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PubMed2001

Reversal of methylation-mediated repression with short-chain fatty acids: evidence for an additional mechanism to histone deacetylation.

We have constructed a stable cell line, human embryonal kidney 293M+, containing a lacZ reporter gene controlled by an in vitro methylated hormone-responsive enhancer. Methylation of the enhancer-promoter abolishes lacZ expression controlled by ponasterone A (an analogue of ecdysone). Ponasterone A-induced expression is restored by the short-chain fatty acids valeric > butyric > propionic > acetic acid, but not by the histone deacetylase inhibitors trichostatin A and suberoylanilide hydroxamic acid (SAHA). lacZ expression is restored to levels approaching that from an unmethylated counterpart. Incubation with short-chain fatty acids alone does not promote demethylation of the lacZ promoter, however, some demethylation (30%) is observed when transcription is triggered by addition of ponasterone A. Similar levels of hyperacetylated histones H3 and H4 were observed in cells treated with short-chain fatty acids, trichostatin A or SAHA. In vivo DNase I footprinting indicates a more open chromatin structure at the promoter region for butyric acid-treated cells. A synergistic effect in reversing the methylation-mediated repression of the lacZ gene is obtained by combined treatments with the normally ineffective compounds trichostatin A and the short-chain fatty acid caproic acid. Our results suggest the existence of an alternative silencing mechanism to histone deacetylation in executing methylation-directed gene silencing.

Animals↗
PubMed2005

Dynamics and lithium binding energies of polyelectrolytes based on functionalized poly(para-phenylene terephthalamide).

Polyelectrolyte materials are an interesting class of electrolytes for use in fuel cell and battery applications. Poly(para-phenylene terephthalamide) (PPTA, Kevlar) is a liquid crystalline polymer that, when sulfonated, is a polyelectrolyte that exhibits moderate ion conductivity at elevated temperatures. In this work, quasi-elastic neutron scattering (QENS) experiments were performed to gain insight into the effect of the presence of lithium counterions on the chain dynamics in the material. It was found that the addition of lithium ions decreases the dynamics of the chains. Additionally, the binding of lithium ions to the sulfonic acids groups was investigated by density functional theory (DFT) calculations. It was found that the local surroundings of the sulfonic acid group have very little effect on the lithium-ion binding energy. Binding energies for a variety of different systems were all calculated to be around 150 kcal/mol. The DFT calculations also show the existence of a structure in which a single lithium ion interacts with two sulfonic acid moieties on different chains. The formation of such "electrostatic cross-links" is believed to be the source of the increased tendency to aggregate and the reduced dynamics in the presence of lithium ions.

Journal Article↗
PubMed1997

Structure of chain d of the gigantic hemoglobin of the earthworm.

The extracellular hemoglobin of the earthworm has four major O2-binding chains, a, b, c and d, together with additional non-heme structural chains that are required for assembly. Although the abc trimer self-associates extensively at least to (abc)10, addition of chain d results in the formation of a discrete 280 kDa complex corresponding to (abcd)4. Thus a primary function of chain d is to cap the abc association and convert an abc trimer that binds O2 with weak cooperativity to a highly cooperative (abcd)4 complex. Amino-acid sequences of the major globin chains a, b, c have been determined previously by peptide and cDNA analysis. However, the peptide sequence reported for the major chain d (Shishikura, F., Snow, J.W., Gotoh, T., Vinogradov, S.N. and Walz, D.A. (1987) J. Biol. Chem., 262. 3123-3131), has a calculated molecular mass 134-167 Da higher than masses for components of chain d determined by mass spectrometry (Owrby, D.W., Zhu, H., Schneider, K., Beavis, R.C., Chait, B.T. and Riggs, A.F. (1993) J. Biol. Chem. 268, 13539-13547). Reverse-phase HPLC confirms the presence of two distinct polypeptides, d1 and d2, together with d'1, a variant of d1, cDNA derived amino acid sequences have been determined for chains d'1 and d2 by application of the polymerase chain reaction with primers based on the NH2-terminal sequences and oligo-dT. Each of the two cDNA-derived sequences has 140 residues and they differ by 28 substitutions. The data show that the sequence originally reported had been derived from peptides generated from both polypeptides.

Amino Acid Sequence↗
PubMed1996

Two glycosyltransferase genes, lgtF and rfaK, constitute the lipooligosaccharide ice (inner core extension) biosynthesis operon of Neisseria meningitidis.

We have characterized an operon required for inner-core biosynthesis of the lipooligosaccharide (LOS) of Neisseria meningitidis. Using Tn916 mutagenesis, we recently identified the alpha-1,2-N-acetylglucosamine (GlcNAc) transferase gene (rfaK), which when inactivated prevents the addition of GlcNAc and alpha chain to the meningococcal LOS inner core (C. M. Kahler, R. W. Carlson, M. M. Rahman, L. E. Martin, and D. S. Stephens, J. Bacteriol. 178:1265-1273, 1996). During the study of rfaK, a second open reading frame (lgtF) of 720 bp was found upstream of rfaK. An amino acid sequence homology search of the GenBank and EMBL databases revealed that the amino terminus of LgtF has significant homology with a family of beta-glycosyltransferases involved in the biosynthesis of polysaccharides and O antigen of lipopolysaccharides. The chromosomal copy of lgtF was mutagenized with a nonpolar antibiotic resistance cassette to minimize potential polar effects on rfaK. Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and composition analysis of the LOS from the nonpolar lgtF mutant showed that this strain produced a truncated LOS structure which contained a LOS inner core of GlcNAc1Hep2KDO2lipid A but without the addition of lacto-N-neotetraose to HepI or glucose to HepII. These results and the amino acid homology with beta-glycosyltransferases suggest that lgtF encodes the UDP-glucose:LOS-beta-1,4-glucosyltransferase which attaches the first glucose residue to HepI of LOS. Reverse transcriptase PCR and primer extension analysis indicate that both lgtF and rfaK are cotranscribed as a polycistronic message from a promoter upstream of lgtF. This arrangement suggests that completion of the LOS inner core and the initiation of the alpha chain addition are tightly coregulated in N. meningitidis.

Amino Acid Sequence↗
PubMed1998

Introduction of a new scale into reversed-phase high-performance liquid chromatography of pyridylamino sugar chains for structural assignment.

Addition of a monosaccharide residue to a pyridylaminated (PA)-N-linked sugar chain results in an increment or decrement in the elution time on reversed-phase HPLC, the difference being defined as the partial elution time of the residue. Based on this principle, an empirical rule was deduced, which states that the elution time is roughly equal to the sum of the partial elution times of the component sugar residues [Anal. Biochem., 167 (1987) 321-326]. In practice, however, some partial elution times obtained from different pairs of mother PA-sugar chains are found to deviate, and consequently the closeness of the elution times of PA-sugar chains calculated therefrom to the observed times is reduced in such cases. To improve the reliability of the additivity rule and to generalize elution times so that they are less dependent on minor alterations in the elution conditions, we have devised a new scale for elution time, which we have named a reversed-phase scale. The elution times on the reversed-phase scale (the R values) are read from a conversion curve constructed using the elution times of eight selected standard PA-sugar chains. The partial elution times on the reversed-phase scale of 22 monosaccharide residues were calculated from the R values of 93 PA-sugar chains. The R values obtained by summing the partial elution times of all the component monosaccharide residues became much closer to the R values obtained from the reversed-phase scale, compared to the results obtained using the previous method. In addition, the R values were less influenced by minor change in the elution conditions. These features of the new scale allow more accurate structural assignment of sugar chains.

Amino Sugars↗
PubMed1989

The intermediate-affinity interleukin (IL)2 receptor expressed on Theileria annulata-infected cells comprises a single IL 2-binding protein. Partial characterization of bovine IL2 receptors.

Bovine high-, intermediate- and low-affinity interleukin 2 receptors (IL2R) were studied by ligand binding and affinity labeling using 125I-labeled IL2 and homobifunctional chemical cross-linking reagents. High- (Kd = 17 pM) and low-affinity (Kd greater than 6 nM) IL 2R were detected on concanavalin A-activated peripheral blood lymphocytes (PBL). Theileria annulata (TA)-infected autonomously growing PBL (TA-PBL) express predominantly intermediate-affinity IL2R (Kd = 1 nM). Affinity-labeling studies revealed that the high-affinity IL2R comprises a 55-kDa (L chain) and an additional 90-kDa IL 2-binding protein (H chain). TA-PBL express predominantly the H chain. In contrast to the human chain, the bovine form was not separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/urea into the two distinct bands H1 and H2 and ran in parallel to the human H2 band. These results suggest (a) that the bovine intermediate-affinity IL2R comprises a single H chain and (b) that the single H chain and the L chain are sufficient to form the functional high-affinity bovine IL2R.

Affinity Labels↗
PubMed1993

Change of paraprotein from IgG lambda to lambda chain in myeloma.

We describe a unique case of IgG lambda myeloma in which a plasma cell leukemia developed and the paraprotein changed from IgG lambda to lambda chain, the latter inducing fatal renal failure. In addition, J-chain was detected in the cytoplasm of the plasma cell leukemia cells.

Female↗
PubMed1996

Mannose receptor dependent uptake of ricin A1 and A2 chains by macrophages.

The ricin A chain, the toxic subunit of ricin, consists of two forms which differ in sugar content. The major component A1 contains one high mannose chain while the minor component A2 contains an additional high mannose chain. Endocytosis of this toxin occurs in macrophages via the mannose receptor. To study the role of the sugar residues in ricin A chain cytotoxicity, we have purified the two forms by ion-exchange chromatography. The uptake of A1 and A2 by a macrophage cell line was concentration and time dependent. The total amount of A2 internalized was approximately twice the amount of A1, indicating a higher affinity of A2 for the mannose receptor. Ricin A2 was four times more toxic to macrophages than A1, in agreement with the higher affinity of A2 compared to the A1. These experiments suggest that the high mannose chains on the A chain promote mannose-receptor-mediated endocytosis by providing the initial binding to the cell surface. Once the toxin is accumulated inside the cell however, the carbohydrates do not seem to influence intracellular transport and/or translocation of the ricin A chain into the cytoplasm.

Animals↗
PubMed1983

Synthesis of hyaluronate in differentiated teratocarcinoma cells. Mechanism of chain growth.

Hyaluronate could be labelled in vivo with [32P]phosphate. [32P]UDP in an alpha-glycosidic linkage constituted the reducing end of membrane-bound hyaluronate. The UDP is liberated during further chain elongation, indicating that chain growth occurs at the reducing end. [3H]Uridine could be incorporated into hyaluronate during synthesis on the isolated membraneous fraction from [3H]UDP-GlcNAc and [3H]UDP-GlcA, confirming the identification of UDP as a constituent of membrane-bound hyaluronate. These results led to a model of hyaluronate chain elongation at the reducing end by alternate addition of the chains to the substrates. Membrane-bound pyrophosphatases or 5'-nucleotidase are suggested as modulators of hyaluronate synthesis.

Cell Membrane↗
PubMed1971

Localization of two additional hypervariable regions in immunoglobulin heavy chains.

Cyanogen bromide fragments were isolated from the heavy chains of three human IgG myeloma proteins of the V(H)III subgroup, sequenced by an automated method, and localized to the variable region. Inspection of these sequences, together with corresponding stretches from both human and animal proteins (studied in other laboratories) led to the detection of two additional hypervariable regions characteristic of the V(H) segment of immunoglobulin heavy chains. These areas of hypervariability, involving heavy-chain residues 86-91 and 101-109, were separated by a region of relative constancy. The close relationship of these two hypervariable regions, and the previously described first heavy-chain hypervariable region (residues 31-37), to the first heavy-chain disulphide bridge implies that the three hypervariable areas might be in close steric approximation in native immunoglobulin molecules. Examination of the sequences of the terminal portion of V(H) of all these proteins (the segment from residue 95 to the beginning of homology region C(H)l) revealed that no subgroup-specific residues could be identified in this area. Thus, heavy-chain subgroup distinctions may not extend through the entire variable region.

Amino Acid Sequence↗
PubMed2002

Heterologous protection induced by the inner capsid proteins of rotavirus requires transcytosis of mucosal immunoglobulins.

Protective immunization against rotavirus (RV) can be achieved with heterologous RV, i.e., virus isolated from another species, and with heterologous inner core VP2 and VP6 proteins assembled as virus-like particles (VLP). Although the antigenically conserved VP6 protein does not induce in vitro-neutralizing antibodies, it may, however, elicit immunoglobulins (Ig) involved in heterologous protection, as some IgA against VP6 prevent RV infection in a backpack mouse model. The protective role of Ig directed to the RV inner core proteins VP2 and VP6 was investigated in J-chain-deficient mice (J chain(-/-)), which have a defect in the polymeric Ig receptor (pIgR)-mediated transcytosis of IgA and IgM. J chain(-/-) mice and wild-type (WT) mice were intranasally vaccinated with bovine RV-derived VLP2/6 and then challenged with highly infectious murine ECw RV. Whereas WT mice were totally protected, immunized J chain(-/-) mice shed RV for several days. In addition, naïve J chain(-/-) mice exhibited a 2-day delay in clearing RV compared with WT mice. The immunized J chain(-/-) mice displayed unaltered VLP2/6-specific B-cell numbers in spleen and in mesenteric nodes and similar levels of serum anti-VLP2/6 Ig, confirming that the adaptive B-cell response is preserved in J chain(-/-) mice. These results indicate that J-chain-mediated transcytosis of Ig participates in the clearance of RV and that epithelial pIgR-mediated transport of Ig is involved in the heterologous protection induced by VLP2/6.

Administration, Intranasal↗
PubMed1998

Analysis of kappa light chain contribution to anti-DNA antibody activity of a human VH4-21-encoded monoclonal antibody (NE-1) by antibody-phage display technique.

Although contribution of light chain to DNA reactivity of some murine anti-DNA antibodies (Abs) has been demonstrated, similar studies on human anti-DNA Abs are limited. To investigate this contribution, we reproduced Fab molecules on the surface of phages from a human B cell line producing IgM anti-DNA monoclonal Ab (NE-1) by an Ab-phage display technique. Expressed Fab molecules (p4-1 clone) were similar to the parental mAb in their binding activities and idiotypic expression. We constructed a light chain shuffled library containing Vkappa genes derived from peripheral blood lymphocytes of a patient with systemic lupus erythematosus (SLE) in combination with the NE-1 heavy chain gene. After panning to ss- or dsDNA, 7 Fab-phage clones which showed significant bindings to ss- or dsDNA were isolated. Many other Fab-phage clones from the library did not bind to ss- nor dsDNA. Sequence analysis revealed that light chains of the 7 clones are derived from diverse Vkappa germline genes including rarely used ones such as the L5 and A30. Most of the Vk germline genes have been used for previously reported anti-DNA antibodies. These findings suggest that diverse Vkappa genes can pair with the NE-1 heavy chain for anti-DNA Ab activity. In addition, kappa light chains seem to modulate DNA binding activities in various ways.

Amino Acid Sequence↗
PubMed1985

Sequence of gene and cDNA encoding murine major histocompatibility complex class II gene A beta 2.

The murine major histocompatibility complex is known to express two class II molecules, A and E. They are composed of one alpha- and one beta-polypeptide chain. Recently, we reported the finding of an additional beta-chain second domain exon tentatively designated A beta 2. We describe here the nucleotide sequence of the A beta 2 gene and an A beta 2 cDNA clone. A beta 2 displays the same exon organization as the known expressed beta-chain genes, and the predicted A beta 2 polypeptide shows all the characteristic features of the expressed beta-chains. The predicted A beta 2 polypeptide shows 49-56% amino acid sequence identity to A beta and E beta chains and to the human DP, DQ, and DR beta-chains. These are 63-79% homologous to each other, indicating that A beta 2 is the most divergent member of the beta-chain family. The A beta 2 gene seems to be transcribed in the same types of cells as other class II genes. Detection of incompletely spliced A beta 2 mRNA and the finding of a cDNA clone containing an intron segment suggest that A beta 2 transcripts are processed slowly. Hybridizations with A beta 2 probes to restriction enzyme-digested genomic DNA indicate that the A beta 2 gene displays lower allelic polymorphism than the A beta gene.

Amino Acid Sequence↗
PubMed1979

Effects of 4-methyl umbelliferyl-beta-D-xylopyranoside on chondrogenesis and proteoglycan synthesis in chick limb bud mesenchymal cell cultures.

When chick limb bud mesenchyme cells from stage 23 to 24 embryos are plated at high density, they rapidly divide and a large proportion initiate chondrogenic expression during the first 2 to 3 days in culture. Between Days 4 and 8, the emergent chondrocytes mature and elaborate a cartilaginous matrix. The proteoglycans synthesized by the newly emergent Day 3 to 4 chondrocytes differ from those synthesized by either the prechondrogenic mesenchyme cells or the mature Day 8 chondrocytes. Cultures were grown from initial plating (Day 0) or from Day 2 in the continuous presence of 1 mM 4-methyl umbelliferyl-beta-D-xyloside, which acts intracellularly as a competitive acceptor with the endogenous core protein of proteoglycans for chondroitin sulfate synthesis. The proteoglycans synthesized by Day 8 cultures which had been maintained on xyloside or to which xyloside was added only 1 h prior to labeling were essentially identical. They were able to form aggregates, and they contained the same number of keratan sulfate chains, but only about 40% as many chondroitin sulfate chains, as normal. Additionally, both the chondroitin sulfate and keratan sulfate chains were 25% shorter than in the normal proteoglycans. The proteoglycans synthesized by cells in a culture maintained on xyloside until Day 8, and then switched to medium with no xyloside 1 h prior to labeling, were characteristic of those synthesized by normal mature Day 8 chondrocytes. These data suggest that stage 23 to 24 mesenchyme cells undergo normal chondrogenic maturation in culture in the presence of xylosides even though (a) most of the polysaccharides are synthesized onto the exogenously supplied xyloside substrate and released into the medium, (b) the proteoglycans that are synthesized are greatly reduced in polysaccharide content, and (c) the extracellular matrix as a consequence is greatly depleted in chondroitin sulfate content and, therefore, is abnormal in general morphology.

Animals↗
PubMed1993

Alterations in the electron transfer chain in mutant strains of Escherichia coli lacking phosphatidylethanolamine.

Inside-out sealed membrane vesicles were prepared from strains of Escherichia coli engineered to be lacking in the major phospholipid of this organism, phosphatidylethanolamine (DeChavigny, A., Heacock, P. N., and Dowhan, W. (1991) J. Biol. Chem. 266, 5323-5332). The energy transducing properties, namely the ability to generate a proton gradient directed inward and to transport electrons to molecular oxygen, were compared to those of membranes isolated from wild type cells containing normal levels of phosphatidylethanolamine. Membranes from both cell types were equal in their ability to oxidize succinate and lactate as well as hydrolyze ATP with the generation of proton gradients of similar magnitude, thus establishing the structural integrity of the membrane barrier and basic functionality of the energy transducing systems in the mutant membranes. However, mutant membranes were reduced by about 80% in their type II NADH dehydrogenase-dependent oxidase activity which resulted in a reduced ability to generate a proton gradient using NADH as an energy source. Use of artificial electron acceptors indicated that the level of type II NADH dehydrogenase activity was normal. Whole chain NADH oxidase activity could be restored by addition of short chain analogs of the naturally occurring Q8, even though the level of the Q8 pool in both cell types was the same. These results suggest that the function of Q8 in linking type II NADH dehydrogenase with the terminal oxidase(s) is dependent on the phosphatidylethanolamine content of the surrounding phospholipid matrix.

Cell Membrane↗
PubMed1995

Circulating dermatan sulfate and heparan sulfate/heparin proteoglycans in children undergoing liver transplantation.

The liver produces dermatan sulfate (DS), heparan sulfate (HS) and heparin glycosaminoglycans (GAG) and in the presence of hepatic disease, tissue levels of the DS GAG increase dramatically. We hypothesized that in children undergoing liver transplantation plasma levels of DS would be increased. Plasma from children undergoing liver transplantation were tested preoperative, intra operative and post operative at 24-48 h, and 1-3 weeks. Fluctuating levels of DS, HS and heparin anticoagulant activity were detected at all timepoints. The anticoagulant activity was purified and gel chromatography of the material displayed a mean Mr 110,000 D. Reductive elimination decreased the mean Mr 24,000 D indicating the activity resides on a proteoglycan (PG). The purified material was subjected to further chromatography and two peaks of anticoagulant activity resolved, compatible with at least two separate PGs, one with DS GAG chains and the additional PG(s) with HS and heparin GAG chains.

Adolescent↗
PubMed1995

Human corneal basement membrane heterogeneity: topographical differences in the expression of type IV collagen and laminin isoforms.

BACKGROUND: The corneal epithelium converges at the peripheral zone (limbus) with the conjunctival epithelium, forming a continuous sheet with phenotypically distinct regions--central, limbal, and conjunctival. The epithelial basement membrane (EBM) is important for corneal functions and cell adhesion, but its regional composition is poorly understood. Current literature is controversial as to the occurrence of type IV collagen in the cornea. The aim of this study was to investigate in detail corneal basement membrane (BM) composition and correlate it with the differentiation state of contributing cells. EXPERIMENTAL DESIGN: Adult human corneas (N = 8) were cryosectioned and analyzed by immunofluorescence with antibodies to 15 BM components and to keratin 3, a marker of corneal epithelial differentiation. RESULTS: A novel type of spatial heterogeneity ("horizontal") in the EBM composition was found between the central cornea, limbus, and conjunctiva. Central EBM had type IV collagen alpha 3-alpha 5 chains, whereas limbal and conjunctival EBM contained alpha 1-alpha 2 chains and also laminin alpha 2 and beta 2 chains. Limbal EBM in addition had alpha 5(IV) chain. Laminin-1 (alpha 1 beta 1 gamma 1), laminin-5 (alpha 3 beta 3 gamma 2), perlecan, fibronectin, entactin/nidogen, and type VII collagen were seen in the entire EBM. Another novel type of BM heterogeneity ("vertical") was typical for the corneal Descemet's membrane: its stromal face had alpha 1(IV) and alpha 2(IV) chains and fibronectin, whereas alpha 3(IV)-alpha 5(IV) chains, entactin/nidogen, laminin-1, and perlecan were present on the endothelial face. CONCLUSIONS: Type IV collagen controversy is the result of the shifts of isoforms in the limbus and conjunctiva. These shifts and the appearance of additional laminins in the limbus may be related to the differentiation state of corneal cells contributing to the EBM formation. Novel types of BM heterogeneity in the human cornea are described: regional (horizontal) in the EBM and vertical in the Descemet's membrane. The first one may be a common feature of converging complex epithelia, whereas the second one may be another unique property of the Descemet's membrane.

Adult↗