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Biomedical subjects

J R Jenkins

Publications and source records attributed to J R Jenkins.

At least 19 recordsLinked to original sources
PubMed1998

Defining the minimal requirements for papilloma viral E6-mediated inhibition of human p53 activity in fission yeast.

The majority of human anogenital carcinomas show evidence of papillomavirus infection. To facilitate viral replication, viruses disable key cellular responses which would otherwise precipitate cell suicide. An obligate factor in one such response is the p53 tumour suppressor protein. p53 gene mutation is an infrequent event in anogenital cancer, apparently due to the action of HPV E6 protein, which inhibits wild-type p53 function by stimulating the degradation of p53 protein. p53 is required for the apoptotic response that is triggered in untransformed cells following inappropriate cell-cycling. E6 directed inhibition of p53 function thus facilitates the survival of transformed cells. We have developed a genetically tractable model that reports E6 protein-mediated human p53 inactivation in the fission yeast Schizosaccharomyces pombe. Functional dissection of the requirements for E6 directed inhibition in this system reveal an absolute requirement for the presence of both E6 protein and the human E3 ubiquitin ligase, E6-AP. Using a defined set of E6 mutants we show that degradation of p53 protein rather than E6/p53 association is likely required for E6-mediated inhibition. This S. pombe based system represents a candidate screen for novel antiviral agents that act by disrupting the E6/E6-AP/p53 interaction.

DNA-Binding Proteins↗
PubMed1998

Use of a rapid throughput in vivo screen to investigate inhibitors of eukaryotic topoisomerase II enzymes.

Topoisomerase II catalyzes the passage of one DNA helix through another via a transient double-stranded break. The essential nature of this enzyme in cell proliferation and its mechanism of action make it an ideal target for cytotoxic agents. Saccharomyces cerevisiae topoisomerase II has been frequently used as a model for testing potential inhibitors of eukaryotic topoisomerase II as antitumor agents. The standard in vivo method of estimating the sensitivity of S. cerevisiae to the antitopoisomerase drugs is via inhibition or kill curves which rely on viable-cell counts and is labor intensive. We present an alternative to this, a high-throughput in vivo screen. This method makes use of a drug-permeable S. cerevisiae strain lacking endogenous topoisomerase II, which is modified to express either human topoisomerase IIalpha or IIbeta or S. cerevisiae topoisomerase II carried on plasmids. Each modified strain expresses a full-length topoisomerase II enzyme, as opposed to the more commonly used temperature-sensitive S. cerevisiae mutant expressing yeast or yeast/human hybrid enzymes. A comparison of this new method with a plating-and-counting method gave similar drug sensitivity results, with increased accuracy and reduced manual input for the new method. The information generated has highlighted the sensitivities of different topoisomerase II enzymes and isoenzymes to several different classes of topoisomerase II inhibitor.

Antineoplastic Agents, Phytogenic↗
PubMed1997

Transinguinal laparoscopic examination of the contralateral groin in pediatric herniorrhaphy.

The authors laparoscopically assessed the contralateral groin (CG) via the symptomatic inguinal hernia in 91 patients to avoid unnecessary CG exploration and to allow for identification of asymptomatic CG hernias. Once the symptomatic hernia sac was opened, a 4.5-mm trocar was placed intraperitoneally and the CG internal ring was inspected with a 4-mm laparoscope. When compared with the authors' previous surgical policy for routine CG exploration in children younger than 2 years of age, laparoscopic findings altered the procedure performed in 42 of 91 patients (46.2%). In patients younger than 2 years of age, the CG did not require repair of an unsuspected hernia in 55.9% of patients based on laparoscopic findings. Conversely, in children 2 years of age or older, 40.4% required CG repairs of unsuspected hernias or patent processus vaginalis (PPV). Transinguinal laparoscopic examination in pediatric herniorrhaphy provides important information about the CG without the need for additional trocar sites.

Adolescent↗
PubMed1996

Human DNA topoisomerases II alpha and II beta can functionally substitute for yeast TOP2 in chromosome segregation and recombination.

The ability of the human DNA topoisomerase II alpha and II beta isozymes to complement functional defects conferred by conditional top2 mutations in Saccharomyces cerevisiae has been investigated. At the restrictive temperature, top2 strains show multiple abnormalities, including an inability to complete mitotic and meiotic division owing to a defect in chromosome segregation, and hyper-recombination within the repetitive rDNA gene cluster. We show that the human topoisomerases II alpha and II beta can each support both vegetative growth and the production of viable spores in a top2-4 mutant at the restrictive temperature. Similarly, both human isozymes can rescue a strain carrying a top2 gene disruption, and suppress hyper-recombination within the rDNA gene cluster. We conclude that the human topoisomerase II alpha and II beta isozymes are functionally interchangeable with yeast topoisomerase II and suggest that any isozyme-specific roles in human cells are likely to be dependent upon factors other than inherent differences in catalytic ability between the alpha and beta isozymes.

Antigens, Neoplasm↗
PubMed1996

Selective loss of endogenous p21waf1/cip1 induction underlies the G1 checkpoint defect of monomeric p53 proteins.

Wild-type p53 protein displays a spectrum of activities including the ability to suppress transformed cell growth to direct apoptotic cell death and to mediate G1 checkpoint in response to cellular DNA damage. Earlier work showed that a self-association defective p53 protein retained transformation suppressor activity in rat embryo fibroblast based assays, but that monomerisation of tumour mutant p53 proteins resulted in loss of dominant transforming activity. In order to acquire a more detailed understanding of the biological consequences attendant on disruption of p53:p53 association we have carried out a study of the wild-type-like activities that are retained by monomeric p53 proteins and which are associated with the suppression of transformation. Here we show that monomeric p53 proteins are G1 checkpoint defective. Although able to stimulate transcription via a p53 DNA binding motif from the p21waf1/cip1 gene promoter in episome based assays these p53 proteins are unable to transactive the chromosomal p21waf1/cip1 gene and are sensitive both to degeneracy of consensus binding site and to half site spacing. Monomeric p53 proteins fail to trigger apoptosis in a BRK cell line transformed with E7 and ras. However, they retain wild type transformation suppressor activity in BRK cell based transformation assays. Our results indicate that p21waf1/cip1 induction and all related p53 dependent G1 checkpoint activities are dispensable for the p53 directed suppression of transformed cell growth, and that such transformation suppression by monomeric p53 proteins may occur in the absence of an apoptotic response.

Animals↗
PubMed1996

Cost-effective evaluation and management of the acute abdomen.

1. Managed care gate-keeper financial responsibility must be balanced with specialist diagnostic and therapeutic responsibility to maximize cost effectiveness and quality of medical service. 2. Cost in health care is closely tied to length of stay and operating room time; extremes of patient age are associated with increased costs. 3. Through years of training and subsequent experience, surgeons are best qualified to direct work-up of patients with an acute abdomen, especially when the work-up requires costly imaging (e.g., CT scan, ultrasonography, angiography). 4. Cost is increased when the surgeon is not involved early in cases of acute abdomen. 5. Surgeons must be willing to enter the evaluation phase of the patient with an acute abdomen patient early and follow during the observation period to best reduce unnecessary laboratory work or tests. 6. Health care teams willing to implement pathways and then document the effect such pathways exert will be most successful in assessing new technologies' cost effectiveness.

Abdomen, Acute↗
PubMed1996

Pavlovian conditioning of agonistic behavior in male threespine stickleback (Gasterosteus aculeatus).

The red coloration of male stickleback (Gasterosteus aculeatus) possesses signal value in male-male interactions. Therefore, it was predicted that males would learn to associate a red signal more readily than a green signal with a conspecific rival in a Pavlovian conditioning experiment. Males were presented red and green signal lights where one signal was always paired with presentation of a rival (excitatory conditioned stimulus, CS+) and one signal was never paired with presentation of a rival (nonreinforced stimulus, CS-). Males learned the task rapidly, showing conditioned approach and zigzag responses, but CS+ vs. CS- differentiation persisted, even after a prolonged extinction period. In addition, there were no differences in learning rates between fish trained to the red signal as the CS+ and fish trained to the green signal as the CS+. The results suggest that, although males may rapidly learn about rivals, they are not predisposed to associated red (over green) with the appearance of a rival under the conditions of this experiment. Because males must establish and maintain territories in order to nest and mate, learning about neighboring rivals may be an adaptive mechanism by which males more effectively defend their territories and thereby increase their reproductive fitness.

Agonistic Behavior↗
PubMed1995

Transcriptional activation plays a role in the induction of apoptosis by transiently transfected wild-type p53.

The p53 tumor suppressor gene has been implicated in the induction of apoptosis in several cell systems. We have recently reported than transiently-transfected wt p53 is capable of inducing apoptosis in certain transformed cell lines. We demonstrated by quantitative analysis using flow cytometry that apoptosis was restricted to the population expressing wt, but not mutant, p53. In the present study we use this model system to analyse the functional domain of p53 in the induction of apoptosis. Several constructs expressing mutations or deletions in the C-terminal oligomerization domain, the N-terminal transactivation domain or the central DNA-binding domain were introduced into HeLa cells, and the ability of the expressed proteins to induce apoptosis was evaluated. All the functional domains were found to be necessary for the induction of apoptosis. In addition, cycloheximide and actinomycin D inhibited wt p53-induced apoptosis. We therefore conclude that p53 acts in this cell system, at least in part, as a transcriptional activator in the induction of apoptosis.

Apoptosis↗
PubMed1995

Oligomerisation of full length p53 contributes to the interaction with mdm2 but not HPV E6.

The tumour suppressor protein p53 normally functions as a tetramer in a defined conformational state. Mutations within p53 which contribute to cancer development frequently induce a conformational shift in the protein which correlates with loss of wild type growth suppressor functions. Both the cell encoded mdm2 protein and the human papillomavirus oncoprotein E6 can regulate p53 function and we have examined the interaction of these proteins with p53. The E6/p53 association is sensitive to conformational alterations in the p53 protein, although oligomerisation is not necessary for this interaction to occur. Analysis of C-terminal p53 truncations has indicated that the region between residues 327 and 347 may play a role in E6 binding. Since monomeric forms of p53 retain transcriptional and transformation suppressor activities, our results indicate that E6 targets p53 proteins which retain these wild type functions. Conversely, the interaction of p53 with mdm2 is not dependent on the conformation of the p53 protein but is significantly impaired by loss of quaternary structure. It is possible that mdm2 plays a role in mediating activities of p53 which, unlike transcriptional activation, depend on oligomerisation.

Base Sequence↗
PubMed1994

The relationship between serum p53 autoantibodies and characteristics of human breast cancer.

Sera from 182 newly diagnosed breast cancer patients were assayed for antibodies to p53 using an enzyme-linked immunosorbent assay (ELISA) method, and antibodies were detected in 48 (26%) compared with 1 out of 76 (1.3%) normal control volunteers (P = 0.0001). In breast cancer patients, autoantibodies were found in all stages of disease progression: carcinoma in situ, primary invasive breast cancer and in metastatic disease. In the subset of patients in whom sequential sera were assessed over a 6 month period, changes in the p53 antibody titres were observed. The presence of antibodies to p53 correlated positively with high histological grade (P = 0.0012) and a history of second primary cancer (six positive out of eight cases). The incidence of autoantibodies was lower in those patients with a first-degree relative with breast cancer (P = 0.046). Out of 68 patients, there was a significant correlation between positive p53 autoantibody status and the detection of p53 protein in the tissue sections by immunocytochemistry (P = 0.002). In the seronegative patients, positive p53 tumour staining was strongly associated with a family history of breast cancer (P = 0.009). The p53 protein overexpressed in heritable breast cancers may therefore be less immunogenic. The presence of p53 autoantibodies provides important additional information to immunochemistry and may identify patients with aggressive histological types of breast cancer.

Adult↗
PubMed1994

Human p53 directs DNA strand reassociation and is photolabelled by 8-azido ATP.

p53 is the most frequent known target for mutation in human cancer. Evidence suggests that p53 protein may be involved variously in transcription and cell cycle control, in DNA replication and in G1 checkpoint control following the cellular response to radiation induced DNA damage. p53 blocks DNA replication of the small DNA tumour virus, simian virus 40, by inhibiting unwinding of the viral origin of replication by the DNA helicase activity of the virally encoded large T antigen protein. Here we report the novel observation that human p53 protein can bind ATP and exhibits an intrinsic ATP stimulated DNA strand reassociation activity. Both activities map to the carboxyl terminal 128 amino acids of p53. Thus, in addition to any role in transcription, our results indicate that p53 is potentially capable of inhibiting mammalian replicative DNA synthesis by blocking the DNA strand separation step during replication origin recruitment. However, the ability of p53 to modulate the topological relationship between complementary nucleotide strands is also compatible with a direct role for p53 in other aspects of DNA synthesis, recombination or repair.

Adenosine Triphosphate↗
PubMed1993

Putative dehydrogenase tms1 suppresses growth arrest induced by a p53 tumour mutant in fission yeast.

Expression of a human tumour-derived p53 His 273 cDNA induced growth arrest in fission yeast Schizosaccharomyces pombe. Based on the p53-induced growth arrest, we cloned an extragenic suppressor, termed tms1, by complementation. The open reading frame of the tms1 gene corresponded to a protein of 347 amino acids with a calculated mass of 37380 Da. The transcriptional start site of the tms1 gene was mapped and, in addition, the corresponding cDNA was isolated and expressed in Escherichia coli. Recombinant tms1 protein served as an antigen to produce specific polyclonal antibodies to aid identification of the tms1-gene-product in total yeast lysates. Comparison of the deduced amino acid sequence of tms1 with available databases revealed significant similarity to dehydrogenases, suggesting that the tms1 protein itself might possess dehydrogenase activity.

Amino Acid Sequence↗
PubMed1993

Human cells express two differentially spliced forms of topoisomerase II beta mRNA.

Screening of a human B-cell cDNA library with a topoisomerase II beta gene-specific probe revealed the presence of two distinct forms of topoisomerase II beta cDNA. One form (designated topoisomerase II beta-1), representing the majority of the clones, would encode the topoisomerase II beta amino acid sequence reported recently [Jenkins, J.R. et al. (1992) Nucleic Acids Res., 20, 5587-5592]. The second form (designated topoisomerase II beta-2) would encode a protein containing an additional 5 amino acids inserted after Valine-23 of the topoisomerase II beta-1 protein sequence. The topoisomerase II beta-1 and beta-2 mRNAs were both widely expressed in human cell lines and tissues. Topoisomerase II beta-2 mRNA was expressed at a lower level than that of the beta-1 form, but the relative expression of the two forms varied in different cell types. Analysis of genomic DNA clones revealed that the two forms of topoisomerase II beta mRNA arose via differential splicing. These data indicate that in addition to the closely related topoisomerase II alpha and beta isozymes, there are two forms of topoisomerase II beta mRNA widely expressed in human cells.

Amino Acid Sequence↗
PubMed1993

The transforming and suppressor functions of p53 alleles: effects of mutations that disrupt phosphorylation, oligomerization and nuclear translocation.

Mutant p53 alleles that have a recessive phenotype in human tumors can, in cooperation with an activated H-ras gene, transform rat embryo fibroblasts (REFs). Mutant p53 proteins differ from wild type, and from each other in conformation, localization and transforming potential. Missense mutations in codons 143, 175 and 275 confer strong transforming potential. A serine 135 p53 mutant has an intermediate transforming potential, while the histidine codon 273 allele transforms weakly, if at all. In contrast to the wild type p53 gene, mutant p53 alleles with strong transforming ability cannot suppress the transformation of REFs by other oncogenes. The His273 allele retains partial suppressor function in this assay. The relevance of p53 oligomerization, phosphorylation and nuclear translocation to the transforming potential of mutant p53 and to wild type p53 suppressor function were examined. The inability of mutant p53 polypeptides to form homodimers correlates with loss of transforming function. Monomeric variants of wild type p53 protein, however, retain the ability to suppress focus formation. Phosphorylation of serine residues 315 and 392 is not required for the transforming function of mutant p53, nor is serine 315 required for suppressor function when these alleles are constitutively expressed in REF assays. Nuclear translocation-defective mutant and wild type p53 proteins retain transforming and suppressor function in REF assays.

Alleles↗
PubMed1993

Two approaches to reading instruction with children with disabilities: does program design make a difference?

This study examined the effects on reading achievement of variation in program design and tested the hypothesis this Distar Reading Mastery's (Engelmann & Bruner, 1988b) demonstrated effects with disadvantaged children in Project Follow Through can be generalized to children with disabilities. We compared the effects of two synthetic phonics reading programs, Direct Instruction (DI) Reading Mastery I and II and Addison Wesley's Meet the Superkids and The Superkids' Club, (Rowland, 1982a, 1982b) for 81 children in transitional kindergarten special education classes. No significant achievement differences were evident for the instructional program either at the end of the treatment year, or on follow-up testing 1 year later. However, among children who made advanced progress, the DI group showed larger reading gains.

Child↗
PubMed1993

Teaching phonological awareness to young children with learning disabilities.

This study examined the feasibility of teaching phonological manipulation skills to preschool children with disabilities. Forty-seven children, 4-6 years old, enrolled in a special education preschool, were randomly assigned to receive training in one of three categories of phonological tasks (rhyming, blending, and segmenting) or a control group. Results indicated that children were able to make significant progress in each experimental category, but that they demonstrated little or no generalization either within a category (e.g., from one type of blending task to another type of blending task) or between categories (e.g., from blending to segmenting). Although the children's level of cognitive development significantly predicted some learning outcomes, it did not appear to limit the learning of phonological tasks.

Awareness↗