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Biomedical subjects

E Palmer

Publications and source records attributed to E Palmer.

At least 19 recordsLinked to original sources
PubMed2006

Systematic in vivo RNAi analysis of putative components of the Drosophila cell death machinery.

Despite the identification of numerous key players of the cell death machinery, little is known about their physiological role. Using RNA interference (RNAi) in vivo, we have studied the requirement of all Drosophila caspases and caspase-adaptors in different paradigms of apoptosis. Of the seven caspases, Dronc, drICE, Strica and Decay are rate limiting for apoptosis. Surprisingly, Hid-mediated apoptosis requires a broader range of caspases than apoptosis initiated by loss of the caspase inhibitor DIAP1, suggesting that Hid causes apoptosis not only by antagonizing DIAP1 but also by activating DIAP1-independent caspase cascades. While Hid killing requires Strica, Decay, Dronc/Dark and drICE, apoptosis triggered by DIAP1 depletion merely relied upon Dronc/Dark and drICE. Furthermore, we found that overexpression of DIAP2 can rescue diap1-RNAi-mediated apoptosis, suggesting that DIAP2 regulates caspases directly. Consistently, we show that DIAP2 binds active drICE. Since DIAP2 associates with Hid, we propose a model whereby Hid co-ordinately targets both DIAP1 and DIAP2 to unleash drICE.

Animals↗
PubMed2002

An improved assay for genotyping the common Alu insertion in the tissue-type plasminogen activation gene PLAT.

An improved assay for genotyping the common Alu insertion in the tissue-type plasminogen activator (PLAT) locus is described in this report. The assay is a valuable asset to clinical researchers interested in exploring disease associations with this allele. The automation and improved accuracy will facilitate future population-based studies, as well as clinical screening.

Alu Elements↗
PubMed2001

Sexual behavior of stallions during in-hand natural service and semen collection: an observation in French studs.

The sexual behavior of 42 stallions from French national and private studs was examined in two contexts: semen collection for artificial insemination (AI) and in-hand natural service (NS). Each stallion was observed twice in the same context. Erection and ejaculation latencies, the number of mounts leading to ejaculation, dismount latency and total breeding time were measured and compared between AI and NS. Mount without erection was rare (6/83 observations). Erection latency was 89+/-11s, and was not different between NS (62+/-22s) and AI (100+/-13s, P=0.128). Stallions ejaculated after either one mount (62/83 observations), or two (11/83 observations) or three mounts (10/83 observations). Ejaculation latency was 85+/-15s (84+/-19 in AI and 86+/-28 in NS). If 1st mount did not lead to ejaculation, then ejaculation latency increased several fold following the 2nd mount during both AI and NS. The results provide reference measures for semen collection in French studs. Difference in erection latency between AI and NS, although not statistically significant, may reflect different contributions of excitatory inputs from the brain and the genital area to the activation of spinal networks controlling erection. In contrast, lack of difference in ejaculation latency between AI and NS suggests that the spinal network that controls ejaculation follows a more rigid motor pattern.

Age Factors↗
PubMed2001

Advances in cryopreservation of stallion semen in modified INRA82.

In the procedure used in this paper, semen was first diluted in INRA82+2% egg yolk (E1) at 37 degrees C. Before or after cooling to 4 degrees C, semen was centrifuged and diluted in E1+2.5% glycerol (E2). Cooled semen was frozen in 0.5-ml straws. Straws were thawed at 37 degrees C for 30s. For fertility trials, frozen ejaculates were used only if total post-thaw motility was above 35%. Most mares were inseminated two times before ovulation with 400 x 10(6) total spermatozoa every 24h. This paper presents post-thaw motility (CASA) and fertility results obtained when some steps of the procedure were evaluated. Use of the first three jets of ejaculate before the centrifugation did not improve post-thaw motility compared to use of the whole semen (25% versus 25%, 2 stallions x 12 ejaculates, P>0.80). When the first dilution was performed in E2 at 22 degrees C instead of in E1 at 37 degrees C, motility was slightly improved (38% versus 36%, n>283 ejaculates per group, P<0.04) but fertility was similar (51% versus 58%, n>196 cycles per group, P>0.10). Coating the spermatozoa with 0.5, 1, 2, 4 and 8mM of Concanavalin A resulted in unchanged post-thaw motility (6 stallions x 3 ejaculates, P>0.05). The extender E2 was modified or supplemented with different substances. Increasing egg yolk concentration from 2 to 4% (v/v) did not increase post-thaw motility (42% versus 34%, 6 stallions x 2 ejaculates, P>0.05). Different glycerol concentrations (range: 1.7-3.7%) had no significant effect on post-thaw motility even though 2.4-2.8% resulted in a nonsignificant higher motility (7 stallions x 2 ejaculates, P>0.05). Glutamine at 50mM in E2 improved post-thaw motility compared with no glutamine (49% versus 46%, n>584 ejaculates per group, P<0.0001) but not fertility (53% versus 54%, n>451 cycles per group, P>0.80). Thawing at 75 degrees C for 10s slightly increased motility after 120 min at 37 degrees C (6 stallions x 1 ejaculate, P<0.05) but no effect on per-cycle fertility was noted (32% (19 cycles) versus 41% (17 cycles), P>0.50). When post-thaw dilution was performed using a fixed molarity multi-step system (25 mOsm per step) from various osmolarities (900-690 mOsm) to 365 mOsm, motility was unaffected compared with dilution in one step (36% versus 38%, 6 stallions x 1 ejaculate, P>0.20).

Animals↗
PubMed2001

Quantifying the frequency of alloreactive T cells in vivo: new answers to an old question.

Alloreactive T cell precursor frequency was measured in vivo using fluorescent dye labeling in combination with novel models based on lymphocyte activation and recovery. CFSE-labeled C57BL/6 (H-2(b)) spleen and lymph node cells were adoptively transferred to C57BL/6xDBA F(1) (H-2(b/d)) recipients, a parent-->F(1) MHC mismatch in which only donor cells respond. Recipients were sacrificed at serial time points to assess engraftment efficiency, and the extent of donor cell activation and proliferation. These data were used to calculate alloreactive T cell frequencies that varied 30-fold (0.71 +/- 0.31% to 21.05 +/- 3.62%), depending upon whether it was assumed that all donor cells injected became established and were capable of responding, or that only those present at later time points (24-72 h) were available to respond. By measuring the number of cells established in the recipient 24 h after transfer, before proliferation, we calculated an in vivo alloreactive frequency of approximately 7%. Using CD69 expression at 48 h to quantify activation, we found that 40-50% of the alloactivated CD4(+) donor T cells do not divide. Studies of cotransferred congenic and allogeneic cells demonstrated that bystander proliferation does not occur. We conclude that accurate calculations of alloreactive precursor frequency must account for both proliferation and cell engraftment. When this is done, a high percentage of alloreactive T cells exists across an MHC mismatch, but not all alloreactive cells proliferate in vivo. Bystander proliferation is negligible, revealing exquisite specificity to the alloresponse. These data provide a novel approach to quantify alloreactive T cell responses during specific immunomodulatory strategies in vivo.

Adoptive Transfer↗
PubMed2001

A critical role for the T cell receptor alpha-chain connecting peptide domain in positive selection of CD1-independent NKT cells.

Natural killer T (NKT) cells are a subset of mature alpha beta TCR(+) cells that co-express NK lineage markers. Whereas most NKT cells express a canonical Valpha14/Vbeta8.2 TCR and are selected by CD1d, a minority of NKT cells express a diverse TCR repertoire and develop independently of CD1d. Little is known about the selection requirements of CD1d-independent NKT cells. We show here that NKT cells develop in RAG-deficient mice expressing an MHC class II-restricted transgenic TCR (Valpha2/Vbeta8.1) but only under conditions that lead to negative selection of conventional T cells. Moreover development of NKT cells in these mice is absolutely dependent upon an intact TCR alpha-chain connecting peptide domain, which is required for positive selection of conventional T cells via recruitment of the ERK signaling pathway. Collectively our data demonstrate that NKT cells can develop as a result of high avidity TCR/MHC class II interactions and suggest that common signaling pathways are involved in the positive selection of CD1d-independent NKT cells and conventional T cells.

Animals↗
PubMed2001

The impact of duration versus extent of TCR occupancy on T cell activation: a revision of the kinetic proofreading model.

The widely accepted kinetic proofreading theory proposes that rapid TCR dissociation from a peptide/MHC ligand allows for stimulation of early but not late T cell activation events, explaining why low-affinity TCR ligands are poor agonists. We identified a low-affinity TCR ligand which stimulated late T cell responses but, contrary to predictions from kinetic proofreading, inefficiently induced early activation events. Furthermore, responses induced by this ligand were kinetically delayed compared to its high-affinity counterpart. Using peptide/MHC tetramers, we showed that activation characteristics could be dissociated from TCR occupancy by the peptide/MHC ligands. Our data argue that T cell responses are triggered by a cumulative signal which is reached at different time points for different TCR ligands.

Animals↗
PubMed2001

Improved student learning in ophthalmology with computer-aided instruction.

PURPOSE: Recent changes in the medical school curriculum and in teaching styles have stimulated the development of novel learning resources. We investigated the impact of computer-aided learning in the field of ophthalmology at a large Australian undergraduate medical school. METHODS: Clinical case studies describing common ophthalmic conditions were incorporated into a computing program which uses a problem-solving format. The program was made available to 85 medical students for a 2 week period. A control group of 20 medical students did not have access to this material. Acquisition of new knowledge was assessed as the improvement in score on a knowledge-based multiple-choice examination which students took both before and following the learning interval. RESULTS: Students in the control group showed no improvement over the test period, whilst the students who had access to the computer significantly increased their knowledge base, both for material provided on the program (p = 0.0001) and in related areas (p = 0.0015). CONCLUSIONS: Suitably constructed material on the computer is a useful aid in teaching ophthalmology to medical undergraduates. The material and the medium may also act as a stimulus for further learning.

Computer-Assisted Instruction↗
PubMed2001

Open donor, laparoscopic donor and hand assisted laparoscopic donor nephrectomy: a comparison of outcomes.

PURPOSE: In experienced hands laparoscopic surgery has been shown to be safe for procuring kidneys for transplantation that function identically to open nephrectomy controls. While searching for a safer and easier approach to laparoscopic donor nephrectomy, hand assisted laparoscopic techniques have been added to the surgical armamentarium. We compare allograft function in patients with greater than 1-year followup who underwent open donor (historic series), classic laparoscopic and hand assisted laparoscopic nephrectomy. MATERIALS AND METHODS: The charts of 48 patients who underwent open donor, laparoscopic donor or hand assisted laparoscopic nephrectomy were reviewed. Only patients with greater than 1-year followup and complete charts were included in our study. Of these patients 34 underwent consecutive laparoscopic live donor nephrectomy and 14 underwent open donor nephrectomy. Mean patient age plus or minus standard deviation (SD) was 36.5 +/- 8.4 years for donors and 29 +/- 17 for recipients at transplantation (range 13 months to 69 years). In the laparoscopic group 11 patients underwent the transperitoneal technique, and 23 underwent hand assisted laparoscopic nephrectomy. RESULTS: Total operating time was significantly reduced with the hand assisted laparoscopic technique compared with classic laparoscopy, as was the time from skin incision to kidney removal and warm ischemic time. Average warm ischemic time plus or minus SD was 3.9 +/- 0.3 minutes for laparoscopic nephrectomy and 1.6 +/- 0.2 for hand assisted laparoscopy (p <0.05). Long-term followup of serum creatinine levels revealed no significant differences among the 3 groups. Comparison of those levels for recipients of open nephrectomy versus laparoscopic and hand assisted laparoscopic techniques revealed p values greater than 0.5. No blood transfusions were necessary. Complications included adrenal vein injury in 1 patient, small bowel obstruction in 2, abdominal hernia at the trocar site in 1 and deep venous thrombosis in 1. CONCLUSIONS: Classic laparoscopic donor and hand assisted laparoscopic donor nephrectomies appear to be safe procedures for harvesting kidneys. The recipient graft function is similar in the laparoscopic and open surgery groups.

Adolescent↗
PubMed2000

Defective signaling to Fyn by a T cell antigen receptor lacking the alpha -chain connecting peptide motif.

A key role in the communication between the alphabetaTCR and the CD3/zeta complex is played by a specific motif within the connecting peptide domain of the TCR alpha chain (alpha-CPM). T cell hybridomas expressing an alpha-CPM-mutated TCR show a dramatic impairment in antigen-driven interleukin-2 production. This defect can be complemented by a calcium ionophore, indicating that activation of the calcium pathway is impaired. Several lines of evidence implicate Fyn in the regulation of calcium mobilization, at least in part through the activation of phospholipase Cgamma. Here we have investigated the potential involvement of Fyn in the TCR alpha-CPM signaling defect. Using T cell hybridomas expressing either a wild-type TCR or an alpha-CPM mutant, we show that Fyn fails to be activated by the mutant receptor following SEB binding and fails to generate tyrosine-phosphorylated Pyk2, a member of the focal adhesion kinase family. This defect correlated with an impairment in phospholipase Cgamma phosphorylation. Production of interlukin-2 and activation of the transcription factor NF-AT in response to triggering of the TCR alpha-CPM mutant with SEB were fully restored in the presence of constitutively active Fyn. Hence the signaling defect generated by the TCR alpha-CPM mutation results at least in part from an impaired coupling of the TCR.CD3 complex to Fyn activation.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Centrifugation and addition of glycerol at 22 degres C instead of 4 degrees C improve post-thaw motility and fertility of stallion spermatozoa.

The aims of this study were to evaluate the effects of cooling rate to 4 degrees C and temperature at the time of centrifugation/glycerol-addition (freezing extender: INRA82 + 2% egg yolk + 2.5% glycerol) on postcentrifugation recovery rate, post-thaw motility and per-cycle fertility. When centrifugation/glycerol-addition was performed at 4 degrees C (14 ejaculates), a moderate cooling rate (37 degrees C to 4 degrees C in I h) resulted in higher post-thaw motility (45%) than when using a slow cooling rate (37 degrees C to 4 degrees C in 4 h) (39%; P<0.05). When centrifugation/glycerol-addition was performed at 22 degrees C (37 degrees C to 22 degrees C in 10 min) (10 ejaculates), post-thaw motility was lower when spermatozoa were frozen directly from 22 degrees C (23%) than when spermatozoa were cooled to 4 degrees C (22 degrees C to 4 degrees C in 1 h) before freezing (47%; P<0.0001). When centrifugation/glycerol-addition was performed at 22 degrees C (before cooling at a moderate rate), as opposed to 4 degrees C (after cooling at a moderate rate), a significant improvement of 1) recovery of spermatozoa after centrifugation (P<0,0001), 2) post-thaw motility of spermatozoa at thawing (40% vs 36% (n < or = 291 ejaculates/group), P<0.0001) and 3) per-cycle fertility (56% vs 42% (n > or = 190 cycles/group), P<0.01) was observed. In conclusion, centrifugation/glycerol-addition at 22 degrees C followed by cooling to 4 degrees C at a moderate rate results in an improvement of post-thaw motility, spermatozoa recovery rate and per cycle fertility.

Animals↗
PubMed2000

Normal thymic architecture and negative selection are associated with Aire expression, the gene defective in the autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED).

T cell development is tightly controlled by thymic stromal cells. Alterations in stromal architecture affect T cell maturation and the development of self-tolerance. The monogenic autoimmune syndrome APECED (autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy) is characterized by the loss of self-tolerance to multiple organs. Although mutations in the autoimmune regulator (AIRE) gene are responsible for this disease, the function of AIRE is not known. Here we report on the spatial and temporal pattern of murine Aire expression during thymic ontogeny and T cell selection. Early during development, thymic Aire transcription is critically dependent on RelB and occurs in epithelial cells in response to lymphocyte-mediated signals. In adult tissue, Aire expression is confined to the medulla and the corticomedullary junction, where it is modulated by thymocytes undergoing negative selection. Aire may determine thymic stromal organization and with it the induction of self-tolerance.

Animals↗
PubMed2000

Essential role of CD8 palmitoylation in CD8 coreceptor function.

To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.

4-Aminobenzoic Acid↗
PubMed2000

A motif in the alphabeta T-cell receptor controls positive selection by modulating ERK activity.

Positive selection allows thymocytes that recognize an individual's own major histocompatibility complex (self-MHC) molecules to survive and differentiate, whereas negative selection removes overtly self-reactive thymocytes. Although both forms of thymic selection are mediated by the alphabeta T-cell receptor (TCR) and require self-MHC recognition, an important question is whether they are controlled by distinct signalling cascades. We have shown that mutation of an essential motif within the TCR alpha-chain-connecting peptide domain (alpha-CPM) profoundly affects positive but not negative selection. Using transgenic mice expressing a mutant alpha-CPM TCR we examined the contribution of several mitogen-activated protein kinase (MAPK) cascades to thymic selection. Here we show that in thymocytes expressing a mutant alpha-CPM receptor, a positively selecting peptide failed to activate the extracellular signal-regulated kinase (ERK), although other MAPK cascades were induced normally. The defect in ERK activation was associated with impaired recruitment of the activated tyrosine kinases Lck and ZAP-70, phosphorylated forms of the TCR component CD3zeta and the adaptor protein LAT to detergent-insoluble glycolipid-enriched microdomains (DIGs). Therefore, an intact DIG-associated signalosome is essential for sustained ERK activation, which leads to positive selection.

Amino Acid Motifs↗
PubMed2000

Clinicians and computers: friends or foes?

BACKGROUND: Computer-aided learning is accepted by students as a learning resource, but the views of the teaching community are largely unknown. PURPOSE: To document clinicians' experience with computers and to record their attitudes toward computer usage in clinical practice and student education. METHODS: Questionnaire mailed out to all clinicians, including interns and residents, fellows, and attending physicians in 3 major teaching hospitals in South Australia, with a total of 646 clinical staff. RESULTS: Replies were received from 246 staff. Eighty percent of clinicians had at least 2 years of experience with computers and used computers for at least 2 hr each week. Despite this, there was an obvious lack of conviction among clinicians that computer-aided learning was of use in student education and assessment. This may reflect their lack of experience with this medium as an educational tool. CONCLUSIONS: If computer-aided learning is to make any significant impact on medical student education, it must be carefully and objectively evaluated, and its benefit must be clearly demonstrated to clinical teachers.

Adult↗
PubMed2000

Clinical studies on daily low dose oxytocin in mares at term.

The aim of this study was to test whether low dose oxytocin i.v. injection once a day to mares diagnosed as being ready for birth by mammary secretion calcium strip test measurements could be used as a reliable method to induce parturition and/or predict the mare would not foal during the following night if parturition did not occur within 2 h of treatment. Fifty-one near-term Haflinger mares were used and a single injection of 2.5 iu oxytocin was given between 1700 and 1900 h, including 10 mares used as controls which were administered a placebo. Administration of oxytocin resulted in the delivery of a normal foal within 120 min in 95% of mares. Twenty-four out of 38 (63%) treated animals foaled in response to the first oxytocin injection, 9 out of 38 (24%) in response to the second injection and 3 out of 38 (8%) in response to the third treatment. Two out of 38 (5%) treated mares foaled during the night irrespective of treatment whereas 7 out of 10 (70%) control mares foaled during the night. It was concluded that the major advantage of injecting a daily low dose of oxytocin appears to be that such a low dose induces delivery only in mares carrying a mature fetus and which are ready to foal.

Animals↗
PubMed1999

Effect of time during transport of excised mare ovaries on oocyte recovery rate and quality after in vitro maturation.

In the mare only a limited number of oocytes can be successfully collected in vivo, so that when large numbers of oocytes are needed for experimentation, ovaries harvested from slaughtered mares must be used. The resulting temperature changes and time intervals mandated by handling and transport of ovaries from the slaughterhouse to the laboratory adversely affect the rate of oocyte recovery and their quality after IVF and maturation. We chose to study the effect of temperature and time in transit of excised ovaries by evaluating rate of oocyte recovery, nuclear maturation stage reached before, and cleavage rate reached after IVF, following short (1.5 to 4 h) and long (6 to 8 h) storage. Temperatures in the storage container decreased from 37-C to 32 degrees and 27.5 degrees C during the short and long interval, respectively. The cumulus-oocytes complexes (COCs) were classified as having a compact cumulus, completely or partially surrounding the oocyte (compact); those having only a corona radiata surrounding the oocyte (corona); those having a completely or partially expanded cumulus, showing a cellular or sparsely cellular, gelatinous cloud around the oocyte (expanded); and those that were completely denuded of both cumulus and corona cells (denuded). All COCs, except the denuded ones, which were discarded, were matured in vitro for 30 h at 38.5 degrees C in 5% CO2. The recovery rate of oocytes was significantly higher after long vs short storage (48 vs 35%; P < 0.01), but the distribution of the collected COCs into the 4 classes was not affected by the storage time. After in vitro maturation nuclear maturity was not affected by the storage time, but oocytes with intact cytoplasmic membranes were more frequently found after short than after long storage (54 vs 34%; P = 0.07), and fully matured oocytes were more often seen with intact membrane (P < 0.01). Moreover, oocytes with intact membranes in metaphase II (MII) were associated with short storage intervals and the corona COC class, while damaged membranes and incomplete maturation were associated with the long storage and the compact COC class.

Animals↗