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Biomedical subjects

C J Watson

Publications and source records attributed to C J Watson.

At least 37 records · Page 2Linked to original sources
PubMed2001

p53-dependent apoptosis induced by proteasome inhibition in mammary epithelial cells.

We have examined the effects of inhibition of the 26S proteasome in a murine mammary cell line, KIM-2 cells using the peptide aldehyde inhibitor MG132. These studies have demonstrated a clear requirement for proteasome function in cell viability. Induction of apoptosis was observed following MG132 treatment in KIM-2 cells and this death was shown to be dependent on the cell actively traversing the cell cycle. KIM-2 cells were generated using a temperature sensitive T-antigen (Tag) and studies at the permissive temperature (33 degrees C) have shown that a Tag binding protein was essential for this apoptotic response. Studies in two additional cell lines, HC11, which is a mammary epithelial cell line carrying mutant p53 alleles and p53 null ES cells suggest that p53 is actively required for the apoptosis induced as a consequence of proteasome inhibition. These results suggest a pivotal role for the 26S proteasome degradation pathway in progression through the cell cycle in proliferating cells.

Animals↗
PubMed2001

Rising incidence of post-transplant lymphoproliferative disease in kidney transplant recipients.

BACKGROUND: The purpose of this study was to determine whether the incidence of post-transplant lymphoproliferative disease (PTLD) has been increasing in renal transplant recipients in this centre. METHODS: Prospectively gathered data were analysed to establish trends in the epidemiology of PTLD in 1537 patients. RESULTS: Overall, PTLD occurred in 2.3 per cent of renal transplant recipients. An increase in its incidence coincided with the introduction of cyclosporin in the 1980s. However, there was a further increase in the incidence of PTLD in the 1990s when the only change in immunosuppressive policy was the abandonment of pretransplantation blood transfusion. The latter increase was particularly pronounced in patients with early-onset PTLD in whom it presented within 600 days after transplantation. CONCLUSION: The incidence of PTLD has been increasing in renal transplant recipients. The recent increase appears to be independent of cyclosporin and may reflect the reduction in pretransplant blood transfusion. Changes in the incidence of PTLD may also mirror changes in the epidemiology of non-Hodgkin lymphoma in the general population.

England↗
PubMed2001

Localization of CYP86B1 in the outer envelope of chloroplasts.

CYP86B1 was cloned from a cDNA library and the protein expressed in E. coli. The protein gave the expected carbon monoxide difference spectrum. Using in vitro import assays with isolated pea chloroplasts, CYP86B1 was shown to be associated with the outer chloroplastic envelope membrane. This study provides the first direct evidence for a chloroplast-localized cytochrome P450-dependent monooxygenase.

Amino Acid Sequence↗
PubMed2001

Functional modeling of tight junctions in intestinal cell monolayers using polyethylene glycol oligomers.

Despite significant advances in the characterization of tight junction (TJ) proteins, little is known about how molecular changes relate to function due primarily to the limitations of conventional paracellular probes. To address this, the paracellular pathway in Caco-2 and T84 cell lines was profiled by measuring the permeabilities of 24 polyethylene glycols (PEG) of increasing molecular radius (3.5--7.4 A) analyzed by mass spectrometry. When combined with a paracellular sieving model, these data provided quantitative descriptors of the pathway under control conditions and after exposure to TJ modulators. PEG profiles in both cell lines conformed to a biphasic process involving a restrictive pore (radius 4.3--4.5 A) and a nonrestrictive component responsible for permeability of larger molecules. PEG profiling revealed significant differences between the effects of EGTA and sodium caprate (C10). The restrictive component of EGTA-treated cells lost all size discrimination due to an increase in pore radius. Sodium caprate had no effect on pore radius but increased permeability via a different mechanism possibly involving increased numbers of functional pores. PEG profiling provides a useful tool for probing the functional regulation of the paracellular route.

Biomarkers↗
PubMed2001

Implant complications and failures: the complete overdenture.

The implant-retained overdenture has been advocated as an effective method of restoring the edentulous patient with an appliance that offers significant benefits over conventional prosthetics. However, the success and survival of such appliances have been shown to vary considerably, depending on which jaw is treated: implants supporting overdentures in the maxilla have a significantly higher failure rate. The restoration also requires considerable maintenance, which is both time consuming and costly. The purpose of this paper is to look at common restorative complications and maintenance problems following the placement of dental implants to support an overdenture. A number of implant systems have been reviewed and the results of the authors' clinical experiences with referred and their own cases are reported.

Bite Force↗
PubMed2001

Reliability of the lateral pull test and tilt test to assess patellar alignment in subjects with symptomatic knees: student raters.

STUDY DESIGN: Test-retest reliability with blinded testers. OBJECTIVES: To determine the inter- and intra-rater reliability of the lateral pull test and patellar tilt test. BACKGROUND: If patellar malalignment can be detected by clinical examination, then condition-specific treatment interventions may be implemented in patients with patellofemoral pain syndrome. However, several clinical tests used to assess patellar mobility have recently been shown to have poor to fair reliability. Because the lateral pull test and the patellar tilt test are widely used clinically as diagnostic tests for patellofemoral pain syndrome but have not been previously tested for reliability, we examined these tests. METHODS AND MEASURES: Fifty-two subjects (age range, 21-48 years) provided 95 knees (19 symptomatic and 76 asymptomatic) for assessment of the lateral pull test. Two testers, blinded to the presence or absence of symptoms, independently performed the lateral pull test in random order. Fifty-five subjects (age range, 22-42 years) provided 99 knees (73 asymptomatic and 26 symptomatic) for assessment of the patellar tilt test. Three blinded testers independently performed the patellar tilt test in random order. All subjects were tested and retested within 3-5 days. A kappa (kappa) statistic was used to assess the agreement of findings within each tester and between testers. RESULTS: The kappa coefficients for intrarater reliability varied from 0.39 to 0.47 for the lateral pull test and from 0.44 to 0.50 for the patellar tilt test, while the coefficients for interrater reliability were 0.31 for the lateral pull test and varied from 0.20 to 0.35 for the tilt test. CONCLUSIONS: Repeated lateral pull tests and patellar tilt tests had fair intrarater and poor interrater reliability. Our results suggest that care must be taken in placing too much emphasis on these tests when making clinical decisions.

Adult↗
PubMed2001

Case report: the transmandibular implant system.

This series of case reports outlines the use of the transmandibular implant system to rehabilitate 10 patients with severely resorbed mandibles. The implants were placed according to manufacturers' guidelines with the patient under a general anaesthetic and reviewed bi-annually thereafter. Three patients experienced problems: in two of these this involved the loss of integration of the distal posts, in the other patient the transmandibular implant was removed in its entirety. Distal locking screws became loose shortly after placement in three patients. Prosthetic complications included difficulty in obtaining passive fit of the superstructure in 7 patients and a high long-term maintenance commitment.

Aged↗
PubMed2000

A novel role for IRF-1 as a suppressor of apoptosis.

The tumour suppressor IRF-1 is a transcription factor involved in the induction of apoptosis in several in vitro systems. Post-lactational involution of the mammary gland is characterized by extensive apoptosis of the epithelial cells. We have previously shown that signal transducer and activator of transcription (Stat) 3 drives apoptosis and involution in the mouse mammary gland. Since one of the downstream targets of the Stat signalling pathway is IRF-1, we have used IRF-1 knockout mice to address the potential role of this transcription factor in involution. Surprisingly, in the absence of IRF-1 significantly higher numbers of apoptotic cells were found in involuting glands at 48 h compared to control glands. In addition, the alveolar structure in IRF-1 null mammary glands had collapsed whereas in control glands the alveoli remained intact and distended. However, by 72 h control and null glands were morphologically similar suggesting that IRF-1 suppresses apoptosis only during the early, reversible, stage of involution. This suggests a survival role for IRF-1 in mammary epithelia and demonstrates a novel role for IRF-1 in vivo--suppression of premature epithelial apoptosis during mammary gland involution.

Animals↗
PubMed2000

Transient inhibition of interleukin 4 signaling by T cell receptor ligation.

Interleukin (IL)-4 and IL-12 together with T cell receptor (TCR) engagement are crucial for the differentiation of CD4(+) T cells into T helper (Th)2 or Th1 cells, respectively. Although IL-4 receptors (IL-4Rs) but not IL-12Rs are expressed on naive CD4(+) T cells, IL-4 has no apparent advantage over IL-12 in driving naive T cell differentiation when the cells are primed with both IL-4 and IL-12 in vitro. It was found that IL-4-induced phosphorylation of Janus kinases 1 and 3, IL-4R alpha, signal transducer and activator of transcription 6, and insulin receptor substrate 2 was strikingly but transiently inhibited by TCR ligation both in conventional and TCR transgenic T cells. TCR engagement also blocked the expression of an IL-4-inducible gene. Signals induced by other cytokines, including IL-2, IL-6, and interferon alpha, but not by insulin-like growth factor 1, were also blocked by TCR engagement. The capacity of various inhibitors to reverse TCR-mediated inhibition of IL-4 signaling suggested that activation of the Ras-mitogen-activated protein kinase pathway and of the calcineurin pathway contribute to desensitizing IL-4R. IL-4 responsiveness returned at about the time ( approximately 12 h) that IL-12-mediated signaling was first observed. Thus, through different mechanisms, neither IL-4R nor IL-12R has any clear advantage in polarizing cells; rather, the availability of cytokine is probably the limiting factor in this process.

Animals↗
PubMed2000

NF-kappaB inhibits apoptosis in murine mammary epithelia.

The transcription factor NF-kappaB is a key modulator of apoptosis in a variety of cell types, but to date this specific function of NF-kappaB has not been demonstrated in epithelia. Here, we describe the activation of NF-kappaB during post-lactational involution of the mouse mammary gland, a period of extensive apoptosis of luminal epithelial cells. Significantly, active NF-kappaB localized exclusively to nonapoptotic epithelial cells both in vivo and in the mammary epithelial cell line, KIM-2, transduced with an NF-kappaB-dependent green fluorescent protein reporter. Activation of NF-kappaB in vitro coincided with a decrease in the cytosolic repressor, IkappaBalpha. Furthermore, induction of NF-kappaB either by extracellular ligands or, more specifically, by inhibition of the IkappaB repressor with adenoviral constructs expressing antisense mRNA, resulted in enhanced survival of KIM-2 cells. Therefore, although coincident with induction of apoptosis both in vivo and in vitro, NF-kappaB appeared to exert a selective survival function in epithelial cells. This study highlights for the first time a role for NF-kappaB in modulating apoptosis in epithelium.

Adenoviridae↗
PubMed2000

A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland.

BACKGROUND: This paper describes the derivation and characterization of a novel, conditionally immortal mammary epithelial cell line named KIM-2. These cells were derived from mid-pregnant mammary glands of a mouse harbouring one to two copies of a transgene comprised of the ovine beta-lactoglobulin milk protein gene promoter, driving expression of a temperature-sensitive variant of simian virus-40 (SV40) large T antigen (T-Ag). RESULTS: KIM-2 cells have a characteristic luminal epithelial cell morphology and a stable, nontransformed phenotype at the semipermissive temperature of 37 degrees C. In contrast, at the permissive temperature of 33 degrees C the cells have an elongated spindle-like morphology and become transformed after prolonged culture. Differentiation of KIM-2 cells at 37 degrees C, in response to lactogenic hormones, results in the formation of polarized dome-like structures with tight junctions. This is accompanied by expression of the milk protein genes that encode beta-casein and whey acidic protein (WAP), and activation of the prolactin signalling molecule, signal transducer and activator of transcription (STAT)5. Fully differentiated KIM-2 cultures at 37 degrees C become dependent on lactogenic hormones for survival and undergo extensive apoptosis upon hormone withdrawal, as indicated by nuclear morphology and flow cytometric analysis. KIM-2 cells can be genetically modified by stable transfection and clonal lines isolated that retain the characteristics of untransfected cells. CONCLUSION: KIM-2 cells are a valuable addition, therefore, to currently available lines of mammary epithelial cells. Their capacity for extensive differentiation in the absence of exogenously added basement membrane, and ability to undergo apoptosis in response to physiological signals will provide an invaluable model system for the study of signal transduction pathways and transcriptional regulatory mechanisms that control differentiation and involution in the mammary gland.

Animals↗
PubMed2000

Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei.

The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a K(m) of 9.4 +/- 2.8 microM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (K(m) = 0.38 +/- 0.06 microM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5-15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (K(m) remained unchanged) and inhibited by cycloheximide (10 microM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (K(m) approximately 0.2 microM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (K(m) approximately 10 microM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.

Adenosine↗
PubMed2000

Identification of polymorphisms within the vascular endothelial growth factor (VEGF) gene: correlation with variation in VEGF protein production.

Dysregulated vascular endothelial growth factor (VEGF) expression has been implicated as a major contributor to the development of a number of common disease pathologies. The aim of this study was to establish the extent of genetic variability within the VEGF gene and to determine whether this genetic variation influenced levels of VEGF protein expression. The promoter region and exon 1 of the VEGF gene were screened for polymorphisms using single-stranded conformation (SSCP) polymorphism analysis and direct PCR-sequencing. We identified 15 novel sequence polymorphisms most of which were rare. Eleven of these polymorphisms were single base substitutions, three were single base insertions and one was a two base deletion. Thirteen of the polymorphisms were located within the promoter and two in the 5' untranslated region (5'UTR) of the gene. We established PCR-RFLP typing systems for ten of the polymorphisms. For the two common polymorphisms at -460 and +405, we developed a combined sequence specific priming (SSP) PCR typing system to determine the cis/trans orientation of each allele and hence, ascertain haplotypes. A significant correlation was observed between lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cell (PBMC) VEGF protein production and genotype for the +405 polymorphism.

5' Untranslated Regions↗
PubMed2000

The role of Stat3 in apoptosis and mammary gland involution. Conditional deletion of Stat3.

STATs (signal transducer and activator of transcription) are a family of latent transcription factors which are activated in response to a variety of cytokines and growth factors. This family of signalling molecules have been implicated in growth, differentiation, survival and apoptosis. In this article, we will review work which highlights the role of individual STAT factors in mammary gland and demonstrate the value of genetically modified mice in defining the function of STAT3. Involution of the mouse mammary gland is characterised by extensive apoptosis of the epithelial cells and the activation of STAT3. STATs 3 and 5 have reciprocal patterns of activation throughout a mammary developmental cycle suggesting that STAT5 may be a survival factor and STAT3 a death factor for differentiated mammary epithelium. To clarify the role of STAT3 in mammary epithelial apoptosis, we have generated a conditional knockout using the lox/Cre recombination system. Mammary glands from crosses of transgenic mice expressing Cre recombinase under the control of the beta-lactoglobulin milk protein gene promoter with mice harbouring one floxed STAT3 allele and one null STAT3 allele, showed a decrease in epithelial apoptosis and a dramatic delay of the involution process upon forced weaning. This was accompanied by precocious activation of STAT1 and increases in p53 and p21 levels--these may act as a compensatory mechanism for initiating the eventual involution which occurs in STAT3 null mammary glands. This demonstrates for the first time the importance of STAT factors in signalling the initiation of physiological apoptosis in vivo and highlights the utility of the lox/Cre system for addressing the function of genes, which have an embryonic lethal phenotype, specifically in mammary gland.

Animals↗
PubMed2000

Placenta growth factor (PlGF) induces vascular endothelial growth factor (VEGF) secretion from mononuclear cells and is co-expressed with VEGF in synovial fluid.

The aims of this study were (i) to determine whether PlGF, VEGF and PlGF/VEGF heterodimers are detected in synovial fluid (SF) and plasma samples from patients with a range of arthropathies; (ii) to describe whether any correlation exists between SF PlGF, VEGF and PlGF/VEGF heterodimer levels and the total and differential SF leucocyte counts; and (iii) to investigate the regulation of peripheral blood mononuclear cell (PBMC) VEGF secretion by stimuli relevant to inflammatory joints. PlGF, VEGF and PlGF/VEGF heterodimer levels were measured in the SF and plasma of patients with a range of arthropathies and normal controls by ELISA. Western blotting for PlGF was performed on SF from three patients with rheumatoid arthritis (RA) and primary inflammatory arthropathies. VEGF was quantified in cell culture supernatants after stimulation with lipopolysaccharide (LPS), PlGF or cobalt ions of PBMC isolated from RA patients and controls. PlGF and VEGF were detected in all SF samples. PlGF/VEGF heterodimers were detected in 10.2% of SF samples, most frequently in RA samples. Western blotting confirmed the presence of PlGF in RA SF. PlGF was detected in 52% of RA and 31% of control plasma samples, and VEGF was detected in 38% of RA and 38% of control plasma samples. PlGF/VEGF heterodimers were detected in 21% of RA samples and none of the control samples. In primary inflammatory arthropathy patients, SF PlGF and VEGF levels correlated significantly with the SF total leucocyte count and the neutrophil count. PlGF was the most potent inducer of PBMC VEGF production in both RA and control subjects. This is the first report of the detection of PlGF and PlGF/VEGF heterodimers in the SF of patients with inflammatory arthropathies, and we have shown for the first time that PlGF up-regulates PBMC VEGF production. PlGF may therefore play a key role in the production of VEGF in the inflammatory joint.

Adolescent↗
PubMed2000

Retroviral vectors.

Traditionally, the retrovirus is regarded as an enemy to be overcome. However, for the past two decades retroviruses have been harnessed as vehicles for transferring genes into eukaryotic cells, a process known as transduction. During this time, the technology has moved from being a scientific laboratory tool to a potential clinical molecular medicine to be used in gene therapy. This review explains the strategy for harnessing the retrovirus life cycle, the scientific research and clinical applications of this methodology, and its limitations, as well as possible future developments.

Gene Transfer Techniques↗
PubMed2000

Successful treatment of Rhodococcus equi pulmonary infection in a renal transplant recipient.

The rhodococcus is a mycobacterium-like organism which is normally a pathogen in foals. It usually spreads by direct contact or by aerosol from horse faeces and causes pyogranulomatous pulmonary infections. Occasionally, it acts opportunistically to infect immuno-compromised human hosts, most commonly those with the acquired immune deficiency syndrome (AIDS). Here we report a pulmonary infection by Rhodococcus equi in a renal transplant recipient who was successfully treated. The literature on this infection in transplant recipients is also reviewed with respect to manifestations and treatment.

Actinomycetales Infections↗