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PubMed1992

Molecular cloning, expression and characterization of the ovine IL-2R alpha chain.

Interleukin-2 (IL-2) stimulates the proliferation of activated antigen-specific T cells through its interaction with high affinity receptors. This event is largely regulated by the inducible expression of the alpha-chain (CD25) which, in combination with the beta-chain and possibly additional chains, forms the high affinity IL-2 receptor (IL-2R) complex. From a concanavalin A (Con A)-activated ovine T-cell complementary DNA (cDNA) library we have isolated two cDNA clones which together constitute a 2650 base pair (bp) messenger RNA (mRNA) species encoding the ovine IL-2R alpha chain. The nucleotide sequence has high homology with analogous cDNA from other species and predicts a mature protein of 254 amino acids. In addition to the predominate 2.6 kilobase (kb) ovine IL-2R alpha chain mRNA species. Northern blot analysis of activated T-cell RNA revealed two larger mRNA species. The ovine IL-2R alpha chain cDNA was transfected into CHO cells and low affinity binding of human recombinant IL-2 demonstrated. Polyclonal antisera generated against the transfected cells cross-reacted with Con A-activated ovine lymphocytes. In addition these antisera were used to immunoprecipitate a unique 50,000 MW protein from the transfected cells. It is likely that this protein represents the expressed ovine IL-2R alpha chain cDNA which is heavily glycosylated as distinct from the 30,869 MW primary translation product. Southern blot analysis of ovine genomic DNA suggests that the ovine IL-2R alpha chain is encoded by a single copy gene.

Animals↗
PubMed1998

Modification of ricin A chain, by addition of endoplasmic reticulum (KDEL) or Golgi (YQRL) retention sequences, enhances its cytotoxicity and translocation.

A pKK expression system in Escherichia coli was used to produce recombinant ricin A chain (rRTA) and rRTA modified by addition of organelle-specific amino acid retention sequences, including KDEL (an endoplasmic reticulum, ER, lumen retention signal), KKMP (an ER membrane retention signal), YQRL (a trans-Golgi network retention signal) and KFERQ (a lysosome-targeting signal) to the C terminus of rRTA. The toxicities of these RTA mutants were assessed in Jurkat cells following fluid-phase endocytosis. rRTA-KDEL and rRTA-YQRL were significantly more cytotoxic for Jurkat cells than rRTA, rRTA-KKMP or rRTA-KFERQ. This difference did not result from signal(KDEL or YQRL)-mediated binding of these RTA mutants to the cell surface. Reconstituted ER and Golgi vesicles have been employed to assess translocation of rRTA and mutant rRTA. RTA-KDEL and RTA-YQRL respectively exhibited 6.7-fold and 6.1-fold more protection against papain digestion in reconstituted ER vesicles and 2.2-fold and 1.8-fold more protection in reconstituted Golgi vesicles, than unmodified rRTA. These mutants were reassociated with ricin B chain to form holotoxins. The mutant RTA-KDEL and RTA-YQRL holotoxins were 3.8-fold and 1.5-fold more cytotoxic for target cells, respectively, than ricin produced using unmodified rRTA. Our results suggest that both ER and the trans-Golgi network may play important roles in the intracellular trafficking and translocation of ricin A chain.

Cytosol↗
PubMed1999

Non-volatile products of triolein produced at frying temperatures characterized using liquid chromatography with online mass spectrometric detection.

Oxidation products from triolein under model heated frying conditions have been analyzed using liquid chromatography with an evaporative light scattering detector and atmospheric pressure chemical ionization (APCI) mass spectrometric detection. Triolein was heated at 190 degrees C with 2% water added each hour, to simulate the moisture of a frozen product, until polar components reached approximately 30%. The samples were separated using reversed-phase high-performance liquid chromatography with APCI-MS detection. Triolein oxidation products included hydroperoxides, epoxides and a ketone. Other products were formed by shortening of an acyl chain on the intact triolein. Normal and oxygen-containing products formed by the dimerization of triolein were also observed. Other products included chain addition products formed by addition of acyl chain subunits to intact triolein to form higher molecular weight products.

Chromatography, High Pressure Liquid↗
PubMed1983

Yeast mutants deficient in protein glycosylation.

The synthesis of asparagine-linked oligosaccharides involves the formation of a lipid-linked precursor oligosaccharide that has the composition Glc3Man9GlcNAc2. We have used a [3H]mannose suicide selection to obtain mutants in yeast that are blocked in the synthesis of this precursor oligosaccharide. The alg1 mutant accumulated lipid-linked GlcNAc2, alg2 mutants accumulated Man1-2GlcNAc2, alg3 mutants accumulated Man5GlcNAc2, alg4 mutants accumulated Man1-8GlcNAc2, and alg5 and alg6 mutants accumulated Man9GlcNAc2. Some of these mutants appeared to transfer oligosaccharides other than Glc3Man9GlcNAc2 from the lipid carrier to invertase. These aberrant protein-linked oligosaccharides were processed by the addition of outer chain residues in the alg3, alg5, and alg6 mutants. There was virtually no outer chain addition in the alg2 and alg4 mutants. alg4 was the only mutant that failed to secrete invertase.

Alleles↗
PubMed2006

SPINFAST: using structure alignment profiles to enhance the accuracy and assess the reliability of protein side-chain modeling.

We present a novel, knowledge-based method for the side-chain addition step in protein structure modeling. The foundation of the method is a conditional probability equation, which specifies the probability that a side-chain will occupy a specific rotamer state, given a set of evidence about the rotamer states adopted by the side-chains at aligned positions in structurally homologous crystal structures. We demonstrate that our method increases the accuracy of homology model side-chain addition when compared with the widely employed practice of preserving the side-chain conformation from the homology template to the target at conserved residue positions. Furthermore, we demonstrate that our method accurately estimates the probability that the correct rotamer state has been selected. This interesting result implies that our method can be used to understand the reliability of each and every side-chain in a protein homology model.

Amino Acid Sequence↗
PubMed1983

Mouse monoclonal anti-Ia antibodies recognize cross-reacting determinants expressed on distinct subsets of human Ia-like cell-surface molecules.

Human Ia-like cell-surface molecules from a homozygous HLA-DR (6/6) B lymphoblastoid cell line have been analyzed using five mouse anti-Ia m.Ab cross-reacting with HLA-DR antigens. The surface-iodinated molecules immunoprecipitated by these m.Ab were analyzed by SDS-PAGE under reducing conditions and by SDS-PAGE followed by isoelectrofocusing. As read from the different migration patterns, three distinct combinations of human Ia-like molecules were identified by these m.Ab. Three anti-I-E-reactive m.Ab immunoprecipitated two-chain molecules whose apparent mol. wt (32K, 29K) corresponded to those of the classical HLA-DR antigens. One m.Ab which on mouse cells recognized a determinant shared by the I-A and I-E molecules precipitated not only the 32-29K bands, but also a 26K band from human cell extracts. Finally, an I-A reactive m.Ab precipitated a complex set of polypeptides including in addition to the 32-29K bands, three additional chains of 30, 28 and 26K. Sequential immunoprecipitation demonstrated that removal of the classical 29-32K HLA-DR chains by an anti-I-E m.Ab did not affect the subsequent immunoprecipitation of the additional chains by the anti-I-A or the anti-I-A + I-E m.Abs. These patterns and those obtained by 2D-gels analysis which demonstrated the complexity of the 26K band are compatible with the coexpression of at least three different subsets of molecules: (1) Ia-like molecules of 29-32K, recognized by all the m.Ab used; (b) molecules of 28-30K recognized by the anti-I-A m.Ab and (c) molecules apparently constituted by 26K chains, precipitated by the anti-I-A m.Ab and by the anti-I-A + I-E m.Ab.

Animals↗
PubMed1993

Structure of the N-linked oligosaccharides that show the complete loss of alpha-1,6-polymannose outer chain from och1, och1 mnn1, and och1 mnn1 alg3 mutants of Saccharomyces cerevisiae.

The periplasmic invertase was purified from Saccharomyces cerevisiae och1::LEU2 disruptant cells (delta och1), which have a defect in elongation of the outer chain attached to the N-linked core oligosaccharides (Nakayama, K., Nagasu, T., Shimma, Y., Kuromatsu, J., and Jigami, Y. (1992) EMBO J. 11, 2511-2519). Structural analysis of the pyridylaminated (PA) neutral oligosaccharides released by hydrazinolysis and N-acetylation confirmed that the och1 mutation causes a complete loss of the alpha-1,6-polymannose outer chain, although the PA oligosaccharides (Man9GlcNAc2-PA and Man10GlcNAc2-PA), in which one or two alpha-1,3-linked mannose(s) attached to the endoplasmic reticulumn (ER)-form core oligosaccharide (Man8GlcNAc2) were also detected. Analysis of the delta och1 mnn1 strain oligosaccharides released from total cell mannoprotein revealed that the delta och1 mnn1 mutant eliminates the alpha-1,3-mannose attached to the core and accumulates predominantly a single ER-form oligosaccharide species (Man8GlcNAc2), suggesting a potential use of this strain as a host cell to produce glycoproteins containing mammalian high mannose type oligosaccharides. The delta och1 mnn1 alg3 mutants accumulated Man5GlcNAc2 and Man8GlcNAc2 in total cell mannoprotein, confirming the lack of outer chain addition to the incomplete corelike oligosaccharide and the leaky phenotype of the alg3 mutation. All the results suggest that the OCH1 gene encodes an alpha-1,6-mannosyltransferase that is functional in the initiation of alpha-1,6-polymannose outer chain addition to the N-linked core oligosaccharide (Man5GlcNAc2 and Man8GlcNAc2) in yeast.

Carbohydrate Conformation↗
PubMed1996

Formation of Three-Membered Rings by S(H)i Displacement. Reverse of Cyclopropyl Ring Opening(1).

The general methods, photoinitiated or peroxide-initiated free radical chain additions of halomethanes to olefins, yield 1,2-addition products at temperatures ranging from 20 to 100 degrees C. At lower temperatures, -42 to -104 degrees C, a competitive reaction, subsequent to the addition of CCl(2)X(*), yields alkylcyclopropanes. The reactions of 1-octene or 1-hexene and 1-methylcyclohexene with atomic hydrogen carried out in the presence of several transfer agents (CCl(4), CCl(3)Br, CCl(2)Br(2)) initiate a radical chain addition of CCl(2)X(*) and yield cyclized materials resulting from the S(H)i displacement of halogen by a carbon-centered radical. The radical displacement of a halogen on carbon, the reverse of homolytic displacement on cyclopropyl carbon, is dominant at low temperatures. The rate constants for cyclization (k(c)) vs transfer with halomethane (k(t)) showed isokinetic temperatures of -46 degrees C (CCl(4), 1-hexene); -35 degrees C (CCl(4), 1-methylcyclohexene). The isokinetic temperatures for the reactions of the two substrates carried out in the presence of BrCCl(3) were calculated as -204 degrees C (1-octene) and -109 degrees C (1-methylcyclohexene).

Journal Article↗
PubMed1992

Molecular analyses of a human sex hormone-binding globulin variant: evidence for an additional carbohydrate chain.

Genomic DNA was isolated from an individual who is homozygous for a sex hormone-binding globulin (SHBG) variant that resolves into three molecular weight forms of 56K, 52K, and 48K during electrophoresis under denaturing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). This material was amplified using intron-specific oligonucleotide primers in a polymerase chain reaction to obtain the eight exons encoding SHBG. Sequence analysis of these exons revealed a point mutation encoding an amino acid substitution (Asp --> Asn) at residue 327 in the SHBG polypeptide, and the same mutation was identified in three siblings who also appear to be homozygous for this trait. This mutation introduces an additional consensus site for N-glycosylation at this position, and to confirm its utilization we introduced it into a human SHBG complementary DNA. The mutated complementary DNA was inserted into the pRc/CMV expression vector, and transfected into Chinese hamster ovary (CHO) cells. The product was secreted normally but proportionally less of it (54%) bound to concanavalin A when compared to normal SHBG produced by CHO cells (85%), or SHBG in the serum of either a normal individual or those who produce an electrophoretic variant (98%). Furthermore, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, the SHBG variant produced by CHO cells consisted of a 60K subunit as well as the heavy (52K) and light (48K) subunits associated with normal SHBG produced by CHO cells or in serum. This additional subunit is larger than the variant in serum and probably reflects a greater degree of complexity in the carbohydrate structures added to recombinant SHBG during synthesis in CHO cells. Nevertheless, its steroid-binding affinity was equal to normal SHBG produced by CHO cells or SHBG in serum.

Amino Acid Sequence↗
PubMed1983

Demonstration of a minor hemoglobin with modified alpha chains and additional modified hemoglobins in normal and diabetic adults.

We have attempted to separate various hemoglobins in 10 normal and 11 diabetic persons by Bio-Rex 70 chromatography at 4 degrees C with an exponentially increasing sodium phosphate gradient. Minor hemoglobins, namely AIa1, AIa2, AIb1, AIb2, AIb3, AIc, AId1, AId2 and AId3, have been separated and eluted in that order. Most of these minor hemoglobins were newly observed and could not be detected by the previous chromatographic techniques. On the basis of colorimetric assay protein-bound ketoamine was present in all the minor hemoglobins, which was confirmed by chromatographic separation of hemoglobins after reduction with NaB3H4. All the minor hemoglobins, with the exception of Hb AIa1, HbIa2, and Hb AIb1, showed a 2-fold increase in the diabetic patients. Hb AIc (5.0% in normals; 9.0% in diabetics) and Hb AId3 (1.9% in normals; 3.2% in diabetics) were present in the largest amounts. Both Hb AId3 and Hb Ao showed a decrease in oxygen affinity in the presence of 2,3-diphosphoglycerate, whereas Hb AIc showed no effect. Separation of globin chains by cellulose acetate electrophoresis at pH 8.6 showed that the cathodal mobility of the alpha chains of Hb AId3 was slower than those of Hb Ao. Glycosylation of Hb with [14C]glucose followed by separation of hemoglobins by two Bio-Rex 70 chromatographic methods indicated that the minor Hb formed by glycosylation of the alpha-chain amino-terminus was separated from Hb Ao, while the minor hemoglobins formed by the glycosylation of epsilon-NH2 groups chromatographed with Hb Ao.

Chromatography, Ion Exchange↗
PubMed2005

Conductance oscillations in zigzag platinum chains.

Using first principles simulations we perform a detailed study of the structural, electronic, and transport properties of monatomic platinum chains, sandwiched between platinum electrodes. First, we demonstrate that the most stable atomic configuration corresponds to a zigzag arrangement that gradually straightens as the chains are stretched. Second, we find that the averaged conductance shows slight parity oscillations with the number of atoms in the chain. Additionally, the conductance of chains of fixed oscillates as the end atoms are pulled apart, due to the gradual closing and opening of conductance channels as the chain straightens.

Journal Article↗
PubMed1984

Subcellular localization of the synthesis and glycosylation of chondroitin sulfate proteoglycan core protein.

In order to study the temporal and topological events involved in the processing and assembly of chondroitin sulfate proteoglycan, a fractionation scheme involving differential centrifugation and discontinuous sucrose density gradient centrifugation was developed for homogenates of chick embryo sternal chondrocytes. The precursors in these subcellular fractions were examined by a series of pulse, pulse-chase, and continuous labeling experiments. When chondrocytes were pulsed for 20 min with [35S]methionine, an immunoprecipitable core protein precursor with an approximate molecular size of 376,000 Da was localized to the rough endoplasmic reticulum fractions. Further incubation under chase conditions showed the presence of the 376,000-Da species as well as two additional polypeptides of higher molecular masses in the smooth membrane-enriched fractions within the next 2 h. This translocation did not occur in the presence of the energy transfer inhibitor carbonyl cyanide-m-chlorophenylhydrazone. The labeling pattern of the newly synthesized core protein precursor with either [3H] mannose or [3H]glucosamine showed that N-linked oligosaccharide addition was found on the earliest synthesized product in the rough endoplasmic reticulum, and the addition of this oligosaccharide was inhibited by co-incubation with tunicamycin. Furthermore, the high mannose oligosaccharide was susceptible to cleavage by endo-beta-N-acetylglucosaminidase H, while upon chase approximately 56 and 31% of the glucosamine- and mannose-labeled oligosaccharides, respectively, were processed to resistant forms, presumably in the Golgi complex. Both direct assay of glycosyl- and sulfotransferases requisite for addition of chondroitin sulfate chains and sensitivity of intracellular precursors to chondroitinase, keratanase , and endoglycosidase H suggest that only the N-linked oligosaccharides are added in the rough endoplasmic reticulum and glycosaminoglycan chain addition occurs predominantly in smooth membranes.

Animals↗
PubMed1994

The integrin chains beta 1 and alpha 6 associate with the chaperone calnexin prior to integrin assembly.

Integrins are alpha beta heterodimers that mediate cell-cell adhesion, as well as cell-substrate adhesion. The largest subclass is formed by 10 heterodimers (the very late antigens) that all share the beta 1-chain. We have found a 90-kDa protein that co-isolates with mouse integrin beta 1-chain. This 90-kDa protein was identified as the mouse homolog of calnexin, a membrane-bound chaperone and resident protein of the endoplasmic reticulum. First, the sequence of the 15 NH2-terminal amino acids of the 90-kDa protein is 80% and 87% identical to the corresponding sequences of canine and human calnexin, respectively. Second, the 90-kDa protein was recognized by a monoclonal antibody against calnexin/IP90. Association of calnexin with the integrin beta 1-chain was directly demonstrated by chemical cross-linking. As pulse-chase experiments revealed, the association of the beta 1-chain with calnexin occurred prior to the assembly with integrin alpha 6-chain. Additionally, the alpha 6-chain bound to calnexin before integrin assembly and dissociated again at the time of integrin-assembly. Our data suggest that calnexin is involved in the assembly of beta 1 integrins, as well as in the retention of a pool of immature integrin beta 1-chains in the ER.

Amino Acid Sequence↗
PubMed1985

Miscibility, chain packing, and hydration of 1-palmitoyl-2-oleoyl phosphatidylcholine and other lipids in surface phases.

The miscibility of 1-palmitoyl-2-oleoyl phosphatidylcholine with triolein, 1,2-diolein, 1,3-diolein, 1(3)-monoolein, oleyl alcohol, methyl oleate, oleic acid, and oleyl cyanide (18:1 lipids) was studied at the argon-water interface. The isothermal phase diagrams for the mixtures at 24 degrees were characterized by two compositional regions. At the limit of miscibility with lower mol fractions of 18:1 lipid, the surface pressure was composition-independent, but above a mixture-specific stoichiometry, surface pressure at the limit of miscibility was composition-dependent. From the two-dimensional phase rule, it was determined that at low mol fractions of 18:1 lipids, the surface consisted of phospholipid and a preferred packing array or complex of phospholipid and 18:1 lipid, whereas, above the stoichiometry of the complex, the surface phase consisted of complex and excess 18:1 lipids. In both regions of the phase diagram, mixing along the phase boundary was apparently ideal allowing application of an equation of state described earlier (J. M. Smaby and H. L. Brockman, 1984, Biochemistry, 23:3312-3316). From such analysis, apparent partial molecular areas and hydrations for phospholipid, complex, and 18:1 lipid were obtained. Comparison of these calculated parameters for the complexed and uncomplexed states shows that the aliphatic moieties behave independently of polar head group. The transition of each 18:1 chain to the complexed state involves the loss of about one interfacial water molecule and its corresponding area. For 18:1 lipids with more than one chain another two water molecules per additional chain are present in both states but contribute little to molecular area. In contrast to 18:1 lipids, the phospholipid area and hydration change little upon complexation. The uniformity of chain packing and hydration behavior among 18:1 lipid species contrasts with complex stoichiometries that vary from 0.04 to 0.65. This suggests that the stoichiometry of the preferred packing array is determined by interactions involving the more polar moieties of the 18:1 lipids and the phospholipid.

Glycerides↗
PubMed2000

Micromechanics of magnetorheological suspensions.

We apply optical trapping techniques in a magnetorheological (MR) suspension, allowing us to directly measure the mechanical properties of single dipolar chains, such as the rupturing tensions and strains under tensile and lateral deformations. Our results are in excellent agreement with calculations of the rupturing tensions using a self-consistent point dipole model of the particle interaction that accounts for induction and multibody effects along the chain. Additionally, we observe the annealing of chain defects under an applied stress, such as the inclusion of neighboring particles into the chain. The micromechanical properties of single chains offers important insight into magnetorheology and electrorheology, especially the yield stress behavior.

Journal Article↗
PubMed1986

Proposed alignment of helical interruptions in the two subunits of the basement membrane (type IV) collagen.

We have isolated a cDNA clone for part of the alpha 2 type IV collagen (pCIV-2-176). Deoxynucleotide sequence analysis shows that this clone codes for 439 amino acids from the helical domain adjacent to the C-terminal globular domain of the alpha 2 (IV) chain. By aligning the deduced amino acid sequence of the alpha 2 (IV) chain with the published sequence for the alpha 1 (IV) chain, we find that all interruptions in the alpha 1 (IV) chain coincide with an interruption in the alpha 2 (IV) chain. Additional interruptions in the alpha 2 (IV) chain exist, however, three out of the four analysed only slightly disturb the collagen triple helix.

Amino Acid Sequence↗