Search PubMed⌕ Search

Biomedical subjects

J S Mills

Publications and source records attributed to J S Mills.

At least 19 recordsLinked to original sources
PubMed1992

Intrinsic activity of precursor forms of HIV-1 proteinase.

The wild-type -Phe*Pro- bond located at the N-terminus of the mature aspartic proteinase of HIV-1 was replaced by -Ile-Pro- or -Val-Pro-. By this means, processing at this cleavage junction was prevented and so, extended or precursor forms of HIV-proteinase were generated. These constructs were expressed in Escherichia coli, purified therefrom, and their specificity, activity at different pH values and susceptibility to the potent inhibitor, Ro31-8959, was assessed. A hitherto unobserved cleavage junction (at approximately Ala-Phe*Leu-Gln approximately) in the frame-shift region of the gag-pol viral genome was identified and confirmed by demonstrating cleavage of a synthetic peptide corresponding to this region. The implications for viral replication of self-processing at neural pH by proteinase whilst still present (in a precursor form) as a component of the polyprotein are considered; such reactions, however, are still blocked even at pH values as high as 8.0 by Ro31-8959.

Amino Acid Sequence↗
PubMed1992

Sites of interaction between rod G-protein alpha-subunit and cGMP-phosphodiesterase gamma-subunit. Implications for the phosphodiesterase activation mechanism.

In photoreceptor cells of vertebrates light activates a series of protein-protein interactions resulting in activation of a cGMP-phosphodiesterase (PDE). Interaction between the GTP-bound form of rod G-protein alpha-subunit (alpha t) and PDE inhibitory gamma-subunit (P gamma) is a key event for effector enzyme activation. This interaction has been studied using P gamma labeled with the fluorescent probe, lucifer yellow vinyl sulfone, at Cys-68 (P gamma LY) and sites of interaction on alpha t and P gamma have been investigated. Addition of alpha tGTP gamma S to P gamma LY produced a 3.2-fold increase in the fluorescence of P gamma LY. The Kd for alpha tGTP gamma S.P gamma LY interaction was 36 nM. Addition of 1 microM alpha tGDP had no effect, but in the presence of A1F4-, alpha tGDP increased P gamma LY fluorescence by 85%. When P gamma LY was reconstituted with P alpha beta to form fluorescent holo-PDE, alpha tGTP gamma S increased the fluorescence of holo-PDE with a K0.5 = 0.7 microM. Also, alpha tGTP gamma S stimulated the activity of this PDE over an identical range of concentrations with a similar K0.5 (0.6 microM). alpha tGTP gamma S enhanced the fluorescence of a COOH-terminal P gamma fragment, P gamma LY-46-87, as well (Kd = 1.5 microM). We demonstrate that an alpha t peptide, alpha t-293-314, which activated PDE (Rarick, H. M., Artemyev, N. O., and Hamm, H. E. (1992) Science 256, 1031-1033), mediates PDE activation by interacting with the P gamma-46-87 region. Peptide alpha t-293-314 bound to P gamma LY (K0.5 = 1.2 microM) as well as to the carboxyl-terminal P gamma fragment, P gamma LY-46-87 (K0.5 = 1.7 microM) as measured by fluorescence increase, while other alpha t peptides had no effect. A peptide from the P gamma central region, P gamma-24-46, blocked the interaction between alpha tGTP gamma S and P gamma LY. The Kd for alpha tGTP gamma S.P gamma-24-46 interaction was 0.7 microM. On the other hand, P gamma-24-46 had no effect on alpha t-293-314 interaction with P gamma LY. Our data suggest that there are at least two distinct sites of interaction between alpha tGTP gamma S and P gamma. The interaction between alpha t-293-314 and P gamma-46-87 is important for PDE activation.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Identification and purification of calcium-independent phospholipase A2 from bovine brain cytosol.

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Mutating P2 and P1 residues at cleavage junctions in the HIV-1 pol polyprotein. Effects on hydrolysis by HIV-1 proteinase.

Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-Leu*Phe-) and (b) between the p51 and RNase H domain (-Phe*Tyr-) within p66 of RT in the HIV-1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a beta-branched (Ile) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a beta-branched (Val) residue in the P2 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.

Base Sequence↗
PubMed1991

Antiviral properties of Ro 31-8959, an inhibitor of human immunodeficiency virus (HIV) proteinase.

Ro 31-8959 inhibits the spread of HIV infection and the production of cytopathic effects in cultures of acutely infected cells. IC50 values for these effects are in the range 0.5-6.0 nM and IC90 values are in the range 6.0-30.0 nM. This inhibitor is effective even when added to cultures at a late stage of infection, after syncytia have started to form. Virus antigen, virus particles and virus cytopathic effects can largely be cleared from cultures treated with compound from 3 days until 6 days post infection. In chronically-infected cells, inhibition of virus maturation can be detected after 24 hours' treatment with 10 nM Ro 31-8959. In addition, a significant reduction of the proteolytic processing of p56 to p24 can be demonstrated in these cells with compound at picomolar concentrations. These properties indicate that Ro 31-8959 is highly effective against HIV with the potential to inhibit acute, established acute and chronic infections.

Antiviral Agents↗
PubMed1990

Phospholipase A2 activities with a plasmalogen substrate in brain and in neural tumor cells: a sensitive and specific assay using pyrenesulfonyl-labeled plasmenylethanolamine.

We have developed a new assay method for phospholipase A2 (EC 3.1.1.4.), towards ethanolamine plasmalogen using pyrenesulfonyl-labeled plasmenylethanolamine as the substrate. This procedure is sensitive to about 3 pmol/ml per min and is absolutely specific for plasmalogen. In this method, the product of phospholipase A2, pyrenesulfonyl-labeled lysoplasmalogen, is hydrolyzed to aldehyde and labeled glycerophosphoethanolamine with hydrochloric acid exposure, and after TLC separation, the pyrenesulfonyl-glycerophosphoethanolamine is quantitated spectrofluorometrically. The excitation and emission wave lengths were 340 and 376 nm, respectively. The activity of bovine brain homogenate was 44.1 +/- 6.47 pmol/min per mg protein (n = 3). Among bovine brain subcellular fractions, the distribution and specific activity of the enzymes were highest in cytosol (38.7 +/- 1.58% and 102.6 +/- 16.2 pmol/min per mg protein, n = 3). The activities of neural tumor cells, PC12 pheochromocytoma, Neuro2A and SKNSH neuroblastoma and U1242MG glioblastoma, were 34.4 +/- 6.83 (n = 5), 7.05 +/- 0.97 (n = 4), 5.25 +/- 1.69 (n = 5), and 9.68 +/- 1.35 (n = 4), pmol/min per mg protein (M +/- S.E.M.), respectively.

Adrenal Gland Neoplasms↗
PubMed1990

Calcium binding to extracellular sites of skeletal muscle calcium channels regulates dihydropyridine binding.

The binding of dihydropyridine (PN200-110) to skeletal muscle microsomes (which were 84% sealed inside-out vesicles) was not influenced by the addition of calcium or magnesium nor by addition of their chelators (EDTA or EGTA) unless the vesicles were pretreated with the calcium-magnesium ionophore A23187 and EDTA to remove entrapped cations. Separation of inside-out vesicles from right-side-out vesicles by wheat germ agglutinin chromatography revealed that only the right-side-out vesicles exhibited a calcium-, magnesium-, and chelator-dependent binding of PN200-110. Dihydropyridine binding to cardiac sarcolemma membranes (which were 46% inside-out) and to solubilized skeletal muscle membranes was inhibited by EDTA and could be fully restored by 10 microM calcium or 1 mM magnesium. Calcium increased PN200-110 binding to partially purified rabbit skeletal muscle calcium channels from 3.9 pmol/mg protein to 25.5 pmol/mg protein with a pK0.5 = 6.57 +/- 0.059 and a Hill coefficient of 0.56 +/- 0.04. Magnesium increased binding from 0.7 pmol/mg protein to 16.8 pmol/mg protein with a pK0.5 = 3.88 +/- 0.085 and a Hill coefficient of 0.68 +/- 0.074. These studies suggest that calcium binding to high affinity sites or magnesium binding to low affinity sites on the extracellular side of skeletal muscle T-tubule calcium channels regulates dihydropyridine binding. Further, similar calcium and magnesium binding sites exist on the cardiac calcium channel and serve to allosterically regulate dihydropyridine binding.

Animals↗
PubMed1990

Effect of two novel inhibitors of the human immunodeficiency virus protease on the maturation of the HIV gag and gag-pol polyproteins.

The ability of two novel synthetic compounds to inhibit the HIV protease-mediated processing of HIV-1 precursor polyproteins was investigated in an in vitro gag-protease mixed lysate assay system and in an assay using recombinant baculoviruses engineered to express the HIV-1 gag and pol genes in cultured insect cells. With the in vitro mixed lysate assay we have shown that both compounds at 1 microM can completely inhibit the HIV-1 and HIV-2 protease-mediated release of p24 from the HIV-1 gag precursor at pH 5.5 and pH 7.0. In the intracellular baculovirus system these compounds were shown to inhibit the protease-mediated maturation of gag and also the excision of the protease moiety from its precursor.

Animals↗
PubMed1989

Solubilization, purification and characterization of lysoplasmalogen alkenylhydrolase (lysoplasmalogenase) from rat liver microsomes.

Alkenylhydrolase (EC 3.3.2.2; EC 3.3.2.5) has been purified 200-fold to a specific activity of 8.0 mumol/min per mg from rat liver microsomes with 51% of the activity recovered. Purification was accomplished by solubilization of the membrane-associated enzyme with octylglucoside and chromatographic resolution on sequential DEAE cellulose and hydroxylapatite (HPLC) columns in the presence of octylglucoside. The partially purified enzyme, specific for the 2-deacylated plasmalogen, lysoplasmalogen (1-alk-1'-enyl-sn-glycero-3-phosphocholine or -ethanolamine), had no hydrolytic activity with intact plasmalogens or 1-acyl-sn-glycero-3-phosphoethanolamine. Kinetic analyses of enzymic activity demonstrated apparent Km values of 5.5 and 42 microM for 1-alk-1'-enyl-sn-glycero-3-phosphocholine and 1-alk-1'-enyl-sn-glycero-3-phosphoethanolamine, respectively. The Vmax values were 11.7 and 13.6 mumol/min per mg with the choline and ethanolamine substrates, respectively. The optimal pH range was between 6.6 and 7.1 with both substrates; the energy of activation for the purified enzyme was 15,200 cal. The enzyme required no cofactors and was unaffected by low millimolar concentrations of Ca2+, Mg2+, Mn2+ or EDTA. It was inhibited by the sulfhydryl-reacting reagent, p-chloromercuribenzoate. Mono- or diradylglycerophospholipids or sphingomyelin did not affect the enzymic activity at 37 degrees C. Activity of the purified enzyme, destroyed by freezing at -20 degrees C, was preserved if stored at this temperature in the presence of 300-600 microM diradylglycerophosphocholine or 50% glycerol. A continuous spectrophotometric assay, adapted in our laboratory for the assay of liver alkenylhydrolase, facilitated this purification. This is the first reported purification of alkenylhydrolase.

Animals↗
PubMed1988

Biologically active fluorescent derivatives of spinach calmodulin that report calmodulin target protein binding.

Spinach calmodulin (CaM) has been labeled at cysteine-26 with the sulfhydryl-selective probe 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) to produce MIANS-CaM. The interaction of MIANS-CaM with CaM binding proteins was studied by fluorescence enhancement accompanying the protein-protein interactions. MIANS-CaM bound to smooth muscle myosin light-chain kinase with a Kd of 9 nM, causing a 4.6-fold fluorescence enhancement. Caldesmon bound with a Kd of 250 nM, causing a 2-fold fluorescence enhancement. Calcineurin (CaN) bound to MIANS-CaM with a Kd less than 5 nM, causing an 80% increase in fluorescence. On the other hand, binding of the CaM antagonist drugs prenylamine and calmidazolium or the potent peptide antagonist melittin did not alter MIANS fluorescence. MIANS-CaM activated brain cGMP phosphodiesterase and CaN as effectively as unlabeled CaM. Spinach CaM was also labeled with three other sulfhydryl reagents, 6-acryloyl-2-(dimethylamino)naphthalene, (2,5-dimethoxy-4-stilbenyl)maleimide, and rhodamine X maleimide. CaN bound to the highly fluorescent rhodamine X maleimidyl-CaM with a Kd of 1.4 nM, causing a 25% increase in polarization. Both MIANS-CaM and rhodamine X-CaM were used to monitor the Ca2+ dependence of the interaction between CaM and CaN. Half-maximal binding occurred at pCa 6.7-6.8 in the absence of Mg2+, or at pCa 6.3 in the presence of 3 mM Mg2+. In both cases, the dependence of the interaction was cooperative with respect to Ca2+ (Hill coefficients of 1.7-2.0). Use of these fluorescent CaMs should allow accurate monitoring of CaM interactions with its target proteins and perhaps their localization within the cell.

Anilino Naphthalenesulfonates↗
PubMed1988

Analysis of regulatory sequences in androgen-responsive genes.

We have analysed the effect of androgens on the activity of promoters from MMTV, and the rat prostate C3(1) and mouse secretory protease inhibitor genes. MMTV promoter activity was stimulated by testosterone as well as progesterone and dexamethasone but not by oestradiol. Deletion analysis indicated that the three steroids acted through DNA sequences between nucleotides -201 and -69 upstream of the MMTV cap site. In contrast, the promoters for the C3(1) gene and the protease inhibitor gene were unaffected by testosterone in a number of cell types, including prostate cells, despite the fact that the MMTV promoter was stimulated in such cells.

Androgens↗
PubMed1987

A secretory protease inhibitor requires androgens for its expression in male sex accessory tissues but is expressed constitutively in pancreas.

A full length cDNA clone encoding a mouse prostatic secretory glycoprotein (p12) whose synthesis is dependent upon testicular androgens has been cloned and characterized. The predicted amino acid sequence of p12 shares extensive homology with several members of the Kazal family of secretory protease inhibitors, in particular the pancreatic secretory trypsin inhibitors. In agreement with sequence data, prostatic secretory p12, purified from mouse ventral prostate secretion, exhibits anti-trypsin activity. Steady-state levels of protease inhibitor mRNA in ventral prostate are reduced from approximately 0.06% in normal mice to undetectable after androgen withdrawal but are inducible within 4 h by re-administration of testosterone. Androgen-dependent expression of the secretory protease inhibitor mRNA was also observed in coagulating gland and seminal vesicle. In seminal vesicle, a tissue of different embryonic origin to the prostate, the kinetics of secretory protease inhibitor mRNA loss after castration are not as rapid as in the ventral prostate and coagulating gland. Low-level androgen independent expression was also observed in the pancreas. There appears to be a single gene for this secretory protease inhibitor and yet expression is markedly stimulated by testosterone in the sex accessory tissues and unaffected by this hormone in the pancreas.

Amino Acid Sequence↗
PubMed1987

Androgen regulated expression of a spermine binding protein gene in mouse ventral prostate.

A full length cDNA (MP25) encoding the major mouse prostatic secretory glycoprotein (p25), whose expression is androgen dependent, has been cloned and characterised. Steady-state levels of mRNA are decreased approximately 100-fold after 3 days castration but are restored progressively over 4 days with testosterone treatment. The secreted glycoprotein appears to be a spermine binding protein since the nucleotide and predicted amino acid sequence of MP25 shares extensive homology with a spermine binding protein (SBP) found in rat ventral prostate. Genomic clones indicate that there is a single gene for SBP which consists of 4 exons, the first of which is only 11bp in length. The second exon encodes the signal peptide, the third contains a portion of the spermine binding protein unique to the mouse and the largest exon encodes the bulk of the secreted protein.

Amino Acid Sequence↗
PubMed1987

A continuous fluorescence assay for cyclic nucleotide phosphodiesterase hydrolysis of cyclic GMP.

The fluorescent 2'-methylanthraniloyl derivative of cyclic GMP undergoes a 45% decrease in fluorescence when it is cleaved by brain phosphodiesterase in the presence of calmodulin. This fluorescence decrease is dependent upon calcium, calmodulin, and phosphodiesterase, and correlates well (r = 0.996) with the disappearance of substrate as monitored by high-performance liquid chromatography. The Kd values determined by this fluorescence method and HPLC suggest that cyclic GMP and its fluorescent derivative exhibit similar kinetic parameters in their hydrolysis.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1987

Androgen-regulated expression of secretory protein synthesis in mouse ventral prostate.

Two proteins of molecular weights 25 and 12 kDa (p25 and p12 respectively), whose expression is regulated by testosterone, were identified in mouse ventral prostate. An antiserum raised to mouse ventral prostate secretion was used to demonstrate that p25 corresponds to the major secretory glycoprotein in mouse prostatic fluid. This antiserum does not cross-react with the major secretory proteins of the rat ventral prostate. Western blot analysis of mouse ventral prostate proteins using the prostatic secretion antiserum demonstrates that p12 and p25 are detectable at 3 weeks of age, but the maximum level of both proteins is not attained until 5 weeks of age. In addition, synthesis of p25 was also observed in prostate tissue derived from differentiated embryonic urogenital sinus tissue growing as implants under the renal capsule of syngeneic male hosts.

Animals↗
PubMed1987

The interaction of felodipine with calcium-binding proteins.

Felodipine is unique among the dihydropyridine calcium antagonists in that it is the most potent in relaxing porcine coronary arteries (IC50 = 1.5 X 10(-10) M); it is not as sensitive to photoinactivation as nifedipine and nisoldipine, and it is fluorescent. The fluorescence of felodipine has allowed us to study many aspects of its interaction with various calcium-binding proteins in muscle, including calmodulin, skeletal troponin C, and cardiac troponin C. Calcium binding to the calcium-specific regulatory sites on these proteins exposes allosterically related felodipine-binding sites. The binding of other calmodulin antagonists and calcium antagonists, including prenylamine, R24571, and diltiazem, to these calcium-binding proteins abolishes the cooperativity between two felodipine-binding sites, resulting in felodipine binding to the remaining site with a 20-25-fold greater affinity. In addition, the binding of high-affinity drugs to these calcium-dependent hydrophobic sites on these calcium-binding proteins produces dramatic increases (40-50-fold) in their affinity for calcium. The affinity of felodipine for these calcium-binding proteins is 100-1,000 times lower than felodipine's IC50 for relaxing tension in coronary arteries, indicating that these calcium-binding proteins are probably not the primary receptors for felodipine. Similarities between the binding of dihydropyridines to the calcium channel and to these calcium-binding proteins have led us to suggest that a "calmodulin-like" calcium-binding protein on the calcium channel is the actual pharmacological receptor for dihydropyridine calcium channel antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Drosophila ARSs contain the yeast ARS consensus sequence and a replication enhancer.

A number of restriction fragments that function as autonomously replicating sequences (ARSs) in yeast have been isolated from Drosophila melanogaster DNA. The behaviour in yeast of plasmids containing Drosophila ARS elements was studied and compared to that exhibited by the archetypal yeast ARS-1 plasmid. ARS functions were localised by subcloning and BAL-31 deletion analysis. These studies demonstrated the structural and functional complexity of Drosophila ARSs. Each Drosophila ARS element has at least two domains, one essential for replication (the replication sequence, RS) and a second (the replication enhancer, RE) which is essential for maximum function of the RS. The RS of three Drosophila ARSs was shown to contain a sequence identical to an 11 bp yeast ARS consensus sequence (5' A/T TTTATPuTTT A/T 3'). These observations lend support to the hypothesis that heterologous ARS elements may be of biological significance.

Animals↗