Distinct mutational signatures characterize concurrent loss of polymerase proofreading and mismatch repair.

Fidelity of DNA replication is maintained using polymerase proofreading and the mismatch repair pathway. Tumors with loss of function of either mechanism have elevated mutation rates with characteristic mutational signatures. Here we report that tumors with concurrent loss of both polymerase proofreading and mismatch repair function have mutational patterns that are not a simple sum of the signatures of the individual alterations, but correspond to distinct, previously unexplained signatures: COSMIC database signatures 14 and 20. We then demonstrate that in all five cases in which the chronological order of events could be determined, polymerase epsilon proofreading alterations precede the defect in mismatch repair. Overall, we illustrate that multiple distinct mutational signatures can result from different combinations of a smaller number of mutational processes (of either damage or repair), which can influence the interpretation and discovery of mutational signatures.

Ventilation-perfusion scintigraphic evaluation of pulmonary clot burden after percutaneous thrombolysis of clotted hemodialysis access grafts.

The objective of this study is to determine, by using rigorous methods, if pulmonary perfusion defects were detectable by ventilation-perfusion scintigraphy after percutaneous thrombolysis of clotted hemodialysis access grafts. Thirteen patients were studied. Four patients underwent pharmacomechanical thrombolysis with urokinase and the remainder had mechanical thrombolysis alone. Pre- and postthrombolysis scintigraphic studies were performed on all patients. Perfusion defects were described as vascular (well-defined borders confined to segmental boundaries) or nonvascular. Vascular defects were graded by severity (0 to 3) and area (0 to 3) for each involved segment. Nonvascular defects were graded by severity (0 to 1) and area (0 to 1). Two experienced readers evaluated the scans blinded to each other's results and all other clinical data, including thrombolysis outcomes. Twelve patients did not have any significant worsening of their perfusion defect scores postthrombolysis. In only one patient did a study show a new nonvascular perfusion defect with a matching ventilation abnormality. The defect was believed to be caused by mucus plugging. The patient had no evidence of pulmonary embolism. Our study suggests emboli that resulted from the pharmacomechanical or mechanical thrombolysis procedure were either small, underwent lysis before impacting the lung, or were below the limit of detection of ventilation-perfusion scintigraphy.

Influence of polymolecular events on inactivation behavior of xylose isomerase from Thermotoga neapolitana 5068.

The inactivation behavior of the xylose isomerase from Thermotoga neapolitana (TN5068 XI) was examined for both the soluble and immobilized enzyme. Polymolecular events were involved in the deactivation of the soluble enzyme. Inactivation was biphasic at 95 degrees C, pH 7.0 and 7.9, the second phase was concentration-dependent. The enzyme was most stable at low enzyme concentrations, however, the second phase of inactivation was 3- to 30-fold slower than the initial phase. Both phases of inactivation were more rapid at pH 7.9, relative to 7.0. Differential scanning calorimetry of the TN5068 XI revealed two distinct thermal transitions at 99 degrees and 109 degrees C. The relative magnitude of the second transition was dramatically reduced at pH 7.9 relative to pH 7.0. Approximately 24% and 11% activity were recoverable after the first transition at pH 7.0 and 7.9, respectively. When the TN5068 XI was immobilized by covalent attachment to glass beads, inactivation was monophasic with a rate corresponding to the initial phase of inactivation for the soluble enzyme. The immobilized enzyme inactivation rate corresponded closely to the rate of ammonia release, presumably from deamidation of labile asparagine and/or glutamine residues. A second, slower inactivation phase suggests the presence of an unfolding intermediate, which was not observed for the immobilized enzyme. The concentration dependence of the second phase of inactivation suggests that polymolecular events were involved. Formation of a reversible polymolecular aggregate capable of protecting the soluble enzyme from irreversible deactivation appears to be responsible for the second phase of inactivation seen for the soluble enzyme. Whether this characteristic is common to other hyperthermophilic enzymes remains to be seen.

Thermotoga neapolitana homotetrameric xylose isomerase is expressed as a catalytically active and thermostable dimer in Escherichia coli.

The xylA gene from Thermotoga neapolitana 5068 was expressed in Escherichia coli. Gel filtration chromatography showed that the recombinant enzyme was both a homodimer and a homotetramer, with the dimer being the more abundant form. The purified native enzyme, however, has been shown to be exclusively tetrameric. The two enzyme forms had comparable stabilities when they were thermoinactivated at 95 degrees C. Differential scanning calorimetry revealed thermal transitions at 99 and 109.5 degrees C for both forms, with an additional shoulder at 91 degrees C for the tetramer. These results suggest that the association of the subunits into the tetrameric form may have little impact on the stability and biocatalytic properties of the enzyme.

A model for pharmacological research-treatment of cocaine dependence.

Major problems for research on pharmacological treatments for cocaine dependence are lack of comparability of results from different treatment research programs and poor validity and/or reliability of results. Double-blind, placebo-controlled, random assignment, experimental designs, using standard intake and assessment procedures help to reduce these problems. Cessation or reduction of drug use and/or craving, retention in treatment, and medical and psychosocial improvement are some of the outcome variables collected in treatment research programs. A model to be followed across different outpatient clinical trials for pharmacological treatment of cocaine dependence is presented here. This model represents an effort to standardize data collection to make results more valid and comparable.

Effects of combined fluoxetine and counseling in the outpatient treatment of cocaine abusers.

Three methods of analysis were used to determine the effects of the combination of counseling with fluoxetine (20, 40, or 60 mg) and "active" placebo (diphenhydramine, 12.5 mg) randomly assigned. Forty-five cocaine-only dependent subjects were treated as outpatients with "interpersonal" counseling, medication, and drug use monitoring three times per week for up to 12 weeks. Treatment effects were analyzed: first, by comparing the three original assignments and placebo; second, by comparing the placebo group to fluoxetine subjects with detectable fluoxetine/norfluoxetine blood levels and those with no detectable medication blood level; third, by examining relapse prevention versus use cessation through stratifying the subjects into four groups according to fluoxetine or placebo assignment and initial urine cocaine positivity or negativity. All three analyses showed improvement on some measures over time regardless of group assignment. The 60-mg fluoxetine group showed least effectiveness, the group with detectable blood levels had less cravings, and the fluoxetine subjects who were abstinent at the start of treatment were somewhat less likely to avoid relapse than those on placebo.

xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana.

The xylA gene coding for xylose isomerase from the hyperthermophile Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 444 residues with a calculated molecular weight of 50,892. The native enzyme was a homotetramer with a molecular weight of 200,000. This xylose isomerase was a member of the family II enzymes (these differ from family I isomerases by the presence of approximately 50 additional residues at the amino terminus). The enzyme was extremely thermostable, with optimal activity above 95 degrees C. The xylose isomerase showed maximum activity at pH 7.1, but it had high relative activity over a broad pH range. The catalytic efficiency (kcat/Km) of the enzyme was essentially constant between 60 and 90 degrees C, and the catalytic efficiency decreased between 90 and 98 degrees C primarily because of a large increase in Km. The T. neapolitana xylose isomerase had a higher turnover number and a lower Km for glucose than other family II xylose isomerases. Comparisons with other xylose isomerases showed that the catalytic and cation binding regions were well conserved. Comparison of different xylose isomerase sequences showed that numbers of asparagine and glutamine residues decreased with increasing enzyme thermostability, presumably as a thermophilic strategy for diminishing the potential for chemical denaturation through deamidation at elevated temperatures.

Characterization of the autoreactive T cell repertoire in cyclosporin-induced syngeneic graft-versus-host disease. A highly conserved repertoire mediates autoaggression.

Syngeneic graft-vs-host disease (SGVHD) is a MHC class II-restricted T cell-mediated autoimmune syndrome that occurs following syngeneic bone marrow transplantation and the administration of cyclosporin (CsA). The present studies evaluated the V beta repertoire of T lymphocytes that mediate SGVHD. To facilitate analysis, SGVHD effector cells were adoptively transferred into thymectomized syngeneic recipients reconstituted with T cell-depleted bone marrow to provide an environment that allows for the selective clonal expansion of autoreactive T cells. Analysis of target tissues and PBL by reverse transcriptase PCR using oligonucleotide V beta-specific primers revealed a predominance of V beta 8.5+ T cells and a minor population expressing V beta 10. The majority of infiltrating lymphocytes in target tissues was confirmed to be V beta 8.5+ by in situ hybridization and by immunoperoxidase staining. A small population of V beta 10+ cells could also be detected. Furthermore, SGVHD effector T splenocytes depleted of lymphocytes expressing either the TCR-alpha beta or the V beta 8.5 determinant could not adoptively transfer SGVHD. Depletion of T cells expressing the V beta 10 determinant delayed the onset of this autoaggression syndrome. Subset analysis of the autoreactive T cell compartment revealed that the V beta 8.5 determinant was expressed on both CD4+ and CD8+ lymphocytes whereas the V beta 10 determinant was principally expressed on a minor population of CD4+ autoreactive T cells. These data were confirmed by limiting dilution analysis. Additional studies examining the effect of CsA on thymic differentiation revealed that although V beta 8.5 is not normally clonally deleted, there was a pronounced shift in the expression of this determinant between CD4 and CD8 single positive thymocytes, suggesting that CsA may inhibit normal positive selection processes for MHC class I and class II reactive T cells.

Psychological reactions and retention by cocaine addicts during treatment according to HIV-serostatus: a matched-control study.

We compared retention in treatment and psychological reactions during drug abuse treatment by 22 HIV-antibody positive, physically asymptomatic cocaine addicts to 22 matched HIV-seronegative cocaine addicts. All subjects participated in an outpatient clinical research project. There were no significant differences between groups in sociodemographics and psychiatric symptom scores on entrance or cocaine use except for route of administration (chi 2 = 11.59, df = 2, p less than .005). There were no significant differences among groups regarding being informed of serostatus and beginning treatment. There was a trend (p = .079) for more seropositives to complete treatment. Using end-point analysis to compare 11 seropositive subjects who completed a minimum of 2 weeks of treatment to a matched seronegative comparison groups, there were no significant differences in mood states except for "anger/hostility" (interaction of group x time; F = 2.24, df = 13/260, p less than .05). Informing drug abusers in treatment regarding positive HIV-serostatus was not associated with a lower treatment-retention rate or adverse psychological reactions when counseling regarding HIV issues was integrated with drug abuse treatment.

Comparison of amantadine and desipramine combined with psychotherapy for treatment of cocaine dependence.

We conducted a single-blind, random assignment, placebo-controlled, 12-week comparison of desipramine hydrochloride and amantadine hydrochloride as adjunctive treatments to counseling for cocaine dependence. Subjects were 54 outpatients who met DSM III-R criteria for active cocaine dependence and who completed a minimum of 2 weeks of treatment. Subjects treated with fixed doses of 200 mg/day desipramine (N = 17), 400 mg/day amantadine-placebo (N = 16), and placebo (N = 21) did not differ for lifetime cocaine use, lifetime histories of psychopathology, admission scores on psychometric assessments, and sociodemographics. All treatment groups demonstrated dramatic and persistent decreases in cocaine use, craving for cocaine, and psychiatric symptoms consequent to treatment. Although there was a trend for more dropouts by subjects taking desipramine, there were no significant differences among treatment groups regarding retention in treatment, craving for cocaine, and decreased cocaine use confirmed by urine toxicology. There was a trend for subjects treated with desipramine to maintain longer periods of cocaine abstinence. Mean plasma concentration of desipramine in a subsample of our subjects was less than that recommended for treatment of depression, thus the dosage of desipramine may have been subtherapeutic.