Biomedical subjects

P Russell

Publications and source records attributed to P Russell.

At least 73 records · Page 4Linked to original sources

PubMed2000

Use of a lipophilic cation to monitor electrical membrane potential in the intact rat lens.

PURPOSE: Tetraphenylphosphonium (TPP+) is a permeant lipophilic cation that accumulates in cultured cells and tissues as a function of the electrical membrane potential across the plasma membrane. This study was undertaken to determine whether TPP+ can be used for assessing membrane potential in intact lenses in organ culture. METHODS: Rat lenses were cultured in media containing 10 microM TPP+ and a tracer level of 3H-TPP+ for various times. 3H-TPP+ levels in whole lenses or dissected portions of lenses were determined by liquid scintillation counting. Ionophores, transport inhibitors, and neurotransmitters were also added to investigate their effects on TPP+ uptake. RESULTS. Incubation of lenses in low-K+ balanced salt solution and TC-199 medium, containing physiological concentrations of Na+ and K+, led to a biphasic accumulation of TPP+ in the lens that approached equilibrium by 12 to 16 hours of culture. The TPP+ equilibrated within 1 hour in the epithelium but penetrated more slowly into the fiber mass. The steady state level of TPP+ accumulation in the lens was depressed by 90% when the lenses were cultured in a medium containing high K+. The calculated membrane potential for the normal rat lens in TC-199 was -75 +/- 3 mV. Monensin (1 microM) and nigericin (1 microM), Na+H+ and K+H+ exchangers respectively, as well as the protonophore carbonylcyanide-m-chlorophenylhydrazone (CCCP, 10 microM) and the calcium ionophore A23187 (10 microM), abolished TPP+ accumulation and caused cloudiness of the lenses. The neurotransmitter acetylcholine at 50 microM decreased TPP+ accumulation in the lens, but this effect could be prevented by simultaneous application of 1 mM atropine. CONCLUSIONS: TPP+ accumulation can be used as an indicator of changes in membrane potential in intact lenses, but because of the long time required to reach steady state, its utility is limited. The slow accumulation of TPP+ and its slow efflux from the lens under conditions known to depolarize membranes are consistent with a diffusion barrier in the deep cortex and nucleus of the lens.

Animals↗

PubMed2000

Localization of myocilin/trabecular meshwork--inducible glucocorticoid response protein in the human eye.

PURPOSE: To study distribution and cellular localization of myocilin/trabecular meshwork-inducible glucocorticoid response protein (TIGR) in the human eye. METHODS: A peptide antibody against a portion of the myosin-like domain of myocilin/TIGR was developed. Different ocular tissues from three human donors were investigated by one- and two-dimensional gel electrophoresis and Western blot analysis. Immunohistochemistry was performed on 25 human eyes enucleated because of posterior choroidal melanoma and on 7 normal human donor eyes. RESULTS: By Western blot analysis, a band at approximately 57 kDa was visualized in cornea, trabecular meshwork, lamina cribrosa, optic nerve, retina, iris, ciliary body, and vitreous humor. By immunohistochemistry, immunoreactivity for myocilin/TIGR was observed in cells of the corneal epi- and endothelium and extracellularly in the corneal stroma and sclera. In the trabecular meshwork, cells of the uveal and corneoscleral meshwork were stained, as was the cribriform area directly adjacent to Schlemm's canal. Positive staining was seen in cells of the ciliary epithelium, ciliary muscle, lens epithelium, and in stromal and smooth muscle cells of the iris. Throughout the entire vitreous body, fine filamentous material was positively labeled. In the retina, staining was seen along the outer surface of rods and cones, in neurons of the inner and outer nuclear layer, and in the axons of optic nerve ganglion cells. Optic nerve axons were stained in the prelaminar, laminar, and postlaminar parts of the nerve. In the region of the lamina cribrosa, astrocytes in the glial columns and cribriform plates were positively labeled. CONCLUSIONS: Myocilin TIGR is expressed in almost every ocular tissue. Depending on the respective tissue, it is observed extra- or intracellularly. The presence of myocilin/TIGR in optic nerve axons and lamina cribrosa astrocytes indicates that the trabecular meshwork might not be the only target of abnormal myocilin/TIGR in GLC1A-linked open-angle glaucoma.

Aged↗

PubMed1999

DNA replication checkpoint control.

The eukaryotic cell cycle comprises two critical phases, DNA replication (S phase) and the subsequent distribution of an equivalent genome to each of two daughter cells at mitosis (M phase). A signal transduction cascade called the replication checkpoint has evolved to ensure that M phase does not occur prior to the completion of S phase. The mitotic regulators targeted by this checkpoint have recently been identified in the fission yeast Schizosaccharomyces pombe. As was the case for the DNA damage checkpoint, studies on the replication checkpoint in fission yeast promise to provide an excellent framework for analogous studies in mammalian cells.

Animals↗

PubMed1999

Diaphragmatic endometriosis.

Endometriosis is a relatively common condition usually found in the pelvis. However, lesions do occur outside the pelvis and, more rarely, in the upper abdomen. In the case reported here, the patient presented with chronic right shoulder tip pain. The diagnosis of extrapelvic endometriosis is often not considered in such circumstances. This patient's symptoms were relieved by surgical excision of the diaphragmatic lesion.

Adult↗

PubMed1999

A new inducible protein expression system in fission yeast based on the glucose-repressed inv1 promoter.

Studies of the fission yeast Schizosaccharomyces pombe have made major contributions towards understanding cell-cycle control and many other important aspects of cell biology. A series of pREP expression vectors that utilize the thiamine-repressible nmt1 promoter are used routinely to manipulate the expression of genes in fission yeast. A shortcoming of the nmt1 promoter is that it is very slowly induced following removal of thiamine from the growth medium, requiring approx. 16h for full induction. Invertase, an enzyme responsible for sucrose metabolism, is regulated transcriptionally by glucose derepression in S. pombe. Using the inv1 promoter, we have developed the pINV1 set of inducible protein expression vectors. A shift from glucose to sucrose-based culture medium leads to a very rapid induction of the inv1 promoter. Genes that are regulated by the inv1 promoter are fully induced within 1h of the shift to sucrose-based medium. The pINV1 vectors utilize a simple induction protocol and enable studies in fission yeast requiring tight and rapid regulation of protein synthesis.

Culture Media↗

PubMed1999

Nuclear localization of Cdc25 is regulated by DNA damage and a 14-3-3 protein.

DNA damage activates a cell-cycle checkpoint that prevents mitosis while DNA repair is under way. The protein Chk1 enforces this checkpoint by phosphorylating the mitotic inducer Cdc25. Phosphorylation of Cdc25 by Chk1 creates a binding site in Cdc25 for 14-3-3 proteins, but it is not known how 14-3-3 proteins regulate Cdc25. Rad24 is a 14-3-3 protein that is important in the DNA-damage checkpoint in fission yeast. Here we show that Rad24 controls the intracellular distribution of Cdc25. Elimination of Rad24 causes nuclear accumulation of Cdc25. Activation of the DNA-damage checkpoint causes the net nuclear export of Cdc25 by a process that requires Chk1, Rad24 and nuclear-export machinery. Mutation of a putative nuclear-export signal in Rad24 impairs the nuclear exclusion of Rad24, the damage-induced nuclear export of Cdc25 and the damage checkpoint. Thus, Rad24 appears to function as an attachable nuclear-export signal that enhances the nuclear export of Cdc25 in response to DNA damage.

14-3-3 Proteins↗

PubMed1999

A longitudinal study of changes in endometrial microvascular density in Norplant implant users.

This study aimed to investigate the effects of the subdermal levonorgestrel contraceptive implant Norplant on endometrial vascular density at different durations of exposure, and the relationship between endometrial histology, vascular density, and bleeding patterns. A prospective controlled trial of Norplant implant users compared endometrial vascular density in biopsies taken before and after Norplant implant insertion. A total of 34 women with regular menstrual cycles requesting long-term contraception were recruited at the Sydney Centre for Reproductive Health Research, Australia. A significant increase in mean endometrial microvascular density was observed from as early as 3 weeks after insertion of Norplant implants. Vascular density was increased from a control secretory phase value of 189.6 (7.0 vessels/mm2 (+/- SEM) to 253.80 +/- 7 vessels/mm2 at 2-13 weeks of Norplant implant exposure (t ratio = 2.08, p = 0.01) and 212.7 +/- 12.9 vessels/mm2 at 14-42 weeks of exposure (t ratio = 2.03, p = 0.02). In those with atrophic endometrium, or in whom myometrium and basalis only were found in biopsies (20 of 66, 30%), mean endometrial vascular density was increased at 273.1 +/- 16.1 vessels/mm2 compared with 210.9 +/- 11.7 vessels/mm2 in other histological groups (F ratio = 9.74, p = 0.0028). Bleeding and spotting in the previous 30 days were less common in those with this histological appearance at a mean of 4.95 days compared with 8.22 days. This is the first study to assess endometrial vascular density in the early months of Norplant implant use. The findings suggest that the endometrial vasculature is profoundly altered in the early months of Norplant implant exposure when bleeding problems are most common.

Adolescent↗

PubMed1999

Hormone replacement therapy and risk of epithelial ovarian cancer.

It has been suggested that oestrogen replacement therapy is associated with risk of epithelial ovarian cancer of the endometrioid type. Using data from an Australian population-based case-control study, the relation between unopposed oestrogen replacement therapy and epithelial ovarian cancer, both overall and according to histological type, was examined. A total of 793 eligible incident cases of epithelial ovarian cancer diagnosed from 1990 to 1993 among women living in Queensland, New South Wales and Victoria were identified. These were compared with 855 eligible female controls selected at random from the electoral roll, stratified by age and geographic region. Trained interviewers administered standard questionnaires to obtain detailed reproductive and contraceptive histories, as well as details about hormone replacement therapy and pelvic operations. No clear associations were observed between use of hormone replacement therapy overall and risk of ovarian cancer. Unopposed oestrogen replacement therapy was, however, associated with a significant increase in risk of endometrioid or clear cell epithelial ovarian tumours (odds ratio (OR) 2.56; 95% confidence interval (CI) 1.32-4.94). In addition, the risk associated with oestrogen replacement therapy was much larger in women with an intact genital tract (OR 3.00; 95% CI 1.54-5.85) than in those with a history of either hysterectomy or tubal ligation. Post-menopausal oestrogen replacement therapy may, therefore, be a risk factor associated with endometrioid and clear cell tumours in particular. Additionally, the risk may be increased predominantly in women with an intact genital tract. These associations could reflect a possible role of endometriosis in the development of endometrioid or clear cell ovarian tumours.

Adenocarcinoma↗

PubMed1999

The contribution of germline BRCA1 and BRCA2 mutations to familial ovarian cancer: no evidence for other ovarian cancer-susceptibility genes.

To establish the contribution of germline BRCA1 and BRCA2 mutations to familial ovarian cancer, we have analyzed both genes in DNA samples obtained from an affected individual in each of 112 families containing at least two cases of epithelial ovarian cancer. Germline mutations were found in 43% of the families; BRCA1 mutations were approximately four times more common than BRCA2 mutations. The extent of family history of ovarian and breast cancers was strongly predictive of BRCA1-mutation status. Segregation analysis suggests that a combination of chance clustering of sporadic cases and insensitivity of mutation detection may account for the remaining families; however, the contribution of other genes cannot be excluded. We discuss the implications for genetic testing and clinical management of familial ovarian cancer arising from the data presented in these studies.

BRCA2 Protein↗

PubMed1999

Cdc25 inhibited in vivo and in vitro by checkpoint kinases Cds1 and Chk1.

In the fission yeast Schizosaccharomyces pombe, the protein kinase Cds1 is activated by the S-M replication checkpoint that prevents mitosis when DNA is incompletely replicated. Cds1 is proposed to regulate Wee1 and Mik1, two tyrosine kinases that inhibit the mitotic kinase Cdc2. Here, we present evidence from in vivo and in vitro studies, which indicates that Cds1 also inhibits Cdc25, the phosphatase that activates Cdc2. In an in vivo assay that measures the rate at which Cdc25 catalyzes mitosis, Cds1 contributed to a mitotic delay imposed by the S-M replication checkpoint. Cds1 also inhibited Cdc25-dependent activation of Cdc2 in vitro. Chk1, a protein kinase that is required for the G2-M damage checkpoint that prevents mitosis while DNA is being repaired, also inhibited Cdc25 in the in vitro assay. In vitro, Cds1 and Chk1 phosphorylated Cdc25 predominantly on serine-99. The Cdc25 alanine-99 mutation partially impaired the S-M replication and G2-M damage checkpoints in vivo. Thus, Cds1 and Chk1 seem to act in different checkpoint responses to regulate Cdc25 by similar mechanisms.

Cell Cycle↗

PubMed1999

Active nucleocytoplasmic shuttling required for function and regulation of stress-activated kinase Spc1/StyI in fission yeast.

Transcriptional induction of many stress-response genes is dependent on stress-induced nuclear accumulation of stress-activated protein kinases (SAPKs). In the fission yeast Schizosaccharomyces pombe, nuclear accumulation of the SAPK Spc1 (also known as StyI) requires activating phosphorylation catalyzed by the SAPK kinase Wis1; however, it is unknown whether the localization of Spc1 is regulated by nuclear transport factors. Herein are reported studies that show that Spc1 localization is regulated by active transport mechanisms during osmotic stress. Nuclear import of Spc1 requires Pim1, a homologue of the guanine nucleotide exchange factor RCC1 that is essential for nucleocytoplasmic shuttling of proteins. Nuclear export of Spc1 is regulated by the export factor Crm1. An Spc1-Crm1 complex forms as Spc1 is exported from the nucleus. Wis1 and the tyrosine phosphatases Pyp1 and Pyp2 that inactivate Spc1 are excluded from the nucleus by a Crm1-independent mechanism; hence the nuclear import of Spc1 leads to transient isolation from its regulatory proteins. Thus, active nucleocytoplasmic shuttling is required for both the function and regulation of Spc1 during the osmotic shock response.

Antigens, Viral, Tumor↗

PubMed1999

Vacuole fusion regulated by protein phosphatase 2C in fission yeast.

The gene ptc4+ encodes one of four type 2C protein phosphatases (PP2C) in the fission yeast Schizosaccharomyces pombe. Deletion of ptc4+ is not lethal; however, Deltaptc4 cells grow slowly in defined minimal medium and undergo premature growth arrest in response to nitrogen starvation. Interestingly, Deltaptc4 cells are unable to fuse vacuoles in response to hypotonic stress or nutrient starvation. Conversely, Ptc4 overexpression appears to induce vacuole fusion. These findings reveal a hitherto unrecognized function of type 2C protein phosphatases: regulation of vacuole fusion. Ptc4 localizes in vacuole membranes, which suggests that Ptc4 regulates vacuole fusion by dephosphorylation of one or more proteins in the vacuole membrane. Vacuole function is required for the process of autophagy that is induced by nutrient starvation; thus, the vacuole defect of Deltaptc4 cells might explain why these cells undergo premature growth arrest in response to nitrogen starvation.

Amino Acid Sequence↗

PubMed1999

Changes in vascular basement membrane in the endometrium of Norplant users.

Progestogen-only contraception is almost invariably associated with changes in menstrual bleeding patterns. Changes in the endometrial vasculature, and in particular an increase in vascular fragility, may contribute to this bleeding. In this study, endometrial vascular density and endothelial cell basement membrane components were examined using immunohistochemistry before and after insertion of Norplant. Endometrial vascular density was increased from a mean (+/- SEM) of 189.6 +/- 7.0 vessels/mm2 during the control cycle to 253.9 +/- 80.7 vessels/mm2 at 2-13 weeks of Norplant exposure, and to 212.7 +/- 12.9 vessels/mm2 at 14-42 weeks. During the control cycle, a mean of 161.4 +/- 4.5 vessels/mm2 stained for collagen IV (85% of all vessels), while at 2-13 weeks, 144.5 +/- 13.0 vessels/mm2 stained for collagen IV (57% of all vessels) (t ratio = 2.08, P = 0.0057). By 14-42 weeks, 71% of vessels (151.0 +/- 9.8) vessels/mm2 were surrounded by collagen IV. This was not significantly different from control values (t ratio = 2.03). Endometrial vascular laminin was also reduced following Norplant insertion, from a mean of 176.0 +/- 4.2 vessels/mm2 in the control cycle (93% of vessels), to 156.3 +/- 6.7 vessels/mm2 at 2-13 weeks of exposure (57% of vessels) (t ratio = 2.08, P = 0.01). By 14-42 weeks of exposure to Norplant, 162.5 +/- 9 vessels/mm2 (76%) stained for laminin. This was not significantly different from control values (t ratio = 2.04). Endometrial vascular heparan sulphate proteoglycan (HSPG) was reduced from 58.6 +/- 3.0 vessels/mm2 during the control cycle (31% of vessels) to 43.6 +/- 5.6 vessels/mm2 (only 17% of vessels) at 2-13 weeks (t ratio = 2.08, P = 0.025). At 14-42 weeks, only 19% of vessels stained for HSPG (41.3 +/- 5.8 vessels/mm2; t ratio = 2.04, P = 0.009).

Adolescent↗

PubMed1999

Proliferating (LMP) mucinous tumors of the ovaries with microinvasion: morphologic assessment of 13 cases.

From a consecutive series of 124 proliferating (low malignant potential) mucinous ovarian tumors, 13 FIGO stage Ia tumors exhibited 1 or more microfoci of stromal invasion. The proliferating mucinous tumors with microinvasion were of intestinal (7/88), endocervical-like (4/26), or mixed epithelial differentiation (2/10) and were more likely to show high-grade (10/42) than low-grade (3/82) epithelial proliferative changes. Morphologically, foci of microinvasion were characterized by single or small groups of epithelial cells in the tumor stroma. These cells were accompanied by a sparse lymphocytic infiltrate and stromal desmoplasia in their immediate vicinity and by a greater degree of nuclear anaplasia in the invading cells and in the lining cells of the nearby glands or cysts than was present elsewhere in the tumors. Focal inflammatory and degenerative changes, commonly seen in ovarian mucinous tumors, were discriminated from microinvasive carcinoma by the nature of the inflammatory response, the presence of stromal mucin deposits, and the absence of nuclear anaplasia. Follow-up was available in 10 of 13 cases (mean 7.5 years) and did not identify any adverse clinical consequences for the patients.

Adult↗

PubMed1999

The SCID/Beige mouse as a model to investigate protection against Yersinia pestis.

In this study, we have shown that severe combined immunodeficient/beige mice reconstituted with hyperimmune Balb/c lymphocytes can be used as a model to demonstrate adoptive and passive protection against plague infection. Reconstitution of severe combined immunodeficient/beige mice was successful in nine out of ten mice as demonstrated by spleen colonisation and sustained circulating immunoglobulin titres. Furthermore, an increase in antibody titre was evident after a booster immunisation of reconstituted mice. Presence of circulating antibody correlated with protection against a systemic plague challenge and indicated that in reconstituted mice adoptive transfer of a functional immune system had occurred. The severe combined immunodeficient/beige mouse was also used to demonstrate passive protection against inhaled and systemic plague infection. The reconstituted severe combined immunodeficient/beige mouse model demonstrating protective immunity against plague will be further developed to identify the immune cell subsets responsible for this protection.

Adoptive Transfer↗

PubMed1999

In vitro susceptibilities of Burkholderia mallei in comparison to those of other pathogenic Burkholderia spp.

The in vitro antimicrobial susceptibilities of isolates of Burkholderia mallei to 16 antibiotics were assessed and compared with the susceptibilities of Burkholderia pseudomallei and Burkholderia cepacia. The antibiotic susceptibility profile of B. mallei resembled that of B. pseudomallei more closely than that of B. cepacia, which corresponds to their similarities in terms of biochemistry, antigenicity, and pathogenicity. Ceftazidime, imipenem, doxycycline, and ciprofloxacin were active against both B. mallei and B. pseudomallei. Gentamicin was active against B. mallei but not against B. pseudomallei. Antibiotics clinically proven to be effective in the treatment of melioidosis may therefore be effective for treating glanders.

Anti-Bacterial Agents↗

PubMed1999

Basis for the checkpoint signal specificity that regulates Chk1 and Cds1 protein kinases.

Six checkpoint Rad proteins (Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1) are needed to regulate checkpoint protein kinases Chk1 and Cds1 in fission yeast. Chk1 is required to prevent mitosis when DNA is damaged by ionizing radiation (IR), whereas either kinase is sufficient to prevent mitosis when DNA replication is inhibited by hydroxyurea (HU). Checkpoint Rad proteins are required for IR-induced phosphorylation of Chk1 and HU-induced activation of Cds1. IR activates Cds1 only during the DNA synthesis (S) phase, whereas HU induces Chk1 phosphorylation only in cds1 mutants. Here, we investigate the basis of the checkpoint signal specificity of Chk1 phosphorylation and Cds1 activation. We show that IR fails to induce Chk1 phosphorylation in HU-arrested cells. Release from the HU arrest following IR causes substantial Chk1 phosphorylation. These and other data indicate that Cds1 prevents Chk1 phosphorylation in HU-arrested cells, which suggests that Cds1 actively suppresses a repair process that leads to Chk1 phosphorylation. Cds1 becomes more highly concentrated in the nucleus only during the S phase of the cell cycle. This finding correlates with S-phase specificity of IR-induced activation of Cds1. However, constitutive nuclear localization of Cds1 does not enhance IR-induced activation of Cds1. This result suggests that Cds1 activation requires DNA structures or protein activities that are present only during S phase. These findings help to explain how Chk1 and Cds1 respond to different checkpoint signals.

Cell Cycle↗

PubMed1999

Na+, K+-ATPase inhibitors down-regulate gene expression of the intracellular signaling protein 14-3-3 in rat lens.

To identify genes that are differentially expressed by Na+, K+-ATPase inhibitors, we used the differential display technique to compare mRNA expression patterns in rat lens. Lenses were treated with 10 microM ouabain, bufalin, or 19-norbufalin derivative for 24 h and were compared with control lenses. Differential display analysis revealed that one of the down-regulated genes was 14-3-3 theta. Down-regulation was confirmed by Northern blot and by reverse transcription-polymerase chain reaction analysis. Reverse transcription-polymerase chain reaction of additional 14-3-3 isoforms revealed that the eta and gamma isoforms of 14-3-3 are also down-regulated by ouabain, bufalin, and 19-norbufalin derivative, whereas the zeta isoform is down-regulated only by bufalin. Down-regulation of the 14-3-3 isoforms occurred without a significant change in gamma-crystallin gene expression. These results demonstrate that one of the consequences of Na+, K+-ATPase inhibition by exogenous or endogenous inhibitors is the down-regulation of mRNA transcripts encoding several isoforms of 14-3-3. Because the 14-3-3 proteins are multifunctional regulatory proteins, the reduction in the abundance of various isoforms will have profound effects on cell function.

14-3-3 Proteins↗