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D Walker

Publications and source records attributed to D Walker.

At least 199 records · Page 11Linked to original sources
PubMed1996

Ricin A chain fused to a chloroplast-targeting signal is unfolded on the chloroplast surface prior to import across the envelope membranes.

The initial stages of chloroplast protein import involve the binding of precursor proteins to surface-bound receptors prior to translocation across the envelope membranes in a partially folded conformation. We have analyzed the unfolding process by examining the conformation of a construct, comprising the presequence of a chloroplast protein linked to ricin A chain, before and after binding to the chloroplast surface. We show that the presequence is highly susceptible to proteolysis in solution, probably reflecting a lack of tertiary structure, whereas the A chain passenger protein is resistant to extremely high concentrations of protease, unless deliberately unfolded using denaturant. The A chain moiety is furthermore active, indicating that the presence of the presequence does not prevent formation of a tightly folded, native state. In contrast, receptor-bound p33KRA (fusion protein comprising the 33-kDa presequence plus 22 residues of mature protein, linked to the A chain of ricin) is quantitatively digested by protease concentrations that have little effect on the A chain in solution. We conclude that protein unfolding can take place on the chloroplast surface in the absence of translocation and without the aid of soluble factors.

Chloroplasts↗
PubMed1996

Hepatitis B virus mutations in the pre-S genome before and after liver transplantation.

Mutational changes in the pre-S region of hepatitis B virus (HBV) were analyzed in 20 patients who experienced HBV reinfection after orthotopic liver transplantation (OLT). HBV DNA was extracted from patient sera before and after OLT. The pre-S sequence was amplified via polymerase chain reaction, subcloned, sequenced, and analyzed. In 18 of 20 patients, mutational changes were found in the pre-S region pre- or post- OLT; 11 showed point mutations (1-10) and 7 cases major changes (insertions/deletions). For the point mutations, there was no trend in the selection of wild-type (wt) HBV before or after OLT in the pre-S region. Additional HBV reinfection during hepatitis B surface antigen antibody (anti-HBS) administration had no influence on selection pressure in the pre-S region. In contrast, insertions/deletions were more frequently found before OLT. In the 7 patients with deletions/insertions, changes in the hepatocyte attachment site were not seen after OLT. Interestingly, the only patient with changes in a major virus population after OLT had changes in the CCAAT-box of the S-promoter. As shown by gel shift analysis, this mutation was associated with loss of specific binding to this element and thus probably led to dysregulation of S-gene transcription. Major changes in the pre-S genome are mainly seen before OLT, and HBV reinfection does occur with the intact hepatocyte attachment sites after OLT. Anti-HBs (hepatitis B immune globulin [HBIg]) creates no selection pressure on the pre-S region. The mutation in the CCAAT-box of the S-promoter potentially leads to its dysregulation and may be associated with the occurrence of fibrosing cholestatic hepatitis after OLT.

Adult↗
PubMed1996

Sacrospinous ligament fixation by palpation: variation of the Richter procedure.

OBJECTIVE: To present our method of sacro-spinous ligament fixation by palpation and compare it to the classical approach described by Richter. MATERIAL: In addition to the standard instruments needed for vaginal surgery, we use a Rasemond dissector, a small O'Shaugnessy dissector with smooth branches. We also use a Bengolea forceps and a monothread-nylon (Ethilon), decimal 4 suture loaded on a needle with a 30-mm curve. PATIENTS: Twenty patients underwent this procedure from 03/15/1978 to 05/19/1995. Their ages ranged from 46 to 86 years with a mean age of 64.7 years. METHOD: This was a retrospective study of the indications, results and complications associated with this technique. RESULTS: With an average follow-up of 7 years, we observed 90% success, 10% recurrences, and no complication directly attributable to this technique. This technique is valuable because of its effectiveness and simplicity. CONCLUSIONS: Sacrospinous fixation by palpation is more simple and provides the same results as the classic exposure technique. We describe the technique in this text. The efficacy of sacro-spinous ligament fixation by palpation would be improved by its systematic and bilateral use. Its value must be confirmed by a controlled prospective study to confirm our impression that our technique carries fewer risks.

Aged↗
PubMed1996

The development of diurnal rhythmicity in fetal suprachiasmatic neurons as demonstrated by fos immunohistochemistry.

Using Fos immunohistochemistry as a marker of cellular activity, we have shown that neurons in the suprachiasmatic nucleus of fetal sheep are active by 75 days gestation. From at least 90 days gestation (term is 146 days), these neurons are more active during the day (12.00) than at night (03.00) when pregnant ewes are exposed to a 12-h light-dark cycle with lights on at 07.00. The day-night difference in Fos immunoreactivity persisted when the lighting schedule was extended by 8 h to 03.00, although neurons were now more active at 03.00 than they were in fetuses maintained on the normal light-dark cycle. When ewes were maintained in constant light from 133 to 138 days, the day-night difference in Fos immunoreactivity in the fetal suprachiasmatic nucleus was abolished, suggesting that diurnal activity of the fetal suprachiasmatic nucleus is maintained by a signal related to the external lighting regime. In a twin pregnancy where one fetus was optically enucleated at 100 days gestation, the density of Fos-immunoreactive neurons in the suprachiasmatic nucleus during the day at 138 days was similar to the unoperated twin. This suggests that the effects of dim light in the uterus on the fetal retina do not account for the high level of Fos immunoreactivity in the suprachiasmatic nucleus in the daytime. We propose that a chemical messenger of maternal origin, possibly melatonin, suppresses the activity of fetal suprachiasmatic neurons during the night, and that fetal suprachiasmatic neurons have endogenous activity which is expressed fully during the daytime.

Animals↗
PubMed1996

Utility of clinical trial batteries in the measurement of Alzheimer's and Huntington's dementia.

Tests used as outcome measures in clinical trials of antidementia agents are not typically employed as part of diagnostic evaluations, and little information exists as to the sensitivity of these tests in terms of either differentiating demented patients from normal individuals or in distinguishing dementias of various types and etiologies. Sensitivity to mild dementia and sensitivity to impairment of various neuropsychological domains are, however, prerequisites for valid use of an instrument as an outcome measure in this context. The present study was undertaken to directly compare six different tests (three traditional psychometric tests and three clinical trial batteries) in terms of their sensitivity to detect and distinguish between mild dementia in patients with either Alzheimer's disease (n = 15) or Huntington's disease (n = 15), when compared to normal controls (n = 15). Tests included the Mattis Dementia Rating Scale, the Mini-Mental State Examination, the Wechsler Memory Scale-Revised, the Alzheimer's Disease Assessment Scale-Cognitive Subscale, the Computerized Drug Research (CDR) Cognitive Assessment System, and the Repeatable Battery for the Assessment of Dementia (RBAD). All of the tests were roughly equivalent in terms of their ability to discriminate normal subjects from mildly demented patients. Only the CDR and RBAD, however, were able to reliably discriminate between the two patient groups. The results are discussed in terms of the applicability of these tests as outcome measures for clinical trials in dementing disorders.

Adult↗
PubMed1996

Comparison of HT29-18-C1 and Caco-2 cell lines as models for studying intestinal paracellular drug absorption.

PURPOSE: To compare the permeability characteristics of HT29-18-C1 colonic epithelial cell line with Caco-2, an established model of intestinal drug transport. METHODS: Cell lines were grown as epithelial monolayers. Permeability was measured over a range of transepithelial electrical resistance (Rt) using a group of drug compounds. RESULTS: HT29-18-C1 develop Rt slowly when grown in culture, allowing permeability to be measured over a wide range (80-600 Omega x cm2). In contrast, Caco-2 monolayers rapidly develop Rt of approximately equal 300 Omega x cm2 and require Ca2+ -chelation to generate Rt equivalent to human intestine (60-120 Omega x cm2). Permeability of atenolol, ranitidine, cimetidine, hydrochlorothiazide and mannitol across HT29-18-C1 decreased 4-5 fold as Rt developed from 100-300 Omega x cm2 indicating they permeate via the paracellular route. In contrast, ondansetron showed no difference in permeability with changing Rt consistent with transcellular permeation. Permeability profiles across low Rt HT29-18C1 and pulse EGTA-treated Caco-2 monolayers were the same for all 5 paracellular drugs suggesting that transient Ca2+ removal does not alter selectivity of the tight junctions. Permeabilities of cimetidine, hydrochlorothiazide and atenolol across 100 Omega x cm2 HT29-18-C1 monolayers reflect more closely those reported for the human ileum in vivo than did mature Caco-2 monolayers. CONCLUSIONS: HT29-18-C1 monolayers can be used to study drug permeability at Rt values similar to human intestine without the need for Ca2+ chelation. As such, they offer a useful alternative to Caco-2 for modelling intestinal drug absorption.

Atenolol↗
PubMed1996

Lack of a temporal gradient of retrograde amnesia following NMDA-induced lesions of the basolateral amygdala assessed with the fear-potentiated startle paradigm.

M. Kim and M. Davis (1993b) previously reported that electrolytic lesions of the central nucleus of the amygdala, made 6 or 30 days after training, completely blocked the expression of fear-potentiated startle in rats. The present study shows that excitotoxic lesions of the basolateral amygdala also block fear-potentiated startle and do so whether the lesions are made soon (i.e., 6 days) or long (i.e., 30 days) after training. The relevance of these findings to various theories of amygdala function is discussed.

Amygdala↗
PubMed1996

Structure and function of the Bacillus SpoIIE protein and its localization to sites of sporulation septum assembly.

Functioning of the spoIIE locus of Bacillus subtilis is required for formation of a normal polar septum during sporulation and for activation of the transcription factor sigma F, which directs early forespore-specific gene expression. We have determined the DNA sequence of the wild type and several mutant alleles of the spoIIE gene of B. subtilis and sequenced a substantial portion of its presumptive homologue in Bacillus megaterium. We show that the spoIIE locus encodes a single large protein with a predicted molecular mass of 92 kDa. Each of five point-mutation alleles, which have traditionally defined the locus, and two transposon-generated mutations were shown to fall within the coding sequence for the 92 kDa gene product or within sequences expected to be required for its expression. The amino-terminal portion of the predicted SpoIIE gene product, comprising approximately 40% of the protein, is extremely hydrophobic and is expected to contain up to 12 membrane-spanning segments. The remainder of the protein contains no hydrophobic segments long enough to span a lipid bilayer and is therefore presumed to comprise one or more globular, aqueous-phase exposed domains. An in-frame fusion joining the 3' end of the B. megaterium spoIIE coding sequence to the 5' end of gfp, a gene encoding the green fluorescent protein (GFP) of Aquorea victoria, resulted in a strong, sporulation-specific fluorescent signal localized to the sites of sporulation septum assembly. We speculate that SpoIIE plays a role in assembling the sporulation septum, perhaps determining the special properties of the structure that permit intercompartment signalling during development.

Alleles↗
PubMed1996

Choice and accountability in health promotion: the role of health economics.

Choices need to be made between competing uses of health care resources. There is debate about how these choices should be made, who should make them and the criteria upon which they should be made. Evaluation of health care is an important part of this debate. It has been suggested that the contribution of health economics to the evaluation of health promotion is limited, both because the methods and principles underlying economic evaluation are unsuited to health promotion, and because the political and cultural processes governing the health care system are more appropriate mechanisms for allocating health care resources than systematic economic analysis of the costs and benefits of different health care choices. This view misrepresents and misunderstands the contribution of health economics to the evaluation of health promotion. It overstates the undoubted methodological difficulties of evaluating health promotion. It also argues, mistakenly, that economists see economic evaluation as a substitute for the political and cultural processes governing health care, rather than an input to them. This paper argues for an economics input on grounds of efficiency, accountability and ethics, and challenges the critics of the economic approach to judge alternative mechanisms for allocating resources by the same criteria.

Choice Behavior↗
PubMed1996

Intrauterine insemination: evaluation of the results according to the woman's age, sperm quality, total sperm count per insemination and life table analysis.

We report on 332 infertile couples who underwent 1115 cycles of intrauterine insemination (IUI) with washed husband's semen. The indication for IUI was an abnormal post-coital test due to either a male or cervical infertility factor. The mean number of IUI cycles per patients was 3.4, the overall pregnancy rate 18.7%, and the pregnancy rate per cycle 5.6%. The cumulative pregnancy rate calculated by life table analysis showed that 16.0% of pregnancies occurred in the first three treatment cycles, while the cumulative pregnancy rate was 26.9% by the sixth cycle. The outcome of the therapy was adversely affected if the woman's age was > 39 years and/or total motile sperm count per insemination was < 1 x 10(6). No pregnancy occurred in women older than 44 years or in cases with a total motile sperm count before semen preparation of < 1 x 10(6).

Adult↗
PubMed1996

Nuclei number in human embryos co-cultured with human ampullary cells.

A study was undertaken to evaluate the effect on nuclei number in human embryos cultured in vitro with primary cell lines of human Fallopian tube epithelium. The development of 203 surplus human embryos, cultured in standard culture medium (Earle's balanced salt solution + 15% A5) with or without ampullary cells, was observed from day 2 to day 5.5 post-insemination. The expanded blastocysts in both culture conditions were analysed for nuclei numbers per blastocyst. Embryos transferred to co-culture at the 2-cell stage had an average of 120.7 nuclei per blastocyst, which was significantly higher than the average of 62.9 nuclei per blastocyst (P = 0.023) for the embryos transferred to co-culture at the 4-cell stage. The embryos cultured in the control medium had an average of 42.1 nuclei per blastocyst, which was significantly less than co-cultured embryos (P = 0.04). Severely fragmented embryos (grades 3 and 4) did not show recovery in co-culture. Our results show that when human embryos are transferred to co-cultures before the 4-cell stage, the blastulation rate and the cell number per embryo increase significantly compared to the embryos cultured in standard culture medium. The possible effect of co-culture on embryonic gene expression is discussed.

Adult↗
PubMed1996

An intensive multiagent chemotherapy regimen for brain tumours occurring in very young children.

Standard treatment for the majority of malignant brain tumours consists of surgery and radiotherapy. This treatment has late morbidity which is accentuated in the very young child. As part of a strategy to improve quality of life and overall survival of young children with brain tumours, members of the United Kingdom Children's Cancer Study Group (UKCCSG) have piloted an intensive chemotherapy regimen which aims to avoid or delay radiotherapy following surgery. Twenty eight children with a variety of malignant brain tumours have received the regimen, which contains carboplatin, vincristine, cyclophosphamide, methotrexate, and cisplatin. The treatment is toxic, resulting in one death from infection. The bulk of the toxicity was associated with the administration of carboplatin. All but three children eventually required adjuvant radiotherapy and this was given between 1.5 and 27 months from diagnosis (median delay to radiotherapy, 12 months). Using this treatment regimen, overall survival at four years is 35% (confidence intervals 10% to 60%). While there is no evidence from this study that radiotherapy can be abandoned in the management of malignant brain tumours, its introduction may be delayed using suitable chemotherapy, thus allowing time for further CNS development. This treatment strategy has been taken forward as an international clinical trial run through the International Society for Paediatric Oncology, but using a smaller dose of carboplatin to reduce toxicity.

Age Distribution↗
PubMed1996

Analysis of 21 Stargardt's disease families confirms a major locus on chromosome 1p with evidence for non-allelic heterogeneity in a minority of cases.

BACKGROUND: Autosomal recessive Stargardt's disease is a macular degeneration characterised by a juvenile onset and a rapidly progressive course resulting in an atrophic macular area typically surrounded by yellowish retinal flecks. METHOD: The disease locus has previously been assigned to markers from chromosome 1p21-p13 by genetic linkage analysis in eight multiplex Stargardt's disease families. RESULTS: In an extended analysis, the assignment to chromosome 1p was confirmed in the majority of the 21 families with Stargardt's disease who were studied. In addition, a series of recombinant chromosomes further narrowed the Stargardt's disease region to an approximately 3 cM interval between markers at D1S424 and D1S497. CONCLUSION: Multipoint linkage analysis most probably excludes this locus in three of these families suggesting non-allelic heterogeneity with at least one additional minor Stargardt's disease locus.

Chromosomes, Human, Pair 1↗
PubMed1996

Cross-linking the TCR complex induces apoptosis in CD4+8+ thymocytes in the presence of cyclosporin A.

Although it is generally agreed that TCR ligation is a minimal requirement for negative selection in the CD4+8+ double-positive (DP) thymocyte subset, the costimulatory requirements and specific signaling events necessary to induce apoptosis are not well defined. We have explored the consequences of cross-linking CD3/TCR complexes on thymocytes from H-Y TCR transgenic (Tg) mice. In agreement with previous reports, we demonstrate that culturing DP thymocytes with plate-bound anti-TCR antibody induces downregulation of CD4 and CD8 and upregulation of CD69 expression. Nevertheless, the activated cells did not undergo apoptosis, as determined by viable cell recoveries and by quantitation of DNA fragmentation using the TUNEL assay. However, specific depletion of the DP subset occurred within 24 hr when thymocytes were incubated in the presence of both anti-TCR and the immunosuppressant cyclosporin A (CsA). CsA also induced depletion of anti-CD3 stimulated normal DP thymocytes. Using mice homozygous for the lpr or gld mutation, we also have shown that Fas/Fas ligand interactions are not involved in the CsA-induced death of TCR-stimulated DP thymocytes. These data verify that TCR cross-linking alone is insufficient to induce apoptosis of DP thymocytes and further suggest that TCR stimulation activates a CsA-sensitive protective pathway that interferes with signaling events leading to apoptosis in DP thymocytes.

Animals↗