Attitudes of patients with metastatic breast cancer toward research biopsies.

BACKGROUND: Research studies involving human tissue are increasingly common. However, patients' attitudes toward research biopsies are not well characterized, particularly when the biopsies are carried out outside the context of therapeutic trials. PATIENTS AND METHODS: One hundred sixty patients with metastatic breast cancer (MBC) from two academic (n = 80) and two community (n = 80) hospitals completed a 29-item self-administered survey to evaluate their willingness to consider providing research purposes only biopsies (RPOBs) (as a stand-alone procedure) and additional biopsies (ABs) (additional needle passes at the time of a clinically indicated biopsy). RESULTS: Eighty-two (51%) of 160 patients would consider having RPOBs, of which 42 (53%) and 40 (50%) patients were from academic and community hospitals, respectively. Patients who had more prior biopsies were less likely to consider RPOBs (RR = 0.6, 95% CI: 0.4-1.0, P = 0.03). Of 160 patients, 115 (72%) patients would consider having ABs. Of these, 64 (80%) and 51 (64%) patients from academic and community hospitals, respectively, would consider ABs (RR = 1.2, 95% CI: 1.0-1.5, P = 0.03). CONCLUSIONS: Many patients with MBC in both academic and community settings report willingness to consider undergoing biopsies for research. Further research is needed to understand ethical, logistical and provider-based barriers to broader participation in such studies.

Kinetic stability as a mechanism for protease longevity.

The folding of the extracellular serine protease, alpha-lytic protease (alphaLP; EC 3.4.21.12) reveals a novel mechanism for stability that appears to lead to a longer functional lifetime for the protease. For alphaLP, stability is based not on thermodynamics, but on kinetics. Whereas this has required the coevolution of a pro region to facilitate folding, the result has been the optimization of native-state properties independent of their consequences on thermodynamic stability. Structural and mutational data lead to a model for catalysis of folding in which the pro region binds to a conserved beta-hairpin in the alphaLP C-terminal domain, stabilizing the folding transition state and the native state. The pro region is then proteolytically degraded, leaving the active alphaLP trapped in a metastable conformation. This metastability appears to be a consequence of pressure to evolve properties of the native state, including a large, highly cooperative barrier to unfolding, and extreme rigidity, that reduce susceptibility to proteolytic degradation. In a test of survival under highly proteolytic conditions, homologous mammalian proteases that have not evolved kinetic stability are much more rapidly degraded than alphaLP. Kinetic stability as a means to longevity is likely to be a mechanism conserved among the majority of extracellular bacterial pro-proteases and may emerge as a general strategy for intracellular eukaryotic proteases subject to harsh conditions as well.

Unfolded conformations of alpha-lytic protease are more stable than its native state.

alpha-Lytic protease (alphaLP), an extracellular bacterial protease, is synthesized with a large amino-terminal pro-region that is essential for its folding in vivo and in vitro. In the absence of the pro-region, the protease folds to an inactive, partially folded state, designated 'I'. The pro-region catalyses protease folding by directly stabilizing the folding transition state (>26kcal mol(-1)) which separates the native state 'N' from I. Although a basic tenet of protein folding is that the native state of a protein is at the minimum free energy, we show here that both the I and fully unfolded states of alphaLP are lower in free energy than the native state. Native alphaLP is thus metastable: its apparent stability derives from a large barrier to unfolding. Consequently, the evolution of alphaLP has been distinct from most other proteins: it has not been constrained by the free-energy difference between the native and unfolded states, but instead by the size of its unfolding barrier.

Pro region C-terminus:protease active site interactions are critical in catalyzing the folding of alpha-lytic protease.

alpha-Lytic protease is encoded with a large (166 amino acid) N-terminal pro region that is required transiently both in vivo and in vitro for the correct folding of the protease domain [Silen, J. L. , and Agard, D. A. (1989) Nature 341, 462-464; Baker, D., et al. (1992) Nature 356, 263-265]. The pro region also acts as a potent inhibitor of the mature enzyme [Baker, D., et al. (1992) Proteins: Struct.,Funct., Genet. 12, 339-344]. This inhibition is mediated through direct steric occlusion of the active site by the C-terminal residues of the pro region [Sohl, J. L., et al. (1997) Biochemistry 36, 3894-3904]. Through mutagenesis and structure-function analyses we have begun to investigate the mechanism by which the pro region acts as a single turnover catalyst to facilitate folding of the mature protease. Of central interest has been mapping the interface between the pro region and the protease and identifying interactions critical for stabilizing the rate-limiting folding transition state. Progressive C-terminal deletions of the pro region were found to have drastic effects on the rate at which the pro region folds the protease but surprisingly little effect on inhibition of protease activity. The observed kinetic data strongly support a model in which the detailed interactions between the pro region C-terminus and the protease are remarkably similar to those of known substrate/inhibitor complexes. Further, mutation of two protease residues near the active site have significant effects on stabilization of the folding transition state (kcat) or in binding to the folding intermediate (KM). Our results suggest a model for the alpha-lytic protease pro region-mediated folding reaction that may be generally applicable to other pro region-dependent folding reactions.

Inhibition of alpha-lytic protease by pro region C-terminal steric occlusion of the active site.

alpha-Lytic protease, a chymotrypsin-like serine protease, is synthesized with an N-terminal 166 amino acid pro region which is absolutely required for folding of the protease. The pro region is also the most potent inhibitor of the protease known with a Ki of approximately 10(-10) M. Compared to its role in the folding reaction, relatively little is known about the mechanism by which the pro region inhibits the mature protease. While proteinaceous protease inhibitors generally function by occluding the active sites of their respective targets [Bode, W., & Huber, R. (1992) Eur. J. Biochem. 204, 433-451], the pro region of alpha-lytic protease with its dual roles in folding and inhibition might be expected to show a novel mechanism of inhibition. However, experiments that probe both the structural and enzymatic consequences of pro region binding indicate that the pro region does not measurably distort the protease active site. Instead, the catalytic site is fully functional in the complex. Pro region inhibition of the protease is due to simple steric obstruction; the pro region C-terminus lies in the substrate binding sites of the protease. The implications of these results are discussed with regard to alpha-lytic protease maturation and folding. In addition, the proposed mechanism of alpha-lytic protease pro region inhibition is discussed with respect to data from other pro region families.

A protein-folding reaction under kinetic control.

Synthesis of alpha-lytic protease is as a precursor containing a 166 amino-acid pro region transiently required for the correct folding of the protease domain. By omitting the pro region in an in vitro refolding reaction we trapped an inactive, but folding competent state (I) having an expanded radius yet native-like secondary structure. The I state is stable for weeks at physiological pH in the absence of denaturant, but rapidly folds to the active, native state on addition of the pro region as a separate polypeptide chain. The mechanism of action of the pro region is distinct from that of the chaperonins: rather than reducing the rate of off-pathway reactions, the pro region accelerates the rate-limiting step on the folding pathway by more than 10(7). Because both the I and native states are stable under identical conditions with no detectable interconversion, the folding of alpha-lytic protease must be under kinetic and not thermodynamic control.

Absorption of 308-nm excimer laser radiation by balanced salt solution, sodium hyaluronate, and human cadaver eyes.

Absorption of the excimer laser radiations of 193-nm argon fluorine and 308-nm xenon chloride in balanced salt solution, sodium hyaluronate, and human cadaver eyes was measured. The absorption of these materials as considerably different for the two wavelengths; we found that 308-nm light experienced much less absorption than the 193-nm light. The extinction coefficient (k) for 308 nm was k = 0.19/cm for balanced salt solution and k = 0.22/cm for sodium hyaluronate. In contrast to this, the extinction coefficient for 193 nm was k = 140/cm for balanced salt solution and k = 540/cm for sodium hyaluronate. Two 1-day-old human phakic cadaver eyes showed complete absorption with both wavelengths. Using aphakic eyes, incomplete absorption was noted at the posterior pole with 308 nm and complete absorption was noted with 193 nm. The extinction in the anterior part of aphakic eyes (the first 6 mm) was 4.2/cm for 308 nm, meaning that the intensity of the light is reduced by a factor of 10 after traveling the first 5.5 mm. However, we observed that the material in the eye fluoresces, meaning the 308 nm is transformed into other (longer) wavelengths that travel through the total eye with minimal absorption. Conclusions drawn from this experiment are that the use of the 308-nm wavelength may have undesirable side effects, while the use of the 193-nm wavelength should be consistent with ophthalmic use on both the cornea and the lens.

Regulation of an enzyme by phosphorylation at the active site.

The isocitrate dehydrogenase of Escherichia coli is an example of a ubiquitous class of enzymes that are regulated by covalent modification. In the three-dimensional structure of the enzyme-substrate complex, isocitrate forms a hydrogen bond with Ser113, the site of regulatory phosphorylation. The structures of Asp113 and Glu113 mutants, which mimic the inactivation of the enzyme by phosphorylation, show minimal conformational changes from wild type, as in the phosphorylated enzyme. Calculations based on observed structures suggest that the change in electrostatic potential when a negative charge is introduced either by phosporylation or site-directed mutagenesis is sufficient to inactivate the enzyme. Thus, direct interaction at a ligand binding site is an alternative mechanism to induced conformational changes from an allosteric site in the regulation of protein activity by phosphorylation.

Nonlinearity in protein assays by the Coomassie blue dye-binding method.

The Coomassie brilliant blue (CBB) method for protein determination takes advantage of the fact that a low-pH red form of CBB reverts to a blue form when CBB binds to protein. The increase in absorbance at 595 nm of a protein-dye mixture compared to a blank containing only the dye reagent has been used to estimate the total amount of protein present in the sample mixture. A disadvantage of this method of protein determination is that the assay plot of absorbance at 595 nm versus total protein is not linear.

Haloketone transition state analog inhibitors of cholesterol esterase.

The cholesterol esterase-catalyzed hydrolysis of p-nitro-phenyl butyrate is reversibly inhibited by four phenyl haloalkyl ketones. Inhibitor potency is greatest for halogenated acetophenones and parallels the extent of hydration of the various ketones in buffered D2O. These results are consistent with an inhibition mechanism wherein haloketones reversibly form hemiketal adducts at the active site that structurally mimic tetrahedral intermediates of the cholesterol esterase catalytic cycle.