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J W Fox

Publications and source records attributed to J W Fox.

At least 19 recordsLinked to original sources
PubMed1992

Sequence of a cDNA clone encoding the zinc metalloproteinase hemorrhagic toxin e from Crotalus atrox: evidence for signal, zymogen, and disintegrin-like structures.

The sequence of two overlapping cDNA clones for the zinc metalloproteinase hemorrhagic toxin e (also known as atrolysin e, EC 3.4.24.44) from the venom gland of Crotalus atrox, the Western diamondback rattlesnake, is presented. The assembled cDNA sequence is 1975 nucleotides in length and encodes an open reading frame of 478 amino acids. The mature hemorrhagic toxin e protein as isolated from the crude venom has a molecular weight of approximately 24,000 and thus represents the processed product of this open reading frame. From the deduced amino acid sequence, it can be hypothesized that the enzyme is translated with a signal sequence of 18 amino acids, an amino-terminal propeptide of 169 amino acids, a central hemorrhagic proteinase domain of 202 amino acids, and a carboxy-terminal sequence of 89 amino acids. The propeptide has a short region similar to the region involved in the activation of matrix metalloproteinase zymogens. The proteinase domain is similar to other snake venom metalloproteinases, with over 57% identity to the low molecular weight proteinases HR2a and H2-proteinase from the Habu snake Trimeresurus flavoviridis. The carboxy-terminal region, which is not observed in the mature protein, strongly resembles the protein sequence immediately following the proteinase domain of HR1B (a high molecular weight hemorrhagic proteinase from the venom of T. flavoviridis) and the members of a different family of snake venom polypeptides known for their platelet aggregation inhibitory activity, the disintegrins. The cDNA sequence bears striking similarity to a previously reported sequence for a disintegrin cDNA. This report is evidence that this subfamily of venom metalloproteinases is synthesized in a proenzyme form which must be proteolytically activated.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

A new family of proteinases is defined by several snake venom metalloproteinases.

Recently, the complete amino acid sequences have been determined for several snake venom metalloproteinases from the genera Crotalus, Trimeresurus and Lachesis of the Crotalidae family. Among these are both hemorrhagic and nonhemorrhagic metalloproteinases. Despite differences in their molecular weights and activities, they all appear to be related through a single ancestral gene, as observed by the comparison of their amino acid sequences. None of these proteins bear significant similarity to any other known protein, with the exception of the zinc binding site demonstrated in thermolysin and other metalloproteinases. Thus we propose that these proteins define a new family of zinc metalloproteinases which is likely to consist of (but is not necessarily limited to) many of the zinc metalloproteinases found in snake venoms.

Amino Acid Sequence↗
PubMed1991

Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.

Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd-I (positions 1-905) comprising the N-terminal globule and rod-like domain and Nd-II corresponding mainly to the C-terminal globule (position 906-1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N-terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N-terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31-56 kDa) connected by either a rod-like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd-I binding to collagen IV and fragment Nd-II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd-I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.

Animals↗
PubMed1991

Proteolytic digestion of non-collagenous basement membrane proteins by the hemorrhagic metalloproteinase Ht-e from Crotalus atrox venom.

Hemorrhagic toxin e (Ht-e), a metalloproteinase isolated from the venom of the Western Diamondback rattlesnake Crotalus atrox, digests laminin and nidogen, both in their isolated forms and when present in a purified soluble complex. The only common site of cleavage by Ht-e of isolated nidogen and nidogen when complexed with laminin is at amino acid residue 336 in the amino terminal domain. Additionally, nidogen in complex with laminin is also cleaved at sites 322, 351 and 840 as determined by sequence analysis and site 953 as proposed from the molecular mass of a digestion product. Isolated nidogen, on the other hand, was cleaved at amino acid residues 75, 336, 402, and 920, as determined by sequence determinations and approximately at residues 296, 478, 625 and 702 as proposed from the molecular mass values of the generated polypeptide chains. Products from the proteolytic cleavage of the A and B2 chains of laminin were observed with the sites of cleavage determined to be at position 2666 in the laminin A chain and position 1238 in the laminin B2 chain. The laminin digestion products were identical regardless of whether nidogen was present in a complex with the laminin chains.

Amino Acid Sequence↗
PubMed1991

Synthetic and endogenous inhibitors of snake venom metalloproteinases.

The venoms of most Crotalidae snakes contain metalloproteinases which are the agents responsible for the production of venom-induced hemorrhage via proteolytic destruction of capillary basement membranes. Prevention of hemorrhage by administration of antiserum is generally not totally effective against damage at the site of envenomation. Therefore, we have investigated alternate methods for the alleviation of hemorrhage by inhibition of the proteolytic activity of the hemorrhagic toxins. The first approach involves the synthesis of carboxyalkyl peptide inhibitors in which the peptide moiety is modeled on the substrate specificity of the toxins. With this approach we have determined that the carboxypentyl group for interaction with the active site Zn+ +ion is most effective. Also, longer peptide moieties enhance the inhibitors' activity giving Ki's in a range of 10(-6) M. Our second approach to hemorrhagic toxin inhibition was to search for the presence of endogenous inhibitors against the toxins in the venom. From the crude venom we have isolated several pyro-glutamate containing peptides, two of which are relatively good inhibitors of the toxins. The isolation and characterization of the endogenous toxin inhibitors as well as the synthetic inhibitors may ultimately serve as a basis for new, effective treatments against venom-induced hemorrhage.

Amino Acid Sequence↗
PubMed1990

Conformational stability of B cell epitopes on group I and group II Dermatophagoides spp. allergens. Effect of thermal and chemical denaturation on the binding of murine IgG and human IgE antibodies.

The conformational stability of B cell epitopes on the 25-kDa group I and 14-kDa group II mite allergens was compared by using heat-treated or chemically denatured allergens to inhibit the binding of native 125I allergens to murine mAb or to human IgE antibodies. Structural changes after treatment were assessed by SDS-PAGE and circular dichroism spectroscopy. Heating for 1 h at greater than 75 degrees C, treatment at pH 2.0 or pH 12.0, or with 6M guanidine or 6M urea, reduced the binding of the group I allergens to mAb or IgE antibodies by 10- to 1000-fold. The group II allergens were heat stable and even after prolonged heat treatment (5 h at 75 degrees C or 30 min at 100 degrees C) their antibody binding activity was reduced by less than twofold. The group II allergens were also resistant to pH and to denaturation with 6M guanidine. However, after reduction and alkylation, antibody binding sites on both the group I and group II allergens were destroyed. Reduction of disulfide bonds with 2-ME caused a marked shift in the molecular mass of group I allergens on SDS-PAGE, from 25 kDa to 28-31 kDa. Reduction and alkylation also generated two high m.w. forms of Der p I and Der f I. After heating (100 degrees for 30 min), both Der f I and Der f II retained significant secondary structure, as judged by circular dichroism spectroscopy, but on reduction they showed the typical spectra of fully denatured proteins (greater than 85% random structure). The results show clear differences between the susceptibility of B cell epitopes on the group I and group II allergens to denaturation. Despite these differences in stability, both allergens are equally potent immunogens for IgE antibody responses in man. The results support the view that the physical properties of allergens (low m.w. and solubility), limiting low dose exposure (1 to 10 ng/day), and host genetic and immunoregulatory processes, are more important than gross structural features in the induction and maintenance of IgE antibody responses.

Alkylation↗
PubMed1990

Interaction of hemorrhagic metalloproteinases with human alpha 2-macroglobulin.

The interaction between four Crotalus atrox hemorrhagic metalloproteinases and human alpha 2-macroglobulin was investigated. The proteolytic activity of the hemorrhagic toxins Ht-c, -d, and -e against the large molecular weight protein substrates, gelatin type I and collagen type IV, was completely inhibited by alpha 2-macroglobulin. The proteolytic activity of Ht-a against the same substrates was not significantly inhibited. Each mole of alpha 2-macroglobulin bound maximally 2 mol of Ht-e and 1.1 mol of Ht-c and Ht-d. These proteinases interacted with alpha 2-macroglobulin rapidly at 22 degrees C. Rate constants based on intrinsic fluorescence measurements were 0.62 X 10(5) M-1 s-1 for interaction of alpha 2-macroglobulin with Ht-c and -d and 2.3 X 10(5) M-1 s-1 for the interaction of alpha 2-macroglobulin with Ht-e. Ht-a interacted with alpha 2-macroglobulin very slowly at 22 degrees C. Increasing the temperature to 37 degrees C and prolonging the time of interaction with alpha 2-macroglobulin resulted in the formation of Mr 90,000 fragments and high molecular weight complexes (Mr greater than 180,000), in which Ht-a is covalently bound to the carboxy-terminal fragment of alpha 2-M. The identification of the sites of specific proteolysis of alpha 2-macroglobulin shows that the cleavage sites for the four metalloproteinases are within the bait region of alpha 2-macroglobulin. Ht-c and -d cleave only at one site, the Arg696-Leu697 peptide bond, which is also the site of cleavage for plasmin, thrombin, trypsin, and thermolysin. Ht-a cleaves alpha 2-macroglobulin primarily at the same site, but a secondary cleavage site at the His694-Ala695 peptide bond was also identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1979

The hydrodynamic shape, conformation, and molecular model of Escherichia coli ribosomal 5 S RNA.

The structure of ribosomal 5 S RNA has been examined using several physical biochemical techniques. Hydrodynamic measurements yield a s020,omega and [eta] of 5.5 x 10(-13) x and 6.9 ml/g, respectively. Other parameters calculated from these values indicate the shape of 5 S RNA is consistent with that of a prolate ellipsoid 160 A in length and 32 A wide. Sedimentation equilibrium results show that 5 S RNA exists as a monomer in the reconstitution buffer with an apparent molecular weight of 44,000. Ultraviolet absorption difference spectra show that approximately 75% of the bases in 5 S RNA are involved in base pairing, and of these base pairs 70% are G-C and 30% are A-U. These results on the overall shape and secondary structure of 5 S RNA have been incorporated with the results of other investigators as to the possible location of single-stranded and double-stranded helical regions, and a molecular model for 5 S RNA is proposed. The molecular model consists of three double helices in the shape of a prolate ellipsoid, with two of the double helical regions at one end of the molecule. The structure is consistent with the available data on the structure and function of 5 S RNA and bears similarity to the molecular model proposed by Osterberg et al. ((1976) Eur. J. Biochem. 68, 481-487) based on small angle x-ray scattering results and the secondary structure proposed by Madison ((1968) Annu. Rev. Biochem. 37, 131-148).

Escherichia coli↗
PubMed1978

Structural changes of ribosome by the action of ethylene glycol.

The denaturation of ribosome and RNA by ethylene glycol (EG) has been studied in an attempt to further understand the conformation and stability of the ribosome. At high concentrations of EG, the ribosome, its subunits, and 16S RNA undergo drastic structural changes as shown by circular dichroism, ultraviolet absorption spectroscopy, and sedimentation velocity. Two separate conformational transitions were observed for the 30S subunit; one from 30 to 50% EG and another from 60 to 90% EG. This observation suggests the presence of two "domains" in the 30S subunit which differ in their stability. However, the 50S subunit undergoes a single sharp transition at 60 to 90% EG, consistent with the notion of a highly cooperative conformation. Association of the subunits stablizes part of the 30S subunit since the transition curve for the 70S ribosome does not exhibit significant change at the low EG concentration region as seen for the 30S subunit. Removal of proteins from the 30S subunit broadens the transition curve to lower EG concentrations and suggests the role of proteins in stabilizing the conformation of the 16S RNA.

Cell Fractionation↗
PubMed1978

Changes in the conformation and stability of 5 S RNA upon the binding of ribosomal proteins.

The binding of ribosomal proteins L25, L18, and L5 to 5 S RNA results in a conformational change and a destabilization of the 5 S RNA molecule. The changes observed in the near ultraviolet circular dichroism (CD) spectra and in the melting profiles indicate an increase in base stacking uith an accompanying increase in asymmetry of the bases and a decrease in the conformational stability of the 5 S RNA. These results are consistent with the interpretation that the binding of these proteins increases the stacking of specific single-stranded bases in 5 S RNA and aligns them in helical arrays, resulting in a conformation which facilitates base-pairing with nucleotide segment(s) of the ribosomal 23 S RNA or the transfer RNA (or both). The simple and precise difference CD method described here is potentially useful for studying subtle conformational changes of other nucleic acid-protein interactions.

Bacterial Proteins↗
PubMed1977

Nonoperative management of fingertip pulp amputation by occlusive dressings.

Nonoperative management of fingertip pulp amputations has been employed in eighteen adults. After wound cleansing and debridement, the wound was covered by an occlusive dressing. Healing of the amputated fingertip occurred within four weeks. The healed fingertip had an excellent sensory perception, normal range of motion and an acceptable cosmetic appearance. This satisfactory outcome was realized with less than ten days lost from work.

Aluminum↗
PubMed1977

An adjunct to mammoplasty: the pectoral muscle flap.

The pectoralis major muscle has overcome many of the complications of augmentation mammoplasty after subcutaneous mastectomy. It places the implant deep to prevent skin slough and irregularities in skin surface contour. The flap eliminates abnormal implant posture when the pectoralis major is contracted. The short (5 to 7 days) staging of the procedure allows sealing of the pocket from external innoculation of bacteria while preventing unretrievable skin and nipple shrinkage.

Breast↗