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PubMed2001

Purification and characterization of a Ca2+-independent endoprotease activity from peripheral blood lymphocytes: involvement in HIV-1 gp160 maturation.

We have analyzed the calcium requirement for HIV-1 gp160 processing in cultured nonlymphoid (CV-1 and HeLa-CD4) and human-lymphoid [Jurkat, Molt-4 and peripheral blood lymphocytes (PBMCs)] cells. The processing of gp160 in these cells, infected with recombinant vaccinia virus encoding the gp160 gene, was only partially affected by intracellular calcium depletion induced by the calcium ionophore A23187 and calcium chelator EGTA. These observations prompted us to purify the Ca(2+)-independent gp160 processing enzyme from natural targets of HIV-1 PBMCs. The endoprotease was purified to homogeneity by the use of four chromatography fractionation steps and the constant detection of the Ca(2+)-independent activity at each one of them. The enzyme was believed to be a membrane-associated heteromeric 120-kDa protein composed of three subunits of 66, 32, and 24 kDa. It was found to be specifically inhibited by substrate analogues, decanoyl-RVKR-chloromethyl ketone, and serine protease inhibitors including diisopropyl fluorophosphate (DFP) and TLCK. In contrast, no effect was observed with reducing agents including 2-beta-mercaptoethanol, N-ethylmaleimide, L-cysteine, and dithiothreitol. There were significant similarities between inhibition profiles of the purified enzyme in vitro and those of the endogenous endoprotease(s) in cell culture experiments. Therefore, the selectivity of purified endoprotease for the gp160 cleavage site, its requirement for additional residues around this consensus sequence, and its isolation from natural targets of HIV-1, made it a good candidate in the gp160 maturation process. We provide more direct and supporting evidence that HIV-1 gp160 maturation may involve at least two families of divergent endoproteases according to calcium dependence.

Amino Acid Sequence↗

PubMed1999

Urothelial hinge as a highly specialized membrane: detergent-insolubility, urohingin association, and in vitro formation.

Urothelial surface is covered by numerous plaques (consisting of asymmetric unit membranes or AUM) that are interconnected by ordinary looking hinge membranes. We describe an improved method for purifying bovine urothelial plaques using 2% sarkosyl and 25 mM NaOH to remove contaminating membrane and peripheral proteins selectively. Highly purified plaques interconnected by intact hinge areas were obtained, indicating that the hinges are as detergent-insoluble as the plaques. These plaque/hinge preparations contained uroplakins, an as yet uncharacterized 18-kDa plaque-associated protein, plus an 85-kDa glycoprotein that is known to be hinge-associated in situ. Examination of the isolated, in vitro-resealed bovine AUM vesicles by quick-freeze deep-etch showed that each AUM particle consists of a 16-nm, luminally exposed "head" anchored to the lipid bilayer via a 9-mm transmembranous "tail", and that an AUM plaque can break forming several smaller plaques separated by newly formed particle-free, hinge-like areas. These data lend support to our recently proposed three-dimensional model of mouse urothelial plaques. In addition, our findings suggest that urothelial plaques are dynamic structures that can rearrange giving rise to new plaques with intervening hinges; that the entire urothelial apical surface (both plaque and hinge areas) is highly specialized; and that these two membrane domains may be equally important in fulfilling some of the urothelial functions.

Animals↗

PubMed1994

Selective tuberculin anergy: case report and review.

Because of the human immunodeficiency virus epidemic, skin testing for Mycobacterium tuberculosis is now performed in conjunction with control skin tests to assess anergy and thus provide information to aid in the interpretation of a negative tuberculin test. However, anergy can occur selectively to purified protein derivative tuberculin when reactions to other recall antigens are present. We report an illustrative case of selective anergy in a patient with tuberculous meningitis and review the literature on this poorly understood phenomenon. When one suspects M. tuberculosis disease, repeating a 5 unit PPD-tuberculin skin test after four to eight weeks of antituberculous treatment may assist in establishing or excluding the diagnosis of tuberculosis.

Clonal Anergy↗

PubMed1999

Antibodies to a non-repeat region of Plasmodium falciparum antigen Pf155/RESA in individuals from malaria-endemic areas.

Human antibodies to the repeat regions of the Plasmodium falciparum asexual blood stage antigen Pf155/RESA interfere with parasite growth in vitro, but the significance in this respect of antibodies to non-repetitive epitopes is less clear. In this study the levels of antibodies to a non-repetitive part of Pf155/RESA (residue 199-221) in malaria-exposed individuals were analysed, as was the parasite-inhibitory capacity of such antibodies. Residue 199-221 is of particular interest since it includes a sequence homologous to a cytoadherence-related motif from band 3. Sera from donors in Liberia and Tanzania were analysed for reactivity in ELISA with synthetic peptides together overlapping this part of Pf155/RESA. High antibody reactivity was observed in most of the sera with two peptides including residues 199-211 and 202-214, respectively. Specific antibodies were affinity-purified from selected sera using these peptide sequences and were shown to react with Pf155/RESA by immunofluorescence and Western blotting. The purified antibodies were furthermore shown to inhibit parasite growth in vitro. The results suggest that both repeat and non-repeat epitopes in Pf155/RESA elicit antibodies with potential to protect against malaria infection.

Amino Acid Sequence↗

PubMed1992

Identification of equine herpesvirus 4 glycoprotein G: a type-specific, secreted glycoprotein.

Equine herpesvirus 4 (EHV4) glycoproteins of M(r) 63K and 250K were identified in the supernatant of infected cell cultures. The 63K glycoprotein was type-specific; that is, it reacted with monospecific sera from horses that had been immunized or infected with EHV4, but not with monospecific sera from horses immunized or infected with EHV1, a closely related alphaherpesvirus. It was postulated that the secreted protein may be the homologue of similarly secreted glycoproteins of herpes simplex virus 2 glycoprotein G (HSV2 gG) and pseudorabies virus (PRV) gX, which is the homologue of HSV2 gG. The US region of the EHV4 genome, toward the internal repeat structure, was sequenced. Four open reading frames (ORFs) were identified of which ORF4 showed 52% similarity to the gene-encoding PRV gX in a 650-nucleotide region. ORF4 coded for a primary translational product of 405 amino acids which has a predicted size of 44K. The amino acid sequence of ORF4 showed 28% identity with PRV gX and 16% identity with HSV2 gG, although significantly greater identity was observed in the N-terminal region including the conservation of 4 cysteine residues. Accordingly, we designate ORF4 as EHV4 gG. The predicted amino acid sequence of the EHV4 gG showed characteristics of an envelope glycoprotein. Expression of the entire EHV4 gG gene in the bacterial expression vector pGEX-3X produced a type-specific fusion protein of M(r) 70K of which the gG portion composes 43K. Antibody that was affinity purified from selected portions of Western blots containing the 70K gG fusion protein reacted with the 63K secreted glycoprotein. Conversely, antibody affinity purified to the 63K secreted product reacted with the 70K gG fusion protein. These results showed that the EHV4 63K secreted glycoprotein was EHV4 gG, the third alphaherpesvirus gG homologue known to be, at least in part, secreted. The type-specificity of this glycoprotein provides, for the first time, the opportunity to differentiate between antibodies present in polyclonal sera from EHV4, EHV1, and dual-infected horses and this has important implications for understanding the epidemiology of these viruses.

Amino Acid Sequence↗

PubMed2000

[Generation of monoclonal antibody Fab fragments to parathyroid hormone related protein by phage display technology].

OBJECTIVE: To develop monoclonal antibody Fab fragments using display technique. METHODS: The mouse IgG Fab genes of heavy and light chains were amplified from spleen cells of a parathyroid hormone-related protein (PTHrP) immunized mouse. The combinatorial phage antibody library was prepared by inserting both heavy and light chain Fab genes into phagemid vector pComb3 and followed by infection of helper phage. The library was selected by purified recombinant PTHrP. RESULTS: The combinatorial phage antibody library was constructed successfully and the specific mouse Fabs to PThrP were selected and expressed in E. coli. CONCLUSIONS: The selected specific mouse Fabs can recognize PTHrP with high specificity.

Animals↗

PubMed1991

Kinetic properties of phenylalanine ammonia-lyase from sunflower (Helianthus annuus L.) hypocotyls.

Phenylalanine ammonia-lyase (PAL) from sunflower hypocotyls has been partially purified by selective precipitation with ammonium sulfate and molecular gel filtration on Sephacryl S-300. Kinetic assays carried out with this partially purified PAL preparation revealed that the enzyme did not show a homogeneous kinetic behaviour. The observed kinetic pattern and parameters (Km and Vmax) depended on the assay conditions used and the protein concentration added to the assay mixture. PAL displayed Michaelian or negative cooperativity kinetics. Such behaviour can be explained by the existence of an association-dissociation process of PAL-protein subunits. The presence of mono-, tri- and tetrameric forms of PAL has been assessed by molecular gel filtration on Sephacryl S-200, using different elution conditions.

Kinetics↗

PubMed2003

Heterogeneous selection at specific loci in natural environments in Arabidopsis thaliana.

Genetic variation for quantitative traits is often greater than that expected to be maintained by mutation in the face of purifying natural selection. One possible explanation for this observed variation is the action of heterogeneous natural selection in the wild. Here we report that selection on quantitative trait loci (QTL) for fitness traits in the model plant species Arabidopsis thaliana differs among natural ecological settings and genetic backgrounds. At one QTL, the allele that enhanced the viability of fall-germinating seedlings in North Carolina reduced the fecundity of spring-germinating seedlings in Rhode Island. Several other QTL experienced strong directional selection, but only in one site and seasonal cohort. Thus, different loci were exposed to selection in different natural environments. Selection on allelic variation also depended upon the genetic background. The allelic fitness effects of two QTL reversed direction depending on the genotype at the other locus. Moreover, alternative alleles at each of these loci caused reversals in the allelic fitness effects of a QTL closely linked to TFL1, a candidate developmental gene displaying nucleotide sequence polymorphism consistent with balancing selection. Thus, both environmental heterogeneity and epistatic selection may maintain genetic variation for fitness in wild plant species.

Arabidopsis↗

PubMed2006

Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library.

Shark immunoglobulin new antigen receptor (IgNAR, also referred to as NAR) variable domains (Vs) are single-domain antibody (sdAb) fragments containing only two hypervariable loop structures forming 3D topologies for a wide range of antigen recognition and binding. Their small size ( approximately 12kDa) and high solubility, thermostability and binding specificity make IgNARs an exceptional alternative source of engineered antibodies for sensor applications. Here, two new shark NAR V display libraries containing >10(7) unique clones from non-immunized (naïve) adult spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks were constructed. The most conserved consensus sequences derived from random clone sequence were compared with published nurse shark (Ginglymostoma cirratum) sequences. Cholera toxin (CT) was chosen for panning one of the naïve display libraries due to its severe pathogenicity and commercial availability. Three very similar CT binders were selected and purified soluble monomeric anti-CT sdAbs were characterized using Luminex(100) and traditional ELISA assays. These novel anti-CT sdAbs selected from our newly constructed shark NAR V sdAb library specifically bound to soluble antigen, without cross reacting with other irrelevant antigens. They also showed superior heat stability, exhibiting slow loss of activity over the course of one hour at high temperature (95 degrees C), while conventional antibodies lost all activity in the first 5-10min. The successful isolation of target specific sdAbs from one of our non-biased NAR libraries, demonstrate their ability to provide binders against an unacquainted antigen of interest.

Amino Acid Sequence↗

PubMed1984

Rapid purification of a cloned gene product by genetic fusion and site-specific proteolysis.

We have developed a rapid and general technique for purification of a protein encoded by a cistron contained in a recombinant DNA clone. The technique consists of fusing the target cistron DNA in the correct reading frame to a marker cistron via a piece of DNA that codes for a linker peptide. The target cistron in the example presented here is the replication initiator cistron of the plasmid R6K. The linker is a DNA fragment encoding 60 amino acids from the triple helical region of chicken pro alpha-2 collagen, and the marker cistron encodes the beta-galactosidase protein of Escherichia coli. The tripartite hybrid protein was rapidly purified by selective binding to and elution from a beta-galactosidase specific-affinity column. The hybrid protein was then digested with a purified microbial collagenase to cleave the linker, and high-pressure liquid chromatography allowed the rapid isolation of the target protein from the marker protein. Using this technique, we have purified the highly labile R6K replication initiator to homogeneity, and we have resolved the protein into NH2-terminal and COOH-terminal segments. We have further shown, by in vitro binding, that the COOH-terminal segment has at least one DNA-binding domain. The domain binds to the same restriction fragments of the R6K chromosome as the intact or beta-galactosidase-tagged initiator protein.

Bacteriophage lambda↗

PubMed1981

Selective in vitro transcription by purified yeast RNA polymerase II on cloned 2 micron DNA.

The in vitro transcription properties of purified yeast RNA polymerase II have been analyzed on prokaryotic plasmids (pBR322 and pBR313) and chimaeric plasmids bearing yeast 2 micron sequences (BTYP 1, BTYH 2 and BTYH 3). Conditions for selective transcription of the 2 micron DNA sequences in chimaeric plasmids have been determined. pBR322 and pBR313 are not transcribed by the purified RNA polymerase II when not bearing eukaryotic inserts. We show that the agarose gel electrophoretic analysis of ternary transcription complexes allows the localization of nascent RNA chains. The RNA produced has been visualized by electron microscopy (nascent RNA hybridization loops) and by gel electrophoretic analysis. All the observed properties are shared by RNA polymerase II purified by a conventional method (1) and by a rapid alternative procedure described herein. The peculiar properties of a partially purified form of RNA polymerase II are reported.

DNA, Fungal↗

PubMed1988

Pure astrocyte cultures derived from cells isolated from mature brain.

Enriched preparations of oligodendrocytes, isolated either from adult bovine brain or from 30-day-old rat brain, eventually yield cultures in MEM-15% calf serum that contain, in addition to oligodendrocytes, proliferating astrocytes and variable numbers of fibroblast-like cells. If these cultures are switched to a serum-free defined medium during the 1st week, mixed cultures containing only oligodendrocytes and astrocytes are obtained. Bovine cultures can be replated and purified by selective adhesion to yield cultures that are greater than 99% astrocytes; similar procedures were not successful with rat cultures. Cytoskeletal preparations of the purified astrocyte cultures from mature bovine brain contain both vimentin and glial fibrillary acidic protein (GFAP), but vimentin is by far the major intermediate filament protein. Thus, the intermediate filament composition of these astrocytes is similar to that of astrocytes in primary cultures obtained from neonatal rat brain. Immunofluorescent studies of these cultures at 24 hr in vitro show that there are no GFAP+ cells in cultures of either species; the bovine cultures contain greater than 95% GC+ cells; and the rat cultures contain 90% GC+ cells. After a few days in vitro flat cells appear that are vimentin+/GFAP-/GC-. In serum-free medium these cells eventually become vimentin+/GFAP+. We propose that the astrocytes that grow in these cultures arise from a population of glial precursor cells, which are present even in adult brain and are isolated together with oligodendroglia, and that they do not derive from contaminating mature astrocytes. Thus, the astrocytes in our cultures may have the same origin as astrocytes grown in culture from dissociated neonatal brain.

Animals↗

PubMed1998

Expression and characterization of human glutamate-cysteine ligase.

Glutamate-cysteine ligase (GLCL) catalyzes the rate-limiting step in glutathione biosynthesis. GLCL comprises regulatory (GLCLR) and catalytic (GLCLC) subunits. To understand better the structure-function relationship of GLCL subunits and holoenzyme, human GLCLR and GLCLC genes were inserted into the baculovirus genome. Recombinant hGLCLR andhGLCLC were produced in cells infected with recombinant baculoviruses, and homogeneous hGLCL subunits and holoenzyme were purified from cell lysates with a Ni-NTA resin. Purified recombinant hGLCL holoenzyme was catalytically more active than hGLCLC with L-glutamate, L-alpha-aminobutyrate, and ATP as substrates. The selectivity of purified hGLCL holoenzyme for L-glutamate, L-alpha-aminobutyrate, or L-cysteine was significantly higher than for hGLCLC. Glutathione was a noncompetitive inhibitor for both hGLCL holoenzyme and hGLCLC. hGLCLC was more sensitive to inhibition by glutathione than hGLCL holoenzyme. Deletion of the first 25 amino acid residues at the amino terminus of GLCLC dramatically decreased GLCL activity, indicating that the amino terminus of GLCLC is required for full catalytic activity. Expressed and purified hGLCL provides a useful tool to investigate glutathione biosynthesis in vitro.

Animals↗

PubMed1982

Octopine synthase mRNA isolated from sunflower crown gall callus is homologous to the Ti plasmid of Agrobacterium tumefaciens.

We have shown that the structural gene for octopine synthase (a crown gall-specific enzyme) is located in a central portion of the T-DNA that came from the Ti plasmid of agrobacterium tumefaciens and is expressed after it has been transferred to the plant cells. Polyadenylylated RNA was prepared from polysomes isolated from an octopine-producing crown gall callus and purified by selective hybridization to one of five recombinant plasmids. Each such plasmid contained a different fragment of T-DNA of pTi-15955 (octopine-type Ti plasmid). Purified mRNA was translated in vitro in rabbit reticulocyte lysates, and the translation products were immunoprecipitated with antibody against octopine synthase. Total and immunoprecipitated products were characterized by their molecular weights. A polypeptide of M(r) 40,000 (the same as authentic octopine synthase) was synthesized in vitro by crown gall mRNA selectively hybridized to three of the five fragments of T-DNA and precipitated with antibody against octopine synthase. This polypeptide was not immunoprecipitated with normal rabbit antibody nor was it synthesized when mRNA from the habituated callus was substituted. A mRNA 1500 bases long was detected when total mRNA was fractionated on an agarose gel, transferred to nitrocellulose, and used for hybridization to three of the five (32)P-labeled T-DNA fragments. This apparent mRNA for octopine synthase hybridized to the same three fragments of T-DNA as the mRNA for the M(r) 40,000 polypeptide and was not detected in the habituated callus.

Journal Article↗

PubMed2003

Purification and characterization of a prolyl aminopeptidase from Debaryomyces hansenii.

A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45 degrees C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg(2+), which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The K(m) for proline-7-amido-4-methylcoumarin was 40 micro M. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.

Aminopeptidases↗

PubMed2001

Megadose transplantation of purified peripheral blood CD34(+) progenitor cells from HLA-mismatched parental donors in children.

We performed HLA-mismatched stem cell transplantation with megadoses of purified positively selected mobilized peripheral blood CD34(+) progenitor cells (PBPC) from related adult donors in 39 children lacking an otherwise suitable donor. The patients received a mean number of 20.7 +/- 9.8 x 10(6)/kg purified CD34(+) and a mean number of 15.5 +/- 20.4 x 10(3)/kg CD3(+) T lymphocytes. The first seven patients received short term (<4 weeks) GVHD prophylaxis with cyclosporin A, whereas in all the following 32 patients no GVHD prophylaxis was used. In 38 evaluable patients, five patients experienced primary acute GVHD grade I and one patient grade II. In 32 patients, no signs of primary GVHD were seen and GVHD only occurred after T cell add backs. T cell reconstitution was more rapid if the number of transplanted CD34(+) cells exceeded 20 x 10(6)/kg. Of the 39 patients, 15 are alive and well, 13 died due to relapse and 10 transplant-related deaths occurred. We conclude that the HLA barrier can be overcome by transplantation of megadoses of highly purified mismatched CD34(+) stem cells. GVHD can be prevented without pharmacological immunosuppression by the efficient T cell depletion associated with the CD34(+) positive selection procedure. This approach offers a promising therapeutic option for every child without an otherwise suitable donor.

Adolescent↗

PubMed1980

Purification of two DD-carboxypeptidases/transpeptidases with different penicillin sensitivities from Proteus mirabilis.

Two membrane-bound enzymes of Proteus mirabilis with the dual functions of peptidoglycan DD-carboxypeptidase and transpeptidase (named DD-carboxypeptidase/transpeptidase H and L) were isolated and purified by selective solubilization with the nonionic detergent Genapol X-100, affinity chromatography on matrix-bound ampicillin, and preparative isoelectric focusing in the presence of detergent. Purified enzymes H and L were, respectively, penicillin-binding proteins 4 and 5 among seven major penicillin-binding proteins present in P. mirabilis. The enzymes differed in the following properties. Enzyme H had an Mr of 49,000; isoelectric point at pH 8.2; high sensitivity to benzylpenicillin and permanent inactivation because of high stability of the enzyme-antibiotic complex EI* (half-life 300 min); fragmentation of benzylpenicillin with formation of phenylacetylglycine during the slow decay of EI*; it functioned as an endopeptidase on peptide-crosslinked side chains of peptidoglycan. Enzyme L had an Mr of 43 000; isoelectric point at pH 5.9; low sensitivity to benzylpenicillin and low stability of EI* (half-life 7.2 min) with rapid recovery of enzyme activity; no function as an endopeptidase. The properties of enzyme L were identical with those of the single active DD-carboxypeptidase found previously in the spheroplast L-form of P. mirabilis grown in the presence of benzylpenicillin. We conclude that the partial penicillin resistance of P. mirabilis, with growth as L-form and synthesis of peptide-crosslinked peptidoglycan, depends on the continuing fuction of enzyme L as a DD-carboxypeptidase and transpeptidase in the presence of the antibiotic.

Carboxypeptidases↗

PubMed1981

Isolation and characterization of a major glycoprotein from milk-fat-globule membrane of human breast milk.

A major periodate--Schiff-positive component from milk-fat-globule membrane of human breast milk has been purified by selectively extracting the membrane glycoproteins, followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-dissociating agents. The purified glycoprotein, termed epithelial membrane glycoprotein (EMGP-70), has an estimated mol.wt. of 70 000 and yields a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The glycoprotein contains 13.5% carbohydrate by weight, with fucose, mannose, galactose, N-acetylglucosamine and sialic acid 17.2, 17.0, 21.1, 7.9 and 36.6% respectively of the carbohydrate moiety. Aspartic and glutamic acid and serine are the major amino acid residues.

Amino Acids↗