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Biomedical subjects

M Clark

Publications and source records attributed to M Clark.

At least 37 records · Page 2Linked to original sources
PubMed2001

Statistical evaluation of differential expression on cDNA nylon arrays with replicated experiments.

In this paper we focus on the detection of differentially expressed genes according to changes in hybridization signals using statistical tests. These tests were applied to 14 208 zebrafish cDNA clones that were immobilized on a nylon support and hybridized with radioactively labeled target mRNA from wild-type and lithium-treated zebrafish embryos. The methods were evaluated with respect to 16 control clones that correspond to eight different genes which are known to be involved in dorso-ventral axis specification. Moreover, 4608 Arabidopsis thaliana clones on the same array were used to judge statistical significance of expression changes and to control the false positive rates of the test decisions. Utilizing this special array design we show that differential expression of a high proportion of cDNA clones (15/16) and the respective genes (7/8) were identified, with a false positive error of <5% using the constant control data. Furthermore, we investigated the influence of the number of repetitions of experiments on the accuracy of the procedures with experimental and simulated data. Our results suggest that the detection of differential expression with repeated hybridization experiments is an accurate and sensitive way of identifying even small expression changes (1:1.5) of a large number of genes in parallel.

Animals↗
PubMed2001

Expression of the anti-dorsalizing morphogenetic protein gene in the zebrafish embryo.

The BMP3 related anti-dorsalizing morphogenetic protein (ADMP) has been proposed to function in the organizer of chick and Xenopus embryos. We report here the cloning and expression pattern of a zebrafish admp gene. The gene is expressed in involuting cells of the embryonic shield, but not in the non-involuting forerunner cells. During gastrulation, admp transcripts are detected in the posterior prechordal plate, in the notochord primordium and in cells of the dorsal blastoderm margin. Expression is also detectable in the neuroectoderm overlying the posterior prechordal plate. Expression persists in the tail bud until the end of somitogenesis while expression in other areas disappears during early somitogenesis stages.

Amino Acid Sequence↗
PubMed2001

A crucial component of the endoderm formation pathway, CASANOVA, is encoded by a novel sox-related gene.

casanova (cas) mutant zebrafish embryos lack endoderm and develop cardia bifida. In a substractive screen for Nodal-responsive genes, we isolated an HMG box-containing gene, 10J3, which is expressed in the endoderm. The cas phenotype is rescued by overexpression of 10J3 and can be mimicked by 10J3-directed morpholinos. Furthermore, we identified a mutation within 10J3 coding sequence that cosegregates with the cas phenotype, clearly demonstrating that cas is encoded by 10J3. Epistasis experiments are consistent with an instructive role for cas in endoderm formation downstream of Nodal signals and upstream of sox17. In the absence of cas activity, endoderm progenitors differentiate into mesodermal derivatives. Thus, cas is an HMG box-containing gene involved in the fate decision between endoderm and mesoderm that acts downstream of Nodal signals.

Amino Acid Sequence↗
PubMed2001

Presentation of ovalbumin internalized via the immunoglobulin-A Fc receptor is enhanced through Fc receptor gamma-chain signaling.

The mechanism of enhanced presentation of ovalbumin (OVA) internalized as immunoglobulin A (IgA)-OVA via the IgA Fc receptor (FcalphaR) was analyzed by focusing on the role of the FcalphaR-associated gamma chain. Comparison of B-cell transfectants expressing FcalphaR plus wild-type (WT) gamma chain or gamma chain in which the immunoreceptor tyrosine-based activation motif (ITAM) was altered by tyrosine mutation or substitution with the ITAM of FcgammaRIIA showed that signaling-competent ITAM was not required for endocytosis of IgA-OVA. However, antigen presentation was impaired by ITAM changes. Signaling-competent gamma-chain ITAM appeared necessary for transport of ligated FcalphaR to a lamp-1(+) late endocytic compartment for remodeling and/or activation of that compartment and also for efficient degradation of IgA complexes. Moreover, FcalphaR ligation also activated efficient processing of nonreceptor-targeted antigen. The results suggest that gamma-chain signaling activates the antigen processing compartment.

Amino Acid Motifs↗
PubMed2001

Identification of nodal signaling targets by array analysis of induced complex probes.

Nodal signaling controls germ layer formation, left-right asymmetry, and patterning of the brain in the vertebrate embryo. Cellular responses to Nodal signals are complex and include changes in gene expression, cell morphology, and migratory behavior. Only little is known about the genes regulated by Nodal signaling. We designed a subtractive screening strategy by using a constitutively active Nodal receptor to identify putative target genes of Nodal signals in the early gastrula of zebrafish embryos. By quantitative analysis of macro-array hybridizations, 132 genes corresponding to 1.4% of genes on the entire macro-array were identified, which were enriched in the Nodal-induced probe pool. These genes encode components of signal transduction pathways, transcription regulators, proteins involved in protein metabolism but also cytoskeletal components and metabolic enzymes, suggesting dramatic changes of cell physiology in gastrula cells in response to Nodal signals.

Animals↗
PubMed2001

Effects of warming therapy on pressure ulcers--a randomized trial.

Postoperative pressure ulcers are a common and expensive problem. Intraoperative hypothermia also is a common problem and may have a connection with impaired tissue viability. Researchers in this study hypothesized that intraoperative control of hypothermia may reduce the incidence of postoperative pressure ulcers. A randomized clinical trial (n = 338) was used to test the effects of using forced air warming therapy versus standard care. Results indicated an absolute risk reduction in pressure ulcers of 4.8% (i.e., 10.4% to 5.6%) with a relative risk reduction of 46% in patients who received warming therapy. Although not reaching statistical significance, the clinical significance of almost halving the pressure ulcer rate is important. A correlation between body temperature and postoperative pressure ulcers was established.

Adult↗
PubMed2001

Suicidal intent in patients with chronic pain.

Suicidal ideation among individuals suffering from chronically painful conditions has not been widely studied, although rates of completed suicide are believed to be elevated in this population relative to the general population. The psychiatric literature on suicide documents the importance of controlling for the severity of depression when studying factors associated with suicidal ideation, attempts, or completion. The present study examined the relationships between suicidal ideation and the experience of pain, pain-related disability, and pain coping efforts among a sample of individuals experiencing chronically painful conditions. Of 200 patients evaluated on an inpatient rehabilitation unit in a psychiatric service, 13 individuals (6.5%) reported suicidal intent on a commonly used self-report measure of symptoms of depression, the Beck Depression Inventory. This group was compared to a matched (age, sex, pain duration) group of similarly depressed individuals (N=13) and a matched group of non-depressed individuals (N=13) on measures of pain, disability, pain beliefs, and pain coping strategies. A history of a suicide attempt was associated with suicidal intent. Family history of substance abuse was significantly more prevalent among the depressed groups, regardless of suicidal thinking. The depressed/suicidal group and depressed/non-suicidal groups reported higher levels of pain, higher levels of pain-related disability, lower use of active coping, and higher use of passive coping compared to the non-depressed group. The depressed groups did not differ from one another on any of the measures of pain experience. Depression, not suicidal status, consistently predicted level of functioning. The prevalence of suicidal intent was comparable to rates observed in other studies and relatively low. When individuals with chronic pain report suicidal intent, it is imperative that measures preventing self-harm be implemented immediately and the patient's depression be treated aggressively.

Activities of Daily Living↗
PubMed2001

Implementing a psychological intervention to improve lifestyle self-management in patients with type 2 diabetes.

Lifestyle and behavioural factors play an important role in the development of Type 2 diabetes and management of this illness involves a combination of medication and lifestyle change. However, diabetes lifestyle programs have been found unsuccessful unless they are intensive and continued over long periods of time. The present study, which is one of the few randomised controlled trials of lifestyle interventions to be conducted outside of the US, aims to evaluate a brief psychological intervention that can be integrated into routine usual care to assist people to make the recommended lifestyle changes. Subjects are allocated to either an intervention or usual care control group. The intervention includes assessment and a personalised programme in which realistic manageable goals for lifestyle change and overcoming barriers are negotiated using brief motivational interviewing. Maintenance issues are addressed by follow-up telephone contact. Implementation of this study is described and baseline data presented on initial participants, and implications for practice are discussed.

Adult↗
PubMed2001

Expression of brain subtype creatine kinase in the zebrafish embryo.

Creatine kinases (CK) play crucial roles in intracellular energy transfer. We have isolated a cDNA from zebrafish embryos, which encodes a CK highly related to the mammalian brain subtype creatine kinase (BCK). The bck mRNA is expressed maternally in the zebrafish embryo and transcripts are distributed uniformly in blastula and gastrula stages. Expression becomes restricted to the prechordal plate and the nervous system during subsequent somitogenesis stages. bck transcripts are abundant in primary neurons in the developing central nervous system of the 1-day-old embryo. While some bck expression persists in the hindbrain, expression vanishes in the spinal cord of the 2-day-old embryo. In summary, the expression pattern of bck is highly dynamic and suggests a role for bck during gastrulation and neuronal differentiation.

Amino Acid Sequence↗
PubMed2001

Pre-clinical validation of a novel, highly sensitive assay to detect PML-RARalpha mRNA using real-time reverse-transcription polymerase chain reaction.

We have developed a sensitive and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of PML-RARalpha, the fusion oncogene present as a specific marker in >99% of cases of acute promyelocytic leukemia (APL). The assay is linear over at least 5 orders of magnitude of input DNA or RNA, and detects as few as 4 copies of PML-RARalpha plasmid DNA. PML-RARalpha transcripts could be detected in mixtures containing 2 to 5 pg of RNA from fusion-containing cells in a background of 1 microg of RNA from PML-RARalpha-negative cells. Using 1.0 to 2.5 microg of input RNA, the sensitivity of the assay was between 10(-5) and 10(-6). Furthermore, determination of GAPDH copy number in each reaction allowed an accurate assessment of sample-to-sample variation in RNA quality and reaction efficiency, with consequent definition of a detection limit for each sample assayed. Using an internal calibrator, assay precision was high, with coefficients of variation between 10 and 20%. An interlaboratory study using coded samples demonstrated excellent reproducibility and high concordance between laboratories. This assay will be used to test the hypothesis that sensitive and quantitative measurement of leukemic burden, during or after therapy of APL, can stratify patients into discrete risk groups, and thereby serve as a basis for risk-adapted therapy in APL.

Biomarkers, Tumor↗
PubMed2001

Expression and activity of ADAMTS-5 in synovium.

ADAMTS proteinases, belonging to the adamalysin subfamily of metalloproteinases, have been implicated in a variety of cellular events such as morphogenesis, cell migration, angiogenesis, ovulation and extracellular matrix breakdown. Aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5) have been identified in cartilage and are largely responsible for cartilage aggrecan breakdown. We have shown previously that synovium, the membrane lining diarthrodial joints, generates soluble aggrecanase activity. We report here the expression, localization and activity of ADAMTS-5 from human arthritic and bovine synovium. ADAMTS-5 was expressed constitutively in synovium with little or no transcriptional regulation by recombinant human interleukin-1 alpha or all-trans-retinoate, factors previously shown to upregulate aggrecanase activity in cartilage. Aggrecanase activity generated by synovium in vitro and recombinant ADAMTS-5 cleaved aggrecan extensively, resulting in aggrecan fragments similar to those generated by chondrocyte-derived aggrecanases, and the activity was inhibited by heparin. ADAMTS-5 was immunolocalized in human arthritic synovium, where staining was mostly pericellular, particularly in the synovial lining and around blood vessels; some matrix staining was also seen. The possibility that synovium-derived ADAMTS-5 may play a role in cartilage aggrecan breakdown is discussed.

ADAM Proteins↗
PubMed2001

A survey of nocturnal hypoxaemia and health related quality of life in patients with cryptogenic fibrosing alveolitis.

BACKGROUND: A survey of overnight oximetry was conducted to estimate the prevalence of nocturnal hypoxaemia in patients with cryptogenic fibrosing alveolitis and to establish whether nocturnal hypoxaemia is related to quality of life. METHODS: All patients with cryptogenic fibrosing alveolitis attending Nottingham City Hospital were invited to enter the study. Spirometric measurements and capillary blood gas tensions were obtained and overnight oxygen saturation was recorded at home. Quality of life was assessed using the Short Form-36, Chronic Respiratory Questionnaire, Hospital Anxiety Depression Scale, and Epworth Sleepiness Score questionnaires. RESULTS: Sixty seven eligible patients were identified and 50 agreed to enter the study, although two were subsequently excluded because they already used oxygen overnight. In the remaining 48 the mean (SD) overnight oxygen saturation (SaO(2)) was 92.5 (4.3)% and the median number of dips greater than 4% per hour was 2.3 (interquartile range 1.5-5.3). Daytime oxygen level predicted mean overnight SaO(2) (1.94%/kPa, 95% CI 1.22 to 2.66, p<0.001) but percentage predicted forced vital capacity (FVC) did not (0.018%/% predicted FVC, 95% CI -0.04 to 0.08, p=0.5). Nocturnal hypoxaemia was associated with decreased energy levels and impaired daytime social and physical functioning, and these effects were independent of FVC. CONCLUSIONS: Nocturnal hypoxaemia is common in patients with cryptogenic fibrosing alveolitis and may have an impact on health related quality of life.

Aged↗
PubMed2001

Effect of a single-use sterile catheter for each void on the frequency of bacteriuria in children with neurogenic bladder on intermittent catheterization for bladder emptying.

OBJECTIVE: The frequency of bacteriuria is high in children with neurogenic bladder on intermittent catheterization for bladder emptying. In an effort to decrease bacteriuria, we examined whether the method of catheter care was responsible for the high rate of bacteriuria. For this, the frequency of bacteriuria was examined in the same patient on single-use sterile catheters and on reused clean catheters. METHODS: A prospective, randomized, crossover trial was conducted with 10 patients who were randomized to 4 months of a new, sterile catheter for intermittent catheterization and 4 months of reuse of a clean catheter for intermittent catheterization. Each week, a urine sample was collected and symptoms of infection and medication use were recorded. RESULTS: A total of 158 urine samples were collected during 164 patient-weeks on the new catheter method for each void; 115 (73%) were positive for a pathogen. Of the 161 samples collected during 169 patient-weeks on the standard, reuse method for voiding, 123 (76%) were positive (115 [73%] of 158 vs 123 [76%] of 161). Escherichia coli was the most common pathogen detected during both method periods. CONCLUSION: A new, sterile catheter for each void did not decrease the high frequency of bacteriuria in patients with neurogenic bladder on intermittent catheterization.

Adolescent↗
PubMed2000

Isolation of antibodies which neutralise the activity of early pregnancy factor.

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It functions as an autocrine growth factor for tumour cells and as an autocrine or paracrine growth factor for regenerating normal cells. Anti-EPF antibodies have demonstrable anti-tumour activity and, as a result, hybridomas which produce such antibodies are unstable. In this study, the phage display antibody techniques have been investigated as a means of producing recombinant anti-EPF antibodies. Mice were immunised with synthetic peptides which correspond to the N or C terminal regions of EPF, and their splenic tissue was used to make combinatorial antibody libraries. The Fab repertoire was displayed on the surface of phage and panned over recombinant EPF. Reactive Fabs were identified by ELISA and their binding was characterised by BIAcore analysis and functional studies. Three libraries with a size of greater than 5x10(7)cfu were constructed and a total of 26 unique Fabs with specific reactivity against EPF were identified. Three Fabs were purified and of these one demonstrated strong EPF neutralising activity, one had intermediate activity and the other was not neutralising. Phage display has provided the means of circumventing the problems of anti-EPF hybridoma development and has resulted in the production of antibodies with potential applications in the diagnosis of pregnancy and the diagnosis and therapy of cancer.

Amino Acid Sequence↗