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T Edwards

Publications and source records attributed to T Edwards.

At least 19 recordsLinked to original sources
PubMed2001

Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinephosphonic acid. Spectroscopic and crystallographic characterization of the transition state of peptide hydrolysis.

The nature of the interaction of the transition-state analogue inhibitor L-leucinephosphonic acid (LPA) with the leucine aminopeptidase from Aeromonas proteolytica (AAP) was investigated. LPA was shown to be a competitive inhibitor at pH 8.0 with a K(i) of 6.6 microM. Electronic absorption spectra, recorded at pH 7.5 of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] upon addition of LPA suggest that LPA interacts with both metal ions in the dinuclear active site. EPR studies on the Co(II)-substituted forms of AAP revealed that the environments of the Co(II) ions in both [CoZn(AAP)] and [ZnCo(AAP)] become highly asymmetric and constrained upon the addition of LPA and clearly indicate that LPA interacts with both metal ions. The X-ray crystal structure of AAP complexed with LPA was determined at 2.1 A resolution. The X-ray crystallographic data indicate that LPA interacts with both metal centers in the dinuclear active site of AAP and a single oxygen atom bridge is absent. Thus, LPA binds to the dinuclear active site of AAP as an eta-1,2-mu-phosphonate with one ligand to the second metal ion provided by the N-terminal amine. A structural comparison of the binding of phosphonate-containing transition-state analogues to the mono- and bimetallic peptidases provides insight into the requirement for the second metal ion in bridged bimetallic peptidases. On the basis of the results obtained from the spectroscopic and X-ray crystallographic data presented herein along with previously reported mechanistic data for AAP, a new catalytic mechanism for the hydrolysis reaction catalyzed by AAP is proposed.

Aeromonas↗
PubMed2001

Lameness in feedlot cattle.

This article examines the various causes of lameness in feedlot cattle, with an emphasis on clinical signs, treatment, and prevention. Specific conditions are discussed, including interdigital necrobacillosis, laminitis, feedlot injuries, and feedlot lameness associated with Mycoplasma bovis. Immune management of the foot is also reviewed.

Animals↗
PubMed2000

Patterns of changes in human immunodeficiency virus type 1 V3 sequence populations late in infection.

We have used a V3-specific heteroduplex tracking assay (V3-HTA) with probes from two different human immunodeficiency virus type 1 (HIV-1) subtypes to examine the extent and pace of HIV-1 evolution late in infection. Twenty-four subjects with advanced HIV-1 infection (CD4(+) T-cell count, <100/microl) and stable viral loads were studied using blood plasma samples collected over a study period of approximately 9 months, during which time most of the subjects were treated with reverse transcriptase inhibitors. The V3-HTA patterns from the first and last time points were evaluated initially to determine the amounts of change in V3 sequence populations, which were primarily changes in abundance in preexisting sequence populations. Three of the 24 subjects had major changes (greater than 50% total change in the relative abundance of the sequence populations), 11 subjects had intermediate changes (10 to 50% total change), and 10 subjects had minimal changes (less than 10% total change). The average total amount of change was between two- and threefold greater in subjects with X4-like variants, although there was no correlation between average viral load and the presence of X4-like variants. V3-HTA patterns in monthly samples from 11 of the subjects were also compared. In two subjects, the amount of change exceeded 40% in a 1-month period. Overall, the pace of change in V3 populations varied between subjects and was not constant within a subject over time. Sequence analysis of the V3 variants showed that R5-like variants (not containing any X4-associated substitutions) continued to be maintained in three subjects in the presence of X4-like variants, indicating that X4 variants do not always outgrow R5 variants. The coreceptor usage of the V3 sequences from two subjects was determined using a cell fusion assay. One subject had an X4 variant that was maintained at a low level for at least 9 months, during which time the predominant variants were R5X4 (dualtropic), while in the second subject the reverse situation was observed. One of the dualtropic variants had a novel sequence motif in V3, suggesting another evolutionary pathway to altered tropism. These studies begin to probe the complexities and pace of V3 evolution in vivo, revealing dynamic patterns of change among multiple V3 sequence variants in a subset of subjects.

Base Sequence↗
PubMed1999

Two-dimensional crystallization of streptavidin: in pursuit of the molecular origins of structure, morphology, and thermodynamics.

The streptavidin two-dimensional (2D) crystallization model has served as a paradigm for molecular self-assembly at interfaces. We have developed quantitative Brewster angle microscopy for the in situ measurement of spatially resolved relative protein surface densities. This allows investigation of both the thermodynamics and morphologies of 2D crystal growth. For crystal structure analysis, we employ TEM on grown crystals transferred to solid substrates. Comparison of results between commercially available streptavidin, recombinant streptavidin, and site-directed streptavidin mutants has provided insight into the protein protein and protein-lipid interactions that underlie 2D crystallization.

Crystallization↗
PubMed1999

Inhibition of the aminopeptidase from Aeromonas proteolytica by aliphatic alcohols. Characterization of the hydrophobic substrate recognition site.

Seven aliphatic and two aromatic alcohols were tested as reporters of the substrate selectivity of the aminopeptidase from Aeromonas proteolytica (AAP). This series of alcohols was chosen to systematically probe the effect of carbon chain length, steric bulk, and inhibitor shape on the inhibition of AAP. Initially, however, the question of whether AAP is denatured in the presence of aliphatic alcohols was addressed. On the basis of circular dichroism (CD), electronic absorption, and fluorescence spectra, the secondary structure of AAP, with and without added aliphatic alcohols, was unchanged. These data clearly indicate that AAP is not denatured in aliphatic alcohols, even up to concentrations of 20% (v/v). All of the alcohols studied were competitive inhibitors of AAP with K(i) values between 860 and 0.98 mM. The clear trend in the data was that as the carbon chain length increases from one to four, the K(i) values increase. Branching of the carbon chains also increases the K(i) values, but large bulky groups, such as that found in tert-butyl alcohol, do not inhibit AAP as well as leucine analogues, such as 3-methyl-1-butanol. The competitive nature of the inhibition indicates that the substrate and each alcohol studied are mutually exclusive due to binding at the same site on the enzyme. On the basis of EPR and electronic absorption data for Co(II)-substituted AAP, none of the alcohols studied binds to the dinuclear metallo-active site of AAP. Thus, reaction of the inhibitory alcohols with the catalytic metal ions cannot constitute the mechanism of inhibition. Combination of these data suggests that each of these inhibitors bind only to the hydrophobic pocket of AAP and, consequently, block the binding of substrate. Thus, the first step in peptide hydrolysis is the recognition of the N-terminal amino acid side chain by the hydrophobic pocket adjacent to the dinuclear active site of AAP.

1-Propanol↗
PubMed1999

Severe respiratory depression in a patient with gastroparesis while receiving opioids for pain.

OBJECTIVE: To increase awareness of the possibility of severe respiratory depression when oral opioids are used in patients with gastrointestinal motility disorders. SETTING: A major county hospital affiliated with a university. PATIENT: A patient with severe pain from diabetic muscle necrosis with a history of gastroparesis. INTERVENTIONS: Attempted pain control with oral and transdermal opioids. RESULTS AND CONCLUSIONS: Pain control in our patient was attempted using potent oral opioids on two occasions. However, this patient suffered severe respiratory depression after each attempt. Transdermal delivery of fentanyl eventually provided satisfactory pain relief without side effects. We conclude that patients with gastrointestinal motility disorders may be at high risk for side effects of oral opioids due to altered absorption kinetics. Suggestions are made for alternative strategies for opiate delivery in patients with gastrointestinal motility disorders.

Administration, Cutaneous↗
PubMed1998

Trauma 24-hour observation critical path.

BACKGROUND: The 24-hour observation critical pathway for trauma is a clinical tool developed to expedite health care delivery to minimally injured patients. The use of patient care, BS, guidelines and physician-approved standing orders was implemented in a Level I trauma center. METHODS: A retrospective chart review was performed of 122 patients admitted via the emergency department between December 1, 1993, and May 31, 1994. All patients were evaluated in the emergency department by emergency medicine and trauma physicians and deemed appropriate for 24-hour observation. The information collected included patient demographics, hospital charges, injuries, length of stay, diagnostic tests, consultations, and variances from the critical pathway. RESULTS: During the 6-month study period, there were 600 trauma admissions. Of those admissions, 122 patients (20%) were evaluated in the emergency department and deemed appropriate for enrollment in the 24-hour observation pathway. The charts of these patients were reviewed. Fourteen admissions were determined inappropriate for the critical pathway because of the severity of injuries or discharge against medical advice. One hundred eight charts were evaluated further. Eighty-nine patients (80%) completed the critical pathway with a length of stay of 24 hours. CONCLUSION: The 24-hour observation critical pathway was designed and used appropriately as exemplified by an overall 80% completion rate. The critical pathway offers a mechanism to streamline care of the minimally injured trauma patient. It also serves as a quality-improvement tool for increasing efficiency, decreasing utilization of resources, and decreasing length of stay.

Adolescent↗
PubMed1997

Mechanistic studies on the aminopeptidase from Aeromonas proteolytica: a two-metal ion mechanism for peptide hydrolysis.

The aminopeptidase from Aeromonas proteolytica (AAP) is uncompetitively inhibited by fluoride ion at pH 8.0 with an inhibition constant (Ki) of 30 mM. Thus, fluoride inactivates AAP only after substrate binding, and only a single fluoride ion binds to AAP. On the other hand, chloride ion does not inhibit AAP up to concentrations of 2 M at pH 8.0. The pH dependence of fluoride inhibition of AAP was measured over the pH range 6.0-9.5. Between pH values of 6.0 and 9.0, fluoride ion acts as a pure uncompetitive inhibitor of AAP, and the Ki increases from 1.2 to 370 mM. From a plot of pKi vs pH, a pKa value of 7.0 +/- 0.3 was extracted which corresponds to a single deprotonation process. At pH values higher than 9.0, the fluoride inhibition pattern changes to competitive. This change in inhibition pattern was attributed to a change in ionic strength or perhaps pH of the solution since fluoride ion was also found to become a competitive inhibitor of AAP at pH 8.0 in the presence of 2 M NaCl. These data, taken together with previous kinetic studies of mono- and dinuclear hydrolases with fluoride ion, suggest that a Zn(II)-bound water/hydroxide exists at the dimetal active site of AAP with a pKa of 7.0 and that this water/hydroxide acts as the active site nucleophile. The hydrolysis of L-leucine-p-nitroanilide was measured spectrophotometrically in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 5 to 800 microM. From these data, Km values were derived at each temperature studied and were found to increase exponentially with increasing temperature. Moreover, the calculated Vmax values were also found to increase over this temperature range, mimicking the Km values. An Arrhenius plot was constructed from k(cat) values and was found to be linear over the temperature range 25-85 degrees C, indicating that the rate-limiting step in AAP peptide hydrolysis is product formation and does not change as a function of temperature. From the slope of the line, the activation energy (Ea) was calculated to be 36.5 kJ/mol. The enthalpy and entropy of activation at 25 degrees C calculated over the temperature range 298-358 K were found to be 34.0 kJ/mol and -94.2 J/(mol x K), respectively. The free energy of activation at 25 degrees C was found to be 62.1 kJ/mol. Combination of the available X-ray crystallographic data with the present kinetic and thermodynamic results, as well as the previously reported kinetic and spectroscopic data, has allowed a detailed catalytic mechanism for AAP to be proposed.

Aeromonas↗
PubMed1997

Expression of members of a novel membrane linked metalloproteinase family (ADAM) in human articular chondrocytes.

Using resting chondrocytes derived from human articular femoral head and a conditionally immortalised human articular chondrocyte cell line we have studied the expression of members of the novel metalloproteinase/disintegrin family termed ADAM. Using RT-PCR we can detect the expression of ADAM-12 a novel family member isolated from myeloma cells [1]. We also find expression of ADAM 10 a functional metalloproteinase/disintegrin first isolated from bovine brain and ADAM-15 a metallodisintegrin isolated from mammary derived epithelial cells. Northern blotting was used to confirm expression. One main transcript is visible for ADAM-12 whereas both ADAM-10 and ADAM-15 have multiple transcripts indicating possible RNA variants potentially derived from alternative splicing or alternative use of polyadenylation sites. Since chondrocytes are proposed as an important source of metalloproteinase enzymes involved in joint pathology the potential relevance of the expression of these molecules to connective tissue disorders is discussed.

ADAM Proteins↗
PubMed1996

Cloning of a novel membrane-linked metalloproteinase from human myeloma cells.

We have isolated a novel cDNA from human myeloma cells encoding a member of the reprolysin family of metalloproteinases. Derived amino acid sequence predicts a protein of approx. 76 kDa. The open reading frame predicts the presence of a leader peptide, a pro-peptide with a 'cysteine switch', a metalloproteinase domain, a disintegrin-like domain, a cysteine-rich domain, an epidermal growth factor-like domain and a putative transmembrane sequence. Expression of the mRNA for this metalloproteinase has been demonstrated in human myeloma cells.

Amino Acid Sequence↗
PubMed1996

A method of detecting the muscle pain threshold using an objective software-mediated technique.

This paper describes the reliability of a new procedure of applying force to a muscle site using a computer-mediated myometric procedure to measure a discomfort-pain threshold. 24 female subjects were divided into four groups. Each group was tested on three occasions (trial) at four muscle sites (site) repeated daily over four days (day). Each group received one of four different rates of force application (ramp). Analysis of variance (ramp by day by site by trial) showed a significant main effect for trials and discomfort-pain threshold at different muscle sites, but no significant main effect for days or ramps. Intraclass correlations for each muscle site were in the range of R = 0.73 to 0.98. This procedure appears to be a valid, reliable and nonintrusive means of assessing the muscle discomfort-pain threshold.

Adult↗
PubMed1995

Membrane-interactive phospholipids inhibit HIV type 1-induced cell fusion and surface gp160/gp120 binding to monoclonal antibody.

Membrane-interactive phospholipids (PLs), previously evaluated for activity against HIV-1 in vitro, are known to affect late steps in viral replication. Studies were done to determine the effects of PL analogs on post-translational processing of HIV-1 proteins, binding of viral surface gp160/gp120 to CD4 receptor, and HIV-1-induced cell fusion. Results of this investigation indicated that PL alone (1-octadecanamido-2-ethoxypropyl-rac-3-phosphocholine, CP-51) and PL-AZT conjugate (1-octadecanamido-2-ethoxypropyl-rac-3-phospho-3'- azido-3'-deoxythymidine, CP-92) have no effect on HIV-1-induced syntheses or processing of gp160/gp120, pr51, p24, or p17 (including myristoylation) in infected cells. Progeny HIV-1 particles made in CP-92-treated H9IIIB cells contained gp120, pr51, and p24; however, these virus particles had reduced capacity to bind to CD4+ cells. Both CP-51 and CP-92 inhibited syncytium (cell fusion) formation between treated HIV-1-infected cells and uninfected CD4+ cells, and, they reduced HIV-1 gp160/gp120 binding to CD4+ cells and monoclonal antibody. These results suggest that anti-HIV-1 activity of PL compounds involves alteration of cell surface membranes and viral envelopes. Phospholipid compounds are a novel class of membrane interactive compounds with potential use in blocking the spread of HIV-1 infection and pathogenesis in AIDS.

Antibodies, Monoclonal↗
PubMed1994

Evaluation of sensitivity and specificity of six D-dimer latex assays.

The results of six commercially available D-Dimer latex assays were compared with those obtained by two D-Dimer enzyme-linked immunosorbent assay; this comparison was done by assessing the sensitivity and specificity of the assays. Results indicated that five of the six latex assays were unable to detect consistently D-Dimer levels (ie, equal to the claims stated in the manufacturers' package inserts). Performance, in order of decreasing sensitivity ([true positive/(true positive+false negative)] as compared with the enzyme-linked immunosorbent assay results), was as follows: ACCUCLOT D-Dimer (Sigma Diagnostics, St Louis, Mo) (93.8%) greater than Fibrinosticon (Organon Teknika Corp, Durham, NC) (81.3%) greater than D-Di Test (Diagnostica Stago, Asnières, France) (68.8%) greater than Dade Dimer-test Latex Assay (Baxter Diagnostics Inc, Miami, Fla) (59.4%) greater than Dimertest Latex Kit (American Diagnostica Inc, Greenwich, Conn) and DimerKlone (Ortho Diagnostic Systems Inc, Raritan, NJ) (56.3%). The estimated sensitivity for the lowest sensitivity assays (group 1) (American Diagnostica Inc, Ortho Diagnostic Systems, and Dade Diagnostics) was approximately 1.5 mg/L; the estimated sensitivity for the moderate sensitivity assays (group 2) (Organon Teknika Corp and Diagnostica Stago) was approximately 1.0 mg/L; and the estimated sensitivity for the highest sensitivity assay (group 3) (Sigma Chemical Co) approached a detection limit in the 0.25- to 0.50-mg/L range.

Enzyme-Linked Immunosorbent Assay↗