Development of a protein phosphatase-based assay for the detection of phosphatase inhibitors in crude whole cell and animal extracts

Diarrhetic shellfish poisoning (DSP) is a serious and globally widespread phytoplankton-related seafood illness. Although DSP is rarely life-threatening, it causes incapacitating diarrhea and vomiting with no known medical treatments. In addition, phytoplankton producing DSP toxins have been identified in temperate coastal waters worldwide, and their numbers may be increasing as a result of coastal eutrophication. The toxic effects of the major DSP toxins, okadaic acid and dinophysistoxin-1 (35-methylokadaic acid), appear to originate from their inhibitory activity against a family of structurally related serine/threonine protein phosphatases (PSPases). In particular, the inhibition of essential PSPases (e.g. PP1 and PP2A) has catastrophic consequences in most eukaryotic cells. Exploiting the potent inhibitory property of the DSP toxins, we have developed an enzyme-based assay (PP2A assay) capable of detecting both okadaic acid and dinophysis- toxin-1 in nanogram amounts. The assay employs purified PP2A, which has an extremely high affinity for both DSP toxins. This provides the PP2A assay with a level of sensitivity comparable to, or surpassing, that of most monoclonal antibody probes. To evaluate the PP2A assay as a means of detecting contaminated shellfish, a series of spike recovery experiments was conducted. The findings from these studies suggest that the PP2A assay has the potential for development into a rapid and relatively simple method for detecting PSPase inhibitors in crude extracts produced from shellfish.     

Efficacy of low-dose interferon with antiretroviral therapy in Kaposi's sarcoma: a randomized phase II AIDS clinical trials group study.

We wished to evaluate the efficacy and safety of a low and an intermediate daily dose of interferon-alpha2b (IFN-alpha2b) with didanosine in patients with acquired immunodeficiency syndrome (AIDS)-associated Kaposi's sarcoma (KS). HIV-seropositive subjects with biopsy-confirmed cutaneous KS were randomized to receive either a low (1 million IU) or an intermediate (10 million IU) dose of IFN-alpha2b once daily with twice daily doses of didanosine. Treatment assignment was stratified by CD4 count. Response, toxicity, changes in CD4 counts, and survival were evaluated. Sixty-eight eligible subjects were accrued, 35 to low-dose and 33 to intermediate-dose IFN-alpha2b. The response rate was 40% in the low-dose group (95% CI, 24-58) and 55% in the intermediate-dose group (95% CI, 36-72) (p = 0.338). The median response duration was approximately 110 weeks in both groups. Intermediate-dose IFN induced grade 3/4 neutropenia more often (21% vs. 3%, p = 0.048) and grade 3/4 toxicity faster (p = 0.0231) and necessitated treatment discontinuation earlier for drug-related toxicities (p = 0.0416) than low-dose IFN. There were no significant differences in survival between the treatment groups. Baseline CD4 count was the only significant factor predicting response. Once-daily low-dose and intermediate-dose IFN-alpha2b induced similar response rates, which were achieved without optimal antiretroviral therapy. The slightly higher response rate in the intermediate-dose group was offset by its significantly poorer tolerance. These findings justify the use of lower, well-tolerated IFN doses for treatment of KS with currently used antiretroviral regimens.     

Muscle insulin-like growth factor status, body composition, and functional capacity in hemodialysis patients.

BACKGROUND: Hemodialysis (HD) patients typically have reduced muscle mass and diminished functional capacity. The role of the muscle insulin-like growth factors (IGFs), a principal anabolic system that is involved in protein synthesis and that has downregulation that is implicated in muscle loss in animal models of uremia, has previously not been assessed in vivo in HD patients. METHODS: Seventeen HD patients were compared cross-sectionally with 17 age-, sex-, and body mass index-matched healthy controls. Body composition was assessed by dual energy x-ray absorptiometry and bioelectrical impedance spectrometry; functional capacity by hand grip strength, quadriceps strength, and 30-second sit-to-stand test; systemic inflammation by tumor necrosis factor-alpha (TNF-alpha) and TNF receptor 1 (TNFR1); serum and muscle IGF-I and IGFBP-3 by radioimmunoassay; and fragmentation of serum IGFBP-3 by Western immunoblotting. RESULTS: Appendicular lean mass was significantly decreased in HD patients compared with controls (17.6 +/- 0.9 versus 21.5 +/- 1.5 kg, P < .05), as were all measures of functional capacity (P < .01 to .001), and highly significant positive correlations between appendicular lean mass and functional capacity were evident (appendicular lean mass and hand-grip strength, quadriceps strength, 30-second sit-to-stand test, all P < .001). TNF-alpha and TNFR1 were elevated in patients (P < .001). Although serum IGF-I and IGFBP-3 levels did not differ between the groups (P = .295 and .379 respectively), fragmented IGFBP-3 levels were increased (53.1 +/- 16.0 versus 29.81 +/- 15.3%, P < .005). In contrast, muscle IGF-I was substantially diminished in the patient group (n = 7) relative to control (n = 5) levels (0.84 +/- 0.06 versus 2.78 +/- 1.80 pg/microg, P < .05). CONCLUSIONS: We provide evidence of reduced IGF-I in HD patients' skeletal muscle that may be a causal factor in the muscle wasting characteristic of this population. Future research should determine the exact consequences and causes of alterations to the muscle IGF system in HD patients.     

Office-based laryngeal laser surgery: a review of 443 cases using three wavelengths.

BACKGROUND: Unsedated office-based laser surgery (UOLS) of the larynx and trachea has significantly improved the treatment options for patients with laryngotracheal pathology including recurrent respiratory papillomas, granulomas, leukoplakia, and polypoid degeneration. UOLS delivered by flexible endoscopes has dramatically impacted office-based surgery by reducing the time, costs, and morbidity of surgery. OBJECTIVES: To review our experience with 443 laryngotracheal cases treated by UOLS. METHODS: The laser logbooks at the Center for Voice and Swallowing Disorders were reviewed for UOLS, and the medical and laryngological histories were detailed, as were the treatment modalities, frequencies, and complications. RESULTS: Of the 443 cases, 406 were performed with the pulsed-dye laser, 10 with the carbon-dioxide laser, and 27 with the thulium: yttrium-aluminum-garnet laser. There were no significant complications in this series. A review of indications and wavelength selection criteria is presented. CONCLUSION: Unsedated, office-based, upper aerodigestive tract laser surgery appears to be a safe and effective treatment option for many patients with laryngotracheal pathology.