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D Eaton

Publications and source records attributed to D Eaton.

34 records · Page 2Linked to original sources
PubMed1992

Biochemical characterization of matrilysin. Activation conforms to the stepwise mechanisms proposed for other matrix metalloproteinases.

The latent precursor of matrilysin (EC 3.4.24.23; punctuated metalloproteinase (PUMP) was purified from transfected mouse myeloma cell conditioned medium and was found to contain one zinc atom per molecule which was essential for catalytic activity. Promatrilysin could be activated to the same specific activity by (4-aminophenyl)mercuric acetate, trypsin, and incubation at elevated temperatures (heat activation). Active matrilysin hydrolyzed the fluorescent substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond with a maximum value for kcat/Km of 1.3 x 10(4) M-1 s-1 at the pH optimum of 6.5 and pKa values of 4.60 and 8.65. Activity is inhibited by the tissue inhibitor of metalloproteinases-1 in a 1:1 stoichiometric interaction. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with N-terminal sequencing revealed that, as with all other matrix metalloproteinases similarly studied, promatrilysin activation was accompanied by the stepwise proteolytic removal of an M(r) 9000 propeptide from the N-terminus. The intermediates generated were dependent on the mode of activation used but, in all cases studied, activation terminated with an autocatalytic cleavage at E77-Y78 to yield the final M(r) 19,000 active matrilysin. From an analysis of the stability of the various intermediates, we propose that the sequence L13-K33 is particularly important in protecting the E77-Y78 site from autocatalytic cleavage, thereby maintaining the latency of the proenzyme.

Amino Acid Sequence↗
PubMed1992

The C-terminal domain of 72 kDa gelatinase A is not required for catalysis, but is essential for membrane activation and modulates interactions with tissue inhibitors of metalloproteinases.

Recombinant 72 kDa gelatinase A and a truncated form lacking the C-terminal domain were shown to be activated by organomercurials and to possess similar activities towards a number of substrates. The truncated proenzyme differed from the full-length gelatinase in that it could not be activated by a membrane activator and did not bind tissue inhibitor of metalloproteinase (TIMP)-2. Kinetic studies also showed that the inhibition of the activated truncated enzyme, by both TIMP-1 and TIMP-2, was considerably decreased compared with the full-length enzyme. We conclude that the C-terminal domain plays an important role in the regulation of gelatinase A by a potential physiological activator and inhibitors.

Base Sequence↗
PubMed1992

Organophosphate pesticide exposure in a group of Washington State orchard applicators.

As part of a study to investigate the potential for organophosphates to cause chronic neurologic sequelae, we assessed the pesticide exposure experience of a group of Washington State apple orchard applicators. Seasonal monitoring of cholinesterase activity for 48 regular organophosphate applicators and a control group of 40 slaughterhouse workers was performed. A subset of the pesticide applicators participated in an in-depth exposure assessment. This involved observation of spraying activities during 1 spray day, as well as cholinesterase monitoring and dermal exposure assessment using a fluorescent tracer in the pesticide formulation. Comparison of seasonal red blood cell cholinesterase change in pesticide workers according to exposure level, characterized by frequency of pesticide spraying and protective equipment use, showed lower cholinesterase levels among higher exposed groups compared to lesser exposed groups. In-depth exposure assessment revealed exposure primarily on the head and hand regions. Subclinical changes (less than 15% inhibition) in red cell cholinesterase correlated well with dermal exposure calculations. This study suggests that cholinesterase monitoring may be a useful biological marker for even subclinical organophosphate pesticide effects.

Agricultural Workers' Diseases↗
PubMed1991

Purification of a hybrid plasminogen activator protein.

Recombinant DNA technology has been employed to produce a hybrid gene in which the kringle and serine protease domains of tissue plasminogen activator are linked to the heavy-chain Fd region of a fibrin-specific antibody. The hybrid gene is co-expressed with antibody light chains. This communication describes a purification procedure for the hybrid protein, involving affinity and ion-exchange chromatography. The purified hybrid protein has been used in vivo and in vitro clot lysis experiments and has been shown to be effective at clot dissolution.

Animals↗
PubMed1990

Calcified neonatal renal vein thrombosis demonstration by CT and US.

Two cases of calcified renal vein thrombosis (RVT) were diagnosed, incidentally, within the first weeks of life. The CT images present the virtually diagnostic branching pattern of calcification that has been previously noted on pathology specimen radiographs. The CT and US images show peripheral renal vein, central renal vein and inferior vena cava calcification conforming to the two theoretical origins of intravascular calcification. The patients had normal laboratory results and no symptoms related to renal vein or inferior vena cava thrombi.

Calcinosis↗
PubMed1990

The use of gonadotropin-releasing hormone agonist to regulate oocyte retrieval time.

We investigated the use of a gonadotropin-releasing hormone agonist (GnRH-a) to assist in the synchronous entry of a fixed number of subjects into in vitro fertilization (IVF) and gamete intrafallopian tube transfer cycle. Using a protocol in which a GnRH-a was given in the follicular or luteal phase, the clinical pregnancy rate per procedure was 27%. We conclude that the use of a GnRH-a for synchronous cycles in IVF-embryo transfer is efficacious and results in acceptable pregnancy rates.

Embryo Transfer↗
PubMed1987

Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli.

In a lacZ expression vector (pMC1403Plac), all 64 codons were introduced immediately 3' from the AUG initiation codon. The expression of the second codon variants was measured by immunoprecipitation of the plasmid-coded fusion proteins. A 15-fold difference in expression was found among the codon variants. No distinct correlation could be made with the level of tRNA corresponding to the codons and large differences were observed between synonymous codons that use the same tRNA. Therefore the effect of the second codon is likely to be due to the influence of its composing nucleotides, presumably on the structure of the ribosomal binding site. An analysis of the known sequences of a large number of Escherichia coli genes shows that the use of codons in the second position deviates strongly from the overall codon usage in E. coli. It is proposed that codon selection at the second position is not based on requirements of the gene product (a protein) but is determined by factors governing gene regulation at the initiation step of translation.

Base Sequence↗
PubMed1987

Ca2+ responses in cytomegalovirus-infected fibroblasts of human origin.

Cytomegalovirus (CMV) infection of fibroblasts of human origin is associated with a cascade of cellular responses (rounding, "contraction," "relaxation," and enlargement). Since in other systems these cellular responses are regulated by intracellular free Ca2+ activity ([Ca2+]i), we measured intracellular Ca2+ responses to CMV infection. At relatively high multiplicities of infection (m.o.i), an influx of Ca2+ was observed within the first hour after CMV infection (p.i.) at which time it was at its maximum rate. Both the time of occurrence and the magnitude of this Ca2+ influx were dependent on the calculated input m.o.i. In CMV-infected cells, [Ca2+]i rose gradually from 80 nM at 0 hr to 174 nM at 48 hr p.i. (about 2.7 times the [Ca2+]i found in mock-infected cells at this time). At 8 and 12 hr p.i. CMV-infected cells consistently contained a somewhat greater level of 45Ca2+ than mock-infected cells, despite the fact that there was only a small increase in [Ca2+]i between CMV and mock-infected cells in the same period. This observation suggests that there may be significant amounts of Ca2+ taken up into intracellular stores. This Ca2+ in intracellular stores may, at later times after infection, contribute to the increase in [Ca2+]i observed from 12 to 48 hr p.i. Ca2+ influx blockers, such as nifedipine and verapamil, inhibited the rise in [Ca2+]i. The increase in [Ca2+]i in response to CMV infection was shown to be dependent on the m.o.i., require infectious virus, and occur under conditions consistent with the expression of immediate-early CMV genes. The capability of inducing such Ca2+ responses was conserved among three laboratory strains of CMV. The CMV-induced Ca2+ responses may be related not only to the development of CMV cytopathology, but also to the replication of CMV, since in other studies cyclic nucleotide modulators and Ca2+ influx blockers were found to inhibit the replication of CMV.

Calcium↗
PubMed1986

Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule.

The primary structure of factor VIII consists of 2332 amino acids that exhibit 3 distinct structural domains, including a triplicated region (A domains), a unique region of 909 amino acids (B domain), and a carboxy-terminal duplicated region (C domains), that are arranged in the order A1-A2-B-A3-C1-C2. The B domain (residues 741-1648) of factor VIII is lost when factor VIII is activated by thrombin, which proteolytically processes factor VIII to active subunits of Mr 50,000 (domain A1), 43,000 (domain A2), and 73,000 (domains A3-C1-C2). To determine if the B domain is required for factor VIII coagulant activity, a variant was constructed by using recombinant DNA techniques in which residues 797-1562 were eliminated. This shortened the B domain from 909 to 142 amino acids. This variant factor VIIIdes-797-1652 was expressed in mammalian cells and was found to be functional. The factor VIIIdes-797-1562 protein was purified and shown to be processed by thrombin in the same manner as full-length factor VIII. The factor VIIIdes-797-1562 variant also bound to von Willebrand factor (vWF) immobilized on Sepharose. These results indicate that most of the highly glycosylated B domain of factor VIII is not required for the expression of factor VIII coagulant activity and its interaction with vWF.

Amino Acid Sequence↗
PubMed1986

Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity.

Human factor VIII was isolated from commercial factor VIII concentrates and found to consist of multiple polypeptides with molecular weights ranging from 80 000 to 210 000. Immunological and amino acid sequence data identified these polypeptides as subunits of factor VIII. N-Terminal amino acid sequence analysis determined that the Mr 210 000 and 80 000 proteins are derived from the N- and C-terminal portions of factor VIII, respectively; Mr 90 000-180 000 polypeptides are derived from the Mr 210 000 polypeptide by C-terminal cleavages. Treatment of purified factor VIII with thrombin resulted in proteolysis of Mr 80 000-210 000 proteins and the generation of polypeptides of Mr 73 000, 50 000, and 43 000. Maximum coagulant activity of thrombin-activated factor VIII was correlated with the generation of these polypeptides. The proteolysis as well as activation of factor VIII by thrombin was found to be markedly dependent on CaCl2 concentration. Proteolysis of factor VIII with activated protein C (APC) resulted in degradation of the Mr 90 000-210 000 proteins with the generation of an Mr 45 000 fragment. This cleavage correlated with inactivation of factor VIII by APC. The Mr 80 000 protein was not degraded by APC. Factor Xa cleaved the Mr 80 000-210 000 factor VIII proteins, resulting in the generation of fragments of Mr 73 000, 67 000, 50 000, 45 000, and 43 000. Factor Xa was found to initially activate and subsequently inactivate factor VIII.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1980

Streptomyces viridochromogenes spore germination initiated by calcium ions.

Initiation of germination of heat-activated Streptomyces viridochromogenes spore occurs in media containing only calcium ions and organic buffer. The calcium-induced initiation of germination was accompanied by a decrease in absorbance of the spore suspension, an increased rate of endogenous metabolism, the loss of spore carbon, and the loss of heat resistance. Calcium amounts to 0.28% of the dry weight of freshly harvested spores. The amount of calcium remained the same after incubation of spores in water after heat activation. The spore content of calcium doubled after incubation in 0.5 mM CaCl2 for 5 min at 4 degrees C and during calcium-induced germination. Nearly all of the calcim appears to be bound to sites external to the spore membrane, since the chelating agents (ethylenedinitrilo) tetraacetic acid and arsenazo III removed virtually all of the calcium ions. The calcium ions must be present during the entire initiation of germination period. Germination ceases after an (ethylenedinitrilo) tetraacetic acid wash and begins again immediately after addition of calcium ions.

Arsenazo III↗
PubMedDate not supplied

Structure of human factor VIII.

The deduced amino acid sequence of human factor VIII, obtained from the DNA sequence, predicts a mature polypeptide of 2,332 amino acids containing a triplicated domain structure. The polypeptide has 35% sequence homology with the copper-binding plasma protein, ceruloplasmin. Determination of the thrombin cleavage sites in plasma-derived factor VIII polypeptides allows prediction of the domains involved in the associated activation and inactivation of the protein.

Amino Acid Sequence↗