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Biomedical subjects

J Dennis

Publications and source records attributed to J Dennis.

At least 37 records · Page 2Linked to original sources
PubMed1999

Autogenous regulation of transcription termination factor Rho and the requirement for Nus factors in Bacillus subtilis.

The expression and activity of transcription termination factor Rho and the requirement for transcription elongation factors NusA and NusG was investigated in Bacillus subtilis. Rho was present at < 5% of the level found in Escherichia coli, but Rho factors from these two bacteria had similar properties as RNA-activated ATPases and in vitro termination of transcription on the lambda tR1 terminator. The B. subtilis rho gene was autoregulated at the level of transcription; autoregulation required sequences within the rho mRNA leader region and gene. To date, the B. subtilis rho is the only gene from a Gram-positive bacterium found to be regulated by Rho. Rho was not involved in bulk mRNA decay in B. subtilis. The E. coli elongation factors NusA and NusG target Rho, and the importance of these proteins in B. subtilis was examined by gene disruption. The B. subtilis NusG was inessential for both the viability and the autoregulation of Rho, whereas NusA was essential, and the requirement for NusA was independent of Rho. This contrasts with E. coli in which NusG is essential but NusA becomes dispensable if Rho terminates transcription less efficiently.

Adenosine Triphosphate↗
PubMed1998

Recent developments of magnetic beads for use in nucleic acid purification.

The performance of Magarose, an agarose-based bead containing a paramagnetic component has been evaluated. The anion exchanger DEAE-Magarose is effective at binding DNA from a crude cell lysate. The plasmid pBluescript was isolated from 1.5 ml Escherichia coli JM109 cell culture, following alkaline lysis yielding 8.2 micrograms high-quality DNA. Under similar binding conditions 21 micrograms of salmon sperm DNA bound to the ion exchangers. The affinity medium oligo-dT Magarose was demonstrated to bind 75 mumol of an oligo-dA probe/g of medium by hybridization. Under similar conditions mRNA could be isolated from a preparation of baby hamster cell total RNA. The magnetic susceptibility of Magarose is very high, facilitating the use of this separation technique for rapid batch chromatographic processes.

Animals↗
PubMed1998

Biosynthesis of Aedes aegypti lipophorin and gene expression of its apolipoproteins.

The biosynthesis of lipophorin of the yellow fever mosquito, Aedes aegypti, was investigated. Fat bodies were incubated in vitro with radiolabeled methionine and cysteine, and radiolabeled proteins secreted into the medium were analyzed by density gradient ultracentrifugation, SDS-PAGE and fluorography. Lipophorin was synthesized in the fat body and secreted into the medium. Its density was 1.114 g/ml, similar to that of lipophorin circulating in hemolymph. Three peptides of a tryptic digest of apolipophorin II were sequenced and degenerate oligonucleotide primers were designed based on the amino acid sequences. With these primers, a cDNA product of 1.2 kb was amplified by RT-PCR using as template RNA extracted from adult female mosquitoes 24 h after ingestion of a blood meal. This cDNA was cloned, sequenced and used as a probe for Northern blot analysis, which revealed that the apoproteins of lipophorin were coded for by a single mRNA of approximately 10 kb. The expression of the apolipophorins was induced by blood feeding. From the data presented we concluded that Aedes aegypti lipophorin is synthesized in the fat body and that the expression of its apolipophorins is induced by blood feeding.

Aedes↗
PubMed1997

The critical role of p38 MAP kinase in T cell HIV-1 replication.

BACKGROUND: Replication of HIV-1 in human T lymphocytes requires the activation of host cellular proteins. This study identifies p38 mitogen-activated protein kinase (MAPK) as one such kinase necessary for HIV-1 replication in T cells. MATERIALS AND METHODS: Primary human T lymphocytes were infected with the LAI strain of HIV-1 and Jurkat cells were infected with the RF strain of HIV-1. HIV replication was measured by reverse transcriptase activity. Cellular expression of endogenous p38 MAPK protein was analyzed using immunoprecipitation. Blockade of p38 MAPK expression was achieved using antisense oligonucleotides to p38 MAPK and the guanylhydrazone compound CNI-1493, an inhibitor of p38 MAPK activation. RESULTS: HIV-1 infection of both primary human T lymphocytes and a T cell line rapidly activated the cellular p38 MAPK pathway, which remained activated for the duration of the culture. Addition of phosphothioated antisense oligonucleotides to p38 MAPK specifically inhibited viral replication. Blockade of p38 MAPK activation by addition of CNI-1493 also inhibited HIV-1 viral replication of primary T lymphocytes in a dose- and time-dependent manner. Stimulation of p38 MAPK activation did not occur with the addition of heat-inactivated virus, suggesting that viral internalization, and not just membrane binding, is necessary for p38 MAPK activation. CONCLUSIONS: These results indicate that activation of the p38 MAPK cascade is critical for HIV-1 replication in primary T lymphocytes, and that blockade of this signal transduction pathway may be a novel therapeutic approach to the treatment of HIV-1 infection.

Blotting, Western↗
PubMed1996

CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency.

Tumor necrosis factor (TNF) mediates a wide variety of disease states including septic shock, acute and chronic inflammation, and cachexia. Recently, a multivalent guanylhydrazone (CNI-1493) developed as an inhibitor of macrophage activation was shown to suppress TNF production and protect against tissue inflammation and endotoxin lethality [Bianchi, M., Ulrich, P., Bloom, O., Meistrell, M., Zimmerman, G. A., Schmidtmayerova, H., Bukrinsky, M., Donnelley, T., Bucala, R., Sherry, B., Manogue, K. R., Tortolani, A. J., Cerami, A. & Tracey, K. J. (1995) Mol. Med. 1, 254-266, and Bianchi, M., Bloom, O., Raabe, T., Cohen, P. S., Chesney, J., Sherry, B., Schmidtmayerova, H., Zhang, X., Bukrinsky, M., Ulrich, P., Cerami, A. & Tracey, J. (1996) J. Exp. Med., in press]. We have now elucidated the mechanism by which CNI-1493 inhibits macrophage TNF synthesis and show here that it acts through suppression of TNF translation efficiency. CNI-1493 blocked neither the lipopolysaccharide (LPS)-induced increases in the expression of TNF mRNA nor the translocation of nuclear factor NF-kappa B to the nucleus in macrophages activated by 15 min of LPS stimulation, indicating that CNI-1493 does not interfere with early NF-kappa B-mediated transcriptional regulation of TNF. However, synthesis of the 26-kDa membrane form of TNF was effectively blocked by CNI-1493. Further evidence for the translational suppression of TNF is given by experiments using chloram-phenicol acetyltransferase (CAT) constructs containing elements of the TNF gene that are involved in TNF translational regulation. Both the 5' and 3' untranslated regions of the TNF gene were required to elicit maximal translational suppression by CNI-1493. Identification of the molecular target through which CNI-1493 inhibits TNF translation should provide insight into the regulation of macrophage activation and mechanisms of inflammation.

Base Sequence↗
PubMed1996

An exploration of feeding difficulties in children with Down syndrome.

Detailed examination of several aspects of feeding was carried out on a representative sample of a defined geographical population of children with Down syndrome. The examination included standardised assessments both of oral-motor function and of parent-child interaction. The findings suggest that the development of oral-motor function in children with Down syndrome not only lags behind intellectual development, but also follows an aberrant pathway. In particular, specific aspects of tongue and jaw function were impaired together with problems initiating and maintaining a smooth sequence of feeding actions. Also, parent-child interactions, as in studies on play, tended to be more controlling. Parents of children with Down syndrome do not spontaneously report the extent of their child's feeding problems unless specific enquiry is made, preferably accompanied by observation of feeding.

Child, Preschool↗
PubMed1995

Neonatal seizures: long-term outcome and cognitive development among 'normal' survivors.

The authors report the long-term outcome and cognitive development in the late-teenage years of 'normal' survivors of neonatal seizures. The outcome of the children was good, and normal in that they had attended normal schools and had normal overall intelligence test scores as adults. However, all of the sample displayed abnormal neuropsychological development in terms of intelligence test profile and subtest scatter, or development of spelling, or development of memory. This was independent of social and behavioural difficulties, which may also be increased. Neonatal seizures may be indicative of a subtle neurodevelopmental vulnerability which may manifest later in life as specific learning difficulties or poor social adjustment.

Adolescent↗
PubMed1995

Impaired oral-motor function in children with Down's syndrome: a study of three twin pairs.

Three twin births occurring within 18 months in Oxfordshire, in which a girl with Down's syndrome was twin to an unaffected boy, provided an opportunity for a closely matched comparison of ratings on the Schedule for Oral Motor Assessment, which examines oral-motor function in a systematic and reliable way. A detailed description of the video-recording of one twin pair is included. This illustrates abnormalities of oral-motor function which may occur generally in Down's syndrome, as suggested further by a numerical tabulation of the differences found between the twins.

Child, Preschool↗
PubMed1993

Prenatal diagnosis of bovine citrullinaemia.

Holstein/Friesian embryos, progeny of citrullinaemia heterozygotes, were transferred to recipient cows. The citrullinaemia genotype of the fetus/calf was established, at birth, by estimation of citrulline concentration in plasma or by analyses of argininosuccinate synthetase exon 5 sequences in DNA from blood leucocytes. Between 115 and 125 days of gestation amniotic fluid was collected transabdominally from nine recipient cows. The mean citrulline concentration in amniotic fluid from fetuses unaffected with citrullinaemia was 28 microM, SD 9 and range 15 to 38 microM. Citrulline concentration was 107 and 130 microM in amniotic fluid from two fetuses homozygous for the citrullinaemia mutation. Only the normal bovine citrullinaemia sequence was detected in cells cultured from amniotic fluid taken from two homozygous normal and two heterozygous fetuses. Both normal and mutant sequences were found in cells cultured from amniotic fluid collected from three of five heterozygous fetuses. Only the mutant sequence was found in cells cultured from the two citrullinaemia affected fetuses. MHC class II DRB3 haplotyping revealed that in four instances cultured cells were from the surrogate mother, not the fetus, these included samples from two heterozygotes in which only the normal sequence was detected in amniotic fluid.

Amino Acid Metabolism, Inborn Errors↗
PubMed1993

An economic appraisal of alternative pre-natal screening programmes for Down's syndrome.

The objective of this study was to evaluate economically a screening programme within the Oxford Regional Health Authority for Down's syndrome, based on maternal serum alpha fetoprotein, unconjugated oestriol and human chorionic gonadotrophin as well as maternal age (the triple test) against maternal age alone. The design of the study involved cost-effectiveness analysis of the triple test relative to the maternal age screening programme, and the main outcome measure was the cost per Down's birth avoided. It was found that the triple test is more cost-effective over a wide range of assumptions concerning detection rates and procedure costs. Indirect costs are important in considering the cost-effectiveness of the screening programmes. The most efficient detection rate is around 58 per cent for which the cost per Down's birth avoided is approximately 29,600 pounds if only direct costs are evaluated, 20,100 pounds if all NHS costs are considered and -49,800 pounds if all resource consequences are analysed. It may be concluded that screening for Down's syndrome using the triple test is cost-effective over a wide range of assumptions concerning detection rate and procedure costs. If all resource costs are considered, the programme is highly cost-effective in comparison with other health care interventions.

Abortion, Therapeutic↗
PubMed1992

"X Lite": a protective shield for skin grafts on the back.

"X Lite", a thermoplastic material was used in eight patients as a splint for protecting split skin grafts on the back. After an average stay of 4.3 days patients were discharged with no incidence of graft failure. The patients involved felt comfortable whilst remaining supine. We believe time saved by adequate protection leading to successful grafts makes this method economically useful.

Back↗
PubMed1992

Direct detection of Molluscum contagiosum virus in clinical specimens by in situ hybridization using biotinylated probe.

Molluscum contagiosum virus (MCV) is an unclassified poxvirus which has recently become recognized as causing a major sexually transmitted disease. At present no assay is available for specific detection of MCV because the virus cannot be serially propagated in cell culture. Since MCV produces an abortive, limited growth with some cytopathic effect in certain cell lines, we were able to develop an in situ hybridization assay for detection of MCV genome in clinical specimens. Human fetal diploid lung cell monolayers were infected with clinical specimens, and after proper incubation and fixation in paraformaldehyde, hybridization was performed under full stringency conditions with a molecularly cloned biotinylated probe. Only MCV infected cells showed homology to the MCV probe with a purple-brown cytoplasmic staining. Additionally, we have described an in situ hybridization assay for direct detection of MCV genome in formalin-fixed, paraffin-embedded biopsies. Characteristic intracytoplasmic Molluscum bodies (Henderson-Paterson bodies) were detected in stratum spinosum cells of the epidermis. Striking staining similarities have been observed between in situ hybridization and haematoxylin-eosin cytostaining. These procedures are the first successful identification of MCV genome in clinical samples by molecular hybridization, with sensitivity and specificity equal to or greater than electron microscopy.

Blotting, Southern↗
PubMed1992

Long term effects of periconceptional multivitamin supplements for prevention of neural tube defects: a seven to 10 year follow up.

Periconceptional supplementation with Pregnavite Forte F was offered to women who presented consecutively to the Oxford genetic counselling service in the early 1980s who had previously had one or more pregnancies complicated by a neural tube defect. The first 100 children born alive to these women are the subject of this study. Birth weight, gestation, and congenital abnormalities were recorded. At age 2-5 years all 96 children remaining in the United Kingdom were assessed clinically and developmentally and behavioural information was obtained by questionnaire. At age 7-10 years, follow up of 91 children by telephone and postal questionnaire yielded further information about growth, general health, vision, hearing, and educational and behavioural status. Entry criteria excluded single mothers but the social class distribution of the sample was otherwise representative of the Oxfordshire population. There were no recurrences of neural tube defects. One child had radiological evidence of spina bifida occulta affecting only the fifth lumbar vertebra. One had an autosomal recessive disorder. Eight had random minor congenital anomalies. Birth weight for gestational age was significantly greater than for the local population and at age 7-10 years the girls were considerably taller than expected. Health, auditory, visual, and developmental status were no different from the general population. None of the children had special educational needs. None showed a major behaviour disorder but worries, fussiness, and fearfulness were highly significantly over represented.

Adult↗
PubMed1991

Constitutive expression and secretion of proteases in non-metastatic SP1 mammary carcinoma cells and its metastatic sublines.

Malignant tumors are generally characterized by extensive local tissue invasion and destruction of ECM which may be due to increased constitutive expression and activity of secreted proteases. Moreover, a large number of diverse protease activities may be constitutively over-expressed in a simultaneous or co-ordinated fashion, thereby significantly increasing cellular invasive potential of the cells. To explore this relationship, we have measured steady-state levels of mRNA coding for urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), transin and tissue-specific inhibitor of metalloproteinases (TIMP); as well as gelatinolytic, caseinolytic and plasminogen activator activities secreted by SPI, a non-metastatic mouse mammary carcinoma cell line and 4 metastatic sublines derived from it. mRNA encoding metalloproteinase transin was increased 15- to 20-fold, while TIMP transcripts were decreased 3-fold in the metastatic sublines compared to parental SPI tumor cells. Metastatic sublines secreted higher levels of gelatinase (i.e., 92 kDa and 64 kDa) as well as proteases with caseinolytic activity (i.e., 115 kDa and 57 kDa) when compared with SPI cells. Moreover, these enzymes were identified as neutral metalloproteinases. Although the amount of uPA mRNA appeared to be the same in SPI and the metastatic sublines, the latter secreted 1.5-3 times more uPA activity into the culture supernatants. Metastatic competence in the SPI tumor model is therefore associated with increased secretion of several metalloproteinase activities and uPA, as well as decreased TIMP expression, consistent with a more invasive phenotype.

Adenocarcinoma↗