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PubMed1983

Biosynthesis and secretion of procollagenase by rabbit synovial fibroblasts. Inhibition of procollagenase secretion by monensin and evidence for glycosylation of procollagenase.

Monolayer cultures of rabbit synovial fibroblasts stimulated with phorbol myristate acetate to produce large amounts of collagenase (EC 3.4.24.7) were used to study the biosynthesis and secretion of this enzyme. [3H]Leucine was added to cell cultures for pulse-chase and continuous-labelling experiments. The labelled procollagenase synthesized was identified by immunoprecipitation followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The amounts of intracellular and extracellular proenzyme were quantified by measuring radioactivity incorporated into the proteins. procollagenase was synthesized as doublet proteins of Mr 57 000 and Mr 61 000. Immunoprecipitable proenzyme proteins were first detected in culture medium 35 min after [3H]leucine was added to the cells. Monensin treatment of the cells inhibited procollagenase secretion and led to intracellular accumulation of the proenzyme. Cells treated with tunicamycin produced only the 57 000-Mr form, indicating that in rabbit synovial cells the 61 000-Mr form was post-translationally modified by addition of oligosaccharides to asparagine residues. The ratios of glycosylated to unglycosylated forms in cell lysates and in culture medium were 0.22:1 and 0.07:1 respectively.

Animals↗
PubMed1991

Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane-spanning sequences using a novel alkaline phosphatase expression vector.

Treponema pallidum subspecies pallidum is a pathogenic spirochaete for which there are no systems of genetic exchange. In order to provide a system for the identification of T. pallidum surface proteins and potential virulence factors, we have developed a novel expression vector which confers the utility of TnphoA transposition. The relevant features of this plasmid vector, termed pMG, include an inducible tac promoter, a polylinker with multiple cloning sites in three reading frames, and an alkaline phosphatase (AP) gene lacking the signal sequence-encoding region. Library construction with Sau3A-digested T. pallidum genomic DNA resulted in the creation of functional T. pallidum-AP fusion proteins. Analysis of fusion proteins and their corresponding DNA and deduced amino acid sequences demonstrated that they could be grouped into three categories: (i) those with signal peptides containing leader peptidase I cleavage sites, (ii) those with signal peptides containing leader peptidase II cleavage sites, and (iii) those with non-cleavable hydrophobic membrane-spanning sequences. Triton X-114 detergent phase partitioning of individual T. pallidum-AP fusions revealed several clones whose AP activity partitioned preferentially into the hydrophobic detergent phase. Several of these fusion proteins were subsequently shown to be acylated by Escherichia coli following [3H]-palmitate labelling, indicating their lipoproteinaceous nature. DNA and amino acid sequence analysis of one acylated fusion protein, Tp75, confirmed the presence of a hydrophobic N-terminal signal sequence containing a consensus leader peptidase II recognition site. The DNA sequence of Tp75 also indicates that this is a previously unreported T. pallidum lipoprotein. T. pallidum-AP fusion proteins which partitioned into the hydrophobic detergent phase but did not incorporate palmitate were also identified. DNA and amino acid analysis of one such clone, Tp70, showed no cleavable signal but had a significant hydrophobic region of approximately 20 residues, consistent with a membrane-spanning domain. Immunoblot analysis of T. pallidum-AP fusions detected with a monoclonal antibody specific for AP identified several fusion proteins which migrated as doublets separated in apparent electrophoretic mobility by no more than 3 kDa. [35S]-methionine pulse-chase incorporation showed that the doublet AP fusions represented precursor and processed forms of the same protein. DNA and amino acid sequence analysis of clones expressing processed fusion proteins demonstrated hydrophobic N-terminal signal sequences containing consensus leader peptidase I recognition sites.

Alkaline Phosphatase↗
PubMed2005

Florfenicol resistance gene floR is part of a novel transposon.

The florfenicol/chloramphenicol resistance gene floR was found to be part of the novel 4,284-bp transposon TnfloR from Escherichia coli. TnfloR consists of the gene floR, a putative regulatory gene, and the transposase gene tnpA. A circular form of TnfloR was detected and suggested the potential mobility of this transposon.

Anti-Bacterial Agents↗
PubMed1977

Simian virus 40 tumor-specific proteins: subcellular distribution and metabolic stability in HeLa cells infected with nondefective adenovirus type 2-simian virus 40 hybrid viruses.

HeLa cells infected with adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses produce several SV40-specific proteins. These include the previously reported 28,000-dalton protein of Ad2+ND1, and 42,000- and 56,000-dalton proteins of Ad2+ND2, the 56,000-dalton protein of Ad2+ND4, and the 42,000-dalton protein of Ad2+ND5. In this report, we extend the list of SV40-specific proteins induced by Ad2+ND4 to include proteins of apparent molecular weights of 28,000 42,000, 60,000, 64,000, 72,000, 74,000, and a doublet of 95,000. Cell fractionation studies demonstrate that the SV40-specific proteins are detectable in the nuclear, cytoplasmic, and plasma membrane fractions. By pulse-chase and cell fractionation experiments, three classes of SV40-specific proteins can be distinguished with regard to metabolic stability: (i) unstable in the cytoplasmic but stable in the nuclear and plasma membrane fractions; (ii) stable in the nuclear, cytoplasmic, and plasma membrane fractions; and (iii) unstable in all subcellular fractions. Immunoprecipitation of infected cell extracts demonstrates that most of the above proteins share antigenic determinants with proteins expressed in hamsters bearing SV40-induced tumors. Only the 42,000-dalton protein of Ad2+ND5 is not immunoprecipitable.

Adenoviruses, Human↗
PubMed1984

Calcineurin: a member of a family of calmodulin-stimulated protein phosphatases.

Calcineurin, a major calmodulin-binding protein of brain, is a heterodimer composed of a 61,000 Mr calmodulin-binding subunit, calcineurin A, and a 19,000 Mr Ca2+-binding subunit, calcineurin B. The discovery of a calmodulin-regulated protein phosphatase in rabbit skeletal muscle with a similar subunit structure led to the identification of calcineurin as a protein phosphatase (AA Stewart, TS Ingebritsen, A Manalan, CB Klee, P Cohen (1982) FEBS Lett 137:80-84). Using rabbit polyclonal antibodies to bovine brain calcineurin, both subunits of calcineurin can be identified in crude homogenates of bovine brain by an immunoblotting technique. In crude homogenates of bovine skeletal and cardiac muscle, a 59,000-61,000 Mr doublet and a 15,000 Mr species (the electrophoretic mobility of calcineurin B) are also detected by this technique. The cross-reactivity of these species with antibodies to brain calcineurin indicates antigenic similarity between the muscle proteins and calcineurin, and suggests the existence of a family of structurally related calmodulin-stimulated protein phosphatases. Like calcineurin, the 61,000 Mr subunits in skeletal and cardiac muscle bind calmodulin and are detected in crude tissue extracts by 125I-calmodulin gel overlay. Thus, both the 125I-calmodulin gel overlay method and the immunoblotting technique are useful in screening crude preparations, in which detection of calmodulin-stimulated protein phosphatase activity may be complicated by the many phosphatases present.

Animals↗
PubMed1987

Production of procollagenase by cultured human keratinocytes.

Using a collagen film assay utilizing 14C-labeled type I collagen, we demonstrated that cultured human keratinocytes produced a procollagenase after treatment with the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Production of collagenase paralleled alterations in cellular morphology induced by TPA. When procollagenase was immunoprecipitated with antibody to human fibroblast collagenase and analyzed on sodium dodecyl sulfate-polyacrylamide gels, the zymogen was revealed as a 56- and 51-kDa doublet. The keratinocyte-derived collagenase was a neutral metalloprotease, required activation with trypsin for detection in the collagenase assay and produced the characteristic three-quarter and one-quarter length collagen cleavage products when incubated with type I collagen at 25 degrees C. The enzyme was inhibited by serum and cysteine and was largely unaffected by serine, thiol, and carboxyl protease inhibitors. Cycloheximide inhibited the TPA-induced production of collagenase, suggesting that the procollagenase was not stored preformed in the keratinocytes. Keratinocytes treated with a tumor-promoting analogue of TPA also produced collagenase, but cells treated with cytochalasin B, interleukin-1, or two non-tumor promoting phorbol esters did not. Keratinocyte-derived collagenase may play a role in wound healing and morphogenesis.

Adult↗
PubMed1994

Identification of subspecies- and serotype 1-specific epitopes on the 80- to 90-kilodalton protein region of Chlamydia psittaci that may be useful for diagnosis of chlamydial induced abortion.

Genus-, subspecies-, and serotype 1-specific antigens of Chlamydia psittaci were characterized by immunoblot analysis, using monoclonal antibodies that recognize 2 C psittaci strains: AB7 isolated from an ewe that had aborted, and iB1 isolated from feces of a healthy ewe. Genus-specific epitopes were detected on lipopolysaccharide, on a 47-kd protein, and on a 27- to 30-kd doublet. Subspecies-specific epitopes were located on a 30-kd protein, and a 80- to 90-kd protein region was identified, which bore subspecies- and serotype 1-specific epitopes. These 80- to 90-kd proteins were highly reactive with serum from ewes that had aborted and could be a useful antigen for diagnosis of chlamydial induced abortion of ruminants.

Abortion, Veterinary↗
PubMed1998

The presence of an unusual PKC isozyme profile in rat liver cells.

We have previously shown that protein kinase C (PKC) is involved in the mitogenic response of T51B cells to epidermal growth factor. In fact, epidermal growth factor was an excellent mitogen, even after prolonged pretreatment of cells with TPA, suggesting that the PKC isoform implicated in proliferation is not down-regulated by 12-O-tetradecanoyl phorbol-13-acetate (TPA). We have now determined that the PKC isozymes -alpha, -beta, -delta, -epsilon, and -zeta are present in T51B cells. All five isoforms are associated with the plasma membrane and the cytoplasm and are either in or around the nucleus. PKC-beta I has a slightly different subcellular profile from that of the other isoforms in that it is clearly and strongly associated with the nuclear membrane. Also, a unique and novel pattern is obtained from immunoblots with anti-PKC-beta I. PKC-beta I is detected as a single band of 70 kDa in the cytosolic fraction and as a doublet of 65 and 77 kDa in the membrane fraction. PKC-alpha, -delta, and -epsilon were down-regulated by pretreatment of cells with TPA, while PKC-zeta was unaffected. Of particular interest was the fact that TPA did not down-regulate PKC-beta I. In fact, the amount of this isoform associated with the plasma membrane increased. These findings indicate that it is probably PKC-beta I that is involved in the mitogenic response of T51B cells to epidermal growth factor. Since PKC-zeta is also not down-regulated by TPA, the possible involvement of this isoform needs to be resolved.

Animals↗
PubMed1993

Modulation of protein tyrosine phosphorylation during G1/S transition in activated human T-lymphoblasts.

We report that in activated human T-lymphoblasts synchronized early in the G1-phase of the cell cycle, addition of interleukin 2 (IL-2) stimulates transition into S-phase in conjunction with specific and reproducible protein tyrosine phosphorylation events. Prominent among these was de novo phosphorylation of the p56lck tyrosine kinase, which appeared as a single polypeptide on SDS-polyacrylamide gel electrophoresis prior to the addition of IL-2, but as a doublet of approximately 56 and 59 kDa 3 h after IL-2 addition. Although the lck polypeptide doublet persisted into S-phase, the 59-kDa form was virtually undetectable in T-lymphoblasts in log phase growth. In T-lymphoblasts metabolically labeled with 32Pi, antiphosphotyrosine antisera identified a major 56-kDa tyrosine-phosphorylated protein, whereas antisera to lck showed that phosphorylation of lck occurred on both the 56- and 59-kDa forms. Phosphoamino acid analyses identified phosphoserine as the major phosphoamino acid on both of the lck polypeptides, although small amounts of phosphotyrosine were also detected on the 56-kDa form. Immune complex kinase assays revealed that only the lower band of the lck doublet exhibited autocatalytic activity. Furthermore, the 59-kDa form neither displayed autocatalytic activity, nor was it a substrate for phosphorylation by the 56-kDa form. We conclude that entry into S-phase in activated human T-lymphoblasts is associated with tyrosine phosphorylation of a limited number of proteins and suggest that p56lck activity is regulated during G1/S.

Antigen-Antibody Complex↗
PubMed1991

The presence of two forms of the phosphocarrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system in streptococci.

The protein, HPr, a necessary component of the phosphoenolpyruvate phosphotransferase system (PTS) in bacteria, was purified from Streptococcus salivarius by column chromatography. The purified preparation gave only one band when analyzed by sodium dodecylsulfate gel electrophoresis or by isoelectric focusing in polyacrylamide gel (pI = 4.85). However, electrophoresis in Tris-containing buffers under non-denaturing conditions revealed 2 bands that could be phosphorylated by PEP in the presence of enzyme I of the PTS or by ATP with the HPr kinase. Homogeneous preparations of these 2 forms could be obtained by preparative electrophoresis. Each preparation exhibited only 1 band when analyzed by electrophoresis under non-denaturing conditions, indicating that the doublet observed before preparative electrophoresis was not an electrophoretic artefact. The electrophoretic mobility of each protein was not modified following heat-treatment at 100 degrees C for 20 min or storage at -40 degrees C for several months. Both HPr proteins catalyzed in vitro the PEP-dependent phosphorylation of glucose, but at a rate slightly lower than that observed with a preparation of HPr containing both forms of the protein. Both forms were also able to transfer the phosphate group from PEP to the other specific PTS proteins known in S salivarius. Rabbit polyclonal antibodies directed against each form reacted with both proteins. The presence of the 2 forms of HPr was detected in fresh cellular extracts of S salivarius; however, their intracellular ratio varied according to growth conditions. A doublet was also found in many other streptococcal species tested (S mutans, S sobrinus, S sanguis, S thermophilus, S bovis, S rattus) and also in L lactis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Derivatization for liquid chromatography/electrospray mass spectrometry: synthesis of tris(trimethoxyphenyl)phosphonium compounds and their derivatives of amine and carboxylic acids.

A simple method for the derivatization of primary amines and carboxylic acids with tris(trimethoxyphenyl)phosphonium (TMPP) reagents to enhance their detection by electrospray mass spectrometry (ESI-MS) has been developed. The synthesis of novel TMPP reagents and their stable isotopically labelled analogues is described. Through the use of stable isotopically labelled TMPP "tags", incorporation of a doublet (1:1, (1)H/(2)H or (12)C/(13)C) into the target molecule can be achieved, enabling the use of isotopic target analysis to detect compounds of unknown molecular weight but with a characteristic isotope pattern and accurate mass difference.

Journal Article↗
PubMed2000

Use of antibodies in the analysis of connexin 43 turnover and phosphorylation.

A series of antipeptide antibodies designed to recognize specific sequences of the gap junction protein connexin 43 (Cx43) were developed and characterized immunochemically and immunohistologically. These antibodies bound to gap junctions and, on Western blots, to 43-kDa (often resolved as a doublet) and 41-kDa proteins in samples from heart, leptomeningeal cells, and brain. Relatively little of the 41-kDa protein was detectable in heart homogenates. Cultured rat leptomeningeal cells expressed high levels of the gap junction protein Cx43 and were used to analyze its turnover and phosphorylation. Pulse-chase experiments in leptomeningeal cells with [(35)S]methionine indicated that the 41-kDa form of connexin 43 was the first immunoprecipitable translation product. Radiolabel subsequently appeared in the lower band of the doublet at 43 kDa, followed by a shift into the higher band and turnover of the protein with a t(1/2) of 2.7 h. Pulse-chase labeling with [(32)P]P(i) indicated that phosphorylation of connexin 43 was limited to the 43-kDa protein, with a t(1/2) of 1.7 h. Treatment with alkaline phosphatase shifted the apparent molecular mass of the 43-kDa protein doublet such that it comigrated with the 41-kDa form. Hence, the 43-kDa protein observed on Western blots of both leptomeningeal cells and heart arises by phosphorylation of the 41 kDa precursor. Phosphorylation of serine residues accounts for most, if not all, of Cx43 phosphorylation in this system.

Amino Acid Sequence↗
PubMed1986

An endopeptidase associated with bovine neurohypophysis secretory granules cleaves pro-ocytocin/neurophysin peptide at paired basic residues.

The octacosapeptide sequence [Tyr18] pro-ocytocin/neurophysin (1-18)NH2 [pro-OT/Np(1-18)NH2] was synthesized and used as substrate to detect endoprotease(s) possibly involved in the processing of this precursor in bovine hypothalamo-neurohypophyseal tract. An endopeptidase (58 Kda) was detected in Lysates made from highly purified neurosecretory granules. This protease which cleaves the peptide bond on the carboxyl side of the Lys-Arg doublet, and no single basic residue, generates both OT-Gly10-Lys11-Arg12+Ala13-Val-Leu-Asp-Leu-Tyr18 (NH2) from the octacosapeptide substrate. In addition, a carboxypeptidase B-like activity converting OT-Gly10-Lys11-Arg12 into OT-Gly10 was detected in the same granule Lysates. It is hypothesized that a combination of these endoprotease and carboxypeptidase B-like activities together with the amidating enzyme of secretory granules might participate in the cleavage and processing of pro-OT/Np in vivo.

Amino Acid Sequence↗
PubMed1992

NO accounts completely for the oxygenated nitrogen species generated by enzymic L-arginine oxygenation.

We have assessed the stoichiometry of the nitric oxide (NO) synthase reaction by using a novel e.p.r. technique. NO generated by crude and partially purified NO synthase from endothelial cells and Escherichia coli-lipopolysaccharide-activated macrophages was trapped by a ferrous diethyldithiocarbamate complex dispersed in yeast. The paramagnetic ferrous mononitrosyl dithiocarbamate complex formed exhibited a characteristic e.p.r. signal at g perpendicular = 2.035 and g parallel = 2.02 with a triplet hyperfine structure (hfs) at g perpendicular. NO, 3-morpholinosydnonimine and S-nitroso-L-cysteine, but not nitrite or hydroxylamine, generated a similar e.p.r. signal. NO generated by NO synthase and by SIN-1 accumulated at a constant rate for 1 h, as measured by continuous e.p.r. registration at 37 degrees C. The formation of e.p.r.-detectable NO by NO synthases was inhibited by NG-nitro-L-arginine. Incubation with [15N]NG-L-arginine caused an e.p.r. signal with doublet hfs, indicating that the nitrosyl nitrogen derived exclusively from the guanidino nitrogen. The amount of NO generated by NO synthase as measured by e.p.r. technique was compared with formation of L-[3H]citrulline from L-[3H]arginine. NO and L-citrulline were detected at a 1:1 ratio with both NO synthase preparations. GSH and thiol depletion did not significantly affect NO synthase activity, excluding S-nitrosothiols as intermediates in the NO synthase reaction. We conclude that NO fully accounts for the immediate oxygenated nitrogen species derived from the enzymic oxygenation of L-arginine.

Amino Acid Oxidoreductases↗
PubMed1992

Nucleolar antigens and autoantibodies in hepatocellular carcinoma and other malignancies.

Patients with hepatocellular carcinoma (HCC), gastrointestinal, lung, and ovarian cancers were shown to have autoantibodies to nuclear and nucleolar antigens as detected by immunofluorescence on cell substrates. The frequency of antinuclear antibodies (ANAs) was significantly higher (P less than 0.001) in patients with HCC (57/184 = 31%) than in patients with chronic hepatitis or liver cirrhosis (25/187 = 13%). Although a range of fluorescence patterns was observed, a higher percentage of nucleolar fluorescence was detected in HCC, and three of these nucleolar antigens were identified. They were NOR-90, nucleolus organizer region doublet polypeptides of 93 and 89 kDa involved in RNA polymerase I transcription; fibrillarin, a 34 kDa protein of the nucleolar U3 ribonucleoprotein particle which is engaged in preribosomal RNA processing; and nucleophosmin/protein B23, a 37 kDa polypeptide which is associated with ribosome maturation and cellular proliferation. All these antigens are nucleolar components that are engaged in some aspect of ribosome biosynthesis. Since autoantibodies to these nucleolar antigens have also been found in systemic autoimmune diseases, they do not represent autoimmune reactions unique to cancer but might reflect reaction pathways related to immune responses that are antigen-driven. The ANA response in HCC appears to be dynamic reactions to this antigen-drive since some patients with chronic liver disease showed seroconversion to ANA positivity, marked increase in titer and/or change in antibody specificity preceding or coincident with clinical detection of HCC. These changes in ANA showed a close temporal relationship with transformation from long-established chronic liver disease to HCC.

Antibodies, Neoplasm↗
PubMed1996

Glutamylated tubulin as a marker of microtubule heterogeneity in the human sperm flagellum.

Four site-directed monoclonal antibodies (mAbs) to tubulin: DM1A and DM1B general anti-alpha and anti-beta tubulin, 6-11B-1 anti-acetylated alpha tubulin and GT335 anti-glutamylated alpha and beta tubulin were used to study the distribution of tubulin isoforms in the human sperm flagellum. Since indirect immunofluorescence (IIF) did not give reliable results, a quantitative study of the immunogold labelling of the flagellum was performed at five levels: the mid-piece, three successive regions of the principal piece and the terminal piece. A uniform labelling was observed with DM1A, DM1B and 6-11B-1 mAbs. In contrast, the labelling of glutamylated tubulin detected with GT335 mAb decreased from the middle piece to the terminal piece both for peripheral doublets and the central pair. The changes in labelling of peripheral doublets were related to the pattern of outer dense fibre (ODF) changes. Thus doublets 1-5-6, associated with the largest number of ODF, were the most heavily labelled. This predominant labelling corresponded to the plane of flagellar beating suggesting a functional heterogeneity of peripheral doublets.

Antibodies, Monoclonal↗
PubMed1999

Characterization of monoclonal antibodies raised against the latent nuclear antigen of human herpesvirus 8.

Human herpesvirus 8 (HHV-8; also designated Kaposi's sarcoma-associated herpesvirus) is the likely etiological agent of Kaposi's sarcoma (KS). HHV-8 encodes a latent nuclear antigen (LNA) which is the product of the viral gene orf 73. LNA is recognized by most infected patient sera and is the basis of current immunofluorescence assays used in epidemiological studies of HHV-8 infection. Here we describe the characterization of four monoclonal antibodies raised to the C-terminal third of LNA-glutathione S-transferase fusion proteins. These monoclonal antibodies recognized discrete linear epitopes within the C terminus and repetitive region of LNA, detected antigen in primary effusion lymphoma (PEL) cells, and precipitated a 220- to 230-kDa protein doublet corresponding to LNA from HHV-8-infected PEL cell lines. In situ immunocytochemistry of KS lesions with these antibodies show that LNA is extensively expressed in KS spindle cells.

Antibodies, Monoclonal↗
PubMedDate not supplied

Synthesis and secretion of the ovarian cancer antigen CA 125 by the human cancer cell line NIH:OVCAR-3.

To provide further information on the biochemical nature of the cellular and secreted forms of the mucin-like CA 125 ovarian cancer antigen. Pulse-chase experiments were performed in the NIH:OVCAR-3 ovarian cancer cell line with [(35)S]Met/Cys radiolabeling. After pulsing the cells with radioisotope for 30 min and analyzing cell lysates by immunoprecipitation with anti-CA 125 antibodies, a doublet species (form B, approximately 400 kD) and a ladder of slower-moving components were detected by SDS-PAGE and autoradiography. After a 4-hour chase period, a much larger species (form A) became evident. With further culture, the B form disappeared and the A form accumulated, suggesting a 'precursor-product' relationship between the two forms. The putative precursor species did not appear in the culture supernatant, but secretion of the mature species (form A) began after about 1 h of synthesis. Lectin-binding experiments demonstrated that the B form is a glycoprotein and not an early apomucin precursor. In contrast to other reports, no smaller species of the mature form of CA 125 were detected in this study. Trypsin digestion severely degraded the antigen but discrete smaller fragments were not formed. CA 125 antigen is synthesized through a glycosylated 400-kD precursor species. The mature form of the antigen appears in the cell after about 1 h of synthesis and in the culture medium after 1-4 h.

Animals↗