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Biomedical subjects

J Moffett

Publications and source records attributed to J Moffett.

At least 19 recordsLinked to original sources
PubMed2005

The role of physiotherapy in the management of non-specific back pain and neck pain.

This paper provides an overview of best practice for the role of physiotherapy in managing back pain and neck pain, based mainly on evidence-based guidelines and systematic reviews. More up-to-date relevant primary research is also highlighted. A stepped approach is recommended in which the physiotherapist initially takes a history and carries out a physical examination to exclude any potentially serious pathology and identify any particular functional deficits. Initially, advice providing simple messages of explanation and reassurance will form the basis of a patient education package. Self-management is emphasized throughout. A return to normal activities is encouraged. For the patient who is not recovering after a few weeks, a short course of physiotherapy may be offered. This should be based on an active management approach, such as exercise therapy. Manual therapy should also be considered. Any passive treatment should only be used if required to relieve pain and assist in helping patients get moving. Barriers to recovery need to be explored. Those few patients who have persistent pain and disability that interferes with their daily lives and work need more intensive treatment or a different approach. A multidisciplinary approach may then be optimal, although it is not widely available. Liaison with the workplace and/or social services may be important. Getting all players on side is crucial, especially at this stage.

Back Pain↗
PubMed2001

The keratin 19 promoter is potent for cell-specific targeting of genes in transgenic mice.

BACKGROUND & AIMS: Keratins are intermediate filaments that are critical in cytoskeletal organization. Their roles in cellular processes are underscored by inherited human diseases in which germline mutations of keratins are found, as well as by transgenic and knockout mouse models that recapitulate those diseases. Keratin 19 (K19) has unique structural properties and developmental and spatial expression patterns. This suggests that K19 expression may correlate with important cell fate decisions in gastrointestinal tract epithelia. METHODS: We used mouse K19 5' untranslated region and promoter sequences and fused it to the lacZ reporter gene in a transgene construct. Characterization was by beta-galactosidase expression and X-gal histochemistry in gastrointestinal epithelia. Because endogenous K19 protein is transcriptionally regulated by the Kruppel-like transcription factor 4 (KLF4), we determined the spatial expression patterns of KLF4 and K19 in relationship to the lacZ reporter gene product. RESULTS: K19-lacZ transgenic mice were found to have reporter gene expression in an epithelial-specific pattern. Expression was restricted to ductal epithelial cells in the pancreas, surface colonocytes, small intestinal villi, and gastric isthmus cells. Transgene expression correlated with K19 and KLF4 protein expression in the pancreas and stomach and was overlapping in the small and large intestine. CONCLUSIONS: The K19 promoter may be a useful tool to study epithelial cell biology and subsequent transdifferentiation programs, particularly the pancreas and stomach.

Animals↗
PubMed2001

Phosphorylation of the serine 60 residue within the Cdx2 activation domain mediates its transactivation capacity.

BACKGROUND & AIMS: Cdx2 is critical in intestinal proliferation and differentiation. Modulation of Cdx2 function in response to cellular signaling is to be elucidated. We hypothesize that phosphorylation of the Cdx2 activation domain can modulate its function. METHODS: The Cdx2 activation domain was delineated in transient transfections using different portions of Cdx2 fused to the Gal4-DNA binding domain. In vivo phosphorylation was studied by metabolic labeling with (32)P-orthophosphate. To study a potential phosphorylation site, polyclonal antibodies were generated: CNL was raised against amino acids 54-66 of Cdx2 and P-Cdx2-S60 against the same epitope in which serine 60 was phosphorylated. RESULTS: A critical region for transactivation resides within amino acids 60-70. Substitution of serine 60 with alanine reduces incorporation of (32)P-orthophosphate substantially. S60-phosphorylation decreases Cdx2 transactivation. Phosphorylation of serine 60 can be inhibited with the mitogen-activated protein kinase inhibitors PD98059 or UO126. P-Cdx2-S60 recognizes phosphorylated serine 60 mainly in proliferative compartment of the intestinal epithelial layer. In contrast, CNL recognizes Cdx2 predominantly in the differentiated compartment. CONCLUSIONS: The Cdx2 activation domain is phosphorylated at serine 60 via the mitogen-activated protein kinase pathway. S60-phosphorylated and S60-nonphosphorylated Cdx2 have different transcriptional activity, as well as different spatial expression patterns in the intestinal epithelium.

Amino Acid Sequence↗
PubMed2001

Novel nuclear signaling pathway mediates activation of fibroblast growth factor-2 gene by type 1 and type 2 angiotensin II receptors.

In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1',3'-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.

Adrenal Medulla↗
PubMed1999

Adenoviral vector-mediated gene therapy in the mouse lung: no role of Fas-Fas ligand interactions for elimination of transgene expression in bronchioepithelial cells.

The early successes of adenoviral vector-mediated gene therapies in the lung have been hampered by the immune response directed against viral proteins and transgene product. Intratracheal administration of adenovirus vector in immune-competent mice transduces bronchioepithelial cells of lung extremely efficiently; however, transgene expression is eliminated within 2 weeks. Extinction of transgene expression has been attributed to infiltrating cytotoxic T lymphocytes (CTLs). Fas-Fas ligand (Fas-FasL) interactions play a critical role in the effector function of the CTL killing. We have previously demonstrated that this interacting pair of molecules plays a critical role in elimination of transgene expression in mouse liver. In this study we investigated the role of Fas-FasL interactions in CTL effector functions in vivo in mouse lung. Analyses of these molecules in lung showed constitutive expression of Fas in bronchiooepithelial cells. On the other hand, FasL was inducible in the bronchioepithelial cells after adenovirus vector treatment. The in vivo role of the Fas-FasL interactions was determined by adoptive transfer of splenocytes from normal immune-competent mice to Fas-deficient mice (B6-lpr); likewise, the function of FasL on CTLs was analyzed by the ability of splenocytes from FasL-deficient mice (B6-gld) to eliminate transgene expression in Rag1-deficient mice. These studies demonstrate that despite expression of Fas and FasL in bronchioepithelial cells and CTLs, these interacting molecules do not play a critical role in elimination of transgene expression in the lung.

Adenoviridae↗
PubMed1998

Increased tyrosine phosphorylation and novel cis-acting element mediate activation of the fibroblast growth factor-2 (FGF-2) gene by nicotinic acetylcholine receptor. New mechanism for trans-synaptic regulation of cellular development and plasticity.

FGF-2, a mitogenic/neurotrophic protein, controls the development and plasticity of many types of neural cells. In neural crest-derived adrenal pheochromatocytes, induction of FGF-2 coincides with the establishment of functional innervation and is reproduced in vitro by stimulating acetylcholine receptors (AChR). The mechanisms by which AChR activate the FGF-2 gene were examined in cultured bovine adrenal medullary chromaffin (BAMC) cells in which AChR induce expression and nuclear accumulation of growth-promoting FGF-2 and FGF-2 receptors. Carbachol or nicotine increased expression of transfected FGF-2 gene promoter-luciferase constructs and were more potent than the muscarinic agonist ABMCB. Deletion analysis has identified a unique -555/-512 bp element that confers AChR stimulation and basal activity to the downstream FGF-2 promoter, and a separate protein kinase C/cAMP-responsive sequence (-625/-555 bp). Stimulation of AChR increased in vitro formation of protein complexes with the AChR-responsive element which were not displaced by target oligonucleotides for common trans-activators. Southwestern analysis identified 50-55, 125, 140 and 170 kDa proteins that interact with the AChR-responsive element in a manner stimulated by AChR. Nicotine increased tyrosine phosphorylation of cytoplasmic and nuclear proteins, including 50-55 kDa promoter-binding factors. Activation of the FGF-2 promoter was reduced by genistein. Thus, nicotinic AChR activate the FGF-2 gene via a new signaling mechanism separate from the cAMP/PKC pathways. It utilizes tyrosine phosphorylation and interaction of trans-activating factors with a novel cis-acting element. It offers a new pathway through which trans-synaptic signals may control neural development and plasticity.

5' Untranslated Regions↗
PubMed1998

Transcriptional regulation of fibroblast growth factor-2 expression in human astrocytes: implications for cell plasticity.

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1beta, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter-luciferase constructs identified a unique -555/-513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (-624/-556 bp) essential for PKC and cAMP stimulation. DNA-protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.

Astrocytes↗
PubMed1997

Nuclear accumulation of fibroblast growth factor receptors in human glial cells--association with cell proliferation.

In this study we describe the presence of high affinity FGF-2 binding sites in the nuclei of U251MG glioma cells (K(d)=7 pM). Immunoprecipitation of total cell extracts with FGF receptor (FGFR) 1-4 antibodies showed that U251MG glioma cells express only FGFR1. [125I]FGF-2 cross linking to nuclear extracts followed by FGFR1 immunoprecipitation showed that FGFR1 may account for the nuclear FGF-2 binding sites. Western blot analysis demonstrated the presence of 103, 118 kDa and small amounts of 145 kDa FGFR1 isoforms in the nuclei of glioma cells. All isoforms contain both the C- and N-terminal domains. Nuclear FGFR1 retains kinase activity. Immunocytochemistry using confocal microscopy showed specific FGFR1 immunoreactivity within the nuclear interior. In continuously proliferating glioma cells, nuclear FGFR1 is constitutively expressed, independent of cell density. In contrast, in nontransformed human astrocytes, nuclear FGFR1 levels fluctuate with the proliferative state of the cell. In quiescent, confluent astrocytes nuclear FGFR1 protein was depleted. An accumulation of nuclear FGFR1 was observed following the transition to a subconfluent, proliferating state. Transfection of a pcDNA3.1-FGFR1 expression vector into glioma cells that do not express FGFR1 resulted in the nuclear accumulation of FGFR1, increased cell proliferation, and stimulated transition from the G0/G1 to the S-phase of the cell cycle. The increased proliferative rate was resistant to inhibition by the cell-impermeable FGF binding antagonist, myoinositol hexakis [dihydrogen phosphate]. Our results suggest that the constitutive nuclear presence of FGFR1 contributes to the increased proliferation of glioma cells while the transient nuclear accumulation of FGFR1 in normal astrocytes may play a role in the transition to a reactive state.

Astrocytes↗
PubMed1997

Nuclear accumulation of FGF-2 is associated with proliferation of human astrocytes and glioma cells.

FGF-2 has been implicated in the neoplastic transformation of glioma cells and in the transition of normal quiescent astrocytes to a proliferating, reactive state. In the present study we have observed that in human glial cells, levels and subcellular localization of FGF-2 are different in quiescent and proliferating cells. FGF-2 was detected in the cytoplasm of non-reactive astrocytes in human brain sections. In contrast FGF-2 was located within the cytoplasm and nuclei of reactive astrocytes in gliotic brain tissue and in neoplastic cells of glioma tumors. In vitro, FGF-2 was found predominantly in the nucleus of subconfluent proliferating astrocytes, but was detected only in the cytoplasm of density arrested quiescent astrocytes. Our results suggest that reduced cell contact stimulates nuclear accumulation of FGF-2, accompanying mitotic activation of reactive human astrocytes. FGF-2 was constitutively localized to the nucleus of continuously proliferating glioma cells independent of cell density. A role for intracellular FGF-2 was further suggested by the observation that glioma cells that are not stimulated to proliferate by extracellular FGF-2 proliferated faster when transfected with FGF-2 expressing vectors. This increased proliferation correlated with nuclear accumulation of FGF-2. Cell proliferation was attenuated by 5'-deoxy-5'-methylthioadenosine, a FGF-2 receptor tyrosine kinase inhibitor that acts within the cell, but was unaffected by myo-inositol hexakis [dihydrogen phosphate] that disrupts FGF-2 binding to plasma membrane receptors. Our results indicate that FGF-2 serves as a nuclear regulator of proliferation in astrocytic cells. In glioma cells, the constitutive presence of FGF-2 in the nucleus may promote proliferation that is insensitive to cell contact inhibition.

Astrocytes↗
PubMed1997

Growth factor regulation of cell growth and proliferation in the nervous system. A new intracrine nuclear mechanism.

This article discusses a novel intracrine mechanism of growth-factor action in the nervous system whereby fibroblast growth factor-2 (FGF-2) and its receptor accumulate in the cell nucleus and act as mediators in the control of cell growth and proliferation. In human and rat brain the levels and subcellular localization of FGF-2 differ between quiescent and reactive astrocytes. Quiescent cells express a low level of FGF-2, which is located predominantly within the cytoplasm. In reactive astrocytes, the expression of FGF-2 increases and the proteins are found in both the cytoplasm and nucleus. In glioma tumors, FGF-2 is overexpressed in the nuclei of neoplastic cells. Similar changes in FGF-2 expression and localization are found in vitro. The nuclear accumulation of FGF-2 reflects a transient activation of the FGF-2 gene by potentially novel transactivating factors interacting with an upstream regulatory promoter region. In parallel with FGF-2, the nuclei of astrocytes contain the high-affinity FGF-2 receptor, FGFR1. Nuclear FGFR1 is full length, retains kinase activity, and is localized within the nuclear interior in association with the nuclear matrix. Transfection of either FGF-2 or FGFR1 into cells that do not normally express these proteins results in their nuclear accumulation and concomitant increases in cell proliferation. A similar regulation of nuclear FGF-2 and FGFR1 is observed in neural crestderived adrenal medullary cells and of FGF-2 in the nuclei of cerebellar neurons. Thus, the regulation of the nuclear content of FGF-2 and FGFR1 could serve as a novel mechanism controlling growth and proliferation of glial and neuronal cells.

Animals↗
PubMed1996

Promoter regions involved in density-dependent regulation of basic fibroblast growth factor gene expression in human astrocytic cells.

Expression of mitogenic basic fibroblast growth factor (bFGF) in the central nervous system is inhibited by direct cell contact and is implicated in reactive and neoplastic transformation of astrocytes. The molecular mechanisms controlling expression of bFGF were examined in cultures of human astrocytes. Cell-density-dependent depletion of bFGF mRNA levels parallels changes in bFGF gene protein. Regulation of transcription of a bFGF luciferase reporter gene containing an upstream region (bp -1800 to +314) of the bFGF gene promoter mimicks the density-dependent regulation of the endogenous bFGF gene in transfected astrocytes. Deletion analysis has identified a fragment (bp -650 to -513) and sequences further downstream (bp -274 to +314) as the regions required for the regulation of bFGF gene activity by cell density. Unlike in astrocytes, changing the cell density of glioma cell cultures does not affect the levels of bFGF protein and mRNA. bFGF luciferase constructs were expressed at the same level in high- or low-density cultures of glioma cells, indicating altered regulation of the bFGF gene promoter. Electrophoretic mobility shift assays showed binding of nuclear proteins to a fragment of bFGF gene promoter from bp -650 to -453. This binding was abolished by a deletion of the upstream cell-density-responsive region (bp -650 to -512). Binding was observed with nuclear extracts from subconfluent astrocytes but was reduced in extracts from confluent astrocytes. Our results indicate that induction of bFGF in astrocytes upon reduction of cell density is mediated transcriptionally by positive trans-acting factors interacting with bFGF promoter. In contrast, nuclear proteins from glioma cells bind to the promoter region from bp -650 to -453 independent of cell density. Thus, the constitutive binding of trans-acting factor(s) to the region of the bFGF promoter from bp -650 to -453 may be responsible for the continuous expression of bFGF that leads to the uncontrolled growth of glioma cells.

Astrocytes↗
PubMed1994

Regulation of bFGF gene expression and subcellular distribution of bFGF protein in adrenal medullary cells.

Basic fibroblast growth factor (bFGF), a potent mitogenic/neurotrophic factor, controls the development and plasticity of many types of neural cells. In adrenal chromaffin cells, the appearance of bFGF protein coincided with the establishment of functional innervation, suggesting induction by trans-synaptic signals. In cultured bovine adrenal medullary cells Western blot analysis revealed 18-, 23-, and 24-kD bFGF isoforms in the cytosolic and nuclear fractions. Stimulation of acetylcholine nicotinic receptors or hormonal angiotensin II receptors or the direct stimulation of adenylate cyclase with forskolin or protein kinase C (PKC) with PMA increased the content of all bFGF isoforms. Increases in the levels of intracellular bFGF did not result in detectable presence of bFGF proteins in culture medium. Instead, bFGF proteins accumulated in the cytoplasm or the nucleus depending on whether PKC or cAMP pathways were activated. The long-term nuclear forskolin-induced accumulation of bFGF was prevented by cycloheximide or by antisense bFGF oligonucleotide and was also accompanied by an increase in bFGF mRNA. We used luciferase reporter plasmids containing the human bFGF promoter to show that the induction of bFGF resulted from transcriptional activation of the bFGF gene and was mediated by regulatory sequences located upstream from its transcription start site. Stimulation of bFGF gene expression by forskolin and PMA was synergistic and was mediated through different promoter regions. The results suggest that stimulation by cAMP and PKC is mediated through novel cis elements. The regulation of bFGF protein content also involves posttranscriptional mechanisms since changes in the levels of individual bFGF isoforms were different depending on whether cells were treated with carbachol or angiotensin II, forskolin, or PMA. The present study indicates that bFGF is an intracrine cytoplasmic-nuclear factor, whose expression is regulated by trans-synaptic and hormonal stimuli and which may act as a direct mediator of genomic responses to afferent stimulation.

Adrenal Medulla↗
PubMed1988

Two membrane-bound proteins associated with alanine resistance and increased A-system amino acid transport in mutants of CHO-K1.

Growth of CHO-K1, a proline auxotroph, is inhibited by amino acids that prevent proline transport. From a hydroxyurea-treated, alanine-resistant, constitutive mutant, alar4, we isolated, in a stepwise fashion, mutants, resistant to higher concentrations of alanine, that have increased velocity of amino acid transport through the A system. Two such mutants, alar4-H2.1 and alar4-H3.9, isolated as resistant to 50 mM and 125 mM alanine, respectively, showed increases in Vmax of proline transport through the A system that are directly proportional to their resistance to alanine. Alar4-H3.9, as compared to alar4 and CHO-K1, has six and 29 times the Vmax of proline transport through the A system and two and five times the velocity of transport through the combined ASC and P systems, respectively, and no change in system L. No double-minute or homologous staining regions were detectable in alar4-H3.9. A-system activity of alar4-H2.1 and alar4-H3.9, when grown under nonselective conditions, was stable for 20 generations and then declined. The phenotype of alar4-H3.9 is codominant with that of alar4 and partially recessive to that of CHO-K1. Membrane vesicles prepared from alar4-H3.9 show increases mainly in A-system transport. In sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis of A-system active membrane vesicles and endoplasmic reticulum, two bands of molecular weight of approximately 62-66 kd and 29 kd are present in higher concentrations in alar4-H3.9 than in CHO-K1. These results are compatible with the hypothesis that the phenotype of alar4-H3.9 is the result of gene amplification of an A-system transporter structural gene and that the two bands may represent this transporter.

Alanine↗
PubMed1987

Amino acid transport in membrane vesicles from CHO-K1 and alanine-resistant transport mutants.

Membrane vesicles were prepared from CHO-K1 and alanine-resistant transport mutants, alar4 and alar4-H3.9. Alar4 is a constitutive mutant of the A system, and alar4-H3.9, derived from alar4, may be the result of amplification of a gene coding for an A-system transporter. Under conditions in which the same membrane potential (interior negative) and Na+ gradient were employed, the mutant vesicles show increases in the A system over that of the parental CHO-K1 cell line, paralleling, but not equivalent to, that found in whole cells. L-system and 5'-nucleotidase activities of these vesicles were similar, indicating that the increased A-system activity of the mutant vesicles is not due to the differential enrichment of the A system in these vesicles. The membrane potential was produced by a K+ diffusion gradient (internal greater than external) in the presence of valinomycin or by the addition of a Na+ salt of a highly permeant anion such as SCN-. Monensin was employed to study the effect of the Na+ gradient on transport and membrane potential. The latter was determined by measuring the uptake of tetraphenylphosphonium ion. A negative membrane potential determines the concentrative ability and the initial velocity of the A system in these vesicles. The concentration of external Na+ has a stimulatory effect on the initial velocity of this system. However, the Na+ gradient (external greater than internal) has no effect on the initial velocity or the membrane potential when the potential is set by valinomycin and high internal K+. Little if any ASC system could be detected in vesicles from CHO-K1.

Alanine↗
PubMed1987

Control of A-system amino acid transport by a second regulatory gene R2 in Chinese hamster ovary cells CHO-K1 and the possible connection of this gene with insulin activity.

Evidence based on a study of alanine-resistant (Alar), constitutive mutants of CHO-K1 cells and the conditions that favor stimulation of the A system of amino acid activity supports the model that the A system of amino acid transport in these cells is repressible and under negative control of regulatory gene R1. In this study, we show that mutant Alar6, when grown under conditions of repression, has an A system of amino acid transport activity similar to that of the derepressed parental cell line, CHO-K1 (wild type) and of the fully constitutive mutant in gene R1, Alar4. However, the A system of Alar6 is further derepressible. The Vmax for proline transport through this system in mutant Alar6 is four times that of the parental culture, with no significant change in Km. Analysis of hybrids produced by crossing mutant Alar6 with the parental culture and with Alar4 shows that mutant Alar6 is recessive to wild type and complements mutant Alar4. Although the amino acid transport A system of CHO-K1 is stimulated by insulin, mutant alar6 is not stimulated by insulin. These results support the hypothesis that mutant alar6 results from mutation in another regulatory gene, R2, that, in conjunction with gene R1, negatively controls the expression of a structural gene for the A-system transport. Evidence also indicates that R2 gene product is not responsive to amino acids and that insulin stimulation of the A system may result from insulin inactivation of this repressor.

Amino Acids↗