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J D Kaufman

Publications and source records attributed to J D Kaufman.

At least 37 records · Page 2Linked to original sources
PubMed1996

Mapping hydration water molecules in the HIV-1 protease/DMP323 complex in solution by NMR spectroscopy.

A tetrahedrally hydrogen-bonded structural water molecule, water 301, is seen in the crystal structure of nearly every HIV-1 protease/inhibitor complex. Although the urea oxygen of the designed inhibitor, DMP323, mimics and replaces water 301, other water molecules are seen in the protease/DMP323 crystal structure. As a first step toward understanding how water molecules may contribute to inhibitor potency and specificity, we have recorded water-NOESY and water-ROESY spectra of the protease/ DMP323 complex. Cross relaxation rates derived from these spectra, together with interproton distances calculated from the crystal structure of the complex, were used to classify the exchange cross peaks as follows: (A) a direct NOE with a water proton, (B) an indirect NOE with water through a labile protein proton, and (C) direct exchange of an amide proton with water. Type A and B cross peaks were analyzed using three models of water dynamics: (1) two-site exchange, with water molecules randomly hopping between bound and free states, (2) bound water with internal motion, and (3) free diffusion. Using the two-site exchange model to analyze the relaxation data of the type A cross peaks, it was found that the water molecules had short residence times, ca. 500 ps. in contrast with the > 9 ns residence time estimated for water 301 in the protease/P9941 complex [Grzesiek et al. (1994) J. Am. Chem. Soc. 116, 1581-1582]. The NMR data are consistent with the X-ray observation that two symmetry-related water molecules, waters 422 and 456, are bound at the DMP323 binding site. Hence, these water molecules may help to stabilize the structure of the complex. Finally, it was found that three buried and hydrogen-bonded Thr hydroxyl protons were in slow exchange with solvent. In contrast, it was found that the DMP323 H4/H5 hydroxyl protons and the Asp25/125 carboxyl protons, which form a buried hydrogen-bonded network at the catalytic site of the protease, are in rapid exchange with solvent, suggesting that solvent can penetrate into the buried protein/inhibitor interface on the millisecond to microsecond time scale.

Azepines↗
PubMed1996

Solution NMR evidence that the HIV-1 protease catalytic aspartyl groups have different ionization states in the complex formed with the asymmetric drug KNI-272.

In order to improve the design of HIV-1 protease inhibitors, it is essential to understand how they interact with active site residues, particularly the catalytic Asp25 and Asp125 residues. KNI-272 is a promising, potent HIV-1 protease inhibitor (K(i) approximately 5 pM), currently undergoing phase 1 clinical trials. Because KNI-272 is asymmetric, the complex it forms with the homodimeric HIV-1 protease also lacks symmetry, and the two protease monomers can have distinct NMR spectra. Monomer specific signal assignments were obtained for amino acid residues in the drug binding site as well as for six of the eight Asp residues in the protease/KNI-272 complex. Using these assignments, the ionization states of the Asp carboxyl groups were determined from measurements of (a) the pD dependence of the chemical shifts of the Asp carboxyl carbons and (b) the H/D isotope effect upon the Asp carboxyl carbon chemical shifts. The results of these measurements indicate that the carboxyl of Asp25 is protonated while that of Asp125 is not protonated. These findings provide not only the first experimental evidence regarding the distinct protonation states of Asp25/125 in HIV-1 protease/drug complexes, but also shed light on interactions responsible for inhibitor binding that should form the basis for improved drug designs.

Amino Acid Sequence↗
PubMed1996

Fatal and nonfatal injuries related to violence in Washington workplaces, 1992.

This study describes injuries related to assaults and violence that occurred in Washington State workplaces in 1992. Nonfatal injuries are emphasized. High-risk industries and occupations are described. Fatalities caused by work-related violence were identified using the 1992 U.S. Department of Labor Bureau of Labor Statistics (BLS) Census of Fatal Occupational Injuries. Nonfatal injuries were identified using the BLS Annual Survey of Occupational Injuries and Illnesses and the Washington State workers' compensation system. Thirteen occupational fatalities resulted from assaults or violent acts in 1992. BLS data identified 784 nonfatal injuries that resulted in one or more day off work; workers' compensation data identified 2,395 claims. Industries experiencing the highest claim rates were Social Services (148 per 10,000) and Health Services (106 per 10,000). Nonfatal violent injuries were common and appeared to have different risk factors than fatal injuries. Industries in which injuries occurred were often predictable and suggested specific strategies for prevention efforts.

Absenteeism↗
PubMed1996

Three-dimensional solution structure of the HIV-1 protease complexed with DMP323, a novel cyclic urea-type inhibitor, determined by nuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of the HIV-1 protease homodimer, MW 22.2 kDa, complexed to a potent, cyclic urea-based inhibitor, DMP323, is reported. This is the first solution structure of an HIV protease/inhibitor complex that has been elucidated. Multidimensional heteronuclear NMR spectra were used to assemble more than 4,200 distance and angle constraints. Using the constraints, together with a hybrid distance geometry/simulated annealing protocol, an ensemble of 28 NMR structures was calculated having no distance or angle violations greater than 0.3 A or 5 degrees, respectively. Neglecting residues in disordered loops, the RMS deviation (RMSD) for backbone atoms in the family of structures was 0.60 A relative to the average structure. The individual NMR structures had excellent covalent geometry and stereochemistry, as did the restrained minimized average structure. The latter structure is similar to the 1.8-A X-ray structure of the protease/DMP323 complex (Chang CH et al., 1995, Protein Science, submitted); the pairwise backbone RMSD calculated for the two structures is 1.22 A. As expected, the mismatch between the structures is greatest in the loops that are disordered and/or flexible. The flexibility of residues 37-42 and 50-51 may be important in facilitating substrate binding and product release, because these residues make up the respective hinges and tips of the protease flaps. Flexibility of residues 4-8 may play a role in protease regulation by facilitating autolysis.

Azepines↗
PubMed1996

Semen quality of men employed at a lead smelter.

OBJECTIVE: To evaluate the effects of recent and long term occupational lead exposure on indicators of male reproductive health. METHODS: In a cross sectional study of male employees of a lead smelter (n = 2469), blood samples were obtained from 152 workers including 119 who also provided semen samples. Semen analysis and serum concentrations of testosterone, follicle stimulating hormone, and luteinising hormone were used as indicators of reproductive health. Semen and hormone variables were examined in relation to measures of current and long term body lead burden estimated from current blood lead concentrations and historical blood lead monitoring data. RESULTS: For current blood lead concentration groups of < 15, 15-24, 25-39, > 40 micrograms/dl, the geometric mean sperm concentrations were, respectively, 79.1, 56.5, 62.7, and 44.4 million cells/ml and geometric mean total sperm counts were 186, 153, 137, and 89 million cells (P for trend 0.04). Compared with workers with blood lead concentrations less than 15 micrograms/dl, workers with current blood lead concentrations of 40 micrograms/dl or more had an increased risk of below normal sperm concentration (odds ratio (OR) 8.2, 95% confidence interval (95% CI) 1.2-57.9) and total sperm count (OR 2.6, 95% CI 0.4-15.7), based on World Health Organisation standards. Independent of current lead exposure, sperm concentration, total sperm count, and total motile sperm count were inversely related to measures of long term lead exposure. No association was found between lead exposure and measures of sperm motility, sperm morphology, or serum concentrations of reproductive hormones. CONCLUSIONS: Blood lead concentrations below the currently accepted worker protection criteria seem to adversely affect spermatogenesis.

Adult↗
PubMed1995

Flexibility and function in HIV-1 protease.

HIV protease is a homodimeric protein whose activity is essential to viral function. We have investigated the molecular dynamics of the HIV protease, thought to be important for proteinase function, bound to high affinity inhibitors using NMR techniques. Analysis of 15N spin relaxation parameters, of all but 13 backbone amide sites, reveals the presence of significant internal motions of the protein backbone. In particular, the flaps that cover the proteins active site of the protein have terminal loops that undergo large amplitude motions on the ps to ns time scale, while the tips of the flaps undergo a conformational exchange on the microsecond time scale. This enforces the idea that the flaps of the proteinase are flexible structures that facilitate function by permitting substrate access to and product release from the active site of the enzyme.

Amino Acid Sequence↗
PubMed1995

Health symptoms and the work environment in four nonproblem United States office buildings.

OBJECTIVES: The objective of this study was to quantify health symptom reports in four "nonproblem" buildings and to assess the relationship between symptoms and air quality measures, workstation characteristics, and psychosocial aspects of the workplace. METHODS: Environmental sampling was conducted in four office buildings occupied by employees working for the state of Washington in March of 1992. A questionnaire was concurrently administered to building occupants. RESULTS: While measured contaminant levels were low, symptoms were frequent. Fifty-five percent of the 646 respondents reported recent symptoms which affected the eyes, nose, or throat and improved when away from work. Symptoms were not associated with measured contaminant levels, but, rather, with perceptions about air movement, dryness, odors, and noise. Psychosocial factors were less strongly associated with symptoms. CONCLUSIONS: Even in nonproblem buildings, symptom reports can be frequent and may represent overall satisfaction with the work environment. In response to symptoms ascribed to air quality problems, it may be appropriate to address employee perceptions regarding the work environment in addition to evaluating environmental characteristics relating to chemicals, biological contamination, air movement, temperature, and humidity.

Adult↗
PubMed1994

Secondary structure and signal assignments of human-immunodeficiency-virus-1 protease complexed to a novel, structure-based inhibitor.

We report comprehensive NMR studies in solution of the human-immunodeficiency-virus (HIV)-1 protease. Stable solutions of the protease were obtained by complexing the protein to a designed cyclic urea inhibitor DMP 323. A variety of triple-resonance experiments provided essentially complete 1H, 13C and 15N NMR signal assignments of the protease. These assignments, together with short-range NOE constraints, coupling constants and hydrogen-exchange data, yielded the secondary structure of the protease in solution. The results reported herein open the way to the determination of the high-resolution three-dimensional solution structures of protease/inhibitor complexes, as well as to studies of protease dynamics and solvent interactions.

Amino Acid Sequence↗
PubMed1994

Atrial fibrillation and sudden death related to occupational solvent exposure.

Two cases of atrial fibrillation and one case of sudden death occurred in workers exposed to trifluorotrichloroethane (CFC 113) as a solvent/degreasing agent. This agent and related halogenated hydrocarbons are widely used in industry as solvents and degreasing agents, and have been previously linked to ventricular arrhythmias and cardiac sudden death when inhaled in excessive concentrations. We suggest that occupational overexposure to halogenated hydrocarbons should be considered a potential precipitant for atrial as well as ventricular arrhythmias.

Adult↗
PubMed1994

Occupational lead poisoning: can it be eliminated?

The continued occurrence of occupational lead poisoning and overexposure in the United States represents a serious challenge to the occupational health community. We outline a proposed action strategy which integrates case-based surveillance, hazard surveillance, increased requirements for biological monitoring, and targeted educational activities, with a goal of eliminating occupational lead poisoning. The system provides a simple mechanism for monitoring compliance in lead-using employers, in order to identify employers for enforcement action. Lead poisoning should be viewed as an eradicable condition; successfully solving the persistent lead overexposure problem can serve as a model for approaching other occupational diseases.

Case-Control Studies↗
PubMed1994

Construction of an enzymatically active ribonuclease H domain of human immunodeficiency virus type 1 reverse transcriptase.

The isolated ribonuclease (RNase) H domain of human immunodeficiency virus type 1 (HIV-1) is enzymatically inactive. The incorporation of the putative substrate binding site of Escherichia coli RNase HI (amino acid residues 76-102, the alpha c-helix and adjacent loop region) into the equivalent position of the RNase H domain of HIV-1 resulted in a highly active hybrid protein dependent on Mn2+. Similar restoration of RNase H activity has been observed when histidine residues are added to either the N- or C-terminus of the HIV-1 RNase H domain. The hybrid HIV-1/E. coli RNase H protein is approximately 10-fold more active than HIV-1 reverse transcriptase and 30-fold more active than the histidine-tagged proteins, indicating that the alpha c-helix and adjacent loop region of E. coli RNase HI is an excellent substrate binding region because of its sequence and/or location. The RNase H hybrid produced the same specific cleavage in the model tRNA(Lys3) primer removal assay as HIV-1 reverse transcriptase, showing that substrate binding and specificity are separable and that the specificity determinants are at least partially, if not totally, contained in the amino acid sequence of the hybrid protein derived from HIV-1 reverse transcriptase.

Amino Acid Sequence↗
PubMed1992

A study of the cardiac effects of bromochlorodifluoromethane (halon 1211) exposure during exercise.

Bromochlorodifluoromethane (halon 1211, a fire extinguisher), like other fluorocarbons, has been linked with ventricular arrhythmias and myocardial depression. Ten healthy firefighters, aged 40-50, were exposed to 1,000 ppm halon while exercising, in a double-blind, placebo-controlled crossover experiment, and were monitored during and after exposure. Complex ectopy (ventricular couplets and idioventricular rhythm) occurred in two subjects with halon, but none with placebo. One subject had 49.5 ventricular premature beats (VPB)/hour during the period of halon exposure and subsequent 8 hours and only 8.7 VPB/hour during the same period of placebo. In addition, 8 of the 10 subjects had a smaller systolic blood pressure rise during exercise with halon than with placebo. None of the observed differences was statistically significant. These results are consistent with findings in other investigations, suggesting that occupational fluorocarbon exposures may be cardiotoxic in certain individuals, although the small sample sizes used in this and other studies have resulted in limited statistical power to demonstrate this effect.

Adult↗
PubMed1992

The 10-year experience of an academically affiliated occupational and environmental medicine clinic.

Occupational and environmental diseases are underrecognized. Among the barriers to the successful diagnosis, treatment, and prevention of these conditions are inadequate consultative and information resources. We describe the 10-year clinical and training experiences of an academically affiliated referral center that has as its primary goal the identification of work-related and other environmental diseases. The University of Washington Occupational and Environmental Medicine Program has evaluated 6,048 patients in its diagnostic and screening clinics. Among the 2,841 seen in the diagnostic clinics, 1,553 (55%) had a work-related condition. The most prevalent diagnoses included asbestos-related lung disease (n = 603), toxic encephalopathy (n = 160), asthma (n = 119), other specific respiratory conditions (n = 197), carpal tunnel syndrome (n = 86), and dermatitis (n = 82). The clinics serve as a training site for fellows in the specialty training program, primary care internal medicine residents, residents from other medical specialties, and students in industrial hygiene, toxicology, and occupational health nursing. The program serves two additional important functions: providing consultative services to community physicians and training specialists and other physicians in this underserved area of medicine.

Adolescent↗
PubMed1991

IL-1 expression in human monocytes is transcriptionally and posttranscriptionally regulated by IL-4.

The T cell-derived lymphokine, IL-4, inhibits production of IL-1 beta by normal human monocytes. To determine whether IL-4 suppresses IL-1 expression by a transcriptional and/or posttranscriptional mechanism, we evaluated the half-life of LPS-induced IL-1 beta message and transcriptional rate of the pro-IL-1 beta gene in human monocytes after treatment with IL-4. Although the initial steady-state IL-1 mRNA levels in control and IL-4-treated monocytes were comparable during the first 2 h after stimulation with LPS, IL-1 message levels subsequently decreased at a significantly greater rate in the IL-4-treated cells. Thus, IL-4 did not prevent the initial expression of IL-1 message, but did accelerate down-regulation of IL-1 mRNA in LPS-stimulated monocytes. The initial 2 to 3 h lag period may be necessary for production of a protein(s) that mediates this inhibitory effect because treatment with the protein synthesis inhibitor, cycloheximide, blocked the marked reduction of IL-1 message levels induced by IL-4. Nuclear run-on analyses demonstrated that IL-4 decreases IL-1 mRNA levels, in part, by repressing IL-1 gene transcription. Furthermore, mRNA half-life studies showed that IL-4 also significantly increases the rate of IL-1 message turnover in these cells. Together, these findings demonstrate that IL-4 inhibits IL-1 production in human monocytes by suppressing the formation of new IL-1 transcripts as well as by decreasing IL-1 message stability. In addition, the kinetics of inhibition and the fact that cycloheximide blocks this process suggest that IL-4 induces or enhances synthesis of a protein(s) that mediates these effects.

Cells, Cultured↗
PubMed1987

Phorbol ester enhances human immunodeficiency virus-promoted gene expression and acts on a repeated 10-base-pair functional enhancer element.

T-cell activation pathways are involved in the regulation of human immunodeficiency virus (HIV) expression. Phorbol 12-myristate 13-acetate (PMA) is a potent inducer of T-cell immune functions and has recently been demonstrated to increase viral replication in cell lines infected with HIV. To define sequences required for viral induction by PMA. T-cell lines were transiently transfected with viral long terminal repeat (LTR) sequences directing chloramphenicol acetyltransferase (CAT) gene expression. PMA added to transfected cell cultures 24 h before harvest reproducibly increased both CAT mRNA and enzyme expression 2- to 2-fold. Sequences necessary for basal and PMA-induced levels of CAT expression were determined by deletion and enhancer reconstitution constructs with fragments and oligonucleotides from the original LTR-CAT expression plasmid. PMA-inducible and basal activity required tandem repeats of a core enhancer element (GGGACTTTCC) located in the LTR between -105 and -82 relative to the RNA start site. The enhancerlike sequence could be inserted at a site distant to the CAT gene open reading frame and functioned in a position- and orientation-independent manner. The data thus define a transcriptionally active regulatory-enhancer element critical to the control of HIV gene expression.

Chromosome Mapping↗
PubMed1985

Chloramphenicol-induced translation of cat-86 mRNA requires two cis-acting regulatory regions.

Sequences essential to the chloramphenicol-inducible expression of cat-86, a chloramphenicol acetyltransferase gene, reside in a 144-base pair (bp) regulatory region that intervenes between the cat-86 coding sequence and its promoter. A key regulatory element within the 144-bp segment consists of a pair of inverted-repeat sequences that immediately precede the cat-86 coding region and span the ribosome-binding site for the gene. Because of the location of the inverted repeats, cat-86 transcripts are predicted to sequester the ribosome-binding site in a stable RNA stem-loop structure which should block translation of cat-86 mRNA. Chloramphenicol induction of gene expression is believed to result from ribosome-mediated destabilization of the RNA stem-loop structure, which frees the cat-86 ribosome-binding site, thereby allowing translation. In this study we demonstrated that deletion of 85 bp from the 5' end of the 144-bp regulatory region abolishes inducible expression of cat-86, although the gene is transcribed. This deletion leaves intact both the inverted repeats and the cat-86 coding sequence, and the deletion mutation is not complementable. Therefore, inducible regulation of cat-86 requires the inverted repeats plus an upstream, cis-acting regulatory region. The cis-acting region is believed to control translation of cat-86 mRNA by its essential participation in chloramphenicol-induced opening of the RNA stem-loop. cat-86 deleted for the 85-bp regulatory region and therefore virtually unexpressed was used to select for mutations that restore expression to the gene. An analysis of one mutant plasmid showed that the cat-86 gene is constitutively expressed and that this results from a duplication of the DNA sequence that spans the ribosome-binding site. The duplication provides cat-86 with two ribosome-binding sites. One of these sites is predicted to be sequestered in an RNA stem-loop, and the other is not involved in RNA secondary structure.

Acetyltransferases↗