Chromatographic assay and peptide substrate characterization of partially purified farnesyl- and geranylgeranyltransferases from rat brain cytosol

The dose-response relationships for DNA fragmentation (assessed by pulsed-field gel electrophoresis, PFGE) and for viability (evaluated by measuring the reduction of MTT dye which can be accomplished by viable cells only) were investigated in order to discriminate between genotoxicity and cytotoxicity in the pathogenesis of DNA double-strand breaks (DSB). Cultured human lung epithelial cells (A549) were treated with the DNA-intrastrand crosslinker cisplatin, the DNA-interstrand crosslinker melphalan and the topoisomerase II inhibitor etoposide. The cytotoxic mode of DSB induction was investigated by using the mitochondrial respiratory chain toxin potassium cyanide (KCN) and the detergent Triton X-100. gamma-Irradiation induced a linear dose response for DSB which were efficiently repaired and did not cause reduction in cell survival over a period of 72 h. With etoposide and melphalan a significant increase in DSB was seen 8 h after treatment initiation with concentrations that did not affect cell survival, implicating genotoxicity as the causal event. In contrast, induction of DSB by KCN and Triton X-100, and also by cisplatin, was seen only after cell viability was reduced to less than about 60%, indicating that DSB were the consequence of extragenomic damage. This mechanistic distinction of the two classes was supported by DNA fragment length analysis. In line with a genotoxic mechanism and absence of additional cytotoxic effects, the DNA fragments generated by gamma-irradiation as well as by etoposide and melphalan displayed a distribution between 1 and 4 Mbp with a peak around 2 Mbp. In contrast, DNA fragments induced by Triton X-100 and KCN peaked below 0.5 Mbp, implicating activation of DNA-degrading enzymes. This type of investigation is suggested for the study of chemicals for potential DNA interstrand crosslinking, an important promutagenic type of DNA damage. To avoid false positive results in genetic toxicity testing it is suggested that all assays include a dose-response relationship for both genotoxicity and viability.     

A survey of current clinical practice of permanent prostate brachytherapy in the United States

PURPOSE: To help establish standards of care for transperineal interstitial permanent prostate brachytherapy (TIPPB) by obtaining data regarding current clinical practice among the most experienced TIPPB brachytherapists in the United States. METHODS AND MATERIALS: The 70 brachytherapists who performed the greatest number of TIPPB cases in 1995 in the U.S. were surveyed. Each received a comprehensive four page questionnaire that included sections on training and experience, patient and isotope selection criteria, manpower, technique, and follow-up. Thirty-five (50%) surveys were ultimately returned after three mailings and follow-up phone calls. The cumulative experience of the 35 respondents represented approximately 45% of the total TIPPB volume in the U.S. for 1995. Respondents included 29 from the private sector and six from academic programs. RESULTS: The median physician experience with TIPPB was reported as 4.9 years. Each performed an average of 73 TIPPB procedures in 1995 (range 40-300). This represented an increase in volume for most (74%) of the respondents. Sixty-three percent of the respondents attended a formal training course, 54% had TIPPB-specific residency training, and 31% had been proctored (16 had received two or more types of training experience). The most commonly reported selection criteria for implant alone was on Gleason score < or = 7, PSA < 15, < or = Stage T2a, and gland size < or = 60 cc, although no clear consensus was found. Fifty-four percent considered a history of TURP to be a relative contraindication, while 34% considered TURP to have no impact on patient selection. Eighty-six percent of respondents combine brachytherapy with external beam radiation in an average of 32% of their patients. Boosts were given with both 125I prescribed to 120 Gy (75%) or 103Pd to 90 Gy (50%). Sixty percent reported using a Mick applicator, 46% prefer using preloaded needles, and (11%) use both techniques. Real-time imaging was usually performed with ultrasound (94%); most included fluoroscopy (60%). Definitions of PSA control varied widely. CONCLUSIONS: TIPPB clinical practice in the U.S. demonstrates similarities in technique, but differences in patient selection and definitions of biochemical control. It is, therefore, incumbent on those beginning TIPPB programs to carefully review the specific practice details of those institutions with a broad experience.     

Promiscuous binding of synthetic copolymer 1 to purified HLA-DR molecules

Copolymer 1 (Cop 1) is a random synthetic amino acid copolymer of L-alanine, L-glutamic acid, L-lysine, and L-tyrosine, effective both in suppression of experimental allergic encephalomyelitis and in the treatment of relapsing forms of multiple sclerosis. Cop 1 binds promiscuously and very efficiently to living APCs of various HLA haplotypes. In the present study, a substantial part of the whole mixture of random polypeptides that compose Cop 1 was shown to bind to purified human HLA-DR1, DR2, and DR4 with high affinity in a temperature- and time (and, in the case of DR4, pH)-dependent manner, and was competitively inhibited by DR-restricted peptides, but not by peptide derivatives that bind with low affinity. Bacterial superantigens inhibited Cop 1 binding only at very high concentrations. The formation of the Cop 1-DR1 complex was also shown by SDS-PAGE. These findings represent the first direct evidence for interactions of Cop 1 with purified DR molecules, and suggest that its effectiveness in experimental allergic encephalomyelitis and multiple sclerosis may be directly related to its binding in the groove of HLA-DR proteins.