A new and efficient DNA enzyme for the sequence-specific cleavage of RNA.

A new DNA enzyme, the "Bipartite DNAzyme", suitable for the sequence-specific cleavage of RNA, was obtained from a random DNA library by in vitro selection. Only a single family of catalytic molecules emerged from the selection, and a 22 nucleotide consensus sequence common to all clones defined a putative catalytic core. The most abundant clone self-cleaved at a single internal ribonucleotide phosphodiester with a relatively fast k(obs) value of 1.7 min(-1), in 10 mM MgCl(2) at 23 degrees C. This DNAzyme ("Bipartite I") required divalent cations, with magnesium and manganese most optimally supporting cleavage. A reselection from a mutagenized DNAzyme pool for the ability to cleave at extended RNA substrates yielded an unchanged catalytic core sequence. From this re-selection a DNAzyme ("Bipartite II") capable of sequence-specifically cleaving extended stretches of RNA was derived. A rate versus pH analysis of the Bipartite II DNAzyme revealed a two-phase profile, similar to that reported for the hepatitis delta virus (HDV) ribozyme, suggesting that the Bipartite II DNAzyme and the HDV ribozyme may share similar catalytic strategies. Multiple-turnover kinetics, measured in 30 mM MgCl(2), at 37 degrees C, with an HIV-1-derived RNA substrate, yielded a k(cat) value of approximately 1.4 min(-1) and a K(M) value of approximately 230 nM, which were of the same order as k(cat) and K(M )values measured for other ribozymes and DNAzymes in general use for RNA cleavage. The Bipartite DNAzyme therefore represents a new and potentially useful reagent, both for the processing of RNA transcripts in vitro and for mRNA ablation procedures in vivo.

Prostate-specific membrane antigen is a hydrolase with substrate and pharmacologic characteristics of a neuropeptidase.

This report demonstrates that the investigational prostatic carcinoma marker known as the prostate-specific membrane antigen (PSM) possesses hydrolytic activity with the substrate and pharmacologic properties of the N-acetylated alpha-linked acidic dipeptidase (NAALADase). NAALADase is a membrane hydrolase that has been characterized in the mammalian nervous system on the basis of its catabolism of the neuropeptide N-acetylaspartylglutamate (NAAG) to yield glutamate and N-acetylaspartate and that has been hypothesized to influence glutamatergic signaling processes. The immunoscreening of a rat brain cDNA expression library with anti-NAALADase antisera identified a 1428-base partial cDNA that shares 86% sequence identity with 1428 bases of the human PSM cDNA [Israeli, R. S., Powell, C. T., Fair, W. R. & Heston, W.D.W. (1993) Cancer Res. 53, 227-230]. A cDNA containing the entire PSM open reading frame was subsequently isolated by reverse transcription-PCR from the PSM-positive prostate carcinoma cell line LNCaP. Transient transfection of this cDNA into two NAALADase-negative cell lines conferred NAAG-hydrolyzing activity that was inhibited by the NAALADase inhibitors quisqualic acid and beta-NAAG. Thus we demonstrate a PSM-encoded function and identify a NAALADase-encoding cDNA. Northern analyses identify at least six transcripts that are variably expressed in NAALADase-positive but not in NAALADase-negative rat tissues and human cell lines; therefore, PSM and/or related molecular species appear to account for NAAG hydrolysis in the nervous system. These results also raise questions about the role of PSM in both normal and pathologic prostate epithelial-cell function.

Efficacy and safety of fluconazole prophylaxis for fungal infections after marrow transplantation--a prospective, randomized, double-blind study.

A randomized, double-blind, placebo-controlled trial assessed the efficacy and toxicity of 400 mg/day fluconazole in preventing fungal infections during the first 75 days after marrow transplantation. During prophylaxis, systemic fungal infections occurred in 10 (7%) of 152 fluconazole-treated patients compared with 26 (18%) of 148 placebo-treated patients (P = .004). There were no Candida albicans infections in fluconazole recipients compared with 18 in placebo recipients (P < .001) and no significant increase in Candida infections other than C. albicans. Fluconazole also significantly reduced the incidence of superficial fungal infections (P < .001), fungal colonization (P = .037), and empiric amphotericin B use (P = .005). The probability of survival was improved in fluconazole recipients, in whom 31 deaths occurred up to day 110 after transplantation compared with 52 deaths in placebo recipients (P = .004). No clinically significant toxicity was detected with fluconazole use. Prophylactic fluconazole was safe and significantly reduced systemic fungal infections with other benefits, including improved survival at day 110 after marrow transplantation.

Focal myositis in a patient with bilateral painful nodules.

A 30-year-old African-American man presented with painful lower extremities and bilaterally symmetrical dermal nodules. Examination of a biopsy specimen of one of the nodules showed focal myositis overlying a deeper defect. Subsequent excisions produced multifocal eruptive lipomas. The histopathologic findings of focal myositis are reviewed along with this previously unreported association.

Cutaneous manifestation of atrial myxoma.

A left atrial myxoma may present in a manner that diverts attention from its cardiac origin. This case report documents a cutaneous clinical presentation that initially was thought to represent a peripheral vasculitis or connective tissue disease. Biopsy specimens revealed a peripheral arterial myxomatous embolus in the skin, and echocardiogram results were diagnostic for atrial myxoma. Embolic manifestations may occur in up to 40% of cases. We hope that description of this patient will alert others to suspect this tumor in unexplained or peculiar forms of vasculitis and to consider an echocardiogram as part of the diagnostic workup.

Phaeohyphomycotic cutaneous disease caused by Pleurophoma in a cardiac transplant patient.

A 56-y-old female cardiac transplant patient treated with cyclosporine and prednisone noted the onset of three nontender red nodules on the legs and arms after gardening. Biopsy of all lesions revealed inflammatory cells and hyphal elements in the dermis. The dematiaceous fungus cultured from biopsy tissue was a pycnidial-forming organism of the genus Pleurophoma. The lesions responded to the topical application of miconazole.

Selection factors in clinical trials: results from the Community Clinical Oncology Program Physician's Patient Log.

Between August 1984 and November 1985, data concerning 44,156 newly diagnosed cancer patients were entered in the Community Clinical Oncology Program Physician's Patient Log. Forty percent (17,773 patients) had a National Cancer Institute-approved protocol available for site and stage of disease. Fifty-six percent of patients with a protocol available were clinically eligible and 19% were entered on protocol. Thus, one-third of clinically eligible patients were entered in the study. Age was a major factor influencing eligibility and entry on study. Eighty percent of patients less than 25 years of age with a protocol available were clinically eligible for clinical trials participation, and 58% were placed on protocol. Patients greater than or equal to 65 years of age with a protocol available were less likely to be clinically eligible (48%) or placed on study (14%). A physician's preference for an alternate treatment and patient refusal for protocol treatment were also important reasons that clinically eligible patients were not registered on protocol. Selection for clinical trials participation affects the characteristics of patients under study and may have an impact on the ability to generalize the results of clinical trials.

The prevalence of cancer. Estimates based on the Connecticut Tumor Registry.

Cancer incidence and mortality do not fully reflect the effect of cancer. To estimate the number of persons alive who have a history of cancer, we derived prevalence rates based on data from the Connecticut Tumor Registry. We did not attempt to distinguish between people who had been cured of cancer and those who still had the disease. In 1982 the age-adjusted prevalence rates of cancer among males and females were 1,789 and 2,222, respectively, per 100,000. Age-specific prevalence rates were highest among the elderly; 12 percent of men and 11 percent of women over 70 had previously been given a diagnosis of cancer. Breast cancer in females and prostate cancer in males were the two most prevalent malignant diseases. We estimate that about 5 million persons alive in the United States today have at one time received a diagnosis of cancer.