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Pseudomonas aeruginosa is a Gram-negative bacterium that is ubiquitous in the environment and can cause a variety of diseases in compromised patients. The genome of P. aeruginosa strain PAO1 has been reported to contain 5570 potential proteins. The value of this genomic database is that new proteins can be recognized to use as diagnostic markers, novel drug targets, and to better understand the physiology of this organism. However, similar to what has been observed in other sequenced bacterial genomes, approximately one third of the potential proteins have no known function. This is somewhat surprising given the long-standing interest in P. aeruginosa as an opportunistic pathogen. Obviously new tools, in addition to sequence similarity analysis, are needed to determine the role of these proteins. Proteomics using two-dimensional gel electrophoresis followed by mass spectrometry to detect and identify P. aeruginosa proteins represents a novel approach to address this gap.

We utilized electrospray ionization mass spectrometry (ESI-MS) and hydrogen-deuterium exchange (HX) to detect unfolding of hen egg white lysozyme during salt-induced precipitation. Deuterated lysozyme was dissolved in protonated buffer at pH 2.16 and precipitated with ammonium sulfate, sodium chloride, and potassium thiocyanate. ESI-MS was used to detect mass differences in lysozyme due to the loss of deuterons for solvent protons, providing insight on the conformational history of the protein during the labeling experiment. Precipitation with ammonium sulfate and sodium chloride did not unfold lysozyme, consistent with the known stabilizing effects of kosmotropic salts. Potassium thiocyanate, an aggressive chaotrope, was an effective precipitant at 0.2 M, but also disrupted lysozyme structure and caused the formation of precipitate fractions that did not readily redissolve into aqueous solution without the use of a chemical denaturant. Precipitation with 1.0 M thiocyanate resulted in faster rates of unfolding and larger amounts of the insoluble precipitate. The unfolding kinetics were biphasic, exhibiting a slow phase after a few hours that presumably reflected a smaller propensity for lysozyme to unfold in the precipitated state. Bimodal mass distributions in the ESI-MS spectra for the thiocyanate precipitates indicate two states for lysozyme in this system, a native and a molten globule-like partially unfolded state. ESI-MS analysis of the insoluble precipitates indicated that they consisted primarily of protein molecules that had unfolded. Investigation of the HX behavior of lysozyme in a KSCN solution at low protein concentrations confirmed the destabilizing effect of the salt on the protein structure, even when there was almost no solid phase present. The HX/ESI-MS results provide insight into the mechanism combining precipitation and denaturation for such a system, both in terms of obtaining quantitative kinetic and stability information and the identification of the conformers present., (Copyright 2001 John Wiley & Sons, Inc.)

The influence of local control on metastatic dissemination was analyzed in 601 patients with clinically staged A2 to C prostate cancer treated by high-energy external beam radiation therapy who did not undergo hormonal manipulation before disease progression. Median follow-up for surviving patients was 7.7 years. Ninety-three patients had locally recurrent disease. The actuarial incidence of metastases in these patients (70% at 13 years) was significantly higher than in the 508 patients without local failure (40% at 13 years, P less than 0.001). High stage, high grade, prior transurethral resection, elevated acid phosphatase, disease fixation to the pelvic sidewall, and failure to perform a baseline bone scan correlated positively with the occurrence of metastases. However, except for a slight excess of Stage C, none of the metastatic predictors were more common in patients who failed locally than in those who did not. The Stage C preponderance does not account for the difference in incidence of metastases between the two groups, in as much as metastases were significantly more common in Stage C when disease recurred locally than when it did not. Thus, local control of prostate cancer does decrease the likelihood of metastatic disease. Moreover, patients with local control experienced a significantly better disease-specific survival than patients who failed locally.

This study of 133 patients with localized prostate cancer (Stages A2 to C), treated by external beam radiation therapy (XRT), was undertaken for two reasons: (1) to investigate the usefulness of pretreatment serum prostate-specific antigen (PSA) levels in evaluating patients before XRT; and (2) to investigate post-XRT changes in PSA values and their likely clinical significance. It was found that pretreatment PSA values in patients with localized disease exhibit wide patient to patient variability with a greater than 100-fold difference between the lowest and highest values. Although mean PSA values were significantly higher in Stage C disease (51 patients; mean PSA, 17.3 ng/ml) than in Stage A2 disease (31 patients; mean PSA, 9.0 ng/ml), Stage B1 disease (23 patients; mean PSA, 9.1 ng/ml), or Stage B2 disease (28 patients; mean PSA, 10.6 ng/ml), individual values were of virtually no help in assigning individual patients to a clinical stage. PSA levels did not correlate with grade. After XRT, PSA values fell significantly and dramatically in virtually all patients (98%) by 3 months follow-up. Mean PSA fell from 12.5 to 2.6 ng/ml, and median PSA fell from 6.6 to 1.9 ng/ml. In most patients, PSA continued to fall up to 12 months after XRT and then stabilized at 21 months. Although PSA values fell dramatically after XRT, PSA was detectable in the serum of all patients. PSA values tended to transiently and mildly elevate during XRT. In a small proportion of patients, rising PSA values were observed after 6 months. The full significance of this requires further follow-up, of four such patients, one has relapsed. PSA is a more sensitive marker of prostatic radiation than prostatic acid phosphatase.

Seventy-eight patients with soft tissue sarcoma (STS) arising in the distal extremities--wrist, hand, finger, ankle, foot, and toe--who were treated with conservation surgery and radiation therapy were studied retrospectively with respect to survival, local recurrence, functional limb preservation, complications, and distant metastasis. After a median follow-up of 7.9 years, actuarial 5-year and 10-year survival rates were 80% and 69%, respectively, and disease-free rates were 61% and 51% at the same times. Actuarial local control rates were 80% and 74% at 5 and 10 years, respectively. Fifteen patients (19%) had local recurrence, but 12 of these were salvaged. Ultimately, 53 patients (68%) retained a normal or fairly normal extremity, six (8%) needed amputation for complications, and 13 (17%) needed amputation to control recurrent disease. The functional outcome was significantly better for patients with upper extremity lesions than for those with lower extremity tumors; even for the latter, this treatment strategy was preferable to amputation. The incidence of hematogenous metastases from distal extremity sarcomas depends on the size of the primary tumor. It was concluded that conservation surgery and radiation therapy (XRT) is an acceptable treatment strategy for STS arising in distal extremities; it yielded a high rate of disease control and functional limb preservation.