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PubMed1988

Localizing the nitroxide group of fatty acid and voltage-sensitive spin-labels in phospholipid bilayers.

The intramembrane locations of several spin labeled probes in small egg phosphatidylcholine (egg PC) vesicles were determined from the enhancement of the 13C nuclear spin lattice relaxation of the membrane phospholipid. Electron paramagnetic resonance (EPR) spectroscopy was also used to measure the relative environmental polarities of the spin labels in egg PC vesicles, ethanol and aqueous solution. The binding location of the spin label group was determined for a pair of hydrophobic ion spin labels, a pair of long chain amphiphiles, and three stearates containing doxyl groups at the 5, 10 and 16 positions. The nuclear relaxation results indicate that the spin label groups on the stearates are located nearer to the membrane exterior than the analogous positions of the unlabeled phospholipid acyl chains. In addition, the spin label groups of the hydrophobic ions and long chain amphiphiles are located near the acyl chain methylene immediately adjacent to the carboxyl group. The relative polarities, determined by the EPR technique, are consistent with the nuclear relaxation results. This information, when combined with information on their electrical properties, allows for an assessment of the conformation and position of these voltage sensitive probes in membranes.

Electron Spin Resonance Spectroscopy↗

PubMed1991

Biochemical and structural adaptation of autologous skeletal muscle used for counterpulsation.

We have studied in a normal animal model (sheep), the biochemical and morphological adaptation of electrically stimulated skeletal muscle used for extra aortic counterpulsation. Immunocytochemical analysis of latissimus dorsi, using monoclonal antibodies to slow and fast myosin heavy chains, indicated an increase in the population of mixed fibres after stimulation for one week. By one month, up to 70% of fibres expressed both slow and fast myosin heavy chains in addition to the 15% of fibres expressing only slow myosin heavy chains. After 4 months, the population of mixed fibres was further transformed towards purely slow fibres to give values of 40 and 67% of fibres expressing only slow myosin heavy chain at 4 and 6 months, respectively. Increased staining, both in intensity and area, for NADH tetrazolium reductase activity (an enzyme of the oxidative metabolic pathway) was detected by 28 days. An increase in mitochondrial number was observed also by 28 days, further indicating a shift towards an oxidative metabolism. The molecular adaptation of latissimus dorsi was achieved by stimulation every fourth cardiac cycle at 35 Hz, 3 V, initiated 48 hours after the operation; this being a marked reduction in the delay from operation prior to stimulation. Evaluation of other regimes indicated that more frequent modes, or an increase in voltage or frequency, caused damage to the muscle during the early phase of molecular adaptation. A thorough understanding of the time sequence of the different adaptive processes is required to determine the ideal regime of stimulation initiated promptly after mobilisation of the muscle; aimed at harvesting the maximum amount of energy from the autologous muscle.

Adaptation, Physiological↗

PubMed2003

Lipid-binding proteins: structure of the phospholipid ligands.

Dihedral angles are evaluated for the phospholipid ligands of the lipid-binding proteins found in the Protein Data Base (PDB). Phospholipid structures occur with a trans C1-C2 configuration of the glycerol backbone and oppositely extended chains, in addition to the gauche C1-C2 rotamers found in membranes. Headgroup conformations are not restricted to the single bent-down configuration and gauche-gauche configuration of the phosphodiester that is found in phospholipid crystals. Additionally, fully extended headgroups and orientations directed away from the lipid chains are found for phospholipids in the protein binding pockets. On average, the hydrocarbon chains of the protein-bound lipids are conformationally more disordered than in fluid bilayer membranes. However, much of this configurational disorder arises from energetically disallowed skew conformations. This suggests a configurational heterogeneity in the lipids at a single binding site: Eclipsed conformations occur also in some lipid headgroups and glycerol backbones. Stereochemical violations appear for some of the ester carboxyl groups of the protein-bound phospholipids in the PDB, and two glycerol backbones have the incorrect enantiomeric configuration.

Animals↗

PubMed2001

A frameshift mutation in the human fibrinogen Aalpha-chain gene (Aalpha(499)Ala frameshift stop) leading to dysfibrinogen San Giovanni Rotondo.

We have investigated a 53-yr-old asymptomatic white man with decreased functional, but not immunologic, fibrinogen plasma levels together with prolonged thrombin and reptilase times, detected through routine coagulation studies prior to a surgical procedure. A new heterozygous single nucleotide deletion (C) at position Ala499 within the Aalpha-chain gene was identified, which predicted changes of the corresponding amino acids encoded by the subsequent portion of the exon V and the appearance of a premature stop codon at position 518 (Aalpha[499]Ala frameshift stop). The new dysfunctional fibrinogen, San Giovanni Rotondo variant, was confirmed in vivo by SDS-PAGE analysis of HPLC-purified fibrinogen chains. Mass spectrum examination of the abnormal HPLC-purified peak gave an estimated mass (56,088 Da) similar to that predicted by DNA analysis of the mutated Aalpha-chain gene (56,088 Da) and, after tryptic digestion, the truncated Aalpha-chain was shown only in the propositus, who also carried normal Aalpha-chain. In addition, mass spectrum analysis of the tryptic digest of the abnormal chain confirmed the presence of a new and unpaired cysteine at the last position that was predicted to form a disulfide bridge with human serum albumin. Immuno-blot analysis confirmed that fibrinogen San Giovanni Rotondo variant, but not normal fibrinogen. contained substantial amounts of albumin. Present findings confirm that truncated Aalpha-chain lacking part of the terminal domain may be incorporated into mature fibrinogen molecules and normally secreted in the bloodstream.

Afibrinogenemia↗

PubMed1984

Structures of the oligosaccharide chains of two forms of alpha 1-acid glycoprotein purified from liver metastases of lung, colon, and breast tumors.

Two forms of alpha 1-acid glycoprotein with common immunological determinants and almost identical amino acid compositions but different amounts of carbohydrate were isolated from liver metastases of primary colon, lung, and breast tumors by extraction with perchloric acid, gel filtration on Sepharose CL-6B and Sephadex G-200, and affinity chromatography on concanavalin A:agarose and Ricinus communis agglutinin l:agarose. Both forms of the antigen yielded single bands which stained for protein and carbohydrate when examined by disc gel electrophoresis and immunodiffusion. The molecular weights of the two forms were 45,000 and 37,000 respectively. The larger form contained about five to six oligosaccharide chains, whereas the smaller form had only three to four chains. The composition and structures of the oligosaccharide chains in the two forms of this glycoprotein were very similar. Each contained di-, tri-, and tetraantennary complex-type oligosaccharide chains. The diantennary oligosaccharide chains caused both forms of alpha 1-acid glycoprotein to be retained by concanavalin A-agarose columns. The lower-molecular-weight form contained fewer chains and correspondingly fewer terminal galactosyl residues. This resulted in the separation of this species from the higher-molecular-weight form on columns containing R. communis agglutinin I. Three types of reduced oligosaccharides were released from the light and heavy forms of alpha 1-acid glycoprotein by treatment with alkaline borohydride or by hydrazinolysis. These chains were isolated by chromatography on concanavalin A:agarose and Bio-Gel P-6 columns. The arrangement and linkage of sugars in the purified oligosaccharides were determined by periodate oxidation, sequential hydrolysis with glycosidases, and methylation analysis. The major oligosaccharide chain, comprising 50 to 55% of the carbohydrate, had a triantennary structure as shown in the structure: (formula; see text) in which NeuNAc is N-acetylneuraminic acid, Gal is galactose, GlcNAc is N-acetylglucosamine, Man is mannose, GlcNAcol is N-acetylglucosaminitol, and Fuc is fucose. Tetraantennary chains comprised about 25 to 30% of the carbohydrate, and the additional outer chain was attached to the alpha 1,6-mannosyl residue through a beta 1,6-linked GlcNAc unit. The remaining 15 to 20% of the oligosaccharide chains had a diantennary structure. The extent of sialylation of these chains varied in samples isolated from tumors of the same histological type from different individuals. However, a relatively constant proportion of the three types of chains was present in different forms of the glycoprotein isolated from liver metastases.

Amino Acids↗

PubMed1986

Peptide-specific antibodies identify the alpha 2 chain as the proteoglycan subunit of type IX collagen.

Type IX collagen is a recently characterized product of chondrocytes. The molecules of this collagen are heterotrimers of three genetically distinct polypeptide chains. One of the three chains contains chondroitin and/or dermatan sulfate glycosaminoglycan chains, giving the molecule a proteoglycan character. In fact, Type IX collagen has been identified with the proteoglycan Lt (PG-Lt), first isolated by Noro, A., Kimata, K., Oike, Y., Shinomura, T., Maeda, N., Yano, S., Takahashi, N., and Suzuki, S. (1983) J. Biol. Chem. 258, 9323-9331 from chick embryonic tibia and femur. Based on amino acid sequences predicted from the nucleotide sequences of cDNA and genomic clones specific for two of the chains of Type IX collagen, we have synthesized oligopeptides representing portions of the two chains. In addition, an oligopeptide has been made based on a partial amino acid sequence of the third chain. Antibodies against the synthetic peptides have been generated in rabbits, and the polyclonal sera have allowed identification of the three genetically distinct polypeptide subunits of Type IX collagen. In addition, labeling with [35S]sulfate and treatment with chondroitinase ABC demonstrates that glycosaminoglycan chains are present on the subunit that has been given the designation alpha 2(IX).

Amino Acid Sequence↗

PubMed1986

Ammonium chloride interferes with a distinct step in the biosynthesis and cell surface expression of human melanoma-type chondroitin sulfate proteoglycan.

Human melanoma cells synthesize a cell-associated chondroitin sulfate-rich proteoglycan, whose core protein is recognized by monoclonal antibody 9.2.27. We report that the core protein is present on the surface of melanoma cells in two forms, either free or modified by the addition of chondroitin sulfate chains, suggesting that the addition of glycosaminoglycan chains may not be a prerequisite for cell surface expression of the proteoglycan core protein. Free core protein found at the cell surface does not seem to represent an overflow of the proteoglycan synthetic pathway, since experiments using a beta-D-xyloside acceptor suggest that core protein is, in fact, limiting proteoglycan synthesis. NH4Cl inhibits the synthesis of melanoma-type proteoglycan, shifting the balance of surface core protein toward the free form. The inhibition of proteoglycan synthesis is apparently not due to a disruption of enzymes and precursors involved in glycosaminoglycan synthesis, since cells treated with NH4Cl retain their ability to initiate and elongate chondroitin 4-sulfate chains on a beta-D-xyloside acceptor. In contrast, the divalent ionophore monensin inhibited core protein maturation and synthesis of glycosaminoglycan chains. The effects of both NH4Cl and monensin were reversible; thus, experiments using the drugs sequentially indicated that monensin temporally precedes NH4Cl in interfering with proteoglycan biosynthesis. Since the NH4Cl and monensin share the property of inhibiting the acidification of intracellular vesicles within cells, the present findings raise the possibility that the accessibility of proteoglycan core protein to the Golgi site of glycosaminoglycan addition is regulated in melanoma cells by acidification of intracellular compartments.

Aggrecans↗

PubMed1993

Exocrinopathy resembling Sjögren's syndrome induced by a murine retrovirus.

BACKGROUND: Sjögren's syndrome (SS) is characterized by lymphocytic infiltration into, and destruction of exocrine glands, resulting in dryness of the mouth and eyes. The disease is considered to have an autoimmune etiology, however, its etiopathogenesis remains largely unknown. Recently, retrovirus is suggested to participate in the pathogenesis of SS, because SS-like lesions are reported in HIV infection or in human T cell leukemia virus type I infection. Moreover, human intracisternal A-type retroviral particles are reported to be detected in SS patients. During the course of our study on the histopathology of mice infected with a murine retrovirus, we happened to find SS-like exocrinopathy in those mice. EXPERIMENTAL DESIGN: Four-week-old C57BL/6 (B6) mice were injected intraperitoneally with LP-BM5 murine leukemia virus. This virus is known to induce splenomegaly, lymphadenopathy followed by lymphoid malignancy, and profound immunodeficiency in sensitive strains of mice. From 4 to 16 weeks after the virus inoculation, the infected mice were sacrificed and their submandibular and lacrimal glands were analyzed light and electron microscopically and immunohistochemically. The existence of the virus in the lesion in situ was also analyzed by the same method, and additionally by a polymerase chain reaction method. RESULTS: Periductal lymphocytic infiltration into the submandibular and lacrimal glands was observed in all the virus-infected mice at 4 weeks after the infection and progressed with time. Extraglandular lymphocytic infiltration was also observed in liver, kidney, lung, and pancreas. Immunohistochemical examination revealed that most infiltrating cells into the glands were composed of CD3+ T cells (CD4-dominant), Mac-1+ cells, and B220+ cells. The virus genome was detected in submandibular glands by immunohistochemistry or by polymerase chain reaction. In addition, retroviral particles were secreted into the lumen of exocrine ducts of submandibular glands. CONCLUSIONS: This might be an SS animal model that is induced by a certain defined retrovirus. This experimental system might provide us with valuable information for analyzing the mechanisms of how a retrovirus could induce SS.

Animals↗

PubMed2000

Locking regulatory myosin in the off-state with trifluoperazine.

Scallop striated adductor muscle myosin is a regulatory myosin, its activity being controlled directly through calcium binding. Here, we show that millimolar concentrations of trifluoperazine were effective at removal of all regulatory light chains from scallop myosin or myofibrils. More important, 200 microM trifluoperazine, a concentration 10-fold less than that required for light-chain removal, resulted in the reversible elimination of actin-activated and intrinsic ATPase activities. Unlike desensitization induced by metal ion chelation, which leads to an elevation of activity in the absence of calcium concurrent with regulatory light-chain removal, trifluoperazine caused a decline in actin-activated MgATPase activity both in the presence and absence of calcium. Procedures were equally effective with respect to scallop myosin, myofibrils, subfragment-1, or desensitized myofibrils. Increased alpha-helicity could be induced in the isolated essential light chain through addition of 100-200 microM trifluoperazine. We propose that micromolar concentrations of trifluoperazine disrupt regulation by binding to a single high-affinity site located in the C-terminal domain of the essential light chain, which locks scallop myosin in a conformation resembling the off-state. At millimolar trifluoperazine concentrations, additional binding sites on both light chains would be filled, leading to regulatory light-chain displacement.

Adenosine Triphosphatases↗

PubMed1991

Gly-238-Ser substitution changes the substrate specificity of the SHV class A beta-lactamases.

The SHV-type beta-lactamase SHV-2A is related to SHV-1 by a Gly-238-Ser replacement. Strains carrying SHV-2A are resistant to the third generation cephems cefotaxime and ceftizoxime, whereas those that carry SHV-1 are sensitive to these drugs. We present a kinetic analysis of a SHV-1 and SHV-2A enzymes, with the goal of gaining insight into the role of residue 238 in hydrolyzing cefotaxime and ceftizoxime. SHV-2A shows altered kinetic properties for a number of other cephems that also have heterocyclic side chains at the amino position of the 7-aminocephalosporanic acid nucleus (R1 side chain), including a significantly higher kcat/Km than does SHV-1 for cephaloridine, cephalothin, and cefotiam. Two cephems with straight chain R1 substitutions, cephalosporin C and cephacetrile, are not hydrolyzed more efficiently by SHV-2A. These results indicate that the Ser-238-Gly substitution increases the affinity toward cephems with a heterocyclic ring in the R1 side chain. In addition, the data for ampicillin and benzylpenicillin show that addition of a nitrogen to the second carbon of the R1 side chain of a penem results in a lower kcat/Km for SHV-2A relative to SHV-1. These data strongly suggest that the previously proposed hydrogen bond formation between Ser-238 and the second carbon nitrogen of cefotaxime is not an important factor in hydrolysis by SHV-2A. We propose that the Gly-238 to Ser-238 replacement in SHV-2A has altered the hydrophobic pocket so that it can better accommodate cephems with bulky R1 side chains.

Amino Acid Sequence↗

PubMed1991

Immunohistochemistry and gene rearrangement studies in the diagnosis of malignant lymphomas: a comparison of 152 cases.

One hundred fifty-two cases (155 specimens) of lymphoproliferative disorders were studied by immunohistochemistry and gene rearrangement analysis. Ninety-five of 96 B-cell lymphomas (99%) showed genotypic B-cell monoclonality. Of these, five cases had rearranged T-cell receptor (TCR) beta chain gene in addition to immunoglobulin heavy chain (IgH) and kappa light chain (Ig-K), one case had rearranged IgH and TCR-gamma chain but not Ig-K or TCR-beta, and two cases had only Ig-K rearrangement. One exceptional case in the B-cell lymphoma group had unrearranged, germline genotypes. In contrast, only 10 of 19 (53%) phenotypic T-cell lymphomas had rearranged TCR-beta, eight with concurrent TCR-gamma rearrangement. Of the remaining nine cases, six had germline configuration, two had rearranged Ig-K only, and one had both IgH and Ig-K rearrangement. This last case was reclassified as T-cell predominant, B-cell lymphoma. Thirteen of 16 cases of Hodgkin's disease had germline configuration; three cases had rearranged IgH and Ig-K, of which two were lymphocyte predominant with light chain monoclonality and one was a recurrence. Among 21 reactive lesions, 17 had germline configuration and four had rearranged IgH and Ig-K genes. Of these four cases, two were orbital lesions, one was a partially involved lymph node, and one developed a nodular lymphoma 9 months later. Our results indicate that almost all B-cell lymphomas have IgH and/or Ig-K rearrangement. In contrast, peripheral T-cell lymphomas have greater genotypic heterogeneity, and germline patterns for TCR genes are not uncommon. Reactive lesions and Hodgkin's disease tend to retain germline configuration, and any exception is often associated with an unusual clinical setting and/or histology. Genotypic analysis is thus most indicated in B-cell lymphomas with equivocal immunohistochemistry findings, T-cell lymphomas, and atypical cases of Hodgkin's disease and reactive lesions.

Antigens, Differentiation↗

PubMed1994

Nitric oxide decreases [Ca2+]i in vascular smooth muscle by inhibition of the calcium current.

Endothelium derived relaxing factor (nitric oxide, or NO) activates cytoplasmic guanylate cyclase in vascular smooth muscle and decreases vascular tone through cGMP-dependent mechanisms that are not yet understood fully. In cultured vascular smooth muscle cells (A7r5 cell line) sodium nitroprusside (NP), a vasodilator that decomposes into nitric oxide, lowered [Ca2+]i in cells in which [Ca2+]i was elevated after depolarization. NP decreased current through voltage-gated calcium channels, but did not affect release of calcium from intracellular stores. Hemoglobin, a scavenger of NO, reversed the effect of NP on [Ca2+]i and 8-Br-cGMP, a membrane permeant form of cGMP, mimicked the effect of NP on [Ca2+]i and on calcium currents. Thus, the signal transduction mechanism of endothelium dependent relaxation of vascular smooth muscle involves a decrease in [Ca2+]i by inhibition of Ca2+ entry. Relaxation or vasodilation would then result from decreased activity of myosin light chain kinase, in addition to myosin light chain dephosphorylation.

Animals↗

PubMed1991

Structural study of the sugar chains of human leukocyte cell adhesion molecules CD11/CD18.

Leu-CAMs (CD11/CD18) consisting of LFA-1, Mac-1, and p150/95 are leukocyte cell surface glycoproteins that are involved in various leukocyte functions. The asparagine-linked sugar chains were released as oligosaccharides from Leu-CAMs by hydrazinolysis. About 12 mol of sugar chains was released from 1 mol of Leu-CAMs. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. All of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. The neutral and sialdase-treated acidic oligosaccharides were fractionated by chromatography on lectin columns followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that Leu-CAMs contain mainly high mannose type and high molecular weight complex type sugar chains. The latter sugar chains were of bi-, tri-, and tetraantennary complex types with the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and/or the Gal beta 1----3GlcNAc beta 1----groups together with the Gal beta 1----4GlcNAc group in their outer-chain moieties. In addition to these sugar chains, a small amount of monoantennary complex type and hybrid type sugar chains was found in Leu-CAMs. Furthermore, analysis of the asparagine-linked sugar chains released from the beta-subunit of Leu-CAMs by a series of lectin chromatography showed that subunit-specific glycosylation is not observed between the alpha- and beta-subunits of Leu-CAMs.

Antigens, CD↗

PubMed2006

A new approach for the detection and identification of protein impurities using combinatorial solid phase ligand libraries.

We propose a novel method for detection of protein impurities present in plasma-derived and recombinant purified injectable biopharmaceuticals by enhancing the concentration of protein impurities, in essence "amplifying" their presence to detectable levels. The method is based on the capture of proteins using a combinatorial solid-phase hexapeptides ligand library previously described for the reduction of protein concentration difference in biological fluids. Three proteins have been investigated: Staphylococcus aureus Protein A, expressed in Escherichia coli and supplied as 99% pure, recombinant human albumin, expressed in Pichia pastoris and certified as 95% pure, and therapeutic albumin supplied as 96-98% pure injectable solution. In all cases, after treatment with the ligand libraries, a number of additional polypeptide chains, not visible in the control, could be detected and obtained in sufficient amounts for MS analysis. In the cases of the two recombinant proteins, it could be demonstrated that a number of these polypeptide chains were host cell proteins still present in the purified product. In addition, a substantial number of these spots were found to be cleavage products of the original recombinant DNA species. Such cleavage products were particularly abundant in the recombinant human albumin preparation. From pure injectable serum albumin, a number of human plasma protein impurities were also identified by LC-MS/MS analysis. Treatment with ligand libraries of purified proteins is thus seen as a very powerful method of capture and concentration of host proteins and cleaved products for further analysis to control better the quality of industrial biotechnology products.

Albumins↗

PubMed1998

Analysis of the effect of pressure on compression moulding of UHMWPE.

Ultra-high molecular weight polyethylene (UHMWPE) powder is effectively processed by compression moulding due to its very high melt viscosity. Compression moulding involves application of temperature and pressure as a function of time. The pressure applied during processing has a significant influence on the part properties. The effect of pressure applied during compression moulding was studied by moulding parts at different pressures. Increase in the applied pressure causes increase in the melting and recrystallization temperatures. An increase in the pressure applied at the melt temperature ( approximately 140 degrees C) from 7.8 MPa to 15.6 MPa caused the crystallinity to increase from 54% to 61%, the stiffness of the moulded part to increase from 257 MPa to 435 MPa and oxidative index to increase from 0.055 to 0.059. Further increase in the pressure applied at the melt to 23 MPa caused the crystallinity to fall to 49%, the modulus to reduce significantly to 302 MPa and the oxidative index to change to 0.063. Increase in the pressure applied at the recrystallization temperature ( approximately 91 degrees C) from 38 MPa to 78 MPa increased the crystallinity from 54% to 65%, increased the modulus from 257 MPa to 279 MPa and increased the oxidative index from 0.055 to 0.065. Further increase in the applied pressure to 97 MPa, caused the crystallinity to drop slightly to 61% the modulus to reduce to 269 MPa, and the oxidative index to reduce to 0.057. The experiments showed that for obtaining maximum crystallinity and stiffness, the applied pressure should be within a narrow range. The highest recrystallization pressure (97 MPa) indicated the formation of extended-chain crystals in addition to the chain-folded crystals. The change in pressure applied at the melt temperature had a significantly greater effect on Young's modulus, as compared to change in pressure applied at the recrystallization temperature. Fourier transform-infrared spectroscopy analysis of the samples moulded at different pressures revealed that the increase in crystallinity and stiffness was accompanied by increase in oxidation within the part. By filling the die in a nitrogen atmosphere instead of air, the oxidation level in the moulded parts was reduced by almost 60%, without adversely affecting the crystallinity and the modulus.

Journal Article↗

PubMed2005

Aqueous compatible polymers in bionanotechnology.

Core-shell molecularly imprinted particles (CS-MIPs) have been synthesised using the technique of emulsion polymerisation with caffeine and theophylline being used in the surface template polymerisation with ethylene glycol dimethacrylate and oleylphenyl hydrogen phosphate. A radiolabelling study with caffeine-8-14C showed that the template was completely located at the particle surface during polymerisation. Caffeine could be specifically bound to a caffeine-imprinted CS-MIP to give a biphasic Scatchard binding curve, whereas the binding profile to a theophylline-imprinted CS-MIP was monophasic. The nanoparticles have the potential to be used in the molecular recognition of small molecules in a complex biological matrix. Water soluble highly-branched imidazole end-chain functionalised polymers of nanodimensions have also been synthesised via reversible addition-fragmentation chain transfer polymerisation. The polymers have lower critical solution temperatures which occur at sub-ambient temperatures and have proven useful in the affinity precipitation of proteins which are particularly temperature sensitive, e.g. the histidine-tagged protein fragment BRCA1. An overview of both of these areas of research is described outlining the diversity of these aqueous compatible polymers in molecular recognition processes at the nanoscale.

Biopolymers↗

PubMed1999

Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns.

beta1,4-Galactosyltransferase (UDP galactose: beta-N-acetylglucosaminide: beta1,4-galactosyltransferase; EC 2.4.1. 22) catalyzes the transfer of galactose from UDP-Gal to N-acetylglucosamine in the penultimate stages of the terminal glycosylation of N-linked complex oligosaccharides in mammalian cells. Tobacco BY2 cells lack this Golgi enzyme. To determine to what extent the production of a mammalian glycosyltransferase can alter the glycosylation pathway of plant cells, tobacco BY2 suspension-cultured cells were stably transformed with the full-length human galactosyltransferase gene placed under the control of the cauliflower mosaic virus 35S promoter. The expression was confirmed by assaying enzymatic activity as well as by Southern and Western blotting. The transformant with the highest level of enzymatic activity has glycans with galactose residues at the terminal nonreducing ends, indicating the successful modification of the plant cell N-glycosylation pathway. Analysis of the oligosaccharide structures shows that the galactosylated N-glycans account for 47.3% of the total sugar chains. In addition, the absence of the dominant xylosidated- and fucosylated-type sugar chains confirms that the transformed cells can be used to produce glycoproteins without the highly immunogenic glycans typically found in plants. These results demonstrate the synthesis in plants of N-linked glycans with modified and defined sugar chain structures similar to mammalian glycoproteins.

Agrobacterium tumefaciens↗

PubMed1984

The HLA-DR4 family of haplotypes consists of series of distinct DR and DS molecules.

Among DR4-associated HLA-D antigens, distinct and consistent structural variations were found for the products of two human "Ia-like" loci, DR and DS. Analysis of neuraminidase-treated immunoprecipitated DR molecules from 15 HLA-DR4-associated HLA-D homozygous B-lymphoblastoid cell lines by two dimensional polyacrylamide gel electrophoresis identified five distinct DR beta chains. In addition, gel analysis of immunoprecipitated DS molecules identified three distinct DS beta chains. Altogether, five distinct DR4 haplotypes were defined according to the observed structural diversity of the DR and DS beta chains. These gene products presumably contribute the dominant polymorphisms recognized by T cells in mixed lymphocyte reaction (MLR). Thus, these studies indicate that the serologic specificity known as HLA-DR4 is not a single haplotype, but a determinant present on products of individual loci arrayed into distinctly different haplotypes. These findings suggest that distinct products of individual loci, rather than conventional HLA specificities defined by alloimmune sera, may represent the genetic markers relevant to HLA-D/DR associated diseases.

Antibodies, Monoclonal↗