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Biomedical subjects

P Roberts

Publications and source records attributed to P Roberts.

At least 55 records · Page 3Linked to original sources
PubMed2000

Nucleus-vacuole junctions in Saccharomyces cerevisiae are formed through the direct interaction of Vac8p with Nvj1p.

Vac8p is a vacuolar membrane protein that is required for efficient vacuole inheritance and fusion, cytosol-to-vacuole targeting, and sporulation. By analogy to other armadillo domain proteins, including beta-catenin and importin alpha, we hypothesize that Vac8p docks various factors at the vacuole membrane. Two-hybrid and copurfication assays demonstrated that Vac8p does form complexes with multiple binding partners, including Apg13p, Vab2p, and Nvj1p. Here we describe the surprising role of Vac8p-Nvj1p complexes in the formation of nucleus-vacuole (NV) junctions. Nvj1p is an integral membrane protein of the nuclear envelope and interacts with Vac8p in the cytosol through its C-terminal 40-60 amino acids (aa). Nvj1p green fluorescent protein (GFP) concentrated in small patches or rafts at sites of close contact between the nucleus and one or more vacuoles. Previously, we showed that Vac8p-GFP concentrated in intervacuole rafts, where is it likely to facilitate vacuole-vacuole fusion, and in "orphan" rafts at the edges of vacuole clusters. Orphan rafts of Vac8p red-sifted GFP (YFP) colocalize at sites of NV junctions with Nvj1p blue-sifted GFP (CFP). GFP-tagged nuclear pore complexes (NPCs) were excluded from NV junctions. In vac8-Delta cells, Nvj1p-GFP generally failed to concentrate into rafts and, instead, encircled the nucleus. NV junctions were absent in both nvj1-Delta and vac8-Delta cells. Overexpression of Nvj1p caused the profound proliferation of NV junctions. We conclude that Vac8p and Nvj1p are necessary components of a novel interorganelle junction apparatus.

Cell Fractionation↗
PubMed2000

Can 16-18-year-old elite ballet dancers improve their hip and ankle range of motion over a 12-month period?

OBJECTIVE: To determine the effect of a 12-month intensive ballet training regimen on hip and ankle range of motion in male and female, first- and second-year professional dancers. DESIGN: 12-month longitudinal follow-up. SETTING: National classical ballet school in Australia. PARTICIPANTS: 28 female and 20 male full-time ballet students with a mean +/- 1 SD, ages 16.8 +/- 0.8 and 17.7 +/- 1.2 years, respectively. MAIN OUTCOME MEASURES: Degrees of range of motion of left and right sides for the following movements: standing plié in parallel-passive ankle dorsiflexion (DF); standing turnout in the balletic first position--lower leg external rotation (LLER); supine hip external rotation (ER); supine hip internal rotation (IR). An additional range of motion was calculated: external rotation below the hip joint (BHER) derived by subtracting hip ER from LLER. MAIN RESULTS: In all subjects combined, hip and ankle ranges increased statistically on the right. However, the amount was generally minimal and most at the borderline of the amount of error associated with the measurement tool. While there was no change in LLER, there was a decrease in BHER. There were no overall gender differences, and year differences existed only for left hip ER and total hip ER with first-year dancers showing significant improvements in these ranges. For DF and sum of hip IR, first-year males and second-year females had increases in range. There was a negative relationship between baseline range and the amount of change over the 12 months. CONCLUSIONS: Dancers ages 16-18 years who enter full-time ballet training did not augment their ankle dorsiflexion to any appreciable degree. Some, but certainly not all, increased their hip active external rotation over 12 months without increasing their total lower limb turnout. Hip ER was more likely to improve in the first-year rather than second-year student in this elite full-time training school.

Adolescent↗
PubMed2000

Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension.

Epidemiological studies have indicated that susceptibility of human adults to hypertension and cardiovascular disease may result from intrauterine growth restriction and low birth weight induced by maternal undernutrition. Although the 'foetal origins of adult disease' hypothesis has significant relevance to preventative healthcare, the origin and biological mechanisms of foetal programming are largely unknown. Here, we investigate the origin, embryonic phenotype and potential maternal mechanisms of programming within an established rat model. Maternal low protein diet (LPD) fed during only the preimplantation period of development (0-4.25 days after mating), before return to control diet for the remainder of gestation, induced programming of altered birthweight, postnatal growth rate, hypertension and organ/body-weight ratios in either male or female offspring at up to 12 weeks of age. Preimplantation embryos collected from dams after 0-4.25 days of maternal LPD displayed significantly reduced cell numbers, first within the inner cell mass (ICM; early blastocyst), and later within both ICM and trophectoderm lineages (mid/late blastocyst), apparently induced by a slower rate of cellular proliferation rather than by increased apoptosis. The LPD regimen significantly reduced insulin and essential amino acid levels, and increased glucose levels within maternal serum by day 4 of development. Our data indicate that long-term programming of postnatal growth and physiology can be induced irreversibly during the preimplantation period of development by maternal protein undernutrition. Further, we propose that the mildly hyperglycaemic and amino acid-depleted maternal environment generated by undernutrition may act as an early mechanism of programming and initiate conditions of 'metabolic stress', restricting early embryonic proliferation and the generation of appropriately sized stem-cell lineages.

Animal Nutritional Physiological Phenomena↗
PubMed2000

N;-3 and n;-6 polyunsaturated fatty acids induce cytostasis in human urothelial cells independent of p53 gene function.

The role of long-chain polyunsaturated fatty acids (PUFA) in the etiopathology and treatment of cancer is poorly understood. We have studied the effects of n;-3 and n;-6 PUFA on the proliferation and survival of normal human uroepithelial (NHU) cells, cells with disabled p53 function after stable transfection with the human papillomavirus 16 (HPV16) E6 gene (HU-E6), and p53-disabled cells that had passed through crisis and acquired karyotypic abnormalities (HU-E6P). The n;-3 and n;-6 PUFA had distinct reversible antiproliferative and irreversible cytostatic effects according to concentration and exposure time. The reversible antiproliferative effect was partly due to the production of lipoxygenase metabolites. NHU and HU-E6 cells were equally sensitive to n;-3 and n;-6 PUFA, but HU-E6P cells were more resistant to both the antiproliferative and cytostatic effects. Cytostatic concentrations of n;-3 and n;-6 PUFA did not induce apoptosis, but caused permanent growth arrest ("interphase" or "reproductive" cell death) and mRNA levels for genes involved in cell cycle control (p21, p16, p27, cdk1, cdk2, and cdk4) were not altered. Neither n;-3 nor n;-6 PUFA promoted acquisition of karyotypic abnormalities in HU-E6 cells, suggesting that n;-3 and n;-6 PUFA do not cause genotoxic damage. In conclusion, our studies show that the antiproliferative and cytostatic effects of n;-3 and n;-6 PUFA are not dependent on p53 function and, further, that transformation results in a loss of sensitivity to n;-3 and n;-6 PUFA-mediated growth inhibition.

Apoptosis↗
PubMed2000

Evolution of combined modality therapy for stage III non-small-cell lung cancer.

A number of randomized clinical trials and meta-analyses now support the conclusion that combined modality regimens that include cisplatin (Platinol)-based chemotherapy improve survival in stage III non-small-cell lung cancer (NSCLC) more effectively than radiotherapy or surgery alone. Depending on the therapy, chemotherapy may play a cytoreductive role by eradicating distant micrometastases, a radiosensitizing role by improving local control, or do both. In general, sequential therapies in which platinum-based chemotherapy precedes thoracic radiation or surgery have improved outcome by affecting distant metastases. In contrast, concurrent chemoradiotherapy utilizing low-dose cisplatin improves survival by reducing local recurrence without an effect on distant failure rates. In view of these observations, chemoradiotherapy strategies that integrate both radiosensitizing agents and dose levels of chemotherapy that are effective against micrometastases may prove to be most efficacious. Since distant metastases remain the major site of failure, it is also likely that more effective chemotherapy or other systemic antitumor agents are necessary to increase the current level of response and survival. Fortunately, several new chemotherapeutic agents are highly effective against NSCLC as well as potent radiosensitizers.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1999

Redox cycles in trimethylamine dehydrogenase and mechanism of substrate inhibition.

The steady-state reaction of trimethylamine dehydrogenase (TMADH) with the artificial electron acceptor ferricenium hexafluorophosphate (Fc(+)) has been studied by stopped-flow spectroscopy, with particular reference to the mechanism of inhibition by trimethylamine (TMA). Previous studies have suggested that the presence of alternate redox cycles is responsible for the inhibition of activity seen in the high-substrate regime. Here, we demonstrate that partitioning between these redox cycles (termed the 0/2 and 1/3 cycles on the basis of the number of reducing equivalents present in the oxidized/reduced enzyme encountered in each cycle) is dependent on both TMA and electron acceptor concentration. The use of Fc(+) as electron acceptor has enabled a study of the major redox forms of TMADH present during steady-state turnover at different concentrations of substrate. Reduction of Fc(+) is found to occur via the 4Fe-4S center of TMADH and not the 6-S-cysteinyl flavin mononucleotide: the direction of electron flow is thus analogous to the route of electron transfer to the physiological electron acceptor, an electron-transferring flavoprotein (ETF). In steady-state reactions with Fc(+) as electron acceptor, partitioning between the 0/2 and 1/3 redox cycles is dependent on the concentration of the electron acceptor. In the high-concentration regime, inhibition is less pronounced, consistent with the predicted effects on the proposed branching kinetic scheme. Photodiode array analysis of the absorption spectrum of TMADH during steady-state turnover at high TMA concentrations reveals that one-electron reduced TMADH-possessing the anionic flavin semiquinone-is the predominant species. Conversely, at low concentrations of TMA, the enzyme is predominantly in the oxidized form during steady-state turnover. The data, together with evidence derived from enzyme-monitored turnover experiments performed at different concentrations of TMA, establish the operation of the branched kinetic scheme in steady-state reactions. With dimethylbutylamine (DMButA) as substrate, the partitioning between the 0/2 and 1/3 redox cycles is poised more toward the 0/2 cycle at all DMButA concentrations studied-an observation that is consistent with the inability of DMButA to act as an effective inhibitor of TMADH.

Diethylamines↗
PubMed1999

Maintenance of steroid metabolism and hormone responsiveness in cryopreserved dog, monkey and human hepatocytes.

The efficient and effective use of hepatocytes from larger species and rare human material requires a reliable storage method for cells not needed on the day of preparation. Cryopreservation would seem to be the only viable alternative. In this study the suitability of a published cryopreservation technique on dog, monkey and human hepatocytes has been examined and the cells were tested for functionality directly after thawing and subsequent to culture using steroid metabolism and hormone responsiveness of glycogen phosphorylase a. Monkey and human hepatocytes appear to survive the freezing and thawing process better than dog cells-the latter losing the ability to respond to adrenergic stimuli and their ability to maintain steroid metabolism in culture. Although monkey and human cells do preserve their steroid metabolising capacity after freeze/thawing, there is not the significant increase in enzyme activity seen during culturing freshly isolated cells. It would appear, therefore, that some damage has occurred to the cells during the freeze/thaw process. As previously noted, Williams' medium E is superior to Ham's F-10 in maintaining enzyme activities in culture. It is suggested that cryopreservation is the way forward for the development of stockpiles of viable hepatocytes for biomedical and toxicological research and development but that further modifications to the process are still necessary to optimise the maintenance of liver-specific functions in the thawed cells.

Androstenedione↗
PubMed1999

A multi-laboratory evaluation of cryopreserved monkey hepatocyte functions for use in pharmaco-toxicology.

Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols, we validated them through a multi-laboratory study. Dissociation of the whole animal liver resulted in obtaining 1-5 billion parenchymal cells with a viability of about 86%. An appropriate fraction (around 20%) of the freshly isolated cells was immediately set in primary culture and various hepato-specific tests were performed to examine their metabolic, biochemical and toxicological functions as well as their ultrastructural characteristics. The major part of the hepatocytes was frozen and their functionality checked using the same parameters after thawing. The characterization of fresh and thawed monkey hepatocytes demonstrated the maintenance of various hepato-specific functions. Indeed, cryopreserved hepatocytes were able to survive and to function in culture as well as their fresh counterparts. The ability for synthesis (proteins, ATP, GSH) and conjugation and secretion of biliary acids was preserved after deep freeze storage. A better stability of drug metabolizing activities than in rodent hepatocytes was observed in monkey. After thawing, Phase I and Phase II activities (cytochrome P450, ethoxycoumarin-O-deethylase, aldrin epoxidase, epoxide hydrolase, glutathione transferase, glutathione reductase and glutathione peroxidase) were well preserved. The metabolic patterns of several drugs were qualitatively and quantitatively similar before and after cryopreservation. Lastly, cytotoxicity tests suggested that the freezing/thawing steps did not change cell sensitivity to toxic compounds.

Adenosine Triphosphate↗
PubMed1999

Measles antibody seroprevalence rates among immunized Inuit, Innu and Caucasian subjects.

Measles antibody seroprevalence was compared in Innu, Inuit, and Caucasian peoples of northern Newfoundland, Canada, who were immunized with a single dose of M-M-R-II (Merck Research Laboratories) vaccine. Healthy, volunteer schoolchildren (n = 606) were enrolled. Measles antibody was measured with a whole virus measles-specific IgG EIA. Native (Innu and Inuit) schoolchildren (n = 253) had a significantly higher seropositive rate (83%) after a single dose of measles vaccine compared to Caucasian (n = 353) children (76%; p = 0.025), and higher mean antibody levels after immunization compared to Caucasian children (1.74 EIA units, vs. 1.63; p = 0.06). Caucasian children were more likely to have been immunized after age 15 months (20.6% vs. 9.6%; p = 0.001). There was no significant difference in the mean time interval between immunization and blood sampling for natives versus Caucasian (8.0 years vs. 7.95 years; p = 0.49). After adjustment for time from immunization and age at immunization, there remained a marginally significant racial difference in seropositivity (OR = 1.65, 95% CI 0.96, 2.83, p = 0.068). The unadjusted odds ratio for seropositivity (comparing natives vs. non-natives, combining negative and equivocal results) was 1.66 (95% CI 1.06-2.59, p = 0.018). The higher measles-seropositive rate found among native compared to non-native Canadian children suggests that genetic and/or environmental factor(s) affect circulating antibody levels following immunization. The determination of these sources of variability may lead to the development of more efficacious vaccines or delivery strategies.

Adolescent↗
PubMed1999

A combined cytogenetic and molecular approach to diagnosis in a case of desmoplastic small round cell tumor with a complex translocation (11;22;21).

Desmoplastic small round cell tumor (DSRCT) has recently been described as a discrete tumor entity. It is distinguished from other small round cell tumors by its prominent desmoplastic quality, its preponderance in adolescent males, its almost exclusive intraabdominal location, a multi-immunophenotypic profile, and its aggressive nature. Diagnosis on histology alone is not always unequivocal. A recurrent t(11;22)(p13;q12) translocation has recently been described in this tumor, and a chimeric RNA fusion product formed from the WT1 and EWS genes is detectable by reverse transcriptase-polymerase chain reaction (RT-PCR). We describe the use of a multi-faceted approach using conventional G-banding, fluorescence in situ hybridization (FISH) and RT-PCR to assist the diagnosis of a case of DSRCT with a complex variant t(11;22;21)(p13;q12;q22.1) translocation and demonstrate the value of a combined approach to genetic investigation of solid tumors.

Abdominal Neoplasms↗
PubMed1999

Renal medullary carcinoma: prolonged remission with chemotherapy, immunohistochemical characterisation and evidence of bcr/abl rearrangement.

BACKGROUND: Renal medullary carcinoma (RMC), an extremely rare tumour of the kidney, carries a dismal prognosis, with no reports to date of significant response to chemotherapy or radiotherapy. A case of this tumour in a male child, who showed a dramatic response to chemotherapy, is described. PROCEDURE: A detailed histological evaluation of the tumour and cytogenetic analysis using fluorescent in situ hybridisation (FISH) was carried out. The child was treated with multiagent chemotherapy, followed by abdominal radiotherapy. RESULTS: A detailed histopathological and immunohistochemical portrait of this tumour is described, and FISH studies confirmed the presence of a bcr/abl rearrangement. The child obtained complete radiological remission following chemotherapy, although he later relapsed and died of progressive disease despite further attempts at treatment with chemotherapy. CONCLUSIONS: Although there are no previous reports of response of this tumour to chemotherapy, this case illustrates that treatment of this disease is justified. The responses of other cases to similar drug regimens would be of interest to confirm whether the encouraging response described for this case could be reproduced. Cytogenetic analysis of other cases of RMC may clarify whether the abnormalities seen in this case are typical.

Adolescent↗
PubMed1999

The development of NEdSERV: quantitative instrumentation to measure service quality in nurse education.

The political climate of health care provision and education for health care in the latter years of the 20th century is evolving from the uncertainty of newly created markets to a more clearly focused culture of collaboration, dissemination of good practice, with an increased emphasis on quality provision and its measurement. The need for provider units to prove and improve efficiency and effectiveness through evidence-based quality strategies in order to stay firmly in the market place has never been more necessary. The measurement of customer expectations and perceptions of delivered service quality is widely utilized as a basis for customer retention and business growth in both commercial and non-profit organizations. This paper describes the methodological development of NEdSERV--quantitative instrumentation designed to measure and respond to ongoing stakeholder expectations and perceptions of delivered service quality within nurse education.

Attitude of Health Personnel↗
PubMed1999

Identification of the t(15;17) in AML FAB types other than M3: evaluation of the role of molecular screening for the PML/RARalpha rearrangement in newly diagnosed AML. The Medical Research Council (MRC) Adult Leukaemia Working Party.

Acute promyelocytic leukaemia (APL) is characterized by the t(15;17) leading to the formation of PML-RARalpha and RARalpha-PML fusion genes; this rearrangement has been considered both diagnostic for, and restricted to, this subtype of acute myeloid leukaemia (AML FAB M3). We describe two cases of AML with the t(15;17) associated with a PML/RARalpha rearrangement which lacked typical APL morphology, classified as FAB M1 and M2 respectively. In both cases morphological review revealed small populations of cells which exhibited some features associated with APL. In the case classified as M1, PML immunofluorescence studies revealed the classic microparticulate nuclear staining pattern as observed in typical cases of APL with the t(15;17). Similarly, blasts from this case were found to be sensitive to ATRA in vitro as determined by NBT reduction test and by normalization of the PML nuclear body staining pattern. To determine the frequency of PML/RARalpha rearrangements in FAB subtypes other than M3, 530 patients from the MRC AML trials were screened using nested RT-PCR. Only one individual, initially classified as M5 with a normal karyotype, was found to have a PML/RARalpha rearrangement. The diagnosis was revised to M3 variant on subsequent morphological review. In conclusion, this study demonstrates that, in rare cases, the t(15;17) is not restricted to patients with M3 morphology as defined by current FAB criteria. Therefore, although we consider cytogenetic analysis of newly diagnosed cases of AML to be mandatory, our data suggests that routine molecular screening for PML/RARalpha rearrangements is not justified and should be reserved for those cases displaying features which may be suspicious of APL even if such cells comprise only a minority of the total population.

Adolescent↗
PubMed1998

Uroplakin gene expression by normal and neoplastic human urothelium.

cDNA sequences for human uroplakins UPIa, UPIb, UPII, and UPIII were cloned and used to investigate uroplakin transcription by normal and neoplastic urothelial cells. Normal urothelium expressed mRNA for all four uroplakins, although UPIII could be detected only by ribonuclease protection assay. By in situ hybridization, UPIa and UPII were confined to superficial cells and UPIb was also expressed by intermediate cells. Cultured normal human urothelial cells showed a proliferative basal/intermediate cell phenotype and constitutive expression of UPIb only. Uroplakin expression by transitional cell carcinoma cell lines was related to their differentiated phenotype in vitro. RT4 cells expressed all uroplakins, VM-CUB-3 expressed three uroplakins, RT112 and HT1376 cells expressed only UPIb in high abundance, and COLO232, KK47, and EJ cells had no detectable expression. These results correlated with patterns of uroplakin expression in tumors. UPIa and UPII were detected superficially only in well differentiated transitional cell carcinoma papillae. UPIb was positive in seven of nine and overexpressed in five of nine noninvasive transitional cell carcinomas and was also present in four of eight invasive transitional cell carcinomas. Lymph node metastases retained the same pattern of UPIb expression as the primary tumor. Unlike the three differentiation-regulated uroplakins, UPIb may have an alternative role in urothelial cell/tissue processes.

Amino Acid Sequence↗