[Structural bases of the mechanism of lymph formation].
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Absorption↗
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Collagen metabolism in experimental filariasis.
Chronic lymphatic filariasis is accompanied by edema and fibrosis. In order to assess the latter response, the tissue content of collagen was measured in the skin and lymphatic vessels of normal and affected limbs of cats infected by Brugia malayi. Collagen content, expressed per gram wet weight of tissue, was increased in the infected limbs for both kinds of tissues. Moreover, the proportion of acetic acid soluble to total collagen was increased. The degree of crosslinking in soluble collagen was assessed by acrylamide gel electrophoresis and shown to be decreased in infected tissues. These data suggest an increased rate of collagen metabolism in Brugia malayi-infected tissues and provide data for future studies evaluating the efficacy of treatment in chronic experimental filariasis.
Animals↗
Phosphorylation of RNA polymerase I augments its interaction with autoantibodies of systemic lupus erythematosus patients.
Purified RNA polymerase I was phosphorylated by the endogenous protein kinase or dephosphorylated by alkaline phosphatase and used as antigen in a radioimmunoassay with sera from systemic lupus erythematosus patients or serum from an immunized rabbit. Enzyme incubated in the absence of ATP or phosphatase served as control. Three to seven times more of the autoantibodies in the patients' sera reacted with phosphorylated RNA polymerase I than with control enzyme. The reactivity of the dephosphorylated enzyme with lupus autoantibodies was only 50-60% of that observed with control enzyme. Neither phosphorylation nor dephosphorylation of the enzyme had an effect on its reaction with the rabbit antibodies. The effect of phosphorylation on the reaction of each RNA polymerase I subunit (S1-S8; Mr = 190,000-17,000) with the patients' antibodies was determined by an immunoblot procedure following resolution of the subunits on polyacrylamide gels. Prior phosphorylation of the enzyme resulted in a dramatic increase in binding of each patient's antibodies to all polymerase subunits with the exception of S4. Anti-S4 antibody was not detected with either phosphorylated or control enzyme. Strikingly, antibodies in each patients' sera reacted with S6 only after its phosphorylation. Similarly, anti-S5 antibodies in the serum of one patient were only detected with phosphorylated RNA polymerase I. The present data suggest that at least a significant fraction of the anti-RNA polymerase I autoantibodies in the sera of systemic lupus erythematosus patients might be directed against phosphorylated sites on the enzyme and that phosphorylation may have a role in the production of this and other autoimmunogenic nuclear components which are hallmarks of this disease.
Adenosine Triphosphate↗
Isolation of the membrane-binding peptide of NADPH-cytochrome P-450 reductase. Characterization of the peptide and its role in the interaction of reductase with cytochrome P-450.
A peptide identified as the membrane-associated segment of NADPH-cytochrome P-450 reductase has been generated by steapsin protease treatment of vesicle-incorporated reductase and isolated by preparative gel electrophoresis. This peptide remains associated with vesicles when steapsin protease digests of vesicle-incorporated reductase were fractionated by Sepharose 4B chromatography, confirming its identity as the membrane-binding peptide. The molecular weight of the membrane-binding peptide was 6400 as determined by gel filtration in 8 M guanidine hydrochloride, and its amino acid content was not especially hydrophobic. The activity of reconstituted hydroxylation systems consisting of reductase, cytochrome P-446, and dilauroyl phosphatidylcholine was not inhibited by large molar excesses of purified membrane-binding peptide. Moreover, when purified reductase and cytochrome P-446 were added to liposomes which contained the membrane-binding peptide, it was determined that mixed function oxidase activity was reconstituted as effectively as when vesicles without the membrane-binding peptides were used. Similar results were obtained with reductase, cytochrome P-450, and detergent-solubilized liposomes (with or without the membrane-binding peptide). Thus, the membrane-binding peptide does not appear to interact with either of these two forms of the hemoprotein in a site-specific manner to prevent reconstitution of hydroxylation activity.
7-Alkoxycoumarin O-Dealkylase↗
Effects of cryoglobulins and aggregated IGG on in vitro monocyte phagocytosis.
Monocyte yeast cell phagocytosis (YCP) was low in presence of cryoglobulins(CG) from 7 of 7 patients with SLE and RA with vasculitis and from 2 of 5 patients with essential cryoglobulinaemia. IgM and C3 were detected only in CG associated with low YCP. Furthermore, low YCP was only found with CG isolated from hypocomplementaemic sera. The results also showed low YCP when heat-aggregated IgG (HAG) greater than or equal to 19S was added to the culture medium. In the presence of HAG or SLE-sera, preopsonized yeast cells could be adequately phagocytosed by monocytes whereas, in parallel cultures, the phagocytosis of untreated yeast cells was low. It is possible that CG and HAG impair YCP in the same way as SLE-sera. The nature of this presumed common mechanism has not been clarified in this limited study. The findings are, however, compatible with the possibility that immune complexes may interfere with the opsonization of the yeast cells, possibly through inactivation of complement components.
Antigen-Antibody Complex↗
[Lymphatic delivery of drugs and its application to cancer chemotherapy (author's transl)].
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Antineoplastic Agents↗
Characterization of amyloid related protein SAA complexed with serum lipoproteins (apoSAA).
The amyloid related protein SAA was isolated from the serum lipoproteins of three patients with connective tissue disease, one of them having amyloidosis, and from a pool of normal control sera. The bulk of SAA (apoSAA) was complexed to high density lipoprotein (HDL), but significant amounts of apoSAA were also detected in the other lipoprotein fractions. Electrofocusing revealed five-six subspecies of SAA which were distributed in similar proportions in HDL and LDL. Three of these SAA subspecies made up almost all of the apoSAA present in HDL and LDL in the sera from the patients as well as in the control. A small portion of SAA not complexed to lipoproteins was isolated corresponding to a molecular weight higher than 200,000. No particular 'amyloid prone' SAA was found in serum from the patient with amyloidosis.
Amino Acids↗
Autoimmune antiphospholipid antibodies are directed against a cryptic epitope expressed when beta 2-glycoprotein I is bound to a suitable surface.
The antiphospholipid antibodies (aPL) present in autoimmune disorders are associated with thromboembolic episodes, and their binding to phospholipids (PL) is mediated by a plasma cofactor, beta 2-glycoprotein I (beta 2GPI). Both PL and beta 2GPI seem necessary for binding, thus indicating that the two components comprise the epitope against which aPL are directed. Using an anti-beta 2GPI antibody ELISA with the antigen adsorbed onto polyvinylchloride (PVC) plates, we detected high antibody titres in 12 out of 12 plasma from patients with the antiphospholipid syndrome. No or very low positivity was obtained when the same ELISA was carried out in polystyrene (PST) plates, while an increasing positivity was found when processed (i.e. more hydrophilic) or COOH-surface PST plates were used. When beta 2GPI dependent IgG-aPL were purified using agarose-immobilized cardiolipin, 4 out of 4 preparations were highly positive in anti-beta 2GPI antibody ELISA using PVC plates, while beta 2GPI was not fully recognized by aPL-IgG when adsorbed onto PST plates. These findings demonstrate that aPL are, in fact, anti-beta 2GPI antibodies directed against a cryptic epitope which is expressed when beta 2GPI is bound to anionic phospholipid, or another suitable surface.
Adolescent↗
Co-translational trimerization of the reovirus cell attachment protein.
The reovirus cell attachment protein, sigma1, is a trimer with a 'lollipop' structure. Recent findings indicate that the N-terminal fibrous tail and the C-terminal globular head each possess a distinct trimerization domain. The region responsible for N-terminal trimerization (formation of a triple alpha-helical coiled-coil) is located at the N-terminal one-third of sigma1. In this study, we investigated the temporality and ATP requirement of this trimerization event in the context of sigma1 biogenesis. In vitro co-synthesis of the full-length (FL) and a C-terminally truncated (d44) sigma1 protein revealed a preference for homotrimer over heterotrimer formation, suggesting that assembly at the N-terminus occurs co-translationally. This was corroborated by the observation that polysome-associated sigma1 chains were trimeric as well as monomeric. Truncated proteins (d234 and d294) with C-terminal deletions exceeding half the length of sigma1 were found to trimerize post-translationally. This trimerization did not require ATP since it proceeded normally in the presence of apyrase. In contrast, formation of stable FL sigma1 trimers was inhibited by apyrase treatment. Collectively, our data suggest that assembly of nascent sigma1 chains at the N-terminus is intrinsically ATP independent, and occurs co-translationally when the ribosomes have traversed past the midpoint of the mRNA.
Adenosine Triphosphate↗
A nephelometric study of the reaction of monoclonal rheumatoid factor with heat aggregated gamma globulin and sera from patients with immune complex diseases.
Monoclonal rheumatoid factors (MCRF) have previously been used in a variety of assays for the detection of IgG-containing circulating immune complexes. We have isolated a MCRF from a patient with a lymphoproliferative disorder and have used a nephelometric technique to characterize its reaction with heat-aggreagated gammaglobulin (HAGG) used as a source of artificial immune complexes. The method is simple, economical and rapid and will detect as little as 6 microgram/ml of HAGG over a wide range of physicochemical conditions. A clinical study demonstrated that the sera from thirty-five out of fifty-eight patients (59%) with rheumatoid arthritis and twenty-one out of seventy-four patients (28%) with systemic lupus erythematosus (SLE) gave increased precipitation with MCRF compared with 232 blood donors. However, in marked contrast to previous studies, sucrose gradient ultracentrifugal analysis of nine strongly precipitating sera revealed that in eight the MCRF precipitated with material sedimenting in the monomeric IgG position. In only one specimen did the MCRF react with material sedimenting in heavier regions. It is suggested that different MCRFs vary in the specificity for binding IgG complexes and these reagents should be carefully characterized before becoming established in nephelometric assays for circulating immune complexes.
Adult↗
Anti-beta2-glycoprotein I antibodies.
The relationship between presence of anti-beta2-glycoprotein I autoantibodies (abeta2-GPI) and history of thrombosis is now widely known. However, differences in the methodology of abeta2-GPI detection have made the comparison of data from different laboratories extremely difficult. We discuss the significance of abeta2-GPI of the IgG, IgM and IgA isotypes, and our approach to developing an easier and more reproducible method for the detection of this autoantibody. In addition, we present data that shows that commercially available enzyme immunoassay plates differ regarding detectability of abeta2-GPI. Since the clinical significance of this heterogeneity is presently unclear, the set-up of the detection systems and interpretation of data need great care.
Animals↗
The prevalence of a non-phospholipid-binding form of beta2-glycoprotein I in human plasma--consequences for the development of anti-beta2-glycoprotein I antibodies.
The presence of antiphospholipid antibodies (aPL) is strongly correlated with venous and arterial thrombosis, fetal loss and thrombocytopenia. This relation is called the antiphospholipid syndrome (APS). It is well recognized that thrombosis related aPL are not directed against phospholipids alone, but to phospholipid bound plasma proteins like beta2-glycoprotein I (beta2GPI). aPL that need beta2GPI for the binding to negatively charged phospholipids are called anti-beta2GPI-antibodies. Recently, a mutation in the gene encoding beta2GPI has been described, which results in an amino acid substitution Trp316 into Ser316. This Ser316-beta2GPI did not bind to negatively charged phospholipids. Because only phospholipid bound beta2GPI is recognized by human anti-beta2GPI-antibodies, it might be argued that individuals carrying the Trp316Ser mutation are protected against the development of anti-beta2GPI-antibodies. To investigate this hypothesis, the prevalence of the Trp316Ser mutation was measured in 170 systemic lupus erythematosus (SLE) patients and in 18 patients with the primary antiphospholipid syndrome (PAPS) and the mutation was correlated with the presence of anti-beta2GPI-antibodies. In the total patient group 1 homozygous patient and 21 heterozygous patients were found. The allele frequency of the mutation in SLE patients with anti-beta2GPI-antibodies (0.063) was comparable to that found in SLE patients without anti-beta2GPI-antibodies (0.062). These results indicate that the heterozygous presence of Trp316Ser mutation does not prevent an individual from developing anti-beta2GPI-antibodies. We showed that this can be explained by the concentration of Trp316-beta2GPI in heterozygous patients, which is far above the minimal beta2GPI level necessary for optimal phospholipid binding. In our single patient homozygous for the Trp316Ser mutation no binding beta2GPI to the phospholipid surface was detected and no anti-beta2GPI-antibodies were present in the plasma of this patient. In conclusion, heterozygous Trp316Ser beta2GPI persons are not protected against the development of anti-beta2GPI-antibodies. To confirm that homozygotes do not develop anti-beta2GPI-antibodies a very large population is needed, due to the relatively low prevalence of the mutation.
Adolescent↗
Inhibition of lupus anticoagulant activity by hexagonal phase phosphatidylethanolamine in the presence of prothrombin.
We have previously demonstrated that lupus anticoagulant antibodies from patients with systemic lupus erythematosus (SLE) specifically recognize hexagonal (II) phase phosphatidylethanolamine (PE), but not bilayer PE (Thromb Haemost 1989; 62: 892). In those studies, the involvement of proteins in this recognition was not evaluated. To address this issue, we have isolated IgG lupus anticoagulant antibodies from the plasma of SLE patients and evaluated the inhibition of lupus anticoagulant activity by hexagonal (II) phase PE in the presence and absence of purified plasma proteins. All six of the IgG lupus anticoagulant antibodies tested were inhibited by hexagonal (II) phase PE in the presence, but not the absence, of human prothrombin. In contrast, little or no inhibition was observed with prothrombin alone or with PE in combination with either beta2-glycoprotein I or annexin V. These data indicate that, for certain lupus anticoagulant antibodies, inhibition by hexagonal (II) phase PE is dependent on prothrombin, suggesting that these antibodies recognize a complex of PE and prothrombin.
Adult↗
Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems.
The P1 plasmid partition locus, P1 par, actively distributes plasmid copies to Escherichia coli daughter cells. It encodes two DNA sites and two proteins, ParA and ParB. Plasmid P7 uses a similar system, but the key macromolecular interactions are species specific. Homolog specificity scanning (HSS) exploits such specificities to map critical contact points between component macromolecules. The ParA protein contacts the par operon operator for operon autoregulation, and the ParB contacts the parS partition site during partition. Here, we refine the mapping of these contacts and extend the use of HSS to map protein-protein contacts. We found that ParB participates in autoregulation at the operator site by making a specific contact with ParA. Similarly, ParA acts in partition by making a specific contact with ParB bound at parS. Both these interactions involve contacts between a C-terminal region of ParA and the extreme N-terminus of ParB. As a single type of ParA-ParB complex appears to be involved in recognizing both DNA sites, the operator and the parS sites may both be occupied by a single protein complex during partition. The general HSS strategy may aid in solving the three-dimensional structures of large complexes of macromolecules.
Amino Acid Sequence↗
Nucleoprotein-based nanoscale assembly.
A system for addressing in the construction of macromolecular assemblies can be based on the biospecificity of DNA (cytosine-5) methyltransferases and the capacity of these enzymes to form abortive covalent complexes at targeted 5-fluorocytosine residues in DNA. Using this system, macromolecular assemblies have been created using two representative methyltransferases: M-HhaI and M x MspI. When 5-fluorocytosine (F) is placed at the targeted cytosine in each recognition sequence in a synthetic oligodeoxynucleotide (GFGC for M x HhaI or FCGG for M x MspI), we show that the first recognition sequence becomes an address for M x HhaI, while the second sequence becomes an address for M x MspI. A chimeric enzyme containing a dodecapeptide antigen linked to the C terminus of M-HhaI retained its recognition specificity. That specificity served to address the linked peptide to the GFGC recognition site in DNA. With this assembly system components can be placed in a preselected order on the DNA helix. Axial spacing for adjacent addresses can be guided by the observed kinetic footprint of each methyltransferase. Axial rotation of the addressable protein can be guided by the screw axis of the DNA helix. The system has significant potential in the general construction of macromolecular assemblies. We anticipate that these assemblies will be useful in the construction of regular protein arrays for structural analysis, in the construction of protein-DNA systems as models of chromatin and the synaptonemal complex, and in the construction of macromolecular devices.
Base Sequence↗
Determination of macromolecular exchange and PO2 in the microcirculation: a simple system for in vivo fluorescence and phosphorescence videomicroscopy.
We have developed a system with two epi-illumination sources, a DC-regulated lamp for transillumination and mechanical switches for rapid shift of illumination and detection of defined areas (250-750 microm(2)) by fluorescence and phosphorescence videomicroscopy. The system permits investigation of standard microvascular parameters, vascular permeability as well as intra- and extravascular PO2 by phosphorescence quenching of Pd-meso-tetra (4-carboxyphenyl) porphine (PORPH). A Pechan prism was used to position a defined region over the photomultiplier and TV camera. In order to validate the system for in vivo use, in vitro tests were performed with probes at concentrations that can be found in microvascular studies. Extensive in vitro evaluations were performed by filling glass capillaries with solutions of various concentrations of FITC-dextran (diluted in blood and in saline) mixed with different amounts of PORPH. Fluorescence intensity and phosphorescence decay were determined for each mixture. FITC-dextran solutions without PORPH and PORPH solutions without FITC-dextran were used as references. Phosphorescence decay curves were relatively unaffected by the presence of FITC-dextran at all concentrations tested (0.1 microg/ml to 5 mg/ml). Likewise, fluorescence determinations were performed in the presence of PORPH (0.05 to 0.5 mg/ml). The system was successfully used to study macromolecular extravasation and PO2 in the rat mesentery circulation under controlled conditions and during ischemia-reperfusion.
Animals↗
Blu-Ice and the Distributed Control System: software for data acquisition and instrument control at macromolecular crystallography beamlines.
The Blu-Ice and Distributed Control System (DCS) software packages were developed to provide unified control over the disparate hardware resources available at a macromolecular crystallography beamline. Blu-Ice is a user interface that provides scientific experimenters and beamline support staff with intuitive graphical tools for collecting diffraction data and configuring beamlines for experiments. Blu-Ice communicates with the hardware at a beamline via DCS, an instrument-control and data-acquisition package designed to integrate hardware resources in a highly heterogeneous networked computing environment. Together, Blu-Ice and DCS provide a flexible platform for increasing the ease of use, the level of automation and the remote accessibility of beamlines. Blu-Ice and DCS are currently installed on four Stanford Synchrotron Radiation Laboratory crystallographic beamlines and are being implemented at sister light sources.
Crystallography, X-Ray↗
Total parenteral nutrition in germfree colostrum-deprived neonatal miniature piglets: a unique model to study the ontogeny of the immune system.
In order to investigate the regulatory role of the immune system on gastrointestinal macromolecular permeability in neonates, and ontogeny of the neonatal immune system, we have developed a total parenteral nutrition (TPN) program capable of maintaining immunologically "virgin" germfree (GF) neonatal piglets. GF colostrum-deprived piglets obtained by aseptic hysterectomy 3-5 days prior to term were denied any oral feeding and maintained on central venous hyperalimentation in the GF isolators. GF piglets were anesthetized and 20-gauge silicone tubing was inserted into the external jugular vein and advanced to the level of right auricle of the heart. The distal end of the cannula was subcutaneously tunneled out between the shoulders and attached to a light jacket, tether, and swivel assembly that allowed the piglet to move freely inside the cage in a germfree unit. The TPN formula provided 60 Cal/kg/day for the first day and gradually increased up to 165 Cal/kg/day for day-7 and older piglets. The GF piglets were maintained on the TPN formula for 21 days and weight gain of the GF-TPN piglets averaged 23 g/day. The hematologic and serum biochemical parameters of GF-TPN piglets were within the normal range of gnotobiotic animals of their age group. The serum levels of total protein and albumin in the GF-TPN piglets were significantly less than those of colostrum-fed piglets, who absorbed a large quantity of the colostral macromolecules through their gastrointestinal tract into the blood stream.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗