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J S Punzi

Publications and source records attributed to J S Punzi.

11 recordsLinked to original sources
PubMed1999

Serum modulation of neutrophil response to Porphyromonas gingivalis LPS in periodontal disease.

Rapidly progressive periodontitis is a form of early onset periodontitis characterised by severe gingival inflammation and rapid bone loss. Release of lipopolysaccharide (LPS) from the outer membrane of Gram negative bacteria initiates various biological activities including complement activation, cytotoxicity, and bone resorption. Serum from rapidly progressive periodontitis patients has been reported to enhance the superoxide response of normal neutrophils to lipopolysaccharide purified from Porphyromonas gingivalis, compared to control serum. The purpose of this study was to identify the factor in rapidly progressive periodontitis serum which is responsible for the enhancement of lipopolysaccharide activity. Candidate molecules were considered to be lipopolysaccharide binding protein or antibody to lipopolysaccharide. Lipopolysaccharide binding protein was quantified in serum by ELISA, and rapidly progressive periodontitis sera were found to contain two to three fold more lipopolysaccharide binding protein than control sera. Removal of lipopolysaccharide binding protein from either serum by adsorption to an anti lipopolysaccharide binding protein-sepharose affinity column removed the priming activity of normal neutrophils. Addition of exogenous lipopolysaccharide binding protein to control sera enhanced the priming activity in a dose dependent manner. These results suggest that lipopolysaccharide binding protein in rapidly progressive periodontitis serum may be responsible for enhancement of superoxide generation, which may result in more severe tissue damage in these patients.

Acute-Phase Proteins↗
PubMed1998

Purification and sequencing of a 21 kDa and 25 kDa bovine enamel metalloproteinase.

The developing enamel matrix contains metalloproteinases that are presumed to have a role in hydrolysis of enamel matrix proteins. Determination of the identity and function of these proteinases requires further information such as their amino acid composition and sequence. In this study, we purified the 21 kDa and 25 kDa matrix metalloproteinase from secretory stage bovine enamel matrix. After extraction, these proteinases were further purified by sequential separation by ion exchange, Con A affinity chromatography, and reversed phase HPLC. The proteinases were separated by SDS PAGE, transferred to a PVDF membrane and the N-terminus was sequenced. The N-terminal sequences of both proteinases were the same, and showed homology to the porcine enamelysin (MMP 20) cDNA sequence. A cDNA for bovine MMP 20 was isolated from a bovine enamel organ cDNA library using a probe generated by PCR amplification. The coding regions of bovine and porcine MMP 20 cDNAs were highly homologous and contained the same regions of predicted amino acid sequence homology with the proteinase N-terminal sequences. These results suggest that the 21 kDa and 25 kDa enamel matrix metalloproteinases are cleavage products of the initially secreted MMP 20, and that the sequence for MMP 20 is conserved across species.

Amino Acid Sequence↗
PubMed1995

Purification of nonamelogenin proteins from bovine secretory enamel.

The developing enamel matrix is composed of two groups of proteins that can be generally classified as amelogenins and nonamelogenins. The hydrophobic amelogenins represent the majority of the developing enamel matrix proteins, whereas nonamelogenins include the more hydrophilic enamelins, proteinases, and other minor protein components, which represent a small proportion of the matrix. This report describes the purification and partial amino acid sequences of two previously unknown proteins isolated from developing bovine enamel. These proteins were prepared by extracting bovine secretory stage enamel with low ionic strength buffer, followed by ammonium sulfate fractionation. The proteins were purified by ion-exchange, affinity, and reversed-phase chromatography. We propose to designate the proteins BEgp (a glycoprotein) and BEpa. A partial sequence was also obtained from a third protein (BEpb) which was nearly identical to BEpa. Antibodies were prepared to a synthetic peptide based on the N-terminal sequence of BEpa and subsequent immunoblots of various bovine tissues showed a major component of approximately 25 kDa specifically in enamel and ameloblasts. Little or no cross-reactivity of the antibody was found to bovine proteins extracted from heart, lung, kidney, liver, dental pulp, or bone. Similar analyses of both rat secretory stage and maturation stage enamel showed two bands of 28 kDa and 29 kDa. Immunohistochemical localization in rat incisors, showed specific staining of the enamel, secretory granules, and Golgi apparatus in ameloblasts. No sequence homology with known proteins could be demonstrated for BEgp or BEpa, suggesting that these components of developing enamel are novel tooth-specific proteins.

Amino Acid Sequence↗
PubMed1994

Crystallographic studies of isosteric NAD analogues bound to alcohol dehydrogenase: specificity and substrate binding in two ternary complexes.

CNAD (5-beta-D-ribofuranosylnicotinamide adenine dinucleotide) is an isosteric C-glycosidic analogue of NAD(H) containing a neutral pyridine ring. CPAD (5-beta-D-ribofuranosylpicolinamide adenine dinucleotide) is a closely related pyridine-containing analogue with the pyridine nitrogen on the opposite side of the ring. CNAD is a potent and specific inhibitor of horse liver alcohol dehydrogenase (LADH), binding with a dissociation constant in the nanomolar range. CPAD binds LADH with an affinity comparable to that of NAD. Crystal structures of CNAD and CPAD bound to LADH are presented at 2.4 and 2.7 A, respectively. The two complexes are isomorphous, crystallizing in the triclinic system with cell dimensions different from those seen in previous ternary LADH complexes. Structures were solved using the molecular replacement method and refined to crystallographic R values of 18% (CNAD) and 17% (CPAD). Both inhibitors bind to the "closed" form of LADH in the normal cofactor-binding cleft. The conformation of LADH-bound CPAD closely mimics that of LADH-bound NAD(H). The data suggest that alcohol substrate binds directly to the catalytic zinc atom. In the CNAD complex, the pyridine nitrogen replaces alcohol as the fourth coordination ligand to the active site zinc atom, while all other polar interactions remain the same as those of bound NAD(H). The zinc-nitrogen ligand explains the high affinity of CNAD for LADH.

Alcohol Dehydrogenase↗
PubMed1994

Crystallographic studies of two alcohol dehydrogenase-bound analogues of thiazole-4-carboxamide adenine dinucleotide (TAD), the active anabolite of the antitumor agent tiazofurin.

Thiazole-4-carboxamide adenine dinucleotide (TAD) is the active anabolite of the antitumor drug tiazofurin. Beta-methylene TAD (beta-TAD) is a phosphodiesterase-resistant analogue of TAD, active in tiazofurin-resistant cells. Beta-methylene SAD (beta-SAD) is the active selenium derivative of beta-TAD. Both agents are analogues of the cofactor NAD and are capable of acting as general dehydrogenase inhibitors. Crystal structures of beta-TAD and beta-SAD bound to horse liver alcohol dehydrogenase (LADH) are presented at 2.9 and 2.7 A, respectively. Both complexes crystallize in the orthorhombic space group C222(1) and are isomorphous to apo-LADH. Complexes containing beta-TAD and beta-SAD were refined to crystallographic R values of 15% and 16%, respectively, for reflections between 8 A and the minimum d spacing. Conformations of both inhibitors are similar. beta-TAD and beta-SAD bind to the "open" form of LADH in the normal cofactor-binding cleft between the coenzyme and catalytic domains of each monomer. Binding at the adenosine end of each inhibitor resembles that of NAD. However, the positions of the thiazole and selenazole heterocycles are displaced away from the catalytic Zn cation by approximately 4 A. Close intramolecular S-O and Se-O contacts observed in the parent nucleoside analogues are maintained in both LADH-bound beta-TAD and beta-SAD, respectively. These conformational constraints may influence the binding specificity of the inhibitors.

Adenosine Diphosphate↗
PubMed1992

Molecular conformation of estramustine and two analogues.

The crystal and molecular structures of estramustine and two of its analogues have been determined by X-ray crystallographic techniques (a total of three different compounds). The compounds studied are estramustine [1,3,5(10)-estratriene-3,17 beta-diol-3-N,N-bis(2'- chloroethyl)carbamate] and its monohydrate, estromustine [17-oxo-1,3,5(10)-estratriene-3-yl-N,N-bis(2'-chloroethyl)carbamate], and 17-oxo-5-androsten-3 beta-yl-N,N-bis(2'-chloroethyl)carbamate. Three views of estramustine were obtained from the study of its two crystal forms. The main structural features found are as follows: (a) the geometries of the steroid moieties are closely similar to those of the parent steroids, (b) the bonds around the nitrogen atom of the nitrogen mustard grouping lie approximately in a plane in each structure, (c) the plane through the carbon atoms of the steroid A-ring lies approximately perpendicular to the plane through the carbamate atoms in each structure, (d) the carbonyl C-O of the carbamate points to the alpha side of the steroid moiety in each structure, and (e) one chlorine atom of the nitrogen mustard grouping makes a close contact [3.13 A], in each structure, to the nitrogen atom. Hydrogen bonding to the carbamate appears to occur from the alpha side of the steroid; there is no hydrogen bonding to the nitrogen atom of the carbamate group. These structural data provide some steric explanations for the resistance of the carbamate to enzymatic hydrolysis. The long in vivo half-life of the intact estramustine molecule is a result of this stability. This is responsible for the absence of alkylating ability and the propensity of the drug to bind microtubule-associated proteins and express an antimitotic mechanism of action.

Estramustine↗
PubMed1990

[3H]mecamylamine binding to rat brain membranes. Studies with mecamylamine and nicotine analogues.

Mecamylamine, an antagonist to nicotine, does not compete at the nicotinic recognition site, but is believed to block the ion channel of the nicotinic receptor. The present study demonstrates specific, saturable [3H]mecamylamine binding in rat brain membranes. [3H]Mecamylamine binding was destroyed by heating at 100 degrees and trypsin. Scatchard analysis revealed the presence of two sites with Kd values of 9.6 x 10(-8) and 1.1 x 10(-6) M and Bmax values of 7 x 10(-12) and 3 x 10(-11) mol/mg protein respectively. A good correlation was observed between the Ki values for [3H]mecamylamine binding of a number of mecamylamine and related analogues and their ability to block nicotine-induced prostration in rats and seizures in mice. Inorganic cations, particularly divalent, and various ion channel blockers, such as phencyclidine and verapamil, exhibited a high affinity for the [3H]mecamylamine site. Although mecamylamine did not block nicotine binding, nicotine and its analogues exhibited a high affinity for the [3H]mecamylamine site, a finding which suggests that nicotine acts directly on ion channels as well as the nicotinic cholinergic recognition sites. The data are consistent with the notion that mecamylamine interacts with the open ion channel of the nicotinic receptor.

Animals↗
PubMed1988

Crystallization of prostatic binding protein.

Prostatic binding protein is a dimeric glycoprotein capable of binding a variety of steroids. This protein is a major component of rat prostate cytosol making it possible to purify milligram quantities. Hexagonal crystals of X-ray diffraction quality have been grown from phosphate buffered ammonium sulfate solution by vapor diffusion methods. These crystals which are reasonably stable to X-rays, show diffraction to 6.3 A and belong to space group P6(1) or P6(1)22 or the enantiomorphs. The unit cell has dimensions a = 88.7(5) A, c = 405(2) A, contains 24 molecules and has a specific volume of 2.8 A3/Dalton.

Ammonium Sulfate↗
PubMed1986

Crystals of active tetramers of 3 alpha, 20 beta-hydroxysteroid dehydrogenase.

The NADH-dependent steroid metabolizing enzyme 3 alpha, 20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53), from Streptomyces hydrogenans, has been crystallized in the active tetrameric form. Single crystals (approximately 0.75 X 0.40 X 0.40 mm) of square bipyramid shape have been grown reproducibly at room temperature in the presence of excess NADH. Diffraction experiments have been performed at the Cornell High Energy Synchrotron Source. The space group is P43212 or its enantiomorph, and the cell dimensions are a = 106.0(5) A and c = 204(1) A. The asymmetric unit is a tetramer of identical subunits of approximately 25,000 daltons each. The specific volume is 2.8 A3/dalton. A native data set at 2.5-A resolution has been collected. Two potential heavy atom derivatives, with K2Pt(CN)4 and KAu(CN)2, have been identified from the diffraction photographs.

20-Hydroxysteroid Dehydrogenases↗
PubMed1984

Crystallization and preliminary crystallographic study of 3 alpha, 20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans.

3 alpha, 20 beta-Hydroxysteroid dehydrogenase, an NADH-dependent oxidoreductase isolated from Streptomyces hydrogenans , is a tetramer containing four subunits each of Mr 25,000. The enzyme has been crystallized by the vapor diffusion technique using either phosphate or borate buffered ammonium sulfate (pH between 6.0 and 8.7) as the precipitant. The crystals are hexagonal bipyramids ; they have the symmetry of space group P6(4)22 (or P6(2)22), with unit cell dimensions a = 127.3 A, c = 112.2 A. Volume and density considerations imply that the crystallographic asymmetric unit contains two monomers, and therefore that the tetramer possesses a 2-fold axis of symmetry that is coincident with a crystallographic 2-fold symmetry element.

20-Hydroxysteroid Dehydrogenases↗