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Biomedical subjects

G T Wang

Publications and source records attributed to G T Wang.

At least 19 recordsLinked to original sources
PubMed1996

Efficacy of moxidectin pour-on against gastrointestinal nematodes and Dictyocaulus viviparus in cattle.

Twenty crossbred beef heifer calves were used for an efficacy evaluation of 0.5% moxidectin pour-on against gastrointestinal nematodes and Dictyocaulus viviparus. The latter parasite and Bunostomum phlebotomum were the target species. The calves were exposed to natural infection on pasture from late September to December 22. Additionally, all calves were experimentally infected with B. phlebotomum at 71 and 29 days before treatment and with D. viviparus at 29 days before treatment. The 20 calves were randomly allocated into two groups of ten, based on presence of patent lungworm and hookworm infections on days -6 and -1. Treatments were as follows: Group A, moxidectin 0.5% pour-on (PO) at 500 micrograms kg-1 BW; Group B, moxidectin vehicle (controls) by PO application. General strongyle and hookworm egg counts and lungworm larval counts were reduced to zero at 13 days after moxidectin treatment; treatment effect was significant (P < 0.05) only for the strongyle and hookworm counts. Percentage reduction for adult hookworms and mature-immature adult lungworms was 100.0 and also for adults and L4 Haemonchus placei and Ostertagia ostertagi and Trichostrongylus axei adults, adult males of Cooperia pectinata and C. spatulata, and Oesophagostomum radiatum adults. Efficacy values for C. punctata adult males, Cooperia spp. adult females, and Cooperia spp. L4 were > 99.9%, > 99.9%, and 92.4%, respectively. All efficacy values were significant (alpha = 0.05, 1-sided) except for Cooperia spp. L4.

Administration, Topical↗
PubMed1995

Synthetic chemical diversity: solid phase synthesis of libraries of C2 symmetric inhibitors of HIV protease containing diamino diol and diamino alcohol cores.

Solid phase synthesis of non-oligomeric organic compounds has been pursued for high-efficiency generation of large numbers of structurally diverse compounds for drug screening. Known as chemical diversity libraries or combinatorial libraries (when the synthesis is carried out in a combinatorial fashion), these compounds can be used for de novo discovery of drug leads or for expedient structure--activity relationship (SAR) studies. To expand the scope of solid phase synthesis beyond the capability of the traditional method of solid phase synthesis for peptides, a strategy was developed for bi-directional solid phase synthesis starting with diamino alcohol or diamino diol core structures. The strategy relies on using bifunctional linkers to modify the core structures, simultaneously protecting the hydroxyl group or the diol moiety of the core and providing a carboxyl group for attachment of the modified cores to a solid support. The two NH2 groups of the modified cores attached to the solid support were then deprotected and reacted with a wide variety of amine-reactive reagents (carboxylic acids, sulfonyl chlorides, isocyanates, chloroformates, etc.) to extend the molecule in both directions. This strategy was successfully applied to automated parallel synthesis of a library of C2 symmetric inhibitors of HIV protease containing the known symmetry-based diamino diol and diamino alcohol core structures, thus enabling expedient access of large numbers of analogs in this series. A library of over 300 discrete compounds was synthesized using this methodology in order to identify potent (IC50 < 100 nM) HIV protease inhibitors with reduced size. This paper describes the technical aspects of this technology.

Alcohols↗
PubMed1995

Cathepsin D from Alzheimer's-diseased and normal brains.

An acid protease activity from human brain was found to cleave a fluorogenic peptide substrate encompassing the amino terminus of Alzheimer's amyloid-beta peptide (A beta). The protease was isolated and determined to be cathepsin D based on chromatographic, immunological, and enzymatic data. Analysis of the cleavage sites indicated that cathepsin D hydrolyzed the methionine--aspartate bond generating the in vivo amino terminus of A beta. These data suggested that cathepsin D could be involved in amyloidogenic processing of the amyloid precursor protein. Consequently, cathepsin D from both Alzheimer's-diseased and control brains was compared to determine whether there were any differences which could account for an increase in A beta production in Alzheimer's disease. No differences were detected in isoform composition or tissue content of cathepsin D as measured by 2-D IEF-SDS-PAGE. Enzymological characterization of brain cathepsin D demonstrated that it could undergo a previously undescribed pH-dependent reversible activation. However, that activation appeared identical for both AD and normal brain enzymes. These data demonstrate that concentration, isoform distribution, and several enzymological characteristics of cathepsin D are not distinguishable between AD and normal brain. The pH dependence of cathepsin D activity suggests, however, that its intracellular localization may be important in considering the potential role of cathepsin D in Alzheimer's disease.

Aged↗
PubMed1994

Cleavage at the amino and carboxyl termini of Alzheimer's amyloid-beta by cathepsin D.

Amyloid beta (A beta) is a 39-43-residue protein that originates from proteolysis of the beta-protein precursor (beta PP) and accumulates in senile plaques in brains of Alzheimer's disease (AD) patients. Mutant beta PP, which incorporates an AD-causing double mutation at positions 687-688, has been shown to enhance A beta production in transfected cells. In this work we investigate the susceptibility of the mutant beta PP sequence to proteolytic cleavage by proteinases from human brain. Internally quenched fluorogenic substrates were used that encompass the NH2-terminal sequence of A beta from wild-type beta PP, the double mutant, and the two single substitutions. Proteinase activity in brain extract cleaved the mutant substrate 100-fold faster than the wild-type substrate and the partial mutants 25-fold faster. The major cleavage site in all substrates was at the amyloidogenic Asp1 site. The brain activity appeared to be cathepsin D (CD), as indicated by similarities to purified CD in 1) the rate and site of substrates cleavage, 2) the pH optima, and 3) the sensitivity to pepstatin A. The increased activity against the mutant substrate was not shared by cathepsins B and C, pepsin, HIV proteinase, and Candida albicans Asp-proteinase. Furthermore, CD cleaved a substrate that incorporates the COOH terminus of A beta at positions equivalent to Thr43 and Ala42, at ratios of 68% and 32%, respectively. CD degraded A beta 1-40 into six fragments but A beta 1-42 was completely resistant to digestion, probably because of its aggregation characteristics. These results indicate that CD is capable of producing the cleavages resulting in A beta production and that it may prove to be a suitable therapeutic target.

Amino Acid Sequence↗
PubMed1994

Complement C1q does not bind monomeric beta-amyloid.

The tendency of both labeled and unlabeled beta-amyloid to bind in solution to C1q, the recognition species in the complement cascade, was examined using both hydrodynamic and spectroscopic methods. Potential binding interactions were evaluated using a purified synthetic beta-amyloid 1-40 sequence, alone, and selectively labeled at the amino terminus with spectroscopic probes. The probes permitted both absorbance and fluorescence analyses of beta-amyloid binding interactions. Under conditions used for the analyses beta-amyloid exists exclusively as a monomer in solution, and C1q retains an intact quaternary structure and is capable of binding to IgM. When mixed together the monomeric beta-amyloid does not bind to, or interact with, the complement C1q at concentrations below approximately 100 microM. The data suggest that if beta-amyloid toxicity is associated with complement activation in Alzheimer's disease then monomeric beta-amyloid is likely not responsible for activation through the classical complement pathway.

Amyloid beta-Peptides↗
PubMed1994

Potential beta PP-processing proteinase activities from Alzheimer's and control brain tissues.

Fluorogenic peptide substrates designed to encompass the reported alpha-secretory and amyloidogenic cleavage sites of the amyloid-beta precursor protein (beta PP) were used to analyze proteinase activities in brain extracts from control patients and those with Alzheimer's disease (AD). Activity against the secretory substrate at pH 7.5 in control and AD brains produced a major endopeptidase cleavage at the Lys687-Leu688 bond (beta PP770 numbering), consistent with the beta PP secretase cleavage. Activity in control brains against the amyloidogenic substrate at pH 7.5 produced one cleavage at the Ala673-Glu674 bond, two residues C-terminal to the amyloidogenic Met-Asp site. However, in three of four AD brains, the major cleavage was at the Asp-Ala bond, one residue from the amyloidogenic site. Both endopeptidase and carboxypeptidase activities in AD brains were lower than in control brains. Proteinase activities against the secretory substrate had a major optimum at pH 3.0-4.0 and another at pH 6.0-7.5. Proteinase activities against the amyloidogenic substrate had a major optimum at or below pH 3.0 and another at pH 6.0. Using both substrates, activities at low pH were higher in AD-brains than in controls, while at pH above 6.5, activities in control brains were higher than in AD. These results indicate that the levels of proteolytic enzymes in AD brains are altered relative to controls.

Aged↗
PubMed1994

Synthetic approaches to continuous assays of retroviral proteases.

This chapter has described a number of approaches for continuous assay of retroviral proteases using either chromogenic or fluorogenic synthetic substrates. The significant progress in this area has been catalyzed by the intense interest in HIV protease as a therapeutic target, but these versatile methods will be used widely in future for studies of many other proteases.

Amino Acid Sequence↗
PubMed1994

Amyloid-beta aggregation: selective inhibition of aggregation in mixtures of amyloid with different chain lengths.

One of the clinical manifestations of Alzheimer's disease is the deposition of the 39-43 residue amyloid-beta (A beta) peptide in aggregated fibrils in senile plaques. Characterization of the aggregation behavior of A beta is one of the critical issues in understanding the role of A beta in the disease process. Using solution hydrodynamics, A beta was observed to form three types of species in phosphate-buffered saline: insoluble aggregates with sedimentation coefficients of approximately 50,000 S and molecular masses of approximately 10(9) Da, "soluble aggregates" with sedimentation coefficients of approximately 30 S and masses of approximately 10(6) Da, and monomer. When starting from monomer, the aggregation kinetics of A beta 1-40 (A beta 40) and A beta 1-42 (A beta 42), alone and in combination, reveal large differences in the tendency of these peptides to aggregate as a function of pH and other solution conditions. At pH 4.1 and 7.0-7.4, aggregation is significantly slower than at pH 5 and 6. Under all conditions, aggregation of the longer A beta 42 was more rapid than A beta 40. Oxidation of Met-35 to the sulfoxide in A beta 40 enhances the aggregation rate over that of the nonoxidized peptide. Aggregation was found to be dependent upon temperature and to be strongly dependent on peptide concentration and ionic strength, indicating that aggregation is driven by a hydrophobic effect. When A beta 40 and A beta 42 are mixed together, A beta 40 retards the aggregation of A beta 42 in a concentration-dependent manner. Shorter fragments have a decreasing ability to interfere with A beta 42 aggregation. Conversely, the rate of aggregation of A beta 40 can be significantly enhanced by seeding slow aggregating solutions with preformed aggregates of A beta 42. Taken together, the inhibition of A beta 42 aggregation by A beta 40, the seeding of A beta 40 aggregation by A beta 42 aggregates, and the chemical oxidation of A beta 40 suggest that the relative abundance and rates of production of different-length A beta and its exposure to radical damage may be factors in the accumulation of A beta in plaques in vivo.

Alzheimer Disease↗
PubMed1994

Evidence against a role for the Kunitz domain in amyloidogenic and secretory processing of the amyloid precursor protein.

The effect of the Kunitz proteinase inhibitor (KPI) on potential beta-amyloid precursor protein (beta PP)-processing activities from control and Alzheimer's disease (AD) brains was examined using fluorogenic substrates designed to mimic the secretory and amyloidogenic cleavages in beta PP. In addition, the level of secretion of KPI-containing beta PP751 and KPI-lacking beta PP695 from transfected cells was examined to assess the effect of the KPI on beta PP secretion. beta PP751 and beta PP695, obtained from conditioned media of transfected cells, had no effect on proteinase activities against the secretory and amyloidogenic substrates in extracts from control and AD brains. At similar concentrations beta PP751, but not beta PP695, completely inhibited the activity of trypsin against these substrates. Serine proteinase inhibitors had only modest effects on activities from brain, whereas cysteine modification completely inhibited them, indicating that these proteinase activities were not of the serine type. Thus, the results do not support a role for the KPI in the secretion of beta PP or in the amyloidogenic cleavage of beta PP. The amounts of beta PP695 and beta PP751 collected from the media of transfected cells after 48 h of growth were similar, indicating an equal rate of secretion. This result suggests that the KPI domain in beta PP751 did not inhibit the secretory cleavage in transfected cells.

Aged↗
PubMed1994

[Sequencing of hepatitis B virus DNA fragment coding major HBsAg of escape mutant].

We amplificated a HBV DNA fragment coding the major HBsAg from a chronic HBsAg carrier's serum with high titer of HBsAg and anti-HBs using polymerase chain reaction (PCR). The direct sequencing of PCR products revealed a single mutation from adnosine to guanosine at the nucleotide position 532 of HBV DNA of the escape mutant. This mutation might result in an amino acid substitution from threonine to alanime at the amino acid position 126 of HBsAg. Since this amino acid is located in the first loop of the "a" determinant of HBsAg, the mutation might result in an alteration of the structural antigenicity of the "a" determinant and induce escape mutant of HBV.

Antigenic Variation↗
PubMed1994

Safety assessment of moxidectin 1% injectable on reproductive performance in beef cows.

The safety of moxidectin 1% injectable anthelmintic (0.6 mg/kg of body weight, 3 times the recommended dose) was evaluated in 145 reproductively sound, beef cows undergoing estrous cycle. Five treatment groups received moxidectin 1% injectable at specific times relative to a synchronized estrus (day 0): preovulatory treatment (day -2, treatment group 1), treatment at ovulation (day 0, group 2), and treatment after ovulation (days 7, 14, and 28, group 3, 4, and 5, respectively). Two groups of control cows received an injection of vehicle only at times corresponding to treatment in the other groups (6 at days -2, 7, and 28; 7 at days 0, 7, and 14). A final control group (8) received neither product or vehicle. Adverse clinical reactions were not observed in moxidectin- or vehicle-treated cows. Cows were bred by artificial insemination between days -2 and 25 and, subsequently, by breeding-sound bulls through day 65 of the study. Treatment and control groups did not differ in pregnancy rate or time to conception. Moxidectin (at 3 times the therapeutic dose) did not have deleterious effects on cow reproductive performance as examined (eg, at folliculogenesis, ovulation, and the early embryonic phase of development).

Analysis of Variance↗
PubMed1993

A continuous fluorescence assay of renin activity.

A sensitive fluorescence assay that employs a new fluorogenic peptide substrate has been developed to continuously measure the proteolytic activity of human renin. The substrate, DABCYL-gaba-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS, has been designed to incorporate the renin cleavage site that occurs in the N-terminal peptide of human angiotensinogen. The assay relies upon resonance energy transfer-mediated, intramolecular fluorescence quenching that occurs in the intact peptide substrate. Efficient fluorescence quenching occurs as a result of favorable energetic overlap of the EDANS excited state and the DABCYL absorption, and the relatively long excited state lifetime of the EDANS fluorophore. Cleavage of the substrate by renin liberates the peptidyl-EDANS fragment from proximity with the DABCYL acceptor, restoring the higher, unattenuated fluorescence of the EDANS moiety. This leads to a time-dependent increase in fluorescence intensity, directly related to the extent of substrate consumed by renin cleavage. The kinetics of renin-catalyzed hydrolysis of this substrate have been shown to be consistent with a simple substrate inhibition model with a substrate Km approximately equal to 1.5 microM at physiological pH; Cleavage of the substrate occurs specifically at the Leu-Val bond and corresponds to the renin cleavage site of angiotensinogen, as reported earlier. In this report, we describe in detail the synthesis of the fluorogenic renin substrate and its application in assays of renin activity. Assay sensitivity has been evaluated by a series of enzyme dilution experiments using the continuous assay format, showing that the assay can detect renin as low as 30 ng/ml after a incubation of only 3-5 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Efficacy of moxidectin against gastrointestinal nematodes of cattle.

Three groups of 11 naturally infected crossbred beef calves were injected subcutaneously with moxidectin 1 per cent injectable at 0.2 or 0.3 mg moxidectin/kg bodyweight or with the unmedicated vehicle. Nematode infections had been acquired during grazing from December to April. Based on the faecal egg counts and total worm counts of the control calves at necropsy (11 to 13 days after treatment) most of the calves had heavy parasitic burdens. Ostertagia ostertagi was predominant and the mean numbers of adults, developing fourth stage larvae (L4) and inhibited early L4 were 45,906, 10,061 and 68,918, respectively. Haemonchus placei and Trichostrongylus axei were also present in the abomasa. Three species of Cooperia, Oesophagostomum radiatum L4 and T colubriformis adults were found in the intestinal tract. Both dosages of moxidectin were equally effective (P < 0.05) against all the abomasal nematodes (99.9 to 100 per cent) and the intestinal tract nematodes (99.4 to 100 per cent). No adverse reactions to the moxidectin treatment were observed. Abomasal pathology characteristic of heavy O ostertagi infection was observed in the control calves, but not in the treated calves.

Animals↗
PubMed1992

Moxidectin: systemic activity against common cattle grubs (Hypoderma lineatum) (Diptera: Oestridae) and trichostrongyle nematodes in cattle.

Moxidectin, a systemic insecticide, was evaluated for its efficacy against the migrating first instars of the common cattle grub, Hypoderma lineatum, and against nematode egg production in beef cattle. It was observed that all three levels (0.1, 0.2 and 0.4 mg moxidectin kg-1) were 100% effective against cattle grubs when administered as a s.c. injection. The same levels of treatment were very effective (90-100%) in reducing trichostrongyle nematode egg production. However, there was a slight indication that at least one species, Cooperia oncophora, was not completely eliminated, as it was observed that small numbers of eggs began to appear after 2 weeks post-treatment when there had been no opportunity for reinfection.

Animals↗
PubMed1992

Efficacy of moxidectin against an ivermectin-resistant strain of Haemonchus contortus in sheep.

The efficacy of moxidectin was determined against ivermectin-susceptible and resistant strains of Haemonchus contortus. At the onset of the trial, 40 lambs were each infected with 5000 third stage larvae of one of two strains of Haemonchus contortus. The lambs were randomly sorted into eight treatment groups 28 days post-infection and were treated as follows: Group 1, susceptible strain with no treatment; Group 2, resistant strain with no treatment; Group 3, susceptible strain treated with 0.2 mg moxidectin kg-1 body weight; Group 4, resistant strain treated with 0.2 mg moxidectin kg-1; Group 5, resistant strain treated with 0.4 mg moxidectin kg-1; Group 6, susceptible strain treated with 0.2 mg ivermectin kg-1; Group 7, resistant strain treated with 0.4 mg ivermectin kg-1; Group 8, resistant strain treated with 0.8 mg ivermectin kg-1. The lambs were killed 1 week post-treatment. Comparisons were made among groups based on the number of eggs per gram of feces on the day of treatment and the numbers of worms recovered from each lamb. Both moxidectin and ivermectin were effective in removing susceptible Haemonchus with efficacies of 100% and 99.7%, respectively. The efficacy of moxidectin against the resistant strain was 99.9% and 100% at 0.2 mg kg-1 and 0.4 mg kg-1, respectively, whereas there were only 38.8% and 53.1% efficacies in the lambs treated with 0.4 mg ivermectin kg-1 and 0.8 mg kg-1 body weight, respectively.

Animals↗
PubMed1992

Efficacy of Cydectin moxidectin 1% injectable against experimental infections of Dictyocaulus viviparus and Bunostomum phlebotomum superimposed on natural gastrointestinal infections in calves.

Twenty male Holstein calves averaging 105 kg in weight and naturally infected with gastrointestinal nematodes and small numbers of lungworm and hookworm, were given experimental infections with the two latter species to provide adult and larval stages for anthelmintic evaluation. Following random allotment, one group of 10 calves was injected subcutaneously with moxidectin at a dosage of 0.2 mg kg-1 of body weight. A second group of 10 was injected subcutaneously with unmedicated blank vehicle at a dosage of 1 ml per 50 kg of body weight. Fecal samples were examined before treatment and at 7 and 13 days after treatment. The 20 calves were necropsied for worm recovery at 13 and 14 days after treatment. All calves were positive for lungworm and hookworm on the treatment date. Treatment was 100% effective in elimination of hookworm eggs and lungworm larvae and 99.9% in reducing total egg counts at both 7 and 13 days after treatment. Moxidectin was 100% effective (P less than 0.01) in eliminating the following 11 species of nematodes. Dictyocaulus viviparus mature and immature adults (E5), Bunostomum phlebotomum adults and L4, Ostertagia ostertagi adults and early L4, Ostertagia lyrata adult males, Haemonchus placei adults. Trichostrongylus axei adults, Cooperia spp., including Cooperia punctata, Cooperia spatulata, and Cooperia pectinata adults, Oesophagostomum radiatum adults and Trichuris discolor adults. No adverse reactions to moxidectin treatment were observed.

Animals↗
PubMed1991

Characterization of recombinant human renin: kinetics, pH-stability, and peptidomimetic inhibitor binding.

The kinetic behavior and pH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P6-P3' renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. The pH-stability profile for renin, measured with the substrate at 25 degrees C, indicated a broad plateau of stability between pH 6.0 and 10.0. Analysis of the pH-activity profile of renin for the substrate indicated a minimum Km (approximately 1.8 microM) at pH approximately 7.4 and a maximum Vm between pH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P2 site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.

Amino Acid Sequence↗