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M Robertson

Publications and source records attributed to M Robertson.

At least 91 records · Page 5Linked to original sources
PubMed1995

Proteolytic activation of protein kinase C delta by an ICE-like protease in apoptotic cells.

These studies demonstrate that treatment of human U-937 cells with ionizing radiation (IR) is associated with activation of a cytoplasmic myelin basic protein (MBP) kinase. Characterization of the kinase by gel filtration and in-gel kinase assays support activation of a 40 kDa protein. Substrate and inhibitor studies further support the induction of protein kinase C (PKC)-like activity. The results of N-terminal amino acid sequencing of the purified protein demonstrate identity of the kinase with an internal region of PKC delta. Immunoblot analysis was used to confirm proteolytic cleavage of intact 78 kDa PKC delta in control cells to the 40 kDa C-terminal fragment after IR exposure. The finding that both IR-induced proteolytic activation of PKC delta and endonucleolytic DNA fragmentation are blocked by Bcl-2 and Bcl-xL supports an association with physiological cell death (PCD). Moreover, cleavage of PKC delta occurs adjacent to aspartic acid at a site (QDN) similar to that involved in proteolytic activation of interleukin-1 beta converting enzyme (ICE). The specific tetrapeptide ICE inhibitor (YVAD) blocked both proteolytic activation of PKC delta and internucleosomal DNA fragmentation in IR-treated cells. These findings demonstrate that PCD is associated with proteolytic activation of PKC delta by an ICE-like protease.

Amino Acid Sequence↗
PubMed1995

Fas-activated serine/threonine kinase (FAST) phosphorylates TIA-1 during Fas-mediated apoptosis.

We have identified a serine/threonine kinase that is rapidly activated during Fas-mediated apoptosis. Fas-activated serine/threonine kinase (FAST) is phosphorylated on serine and threonine residues in Jurkat cells. In response to Fas ligation, it is rapidly dephosphorylated and concomitantly activated to phosphorylate TIA-1, a nuclear RNA-binding protein that has been implicated as an effector of apoptosis. Phosphorylation of TIA-1 precedes the onset of DNA fragmentation, suggesting a role in signaling downstream events in the apoptotic program. Our results introduce Fast and TIA-1 as components of a molecular cascade involved in signaling Fas-mediated apoptosis.

Amino Acid Sequence↗
PubMed1995

Response of colon cancer cell lines to the introduction of APC, a colon-specific tumor suppressor gene.

The APC gene, mutations in which are responsible for the inherited colon cancer syndrome adenomatous polyposis coli (APC), is described as a tumor suppressor gene. A full-length, wild-type APC gene was introduced by transfection into three human colon carcinoma cell lines, each characterized for mutations at loci involved in colon tumor formation. The response of each cell line to the introduction of APC differed with the genotype of the cell line. Some of the cell clones derived from these transfections displayed altered morphologies; some showed suppression of tumorigenicity based on growth in soft agar and tumor formation in nude mice. One cell line, SW480, could not be stably transfected with the APC gene. These results provide the first direct evidence that the APC gene can alter the transformation properties of colon carcinoma cells.

Animals↗
PubMed1995

The RNA-binding protein TIAR is translocated from the nucleus to the cytoplasm during Fas-mediated apoptotic cell death.

We have determined the structure, intracellular localization, and tissue distribution of TIAR, a TIA-1-related RNA-binding protein. Two related isoforms of TIAR, migrating at 42 and 50 kDa, are expressed in primate cells. Unlike TIA-1, which is found in the granules of cytotoxic lymphocytes, TIAR is concentrated in the nucleus of hematopoietic and nonhematopoietic cells. Because TIAR can trigger DNA fragmentation in permeabilized thymocytes, it is a candidate effector of apoptotic cell death. Consistent with this possibility, we have found that the expression and intracellular localization of TIAR change dramatically during Fas-mediated apoptosis. TIAR moves from the nucleus to the cytoplasm within 30 min of Fas ligation. Redistribution of TIAR precedes the onset of DNA fragmentation and is not a nonspecific consequence of nuclear disintegration. Cytoplasmic redistribution of TIAR is not observed during cellular activation triggered by mitogens such as concanavalin A or phytohemagglutinin. Our results suggest that cytoplasmic redistribution of TIAR may be a general feature of the apoptotic program.

Antibodies, Monoclonal↗
PubMed1995

Genomic organization of the neurofibromatosis 1 gene (NF1).

Neurofibromatosis 1 maps to chromosome band 17q11.2, and the NF1 locus has been partially characterized. Even though the full-length NF1 cDNA has been sequenced, the complete genomic structure of the NF1 gene has not been elucidated. The 5' end of NF1 is embedded in a CpG island containing a NotI restriction site, and the remainder of the gene lies in the adjacent 350-kb NotI fragment. In our efforts to develop a comprehensive screen for NF1 mutations, we have isolated genomic DNA clones that together harbor the entire NF1 cDNA sequence. We have identified all intron-exon boundaries of the coding region and established that it is composed of 59 exons. Furthermore, we have defined the 3'-untranslated region (3'-UTR) of the NF1 gene; it spans approximately 3.5 kb of genomic DNA sequence and is continuous with the stop codon. Oligonucleotide primer pairs synthesized from exon-flanking DNA sequences were used in the polymerase chain reaction with cloned, chromosome 17-specific genomic DNA as template to amplify NF1 exons 1 through 27b and the exon containing the 3'-UTR separately. This information should be useful for implementing a comprehensive NF1 mutation screen using genomic DNA as template.

Amino Acid Sequence↗
PubMed1995

A barium enema training programme for radiographers: a pilot study.

We describe a training programme designed to instruct radiographers how to perform the double contrast barium enema (DCBE). The programme was assessed by comparing the first 50 unsupervised examinations with studies performed by senior registrars in radiology. Comparison was made by marking each study blind in terms of barium coating, colonic distension and visualisation of the colon in double contrast. A note was also made of the number of exposures, screening time, examination time and complications. No difference was found in any of these parameters when comparing the two groups. A further 50 patients were assessed at one year and this showed that standards had been maintained in terms of the above criteria. Follow-up at 3 years in the initial group has shown no missed pathology. We believe that delegation of the performance of the DCBE to radiographers is both acceptable to the patient and safe.

Barium Sulfate↗
PubMed1995

Monoclonal antibody-purged bone marrow transplantation therapy for multiple myeloma.

This report describes the clinical characteristics, treatment associated toxicity, and follow-up of fifty-eight patients with plasma cell--dyscrasias treated with high dose chemotherapy and total body irradiation (TBI) at a single institution. Following TBI, 36 patients received anti-B cell monoclonal antibody (MoAb)-treated autologous bone marrow, 21 patients received anti-CD6 cell MoAb-treated allogeneic bone marrow to deplete T cells, and one patient received unpurged bone marrow from a syngeneic donor. Evaluation after high dose chemotherapy and bone marrow transplantation (BMT) demonstrated 26 complete responses (CR), 26 partial responses (PR), 2 non-responders, 1 not yet evaluated, and three toxic deaths. Fourteen of 36 patients who underwent autologous BMT are alive free from progression at 18 (range 5 to 68) months post transplant (post-BMT); of these, 11 remain in continuous complete response at 16 (range 5 to 68) months post-BMT. Seven of 21 patients who underwent allogeneic BMT are alive free from progression at 30 (range 4 to 44) months post-BMT; of these, three patients remain in continuous complete response at 43 (range 33 to 45) months post-BMT. These data suggest that high dose chemotherapy with TBI followed by MoAb purged BM can be performed with acceptable toxicity and high tumor response rates.

Adult↗
PubMed1995

Accuracy of automated DNA sequencing: a multi-laboratory comparison of sequencing results.

A double-stranded (ds)DNA template of "unknown" sequence was distributed to approximately 80 core DNA sequencing laboratories by the Association of Biomolecular Resource Facilities (ABRF) for automated DNA sequence analysis. Forty-four different facilities responded with 83 usable sequence submissions. These sequences were grouped by both sequencing protocol (dye-primer or dye-terminator) and whether manually edited or not. The sequences were aligned with the known sequence, and the number of correct base calls, insertions, deletions, no-calls and miscalls were determined for each group. The dye-primer sequencing protocol provided the longest and most accurate sequence. The edited dye-primer data were > 95% accurate out to 400-450 bp, while the edited dye-terminator data could call only 300-350 bases at this accuracy. However, 75% of the laboratories in this sampling preferred the dye-terminator protocol, presumably because of its versatility and convenience. Laboratories that manually edited the automatically called data were able to obtain an additional 100 bases of good sequence when the dye-primer protocol was used. Surprisingly though, editing of dye-terminator results did not increase the amount of good sequence, although the dye-terminator protocol had a superior base-calling ability within the first 100 bases of called sequence.

Autoanalysis↗
PubMed1994

Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression.

To investigate the activity of candidate regulatory molecules in mammalian embryogenesis, we have developed a general strategy for modifying and reporting resident chromosomal gene expression. The picornaviral internal ribosome-entry site was incorporated into gene targeting constructs to provide cap-independent translation of a selectable marker from fusion transcripts generated following homologous recombination. These promoterless constructs were highly efficient and have been used both to inactivate the stem-cell-specific transcription factor Oct-4 and to introduce a quantitative regulatory modification into the gene for a stem-cell maintenance factor, differentiation-inhibiting activity. In addition, the inclusion of a beta-galactosidase reporter gene in the constructs enabled accurate and sensitive detection of cellular sites of transcription. This has allowed visualization of putative "stem-cell niches" in which sources of elevated expression of differentiation-inhibiting activity were localized to the differentiated cells surrounding colonies of stem cells.

Animals↗
PubMed1994

Search for mutations in the beta 1 GABAA receptor subunit gene in patients with schizophrenia.

As alterations in GABAergic neurotransmission have been indirectly implicated in the pathogenetics of schizophrenia, GABAA receptor subunit genes are plausible candidate genes for the illness. We undertook a search for sequence variations in the coding region of beta 1 subunit gene by designing intron-based primers to amplify its 9 exons. Using single strand conformation polymorphism (SSCP) analysis, we found an exon 9 variant present in 3 of 86 unrelated schizophrenic cases derived from families having at least 2 first-degree relatives with schizophrenia. Direct sequencing of the SSCP variant revealed a C-->G nucleotide transversion at codon 396 predicting a histidine to glutamine substitution in the beta 1 peptide. The predicted amino acid substitution occurs at a highly conserved site, 9 residues from a cAMP-dependent serine phosphorylation consensus sequence. All known GABAA beta 1 subunit genes including human, bovine, and rat, code for histidine at position 396. Although the variant cosegregated with disease in a family with 2 affected sibs, it was only transmitted to 2 of 3 affected sibs in a multiplex family. The variant was not found in an additional sample comprising 155 unrelated schizophrenics and the sequence variant was present at a low frequency (approximately 1.1%) in control groups. Although these results indicate that the sequence variant is likely to be a natural polymorphism, it is possible that the variant may be a predisposing allele in rare instances. It is also possible that the variant may change the function or regulation of the GABAA receptor complex and it may be of pharmacogenetic relevance.

Adolescent↗
PubMed1994

A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression.

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich (core sequence KIKEK-LPG). This antiserum detected a novel M(r) 40,000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequenced differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin. The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance. The M(r) 40,000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.

Abscisic Acid↗
PubMed1994

Can children with Gilles de la Tourette syndrome edit their intentions?

In this paper we describe a cognitive mechanism, the Intention Editor, which is triggered whenever there are several intentions competing in parallel with each other. This mechanism is hypothesized to be a subcomponent of a larger mechanism, the Supervisory Attentional System (SAS: Shallice, 1988) which serves inhibition in general. The Intention Editor interrupts one of several simultaneously activated intentions, preventing it from executing its action, utterance, or thought. This mechanism appears to develop during the first five to six years of life. We propose that an impairment in the development of this mechanism may account for the triad of symptoms in children with Gilles de la Tourette Syndrome (GTS): involuntary movements, involuntary utterances, and obsessive thoughts. This mechanism is tested with normal children aged 3-6 years old, and with children with GTS, in two experiments. In Experiment 1, subjects were required to make one hand movement while inhibiting making a (different) hand movement that the other hand was simultaneously making. In Experiment 2, they were asked to say one thing while inhibiting saying something else. On both tasks, normal 6-year-olds were significantly better than normal 4-year-olds, but children with GTS performed worse than normal 6-year-olds, despite having a mean age of 12 years. These results constitute preliminary evidence for the theory that the Intention Editor is dysfunctional in GTS.

Child↗