Biomedical subjects

J MacDougall

Publications and source records attributed to J MacDougall.

At least 19 recordsLinked to original sources

PubMed2003

Shoot meristem: an ideal explant for Zea mays L. transformation.

We report on a rapid high-frequency somatic embryogenesis and plant regeneration protocol for Zea mays. Maize plants were regenerated from complete shoot meristem (3-4 mm) explants via organogenesis and somatic embryogenesis. In organogenesis, the shoot meristems were directly cultured on a high-cytokinin medium comprising 5-10 mg x L(-1) 6-benzylaminopurine (BAP). The number of multiple shoots produced per meristem varied from six to eight Plantlet regeneration through organogenesis resulted in just four weeks. Callus was induced in five days of incubation on an auxin-modified Murashige and Skoog (MS) medium. Prolific callus, with numerous somatic embryos, developed within 3-4 weeks when cultured on an auxin medium containing 5 mg 2,4-dichlorophenoxyacetic acid x L(-1). The number of multiple shoots varied from three to six per callus. Using R23 (Pioneer, Hi-Bred, Johnston, Iowa), the frequency of callus induction was consistently in excess of 80% and plant regeneration ranged between 47 and 64%. All regenerated plantlets survived in the greenhouse and produced normal plants. Each transgenic plant produced leaves, glumes, and anthers that uniformly expressed green fluorescent protein (GFP). The GFP gene segregated in the pollen. Based on this data it is concluded that the transgenics arose from single-cell somatic embryos. The rate of transfer DNA (T-DNA) transfer to complete shoot meristems of Zea mays was high on the auxin medium and was independent of using super-virulent strains of Agrobacterium.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2001

Construction, expression, and characterization of a baculovirally expressed catalytic domain of human matrix metalloproteinase-9.

We report DNA construction, baculovirus expression, and partial characterization of a minienzyme form of the human matrix metalloproteinase-9 (MMP-9). The MMP-9 minienzyme gene construct consisting of the pre, pro, and catalytic domains of the MMP-9 was introduced into Sf9 insect cells using a baculovirus expression system. The expression of the recombinant MMP-9 minienzyme was estimated to be approximately 0.8 mg/L of cell medium. The recombinant protein was purified using a single-step gelatin-Sepharose affinity column and yielded a highly stable and active minienzyme with gelatinolytic activity. Moreover, two interesting findings related to MMP-9 interactions with heparin and TIMP-1 resulted from our studies. First, the pro and catalytic domains of the human MMP-9 are not sufficient for heparin affinity. Second, in contrast to the prevailing consensus, TIMP-1 blockade of the enzymatic activity of MMP-9 does not require prior binding to the C-terminus of its MMP-9 protein substrate.

Animals↗

PubMed1999

The effects of exercise intensity on post exercise hypotension.

Ten normotensive, recreationally active participants aged 35.0 +/- 16.3 years, volunteered to participate in the study. Average baseline blood pressure (BP) was 132/75 mm Hg for systolic (SBP) and diastolic (DBP) pressure respectively. On two separate days, participants underwent testing in a randomised, repeated measures fashion such that they performed 30-min bouts of cycle ergometry at a power output which elicited 50 or 75% of VO2 Peak. Blood pressure was monitored continuously throughout the session by the Finapres method with 2-min windows recorded at rest, 5, 10, 15, 30, 45 and 60 min post exercise. SBP was similar between the two trials and became hypotensive at 5 through 15 min post exercise. The largest decrement (8 mm Hg) in SBP occurred 5 min post exercise. DBP was also unaffected by the intensity of exercise and was lower than before exercise at 5 and 15 through 45 min post exercise. Similarly, mean arterial pressure (MAP) showed significant decrements at 5 and 15 through 45 min post exercise irrespective of exercise intensity. Heart rate was greater during the 75% intensity than during the 50% intensity trial. Pre-exercise values were re-established by 45 min post exercise. VO2 remained significantly elevated above pre-exercise values in both trials until 15 min post exercise. Haematocrit increased significantly during both exercise bouts but returned to pre-exercise values by 10 min post exercise. This study indicates that cycle ergometry at 50 and 75% of VO2 Peak elicit similar reductions in post exercise BP. Therefore bouts of mild to moderate intensity exercise may be beneficial in the control of hypertension.

Adult↗

PubMed1999

Downregulation of Na+-K+-ATPase pumps in skeletal muscle with training in normobaric hypoxia.

To investigate the effects of training in normoxia vs. training in normobaric hypoxia (fraction of inspired O2 = 20.9 vs. 13.5%, respectively) on the regulation of Na+-K+-ATPase pump concentration in skeletal muscle (vastus lateralis), 9 untrained men, ranging in age from 19 to 25 yr, underwent 8 wk of cycle training. The training consisted of both prolonged and intermittent single leg exercise for both normoxia (N) and hypoxia (H) during a single session (a similar work output for each leg) and was performed 3 times/wk. Na+-K+-ATPase concentration was 326 +/- 17 (SE) pmol/g wet wt before training (Control), increased by 14% with N (371 +/- 18 pmol/g wet wt; P < 0.05), and decreased by 14% with H (282 +/- 20 pmol/g wet wt; P < 0.05). The maximal activity of citrate synthase, selected as a measure of mitochondrial potential, showed greater increases (P < 0.05) with H (1.22 +/- 0.10 mmol x h-1 x g wet wt-1; 70%; P < 0.05) than with N (0.99 +/- 0.10 mmol x h-1 x g wet wt-1; 51%; P < 0.05) compared with pretraining (0.658 +/- 0.09 mmol x h-1 x g wet wt-1). These results demonstrate that normobaric hypoxia induced during exercise training represents a potent stimulus for the upregulation in mitochondrial potential while at the same time promoting a downregulation in Na+-K+-ATPase pump expression. In contrast, normoxic training stimulates increases in both mitochondrial potential and Na+-K+-ATPase concentration.

Adult↗

PubMed1999

Bone density in a population of long term oral contraceptive pill users does not differ from that in menstruating women.

Prevention of osteoporosis is a major public health issue. Amenorrhoeic women have lower bone density than normally menstruating women, which is related to the duration of amenorrhoea and the severity of oestrogen deficiency. Bone mineral density (BMD) in amenorrhoeic women can be improved by oestrogen replacement in the form of the combined oral contraceptive pill (COCP), so increased BMD might be an important non-contraceptive benefit of the COCP in menstruating women. Previous studies have been variably reported, but have used different methodologies for measurement of BMD. We measured BMD using the DEXA technique in long term COCP users and compared this with menstruating women who had never used the COCP. No differences in bone density were found, suggesting that the COCP does not improve bone mass in menstruating women who are adequately oestrogenised by their own ovaries.

Absorptiometry, Photon↗

PubMed1998

A direct estimation of the context effect on the efficiency of termination.

An in vitro assay in which terminating Escherichia coli ribosomes with different stop signals in the A-site compete for a limited amount of a release factor (RF1 or RF2) has been used to estimate the relative termination efficiencies at stop codons with different adjacent downstream nucleotides. The assay allows direct measurements of relative kcat/Km parameters for the productive association of release factors to ribosomes. The kcat/Km parameter is larger for UAA(U) than for UAA(C) programmed ribosomes and the difference in kcat/Km is much larger for RF2 (about 80%) than for RF1 (about 30%). These differences in the kcat/Km parameter are not affected by the addition of release factor RF3. The only discernible effect of RF3 is a considerable acceleration of RF1/2 recycling.The estimated kcat/Km parameters correlate well with the affinities of release factors for ribosomes programmed with different stop signals. These affinities were estimated from the extent of inhibition of ribosomal recycling by high concentrations of release factors in the absence of release factor RF3. The affinity for RF2 depends on the immediate downstream context of the stop codon in the translated mRNA and is about three times higher for UAA(U) than for UAA(C). The corresponding difference in affinities for RF1 is twofold. For all stop signals studied, the estimated affinity of RF2 for terminating ribosomes is much lower than that of RF1. It is also striking that the affinity of ribosomes for a chromosomally expressed RF2 is at least three times higher than for RF2 isolated from an overproducing E. coli strain.

Base Sequence↗

PubMed1998

Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome.

Peptidyl-tRNA dissociation from ribosomes is an energetically costly but apparently inevitable process that accompanies normal protein synthesis. The drop-off products of these events are hydrolysed by peptidyl-tRNA hydrolase. Mutant selections have been made to identify genes involved in the drop-off of peptidyl-tRNA, using a thermosensitive peptidyl-tRNA hydrolase mutant in Escherichia coli. Transposon insertions upstream of the frr gene, which encodes RF4 (ribosome release or recycling factor), restored growth to this mutant. The insertions impaired expression of the frr gene. Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype. Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl-tRNA hydrolase mutant. In vitro measurements of peptidyl-tRNA release from ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl-tRNA release from ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in ribosome recycling. When present together, the three factors were able to stimulate release up to 12-fold. It is suggested that RF4 may displace peptidyl-tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling.

Bacterial Proteins↗

PubMed1997

Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP-dependent manner.

Ribosomes complexed with synthetic mRNA and peptidyl-tRNA, ready for peptide release, were purified by gel filtration and used to study the function of release factor RF3 and guanine nucleotides in the termination of protein synthesis. The peptide-releasing activity of RF1 and RF2 in limiting concentrations was stimulated by the addition of RF3 and GTP, stimulated, though to a lesser extent, by RF3 and a non-hydrolysable GTP analogue, and inhibited by RF3 and GDP or RF3 without guanine nucleotide. With short incubation times allowing only a single catalytic cycle of RF1 or RF2, peptide release activity was independent of RF3 and guanine nucleotide. RF3 hydrolysis of GTP to GDP + P(i) was dependent only on ribosomes and not on RF1 or RF2. RF3 affected neither the rate of association of RF1 and RF2 with the ribosome nor the catalytic rate of peptide release. A model is proposed which explains how RF3 recycles RF1 and RF2 by displacing the factors from the ribosome after the release of peptide.

Bacterial Proteins↗

PubMed1997

Fast recycling of Escherichia coli ribosomes requires both ribosome recycling factor (RRF) and release factor RF3.

A complete translation system has been assembled from pure initiation, elongation and termination factors as well as pure aminoacyl-tRNA synthetases. In this system, ribosomes perform repeated rounds of translation of short synthetic mRNAs which allows the time per translational round (the recycling time) to be measured. The system has been used to study the influence of release factor RF3 and of ribosome recycling factor RRF on the rate of recycling of ribosomes. In the absence of both RF3 and RRF, the recycling time is approximately 40 s. This time is reduced to approximately 30 s by the addition of RF3 alone and to approximately 15 s by the addition of RRF alone. When both RF3 and RRF are added to the translation system, the recycling time drops to <6 s. Release factor RF3 is seen to promote RF1 cycling between different ribosomes. The action of RRF is shown to depend on the concentration of elongation factor-G. Even in the presence of RRF, ribosomes do not leave the mRNA after termination, but translate the same mRNA several times. This shows that RRF does not actively eject mRNA from the terminating ribosome. It is proposed that terminating ribosomes become mobile on mRNA and ready to enter the next translation round only after two distinct steps, catalysed consecutively by RF3 and RRF, which are slow in the absence of these factors.

Bacterial Proteins↗

PubMed1997

Purification of active Escherichia coli ribosome recycling factor (RRF) from an osmo-regulated expression system.

Ribosome release factor (RRF) from Escherichia coli was overproduced from an osmo-expression vector. More than 40% of cell protein was RRF after 6 h of induction. A purification scheme is described that produced 50 mg of RRF from an initial culture of 2 L. The recycling time for ribosomes synthesising the tripeptide fMet-Phe-Leu in vitro in the absence of RF3 was reduced from 40 to 15 s by the addition of purified 1.5 microM RRF.

Bacterial Proteins↗

PubMed1997

Immunochemical determination of the cellular content of polypeptide chain release factor RF3 in Escherichia coli.

Polypeptide chain termination in Escherichia coli is known to require two codon specific release factors, RF1 and RF2. A third factor, RF3, has been described to stimulate the termination. Earlier investigations have estimated the cellular content of factors RF1 and RF2. Two different immunological techniques for measuring the amount of RF3 per cell in crude E coli cell extracts are reported here, using a sensitive immunoblotting method and a sandwich assay by ELISA. Monoclonal murine antibodies and polyclonal rabbit antibodies were raised against extensively purified recombinant E coli RF3. The immunoblotting involves a specific monoclonal antibody (mAb), biotinylated second antibody and finally radioactive iodinated streptavidin. In the sandwich assay polyclonal antibodies are immobilised on a polystyrene surface before addition of crude cell extract; a specific mAb serves as primary antibody and an HRP-labelled anti-mouse Ig as secondary antibody. Both methods are accurate and rapid to perform. The number of RF3 molecules per cell in exponentially growing E coli cells was found to vary considerably according to the K12 strain examined and depended on the culture medium (from 20 to 500 molecules per cell), faster growth being positively correlated with the number of RF3 molecules per cell.

Animals↗

PubMed1995

Physical map of the Treponema denticola circular chromosome.

A physical map of the Treponema denticola ATCC 33520 genome was constructed by pulsed-field gel electrophoresis and DNA hybridization. The organism possesses a single, circular chromosome of approximately 3.0 Mbp and a 2.6-kbp circular plasmid, pTD1. The physical map of the A+T-rich genome was constructed with the rare-cutting restriction enzymes AscI, NotI, and SrfI, which have 8-bp G+C-rich recognition sites. The genes flgE, tdpA, and prtB encoding the flagellar hook protein, a 53-kDa immunogenic protein, and chymotrypsinlike protease, respectively, were located on the map. This treponeme was found to have two copies of each of the rRNA genes, as has been found to be the case for both Treponema phagedenis and Treponema pallidum.

Base Sequence↗

PubMed1995

Health care information services in Ireland.

Against a background of fundamental change in the Irish health care environment, a research project was commissioned to determine the information needs of all health care practitioners (including physicians, allied health professionals, managers, scientists, and technicians) as well as the public, patients, and their caregivers. The research, including a study of international best practice, revealed serious deficiencies in access, awareness, and availability of information for both staff and patients in Ireland. Recommendations include the establishment of a panel to develop a comprehensive national health information strategy and plan to coordinate the future development of health sciences information services.

Caregivers↗

PubMed1993

New opportunities for NHS librarians and information scientists.

The recent fundamental transformation in government policy on the National Health Service has far-reaching implications for all types of health-care information provision and use. This paper highlights the new opportunities, as well as the major issues, these developments have generated.

Health Care Reform↗

PubMed1993

Miscarriage rates following in-vitro fertilization are increased in women with polycystic ovaries and reduced by pituitary desensitization with buserelin.

To assess the risk of miscarriage after in-vitro fertilization (IVF) with respect to age, cause of infertility, ovarian morphology and treatment regimen, a retrospective analysis was performed of the first 1060 pregnancies conceived between June 1984 and July 1990 as a result of 7623 IVF cycles. Superovulation induction was achieved with human menopausal gonadotrophin (HMG) and/or purified follicle stimulating hormone (FSH) together with either clomiphene citrate or the gonadotrophin hormone-releasing hormone (GnRH) agonist buserelin, the latter either as a short 'flare' regimen or as a 'long' regimen to induce pituitary desensitization. There were 282 spontaneous abortions (26.6%) and 54 ectopic pregnancies (5.1%). The mean age of women with ongoing pregnancies was 32.2 (SD 3.9) years compared with 33.2 (SD 4.1) years in those who miscarried, which were significantly different (P = 0.008). There was no relation between the miscarriage rate and the indication for IVF. The miscarriage rate was 23.6% in women with normal ovaries compared with 35.8% in those with polycystic ovaries [P = 0.0038, 95% confidence interval (CI) 4.68-23.10%]. There was no difference in the miscarriage rate between treatment with HMG or FSH. Women whose ovaries were normal on ultrasound were just as likely to miscarry if they were treated with clomiphene or with the long buserelin protocol. Those with polycystic ovaries, however, had a significant reduction in the rate of miscarriage when treated with the long buserelin protocol, 20.3% (15/74), compared with clomiphene citrate, 47.2% (51/108) (P = 0.0003, 95% CI 13.82-40.09%).

Abortion, Spontaneous↗

PubMed1993

The influence of the number of embryos transferred in 1060 in-vitro fertilization pregnancies on miscarriage rates and pregnancy outcome.

To assess the incidence of miscarriage, multiple pregnancy and outcome of pregnancy in relation to the number of embryos transferred during in-vitro fertilization (IVF), an analysis was performed of 1060 pregnancies conceived in a tertiary-referral IVF clinic. There was no difference in the miscarriage rate after transfer of one or two embryos (37.7% and 34.6%), or after three or four embryos (22.5% and 25.2%). The miscarriage rate was, however, higher when one or two embryos were transferred compared with three (P < 0.01) or four embryos (P < 0.02). Of the 724 ongoing pregnancies, 524 (72.3%) were singleton, 164 (22.7%) twin, 33 (4.6%) triplet and three (0.4%) quadruplet. The mean (+/- SD) ages of women with singleton, twin, triplet and quadruplet pregnancies were 32.5 (+/- 3.8), 32.0 (+/- 3.5), 29.76 (+/- 4.3) and 29.67 (+/- 2.5) years respectively. The mean age of women with singleton and twin pregnancies was similar and both were greater than that of triplet pregnancies (P < 0.007). The overall perinatal mortality rate (PNMR) was 39.7/1000. The PNMR for singletons was 17.2/1000, for twins 80.0/1000 and for triplets 30.6/1000. All of the babies from the three quadruplet pregnancies survived. There were more babies lost in the twin pregnancies than any other group, although this only reached significance for singletons versus twins (P < 0.00005). We conclude that the incidence of miscarriage is increased in women in whom one or two embryos are transferred. Multiple pregnancies are more likely to occur in younger women and are associated with a significantly higher rate of perinatal mortality.

Abortion, Spontaneous↗

PubMed1992

Mapping of genes on the linear chromosome of the bacterium Borrelia burgdorferi: possible locations for its origin of replication.

Molecular clones of Borrelia burgdorferi, aetiologic agent of Lyme borreliosis, were isolated and analysed by DNA sequence determination. This procedure yielded B. burgdorferi homologues of gidA, gyrB, gyrA, ftsA and ftsZ. The genes were located on the physical map of the B. burgdorferi linear chromosome. Also mapped were the genes fla and p60 while dnaA was mapped using a heterologous probe. gyrA and gyrB were found to be in tandem and were mapped, along with dnaA at the centre of the chromosome. gidA was located close to the left hand extremity of the chromosome. Because gyrB, dnaA and gidA are normally located within 50 kb of the origin of replication (oriC), we propose two possible sites for oriC in the B. burgdorferi linear chromosome.

Amino Acid Sequence↗

PubMed1992

Polycystic ovaries as a relative protective factor for bone mineral loss in young women with amenorrhea.

OBJECTIVE: To examine the impact of polycystic ovaries (PCO) on bone mineral density in amenorrheic women of reproductive age. DESIGN: A retrospective analysis and comparison of polycystic ovarian syndrome (PCOS) with non-PCOS amenorrheic women. A subgroup of patients with ultrasound (US)-diagnosed PCO was also investigated. SETTING: Specialist clinic in reproductive endocrinology. PATIENTS, PARTICIPANTS: Six hundred ten consecutive cases, mean age of 29.8 +/- 7.5 years, with current history of amenorrhea of various causes. MAIN OUTCOME MEASURE: Bone mineral density in the lumbar spine (L1 to L4) as measured by dual energy x ray absorptiometry, in relation to PCOS, US-diagnosed PCO, and US findings of normal ovaries. RESULTS: Amenorrheic patients with PCOS were found to be younger (P less than 0.001), with higher body mass index (P less than 0.001), were more estrogenized, as measured by endometrial thickness and uterine cross-sectional area (P less than 0.001), and had higher bone mineral density (P less than 0.001) compared with non-PCOS amenorrheic patients. CONCLUSIONS: Patients with amenorrhea because of PCOS and those with US-diagnosed PCO have a higher bone density compared with amenorrheic patients with normal ovaries as detected by US scan.

Adult↗