Mutant PCNA alleles are associated with cdc phenotypes and sensitivity to DNA damage in fission yeast

Twenty-eight site-directed mutations were introduced into the fission yeast gene (pcn1+) that encodes proliferating cell nuclear antigen (PCNA) and their in vivo effects analyzed in a strain with a null pcn1 background. Mutants defective in enhancing processivity of DNA polymerase delta have previously been identified. In this study, we assessed all of the mutants for their sensitivities to temperature, hydroxyurea, UV irradiation and methyl methanesulfonate (MMS), and specific mutants were also tested for sensitivity to gamma irradiation. One cold-sensitive allele, pcn1-3, was characterized in detail. This mutant had a recessive cold-sensitive cdc phenotype and showed sensitivity to hydroxyurea, UV, and gamma irradiation. At the non-permissive temperature pcn1-3 protein was able to form homotrimers in solution and showed increased stimulation of both synthetic activity and processivity of DNA polymerase delta relative to the wild-type Pcn1+ protein. Epistasis analyses indicated that pcn1-3 is defective in the repair pathway involving rad2+ but not defective in the classical nucleotide excision repair pathway involving rad13+. Furthermore, pcn1-3 is either synthetically or conditionally lethal in null checkpoint rad backgrounds and displays a mitotic catastrophe phenotype in these backgrounds. A model for how pcn1-3 defects may affect DNA repair and replication is presented.     

Ligation of medically refracted patent ductus arteriosus (PDA) in an extremely low body weight premature infant

Medically refracted patent ductus arteriosus (PDA) in an extremely low birth weight (ELBW) preterm (gestation 24 weeks 2 days) infant was successfully ligated under general anesthesia in the neonatal intensive care unit (NICU). Pharmacological agents are more effective to close PDA in preterm infants than in full-term infants, although within 48 h three doses of indomethacin were not sufficient to close PDA in this case. At the age of 69 h the infant developed severe symptoms including bradycardia, systemic hypotension, pulmonary hypertension, diastolic steal (reverse distal aorta flow velocity), and anuria. A PDA ligation was thus performed surgically at 72 h of age. General anesthesia and surgical stress were tolerated by this 531 g infant. Postoperatively all symptoms improved dramatically and the general conditions were stable. On the 38th day the endotracheal tube was extubated and on the 50th day nasogastric milk feeding was started. The oxygen supply was weaned on the 78th day. Growth and development until 6 months were within the normal range of very low birth weight infants. A surgical ligation as early as possible in medically refracted PDA in an ELBW infant is thus considered to be a safe and effective treatment. It prevents the development of further complications of cardiopulmonary vascular problems. Color Doppler echocardiography can reliably measure the PDA size, flow velocity, and hemodynamic changes of persistent PDA, even in tiny infants.     

Magnetic resonance images of neuronal migration anomalies

Neuronal migration anomalies are a spectrum of brain malformations caused by insults to migrating neuroblasts during the sixth week to fifth month of gestation. To study the characteristics of MRI findings in migration anomalies, MR images of 36 patients (28 children and 8 adults) with migration anomalies were evaluated. Five patients had lissencephaly, eight had pachygyria, twelve had schizencephaly, six had heterotopias of gray matter, three had hemimegalencephaly, and two had polymicrogyria. The frequency of migration anomalies was 0.51% of all cranial MRI studies and 1.21% of pediatric cranial MRI studies at our hospital. The major clinical presentations of these patients were seizure (64%), development delay (42%), motor deficits (42%) and mental retardation (25%). Twenty-five patients (69%) associated with other brain anomalies, including: other migration anomalies in 12 cases (33%), absence of the septum pellucidum in 10 cases (28%), Dandy-Walker malformation/variant in 5 cases, arachnoid cyst in 4 cases, agenesis of the corpus callosum in 3 cases, holoprosencephaly in 2 cases, mega cisterna magna in 1 case and cephalocele in 1 case. Some of them presented with multiple complicated anomalies. As MR imaging provides superb gray-white matter distinction, details of cortical anatomy and multiplanar capability, it can clearly delineate the detail morphologic changes of the brain caused by neuronal migration disorders as well as the associated anomalies.     

Formal representation of summary tables for health care statistical database management

There is an ever-increasing demand for statistical information in health-care societies, and voluminous statistics are collected every year. When one needs information on survey results, it is generally not allowed to access the original data, and the summary tables are the most important source of information which can be shared. In order to store summary tables as a well-organized database and to perform flexible retrieval, a formal study has been made on the structure of summary tables. We introduce the notion of summary table scheme and define the operations called table transformation. A statistical database management system has been implemented based on the study. In our study, a statistical database is a collection of summary tables obtained from a survey. If a survey is conducted periodically, a database consists of all the tables obtained in the past. System utilities have been implemented for handling time-oriented information.     

Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites

Some cytochrome P450 catalyzed reactions show atypical kinetics, and these kinetic processes can be grouped into five categories: activation, autoactivation, partial inhibition, substrate inhibition, and biphasic saturation curves. A two-site model in which the enzyme can bind two substrate molecules simultaneously is presented which can be used to describe all of these observed kinetic properties. Sigmoidal kinetic characteristics were observed for carbamazepine metabolism by CYP3A4 and naphthalene metabolism by CYPs 2B6, 2C8, 2C9, and 3A5 as well as dapsone metabolism by CYP2C9. Naphthalene metabolism by CYP3A4 and naproxen metabolism by CYP2C9 demonstrated nonhyperbolic enzyme kinetics suggestive of a low Km, low Vmax component for the first substrate molecule and a high Km, high Vmax component for the second substrate molecule. 7, 8-Benzoflavone activation of phenanthrene metabolism by CYP3A4 and dapsone activation of flurbiprofen and naproxen metabolism by CYP2C9 were also observed. Furthermore, partial inhibition of 7, 8-benzoflavone metabolism by phenanthrene was observed. These results demonstrate that various P450 isoforms may exhibit atypical enzyme kinetics depending on the substrate(s) employed and that these results may be explained by a model which includes simultaneous binding of two substrate molecules in the active site.     

Transformation of epithelial Madin-Darby canine kidney cells with p60(v-src) induces expression of membrane-type 1 matrix metalloproteinase and invasiveness

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is expressed both in carcinoma cells and in surrounding stromal fibroblasts. MT1-MMP localizes to the surface of tumor cells and is thought to play an important role in tumor invasion. To analyze the mechanism of MT1-MMP gene expression in epithelial tumor cells, the dog kidney epithelial cell line Madin-Darby canine kidney (MDCK) was transformed by oncogenes, including v-src, and expression of MT1-MMP was examined. Transformation of MDCK cells with v-src resulted in loss of cell-to-cell contacts and morphological change. Expression of MT1-MMP in v-src-transformed cells was identified by Northern and Western blotting. Gelatin zymography analysis showed that progelatinase A in the culture medium was processed from latent to activated form by MDCK cells transformed with v-src. The MDCK cells transformed by v-src were tumorigenic in the subcutis (ectopic) and kidney (orthotopic) of nude mice and spontaneously metastasized to the lung after orthotopic implantation. These results suggest that MT1-MMP induced by v-src transformation may promote invasiveness of transformed cells.     

Treatment of prostate cancer in patients aged 75 or older

In view of the increasing incidence and mortality rate of prostate cancer in Japan, the management of elderly patients with prostate cancer is an important issue now. We therefore analyzed the clinicopathological features and long-term outcomes of 182 patients with prostate cancer, aged 75 or older, in order to establish the treatment strategy for this age group of patients. There were more patients with advanced disease (stage C-D) than those with localized disease (stage A2-B), and the patients with moderate to poorly differentiated tumors were more numerous than those with well-differentiated tumors. The overall survival curve of the patients with localized prostate cancer was in line with the age-matched expected survival curve, while that with advanced prostate cancer was far below the expected survival curve. These results demonstrated that advanced prostate cancer in elderly patients is as harmful as in younger patients, indicating the necessity for early detection and treatment of prostate cancer among the younger generation. On the other hand, localized prostate cancer in elderly patients should be treated less invasively to maintain their quality of life.