Characterizing replication intermediates in the amplified CHO dihydrofolate reductase domain by two novel gel electrophoretic techniques

Using neutral/neutral and neutral/alkaline two-dimensional (2-D) gel techniques, we previously obtained evidence that initiation can occur at any of a large number of sites distributed throughout a broad initiation zone in the dihydrofolate reductase (DHFR) domain of Chinese hamster ovary (CHO) cells. However, other techniques have suggested a much more circumscribed mode of initiation in this locus. This dichotomy has raised the issue whether the patterns of replicating DNA on 2-D gels have been misinterpreted and, in some cases, may represent such noncanonical replication intermediates as broken bubbles or microbubbles. In an accompanying study (R. F. Kalejta and J. L. Hamlin, Mol. Cell. Biol. 16:4915-4922, 1996), we have shown that broken bubbles migrate to unique positions in three different gel systems and therefore are not likely to be confused with classic replication intermediates. Here, we have applied a broken bubble assay developed from that study to an analysis of the amplified DHFR locus in CHO cells. This assay gives information about the number and positions of initiation sites within a fragment. In addition, we have analyzed the DHFR locus by a novel stop-and-go-alkaline gel technique that measures the size of nascent strands at all positions along each arc in a neutral/neutral 2-D gel. Results of these analyses support the view that the 2-D gel patterns previously assigned to classic, intact replication bubbles and single-forked structures indeed correspond to these entities. Furthermore, potential nascent-strand start sites appear to be distributed at very frequent intervals along the template in the intergenic region in the DHFR domain.     

The prognostic value of NCAM, p53 and cyclin D1 in resected non-small cell lung cancer

Expression of the neural cell adhesion molecule, NCAM, in frozen sections has been associated with decreased postoperative survival in non-small cell lung carcinoma. Of the various isoforms of NCAM described, the highly sialylated isoform plays a role in the migration of embryonal cells from the neural crest and is expressed by highly malignant tumours such as small cell lung carcinomas. We investigated the clinical significance of expression of this NCAM isoform as a prognostic factor in a series of 96 non-small cell lung carcinomas resected with curative intent. We also evaluated the effect of microwave pre-treatment of formalin-fixed, paraffin-embedded sections on the NCAM immunostaining and related the outcome to the postoperative clinical course of disease. In addition, in an attempt to extend our search for possible molecular markers of unfavourable prognosis in lung cancer, we evaluated increased immunostaining for p53 and cyclin D1 in the same series. We did not find a significant relation between expression of NCAM or its highly sialylated isoform and the length of postoperative survival. The numbers of positive cases (9 and 14, respectively) were relatively low. Increased p53 and cyclin D1 immunostaining (50 and 55 of the 96 tumours) failed to show a significant relation with postoperative survival. In our material, tumour stage was the only significant prognostic factor.     

Kinetic analysis of the coding properties of O6-methylguanine in DNA: the crucial role of the conformation of the phosphodiester bond

Production by N-nitroso compounds of O6-alkylguanine (O6-alkylG) in DNA directs the misincorporation of thymine during DNA replication, leading to G:C to A:T transition mutations, despite the fact that DNA containing O6-alkylG:T base pairs is less stable than that containing O6-alkylG:C pairs. We have examined the kinetics of incorporation by Klenow fragment (KF) of Escherichia coli DNA polymerase I of thymine (T) and of cytosine (C) opposite O6-MeG in the template DNA strand. Both T and C were incorporated opposite O6-MeG much slower than nucleotides forming regular A:T or G:C base pairs. Using various concentrations of dTTP, dCTP, or their phosphorothioate (Sp)-dNTP alpha S analogues, or a mixture of dTTP and dCTP, the progress of incorporation of a single nucleotide in a single catalytic cycle of a preformed KF-DNA complex was measured (pre-steady-state kinetics). The results were consistent with the kinetic scheme (Kuchta, R. D., Benkovic, P., & Benkovic, S. J. (1988) Biochemistry 27, 6716-6725): (1) binding of dNTP to polymerase-DNA; (2) conformational change in polymerase; (3) formation of phosphodiester between the dNTP and the 3'-OH of the primer; (4) conformational change of polymerase; (5) release of pyrophosphate. The results were analyzed mathematically to identify the steps at which the rate constants differ significantly between the incorporation of T and C. The only significant difference was the 5-fold difference in the rates of formation of the phosphodiester bond (for dTTP, kforward = 3.9 s-1 and kback = 1.9 s-1; for dCTP, kforward = 0.7 s-1 and kback = 0.9 s-1). These pre-steady-state progress curves were biphasic with a rapid initial burst followed by an apparently steady-state rise. Deconvolution of these curves gave direct evidence for the importance of the conformational change after polymerization by showing that the curves represented the sum of the rapid accumulation of the product of step 3 followed by the slow conversion of that to the product of step 5 (because of the rapidity of the release of pyrophosphate there was no significant accumulation of the product of step 4). The equilibrium constants for each step suggest that the greatest change in the Gibbs free energy occurs at the conformational change after polymerization and that while the formation of the phosphodiester bond to T is slightly exothermic, that to C is slightly endothermic.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cloning of cDNAs encoding xenopus neuregulin: expression in myotomal muscle during embryo development

The ontogeny of the myotome was investigated using [3H]thymidine or Brdu treatment in conjunction with 1,1', di-octadecyl-3, 3, 3', 3',-tetramethylindo-carbocyanine perchlorate (DiI) labeling and expression of specific markers. We have identified a subset of early post-mitotic cells that is present in the dorsomedial aspect of epithelial somites and is homogeneously distributed along their entire rostrocaudal extent. The post-mitotic quality of this cell subset enabled us to trace their fate in time-course experiments. Following initial somite dissociation, this epithelial post-mitotic layer bends underneath the medial portion of the nascent dermomyotome. Then, these cells progressively lose epithelial arrangement and migrate in a rostral direction where they accumulate temporarily. Subsequently, these early post-mitotic precursors extend processes that reach both rostral and caudal edges of each segment. Medial somite-derived myofibers also fill the entire mediolateral extent of the segment and reach the dorsomedial lip of the dermomyotome, thus forming the primary myotome. During this process, their large nuclei localize to a narrow stripe in the middle of the nascent myotome. Consistent with the proliferation studies, DiI labeling of the medial epithelial somite cells gave rise to a primary myotomal structure, and continuous pulsing of the DiI-injected embryos with radioactive thymidine revealed that these fibers indeed developed from post-mitotic progenitors. As these early post-mitotic cells that arise prior to somite dissociation are the first wave of progenitors that constitutes the myotome, we have termed them avian muscle pioneers. We propose that the primary myotome formed by the muscle pioneers constitutes a longitudinal scaffold that serves as a substrate for the addition of subsequent waves of myotomal cells.     

Changing clinical practice. Prospective study of the impact of continuing medical education and quality assurance programs on use of prophylaxis for venous thromboembolism

OBJECTIVE: To determine the effect of continuing medical education (CME) with and without a quality assurance component (CME+QA) on physician practices in the prevention of venous thromboembolism. METHODS: A communitywide study was performed in 15 short-stay hospitals in central Massachusetts. The study population included 3158 patients in acute-care hospitals with multiple risk factors for venous thromboembolism. Study hospitals were randomly assigned to one of two educational strategies or to a control group that received no intervention. RESULTS: The proportion of patients at high risk for venous thromboembolism who received effective methods of prophylaxis increased significantly from 29% in 1986 to 52% in 1989 (P < .001). This increase was seen in all study groups: control hospitals, 40% to 51% (P < .001); CME hospitals, 21% to 49% (P < .0001); and CME+QA hospitals, 27% to 55% (P < .0001). The increase in prophylaxis use from 1986 to 1989 was significantly greater among patients cared for in hospitals whose physicians participated in a formal CME program (an increase of 28%) than in control hospitals (an increase of 11%) (P < .001). There was no significant difference in the use of prophylaxis in hospitals whose physicians received CME+QA interventions compared with hospitals whose physicians received CME interventions alone (identical increases of 28%). CONCLUSION: A formal CME program significantly increased the frequency with which physicians prescribed prophylaxis for venous thromboembolism. We believe the key factor in our CME interventions that motivated clinicians to change their practices was the provision of hospital-specific data demonstrating a compelling need for improvement. Despite the substantial investment by hospitals in QA, traditional QA intervention appeared to provide no additional benefit. Even after extensive CME/QA interventions, prophylaxis for venous thromboembolism remained underutilized, suggesting the need to develop new approaches to changing clinical practice.     

Endoscopic management of a case of complete septate uterus with unilateral pyometra

We report a case with an unusual müllerian anomaly: complete uterine septum and pyometra in the right hemicavity and pyocolpos along with a longitudinal vaginal septum. A patient with recurrent low-grade fever and lower abdominal pain was admitted. Pyometra and pyocolpos were detected in the right uterine hemicavity and in the right hemivagina. The septa were resected, the cavities were irrigated, and the patient was treated with antibiotics. The patient conceived 8 months later. Patients with abnormal vaginal findings should be approached with caution; precise knowledge of urogenital anatomy and urogenital anomalies is necessary in the management of these patients.     

Retinal dystrophy in long chain 3-hydroxy-acyl-coA dehydrogenase deficiency

We report a case of Bellini duct carcinoma of the left kidney with invasive growth pattern. A 39-year-old man was admitted to our hospital with the chief complaint of gross hematuria. Ultrasonography showed left renal swelling but normal reniform configuration of the kidney was maintained. Computed tomography demonstrated a low density tumor infiltrating into the renal cortex and with tumor extension into the renal vein. Renal angiography revealed a hypovascular tumor. We suspected a left renal cell carcinoma with tumor extension into the left renal vein, and performed radical nephrectomy. Macroscopically, the resected kidney had a normal outer contour. The tumor with infiltrative growth pattern existed in renal medulla. Histopathologic examination revealed a papillary adenocarcinoma originated in Bellini duct (pT3bN2M0). The patient underwent systemic chemotherapy (M-VAC). This case showed invasive growth pattern, which were different from the usual renal cell carcinoma and Bellini duct carcinoma.