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Biomedical subjects

N C Jones

Publications and source records attributed to N C Jones.

At least 19 recordsLinked to original sources
PubMed1996

E2F-1 but not E2F-4 can overcome p16-induced G1 cell-cycle arrest.

BACKGROUND: The transition from G1 to S phase is the key regulatory step in the mammalian cell cycle. This transition is regulated positively by G1-specific cyclin-dependent kinases (cdks) and negatively by the product of the retinoblastoma tumour suppressor gene, pRb. Hypophosphorylated pRb binds to and inactivates the E2F transcription factor, which controls the expression of genes required for S-phase progression. Hyperphosphorylation of pRb in late G1 phase results in the accumulation of active E2F, a critical event in the progression to S phase. The E2F factor is not a single entity, but rather represents a family of highly related molecules, all of which bind to pRb or the pRb-related proteins p107 and p130. RESULTS: In this study, we have used specific inhibitors of cdks to explore the requirements for cell-cycle progression from G1 to S phase. Expression of p16Ink4, which specifically inhibits cyclin D-directed cdks, blocks cells in G1 phase; this block can be overcome by expression of the viral proteins that inactivate pRb or by E2F-1. Importantly however, the G1 arrest is not overcome by overexpression of E2F-4. By using chimeric E2F proteins, containing amino-acid sequences from E2F-1 and E2F-4, we have shown that their differential abilities to overcome a p16-imposed arrest is determined by their respective amino-terminal regions. We also demonstrate that E2F-1 can promote entry into S phase without concomitant phosphorylation of pRb. In contrast to the p16-mediated G1 block, G1 arrest mediated by the cdk inhibitors p21Cip1 or p27Kip1 cannot be bypassed either by inactivation of pRb or overexpression of E2F family members. CONCLUSIONS: These data demonstrate that the role of the cyclin D-directed cdks in promoting the progression of cells from G1 into S phase is wholly to activate an E2F-1-like activity through phosphorylation, thus preventing the formation of the E2F-pRb complex. The cyclin E-cdk2 complex is also required for the G1/S transition but has a different and as yet undefined role. We also provide evidence for a functional difference between E2F-1 and E2F-4, dependant upon the region that contains the DNA-binding and dimerization domains. These results indicate that these two E2F family members are likely to regulate the expression of different subsets of E2F-responsive promoters.

Base Sequence↗
PubMed1996

The addition of tenoxicam to prilocaine for intravenous regional anaesthesia.

The analgesic effects of tenoxicam 20 mg added to prilocaine in a standard Bier's block (group 2) was studied in 45 patients who had their Colles' fractures reduced under intravenous regional anaesthesia, and compared both to a control group (group 1), and to a group who received a standard Bier's block combined with the same dose of tenoxicam given intravenously into the contralateral arm (group 3). Patients in group 2 obtained significantly better analgesia than group 1, as judged by a longer time before first additional analgesia was required (p < 0.05), less total analgesic consumption (p < 0.01), and lower pain scores (p < 0.01). These benefits were not obtained by patients in group 3.

Adult↗
PubMed1996

Massive arterial air embolism during cardiopulmonary bypass: antegrade blood cardioplegia delivered by the pump--an accident waiting to happen.

Of the many possible causes of air embolism occurring in patients undergoing cardiopulmonary bypass (CPB), human error due to the perfusionist or the surgeon accounts for the vast majority. This case, however, presents a previously unreported, but recognized, cause of air embolism, due to a technical problem encountered during the administration of blood cardioplegia. The nature of some of the older CPB pumps allows the administration of cardioplegia at a time when the main CPB pump is not rotating. A situation may then arise whereby air may be entrained and delivered to the patient. The management of massive air embolism is discussed, and recommendations are made to prevent such an occurrence happening in the future.

Cardioplegic Solutions↗
PubMed1995

The amino acid requirements of Staphylococcus aureus isolated from cases of bovine mastitis.

The amino acid requirements of seven strains of Staphylococcus aureus isolated from cases of bovine mastitis were determined. Arginine, cystine, glycine, leucine, proline and valine were essential for the growth of all isolates. In addition, all isolates required one or more of the following: glutamic acid, histidine, isoleucine, lysine, methionine, phenylalanine, tryptophan and tyrosine.

Amino Acids↗
PubMed1995

Activating transcription factor-1 is a specific antagonist of the cyclic adenosine 3'.5'-monophosphate (cAMP) response element-binding protein-1-mediated response to cAMP.

We have investigated the molecular basis of the variability of the somatostatin cAMP response element (CRE) function in different cell lines. All cells tested contain detectable levels of the CRE-binding protein CREB-1, which mediates transactivation in response to the cAMP-dependent protein kinase (protein kinase-A), in forms that can bind to a somatostatin CRE. Although both responsive and nonresponsive cells contain CREB-1 in heterodimers with activating transcription factor-1 (ATF-1), only cells that allow a cAMP response have a significant proportion of CREB-1 in a homodimeric form. Transfection experiments demonstrate that ATF-1 is capable of antagonizing CREB-1-dependent activation, suggesting that the ability of CREB-1 to mediate a cAMP response is down-regulated by heterodimer formation with ATF-1.

Activating Transcription Factor 1↗
PubMed1994

Different binding specificities and transactivation of variant CRE's by CREB complexes.

The DNA binding specificities of CREB1 and CREB2 homodimers and the CREB2/cJun heterodimer were analyzed with a CASTing technique. All but one of the selected sequences varied from the consensus CRE (TGACGTCA) by three nucleotides or less. The profile of variations selected and the binding affinity for these sequences were unique for each CREB complex. The affinities were not effected by the palindromic nature of the sequences, but were strongly effected by flanking sequences. The strength of DNA binding in vitro correlated with the degree of transactivation observed in JEG-3 cells transfected with reporter plasmids harboring CRE variants, when hybrid CREB proteins fused to the VP16 activation domain were expressed. When native CREB proteins were expressed, the correlation was attenuated by the nature of the variant sequence. A CRE variant (TGACATCA) found in several natural promoters, exhibited the lowest basal transcription rate of the variants and a lower level of induction than expected when compared with the in vitro binding data. These results indicate that transactivation of DNA sequence elements is strongly effected by the strength of transcription factor binding, and that individual sequences can attenuate the level of induction.

Base Sequence↗
PubMed1994

Borrelia burgdorferi decreases hyaluronan synthesis but increases IL-6 production by fibroblasts.

Despite the prevalence of clinical data on human Lyme disease, little is known about the immunopathologic effects of the causative organism on the host. We studied the effect of Borrelia burgdorferi on hyaluronan (hyaluronic acid, HYA) production and the effect on interleukin-6 (IL-6) synthesis by cultured fibroblasts. The cell line employed in this study produced an average of 1406 ng of hyaluronan/ml within 48 h. Using both a morphological staining protocol and a quantitative radiometric assay, we noted that in the presence of a low dose of Borrelia (9.4 x 10(5) cells/ml) the hyaluronan production decreased to an average of 1008 ng/ml, a significant difference (p < 0.05) from the amount of hyaluronan produced by the cells alone. The reduction was even more significant (p < 0.01) when a higher dose of Borrelia (9.4 x 10(6) cells/ml) was used giving an average hyaluronan concentration of 682 ng/ml. In contrast, we found that Borrelia stimulated the cells to produce IL-6 from a baseline of 293 pg/ml to a maximal value of 842 pg/ml (p < 0.01). The spirochetes had no significant effect on cell viability, nor were we able to demonstrate invasion of the cells by the bacteria. Both a decrease in hyaluronan and an increase in IL-6 may correlate with the pathogenicity of Lyme disease in man.

Borrelia burgdorferi Group↗
PubMed1994

A case of Japanese encephalitis.

We report a case of Japanese encephalitis that occurred in a woman who had spent only a few days in an area where she could have been exposed to the virus. The risks and protective efficacy of vaccination against Japanese encephalitis virus for travellers who visit endemic areas for only a short period are discussed.

Acyclovir↗
PubMed1991

Identification and functional characterisation of the cellular activating transcription factor 43 (ATF-43) protein.

The promoter motif CGTCA binds multiple cellular factors that mediate a variety of inducible events, including positive responses to raised cellular levels of cAMP and to the Adenovirus E1a protein. To date, at least ten mammalian cDNA clones have been isolated that encode distinct proteins capable of binding to this motif. However, in most cases the precise stimuli that may regulate these different factors have yet to be determined. We have previously shown that the abundant Hela protein ATF-43 forms a complex in vivo with the cyclic AMP response element binding protein (CREB). In this report we definitively show that ATF-43 is the product of the two published cDNA clones, ATF1 and TREB 36. We confirm that ATF1 efficiently heterodimerises with CREB and demonstrate that even though ATF1 and CREB homodimers, as well as the ATF1/CREB heterodimer efficiently bind to the CGTCA motif, the resulting DNA-protein complexes have significantly different stabilities. A region outside the DNA binding domain of ATF1 contributes to the instability of its interaction with DNA. We further show that despite ATF1's homology to CREB, it responds poorly to activation by protein kinase A. In light of our finding that in Hela cells the majority of CREB protein is heterodimerised with ATF1, we speculate on the functional significance of such heterodimers.

Activating Transcription Factors↗
PubMed1991

Differential regulation of three members of the ATF/CREB family of DNA-binding proteins.

The ATF/CRE binding site can mediate transcriptional activation by cAMP, the adenovirus E1A protein and the human T-cell leukaemia virus 1 (HTLV1) tax protein. A large number of different proteins bind specifically to this element either as homodimers or as heterodimers. Using GAL4-ATF/CREB fusions, we have investigated the regulatory functions of three members of this family. CREB1 (CREB) is strongly activated by cAMP and weakly activated by the E1A protein. In contrast, CREB2 (CRE-BP1, ATF2) is strongly activated by E1A but is insensitive to cAMP stimulation. ATF1 is weakly activated by cAMP but is not activated by E1A. All three proteins are insensitive to activation by the HTLV1 tax protein. The N-terminal region of CREB2, from amino acid residues 19 to 112, is both necessary and sufficient for E1A activation. This region contains a putative C2H2 metal-binding finger, and single amino acid substitutions of the cysteine residues severely decreased CREB2 activity. In contrast, mutations affecting a potential protein kinase A and casein kinase II phosphorylation site within this region had little effect.

Activating Transcription Factors↗
PubMed1990

Activation in vitro of RNA polymerase II and III directed transcription by baculovirus produced E1A protein.

The baculovirus expression system has been successfully used to overproduce a number of different protein products. In this report we describe the construction of a recombinant baculovirus containing the adenovirus E1A 13s cDNA sequence. Infection of insect cells with this virus results in the production of phosphorylated E1A protein. The phosphorylation pattern appears to be similar to the complex pattern associated with E1A protein synthesis in mammalian cells. Purified baculovirus generated E1A protein activated transcription of specific poIIII promoters both in microinjected Xenopus laevis oocytes and in HeLa cell in vitro transcription extracts. The protein also stimulates in vitro transcription of the poIIII transcribed VA1 gene.

Adenovirus Early Proteins↗
PubMed1990

The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We demonstrated that CREB and ATF-47 are identical and that CREB and ATF-43 form protein-protein complexes. We also found that the cis requirements for stable DNA binding by ATF-43 and CREB are different. Using antibodies to ATF-43 we have identified a group of polypeptides (ATF-43) in the size range from 40 to 43 kDa. ATF-43 polypeptides are related by their reactivity with anti-ATF-43, DNA-binding specificity, complex formation with CREB, heat stability, and phosphorylation by protein kinase A. Certain cell types vary in their ATF-43 complement, suggesting that CREB activity is modulated in a cell-type-specific manner through interaction with ATF-43. ATF-43 polypeptides do not appear simply to correspond to the gene products of the ATF multigene family, suggesting that the size of the ATF family at the protein level is even larger than predicted from cDNA-cloning studies.

Activating Transcription Factors↗
PubMed1990

Maintenance of cellular proliferation by adenovirus early region 1A in fibroblasts conditionally immortalized by using simian virus 40 large T antigen requires conserved region 1.

Various mutants of adenovirus E1A were assayed for their ability to complement the growth defect at the nonpermissive temperature for the cell line tsa14 which was isolated by immortalizing rat embryo fibroblasts with the thermolabile large T antigen of tsA58. This cell line grows indefinitely at the permissive temperature but undergoes rapid growth arrest upon shift up to the nonpermissive temperature. Since this growth arrest can be overcome by introduction of wild-type simian virus 40 large T antigen, human papillomavirus 16 E7, and adenovirus E1A, the tsa14 cells provided an excellent system for defining regions of E1A necessary for complementation of the growth defect. We demonstrate that conserved region 1 (CR1) is the region of E1A required for complementation. While CR2 of E1A has been shown to be required for the immortalization of primary cells and is also necessary for the binding of the 105-kDa retinoblastoma protein, mutations within this region did not abrogate complementation of the growth defect. However, since both CR1 and CR2 have previously been shown to be absolutely required for immortalization of primary cells by adenovirus E1A, this evidence suggests that the tsa14 system assays for the maintenance of proliferation and that this requires CR1.

Adenovirus Early Proteins↗
PubMed1990

Transformation by the human adenoviruses.

Oncogenic transformation by the human adenoviruses involves the concerted action of two genes, E1A and E1B. Over the last few years the products of these genes have been characterised in considerable detail using genetic, immunological and biochemical means. The E1A gene by itself can immortalise primary cells and can cooperate to effect full morphological transformation not only with E1B but also with other known oncogenes. The immortalisation and cooperation activities of E1A require multiple functions that are directed by structurally and functionally independent regions of the E1A protein. These regions coincide with sites of protein: protein interaction between E1A and a variety of cellular polypeptides. One of these, the Rb protein, is a known regulator of the mammalian cell cycle. The E1B region encodes two proteins required for transformation, the larger of which binds to the p53 cellular protein. This protein has also been implicated as a negative regulator of cell growth. It appears therefore that E1A and E1B carry out their many functions associated with transformation at least in part by binding to and presumably modulating the activity of key cellular regulators.

Adenoviruses, Human↗
PubMed1990

Heterodimer formation between CREB and JUN proteins.

DNA binding protein families have been identified that contain a leucine zipper dimerization motif preceded by a conserved, highly basic domain involved in direct specific interaction with DNA. Members of two of these families, the Jun and Fos related proteins, have been shown to directly interact and form heterodimeric complexes. A third such family known as the CREB or ATF proteins, bind to a sequence element present in promoters from a number of viral and cellular genes; this element can confer cAMP-inducibility and E1A-inducibility of transcription. In this report we show that one member of the CREB family can efficiently form a heterodimeric complex with the cJun protein. The DNA binding specificity of the heterodimer was indistinguishable from CREB alone. Transfection studies in undifferentiated F9 cells suggest that the CREB/cJun heterodimer can form in vivo, but that the complex does not activate transcription. The heterodimer formation between CREB and Jun proteins is highly specific; only one of the two CREB proteins would heterodimerize with cJun and it would not form dimers with JunB or cFos. The interaction of members of these two families of proteins increases the repertoire of possible regulatory complexes that could play an important role in the regulation of transcription of specific cellular genes.

Base Sequence↗
PubMed1989

C-terminal domain of the adenovirus E1A oncogene product is required for induction of cytotoxic T lymphocytes and tumor-specific transplantation immunity.

Adenovirus genes required for the elicitation of adenovirus group C-specific cytolytic T lymphocytes (CTLs) and for the induction of adenovirus-specific transplantation antigen (TSTA) were identified by immunization with a library of adenovirus mutants. The group C Ad-specific CTL response was elicited by immunization with wild-type adenovirus type 5 (Ad5) or with recombinant adenoviruses containing Ad5 E1A gene. The specific CTL response was also elicited by Ad5 virus constructs which express only the 12 S or 13 S E1A early mRNA, but not with viruses unable to express E1A protein sequences normally encoded by the E1A early messages. The induction of transplantation immunity against tumorigenic Ad-transformed cells was studied next. The product encoded by either 13 S and 12 S E1A mRNA alone was sufficient for strong TSTA activity. A series of viruses with mutations within the first exon of the E1A message also induced strong TSTA, while Ad5 mutants with lesions within the second exon failed to induce syngraft immunity. These results provide strong evidence that amino acid sequence encoded by the second exon of the Ad5 E1A message is required, either directly or indirectly, for the induction of both Ad-specific CTL and Ad TSTA.

Adenovirus Early Proteins↗
PubMed1989

Mammalian cAMP-responsive element can activate transcription in yeast and binds a yeast factor(s) that resembles the mammalian transcription factor ANF.

The human ATF and AP1 transcription factors bind to highly related DNA sequences. Their consensus binding sites differ by a single nucleotide, but this single change is crucial in determining factor binding specificity. We have previously identified an AP1 (yAP1) binding activity in yeast. In this report we identify a yeast ATF (yATF) binding activity whose specificity can be distinguished from that of yAP1 by the same crucial nucleotide that distinguishes binding of human ATF and AP1. The ATF binding site can act as an efficient upstream activating sequence in vivo, suggesting that yATF is a transcriptional activator. The yATF DNA-binding complex is phosphorylated and the binding activity of partially purified yATF can be enhanced in vitro by the addition of protein kinase A, indicating that the phosphorylation state of yATF may be important in determining its ability to bind DNA.

Activating Transcription Factors↗