Biomedical subjects

B R Hubbard

Publications and source records attributed to B R Hubbard.

10 recordsLinked to original sources

PubMed1994

Pentalenene synthase. Purification, molecular cloning, sequencing, and high-level expression in Escherichia coli of a terpenoid cyclase from Streptomyces UC5319.

Pentalenene synthase, which catalyzes the cyclization of farnesyl diphosphate (1) to the tricyclic sesquiterpene hydrocarbon pentalenene (2), was purified from Streptomyces UC5319. A 450-bp hybridization probe, generated by PCR amplification of genomic DNA using primers based on N-terminal and internal tryptic peptide sequence data for pentalenene synthase, was used to screen both plasmid and phage DNA libraries of Streptomyces genomic DNA, resulting in the isolation and sequencing of the complete pentalenene synthase gene. PCR was used to insert the pentalenene synthase gene into the T7 expression vector pLM1. Cloning of the resulting construct in the expression host Escherichia coli BL21 (DE3) gave transformants that expressed pentalenene synthase as greater than 10% of soluble protein. The recombinant enzyme has been purified, and initial physical and kinetic characterization has been performed. The recombinant enzyme appears to be identical in every respect with the native Streptomyces synthase and exhibits the following steady-state kinetic parameters: Km = 0.31 +/- 0.05 microM, kcat = 0.32 +/- s-1, KI(PPi) = 3.2 +/- 0.6 microM. Both enzymes have an absolute requirement of Mg2+ for catalysis and an optimum pH of 8.2-8.4. Both proteins have M(r) values of 41-42 kDa, as determined by SDS-PAGE.

Amino Acid Sequence↗

PubMed1994

Effects of interleukin 12 on immune responses and host protection in mice infected with intestinal nematode parasites.

The cytokine interleukin (IL) 12 stimulates T cell and natural killer cell production of interferon (IFN) gamma and inhibits T cell production of IL-4. We investigated the effects of IL-12 on cytokine gene expression, immunoglobulin (Ig)E, mucosal mast cell, and eosinophil responses, and the course of infection in mice inoculated with the nematode parasite Nippostrongylus brasiliensis, as well as the IFN-gamma dependence of these effects. IL-12 stimulated IFN-gamma and IL-10 gene expression during primary and secondary N. brasiliensis infections and inhibited IL-3, IL-4, IL-5, and IL-9 gene expression during primary infections but had little inhibitory effect during secondary infections. IL-12 inhibited IgE, mucosal mast cell, and blood and tissue eosinophil responses during primary infections, but only eosinophil responses during secondary infections. IL-12 enhanced adult worm survival and egg production during primary, but not secondary infections. IL-12 needed to be administered by day 4 of a primary infection to inhibit IgE and mucosal mast cell responses, and by day 6 to strongly inhibit eosinophil responses and to enhance worm survival and fecundity. Anti-IFN-gamma mAb inhibited the effects of IL-12 on IgE secretion, intestinal mucosal mastocytosis, and parasite survival and fecundity, but did not affect IL-12 inhibition of eosinophilia. These observations indicate that IL-12, if administered during the initiation of eosinophilia. These observations indicate that IL-12, if administered during the initiation of an immune response, can change the response from one that is characterized by the production of T helper (Th)2-associated cytokines to one characterized by the production of Th-1 associated cytokines. However, IL-12 treatment has less of an effect once the production of Th2-associated cytokines has become established. In addition, our results provide evidence that Th2-associated responses protect against, and/or Th1-associated responses exacerbate, nematode infections.

Animals↗

PubMed1994

IL-12 influences intrathymic T cell development.

IL-12 has been implicated in the maturation and activation of peripheral T lymphocytes and NK cells. In the present study we have investigated the potential role of IL-12 in intrathymic T cell development. Treatment of mouse fetal thymic organ culture with IL-12 caused a significant reduction in size and cell number compared with the untreated controls. Flow cytometric analysis of the thymocytes recovered from these lobes showed differential effects on individual thymocyte subsets, most but not all of which were significantly decreased. In contrast, however, we observed an increase in both the percentage and cell number of alpha beta TCR+CD4-CD8+ thymocytes. This effect could be neutralized with an anti-IL-12 antibody, demonstrating the specificity of the influence of IL-12 in the fetal thymic organ culture system. IL-12 caused proliferation of isolated thymocyte subsets, particularly CD3+CD4-CD8+ cells in the presence of IL-2 and IL-4. Additionally, IL-12 induced significant proliferation of early CD3-CD4-CD8- triple negative CD44+CD25+ pro-T cells in combination with stem cell factor. We show that both the p35 and p40 chains of IL-12 are produced in the fetal and adult mouse thymus and that thymic stromal cells are a potential source of this cytokine.

Animals↗

PubMed1994

Effects of IL-12 on in vivo cytokine gene expression and Ig isotype selection.

The effects of murine rIL-12 on cytokine gene expression and Ig secretion were studied in vivo. In untreated mice IL-12 enhanced IFN-gamma and IL-10 gene expression and protein secretion, reduced base line IL-3 and IL-4 gene expression, and increased serum IgG2a concentration. In mice that had been injected with goat anti-mouse IgD antibody (G alpha M delta) to induce increases in IL-3, IL-4, and IL-10 gene expression and serum IgE, IgG1, IgG2a, and IgG3 concentrations, the simultaneous injection of IL-12 enhanced IFN-gamma and IL-10 gene expression and suppressed IL-3 and IL-4 gene expression and serum IgG and IgE responses. Anti-IFN-gamma mAb neutralized most, but not all, IFN-gamma produced by mice treated with G alpha M delta and IL-12. Anti-IFN-gamma mAb enhanced IL-3 and IL-4 gene expression, did not affect IL-10 or IFN-gamma gene expression, and increased serum IgG1, IgG2a, and IgG3 levels, but had relatively little effect on serum IgE in these mice. In contrast to its effects in G alpha M delta-treated mice, IL-12 failed to inhibit the IgE response to G alpha M epsilon antibody, which stimulates mIgE+ B cells to secrete IgE. These observations demonstrate that: 1) IL-12 may limit its own effects by inducing the production of a cytokine (IL-10) that down-regulates both IL-12 production and IL-12-induced IFN-gamma production; 2) IL-12 inhibits the production of at least one cytokine, IL-3, that is not generally regarded to be strictly Th1- or Th2-associated; 3) IL-12 inhibits switching to IgE secretion to a greater extent than it inhibits switching to other Ig isotypes; and 4) the in vivo effects of IL-12 are, to a large extent, IFN-gamma-dependent.

Animals↗

PubMed1994

Administration of recombinant IL-12 to normal mice enhances cytolytic lymphocyte activity and induces production of IFN-gamma in vivo.

IL-12 is a heterodimeric cytokine that has been shown to enhance natural killer (NK) and cytotoxic T lymphocyte (CTL) responses, and to induce IFN-gamma production in vitro. In this study, we have examined the effects in vivo of administering purified murine rIL-12 to normal mice. Daily injections of rIL-12 i.p. (1 ng to 10 micrograms/day) caused dose-dependent enhancement of NK cell lytic activity in the spleens and livers of treated mice. Histologic examination of the livers of IL-12-treated mice revealed focal mononuclear cell infiltrates, and flow cytometry studies indicated that the livers of IL-12-treated mice contained increased numbers of NK cells, CD8+ T cells, and monocytes. Liver and splenic lymphoid cells from IL-12-treated mice, unlike liver and splenic lymphoid cells from control mice, spontaneously secreted IFN-gamma in vitro, suggesting that they had been induced by IL-12 to produce IFN-gamma in vivo. Consistent with this, IFN-gamma could be detected in the serum of IL-12-treated mice. In mice which had been immunized by footpad injection of allogeneic splenocytes, daily administration of rIL-12 i.p. was shown to enhance the specific CTL response in the draining lymph nodes. Thus, these studies demonstrate that IL-12 can enhance NK and CTL activity and induce IFN-gamma production in vivo, as well as in vitro, and suggest possible mechanisms by which IL-12 may exert therapeutic effects in the treatment of some tumors and infectious diseases.

Animals↗

PubMed1993

Antitumor and antimetastatic activity of interleukin 12 against murine tumors.

It has recently been demonstrated that in vivo administration of murine interleukin 12 (IL-12) to mice results in augmentation of cytotoxic natural killer (NK)/lymphocyte-activated killer cell activity, enhancement of cytolytic T cell generation, and induction of interferon gamma secretion. In this study, the in vivo activity of murine IL-12 against a number of murine tumors has been evaluated. Experimental pulmonary metastases or subcutaneous growth of the B16F10 melanoma were markedly reduced in mice treated intraperitoneally with IL-12, resulting in an increase in survival time. The therapeutic effectiveness of IL-12 was dose dependent and treatment of subcutaneous tumors could be initiated up to 14 d after injection of tumor cells. Likewise, established experimental hepatic metastases and established subcutaneous M5076 reticulum cell sarcoma and Renca renal cell adenocarcinoma tumors were effectively treated by IL-12 at doses which resulted in no gross toxicity. Local peritumoral injection of IL-12 into established subcutaneous Renca tumors resulted in regression and complete disappearance of these tumors. IL-12 was as effective in NK cell-deficient beige mice or in mice depleted of NK cell activity by treatment with antiasialo GM1, suggesting that NK cells are not the primary cell type mediating the antitumor effects of this cytokine. However, the efficacy of IL-12 was greatly reduced in nude mice suggesting the involvement of T cells. Furthermore, depletion of CD8+ but not CD4+ T cells significantly reduced the efficacy of IL-12. These results demonstrate that IL-12 has potent in vivo antitumor and antimetastatic effects against murine tumors and demonstrate as well the critical role of CD8+ T cells in mediating the antitumor effects against subcutaneous tumors.

Animals↗

PubMed1992

Dioxygen transfer during vitamin K dependent carboxylase catalysis.

The vitamin K dependent carboxylase of liver microsomes is involved in the posttranslational modification of certain serine protease zymogens which are critical components of the blood clotting cascade. During coupled carboxylation/oxygenation this carboxylase converts glutamate residues, dihydrovitamin K, CO2, and O2 to a gamma-carboxyglutamyl (Gla) residue, vitamin K (2R,3S)-epoxide, and H2O with a stoichiometry of 1:1 for all substrates and products. In this paper we investigate the role of molecular oxygen in the reaction by following the course of the oxygen atoms using 18O2. Two different mass spectroscopic techniques, electron ionization positive ion mass spectrometry and supercritical fluid chromatography-negative ion chemical ionization mass spectrometry, were used to quantitate the amount of 18O incorporation into the various oxygens of the vitamin K epoxide product. We found that 0.95 mol atoms of oxygen were incorporated into the epoxide oxygen, 0.05 mol atoms of oxygen were incorporated into the quinone oxygen of vitamin K epoxide, and the remaining ca. 1.0 mol atoms of oxygen were incorporated into H2O. No incorporation of oxygen into vitamin K epoxide from 50% H2(18)O was observed. Thus, the carboxylase operates as a dioxygenase 5% of the time during carboxylation/oxygenation. The relevance of these findings with respect to the nonenzymic "basicity enhancement" model proposed by Ham and Dowd [(1990) J. Am. Chem. Soc. 112, 1660-1661] is discussed.

Animals↗

PubMed1991

Terpenoid biosynthesis and the stereochemistry of enzyme-catalysed allylic addition-elimination reactions.

Allylic addition-elimination reactions are widely used in the enzyme-catalysed formation of terpenoid metabolites. It has earlier been shown that the isoprenoid chain elongation reaction catalysed by farnesyl pyrophosphate synthase involving successive condensations of dimethylallyl pyrophosphate (DMAPP) and geranyl pyrophosphate (GPP) with isopentenyl pyrophosphate (IPP) corresponds to such an SE' reaction with net syn stereochemistry for the sequential electrophilic addition and proton elimination steps. Studies of the enzymic cyclization of farnesyl pyrophosphate (FPP) to pentalenene have now established the stereochemical course of two additional biological SE' reactions. Incubation of both (9R)- and (9S)-[9-3H,4,8-14]FPP with pentalenene synthase and analysis of the resulting labelled pentalenene has revealed that H-9re of FPP becomes H-8 of pentalenene, while H-9si undergoes net intramolecular transfer to the adjacent carbon, becoming H-1re (H-1 alpha) of pentalenene, as confirmed by subsequent experiments with [10-2H, 11-13C]FPP. These results correspond to net anti-stereochemistry in the intramolecular allylic addition-elimination reaction. The stereochemical course of a second SE' reaction has now been examined by analogous incubations of (4S,8S)-[4,8-3H,4,8-14C]FPP and (4R,8R)-[4,8-3H, 4.8-14C]FPP with pentalenene synthase. Determination of the distribution of label in the derived pentalenenes showed stereospecific loss of the original H-8si proton. Analysis of the plausible conformation of the presumed reaction intermediates revealed that the stereochemical course of the latter reaction cannot properly be described as either syn or anti, since cyclization and subsequent double bond formation require significant internal motions to allow proper overlap of the scissile C-H bond with the developing carbocation.

Carbon Radioisotopes↗

PubMed1989

Vitamin K-dependent carboxylation. In vitro modification of synthetic peptides containing the gamma-carboxylation recognition site.

Synthetic peptides including the gamma-carboxylation recognition site and acidic amino acids were compared as substrates for vitamin K-dependent gamma-carboxylation by bovine liver carboxylase. The 28-residue proPT28 (proprothrombin -18 to +10) and proFIX28 (pro-Factor IX -18 to +10) were carboxylated with a Km of 3 microM. The Vmax of proPT28 was 2-3 times greater than that of proFIX28. An analog of proFIX28 that contained the prothrombin propeptide had a Vmax 2-3-fold greater than an analog of proPT28 that contained the Factor IX propeptide. proFIX28/RS-1, based upon Factor IX Cambridge, proFIX28/RQ-4, based upon Factor IX Oxford 3, and proFIX28 had equivalent Km and Vmax values. Analogs of proPT28 containing Ala6-Glu7 or Glu6-Ala7 were carboxylated at equivalent rates. A peptide containing Asp6-Asp7 was carboxylated at a rate of about 1% of that of Glu carboxylation. Carboxylation of peptides containing Asp6-Glu7 and Glu6-Asp7 yielded results identical with peptides containing Ala6-Glu7 and Glu6-Ala7. Carboxymethylcysteine was not carboxylated when substituted for Glu6 in a peptide containing Asp7. These results indicate that the prothrombin propeptide is more efficient in the carboxylation process than is the Factor IX propeptide, but that both propeptides direct carboxylation; the gamma-carboxylation recognition site does not include residues -4 and -1; aspartic acid and carboxymethylcysteine are poor substrates for the carboxylase, but aspartic acid does not inhibit the carboxylation of adjacent glutamic acids.

Amino Acid Sequence↗

PubMed1989

Vitamin K-dependent carboxylase: affinity purification from bovine liver by using a synthetic propeptide containing the gamma-carboxylation recognition site.

The vitamin K-dependent carboxylase catalyzes the posttranslational modification of specific glutamic acid residues to form gamma-carboxyglutamic acid residues within the vitamin K-dependent proteins. This enzyme recognizes the gamma-carboxylation recognition site on the propeptide of the precursor forms of the vitamin K-dependent blood coagulation proteins. To purify this enzyme to homogeneity, the carboxylase from bovine liver microsomes was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), the protein was fractionated with ammonium sulfate, and then the enzyme was isolated by affinity chromatography using a synthetic peptide based upon the structure of the prothrombin propeptide. Elution with 10 mM propeptide yielded a single major band on SDS gel electrophoresis with a molecular weight of 77,000. In the presence of high concentrations of propeptide, only minimal carboxylase activity was measurable. Antibodies to the protein inhibited the carboxylase activity in crude preparations. In an alternative affinity purification strategy the propeptide was coupled through an NH2-terminal cysteine to an activated thiol-Sepharose column. The carboxylase-propeptide complex was eluted at 25 degrees C by reductive cleavage of the enzyme-propeptide complex in the presence of detergent and phospholipids. The eluted protein (Mr, 77,000) contained both stable vitamin K-dependent carboxylase and vitamin K epoxidase activity. The protein, purified by either method, was detected as a single band (Mr, 77,000) in a Western blot using anti-carboxylase antibodies. A 10,000-fold purification of carboxylase activity from crude microsomes was estimated. Purified bovine liver vitamin K-dependent carboxylase should facilitate the study of its structure and of the mechanism of action of vitamin K as a cofactor in the reaction catalyzed by this enzyme.

Amino Acid Sequence↗