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P Russell

Publications and source records attributed to P Russell.

At least 163 records · Page 9Linked to original sources
PubMed1996

Rapid detection of regionally clustered germ-line BRCA1 mutations by multiplex heteroduplex analysis. UKCCCR Familial Ovarian Cancer Study Group.

Germ-line mutations of the BRCA1 gene are responsible for a substantial proportion of families with multiple cases of early-onset breast and/or ovarian cancer. Since the isolation of BRCA1 last year, >65 distinct mutations scattered throughout the coding region have been detected, making analysis of the gene time consuming and technically challenging. We have developed a multiplex heteroduplex analysis that is designed to analyze one-quarter of the coding sequence in a single-step screening procedure and that will detect approximately 50% of all BRCA1 mutations so far reported in breast/ovarian cancer families. We have used this technique to analyze BRCA1 in 162 families with a history of breast and/or ovarian cancer and identified 12 distinct mutations in 35 families.

BRCA1 Protein↗
PubMed1996

Expression of Crystallins, Pax6, Filensin, CP49, MIP, and MP20 in lens-derived cell lines.

PURPOSE: Cell lines are the systems of choice to analyze cellular functions related to the particular organ system. For lens research, three cell lines are widely used: N/N1003A (derived from rabbit lenses), alpha TN4, and NKR-11 (both of murine origin). The aim of the current study was to characterize these particular cell lines with respect to their expression of genes that are considered to be lens specific or expressed preferentially in the lens, such as crystallins, Pax6, Filensin, CP49, MIP, and MP20. METHODS: alpha A- and alpha B-crystallin cDNA from rabbit lenses were sequenced. The expression of various genes was analyzed by reverse transcription-polymerase chain reaction using specific primers and mRNA from three lens-derived cell lines. For control, the expression of the selected genes was compared in nonlenticular tissues of mouse as well as in non-lens-derived murine cell lines (EF43, NIH-3T3, and L929). RESULTS: None of the transcripts for beta B2-crystallin, gamma-crystallins, MIP, MP20, filensin, and CP49 could be detected in the lens-derived cell lines. Transcripts for alpha A-crystallin were amplified in alpha TN4, but not in N/N1003A and NKR-11 cells. Pax6, a master control gene of eye development, is expressed in all three lens-derived cell lines and, additionally, in cell lines of neuronal origin, but not in corneal endothelial cells and in the currently used control cell lines. CONCLUSIONS: Three cell lines of lenticular origin were tested for expression of genes that were found abundantly in the lens. The observed expression of Pax6 in all lens-derived cell lines allows their use in the analysis of corresponding signal chains.

Amino Acid Sequence↗
PubMed1996

Enzymes protective of oxidative damage present in all decades of life in the trabecular meshwork, as detected by two-dimensional gel electrophoresis protein maps.

Our purpose was to determine whether there are age-related differences in the presence of enzymes protective against oxidative damage in normal human trabecular meshwork (TM). The study was also done to determine whether there are significant changes in proteins related to age, gender, or position around the circumference of the meshwork. Human TM was dissected from both eyes of 16 normal donors ranging in age from 9 to 91 years. Proteins were mapped with two-dimensional (2D) gel electrophoresis. The microanalytical method used allowed analysis of samples as small as one fourth of a single meshwork. Selected proteins were identified by Western blot. Superoxide dismutase (SOD) and glutathione reductase were present in all ages on the 2D maps. Age-related changes in the TM were not apparent, although slight differences may exist between juvenile and adult TM, since differences in the relative intensities of some polypeptides were evident. In any single quadrant of the TM, > or = 160 protein spots could be counted visually. The 2D maps of the various quadrants were very similar, and the protein patterns were analogous between fellow eyes. Distinct differences were noted between maps from TM and adjacent cornea and sclera. Other protein spots identified included actin, albumin, transferrin, and vimentin. The TM samples had a mean cellular protein content of 66.3 +/- 15.4 micrograms.

Adolescent↗
PubMed1996

Human and monkey trabecular meshwork accumulate alpha B-crystallin in response to heat shock and oxidative stress.

PURPOSE: Oxidative stress and other forms of injury to trabecular meshwork (TM) cells may contribute to changes seen with age and primary open-angle glaucoma. This study was designed to investigate if TM expresses alpha B-crystallin, a small heat-shock protein with chaperone activity, and whether it might be overexpressed under stress conditions. METHODS: The TM from human and monkey eyes, as well as organ and primary cell cultures derived from these eyes, were investigated for alpha B-crystallin by immunohistochemistry, two-dimensional gel electrophoresis, Northern and Western blot analysis. The TM cell cultures were stressed by heat shock (44 degrees C for 15 minutes) or hydrogen peroxide (200 mumol for 1 hour). Semiquantitation of alpha B-crystallin messenger RNA (mRNA) or protein was obtained by densitometry. RESULTS: In both species, alpha B-crystallin could be detected in fresh and cultured TM by two-dimensional gel electrophoresis in conjunction with Western blot analysis. Immunohistochemistry of fresh samples showed that alpha B-crystallin was expressed predominantly in the cribriform area. Protein expression was enhanced in 4- to 7-day organ cultures. Primary cultures from human TM cells expressed two sizes (approximately 0.8 and 1.1 kb) of alpha B-crystallin mRNA in Northern blots. In monkey TM cultures, a 0.8-kb band was observed, which comigrated with lens alpha B-crystallin. In both species, heat shock caused a significant increase in alpha B-crystallin mRNA with a peak after 4 hours. An increase in alpha B-crystallin mRNA also was observed after oxidative stress; however, the onset of mRNA induction was slower. After heat shock, but not after oxidative stress, a transient change in mRNA mobility was observed. Western dot blot analysis showed a 3.4-fold increase in protein 24 hours after heat shock and a 20-fold increase after 48 hours. No constitutive mRNA expression and only a minimal increase 4 hours after heat shock could be observed in simian virus 40 transformed cell lines from human TM. CONCLUSIONS: Overexpression of alpha B-crystallin might be an important mechanism for TM to prevent cellular damage associated with various stress conditions.

Aged↗
PubMed1995

Lens hydration in transgenic mice containing HIV-1 protease linked to the lens alpha A-crystallin promoter.

Two constructs of transgenic mice, TG61 and TG72, containing HIV-1 protease linked to lens alpha A-crystallin promoter develop cataract. The TG61 construct exhibits cataractogenesis in utero, while in the TG72 construct frank opacities appear 24 days (homozygotes) and 26 days (hemizygotes) after birth. Differential scanning calorimetry and thermogravimetric analysis studies indicate that the hydration of lenses is strongly correlated with cataractogenesis. In all clear lenses (normal and precataractous) the total water content was the same, 68%, and increased upon opacification. The bound water, measured as percentage nonfreezable water of the total water, decreased upon cataract formation, indicating a syneretic process. On the other hand, the bound water expressed as grams of nonfreezable water per gram dry weight increases upon opacification. This implies that proteolysis and subsequent enhanced hydration is the primary supramolecular event in cataractogenesis and that syneresis in the lens of transgenic mice is of secondary importance.

Animals↗
PubMed1995

Identification of a cdk-activating kinase in fission yeast.

We have identified a second cyclin-dependent kinase (cdk) in fission yeast, crk1, which encodes a 335 amino acid protein that is most closely related to the KIN28 gene product from Saccharomyces cerevisiae and to a cdk activating kinase (CAK) encoded by the MO15 gene from Xenopus laevis, crk1 is essential for viability and delta crk1 cells arrest with septa and condensed chromatin. We show that Crk1 associates with the Mcs2 mitotic catastrophe suppressor, a cyclin H-like molecule, and overexpression of crk1 rescues the cell-cycle arrest defect of a mcs2-75 cdc2-3w cdc25-22 triple mutant at high temperature. The Crk1-Mcs2 complex possesses CAK activity in vitro in that it phosphorylates human Cdk2 on Thr160 which results in its activation in the presence of cyclin A. In addition Crk1-Mcs2 effectively phosphorylates a peptide corresponding to the C-terminal repeat domain (CTD) of RNA polymerase II. We demonstrate that crk1 is allelic to the mcs6 mitotic catastrophe suppressor and that the X.laevis MO15 gene rescues the cell-cycle arrest of an mcs6-13 cdc2-3w cdc25-22 at high temperature. Together these data suggest that the Crk1-Mcs2 complex is a CAK that interacts genetically with Cdc2 in fission yeast.

Alleles↗
PubMed1995

Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast.

In fission yeast the onset of mitosis is brought about by Cdc2/Cdc13 kinase, which is inhibited by the Wee1/Mik1 tyrosine kinases and activated by Cdc25 tyrosine phosphatase. This control network integrates many signals, including those that monitor DNA replication, DNA damage and cell size. We report here that a fission yeast MAP kinase pathway links the cell-cycle G2/M control with changes in the extracellular environment that affect cell physiology. Fission yeast spc1- mutants have a G2 delay that is greatly exacerbated by growth in high osmolarity media and nutrient limitation. A lethal interaction of spc1 and cdc25 mutations shows that Spc1 promotes the onset of mitosis. Spc1 is a MAP kinase homologue that is activated by Wis1 kinase in response to osmotic stress and nutrient limitation. Spc1 is inactivated by Pyp1, a phosphatase previously identified as a mitotic inhibitor. Pyp1 dephosphorylates only tyrosine-173 of Spc1, unlike the dual-specificity phosphatases that have been shown to regulate other MAP kinases.

Amino Acid Sequence↗
PubMed1995

Sequence, initial functional analysis and protein-DNA binding sites of the mouse beta B2-crystallin-encoding gene.

An 800-bp fragment of genomic DNA upstream from the origin of transcription of the mouse beta B2-crystallin-encoding gene (beta B2-Cry) has been isolated and its nucleotide sequence determined. Promoter fragments 275 to +30 or -110 to +30, fused to cat reporter gene, activated transcription in transiently transfected rabbit lens epithelial cells, but not in various non-lens cells. The beta B2-Cry mouse promoter contains a typical TATA-box located approx. 25 bp upstream from the transcription start point. Binding sites (upstream from the TATA-box) for transcription factors possibly involved in the regulation of gene expression have been identified by DNaseI footprinting analysis and lens cell nuclear extracts. Most notably is the binding of the Pax-6 paired domain (PrD) which correlates with the binding of lens cell nuclear proteins at the -80 to -40 region.

Animals↗
PubMed1995

Reproductive and other factors and risk of epithelial ovarian cancer: an Australian case-control study. Survey of Women's Health Study Group.

Of the few factors known to be associated with epithelial ovarian cancer, the most consistently observed relate to women's reproductive function, although even here uncertainties remain. We have undertaken a case-control study involving personal interviews with over 1,600 women, the largest of its kind to date, to investigate further the associations between women's reproductive histories and other factors and the development of ovarian cancer. Cases were drawn from women diagnosed with epithelial ovarian cancer in 3 Australian states, Queensland, New South Wales and Victoria, between August 1990 and December 1993, and controls were drawn at random from the electoral roll, stratified by age and geographic region. Trained interviewers administered standard questionnaires to obtain detailed information about women's reproductive and contraceptive histories and other factors of interest, such as smoking and family history of ovarian or other cancer. Findings were based on data from 824 cases and 860 controls and confirmed the reduced risk of ovarian cancer associated with increasing parity and duration of use of the oral contraceptive pill (OCP), hysterectomy and tubal ligation. The strongest association of all was seen with use of the OCP for 10 years or more. An inverse association between ovarian cancer and age at first birth was observed, but this was not statistically significant. There were no associations between development of ovarian cancer and number of incomplete pregnancies, use of hormone replacement therapy or menstrual history. Among other factors considered, education after leaving school was negatively associated and high body mass index, family history of ovarian cancer, use of talc in the abdominal or perineal region and smoking were positively associated with occurrence of ovarian cancer.

Adolescent↗
PubMed1995

Purification and characterization of the low molecular weight protein tyrosine phosphatase, Stp1, from the fission yeast Schizosaccharomyces pombe.

Genetic screening in fission yeast has identified a gene named stp1+ that rescues cdc25-22 [Mondesert et al. (1994) J. Biol. Chem. 269, 27996-27999]. This gene encodes a 17.4 kDa protein that is 42% identical to members of the low molecular weight protein tyrosine phosphatases (low M(r)PTPases) previously known to exist only in mammalian species. A simple and efficient purification procedure was developed to obtain the homogeneous recombinant yeast low M(r)PTPase, Stp1, in large quantities suitable for kinetic and structural studies. Authentic Stp1 was produced as judged by amino terminal protein sequencing and electrospray ionization mass spectrometry analyses. Stp1 was shown to possess intrinsic phosphatase activity toward both aryl phosphates (such as phosphotyrosine) and alkyl phosphates (such as phosphoserine). Stp1 also dephosphorylated phosphotyrosyl peptide/protein substrates. The yeast enzyme was 6-fold slower than the mammalian enzymes, which made it amenable to pre-steady-state stopped-flow spectroscopic kinetic analysis at 30 degrees C and pH 6.0. Burst kinetics was observed with Stp1 using p-nitrophenyl phosphate as a substrate, suggesting that the rate-limiting step corresponds to the decomposition of the phosphoenzyme intermediate. Interestingly, the bovine heart low M(r)PTPase was capable of removing phosphate groups from both phosphotyrosyl and phosphoseryl/threonyl protein substrates with comparable efficiencies. The low M(r)PTPases, like the Cdc25 family of phosphatases, may represent a new group of dual specificity phosphatases which may be involved in cell cycle control.

Amino Acid Sequence↗
PubMed1995

Cell cycle regulation of human WEE1.

WEE1 kinase negatively regulates entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of CDC2/cyclin B kinase. We report here an investigation of human WEE1. Endogenous WEE1 migrates as an approximately 94 kDa protein in SDS-PAGE, substantially larger than the 49 kDa protein encoded by the original human WEE1 cDNA clone that was truncated at the 5'-end. Antibody depletion experiments demonstrate that WEE1 accounts for most of the activity that phosphorylates CDC2 on Tyr15 in an in vitro assay of HeLa cell lysates, hence it is likely to have an important role in the mitotic control of human cells. WEE1 activity was not found to be elevated in HeLa cells arrested in S phase, suggesting that unreplicated DNA does not delay M phase by hyperactivating WEE1. WEE1 activity is strongly suppressed during M phase, suggesting that negative regulation of WEE1 could be part of the mechanism by which activation of CDC2/cyclin B kinase is promoted during the G2/M transition. M phase WEE1 is re-activated in samples prepared in the absence of protein phosphatase inhibitors, demonstrating that WEE1 is inhibited by a mechanism that requires protein phosphorylation.

CDC2 Protein Kinase↗
PubMed1995

The complete sequence of human lens gamma s-crystallin.

The complete sequence of human gamma s-crystallin has been determined and confirmed using a combination of MS methods, peptide sequencing and cDNA sequencing. Regions 21-35 and 102-107, which were previously assumed to be the same as the bovine sequence, differ from the bovine sequence at residues 22, 28, 31 and 104. An additional six residues were also found to be different from the original sequence determined for Pakastani lenses. Whether these differences represent errors in the original sequence or two different sequences among human lens crystallins is not yet known.

Amino Acid Sequence↗
PubMed1995

Counteractive roles of protein phosphatase 2C (PP2C) and a MAP kinase kinase homolog in the osmoregulation of fission yeast.

With the goal of discovering the cellular functions of type 2C protein phosphatases, we have cloned and analyzed two ptc (phosphatase two C) genes, ptc2+ and ptc3+, from the fission yeast Schizosaccharomyces pombe. Together with the previously identified ptc1+ gene, the enzymes encoded by these genes account for approximately 90% of the measurable PP2C activity in fission yeast cells. No obvious growth defects result from individual disruptions of ptc genes, but a delta ptc1 delta ptc3 double mutant displays aberrant cell morphology and temperature-sensitive cell lysis that is further accentuated in a delta ptc1 delta ptc2 delta ptc3 triple mutant. These phenotypes are almost completely suppressed by the presence of osmotic stabilizers, strongly indicating that PP2C has an important role in osmoregulation. Genetic suppression of delta ptc1 delta ptc3 lethality identified two loci, mutations of which render cells hypersensitive to high-osmolarity media. One locus is identical to wis1+, encoding a MAP kinase kinase (MEK) homolog. The Wis1 sequence is most closely related to the Saccharomyces cerevisiae MEK encoded by PBS2, which is required for osmoregulation. These data indicate that divergent yeasts have functionally conserved MAP kinase pathways, which are required to increase intracellular osmotic concentrations in response to osmotic stress. Moreover, our observations implicate PP2C enzymes as also having an important role in signal transduction processes involved in osmoregulation, probably acting to negatively regulate the osmosensing signal that is transmitted through Wis1 MAP kinase kinase.

Amino Acid Sequence↗
PubMed1995

Computerized consensus diagnosis: a classification strategy for the robust analysis of MR spectra. I. Application to 1H spectra of thyroid neoplasms.

We introduce and apply a new classification strategy we call computerized consensus diagnosis (CCD). Its purpose is to provide robust, reliable classification of biomedical data. The strategy involves the cross-validated training of several classifiers of diverse conceptual and methodological origin on the same data, and appropriately combining their outcomes. The strategy is tested on proton magnetic resonance spectra of human thyroid biopsies, which are successfully allocated to normal or carcinoma classes. We used Linear Discriminant Analysis, a Neural Net-based method, and Genetic Programming as independent classifiers on two spectral regions, and chose the median of the six classification outcomes as the consensus. This procedure yielded 100% specificity and 100% sensitivity on the training sets, and 100% specificity and 98% sensitivity on samples of known malignancy in the test sets. We discuss the necessary steps any classification approach must take to guarantee reliability, and stress the importance of fuzziness and undecidability in robust classification.

Adenocarcinoma, Follicular↗
PubMed1995

Associations between oncogenic human papillomaviruses and local invasive patterns in cervical cancer.

The polymerase chain reaction (PCR) was used to detect human papillomavirus (HPV) DNA in Formalin-acetic acid alcohol (FAA)-fixed paraffin-embedded tissue from 40 patients whose primary cervical cancers showed a tentacular pattern of invasion at their advancing edges, and 40 patients (matched by age, International Federation of Obstetrics and Gynecology (FIGO) stage and histology type) whose tumors showed broad front invasion. The rate of HPV DNA positivity was the same in both the tentacular and the broad front tumors (83%), but the ratios of HPV 16 to HPV 18 in the two groups were markedly different (20:10 versus 27:4, respectively). HPV type 18 was detected more frequently in tentacular than broad front tumors (P = 0.03). The overall rates of recurrence and mortality were 15 and 9%, respectively (18 and 10% in the tentacular group compared with 13 and 8% in the broad front group). Univariate analysis showed statistically significant associations between HPV 18 and tumor recurrence (P = 0.04), but not between a tentacular pattern of invasion and tumor recurrence (P < 0.05). The findings to emerge from this survey indicate that the presence of particular HPV types may, in part, mediate the histological and clinical behavior of cervical cancers.

Adult↗
PubMed1995

Antiproliferative effects of bacillus Calmette-Guerin and interferon alpha 2b on human bladder cancer cells in vitro.

Direct inhibitory effects of bacillus Calmette-Guérin (BCG) and interferon alpha 2b (IFN alpha 2b) on six human bladder carcinoma cell lines, UCRU-BL-13, UCRU-BL-17, UCRU-BL-28, 5637, T24 and J82, were studied using an in vitro proliferation assay. Effects on proliferation following exposure to BCG or IFN alpha 2b were analysed by [3H]thymidine incorporation over 7 days. BCG had an antiproliferative effect on all bladder lines, while sensitivity to IFN alpha 2b varied greatly, being as remarkably low as 1 U/ml for some lines. The antiproliferative effect was greatest when cells were exposed continuously to either agent, but was still evident with a limited exposure. When clinical concentrations were simulated in vitro, BCG+IFN alpha 2b was more effective than BCG alone and as effective as a double BCG concentration. We conclude that, in addition to their immunomodulatory effects, BCG and IFN alpha 2b directly inhibit the proliferation of human bladder cancer cells, and often at extremely low concentrations.

Antineoplastic Agents↗
PubMed1995

Endometrial morphometry in users of intrauterine contraceptive devices and women with ovulatory dysfunctional uterine bleeding: a comparison with normal endometrium.

Copper-bearing intrauterine contraceptive devices (IUCD) are commonly associated with menorrhagia. They cause certain morphological changes in the endometrium. These have been extensively reported and the mechanisms of menorrhagia have been investigated in a number of studies. By contrast, ovulatory dysfunctional uterine bleeding has not been as widely studied and mechanisms of menorrhagia in these patients are still not well understood. In this study, we examined endometrial morphometry in a group of women using IUCD, a group of women with ovulatory dysfunctional bleeding, and a control group of women with entirely normal cycles. There was increased leukocytic infiltration of the endometrium in both groups of women with menorrhagia compared to control cases. In addition, IUCD use was associated with greater luteal phase glandular epithelial height, supporting the concept of a secretory function defect in glandular epithelium. Possible links are postulated between static morphometric observations and dynamic biochemical changes.

Australia↗