Biomedical subjects

N S Thomas

Publications and source records attributed to N S Thomas.

At least 37 records · Page 2Linked to original sources

PubMed2001

BCR-ABL and interleukin 3 promote haematopoietic cell proliferation and survival through modulation of cyclin D2 and p27Kip1 expression.

Although it is evident that BCR-ABL can rescue cytokine-deprived hematopoietic progenitor cells from cell cycle arrest and apoptosis, the exact mechanism of action of BCR/ABL and interleukin (IL)-3 to promote proliferation and survival has not been established. Using the pro-B cell line BaF3 and a BaF3 cell line stably overexpressing BCR-ABL (BaF3-p210), we investigated the proliferative signals derived from BCR-ABL and IL-3. The results indicate that both IL-3 and BCR-ABL target the expression of cyclin Ds and down-regulation of p27(Kip1) to mediate pRB-related pocket protein phosphorylation, E2F activation, and thus S phase progression. These findings were further confirmed in a BaF3 cell line (TonB.210) where the BCR-ABL expression is inducible by doxycyclin and by using the drug STI571 to inactivate BCR-ABL activity in BaF3-p210. To establish the functional significance of cyclin D2 and p27(Kip1) expression in response to IL-3 and BCR-ABL expression, we studied the effects of ectopic expression of cyclin D2 and p27(Kip1) on cell proliferation and survival. Our results demonstrate that both cyclin D2 and p27(Kip1) have a role in BaF3 cell proliferation and survival, as ectopic expression of cyclin D2 is sufficient to abolish the cell cycle arrest and apoptosis induced by IL-3 withdrawal or by BCR-ABL inactivation, while overexpression of p27(Kip1) can cause cell cycle arrest and apoptosis in the BaF3 cells. Furthermore, our data also suggest that cyclin D2 functions upstream of p27(Kip1), cyclin E, and cyclin D3, and therefore, plays an essential part in integrating the signals from IL-3 and BCR-ABL with the pRB/E2F pathway.

Animals↗

PubMed2001

Maternal sex chromosome non-disjunction: evidence for X chromosome-specific risk factors.

Human trisomy is attributable to many different mechanisms and the relative importance of each mechanism is highly chromosome specific. The association between altered recombination and maternal non-disjunction is well documented: reductions in recombination have been reported for maternal meiosis I (MI) errors involving chromosomes 15, 16, 18 and 21 and increased recombination has been reported for meiosis II (MII) errors involving chromosome 21. We therefore investigated maternal X chromosome non-disjunction, to determine whether the effects of recombination are unique to the X chromosome or similar to any of the autosomes thus far studied. We genotyped 45 47,XXX females and 95 47,XXY males of maternal origin. Our results demonstrate that 49% arose during MI, 29% during MII and 16% were postzygotic events; a further 7% were meiotic but could not be assigned as either MI or MII because of recombination at the centromere. Among the MI cases, a majority (56%) had no detectable transitions and so absent recombination is an important factor for X chromosome non-disjunction. However, similar to trisomy 15 and unlike trisomy 21, we observed a significant increase in the mean maternal age of transitional MI errors compared with nullitransitional cases. In our studies of MII errors, recombination appeared normal and there was no obvious effect of maternal age, distinguishing our results from MII non-disjunction of chromosomes 18 or 21. Thus, surprisingly, the risk factors associated with both MI and MII non-disjunction appear to be different for virtually every chromosome that has been adequately studied.

Adult↗

PubMed2001

Linkage analysis of the 5q31-33 candidate region for asthma in 240 UK families.

Atopy and asthma are complex genetic diseases resulting from the interactions of a number of genetic and environmental factors. We had previously reported allelic association between the IL9 marker on chromosome 5q31-33 and atopy. In order to further investigate the role of susceptibility genes on 5q31-33 in the development of atopy and asthma we have studied 240 UK families comprising 131 families selected at random, 60 multiplex families with affected sib pairs, and 49 single proband nuclear families. Polymorphic markers on 5q31-33 were genotyped and both single and multipoint linkage analysis was undertaken using the BETA program. We have used both affection status and quantitative scores for atopy and asthma for phenotypic variables, combining data into scores for asthma and atopy. The strongest suggestion of linkage using multipoint analysis was centred around D5S410 with a maximum Lod of 1.946 at location 171.3 cM and a standard error of 3.3 for the asthma quantitative score. There was no evidence of linkage with atopy, the atopy quantitative score or total serum IgE.

Asthma↗

PubMed2001

The retinoblastoma family member, p107, is active in non-dividing monocyte-derived dendritic cells.

A member of the retinoblastoma family of cell cycle regulatory proteins, p107, is not normally expressed in non-cycling cells. We demonstrate here that p107 is expressed in monocytes as they differentiate into dendritic cells under the influence of granulocyte-macrophage colony-stimulating factor and interleukin 4, a process shown not to involve cellular proliferation. We show that p107 is localized to the nucleus of these cells and is active, in that it binds an E2F-DNA binding site, together with E2F transcription factors. These findings suggest a hitherto unknown role for p107 in non-proliferating dendritic cells that warrants further investigation.

Blotting, Western↗

PubMed2001

Oestriol and oestradiol increase cell to cell communication and connexin43 protein expression in human myometrium.

Oestradiol increases the protein expression of connexin43 (Cx43) gap junctions in myometrium but the effect of oestriol on gap junction expression has not been described previously. Oestriol is the most abundant free oestrogen in pregnant women and there is a marked surge in oestriol concentrations before term and idiopathic preterm labour. In order to determine whether oestriol may have a physiological action on the myometrium, cultured human myometrial cells obtained from non-pregnant hysterectomy specimens were exposed to 10 nmol/l oestradiol or oestriol. Intercellular communication between myometrial cells was investigated by microinjection of confluent cultured cells with the gap junction-permeant tracer Cascade Blue. There was a progressive increase in coupling after exposure to oestradiol or oestriol (P < 0.0005). An increase in Cx43 protein expression was demonstrated by immunocytochemistry after 1 h (P < 0.01) and 3 days (P < 0.01) exposure, and by Western blotting after 1 h (P < 0.01) and 3 days (P < 0.05) exposure, to both oestradiol and to oestriol. We conclude that oestriol increases gap junction communication in human myometrium by increasing gap junction expression. Elevated oestriol concentrations may thus play a role in the initiation of labour in women, by increasing cell-cell communication in the myometrium.

Adult↗

PubMed2001

Antituberculosis drugs and new drug development.

Tuberculosis affects more than 8 million people and has serious repercussions on economic, psychologic, and social status. Since its declaration as a global emergency in 1993 by the World Health Organization, significant development in the treatment and control of tuberculosis has been the implementation of the short-course directly observed treatment along with fixed dose combinations of existing drugs. However, the currently available therapeutic regimens have inherent disadvantages of long treatment duration, patient noncompliance, and risk of drug resistance. Hence, new antituberculosis drugs that are potent, are active against resistant strains and latent forms, and reduce the treatment period are needed to combat this disease. In this review, the authors discuss new chemical entities that in their opinion have a potential to become new antituberculosis drugs. This article emphasizes the role of biopharmaceutics and pharmacokinetics as an indispensable part in the development of new antituberculosis drugs to overcome the common hurdles such as exorbitant cost, time, and attrition rate involved in the process.

Animals↗

PubMed2000

Biopharmaceutics and pharmacokinetics in drug research.

With the synergistic and multiplicative interactions of rational drug design, recombinant biotechnology, combinatorial chemistry and high-throughput screening, millions of compounds are being synthesized by chemists. However, development of these drug candidates has often been impeded, if not terminated, due to biopharmaceutic and/or pharmacokinetic constraints. This has resulted in delays in development time and escalation of cost in the drug research programmes. So, the present emphasis is to reduce development time and cost, which is analogous to added patent life besides the enormous reduction in human suffering. In this compilation the important biopharmaceutic and pharmacokinetic approaches are discussed, which will help in the development of safe and more efficacious drugs with reduced development time and cost.

Biopharmaceutics↗

PubMed2000

Preferential induction of apoptosis of leukaemic cells by farnesol.

Farnesol preferentially inhibits proliferation and induces apoptosis of tumour-derived but not non-transformed cell lines. We investigated whether farnesol induces apoptosis of blasts from patients with acute myeloid leukaemia (AML) and leukaemic cell lines, as compared with normal, human primary haemopoietic cells. We show that 30 microM farnesol causes apoptosis of leukaemic cell lines of T- and B-lymphocyte, myeloid or erythroid lineages and primary blasts obtained from patients with AML. However, the same concentration did not kill primary monocytes, or quiescent or proliferating T-lymphocytes. We conclude that farnesol selectively kills AML blasts and leukaemic cell lines in preference to primary haemopoietic cells.

Apoptosis↗

PubMed2000

Cyclin D3 compensates for loss of cyclin D2 in mouse B-lymphocytes activated via the antigen receptor and CD40.

Cyclin D2 is the only D-type cyclin expressed in mature mouse B-lymphocytes, and its expression is associated with retinoblastoma protein (pRB) and pRB-related protein phosphorylation and induction of E2F activity, as B-cells enter the cell cycle following stimulation via surface IgM and/or CD40. Cyclin D-dependent kinase activity is required for cell proliferation, yet cyclin D2(-/-) mice have normal levels of mature B-lymphocytes. Here we show that B-lymphocytes from cyclin D2(-/-) mice can proliferate in response to anti-IgM and anti-CD40, but the time taken to enter S-phase is longer than for the corresponding cyclin D2(+/+) cells. This is due to the compensatory induction of cyclin D3, but not cyclin D1, which causes pRb phosphorylation on CDK4-specific sites. This is the first demonstration that loss of a D-type cyclin causes specific expression and functional compensation by another member of the family in vivo and provides a rationale for the presence of mature B-lymphocytes in cyclin D2(-/-) mice.

Animals↗

PubMed2000

A reinvestigation of non-disjunction resulting in 47, XXY males of paternal origin.

We have used polymorphisms within the Xp/Yp pseudoautosomal region (PAR 1) to determine the frequency and location of recombination in 80 paternally derived 47, XXY males. Of 64 informative results, there were 10 single cross-overs, one double cross-over and 53 without a cross-over. Therefore 2/3 of 47, XXY males of paternal origin result from meiosis in which the X and Y chromosomes fail to recombine. This failure was not associated with the presence of an increase in recombination in the smaller Xq/Yq pseudoautosomal region (PAR 2) or with the presence of microdeletions within PAR 1.

Adult↗

PubMed2000

Monocyte-derived dendritic cells do not proliferate and are not susceptible to retroviral transduction.

Dendritic cells may be generated ex vivo from CD34+ progenitor cells or peripheral blood mononuclear cells. Initial reports suggested that monocyte-derived dendritic cells (MoDCs) arise from a proliferating precursor and several groups subsequently reported successful retroviral transduction of these cells, again implying that cell division occurs. As this is of importance in the development of immunotherapy protocols, we investigated whether monocytes proliferate as they differentiate into MoDCs and also their susceptibility to retroviral transduction. During MoDC differentiation, there was a 51 +/- 12% reduction in cell number, 98% of cells were in G0/G1, no DNA synthesis was detectable and the cell cycle regulatory proteins pRb and p130 were in the hypophosphorylated forms observed in non-cycling cells. As expected from these results, MoDCs were refractory to transduction with a GALV1 pseudotyped Moloney murine leukaemia virus (MoMLV)-based retroviral vector. In contrast, generation of DCs from purified CD34 progenitors was accompanied by rapid entry into the cell cycle and a 41.1-fold cell expansion at the end of 14 d culture.

Antigens, CD34↗

PubMed2000

Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1).

Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27(KIP1) through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27(KIP1) promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27(KIP1) appears to be critical in the regulation of cell survival since mere ectopic expression of p27(KIP1) was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27(KIP1) null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27(KIP1) transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.

Animals↗

PubMed2000

p107 is active in the nucleolus in non-dividing human granulosa lutein cells.

Cells are maintained in a quiescent state by members of the retinoblastoma protein family, pRb and p130. Both are phosphoproteins and hypophosphorylated forms of pRb and p130 bind and repress the activity of E2F transcription factors, thereby preventing entry into the cell cycle. Mitogenic stimulation causes activation of cyclin dependent kinases (cdk) that phosphorylate both pRb and p130, thereby releasing E2F factors which stimulate the transcription of a number of genes that are required for DNA synthesis and for regulating the cell cycle. In non-dividing cells, cdks are maintained in an inactive state by cdk inhibitor proteins such as p27(Kip1). The aim of our study was to determine how E2F complexes are regulated during the differentiation of human primary granulosa lutein cells (GLC) of the corpus luteum (CL). The CL is formed in the ovary after ovulation at the terminal stage of folliculogenesis after completion of maturation and differentiation of Graafian follicles. As shown by flow cytometry GLC are not dividing, being predominantly in the G(0)/G(1) phase of the cell cycle and, consistent with this, they contain the cdk inhibitor protein, p27(Kip1), but not E2F-1 which is normally expressed only in proliferating cells. The GLC do express E2F-4, hypophosphorylated pRb, p130 forms 1 and 2 and, surprisingly, hypophosphorylated p107. p107 is normally present only in dividing cells where it regulates E2F activity during the cell cycle. These forms of pRb, p130 as well as p107, together with E2F-4 are all active in that they can bind an E2F DNA-binding site in a pull-down assay. Immunocytochemistry shows that these proteins are expressed in almost all GLC but have different sub-cellular distribution: p107 is concentrated in nucleoli, while p130 and E2F-4 show relatively even nuclear and cytoplasmic distributions. Both pRb and p130 have been implicated previously in repressing E2F activity in many different cell types during cell cycle arrest in G(0)/G(1). We conclude that p107 is active in human primary GLC but its nucleolar localisation would suggest that it represses ribosomal RNA synthesis rather than E2F activity.

Cell Cycle↗

PubMed2000

[Genetic factors involved in asthma and atopy. Studies in British families].

Both asthma and atopy run families indicating a strong genetic component. To investigate possible candidate gene regions, we have recruited 131 families without reference to atopy and asthma (random sample) and 60 extended families with two or more members affected by asthma (multiplex sample). Using both candidate and genome screen approaches, we have been able to provide evidence supporting the presence of candidate genes on chromosome 11q (E237G) and 12q but we have been unable to confirm reports of linkage and association for asthma on 5q. Our experience to date suggests that larger numbers of families are needed to increase the confidence of gene localisation and there is a need to improve the phenotypic description of asthma. Finally, it is essential that claims for linkage or association are confirmed in different populations using the same markers.

Asthma↗

PubMed1999

CBP associates with the p42/p44 MAPK enzymes and is phosphorylated following NGF treatment.

The ability of the CBP (CREB binding protein) coactivator to stimulate transcription has previously been shown to be stimulated by treatment of neuronal cells with nerve growth factor (NGF). This effect is dependent upon activation of the p42/p44 MAPK (mitogen activated protein kinase) pathway. Here we show that both CBP and the related p300 protein directly associate with the p42/p44 MAPK enzymes both prior to and following their activation by NGF and that CBP is phosphorylated following NGF treatment. These results indicate that phosphorylation of CBP itself by the p42/p44 MAPK pathway is likely to be critical for its role in NGF-mediated stimulation of gene expression.

Animals↗

PubMed1999

Modulation of E2F complexes during G0 to S phase transition in human primary B-lymphocytes.

The pocket protein-E2F complexes are convergence points for cell cycle signaling. In the present report, we identified and monitored the pocket protein-E2F complexes in human primary B-lymphocytes after activation by phorbol 12-myristate 13-acetate. Consistent with previous data from human and mouse fibroblasts and T-lymphocytes, E2F4 and DP1 form the predominant E2F heterodimers both in G0 and G1 phases of the human B-lymphocyte cell cycle, whereas E2F1 and -3 are first detected in late G1, and their expression levels increase towards S phase. Intriguingly, the major E2F complex that we detected in quiescent human B-lymphocytes is consisted of pRB, E2F4, and DP1. Though the levels of DP1 and -2 increase when cells progress from G0 to S, the proportion of DP1 to DP2 remains relatively constant during the cell cycle. We also observed an increase in electrophoretic mobility of the predominant E2F components, DP1 and E2F4, as B-lymphocytes progressed from G0 into early G1. This increase in mobility was attributable to dephosphorylation, as lambda phosphatase treatment could convert the slower migrating forms into the corresponding faster mobility forms. We further demonstrated that this change in phosphorylation status correlates with a decrease in DNA binding activity. This modulation of DNA binding activity mediated through the dephosphorylation of DP1 and E2F4 could help to explain the lack of in vivo DNA footprinting in late G1 and S phases of gene promoters negatively regulated through E2F sites and suggests a novel mechanism for controlling E2F transcriptional activity during the transition from quiescence to proliferation.

B-Lymphocytes↗

PubMed1999

Xp deletions associated with autism in three females.

We report eight females with small deletions of the short arm of the X chromosome, three of whom showed features of autism. Our results suggest that there may be a critical region for autism in females with Xp deletions between the pseudoautosomal boundary and DXS7103. We hypothesise that this effect might be due either to the loss of function of a specific gene within the deleted region or to functional nullisomy resulting from X inactivation of the normal X chromosome.

Autistic Disorder↗

PubMed1999

Investigation of a cryptic interstitial duplication involving the Prader-Willi/Angelman syndrome critical region.

A 3-year-old female referred with developmental delay, hypotonia and seizures was found to have a cryptic interstitial duplication of the Prader-Willi/Angelman critical region (PWACR). Her clinical features form part of a common phenotype characteristic of PWACR duplications including developmental delay, behavioural problems and speech difficulties. Microsatellite analysis showed that the duplication had arisen de novo, was maternal in origin and involved the entire 4-Mb PWACR between the common deletion breakpoints. The existence of cryptic rearrangements emphasises the need for molecular tests alongside conventional cytogenetics when investigating abnormalities involving this imprinted region.

Angelman Syndrome↗