found in the oligomer spectra beginning with that of hexapeptide (n = 2). So a minimum peptide length when peptide chains are still able to adopt the double-layered sheet conformation in the solid state includes two triplets. The result is similar to that we have revealed early by investigation of the collagen-like triple helix formation in the oligomer series Z-(Gly-Pro-Pro)n-OMe. The double-layered sheet structure of the hexapeptide was found completely regular as that of the polymer. So the formation of the double-layered sheet structure unlike the formation of the triple-helical structure occurs at once without gradual ordering of interpeptide bonds and molecular packing by further increasing of the triplet number in oligomer chains. Additional information concerning specific incorporation of bound water in the double-layered sheet and water influence on peptide packing has been derived from analyses of hydration effect on the oligotripeptide IR-spectra especially on the components of the amide I band.

Amino Acid Sequence↗

PubMed1997

Bisphosphonates: structure-activity relations from a clinical perspective.

Bisphosphonates, synthetic compounds containing two phosphonate groups bound to a central (geminal) carbon (P-C-P) and two additional chains (R1 and R2, respectively) bind strongly to calcium crystals, inhibit their growth and suppress osteoclast-mediated bone resorption. The availability of two side chains allows numerous substitutions and the development of a variety of analogs with different pharmacological properties. The R1 structure together with the P-C-P are primarily responsible for binding to bone mineral and for the physicochemical actions of the bisphosphonates and a hydroxyl group at R1 provides optimal conditions for these actions. The R2 is believed to be responsible for the antiresorptive action of the bisphosphonates and small modifications or conformational restrictions of this part of the molecule may result in marked differences in antiresorptive potency. The presence of a nitrogen function in an alkyl chain or in a ring structure in R2 greatly enhances the antiresorptive potency and specificity of bisphosphonates for bone resorption and most of the newer potent bisphosphonates contain a nitrogen in their structure. Recent evidence, however, suggests that the whole bisphosphonate molecule is essential for antiresorptive action. Thus, although the basic structural requirements for bisphosphonate actions have been defined, precise structure-activity relations have not been defined yet. These are essential for elucidating their molecular mechanism of action and for the rational design of compounds for various clinical indications.

Bone Resorption↗

PubMed1998

Proapoptotic activity of a Trypanosoma cruzi ceramide-containing glycolipid turned on in host macrophages by IFN-gamma.

The effects of glycoinositolphospholipid (GIPL), from the pathogenic protozoan Trypanosoma cruzi, and its isolated glycan and lipid (dihydroceramide) components, were investigated in J774 cells and primary macrophages. Isolated GIPL ceramide, but not intact GIPL or its glycan, induced intense fluid phase endocytosis when added exogenously. In the presence of the cytokine IFN-gamma, GIPL ceramide induced marked apoptosis in J774 cells and macrophages, independent of nitric oxide secretion. When cells were preincubated with the GIPL-derived glycan chain, addition of intact GIPL induced macrophage apoptosis in the presence of IFN-gamma. Synthetic C2-dihydroceramide also induced apoptosis in the presence of IFN-gamma. Induction of apoptosis in T. cruzi-infected macrophages by GIPL ceramide plus IFN-gamma led to increased parasite release compared with IFN-gamma treatment alone. Viable parasites released comprised both infective trypomastigote and spheromastigote forms. These results identify a novel pathway by which T. cruzi glycosylphosphatidylinositol family molecules affect host macrophages, with implications for the infectious process.

Animals↗

PubMed1992

Polymerase chain reaction with additional primers allows identification of amplified DNA and recognition of specific alleles.

The use of additional primers in the standard two primer polymerase chain reaction (PCR) is described. This modification allows detection of a target gene in a single reaction, and identification of the amplification product obtained or recognition of a specific allele. The oligonucleotides used are internal to the original amplification primers and amplification-compatible with one of the original primers. Annealing of an additional primer to the target gene as well as to the primary amplification product will lead to the appearance of an additional smaller amplification fragment upon agarose gel electrophoresis of PCR products. Use of one or more allele-specific oligonucleotides as additional primers, in addition to two gene-specific primers, will allow recognition of different alleles of the target gene in a single PCR without further analysis except gel electrophoresis. The general applicability of the method was determined with several PCR assays for the detection of pathogenic bacteria.

Alleles↗