Biomedical subjects

J Travis

Publications and source records attributed to J Travis.

At least 217 records · Page 12Linked to original sources

PubMed1990

The interplay of population dynamics and the evolutionary process.

Long-term maintenance of genetic diversity is affected by ecological forces that are driven in turn by current levels of genetic variation. The strength of population regulation and the consequent patterns of population fluctuations determine the likelihood of genetic changes considered pivotal for rapid speciation. However, genetic diversity in the susceptibility to regulatory forces can reduce the magnitude of such fluctuations and minimize the likelihood of genetic revolutions. A group of populations that experiences local extinctions and recolonizations may hold lower levels of genetic diversity than in the absence of such extinctions, but local adaption, which provides enhanced genetic diversity, can reduce the likelihood of local extinctions. Tightly regulated populations experience different selection pressures than poorly regulated populations, although tighter regulation itself can evolve. When genotypic variation affects the outcome of interspecific interactions on a local scale, this effect, coupled with appropriate spatial variation, can enhance the resilience of the interactive system.

Animals↗

PubMed1990

Comparative properties of human alpha-1-proteinase inhibitor glycosylation variants.

Variant forms of human alpha-1-proteinase inhibitor (alpha-1-PI), obtained by the treatment of human Hep G2 cells with specific inhibitors of glycosylation were tested for both inhibitory activity and heat stability. All were found to have the same second-order association rate with human neutrophil elastase, indicating a lack of importance of the carbohydrate moiety. In contrast, incompletely glycosylated forms of alpha-1-PI were found to be heat sensitive relative to the mature protein, suggesting a role for carbohydrate in protein stabilization.

1-Deoxynojirimycin↗

PubMed1990

Identification of the primary antimicrobial domains in human neutrophil cathepsin G.

Lysosomal cathepsin G from human neutrophils is a chymotrypsin-like protease which also possesses antimicrobial activity. The antimicrobial activity, however, is independent of protease activity, because treatment of this enzyme with the irreversible serine protease inhibitor diisopropylfluorophosphate has no effect on its antimicrobial action. In this study, we found that digestion of cathepsin G with clostripain caused a loss of proteolytic activity in this neutrophil proteinase. However, bactericidal activity in in vitro assays against Staphylococcus aureus and Neisseria gonorrhoeae was retained. Fractionation of the clostripain-digested cathepsin G mixture yielded two distinct antimicrobial peptides. The sequences of these peptides were IIGGR and HPQYNQR (residues 1-5 and 77-83 in cathepsin G, respectively). Synthetic peptides corresponding to these sequences were also prepared and found to exert broad-spectrum antimicrobial activity in vitro, displaying conditions of temperature- and pH-dependent optima for antimicrobial action resembling that of the full-length enzyme. Depending on the target bacterial strain, these peptides exhibited antimicrobial activity between 5.0 x 10(-5) and 4.0 x 10(-4) M. Significantly, replacement of certain residues within these peptides with either alanine or valine significantly reduced their antibacterial capacities. Our studies suggest that cathepsin G has two antimicrobial sequences, either or both of which may contribute to its bactericidal activity.

Amino Acid Sequence↗

PubMed1990

Plasma inter-alpha-trypsin inhibitor-related urinary glycoprotein EDC1 inhibits the growth of a Burkitt's lymphoma cell line.

A homogeneous preparation of a urinary glycoprotein has been isolated from urine of patients with malignant melanoma and advanced adenocarcinomas of colon and lung. This molecule, Mr 30 kDa, is homologous to EDC1, a proteinase inhibitor antigenically related to plasma inter-alpha-trypsin inhibitor (IATI) originally isolated from the urine of a leukemic patient, E.D. The newly isolated EDC1 inhibits cellular proliferation of a Burkitt's lymphoma cell line, Raji, growing in serum-free medium supplemented with insulin, transferrin, selenium, and linoleic acid. This concentration-dependent inhibitory effect was monitored in terms of change in cell number and 3H-thymidine incorporation. The growth of cells treated with approximately 3.3 pmol EDC1/ml was 50% that of the control group by both assays. EDC1 was not cytotoxic to the cells because the EDC1-treated cells excluded trypan blue and resumed normal growth after removal of EDC1. In addition, EDC1 treatment of Raji cells prelabeled with 3H-labeled DNA did not release more radioactivity into the conditioned medium than the untreated labeled cells. EDC1 did not affect the growth of Hs2B2, a B-lymphoblast cell line, and Hs294T, a human malignant melanoma cell line. Equimolar and larger quantities of other proteinase inhibitors with inhibitory profiles similar to that of EDC1 (alpha-1 proteinase inhibitor, soybean trypsin inhibitor, lima bean trypsin inhibitor, and turkey ovomucoid) did not affect the growth of Raji cells. Raji cells have an absolute requirement of transferrin as a nutrient and require insulin to modulate the expression of transferrin receptors. The cells also synthesize interleukin-1 as an autocrine growth stimulator. EDC1 did not form a detectable complex with transferrin, insulin, or any autocrine factor synthesized by the cells.

Alpha-Globulins↗

PubMed1990

Isolation of nine human plasma proteinase inhibitors by sequential affinity chromatography.

Purification of nine plasma proteinase inhibitors and one zymogen from a single batch of human plasma, using affinity chromatography has been accomplished. Those isolated were plasminogen (lysine-Sepharose), alpha-2-antiplasmin (plasminogen-Sepharose), high and low molecular weight kininogens (CM-papain-Sepharose), alpha-2-macroglobulin (Zn++ chelate-Sepharose), alpha-1-proteinase inhibitor, alpha-1-antichymotrypsin, Cl-inhibitor, inter-alpha-trypsin inhibitor (Blue-Sepharose) and antithrombin III (heparin-Sepharose). Alpha-2-macroglobulin and alpha-1-proteinase inhibitor required gel filtration as additional purification steps. Each protein was recovered in both high yield and purity.

Alpha-Globulins↗

PubMed1990

Serpins: structure and mechanism of action.

The Serpins are a major family of proteins, most of which are involved in the regulation of proteinase activity. Current data indicate that inhibitor function is dependent on formation of tight, but reversible binary complexes, with carbohydrate being unimportant for this function. The reaction takes place in a reactive site loop common to all Serpins, with the key residue for complex formation being in the P1 reactive site position. However, other contact residues are also involved as shown by the variation in specificity in a number of animal proteins with the same P1 residue. The role of other amino acid residues in Serpins which aid in conferring specificity has not yet been established. However, the availability of methods for obtaining site specific mutations should soon make it possible to determine other contact points required for Serpin function, thus allowing for the design of inhibitors which are singularly targeted with a high reaction rate towards a given proteinase.

Animals↗

PubMed1989

Stabilization vs. degradation of Staphylococcus aureus metalloproteinase.

Purified Staphylococcus aureus metalloproteinase contains trace amounts of a serine proteinase which rapidly degrades the metalloproteinase when EDTA is present. However, no degradation occurs when Ca2+ is added or if the serine proteinase is removed by immunoaffinity chromatography. Selective chelation of Zn2+ by o-phenanthroline, which reversibly inactivates the metalloproteinase, does not result in the degradation of the apometalloproteinase, even with excess of serine proteinase. These data are interpreted as follows: EDTA chelates enzyme-bound Ca2+ and Zn2+, causing irreversible inactivation as well as a conformational change in the metal-free protein. This allows proteolysis by the contaminating serine proteinase and explains why the metalloproteinase purified from serine proteinase-deficient strains of S. aureus was previously thought to be stable to autolysis.

Calcium↗

PubMed1989

Inter-alpha-trypsin inhibitor. Inhibition spectrum of native and derived forms.

The conversion of inter-alpha-trypsin inhibitor (I alpha I) into active, acid-stable derivatives by proteolytic degradation has been tested with 10 different proteinases. Of these, only plasma kallikrein, cathepsin G, neutrophil elastase, and the Staphylococcus aureus V-8 proteinase were found to be effective, each releasing more than 50% of this activity. However, a strong correlation between inhibitor degradation and significant release of acid-stable activity could only be found with the V-8 enzyme. Inhibition kinetics for the interaction of native I alpha I, the inhibitory fragment released by digestion with S. aureus V-8 proteinase, or the related urinary trypsin inhibitor, with seven different proteinases indicated that all had essentially identical Ki values with an individual enzyme and, where measurements were possible, nearly identical second order association rate constants. Significantly, none of the five human proteinases tested, including trypsin, chymotrypsin, plasmin, neutrophil elastase, and cathepsin G, would appear to have low enough Ki values to be physiologically relevant. Thus, the role of native I alpha I or its degradation products in controlling a specific proteolytic activity is still unknown.

Alpha-Globulins↗

PubMed1989

The use of alpha 2-antiplasmin as a model for the demonstration of complex reversibility in serpins.

Human alpha 2-antiplasmin readily forms 1:1 complexes with either trypsin or chymotrypsin at independent but overlapping reactive sites. In the absence of alpha 2-macroglobulin, complex dissociation and enzyme release can be demonstrated without regeneration of inhibitory activity. However, in the presence of this inhibitor the dissociation of alpha 2-antiplasmin-chymotrypsin complexes or alpha 2-antiplasmin-trypsin complexs yields functionally active inhibitors which can now inactivate trypsin and chymotrypsin, respectively. These results clearly indicate that Serpin-proteinase complexes can dissociate to give both active inhibitor and enzyme. If the enzyme is trapped by alpha 2-macroglobulin, in vivo, it is possible that the inhibitor may be recycled for further use.

Chymotrypsin↗

PubMed1989

The primary role of the P1 residue (ser359) of alpha-1-proteinase inhibitor.

The replacement of ser359 with ala359 at the P1 position in human alpha-1-proteinase inhibitor results in the production of a variant protein containing 15% of the inhibitory activity of the normal inhibitor. Separation of active from inactive inhibitor on anhydrochymotrypsin-sepharose yields a form which has a second order association rate with neutrophil elastase which is approximately one half that for the native protein. These data indicate that the P1 residue is not of primary importance during the interaction of proteinases with alpha-1-proteinase inhibitor. Since substitution of alanine for serine causes the formation, primarily, of inactive inhibitor the major function of ser359 probably involves proper folding to give a functionally active inhibitory conformation.

Alanine↗

PubMed1989

Antithrombin III activity (residual thrombin activity) in plasma from non-medicated or heparinized horses.

Two synthetic substrate assays (fluorometric and chromogenic) were used to measure antithrombin-III (AT-III) activity (residual thrombin activity) in non-medicated and heparin (sodium) treated horses. In 18 non-medicated horses the fluorometric substrate assay (FSA) values were similar to previous reports but they reflected inconsistent trends and larger deviations in the heparin-treated groups (Group 2: 40 and 100 U/kg IV, n = 6; Group 3: 240 U/kg IV, n = 5; Group 4: 80 U/kg IV followed by 160 U/kg SC, n = 8) when compared to the chromogenic substrate assay (CSA) values. The CSA values for the 18 non-medicated horses indicated a higher AT-III activity (lower residual thrombin activity) than the FSA. AT-III activity was quantified in 18 non-medicated horses (29 mg/dl) and compared well with values for humans (30 mg/dl) and dogs (40 mg/dl). Plasma heparin concentrations, determined by the FSA, correlated well with the 'therapeutic range' (1.5 fold to 2.5 fold prolongation of the activated partial thromboplastin time (APTT) normal value) and values reported for humans. The effect of heparin therapy on AT-III activity in four treatment regimens was evaluated. AT-III activity was not significantly affected (with one exception) by a single dose of intravenous (IV) heparin (40 and 100 U/kg) nor by repeated subcutaneous (SC) injections of heparin (240 U/kg). A transient increase in residual thrombin activity was measured 12 h after an intravenous (80 U/kg) injection of heparin. Large doses of heparin (80 U/kg IV followed by 160 U/kg SC) given every 12 h produced a progressive prolongation of the APTT. In this group the APTT remained prolonged 48 h after the last treatment.

Animals↗

PubMed1989

The effect of RG 12525 on leukotriene D4-mediated pulmonary responses in guinea pigs.

RG 12525 (5-(2-[4-quinolin-2-yl)methoxyl] phenoxymethyl)benzyl tetrazole) is under investigation as a specific inhibitor of leukotriene D4 (LTD4). The present studies examine the effect of orally administered RG 12525 on LTD4 mediated pulmonary responses in three separate guinea pig models. The compound inhibited antigen-induced mortality in the systemic anaphylaxis model with an ED50 (95% confidence interval) = 2.2 (0.8-6.4) mg/kg. In this model, the activity half-life of RG 12525 was shown to be 6.5 hours and the compound offered significant protection within 15 minutes of administration. RG 12525 also protected against LTD4-induced bronchoconstriction in a model measuring changes in pulmonary function with an ED50 = 0.6 (0.4-1.0) mg/kg. The same level of activity was observed in a similar model which monitored changes in pulmonary function in response to exogenous antigen in actively-sensitized guinea pigs. Together, these data indicate that RG 12525 is a potent, orally active LTD4 antagonist which possesses the requisite profile for potential clinical development.

Anaphylaxis↗

PubMed1989

Purification and characterization of insect toxins derived from the mite, Pyemotes tritici.

Three low mol. wt proteins which have contracting-paralyzing activity in insects were isolated from extracts of the straw itch mite, Pyemotes tritici. One of these toxins, referred to as TxP-I, was purified to apparent homogeneity using the following sequence: ion-exchange, affinity, hydroxyapatite and reverse-phase chromatography. The other two toxins, referred to as TxP-II, remained as a mixture. Peptide mapping and immunoblot analysis suggest that TxP-I and TxP-II are probably isoproteins. The apparent mol. wt of native TxP-I and of the two components of TxP-II were 27,000, 28,000 and 29,000, respectively. The apparent mol. wt of the toxins after reductive carboxamidomethylation increased to 38,000, 41,000 and 43,000, respectively. The amino acid composition of TxP-I indicates a high content of Cys (8 mole%). Therefore, several disulfide bonds may impart a very compact tertiary structure to this protein which, upon denaturation, unfolds and increases its Stoke's radius resulting in retarded mobility on a polyacrylamide gel. The N-terminal sequence of TxP-I is not homologous with any other protein for which the sequence is known. The paralysis dose50 of TxP-I (PD50) in wax moth larvae is ca. 500 micrograms/kg and it is not toxic to mice at a dose of 50 mg/kg. A polyclonal antibody, raised against TxP-I, reacted with both TxP-I and TxP-II. The antibody neutralized the rapid, muscle-contracting paralysis of these toxins. Using this antibody and immunocytochemistry, we found the toxins localized in posterior glands which appear to be connected with the stylet through a series of ducts. We conclude that TxP-I and TxP-II are part of a complex mixture of neurotoxins which P. tritici utilizes to capture prey.

Amino Acid Sequence↗

PubMed1989

Effects of 5-lipoxygenase inhibition on cardiac anaphylaxis in isolated guinea pig hearts.

Synthesis of the cardioactive peptidoleukotrienes depends on the activity of 5-lipoxygenase. It is unclear whether inhibition of endogenous leukotriene biosynthesis can alter vasoconstriction and negative inotropic effects associated with inflammatory states in the heart. In an attempt to study this problem, two 5-lipoxygenase inhibitors, the novel compound RG 6866, N-methyl-4-benzyloxyphenyl acetohydroxamic acid, and AA 861 have been examined in Langendorff-perfused guinea pig hearts undergoing cardiac anaphylaxis and compared to indomethacin and LY 171883, a cyclooxygenase inhibitor and a leukotriene antagonist, respectively. In paced hearts perfused at constant pressure, RG 6866 had no apparent direct effects, infused intracardially at 20 micrograms/min, about 3 mumol/l. LY 171883, but not RG 6866 or indomethacin, antagonized coronary vasoconstriction by exogenous leukotriene D4. When hearts were challenged with antigen (ovalbumin 1 mg i.c.) in the presence of antihistamines, a significant reduction in coronary flow occurred within 30 s which was maximal at 2-3 min, -48 +/- 3%, and persisted for at least 10 min. An initial positive intropic effect was followed by a 25% fall in developed pressure. Both 5-lipoxygenase inhibitors blocked effects of antigen challenge on coronary flow: RG 6866, -4 +/- 3%, AA 861, -11 +/- 3%; reduction at 2 min. The late negative inotropic effect was also blocked. The combined 5-lipoxygenase inhibitor/leukotriene antagonist, RG 5901, and the leukotriene antagonist, LY 171883, were also effective, but not indometacin. These results provide further evidence for the contribution of leukotrienes to cardiac anaphylaxis. More importantly, these findings suggest that 5-lipoxygenase inhibitors can be beneficial in situations where endogenous leukotrienes produce coronary vasoconstriction and myocardial depression.

Acetophenones↗

PubMed1989

The human neutrophil elastase gene. Analysis of the nucleotide sequence reveals three distinct classes of repetitive DNA.

DNA sequence analysis reveals the gene encoding human neutrophil elastase to be contained on a 6-kb EcoRI fragment. The gene contains five exons and closely resembles rat mast cell proteinase II and mouse adipsin in its exon structure and intron splice phase. Non-coding regions are very rich in repetitive DNA, containing seven Alu-like segments, three distinct clustered direct repeats with monomer lengths of 53 (six repeats), 23 (three repeats) and 41 (ten repeats) nucleotides, and a 200-nucleotide AT-rich region. Protein sequence analysis, inferred from the coding regions of the gene, indicates that neutrophil elastase may contain an unusual activation peptide similar to that found in the other major neutrophil serine proteinase, cathepsin G.

DNA↗

PubMed1989

Rapid screening for deficiency of alpha 1-proteinase inhibitor.

This rapid screening procedure for detection of low but functional elastase-inhibitory activity in human plasma is based on the fact that incubation of excess porcine pancreatic elastase (EC 3.4.21.36) with plasma results in formation of a complex with active alpha 1-proteinase inhibitor (alpha 1PI, also called alpha 1-antitrypsin). In normal individuals all of the elastase is complexed, leaving no free enzyme to hydrolyze the elastase substrate, and the reaction mixture remains clear. Because individuals homozygous for the Z allele have relatively low concentrations of alpha 1PI, their plasma cannot complex all of the elastase in the assay. The uncomplexed enzyme hydrolyzes the elastase-specific p-nitroanilide substrate, producing a yellow reaction mixture. Use of this simple assay for early screening of individuals for alpha 1PI deficiency may substantially decrease the number of untreated cases of familial emphysema, a disorder that develops as a result of a genetically derived proteinase-proteinase inhibitor imbalance.

Alpha-Globulins↗

PubMed1988

Alpha-2-antiplasmin: a serpin with two separate but overlapping reactive sites.

Although the proteinase inhibitor alpha-2-antiplasmin (alpha 2AP) is known to control the activity of plasmin through rapid formation of stable complexes, it also efficiently inactivates chymotrypsin. These interactions are shown to occur at adjacent, overlapping sites so that plasmin attacks the inhibitor at an Arg364-Met365 peptide bond, while chymotrypsin interacts at a Met365-Ser366 sequence one residue downstream. Thus, a naturally occurring plasma serine proteinase inhibitor can have multiple specificities through interactions at adjacent sites. It also illustrates the potential flexibility of the reactive site loop in this class of inhibitors.

Amino Acid Sequence↗

PubMed1988

Structure, function, and control of neutrophil proteinases.

Elastase and cathepsin G are two of the major enzymes present in and secreted by human neutrophils. These proteinases can rapidly degrade connective tissue proteins. However, they also may be involved in other processes, including the activation or inactivation of protein hormones and the inactivation of plasma proteinase inhibitors. Neutrophil elastase has been implicated in the development of pulmonary emphysema, although a function for cathepsin G has not yet been elucidated. Both enzymes are normally tightly controlled by plasma proteinase inhibitors. However, this proteinase-proteinase inhibitor balance can be perturbed in favor of free enzyme by several methods, with resulting tissue damage. The use of inhibitors from several sources should be helpful in augmenting natural levels so that homeostasis can be maintained.

Blood Proteins↗