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Biomedical subjects

S Wolfe

Publications and source records attributed to S Wolfe.

At least 37 records · Page 2Linked to original sources
PubMed1997

Classical light scattering quantitation of protein aggregates: off-line spectroscopy versus HPLC detection.

This paper describes the development and validation of a new off-line approach to quantitate both covalent and noncovalent, in-solution aggregates present in protein formulations and compares the new assay to established HPLC methods. This off-line analysis is well suited for use in QC release testing, formulation development and stability indicating applications. An inexpensive, continuous source HPLC fluorometer has been adapted with the addition of second order filters for use as a sensitive right-angle scatterometer which can determine the molecular weight of protein aggregates in solution. When used as an HPLC detector, right-angle light scattering is a sensitive method which can determine the molecular weight of peaks separable by HPLC, thus discriminating between monomers of different conformations and aggregates. The weight-averaged molecular weight of aggregate peaks can be calculated with system calibration, yielding the average number of monomers per aggregate. If the protein concentration is high enough for an adequate signal, the off-line technique of right-angle light scattering of protein formulations has advantages of convenience and speed over the HPLC approach. Samples are placed in standard fluorometer cuvettes and toluene is used as a calibrator. Data are presented which show the off-line (static) method to be extremely rapid, rugged and precise. The accuracy of this approach is demonstrated through cross-validation to traditional GPC analysis of protein aggregate distributions. This non-invasive light scattering approach is particularly useful when non-covalent protein aggregation is reversible and readily altered by chromatographic separations typically used for characterizing aggregates.

Calibration↗
PubMed1997

Dissection of the molecular mechanism of action of GW5638, a novel estrogen receptor ligand, provides insights into the role of estrogen receptor in bone.

The estrogen receptor (ER) mixed agonists tamoxifen and raloxifene have been shown to protect against bone loss in ovariectomized rats. However, the mechanism by which these compounds manifest their activity in bone is unknown. We have used a series of in vitro screens to select for compounds that are mechanistically distinct from tamoxifen and raloxifene in an effort to define the properties of an ER modulator required for bone protection. Using this approach, we identified a novel high affinity ER antagonist, GW5638, which when assayed in vitro functions as an ER antagonist, inhibiting the agonist activity of estrogen, tamoxifen, and raloxifene and reversing the "inverse agonist" activity of the pure antiestrogen ICI182,780. Thus, GW5638 appears to function as an antagonist in these in vitro systems, although in a manner distinct from other known ER modulators. Predictably, therefore, GW5638 alone displays minimal uterotropic activity in ovariectomized rats, but will inhibit the agonist activity of estradiol in this environment. Unexpectedly, however, this compound functions as a full ER agonist in bone and the cardiovascular system. These data suggest that the mechanism by which ER operates in different cells is not identical, and that classical agonist activity is not required for the bone protective activity of ER modulators.

Animals↗
PubMed1994

Health care paper chase, 1993: the cost to the nation, the states, and the District of Columbia.

The U.S. health care payment system is an elaborate and increasingly wasteful paper chase. This article presents new state-by-state estimates of health care administrative costs in the United States, and savings that could be realized with single-payer reform. In 1993, health care bureaucracy will consume 24.7 cents of every health care dollar, a total of $232.3 billion. Administration's share of health spending is up from 23.9 percent in 1987, and from 21.9 percent in 1983. Reducing the cost of administration to Canadian levels by adopting a single-payer health care system would cut U.S. health care bureaucracy by more than half (50.7 percent), saving at least $117.7 billion in 1993. The savings achievable with a single-payer system could fund universal access for the uninsured and improve benefits for the tens of millions of Americans who currently have only partial coverage, without any increase in overall health spending. Reform measures such as electronic billing, insurance industry consolidation, and increased competition (including "managed competition") would save little or nothing on administration. Only a single-payer reform that incorporates the "macro-management" approach to cost control, as in Canada, can achieve significant administrative savings.

Cost Savings↗
PubMed1992

Biochemical studies on the activity of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus.

The enzyme activity of purified delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase from Streptomyces clavuligerus was studied biochemically. The dependence of ACV synthetase activity on reaction parameters, including substrates, cofactors, temperature and pH, were determined, resulting in a substantially increased enzyme activity. The activity is very labile to high temperature and is also unstable at acidic pH. The enzyme specificity is strict towards L-alpha-aminoadipate, but rather loose with respect to L-valine; certain modifications of L-cysteine can also be tolerated. Some unnatural tripeptides synthesized by ACV synthetase can be converted into bioactive compounds by isopenicillin N synthase. The only nutrient found to negatively affect ACV synthetase activity is phosphate, but various compounds such as thiol-blocking reagents and ATP-utilization products (AMP and pyrophosphate) are inhibitory to the enzyme.

2-Aminoadipic Acid↗
PubMed1992

Universal access in Canada. Questions of equity remain.

Health/PAC readers will remember Samuel Wolfe's previous Bulletin article about Canada's national health care system ("Importing Health Care Reform? Issues in Transposing Canada's Health Care System to the United States," Summer 1990), in which he described the uniqueness of our northern neighbor's experience. At that time, Wolfe expressed doubt that a Canadian-like model could find sufficient political and social momentum to be propelled south. In this latest article, Wolfe is joined by his long-time colleague, Robin Badgley, to discuss the inequity and inequality they believe still exists within the Canadian system. Wolfe and Badgley's insightful analysis of what they see as shortcomings of the Canadian model and suggestions for their remedy are especially welcome given the questionable validity of most criticism of the Canadian system that Americans hear. Their work helps us understand the real problems that remain in the still-evolving Canadian system. As Americans discuss the various possibilities for progressive single-payer reform, many envision of state-based system. Wolfe and Badgley point out that demographically rooted inequalities and the lingering health burdens of social class may interfere with truly national universal access. They also alert us to the dangers that federal retreat from adequate levels of support would pose to such a state strategy. Samuel Wolfe has a unique background as a country doctor, psychiatrist, and public health official who attended the difficult birth of Saskatchewan's provincial health plan--the prototype for Canada's national health insurance. A respected teacher, researcher, and community health activist, Wolfe has taught in the United States for more than 25 years. He will soon move back to Canada and the welcome security of its universal health care system.

Canada↗
PubMed1991

Purification and characterization of cephalosporin 7 alpha-hydroxylase from Streptomyces clavuligerus.

Cephalosporin 7 alpha-hydroxylase, which catalyses the conversion of cephalosporins into their 7 alpha-hydroxy derivatives, was purified nearly 390-fold from Streptomyces clavuligerus through ion-exchange chromatography, (NH4)2SO4 fractionation, gel filtration and dye chromatography, with the use of h.p.l.c. to monitor enzyme activity. The nearly pure enzyme migrates as a single major band, with an Mr of 32,000 in SDS/PAGE. Its optimum pH is in the range 7.3-7.7. Under our conditions the reaction was fastest at temperatures in the range 20-30 degrees C. The Km for cephalosporin C is 0.72 mM, and the Vmax. is 15.4 mumol of cephalosporin C hydroxylated/min per mg. Cephalosporin 7 alpha-hydroxylase did not show any deacetoxycephalosporin C synthase or deacetoxycephalosporin C hydroxylase activity.

Cephalosporins↗
PubMed1991

Inhibition of leukotriene B4 biosynthesis by disulfiram and A-64077 during carrageenan-induced pleurisy in the rat.

1. The effect of disulfiram and A-64077 on leukotriene B4 biosynthesis was investigated using human polymorphonuclear leukocyte preparations and an in vivo rat pleurisy assay. 2. Disulfiram inhibited the calcium ionophore-induced release of LTB4 by human leukocytes in vitro with an IC50 of 4.6 +/- 0.3 microM, a value similar to that observed with the 5-lipoxygenase inhibitor A-64077 (IC50 = 1.2 +/- 0.3 microM). These inhibitors were at least 100-fold more potent than diethyldithiocarbamate, the primary metabolite of disulfiram. 3. In a rat pleurisy model, the administration of A-64077 (p.o., 2 hr pretreatment) caused a marked decrease in LTB4 levels measureable after ionophore stimulation at doses of 3 and 10 mg kg (67 and 96% inhibition, respectively). Disulfiram was about a 100-fold less potent, inhibiting LTB4 release by 65% at 300 mg kg (p.o., 6 hr pretreatment). 4. In contrast to A-64077, the inhibitory effect of disulfiram on LTB4 production by isolated leukocytes from the pleural cavity was reduced by the addition of the cell-free pleural exudate, suggesting that protein binding or conversion of disulfiram to inactive species contributes to diminish the potency of the drug. 5. The results indicate that disulfiram, after oral administration in rats, causes an inhibition of leukotriene biosynthesis in the pleural cavity and further illustrate the limited specificity of this drug as an inhibitor of aldehyde dehydrogenase at doses generally used to inhibit this enzyme in vivo.

Aldehyde Dehydrogenase↗