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Biomedical subjects

D S Clark

Publications and source records attributed to D S Clark.

At least 37 records · Page 2Linked to original sources
PubMed2000

Posterior capsule opacification.

Posterior capsule opacification (PCO) is the most common complication following primary cataract surgery. Advances in intraocular lens (IOL) designs that have reduced the amount of PCO following surgery have been made. The understanding of how the IOL design effects PCO has also advanced. Lenses that provide a mechanical barrier between it and the posterior lens capsule seem to inhibit PCO to a greater degree. Intracapsular rings are now being explored to test and enhance this barrier effect. Major advances in the elimination of lens epithelial cells at the time of surgery especially by pharmacologic means have also been made. An immunotoxin specific for human lens epithelial cells shows promise and is under latter phase clinical development.

Cataract↗
PubMed2000

Metabolic engineering of an aerobic sulfate reduction pathway and its application to precipitation of cadmium on the cell surface.

The conversion of sulfate to an excess of free sulfide requires stringent reductive conditions. Dissimilatory sulfate reduction is used in nature by sulfate-reducing bacteria for respiration and results in the conversion of sulfate to sulfide. However, this dissimilatory sulfate reduction pathway is inhibited by oxygen and is thus limited to anaerobic environments. As an alternative, we have metabolically engineered a novel aerobic sulfate reduction pathway for the secretion of sulfides. The assimilatory sulfate reduction pathway was redirected to overproduce cysteine, and excess cysteine was converted to sulfide by cysteine desulfhydrase. As a potential application for this pathway, a bacterium was engineered with this pathway and was used to aerobically precipitate cadmium as cadmium sulfide, which was deposited on the cell surface. To maximize sulfide production and cadmium precipitation, the production of cysteine desulfhydrase was modulated to achieve an optimal balance between the production and degradation of cysteine.

Aerobiosis↗
PubMed2000

Engineering hydrogen sulfide production and cadmium removal by expression of the thiosulfate reductase gene (phsABC) from Salmonella enterica serovar typhimurium in Escherichia coli.

The thiosulfate reductase gene (phsABC) from Salmonella enterica serovar Typhimurium was expressed in Escherichia coli to overproduce hydrogen sulfide from thiosulfate for heavy metal removal (or precipitation). A 5.1-kb DNA fragment containing phsABC was inserted into the pMB1-based, high-copy, isopropyl-beta-D-thiogalactopyranoside-inducible expression vector pTrc99A and the RK2-based, medium-copy, m-toluate-inducible expression vector pJB866, resulting in plasmids pSB74 and pSB77. A 3. 7-kb DNA fragment, excluding putative promoter and regulatory regions, was inserted into the same vectors, making plasmids pSB103 and pSB107. E. coli DH5alpha strains harboring the phsABC constructs showed higher thiosulfate reductase activity and produced significantly more sulfide than the control strains under both aerobic and anaerobic conditions. Among the four phsABC constructs, E. coli DH5alpha (pSB74) produced thiosulfate reductase at the highest level and removed the most cadmium from solution under anaerobic conditions: 98% of all concentrations up to 150 microM and 91% of 200 microM. In contrast, a negative control did not produce any measurable sulfide and removed very little cadmium from solution. Energy-dispersive X-ray spectroscopy revealed that the metal removed from solution precipitated as a complex of cadmium and sulfur, most likely cadmium sulfide.

Cadmium↗
PubMed1999

Optimizing the salt-induced activation of enzymes in organic solvents: effects of lyophilization time and water content.

The addition of simple inorganic salts to aqueous enzyme solutions prior to lyophilization results in a dramatic activation of the dried powder in organic media relative to enzyme with no added salt. Activation of both the serine protease subtilisin Carlsberg and lipase from Mucor javanicus resulting from lyophilization in the presence of KCl was highly sensitive to the lyophilization time and water content of the sample. Specifically, for a preparation containing 98% (w/w) KCl, 1% (w/w) phosphate buffer, and 1% (w/w) enzyme, varying the lyophilization time showed a direct correlation between water content and activity up to an optimum, beyond which the activity decreased with increasing lyophilization time. The catalytic efficiency in hexane varied as much as 13-fold for subtilisin Carlsberg and 11-fold for lipase depending on the lyophilization time. This dependence was apparently a consequence of including the salt, as a similar result was not observed for the enzyme freeze-dried without KCl. In the case of subtilisin Carlsberg, the salt-induced optimum value of kcat/Km for transesterification in hexane was over 20,000-fold higher than that for salt-free enzyme, a substantial improvement over the previously reported enhancement of 3750-fold (Khmelnitsky, 1994). As was found previously for pure enzyme, the salt-activated enzyme exhibited greatest activity when lyophilized from a solution of pH equal to the pH for optimal activity in water. The active-site content of the lyophilized enzyme samples also depended upon lyophilization time and inclusion of salt, with opposite trends in this dependence observed for the solvents hexane and tetrahydrofuran. Finally, substrate selectivity experiments suggested that mechanism(s) other than selective partitioning of substrate into the enzyme-salt matrix are responsible for salt-induced activation of enzymes in organic solvents.

Bacillus↗
PubMed1999

Combinatorial biocatalysis: taking the lead from nature.

Combinatorial biocatalysis is an emerging technology in the field of drug discovery. The biocatalytic approach to combinatorial chemistry uses enzymatic, chemoenzymatic, and microbial transformations to generate libraries from lead compounds. Important recent advances in combinatorial biocatalysis include iterative derivatization of small molecules and complex natural products, regioselectively controlled libraries, novel one-pot library syntheses, process automation, and biocatalyst enhancements.

Automation↗
PubMed1999

Pressure-induced thermostabilization of glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus.

In this paper, elevated pressures up to 750 atm (1 atm = 101 kPa) were found to have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases (GDHs): the native enzyme from the hyperthermophile Pyrococcus furiosus (Pf), and a recombinant GDH mutant containing an extra tetrapeptide at the C-terminus (rGDHt). The presence of the tetrapeptide greatly destabilized the recombinant mutant at ambient pressure; however, the destabilizing effect was largely reversed by the application of pressure. Electron spin resonance (ESR) spectroscopy of a spin-label attached to the terminal cysteine of rGDHt revealed a high degree of mobility, suggesting that destabilization is due to weakened intersubunit ion-pair interactions induced by thermal fluctuations of the tetrapeptide. For both enzymes, the stabilizing effect of pressure increased with temperature as well as pressure, reaching 36-fold for rGDHt at 105 degrees C and 750 atm, the largest pressure-induced thermostabilization of an enzyme reported to date. Stabilization of both native GDH and rGDHt was also achieved by adding glycerol. Based on the kinetics of thermal inactivation and the known effects of glycerol on protein structure, a mechanism of pressure-induced thermostabilization is proposed.

Electron Spin Resonance Spectroscopy↗
PubMed1999

Adaptation of proteins from hyperthermophiles to high pressure and high temperature.

Further clarification of the adaptations permitting the persistence of life at temperatures above 100 degrees C depends in part on the analysis of adaptive mechanisms at the protein level. The hyperthermophiles include both Bacteria and Archaea, although the majority of isolates growing at or above 100 degrees C are Archaea. Newly described adaptive features of hyperthermophiles include proteins whose structural integrity persists at temperatures up to 200 degrees C, and under elevated hydrostatic pressure, which in some cases adds significant increments of stability.

Adaptation, Physiological↗
PubMed1998

Malcolm Lilly.

Explore the source record for details and available documents.

Biomedical Engineering↗
PubMed1998

Premerger notification: reporting and waiting period requirements--FTC. Notice of adoption of formal interpretation and request for comments.

The Premerger Notification Office ("PNO") of the Federal Trade Commission ("FTC"), with the concurrence of the Assistant Attorney General in charge of the Antitrust Division of the Department of Justice ("DOJ"), is adopting a Formal Interpretation of the Hart-Scott-Rodino Act, which requires certain persons planning certain mergers, consolidations, or other acquisitions to report information about the proposed transactions to the FTC and DOJ. The Interpretation concerns the reportability of certain transactions involving a Limited Liability Company ("LLC"), a relatively new form of entity authorized by state statutes. Under the Interpretation, the formation of an LLC will be reportable if it will unite two or more pre-existing businesses under common control. Similarly, acquisitions of existing LLC membership interests will be reportable if they would have the effect of uniting two or more pre-existing businesses under common control.

Antitrust Laws↗
PubMed1998

Testing for diffusion limitations in salt-activated enzyme catalysts operating in organic solvents.

The dramatic activation of serine proteases in nonaqueous media resulting from lyophilization in the presence of KCl is shown to be unrelated to relaxation of potential substrate diffusional limitations. Specifically, lyophilizing subtilisin Carlsberg in the presence of KCl and phosphate buffer in different proportions, ranging from 99% (w/w) enzyme to 1% (w/w) enzyme in the final lyophilized solids, resulted in biocatalyst preparations that were not influenced by substrate diffusion. This result was made evident through use of a classical analysis whereby initial catalytic rates, normalized per weight of total enzyme in the catalyst material, were measured as a function of active enzyme for biocatalyst preparations containing different ratios of active to inactive enzyme. The active enzyme content of a given biocatalyst preparation was controlled by mixing native subtilisin with subtilisin preinactivated with PMSF, a serine protease inhibitor, and lyophilizing the enzyme mixture in the presence of different fractions of KCl and phosphate buffer. Plots of initial reaction rates as a function of percent active subtilisin in the biocatalyst were linear for all biocatalyst preparations. Thus, enzyme activation (reported elsewhere to be as high as 3750-fold in hexane for the transesterification of N-Ac-L-Phe-OEt with n-PrOH) is a manifestation of intrinsic enzyme activation and not relaxation of diffusional limitations resulting from diluted enzyme preparations. Similar activation is reported herein for thermolysin, a nonserine protease, thereby demonstrating that enzyme activation due to lyophilization in the presence of KCl may be a general phenomenon for proteolytic enzymes.

Bacillus↗
PubMed1998

Multinuclear NMR study of enzyme hydration in an organic solvent.

Multinuclear NMR spectroscopy has been used to study water bound to subtilisin Carlsberg suspended in tetrahydrofuran (THF), with the water itself employed as a probe of the hydration layer's physicochemical and dynamic characteristics. The presence of the enzyme did not affect the intensity, chemical shift or linewidth of water (up to 8% v/v) added to THF, as measured by 17O- and 2H-NMR. This finding suggests that hydration of subtilisin can be described by a three-state model that includes tightly bound, loosely bound, and free water. Solid-state 2H-NMR spectra of enzyme-bound D2O support the existence of a non-exchanging population of tightly bound water. An important implication is that the loosely-bound water is the same as free water from an NMR viewpoint. This loosely bound water must also be the water responsible for the large increase in catalytic activity observed in previous hydration studies.

Deuterium↗
PubMed1998

Molecular cloning of extremely thermostable esterase gene from hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli.

A genomic library of the hyperthermophilic archaeon Pyrococcus furiosus was constructed in Escherichia coli using pBluescript II SK(+) as a cloning vector. One positive clone exhibiting thermophilic ester-hydrolyzing activity was directly detected by an in situ plate assay using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-acetate. The plasmid isolated from the clone contained a 3.8 kb HindIII fragment from P. furiosus. Expression of active thermostable esterase in E. coli was independent of isopropyl-beta-D-thiogalactopyranoside, suggesting that the archaeal esterase gene was heterologously controlled by its own promoter sequence, not by the vector-located lac promoter. Assays of esterase activity in heat-treated extract of the recombinant E. coli showed the highest temperature optimum (100 degrees C) and thermostability (a half-life of 50 min at 126 degrees C) among esterases reported to date.

Cloning, Molecular↗
PubMed1998

Effect of extracellular glutamine concentration on primary and secondary metabolism of a murine hybridoma: an in vivo 13C nuclear magnetic resonance study.

The effect of changes in extracellular glutamine level on metabolism of a murine hybridoma was examined with in vivo nuclear magnetic resonance (NMR) spectroscopy. Cells were cultured in a hollow-fiber bioreactor at high cell density to allow intracellular metabolite levels to be determined on a metabolically relevant time scale. Steady infusions of [1-13C] glucose were used to label glycolytic and tricarboxylic acid cycle intermediates, which permitted continuous monitoring with NMR spectroscopy during changes in environmental glutamine level. Samples of the extracellular medium were also analyzed to determine the effect of glutamine on other metabolites associated with primary and secondary metabolism. The changes in glutamine concentration had several effects on primary and secondary metabolism, depending on the rate the changes were made. For a brief reduction in feed glutamine concentration from 4 to 0 mM (which produced a rapid change from 0.67 to approximately 0 mM in residual glutamine), large changes were observed in the rate of consumption of metabolites normally associated with energy production. Antibody synthesis was strongly stimulated and nitrogen metabolism was significantly altered. For a more prolonged reduction from 2.4 to 1.2 mM (which produced a slower reduction from 0.30 to 0. 08 mM in residual glutamine), much smaller changes were observed even though the concentration of glutamine at the reduced feed level was very low. Energy metabolism did not appear to be limited by glutamine at 0.08 mM, which suggests that significant futile cycling may occur in energy producing pathways when excess glucose and glutamine are available. However, this concentration of extracellular glutamine appeared to affect some anabolic pathways, which require amino groups from glutamine.

Animals↗
PubMed1998

Combinatorial biocatalysis: a natural approach to drug discovery.

Nature's most potent molecules are produced by enzyme-catalysed reactions, coupled with the natural selection of those products that possess optimal biological activity. Combinatorial biocatalysis harnesses the natural diversity of enzymatic reactions for the iterative synthesis of organic libraries. Iterative reactions can be performed using isolated enzymes or whole cells, in natural and unnatural environments, and on substrates in solution or on a solid phase. Combinatorial biocatalysis is a powerful addition to the expanding array of combinatorial methods for the generation and optimization of lead compounds in drug discovery and development.

Acylation↗
PubMed1998

Purification and functional characterization of a chaperone from Methanococcus jannaschii.

A chaperone from Methanococcus jannaschii has been purified to homogeneity with a single chromatographic step. The chaperone was identified and characterized using activity assays for characteristic chaperone abilities. The M. jannaschii chaperone binds unfolded proteins, protects proteins against heat-induced aggregation, and has a strongly temperature dependent ATPase activity. The chaperone has also been shown to inhibit the spontaneous refolding of a mesophilic protein at low temperatures. The purified chaperone complex has a M(r) of about 1,000,000 and consists of a single type of subunit with an approximate M(r) of 60,000. Analysis of partial sequence data reveals that this chaperone is the predicted protein product of the previously identified chaperonin gene in M. jannaschii (BULT et al., 1996). To our knowledge, this is the first functional characterization of a chaperone from a methanogen.

Adenosine Triphosphatases↗
PubMed1998

Inhibition of posterior capsule opacification with an immunotoxin specific for lens epithelial cells: 24 month clinical results.

PURPOSE: To assess the safety and effectiveness of an immunotoxin, MDX-RA, designed to inhibit posterior capsule opacification (PCO). SETTING: Eleven private practices in the United States. METHODS: This study comprised 63 eyes of 63 patients having extracapsular cataract extraction by phacoemulsification; these patients were enrolled in a Phase I/II clinical investigation of the immunotoxin MDX-RA. At the close of surgery, 21 patients were treated with placebo, 23 patients with 50 units of the immunotoxin, and 19 patients with 175 units of the immunotoxin as an aqueous solution. The patients were monitored for 24 months after primary cataract surgery using external eye and slitlamp examinations, visual acuity assessment, ophthalmoscopy, pachymetry, tonometry, endothelial cell counts, and lens capsule photography. Posterior capsule opacification, recorded on lens capsule photographs, was graded independently by a committee of 3 cataract surgeons. The incidence of neodymium:YAG (Nd:YAG) capsulotomy was projected from the opacification results. RESULTS: The immunotoxin, at the 50 unit dose, was well tolerated and effective in inhibiting PCO. At the 175 unit dose, there was a trend toward increased postoperative inflammation that was transient with no residua. From 6 to 24 months postoperatively, the 50 unit dose significantly inhibited PCO compared with the placebo (P < .05). This significant reduction in PCO translated into a significantly lower projected need for Nd:YAG capsulotomy in the 50 unit than the placebo group (P < .004). About 60% in the placebo group and 4% in the 50 unit group were projected to need an Nd:YAG capsulotomy by 3 years postoperatively. CONCLUSION: The immunotoxin was well tolerated and was effective in reducing PCO for up to 24 months after cataract surgery. Although these preliminary results are encouraging, a larger study is underway to determine whether the reduction in PCO by the immunotoxin decreases the need for Nd:YAG capsulotomy.

Adult↗
PubMed1998

Mathematical model to predict the need for neodymium: YAG capsulotomy based on posterior capsule opacification rate.

PURPOSE: To evaluate a model to project the estimated time required before patients having primary phacoemulsification require neodymium:YAG (Nd:YAG) laser capsulotomy. SETTING: Eleven private practices in the United States. METHODS: Projections of time to capsulotomy were based on assessment of the early development of posterior capsule opacification (PCO) over time. The PCO data were collected during a clinical study to evaluate MDX-RA, an investigational immunotoxin designed to limit epithelial cell growth, preventing postsurgical PCO. From the PCO data, the estimated time to Nd:YAG capsulotomy in a placebo-treated group was compared with the actual time to capsulotomy in a cohort of patients from general practice who had had phacoemulsification. RESULTS: By 6 months, the mean Opacification Index in the MDX-RA group was significantly lower than that in the placebo group (P < .05) and it remained significantly lower at 12 (P < .001), 18 (P < .001), and 24 (P < .016) months. The rate of PCO in the MDX-RA group was approximately 6 times lower than that in the placebo group (P < .0004). Fifty-seven percent in the placebo group and 4% in the MDX-RA group were projected to require an Nd:YAG capsulotomy within 3 years of primary cataract surgery. Projected values for the placebo group were similar to actual values observed in the population-based cohort. CONCLUSIONS: This technique could be used to predict the need for Nd:YAG capsulotomy using early measurements of PCO.

Adult↗