Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway.

The wild-type p53 gene product plays an important role in the control of cell proliferation, differentiation, and survival. Altered function is frequently associated with changes in p53 stability. We have studied the role of the ubiquitination pathway in the degradation of p53, utilizing a temperature-sensitive mutant, ts20, derived from the mouse cell line BALB/c 3T3. We found that wild-type p53 accumulates markedly because of decreased breakdown when cells are shifted to the restrictive temperature. Introduction of sequences encoding the human ubiquitin-activating enzyme E1 corrects the temperature sensitivity defect in ts20 and prevents accumulation of p53. The data therefore strongly indicate that wild-type p53 is degraded intracellularly by the ubiquitin-mediated proteolytic pathway.

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis.

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.

Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity.

JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature sensitive for DNA replication (J. J. Dermody, B. E. Wojcik, H. Du, and H. L. Ozer, Mol. Cell. Biol. 6:4594-4601, 1986). It was chosen for detailed study because of its novel property of inhibiting both polyomavirus and adenovirus DNA synthesis in a temperature-dependent manner. Pulse-labeling studies demonstrated a defect in the rate of adenovirus DNA synthesis. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of uninfected cultures from 33 to 39 degrees C revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shift, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shift, in parallel with the decline in dNTP pool sizes. Moreover, the activity of cell extracts was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. The kinetics of dNTP pool size changes after temperature shift are quite distinct from those reported after inhibition of ribonucleotide reductase with hydroxyurea. An indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since human sequences other than those encoding the enzyme subunits can correct the temperature-sensitive growth defect in the mutant.

Linkage analysis of a candidate locus (HLA) in autosomal dominant sacral defect with anterior meningocele.

Sacral defect with anterior meningocele (SDAM) is a type of caudal dysgenesis. It is present at birth and becomes symptomatic later in life, usually because of obstructive labor in females, chronic constipation, rectal fistula and abscess, or meningitis. The inheritance is autosomal dominant. HLA has been implicated in caudal dysgenesis because of analogy with disorders of the T-locus complex, a tail length determining gene in mice which is linked to the major histocompatibility complex, H-2. Members of a 5-generation family with sacral defect and anterior meningocele (SDAM) were typed with polymorphic markers (dinucleotide repeats D6S89, D6S105, D6S109, and TCTE1) linked to HLA. Two-point and multipoint analysis exclude the HLA region as the location for the SDAM gene in this family.

Inherited thrombophilia genes in minorities.

Mutations in several genes have recently been identified which predispose to thrombosis, specifically Factor V G1691A (Factor V Leiden), Prothrombin G20210A, and Methylene tetrahydrofolate reductase (MTHFR) C677T. The prevalence of these genes in European populations has been studied, but there is little data on their prevalence in minorities. Samples from a predominantly African-American population were analyzed for these mutations. While the G20210A mutation in the prothrombin gene and homozygosity for the C677T mutation of the MTHFR were not found in African-Americans, it appears that the carrier rate for the MTHFR C677T among Hispanics may be higher than in other reported groups.

Prevalence of CTLA-4 polymorphism A49G in Ashkenazi Jews.

Type 1 diabetes has been associated with numerous genetic loci. One locus, IDD12, includes the gene for cytotoxic T lymphocyte antigen (CTLA-4). A polymorphism at position 49 in the CTLA-4 gene, causing a substitution of Thr --> Ala, has been associated with various autoimmune diseases, including diabetes. The frequency of the polymorphism in European and Oriental populations has been recorded, but the frequency among different ethnic groups within the United States has yet to be established. In the present study, we analyzed 100 DNA samples from Ashkenazi Jews to determine the polymorphism's prevalence in that population. The A/A genotype was found in 49% of individuals, 41% were heterozygous A/G, and 10% possessed the G/G genotype. The prevalence of the A/A genotype in Ashkenazi Jewish population is the highest reported to date. The incidence of the homozygous G/G genotype within Spanish controls, 8.8%, is the lowest, followed by the Ashkenazi general population. The frequency of the non-diabetes-associated A/A genotype shows a similarity to the frequency of the diabetes-susceptible HLA haplotype, DR4-DQ8. The low prevalence of the autoimmune-associated G allele among Ashkenazi Jewish and Spanish populations may explain a lower than expected incidence of diabetes in HLA-susceptible populations.

Histological evaluation of repair using a bioresorbable membrane beneath a resin-modified glass ionomer after mechanical furcation perforation in dogs' teeth.

Resorbable barriers have been shown both clinically and histologically to produce favorable periodontal regenerative outcomes. The purpose of the present study was to evaluate the histological response to one such resorbable barrier (Atrisorb) when used as a matrix under a resin-modified glass ionomer (RMGI) sealant in the repair of furcation perforations in experimental animals. After anesthesia, pulp extirpation and filling of radicular canals with zinc oxide-eugenol cement, furcal perforations were made in 30 lower premolar (P2, P3, and P4) teeth of labrador dogs with a 1 mm diameter round bur in a conventional handpiece. The perforated teeth were randomly divided into two groups of 15 teeth. Group 1 teeth were treated with RMGI alone and group 2 with the resorbable barrier under RMGI. Six teeth without perforations served as negative controls. Pulp chambers of all teeth were filled with RMGI cement. Animals were sacrificed after 3 months. The teeth and the surrounding structures were processed for light microscopy. There were no differences found between results for glass ionomer alone and barrier with glass ionomer. The conclusion of this study is that placement of a resorbable barrier at the pulp chamber aspect of a furcation perforation did not result in superior healing, compared with the use of resin-modified glass ionomer alone.

Occupational therapy students' perspectives on occupation as an agent that promotes healthful lifestyles.

OBJECTIVES: This study was conducted to determine occupational therapy students' perspectives on occupation as an agent that promotes healthful lifestyles, what factors influence these perspectives, and the extent to which formal classroom instruction in occupation as a health-promoting agent influenced their perspective transformation. METHOD: Through a precourse and postcourse survey, weekly log sheets, and audiotaped class discussions, data regarding attitudes and beliefs about health promotion were collected from 23 occupational therapy master's students enrolled in the course Wellness Through Occupation. The data were analyzed with a priori concept coding to determine whether perspective transformation occurred as a result of formal classroom instruction. RESULTS: Participants indicated that family values, previous formal and informal instruction, and personal experience were the major factors that influenced their perspectives on occupation as an agent that promotes a healthful lifestyle. The results demonstrated that participants had a perspective transformation, which is the first step in the behavioral change process. CONCLUSION: Formal classroom instruction can be a factor in promoting healthful lifestyles through occupation. Such instruction can be an important step in empowering people to take responsibility for their own health.

Endodontic retreatment: explaining success rates and illustrated cases.

Endodontic retreatment is less successful than conventional root treatment. Factors governing this include difficulty in accessing the root canal system in retreatment cases, problems in negotiating a highly resistant microflora and unfavourable anatomy. However, despite these mitigating factors success is still a realistic goal in the majority of cases.

Why endodontic retreatment is less successful than primary root canal therapy.

Endodontic retreatment has been proven less effective than conventional root treatment. Factors dictating this include difficulty in accessing the root canal system in retreatment cases, problems in negotiating a highly resistant micro flora and unfavourable anatomy. Patients should, therefore, be counselled on the possibility of failure necessitating surgical procedures and possibly extraction. However, despite these mitigating factors success is still a realistic goal in the majority of cases.

Conditional-lethal deoxyribonucleic acid ligase mutant of Escherichia coli.

A new Escherichia coli deoxyribonucleic acid (DNA) ligase mutant has been identified among a collection of temperature-sensitive DNA replication mutants isolated recently (Sevastopoulos, Wehr, and Glaser, Proc. Natl. Acad. Sci. U.S.A. 74:3485-3489, 1977). At the nonpermissive temperature DNA synthesis in the mutant stops rapidly, the DNA is degraded to acid-soluble material, and cell death ensures. This suggests that the mutant may be among the most ligase-deficient strains yet characterized.

Leukocytic infiltration of bovine mammary parenchymal tissue in response to Corynebacterium bovis colonization.

Morphological changes and local leukocyte response to prolonged Corynebacterium bovis colonization were studied in lactating bovine mammary glands. Morphometric analysis of parenchymal tissue demonstrated no adverse effects of colonization on the synthetic and secretory activity of mammary epithelium. Numbers of macrophages, lymphocytes, and plasma cells were higher in tissue from C. bovis-colonized quarters. However, there were no differences in numbers of neutrophils between colonized and uninfected quarters. Results suggest persistent C. bovis colonization may elicit effector cell populations in lactating mammary tissue where leukocyte concentrations tend to be lower.

Learning a clinical method to identify a mandibular reference position.

The teaching of clinical maxillomandibular relationships is an area of weakness in dental school curricula. Most difficulties arise when a reference position is required to adjust or reconstitute the maximum intercuspation of the dentition. The dentist-derived concept of the centric maxillomandibular relation position is used to solve this problem. There is no consensus on the definition of this term. In the descriptions of clinical technique for recording the centric maxillomandibular relation position, clinical criteria to judge whether or not the position required has been achieved usually are not given. The dorsal border paths of the envelope of movement were used to develop a clinical criterion to identify the centric maxillomandibular relation position. Force or "guidance" is not used in this method. Test-patients with normal masticatory systems were randomly selected and fitted with a mechanical tracking apparatus. 50 undergraduate dental students and dental nurses were randomly selected and given instruction in the clinical technique to identify the centric maxillomandibular relation position. The ability of these students to identify the reference position was assessed by having them perform the technique blindfolded on the test-patients on whom tracking apparatus was fitted. Sixty eight percent of the test-clinicians succeeded in using the method to identify the reference position. There was no significant difference in the abilities of subgroups to use the method successfully, e.g., women compared to men, 2nd year students to 3rd year students. It was concluded that the method was a satisfactory way of learning how to identify the centric maxillomandibular relation position in a gentle, non-invasive fashion.

Replication of simian virus 40 DNA in normal human fibroblasts and in fibroblasts from xeroderma pigmentosum.

Simian virus 40 infection of semipermissive human diploid fibroblasts (HF), at early passage in cell culture, was compared with that of permissive established monkey cell lines. Viral DNA can be readily detected at 24 to 48 h postinfection at 37 degrees C with a high multiplicity of infection, approaching 10% of that of monkey cells (TC7). The length of time necessary for replication of an average molecule of viral DNA was found to be indistinguishable in HF and TC7 cells. Strand elongation plus termination were assessed by following the accumulation of DNA I at 40 degrees C from replicative intermediates of tsA30 prelabeled at 33 degrees C, obviating isotope pool problems. Combined initiation and elongation of wild-type viral DNA was measured by density shift experiments involving a 5-bromodeoxyuridine chase of prelabeled [3H]thymidine-labeled viral DNA. Determination of accumulation of viral T and V antigens supports the conclusion that the most likely basis for the reduced virus yield in HF cells results from the inefficiency of an early stage in virus infection, before or during uncoating. Similar results were obtained in fibroblasts derived from patients with xeroderma pigmentosum, suggesting that enzymes of UV repair are not required in unirradiated simian virus 40 DNA synthesis.