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Biomedical subjects

J Buxton

Publications and source records attributed to J Buxton.

At least 19 recordsLinked to original sources
PubMed1998

Patients' experiences using a computerized program with a touch-sensitive video monitor for the assessment of health-related quality of life.

This study assessed patients' experiences using a computerized program with a touch-sensitive video monitor (TSVM) for the assessment of health-related quality of life (HRQoL). A software program was developed for a computerized form of the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire, the QLQ-C30. One hundred and seventy-eight patients completed the QLQ-C30, followed by a structured interview designed to assess perceived difficulties with the use of the TSVM. Patients were asked to evaluate the ease of use of different aspects of the TSVM system (using the touch-sensitive screen, entering the patient identification number, reading the screen and following the on-screen instructions). The majority of patients found all aspects of the TSVM system 'very easy' to use. A few patients (1-2%) admitted finding any aspect of the TSVM use 'somewhat difficult' and none ranked any aspect as 'very difficult'. There were no unanswered items in the QLQ-C30. All patients found the amount of time spent on answering the questionnaire acceptable, (the mean time to complete was 6.8 min with a median of 5 min) and 96% stated they were willing to complete a similar questionnaire on a future occasion. From the patients' perspective the TSVM system appears to be a highly acceptable approach for the collection of HRQoL data in clinical practice.

Aged↗
PubMed1997

Interactions of Drosophila Cbl with epidermal growth factor receptors and role of Cbl in R7 photoreceptor cell development.

The human proto-oncogene product c-Cbl and a similar protein in Caenorhabditis elegans (Sli-1) contain a proline-rich COOH-terminal region that binds Src homology 3 (SH3) domains of proteins such as the adapter Grb2. Cb1-Grb2 complexes can be recruited to tyrosine-phosphorylated epidermal growth factor (EGF) receptors through the SH2 domain of Grb2. Here we identify by molecular cloning a Drosophila cDNA encoding a protein (Drosophila Cbl [D-Cbl]) that shows high sequence similarity to the N-terminal region of human c-Cbl but lacks proline-rich sequences and fails to bind Grb2. Nonetheless, in COS-1 cells, expression of hemagglutinin epitope-tagged D-Cbl results in its coimmunoprecipitation with EGF receptors in response to EGF. EGF also caused tyrosine phosphorylation of D-Cbl in such cells, but no association of phosphatidylinositol 3-kinase was detected in assays using anti-p85 antibody. A point mutation in D-Cbl (G305E) that suppresses the negative regulation of LET-23 by the Cbl homolog Sli-1 in C. elegans prevented tyrosine phosphorylation of D-Cbl as well as binding to the liganded EGF receptor in COS-1 cells. Colocalization of EGF receptors with both endogenous c-Cbl or expressed D-Cbl in endosomes of EGF-treated COS-1 cells is also demonstrated by immunofluorescence microscopy. In lysates of adult transgenic Drosophila melanogaster, GST-DCbl binds to the tyrosine-phosphorylated 150-kDa torso-DER chimeric receptor. Expression of D-Cbl directed by the sevenless enhancer in intact Drosophila compromises severely the development of the R7 photoreceptor neuron. These data suggest that despite the lack of Grb2 binding sites, D-Cbl functions as a negative regulator of receptor tyrosine kinase signaling in the Drosophila eye by a mechanism that involves its association with EGF receptors or other tyrosine kinases.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Clinical-neurologic, cytogenetic and molecular aspects of the Prader-Willi and Angelman syndromes.

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are human neurogenetic disorders involving the imprinting mechanism, at the 15q11-13 chromosome region. The predominant genetic defects in PW are 15q11-13 deletions of paternal origin and maternal chromosome 15 uniparental disomy. In contrast, maternal deletions and paternal chromosome 15 uniparental disomy are associated with a different neurogenetic disorder, the AS. In both disorders, these mutations are associated with parent-of-origin specific methylation at several 15q11-13 loci. We studied 5 patients suspect of PWS and 4 patients suspect of AS who were referred to the Medical Genetics Unit at the University Hospital of Medical School from Ribeirão Preto. Our objective was to establish the correct clinical and etiological diagnosis in these cases. We used conventional cytogenetics, methylation analysis with the probe KB17 (CpG island of the SNRPN gene) by Southern blotting after digestion with the Xba I and Not I restriction enzymes. We studied in patients and their parents the segregation of the (CA)*** repeats polymorphisms by PCR, using the primers 196 and IR4-3R. All the patients had normal conventional cytogenetical analysis. We confirmed 3 cases of PWS: one by de novo deletion, one by maternal chromosome 15 uniparental disomy and one case with no defined cause determined by the used primers. We confirmed 2 cases of AS, caused by de novo deletion at the 15q11-13 region, and one case with normal molecular analysis but with strong clinical characteristics.

Adolescent↗
PubMed1996

Imprint switching on human chromosome 15 may involve alternative transcripts of the SNRPN gene.

Imprinting on human chromosome 15 is regulated by an imprinting centre, which has been mapped to a 100-kb region including exon 1 of SNRPN. From this region we have identified novel transcripts, which represent alternative transcripts of the SNRPN gene. The novel exons lack protein coding potential and are expressed from the paternal chromosome only. We have also identified intragenic deletions and a point mutation in patients who have Angelman or Prader-Willi syndrome due to a parental imprint switch failure. This suggests that imprint switching on human chromosome 15 may involve alternative SNRPN transcripts.

Alternative Splicing↗
PubMed1994

Differential intracellular localizations of GDP dissociation inhibitor isoforms. Insulin-dependent redistribution of GDP dissociation inhibitor-2 in 3T3-L1 adipocytes.

Insulin action on fat cell/skeletal muscle glucose transporter (GLUT4) redistribution to cell surface membranes appears to involve small GTP-binding proteins. It has been recently recognized that at least two GDP dissociation inhibitor (GDI) isoforms, GDI-1 and GDI-2, can bind and release GDP-bound Rab proteins from membranes (Shisheva, A., Südhof, T.C., and Czech, M. P. (1994) Mol. Cell. Biol. 14, 3459-3468). The present studies show that a strikingly higher level of GDI-2 fractionates with total membranes of COS-1 cells, Chinese hamster ovary cells, and 3T3-L1 adipocytes compared to GDI-1, which is virtually totally cytosolic. In 3T3-L1 adipocytes, most of the membrane-bound GDI-2 was present in a low density, intracellular membrane fraction. Immunodepletion of GLUT4-enriched vesicles from this membrane fraction also depleted significant amounts of GDI-2 proteins. Localization of both GDI-2 and GLUT4 in the same perinuclear regions of these cells was established by immunofluorescence microscopy, whereas GDI-1 displayed a diffuse, cytoplasmic distribution. Insulin acutely decreased both GLUT4 and GDI-2 protein levels in the low density microsomes by about 50%. Concomitantly, GLUT4 but not GDI-2 protein content of plasma membranes increased, suggesting release of GDI-2 into the cytoplasm in response to insulin. Taken together, these data suggest functional differences for the GDI-1 and GDI-2 protein isoforms, as well as a potential role of GDI-2 in the action of insulin on membrane movements.

3T3 Cells↗
PubMed1994

Insulin-sensitive association of GLUT-4 with endocytic clathrin-coated vesicles revealed with the use of brefeldin A.

The interaction of the adipocyte/skeletal muscle glucose transporter (GLUT-4) with clathrin lattices may be important in maintaining its intracellular distribution. To better understand the role of clathrin lattices in the sorting of GLUT-4, we have attempted to determine the cellular origin of clathrin-coated vesicles (CCVs) that contain this transporter. The fungal toxin brefeldin A (BFA) causes the selective disassembly of clathrin lattices at the trans-Golgi network (TGN), but not at the plasma membrane (PM), thus providing a way of estimating the proportion of GLUT-4 in PM- versus TGN-derived clathrin lattices. Exposure of 3T3-L1 adipocytes to BFA resulted in a rapid disassembly of clathrin lattices at the TGN, observed by optical sectioning microscopy, and to a pronounced decrease in the yield of CCVs purified from these cells. Thus, CCVs isolated from BFA-treated cells are likely to be derived from the PM. Immunoblotting experiments revealed the presence of GLUT-4 in such CCVs, suggesting that under basal conditions the transporter is continually retrieved from the PM through the CCV pathway. Exposure of both BFA-treated or non-treated cells to insulin resulted in a 4-6-fold increase in the concentration of GLUT-4 at the PM. In parallel, the concentration of GLUT-4 in PM-derived CCVs decreased by 60%. These results suggest (a) that the effect of insulin to increase the cell surface concentration of GLUT-4 is not inhibited by BFA, and (b) that a decreased association of GLUT-4 with endocytic CCVs may be important in facilitating its increased cell surface concentration in response to the hormone.

3T3 Cells↗
PubMed1994

A double leucine within the GLUT4 glucose transporter COOH-terminal domain functions as an endocytosis signal.

The unique COOH-terminal 30-amino acid region of the adipocyte/skeletal muscle glucose transporter (GLUT4) appears to be a major structural determinant of this protein's perinuclear localization, from where it is redistributed to the cell surface in response to insulin. To test whether an underlying mechanism of this domain's function involves glucose transporter endocytosis rates, transfected cells were generated expressing exofacial hemagglutinin epitope (HA)-tagged erythrocyte/brain glucose transporter (GLUT1) or a chimera containing the COOH-terminal 30 amino acids of GLUT4 substituted onto this GLUT1 construct. Incubation of COS-7 or CHO cells expressing the HA-tagged chimera with anti-HA antibody at 37 degrees resulted in an increased rate of antibody internalization compared to cells expressing similar levels of HA-tagged GLUT1, which displays a cell surface disposition. Colocalization of the internalized anti-HA antibody in vesicular structures with internalized transferrin and with total transporters was established by digital imaging microscopy, suggesting the total cellular pool of transporters are continuously recycling through the coated pit endocytosis pathway. Mutation of the unique double leucines 489 and 490 in the rat GLUT4 COOH-terminal domain to alanines caused the HA-tagged chimera to revert to the slow endocytosis rate and steady-state cell surface display characteristic of GLUT1. These results support the hypothesis that the double leucine motif in the GLUT4 COOH terminus operates as a rapid endocytosis and retention signal in the GLUT4 transporter, causing its localization to intracellular compartments in the absence of insulin.

Amino Acid Sequence↗
PubMed1994

Cigarette smoking and histological outcome in women with mildly dyskaryotic cervical smears.

OBJECTIVE: To determine which patient related variables, available at the point of referral, predict the presence of high grade intraepithelial neoplasia when the smear result is mild dyskaryosis with or without co-existent koilocytosis. DESIGN: Multivariate analysis of prospective programme trial. SETTING: Academic Department Colposcopy clinics at Dudley Road Hospital, Birmingham, UK. SUBJECTS: One hundred and sixty-seven women, whose worst ever cervical smear was mild dyskaryosis with or without koilocytosis, referred to colposcopy clinics. All the women had complete data sets, as determined by the format of an ongoing programme, and all were treated by diathermy loop excision of the cervical transformation zone. MAIN OUTCOME MEASURES: Age, parity, contraceptive practice, smoking habit, duration of abnormal cytology and the grade of histology in the excised transformation zone. RESULTS: Forty-seven out of 78 (60%) smokers, compared with 18 out of 73 (25%) nonsmokers, had high grade disease. The other variables considered in the analysis were not significant or very weakly associated with histological grade when analysed in a univariate analysis. Stepwise logistic regression identified cigarette smoking as a powerful independent predictor of high grade disease. CONCLUSION: These data suggest a strong association between smoking and high grade intraepithelial neoplasia in a population of women whose worst ever smear report was mild dyskaryosis. Smoking is a variable that could be built into models to facilitate referral for colposcopic assessment.

Adolescent↗
PubMed1993

Direct diagnosis of myotonic dystrophy with a disease-specific DNA marker.

BACKGROUND: Myotonic dystrophy is the most common inherited form of muscular dystrophy affecting adults. Its symptoms are not confined to muscle, and variability in their nature and in the patient's age at their onset can make diagnosis difficult. A specific unstable DNA sequence associated with myotonic dystrophy has recently been identified. We describe the use of a DNA probe (p5B1.4) that can detect this mutation directly, improving the accuracy and speed of diagnosis. METHODS: We analyzed DNA extracted from the peripheral-blood lymphocytes of 112 unrelated patients with myotonic dystrophy and their families, using molecular genetic techniques. Southern blot analysis and amplification with the polymerase chain reaction were used to determine the extent of expansion of the unstable DNA sequence. RESULTS: Probe p5B1.4 allowed direct identification of the myotonic dystrophy mutation in 108 of the 112 unrelated patients. In three families for whom the clinical and genetic data obtained with linked probes were ambiguous, the probe identified persons at risk for symptoms of this disorder and demonstrated that a possible sporadic case of myotonic dystrophy was familial. In one of these families the size of the unstable myotonic dystrophy-specific fragment decreased on transmission to offspring, who remained asymptomatic. CONCLUSIONS: The diagnosis of myotonic dystrophy is improved by the use of a probe that detects directly the mutation responsible for this disorder.

Adolescent↗
PubMed1993

Isolation and ordering of bacteriophage genomic clones corresponding to two YACs from 19q13.3.

We describe a method for rapidly isolating overlapping bacteriophage clones corresponding to the genomic region cloned in a yeast artificial chromosome (YAC) that does not require sub-cloning or lambda DNA preparation. Purified YACs from 19q13.3 were used to screen a flow-sorted chromosome 19 library, and the resulting positive clones were characterized using inter-Alu PCR. In addition, aliquots of the lambda stocks were gridded out, and hybridized with probes known to be present in the YACs, thereby avoiding having to perform DNA preparations. The application of this technique in the identification of lambda clones which span the myotonic dystrophy (DM) locus on 19q13.3 is presented, and its general advantages are discussed.

Bacteriophage lambda↗
PubMed1993

Pregnancy outcome after large loop excision of the cervical transformation zone.

OBJECTIVE: Our purpose was to determine whether large loop excision of the cervical transformation zone affects the outcome of pregnancy after 20 weeks' gestation. STUDY DESIGN: In a retrospective case control study 40 women who had undergone large loop excision of the cervical transformation zone and were subsequently delivered at Dudley Road Hospital were identified between January 1989 and January 1992. Eighty controls were identified and matched for age, parity, and ethnic group from women delivered immediately before and after index cases. Variables included maternal performance in labor and smoking habits and perinatal outcome. Maternal factors analyzed included pregnancy gestation, length of the first and second stages of labor, use of oxytocin, analgesia, mode of delivery, estimated blood loss, whether labor was spontaneous or induced, and if preterm spontaneous rupture of membranes occurred. Perinatal outcome measures included whether the infant was liveborn, fetal weight, the presence of fetal abnormalities, and admission to the neonatal unit. RESULTS: Women delivered after large loop excision of the cervical transformation zone had infants of significantly lower birth weight than did controls. They were also significantly more likely to have admitted to smoking on admission. CONCLUSION: Previous studies investigating pregnancy outcome after local destructive methods of treating cervical intraepithelial neoplasia have been generally reassuring. However, in this study women who were delivered after large loop excision of the cervical transformation zone had significantly smaller infants. Although this may be related to the characteristics of women who have cervical intraepithelial neoplasia (for example, their smoking habits), larger adequately controlled studies should be performed before colposcopists can be justified in adopting a liberal attitude to treating all women with abnormal smears.

Adult↗
PubMed1993

Exofacial epitope-tagged glucose transporter chimeras reveal COOH-terminal sequences governing cellular localization.

The insulin-regulated adipocyte/skeletal muscle glucose transporter (GLUT4) displays a characteristic steady-state intracellular localization under basal conditions, whereas the erythrocyte/brain transporter isoform (GLUT1) distributes mostly to the cell surface. To identify possible structural elements in these transporter proteins that determine their cellular localization, GLUT1/GLUT4 chimera cDNA constructs that contain the hemagglutinin epitope YPYDVPDYA (HA) in their major exofacial loops were engineered. Binding of monoclonal anti-HA antibody to non-permeabilized COS-7 cells expressing HA-tagged transporter chimeras revealed that expression of transporters on the cell surface was strongly influenced by their cytoplasmic COOH-terminal domain. This method also revealed a less marked, but significant effect on cellular localization of amino acid residues between transporter exofacial and middle loops. The subcellular distribution of expressed chimeras was confirmed by immunofluorescence microscopy of permeabilized COS-7 cells. Thus, HA-tagged native GLUT4 was concentrated in the perinuclear region, whereas a chimera containing the COOH-terminal 29 residues of GLUT1 substituted onto GLUT4 distributed to the plasma membrane, as did native GLUT1. Furthermore, a chimera composed of GLUT1 with a GLUT4 COOH-terminal 30-residue substitution exhibited a predominantly intracellular localization. Similar data was obtained in CHO cells stably expressing these chimeras. Taken together, these results define the unique COOH-terminal cytoplasmic sequences of the GLUT1 and GLUT4 glucose transporters as important determinants of cellular localization in COS-7 and CHO cells.

Amino Acid Sequence↗
PubMed1993

Molecular mechanisms in Angelman syndrome: a survey of 93 patients.

Angelman syndrome (AS) results from a lack of maternal contribution from chromosome 15q11-13, arising from de novo deletion in most cases or rarely from uniparental disomy. These families are associated with a low recurrence risk. However, in a minority of families, more than one child is affected. No deletion has been found in these families, except one. The mode of inheritance in these families is autosomal dominant modified by imprinting. Sporadic cases, with no observable deletion, therefore pose a counselling dilemma as there could be a recurrence risk as high as 50%. We present a series of 93 AS patients, showing the relative contribution of these different genetic mechanisms. Eighty-one AS patients were sporadic cases while 12 cases came from six families. Sixty cases had deletions in 15q11-13 detected by a set of highly polymorphic (CA)n repeats markers and conventional RFLPs. Ten sporadic cases plus all 12 familial cases had no detectable deletion. In addition, two cases of de novo deletions occurred in a chromosome 15 carrying a pericentric inversion. In one of these the AS child had a cousin with Prader-Willi syndrome (PWS) arising from a de novo deletion in an inv(15) inherited from his father. One case arose from a maternal balanced t(9;15)(p24;q15) translocation. There were three cases of uniparental disomy. Five patients were monoallelic for all loci across the minimal AS critical region, but the presence of a deletion cannot be confirmed. In familial cases, all affected sibs inherited the same maternal chromosome 15 markers for the region 15q11-13. Two cases were observed with a de novo deletion starting close to the locus D15S11 (IR4-2R), providing evidence for the development of classical AS with smaller deletions. Cytogenetic analysis proved limited in its ability to detect deletions, detecting only 42 out of 60 cases. However, cytogenetic analysis is still essential to detect chromosomal abnormalities other than deletions such as inversions and balanced translocations since both have an increased risk for deletions. A staged diagnostic strategy based on the use of highly informative (CA)n repeat markers is proposed.

Angelman Syndrome↗
PubMed1992

Detection of an unstable fragment of DNA specific to individuals with myotonic dystrophy.

Myotonic dystrophy (DM) is the most common form of adult muscular dystrophy, with a prevalence of 2-14 per 100,000 individuals. The disease is characterized by progressive muscle weakness and sustained muscle contraction, often with a wide range of accompanying symptoms. The age at onset and severity of the disease show extreme variation, both within and between families. Despite its clinical variability, this dominant condition segregates as a single locus at chromosome 19q13.3 in every population studied. It is flanked by the tightly linked genetic markers ERCC1 proximally and D19S51 distally; these define the DM critical region. We report the isolation of an expressed sequence from this region which detects a DNA fragment that is larger in affected individuals than in normal siblings or unaffected controls. The size of this fragment varies between affected siblings, and increases in size through generations in parallel with increasing severity of the disease. We postulate that this unstable DNA sequence is the molecular feature that underlies DM.

Chromosomes, Human, Pair 19↗