Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.     

Amifostine (WR-2721) shortens the engraftment period of 4-hydroperoxycyclophosphamide-purged bone marrow in breast cancer patients receiving high-dose chemotherapy with autologous bone marrow support

4-Hydroperoxycyclophosphamide (4-HC), a commonly used marrow-purging agent, is active against many tumors, but is also toxic to normal marrow progenitors. Amifostine (WR-2721) is a sulfhydryl compound with chemoprotectant activity. Preclinical studies using suspensions of bone marrow and breast cancer cells demonstrated that ex vivo treatment with amifostine followed by 4-HC resulted in protection of marrow progenitors, with no compromise in the antitumor effect of 4-HC. This fact stimulated the development of a clinical trial. Bone marrow was harvested from 15 poor-prognosis breast cancer patients and randomly assigned to ex vivo treatment with amifostine followed by 4-HC (amifostine + 4-HC), or treatment with 4-HC alone. High-dose chemotherapy was then administered followed by infusion of the purged autologous bone marrow support (ABMS). Leukocyte engraftment, defined as a white blood cell count > or = 1 x 10(9)/L, was achieved in an average of 26 days for patients whose marrow was purged with amifostine + 4-HC versus 36 days for patients whose marrow was purged with 4-HC alone (P = .032). The average number of platelet transfusions (12 v 29; P = .017) and days of antibiotic therapy (28 v 40; P = .012) were significantly less for patients whose marrow was exposed to amifostine + 4-HC, compared with 4-HC alone. Unpurged backup marrow fractions were infused into three patients whose marrow was purged with 4-HC alone, because of inadequate marrow recovery. None of the patients who received amifostine + 4-HC-purged marrow required a backup marrow fraction. Complete remissions were achieved in 83% of patients with measurable disease, with no difference between the two cohorts. Forty-three percent of patients remained alive and progression-free at a mean of 13 months posttransplant. There was no significant difference in the rate or pattern of relapse for patients whose marrow was purged with amifostine + 4-HC compared with those whose marrow was purged with 4-HC alone. Ex vivo treatment of marrow with amifostine significantly shortens the time to marrow recovery, thereby reducing the risk of myelosuppressive complications in breast cancer patients receiving high-dose chemotherapy and 4-HC-purged ABMS. Since supportive care requirements are also significantly decreased, amifostine may reduce the cost of such therapy.     

Fetal experience with milk or an artificial nipple alters appetitive and aversive responses to perioral cutaneous stimuli

Fetal rats exhibit oral grasping of an artificial nipple. The authors examined interactive effects of sensory stimuli normally encountered in the suckling environment on subsequent responses to the nipple. Embryonic Day 20 rat fetuses received an infusion of milk, lemon, or saline through a hollow artificial nipple or an intraoral cannula (producing no nipple stimulation). One minute after sensory pretreatment, behavioral responses of fetuses to an artificial nipple were recorded on videotape for frame-by-frame analysis. Preexposure to the artificial nipple decreased the number of oral grasps and facial wipes directed toward the artificial nipple but increased the duration of grasp responses. Milk uniformly reduced fetal responsiveness to the nipple. Furthermore, the artificial nipple enhanced fetal responses to perioral cutaneous stimulation, whereas milk suppressed perioral responsiveness. These data suggest that the perinatal rat's 1st experience with milk or the nipple can alter subsequent responses to suckling stimuli.     

The influence of breast size on late radiation effects and association with radiotherapy dose inhomogeneity

A prospective assessment of late changes in breast appearance in 559 patients after tumour excision and radiotherapy for early breast cancer noted a strong association with breast size. Only 3/48 (6%) patients with small breasts developed moderate or severe late changes compared with 94/423 (22%) with medium sized breasts and 34/88 (39%) patients with large breasts (p < 0.001). One possibility is that greater radiation changes are related to greater dose inhomogeneity in women with large breasts. To explore this hypothesis, radiation dose distributions were assessed in a separate group of 37 women in whom three-level transverse computer tomographic images of the breast in the treatment position were available. A significant correlation was found between breast size and dose inhomogeneity which may account for the marked changes in breast appearance reported in women with large breasts.     

The granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22: sequence analysis and expression in a heterologous host

BACKGROUND: The granaticins are members of the benzoisochromanequinone class of aromatic polyketides, the best known member of which is actinorhodin made by Streptomyces coelicolor A3(2). Genetic analysis of this class of compounds has played a major role in the development of hypotheses about the way in which aromatic polyketide synthases (PKSs) control product structure. Although the granaticin nascent polyketide is identical to that of actinorhodin, post-PKS steps involve different pyran-ring stereochemistry and glycosylation. Comparison of the complete gene clusters for the two metabolites is therefore of great interest. RESULTS: The entire granaticin gene cluster (the gra cluster) from Streptomyces violaceoruber T-22 was cloned on either of two overlapping cosmids and expressed in the heterologous host, Streptomyces coelicolor A3(2), strain CH999. Chemical analysis of the recombinant strains demonstrated production of granaticin, granaticin B, dihydrogranaticin and dihydrogranaticin B, which are the four known metabolites of S. violaceoruber. Analysis of the complete 39,250 base pair sequence of the insert of one of the cosmids, pOJ466-22-24, revealed 37 complete open reading frames (ORFs), 15 of which resemble ORFs from the act (actinorhodin) gene cluster of S. coelicolor A3(2). Among the rest, nine resemble ORFs potentially involved in deoxysugar metabolism from Streptomyces spp. and other bacteria, and six resemble regulatory ORFs. CONCLUSIONS: On the basis of these resemblances, putative functional assignments of the products of most of the newly discovered ORFs were made, including those of genes involved in the PKS and tailoring steps in the biosynthesis of the granaticin aglycone, steps in the deoxy sugar pathway, and putative regulatory and export functions.     

Reflex sympathetic dystrophy: is the immune system involved?

OBJECTIVE: Evaluation of immune system function in patients with reflex sympathetic dystrophy (RSD). DESIGN: Survey on blood samples obtained from RSD patients and from a randomly selected control group. The lymphocyte populations (T, B, NK cells), and the activated T cells (CD25, and HLA-Dr-positive CD4 and CD8 cells) were analyzed by flow cytometry with dual-color direct immunofluorescence after whole-blood lysis. Clinical chemistry parameters were analyzed in additional serum samples. SETTING: Tertiary care center (outpatient rehabilitation clinic). SUBJECTS: Thirteen patients (nine women) with RSD and a control group of 21 healthy individuals. MAIN OUTCOME MEASURES: The results of the flow cytometry analysis of RSD patients were related to those of the control subjects. Means were analyzed, and confidence intervals for differences of the means were calculated. The means of the clinical chemical analysis were related to local reference values. RESULTS: The flow cytometry analysis did not differ between RSD patients and healthy controls. Although in some patients an individual parameter of clinical chemical analysis differed from its reference value, all of the mean values were within reference limits. Stratification on medications with immunomodulatory effects and on probability of a definite diagnosis of RSD had no influence on the results. CONCLUSION: No association between immunologic indices and RSD was found. This finding is relevant, because recent theories stress that it is not the sympathetic nervous system but a local inflammatory reaction that is fundamental in the pathogenesis of RSD. The results of this study do not support this theory.