Search PubMed⌕ Search

Biomedical subjects

N S Thomas

Publications and source records attributed to N S Thomas.

At least 73 records · Page 4Linked to original sources
PubMed1996

Cloning and characterization of cDNAs encoding the epsilon-subunit of eukaryotic initiation factor-2B from rabbit and human.

A rabbit reticulocyte lysate cDNA library was screened with a polyclonal antiserum directed against eukaryotic initiation factor eIF-2B (eIF-2B). A 2508 base pair cDNA (pA1) was isolated and determined to encode the epsilon-subunit of eIF-2B based on the immunoreactivity of the fusion protein expressed from the cDNA in Escherichia coli and the presence of two peptide sequences obtained from two V8 fragments of purified nonrecombinant eIF-2B epsilon in the deduced amino acid sequence of the cDNA. The open reading frame of the cDNA began with the third nucleotide of the cDNA with the first AUG codon at nucleotide 522. Mutational analysis of pA1 indicated that the cDNA did not code for full-length eIF-2B epsilon. Seven missing codons of the reading-frame and the 71 nucleotide 5' non-coding region of the eIF-2B epsilon mRNA were obtained by 5' RACE. A human eIF-2B epsilon cDNA fragment, which corresponded to a similar 2.3 kb fragment generated by digestion of the rabbit pA1 cDNA with EcoRI, was isolated from a human histiocytic lymphoma (U-937) cell cDNA library constructed in lambda gt10. The nucleotide and amino acid sequences were highly conserved between the rabbit and human cDNAs, showing approx. 90% sequence identity within the open reading frame. Northern and Western blot analyses of reticulocyte lysate and other rabbit tissue extracts indicated that the eIF-2B epsilon polypeptide has a similar apparent molecular weight in all tissues examined, and is coded for by a single approximately 2.8 kilobase mRNA species which is ubiquitously expressed.

Amino Acid Sequence↗
PubMed1996

Oct-1 [corrected] and Oct-2 DNA-binding site specificity is regulated in vitro by different kinases.

The transcription factors Oct-1 and Oct-2 bind differentially to three octamer binding sequences corresponding to the octamer binding site from the H2B promoter [ATGCTAATAA], a simple TAATGARAT motif, found in herpes simplex virus IE4/5 genes [GCGGTAATGAGAT], and a perfect consensus overlapping octamer/TAATGARAT motif [ATGCTAATGAGAT]. By comparing the effects of protein kinase A, protein kinase C and casein kinase 2 in vitro on the binding of Oct-1 and Oct-2 to the three motifs, we show that the actions of these kinases regulate Oct-1 and Oct-2 DNA binding independently of each other in a binding-site-specific manner. Inhibition of cellular phosphatases also regulate Oct-1 and Oct-2 DNA binding in a binding-site-specific manner. Both kinase and phosphatase activity are important for regulating the DNA binding activity of Oct-1 and Oct-2 because, in the presence of phosphatase inhibitors, protein kinase A attenuates the binding of both Oct-1 and Oct-2 to the octamer binding site but enhances binding when phosphatase inhibitors are omitted. Thus the DNA specificity of Oct-1 and Oct-2 can be regulated in vitro by the action of different kinases.

Base Sequence↗
PubMed1996

Variable expression of p16 protein in patients with acute myeloid leukemia without gross rearrangements at the DNA level.

The p16 protein is an inhibitor of cyclin-dependent kinases (cdk) 4 and 6. Both cdk4 and cdk6 phosphorylate the retinoblastoma protein (pRb) and are thought to be required for cell proliferation. Mutations and homozygous deletions in the p16 gene have been found in a number of cell lines but the incidence of abnormalities in primary tumours is controversial. We have studied the p16 gene in 76 cases of acute myeloid leukemia (AML) and 18 hematologically normal controls by quantitative Southern blotting. No deletions or rearrangements were detected. Twenty-five cases also showed no abnormal band patterns by RT-PCR-SSCP analysis of the coding sequence. We analyzed 60 AML samples for p16 protein expression by Western blot analysis. A reduced level of p16 was observed in six cases, however, none of them showed methylation of the 5'-CpG island. Over-expression of p16 protein was detected in six cases. Studies in cell lines have suggested a feedback loop between p16 and pRb with a futile overproduction of p16 protein in cells containing abnormal pRb. In accordance with these findings, four of the AML cases with high levels of p16 had abnormal pRb (two alteration in band size, two absent). From our data we suggest that gross abnormalities in the p16 gene are rare in AML, but that p16 levels are variable and high levels are associated with pRb abnormalities in a subset of cases.

Acute Disease↗
PubMed1996

Expression of two alternatively spliced forms of the 5' untranslated region of the GM-CSF receptor alpha chain mRNA.

The granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is composed of at least two chains (alpha and beta). The alpha chain binds GM-CSF specifically with low affinity, and the binding is converted to high affinity when the alpha chain is associated with the beta chain. To date, there are at least six isoforms described for the GM-CSFR alpha, all involving alternative splicing at the 3' end, which alters the coding region and hence the protein produced. To detect variants at the 5' end of the GM-CSFR alpha mRNA, RNAse protection and reverse transcriptase polymerase chain reaction (RT-PCR) assays were performed using a probe spanning nucleotides 102-392 and pairs of primers covering exons 1-4. in addition to the expected full-length transcript, two mRNAs were detected, one containing a deletion of 24 nucleotides by alternative splicing at the 3' end of exon 2 (exon 2b-deleted isoform) and another in which exon 2 was completely deleted (exon 2-deleted isoform). Together, the isoforms were more highly expressed form). Together, the isoforms were more highly expressed than the full-length sequence (TF-1 cells: full-length 36 +/- 2.8% vs. exon 2-deleted isoforms 64 +/- 5.5%). These isoforms were detected in primary hematopoietic cells, blasts from patients with acute myeloid leukemia (AML), and malignant cell lines and the relative mRNA expression for the isoforms, was always similar to that of TF-1 cells. As sequences in the 5'untranslated region can be involved in the modulation of translational efficiency, translation of constructs constructs corresponding to these exon 2 deleted isoforms was assessed using an in vitro reticulocyte lysate system. Deletion of exon 2 resulted in significantly lower in vitro translation of the receptor protein relative to the full-length sequence (53, 56, and 76% in three separate batches of reticulocytes), while deletion of exon 2b resulted in higher translation of the sequence (164, 128, and 305%; p = 0.01). These data suggest a mechanism by which expression of the GM-CSFR alpha protein may be regulated by alternatively spliced transcripts with different translational efficiencies.

Acute Disease↗
PubMed1995

Down regulation of the octamer binding protein Oct-1 during growth arrest and differentiation of a neuronal cell line.

The octamer binding transcription/DNA replication factor Oct-1 is present in virtually all cell types including proliferating cell lines of neuronal origin but is not detectable in mature non-dividing neurons. Cell cycle arrest in G0/G1 and morphological differentiation of a neuronal cell line is accompanied by a decline in the level of Oct-1 DNA binding, although the level of DNA binding by another octamer binding protein, Oct-2 is unaltered. This effect is paralled by a decline in the level of the Oct-1 mRNA in the non-dividing cells. The decrease in Oct-1 levels occurs only with the production of a mature, non-dividing neuronal phenotype and not when the cells are arrested in late G1 and do not undergo morphological differentiation. The potential role of Oct-1 and other octamer binding proteins in gene regulation in neuronal cells and in their differentiation is discussed.

Animals↗
PubMed1995

Aland island eye disease: clinical and electrophysiological studies of a Welsh family.

Clinical and molecular genetic studies were performed on a single, large, white family, in which congenital nystagmus and moderate to high refractive error segregated as a sex linked trait with manifestation in some female carriers. In this family, affected males demonstrate myopia, but a high proportion of female carriers, and some of the possibly affected males, show hypermetropia. Clinical ophthalmic examination and electrodiagnostic studies of retinal function were fully compatible with a diagnosis of either incomplete congenital stationary night blindness or of Aland island eye disease. Previous studies have mapped both disorders to the proximal short arm of the X chromosome: our molecular studies support this localisation. Incomplete congenital stationary nightblindness and Aland Island eye disease could be considered as a single entity.

Adult↗
PubMed1994

Differentiation-linked changes in tyrosine phosphorylation, functional activity, and gene expression downstream from the granulocyte-macrophage colony-stimulating factor receptor.

The HL-60 model of myeloid maturation was used to test whether changes in signaling from the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor accompany maturation-related changes in cellular responses to GM-CSF. Receptor expression, tyrosine phosphorylation, functional activity, and c-fos gene expression were measured. Functional GM-CSF receptors were present throughout differentiation as both uninduced and dimethyl sulfoxide (DMSO)-induced HL-60 cells responded to GM-CSF, albeit in different ways. Uninduced promyelocytes proliferated in response to GM-CSF, whereas DMSO-induced cells lost the capacity to proliferate but did respond with increased expression of beta 2-integrins, enhanced respiratory burst activity, and metabolism of arachidonic acid. GM-CSF-stimulated upregulation of c-fos mRNA expression was not detected in immature cells but developed after 2 to 4 days with DMSO in line with a marked increase in responsiveness to stimulation with phorbol ester, showing that increased expression of c-fos is predominantly a feature of mature phagocytes. GM-CSF stimulated the tyrosine phosphorylation of a broadly similar range of proteins in both uninduced and DMSO-treated HL-60 cells, but protein bands were more heavily phosphorylated in DMSO-induced cells. Phosphorylation was rapid in onset and very transient in immature cells. Phosphorylation of several proteins, in particular a 130-kD band, was more sustained in DMSO-induced cells. These differences in signaling were not because of numerical differences in receptors, because reduction of GM-CSF concentration to trigger equivalent numbers of high-affinity receptors delayed the onset of phosphorylation in DMSO-induced cells. We conclude that there are maturation-related changes in signaling downstream from the GM-CSF receptor.

Arachidonic Acid↗
PubMed1994

Into and out of G1: the control of cell proliferation.

Progression of cells through G1 is an orderly process which is regulated by a series of key proteins. Over the past few years a number of these proteins have been identified and their mode of action has been studied in detail. This review will concentrate on the current knowledge about the function in G1 of cyclins, cyclin dependent kinases, the retinoblastoma protein and other related 'pocket' proteins. Mutations or deregulation of some of these proteins confer a growth advantage and their role in malignant transformation will be discussed.

Animals↗
PubMed1994

X linked myotubular myopathy (MTM1) maps between DXS304 and DXS305, closely linked to the DXS455 VNTR and a new, highly informative microsatellite marker (DXS1684).

The locus for X linked recessive myotubular myopathy (MTM1) has previously been mapped to Xq28 by linkage analysis. We report two new families that show recombination between MTM1 and either DXS304 or DXS52. These families and a third previously described recombinant family were analysed with two highly polymorphic markers in the DXS304-DXS52 interval, the DXS455 VNTR and a newly characterised microsatellite, DXS1684 (82% heterozygosity). These markers did not recombine with MTM1 in the three families. Together with the recent mapping of an interstitial X chromosome deletion in a female patient with moderate signs of myotubular myopathy, our data suggest the following order of loci in Xq28: cen-DXS304-(DXS455, MTM1)-DXS1684-DXS305-DXS52-tel. This considerably refined localisation of the MTM1 locus should facilitate positional cloning of the gene. The availability of highly polymorphic and very closely linked markers will markedly improve carrier and prenatal diagnosis of MTM1.

Base Sequence↗
PubMed1994

Detection of de novo mutations and analysis of their origin in families with X linked hypohidrotic ectodermal dysplasia.

Hypohidrotic ectodermal dysplasia (EDA) has been localised to the q12-q13.1 region of the X chromosome by both physical and genetic mapping methods. Although linkage analysis using closely linked flanking markers can clarify the carrier status for many females at risk for the disorder, knowledge of the origin of the mutation in instances of possible de novo mutation is critical for accurate genetic counselling of families. Two methods have been used to confirm de novo mutation in families with EDA and to trace their origin. Direct detection of three de novo molecular deletions, one arising during oogenesis and the other two during spermatogenesis, was achieved by Southern analyses using cosmids isolated from the EDA region as probes. Seven de novo mutations arising during spermatogenesis, and two possible de novo mutations during oogenesis, were identified by an analysis of the cosegregation of the disorder with polymorphic markers closely linked to and flanking the EDA locus. The confirmation and analysis of the origin of the 10 de novo mutations greatly assisted genetic counselling in these families. The apparent 3.5:1 excess of male to female origin of mutation in families studied with unidentified types of mutation is similar to other studies of X linked disorders, and suggests that the majority of these mutations may involve single base pair substitutions.

Adult↗
PubMed1993

X chromosome linkage studies in familial Rett syndrome.

Four families, each with two individuals affected by Rett Syndrome (RS), were analysed using restriction fragment length polymorphisms and microsatellite markers from the X chromosome. In two of the families, X-linked dominant inheritance of the RS defect from a germinally mosaic mother could be assumed. Therefore, maternal X chromosome markers showing discordant inheritance were used to exclude regions of the X chromosome as locations of the RS gene. Much of the short arm could be excluded, including regions containing three candidate genes, OTC, synapsin 1 and synaptophysin. Although most of the long arm was inherited in common it was possible to exclude a centromeric region. Inheritance of X chromosome markers is also presented for two families with affected aunt-niece pairs, one of which has not been previously studied at the DNA level.

Chromosome Mapping↗
PubMed1993

Characterisation of molecular DNA rearrangements within the Xq12-q13.1 region, in three patients with X-linked hypohidrotic ectodermal dysplasia (EDA).

A panel of somatic cell hybrids and X-linked hypohidrotic ectodermal dysplasia (EDA) patient-derived cell lines, containing different rearranged X chromosomes, have been used to refine the physical map of the Xq12-q13.1 region. The patient-derived material included genomic DNA from an EDA male (EDA family 1015) with an interstitial deletion, and a cell line GM0705A, obtained from an isolated female patient with a de novo balanced (X;9) translocation, and the somatic hybrid, AnLy, derived from this cell line. This map subdivides the region into at least 6 mapping-intervals. DNA probes from DXS732 and DXS453, identified as the closest flanking marker loci to the EDA locus, were used to identify homologous Yeast Artificial Chromosome (YAC) clones. Two of the DXS732-specific YACs were shown by fluorescent in situ hybridisation (FISH) analysis to bridge the (X;9) translocation breakpoint. These two YACs were also screened against the ICRF human X chromosome cosmid library and identified 36 cosmid clones. Direct cosmid-cosmid hybridisation analysis placed subsets of these clones within four different cosmid contigs. Mapping of anchor clones from each contig, against the mapping panel, localised all these contigs within the Xq12-q13.1 region. One cosmid, ICRFc104C03.184, identified potential junctional-fragments in several restriction digests of AnLy hybrid DNA. This was confirmed by FISH analysis of the GM0705A cell line with total cosmid ICRFc104C03.184, in which both chromosomal elements of the (X;9) translocation were identified. A single-copy probe pC03.184E2, derived from this cosmid, also identified the der(9)-derived junctional fragment when hybridised against AnLy DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1993

Cell cycle arrest of proliferating neuronal cells by serum deprivation can result in either apoptosis or differentiation.

Apoptotic cell death plays a critical role in the development of the nervous system. The death of mature nondividing neurons that fail to receive appropriate input from the target field has been extensively studied. However, the mechanisms mediating the extensive cell death occurring in areas of the developing brain where proliferating neuroblasts differentiate into mature nondividing neurons have not been analyzed. We show here that the cell cycle arrest of a proliferating cell of neuronal origin by removal of serum results in either apoptotic cell death or differentiation to a mature nondividing neuronal cell. The proportion of cells undergoing death or differentiation is influenced in opposite directions by treatment of the cells with cyclic AMP and retinoic acid. This suggests that following the withdrawal of signals stimulating neuroblast cell division, neuronal cells either can cease to suppress a constitutive suicide pathway and hence die by apoptosis or, alternatively, can differentiate into a mature neuronal cell. Regulation of the balance between apoptosis and neuronal differentiation could therefore play a critical role in controlling the numbers of mature neurons that form.

Animals↗
PubMed1993

Detection of a molecular deletion at the DXS732 locus in a patient with X-linked hypohidrotic ectodermal dysplasia (EDA), with the identification of a unique junctional fragment.

X-linked hypohidrotic ectodermal dysplasia (EDA) has been localized to the Xq12-q13.1 region. A panel of genomic DNA samples from 80 unrelated males with EDA has been screened for deletions at seven genetic loci within the Xq12-13 region. A single individual was identified with a deletion at the DXS732 locus by hybridization with the mouse genomic probe pcos169E/4. This highly conserved DNA probe is from locus DXCrc169, which is tightly linked to the Ta locus, the putative mouse homologue of EDA. The proband had the classical phenotype of EDA, with no other phenotypic abnormalities, and a normal cytogenetic analysis. A human genomic DNA clone, homologous to pcos169E/4, was isolated from a human X-chromosome cosmid library. On hybridization with the cosmid, the proband was found to be only partially deleted at the DXS732 locus, with a unique junctional fragment identified in the proband and in three of his maternal relatives. This is the first determination of carrier status for EDA in females, by direct mutation analysis. Failure to detect deletion of the other loci tested in the proband suggests that the DXS732 locus is the closest known locus to the EDA gene. Since the DXS732 locus contains a highly conserved sequence, it must be considered to be a candidate locus for the EDA gene itself.

Adult↗
PubMed1992

Phenotypic evidence for a common pathogenesis in X-linked deafness pedigrees and in Xq13-q21 deletion related deafness.

A structural cochlear abnormality has been observed by high resolution CT scanning in some families where X-linked deafness is segregating. We now present evidence that the same abnormality is present in a deaf patient who has a deletion within Xq21. This observation provides phenotypic evidence that the genotypic basis of deafness is the same in both patient groups. It is also likely that the perilymphatic fluid "gusher" abnormality may be common to both.

Child↗
PubMed1992

The synthesis of p58cyclin A and the phosphorylation of p34cdc2 are inhibited in human lymphoid cells arrested in G1 by alpha-interferon.

Daudi cells arrest in G1 in the presence of alpha-interferon. Such cells have little p58cyclin A, probably due to inhibition of p58cyclin A synthesis. The phosphorylation-associated migration shift of p34cdc2 is not seen in alpha-interferon-arrested cells. Cells arrested in late G1 by aphidicolin have abundant p58cyclin A and phosphorylated p34cdc2. Cell sorting showed that p58cyclin A increases in proliferating cells in late G1 and coincides with phosphorylation of p34cdc2.

Aphidicolin↗
PubMed1992

Down-regulation of human protein kinase C alpha is associated with terminal neutrophil differentiation.

We have established an RNase protection method to quantify the expression of mRNA for the human protein kinase C (PK-C) isoforms alpha, beta 1, beta 2, and gamma. This was used to investigate whether each isoform is differentially expressed during the differentiation of hematopoietic cells. Myeloid and lymphoid cells express PK-C alpha, beta 1, and beta 2 mRNAs in various proportions. PK-C gamma mRNA was detected in human brain, but not in hematopoietic cells. PK-C alpha mRNA decreases as HL-60 cells mature to a neutrophil phenotype in response to retinoic acid, but its abundance does not change during monocytic differentiation in response to vitamin D3. PK-C alpha mRNA and protein were undetectable in peripheral blood neutrophils, but are present in monocytes. The mRNAs for PK-C beta 1 and beta 2 isoforms increase during HL-60 differentiation and are expressed in both neutrophils and monocytes. Therefore, the PK-C alpha isoform is specifically down-regulated during human neutrophil terminal differentiation. These data suggest that mature neutrophil functions do not require the PK-C alpha isoform.

Calcitriol↗