Identifying GFP-transgenic animals by flashlight.
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Animals↗
Biomedical subjects
T Pratt
Publications and source records attributed to T Pratt.
Bone mass and density response to a 12-month trial of calcium and vitamin D supplement in preadolescent girls.
BACKGROUND: Maximal bone acquisition in adolescent girls through dietary and lifestyle practices is advocated to prevent or minimize the development of osteoporosis and its associated complications in later life. Longitudinal investigations of bone acquisition in children and adolescents have utilized areal bone mineral density (BMD, mg/cm(2)) as a measure of bone mass and strength. Peripheral quantitative computed tomography (pQCT), which provides a three-dimensional display of data, separate analyses of bone compartments, and bone mass in terms of volumetric BMD (vBMD, mg/cm(3)), has recently been introduced for clinical use. OBJECTIVE: To assess the impact of a 12-month daily calcium supplement on total and trabecular bone acquisition as measured by pQCT in preadolescent girls. DESIGN: Early adolescent Caucasian girls (aged 12 years, Tanner Stage 2) were enrolled in a randomized trial of daily calcium supplement (TX, 800 mg calcium carbonate and 400 IU vitamin D) or placebo (C). Body weight, height, and distal tibia measurements by pQCT were obtained at enrollment, 6 and 12 months. Pubertal status and physical activity records were assessed at baseline and 12 months. Three-day food intake records were completed every three months. RESULTS: Seventy-one girls completed the 12-month trial (TX=35, C=36). No differences were found for age, weight, height, body mass index, pubertal maturation, or reported physical activity at enrollment or during the study. Average intakes during the study were 1524 mg calcium and 496 IU vitamin D (TX) versus 865 mg calcium and 160 IU vitamin D (C) per day. Baseline total bone values were similar, however, trabecular values were greater in TX girls despite randomization. Percent changes were calculated to adjust for baseline differences. Because of the small cortical thickness at the 10% site (mean values < 1.6 mm), cortical mass and density were not analyzed. The percent changes for trabecular bone mineral content (BMC, mg) and vBMD were significantly greater in TX girls (+4.1% BMC and +1.0% vBMD TX versus -1.6% BMC and -2.0% vBMD C, p<0.006; ANCOVA) after 12 months of supplement. Trabecular bone area (BA, cm(2)) and total bone change, however, did not differ between groups. CONCLUSIONS: Daily calcium and vitamin D supplementation promotes greater trabecular BMC and vBMD acquisition in preadolescent girls. The single site selected for pQCT evaluation in this study did not allow evaluation of the cortical bone compartment. Future studies that utilize the pQCT technique need to incorporate multiple measurement sites to better assess total, cortical, and trabecular bone.
Journal Article↗
Embryonic stem cells and transgenic mice ubiquitously expressing a tau-tagged green fluorescent protein.
We have generated embryonic stem (ES) cells and transgenic mice carrying a tau-tagged green fluorescent protein (GFP) transgene under the control of a powerful promoter active in all cell types including those of the central nervous system. GFP requires no substrate and can be detected in fixed or living cells so is an attractive genetic marker. Tau-tagged GFP labels subcellular structures, including axons and the mitotic machinery, by binding the GFP to microtubules. This allows cell morphology to be visualized in exquisite detail. We test the application of cells derived from these mice in several types of cell-mixing experiments and demonstrate that the morphology of tau-GFP-expressing cells can be readily visualized after they have integrated into unlabeled host cells or tissues. We anticipate that these ES cells and transgenic mice will prove a novel and powerful tool for a wide variety of applications including the development of neural transplantation technologies in animal models and fundamental research into axon pathfinding mechanisms. A major advantage of the tau-GFP label is that it can be detected in living cells and labeled cells and their processes can be identified and subjected to a variety of manipulations such as electrophysiological cell recording.
Animals↗
A role for Pax6 in the normal development of dorsal thalamus and its cortical connections.
The transcription factor Pax6 is widely expressed throughout the developing nervous system, including most alar regions of the newly formed murine diencephalon. Later in embryogenesis its diencephalic expression becomes more restricted. It persists in the developing anterior thalamus (conventionally termed "ventral" thalamus) and pretectum but is downregulated in the body of the posterior (dorsal) thalamus. At the time of this downregulation, the dorsal thalamus forms its major axonal efferent pathway via the ventral telencephalon to the cerebral cortex. This pathway is absent in mice lacking functional Pax6 (small eye homozygotes: Sey/Sey). We tested whether the mechanism underlying this defect includes abnormalities of the dorsal thalamus itself. We exploited a new transgenic mouse ubiquitously expressing green fluorescent protein tagged with tau, in which axonal tracts are clearly visible, and co-cultured dorsal thalamic explants from Pax6(+/+ )or Pax6(Sey/Sey )embryos carrying the transgene with wild-type tissues from other regions of the forebrain. Whereas Pax6(+/+ )thalamic explants produced strong innervation of wild-type ventral telencephalic explants in a pattern that mimicked the thalamocortical tract in vivo, Pax6(Sey)(/Sey) explants did not, indicating a defect in the ability of mutant dorsal thalamic cells to respond to signals normally present in ventral telencephalon. Pax6(Sey)(/Sey) embryos also showed early alterations in the expression of regulatory genes in the region destined to become dorsal thalamus. Whereas in normal mice Nkx2.2 and Lim1/Lhx1 are expressed ventral to this region, in the mutants their expression domains are throughout it, suggesting that a primary action of Pax6 is to generate correct dorsoventral patterning in the diencephalon. Our results suggest that normal thalamocortical development requires the actions of Pax6 within the dorsal thalamus itself.
Alternative Splicing↗
The transcription factor, Pax6, is required for cell proliferation and differentiation in the developing cerebral cortex.
The cerebral cortex develops from the dorsal telencephalon, at the anterior end of the neural tube. Neurons are generated by cell division at the inner surface of the telencephalic wall (in the ventricular zone) and migrate towards its outer surface, where they complete their differentiation. Recent studies have suggested that the transcription factor Pax6 is important for regulation of cell proliferation, migration and differentiation at various sites in the CNS. This gene is widely expressed from neural plate stage in the developing CNS, including the embryonic cerebral cortex, where it is required for radial glial cell development and neuronal migration. We report new findings indicating that, in the absence of Pax6, proliferative rates in the early embryonic cortex are increased and the differentiation of many cortical cells is defective. A major question concerns the degree to which cortical defects in the absence of Pax6 are a direct consequence of losing the gene function from defective cells themselves, rather than being secondary to abnormalities in other cells. Cortical defects in the absence of Pax6 become much more pronounced later in cortical development, and we propose that many result from a compounding of abnormalities in proliferation and differentiation that first appear at the onset of corticogenesis.
Animals↗
Expression of an inter-alpha-trypsin inhibitor heavy chain-like protein in the pig endometrium during the oestrous cycle and early pregnancy.
In pigs, changes in an unidentified endometrial glycoprotein, pGP30, are temporally associated with rapid trophoblast elongation and initial placental attachment on day 12 of gestation. Identification of endometrial pGP30 was undertaken through protein purification, NH2-terminal amino acid sequencing and cDNA sequencing of products generated through reverse transcription-polymerase chain reaction. Sequencing of 35 amino acids from the NH2-terminal end of pGP30 revealed that the 30 kDa glycoprotein is a cleavage product from the C-terminal region of inter-alpha-trypsin inhibitor heavy chain 4 (I alpha IH4), previously known as inter-alpha-trypsin inhibitor heavy chain-like protein. I alpha IH4 is unique compared with the three other inter-alpha-trypsin inhibitor heavy chains as it does not contain a binding site for bikunin that has serine protease inhibitory activity and is sensitive to cleavage by kallikrein. Endometrial gene expression of I alpha IH4 was detected during the oestrous cycle (days 0-18) and early pregnancy (days 10-18). Gene expression of I alpha IH4 appeared to be enhanced during the midluteal phase (days 12 and 15) of the oestrous cycle and the period of trophoblast attachment (days 12-18). Expression of I alpha IH4 was not detected in day 12 conceptus tissue mRNA. Endometrial expression of I alpha IH4 in pigs may function as an acute phase protein for protection of the uterus from the inflammatory response induced by conceptus attachment to the uterine epithelium.
Amino Acid Sequence↗
Characterization and proteolytic activity of a cathepsin L-like polypeptide in endometrium and uterine flushings of cycling, pregnant and steroid-treated ovariectomized gilts.
Cathepsin L has been proposed to be involved with the endothelial-chorial type of placentation in the cat. Little information concerning the presence and secretion of cathepsin L is available for a species with noninvasive epitheliochorial placentation such as the pig. Cathepsin L activity in uterine flushings and endometrium from gilts during different days of the oestrous cycle and early pregnancy was analysed through specific substrate metabolism and Western blot analyses with antiserum against cat endometrial cathepsin L. This antiserum was utilized to determine the cellular localization of the enzyme within porcine endometrium. Cathepsin L activity within uterine flushings was elevated on Day 15 of the oestrous cycle and early pregnancy, with activity declining on Day 18. Cat cathepsin L antiserum cross-reacted with a group of 46, 40 and 38 kDa uterine proteins and detected a product within the surface and glandular epithelium of the endometrium. The appearance of the 40 kDa protein was first detected on Day 10 of the oestrous cycle with the 38 kDa proteins appearing on Day 15 and 18 of pregnancy. The 40 and 38 kDa uterine proteins appear to be steroid regulated as 12 days of progesterone administration is necessary to detect the proteins and cathepsin L activity.
Animals↗
Isolation and characterization of a 30-kDa endometrial glycoprotein synthesized during the estrous cycle and early pregnancy of the pig.
Endometrial polypeptide synthesis, which is regulated through ovarian steroid secretion and steroid production by the developing conceptus, not only provides the necessary secretory components vital to conceptus development but also presents the adhesive changes in the epithelial surface essential for conceptus attachment. In the present study, a 30-kDa, basic endometrial glycoprotein (pGP30) was isolated and characterized during the estrous cycle and early pregnancy of the pig. Uterine flushings and endometrial culture media were obtained from gilts on Days 0, 5, 10, 12, 15, and 18 of the estrous cycle and Days 10, 12, 15, and 18 of pregnancy. A polyclonal antibody was generated to pGP30 after isolation of medium from Day 15 pregnant endometrial cultures separated by gel filtration and PAGE. Western blot analysis indicated that the antiserum reacted with isoforms of pGP30 and cross-reacted with a 90-kDa component in serum that was not removed after cleavage of the oligosaccharide chains from the 90-kDa glycoprotein. Antiserum did not detect a 30-kDa band in media from cultures of kidney, fat, heart, muscle, liver, or serum; however, heart and muscle did contain bands of different molecular masses that cross-reacted with the antiserum. Multiple bands of higher molecular mass (35-40 kDa) were detected in the endometrial cultures from gilts on Days 0 through 10 of the estrous cycle. Treatment of ovariectomized gilts with estradiol-17 beta stimulated a similar response. During the mid- to late luteal phase of the estrous cycle (Days 12-18), the 30-kDa band as well as an additional 32-kDa band was present on Western blots. Administration of progesterone for 14 days stimulated the synthesis of both the 30- and 32-kDa products in ovariectomized gilts. However, only the pGP30 was detected on Days 12-18 of pregnancy. Immunocytochemical localization with antiserum to pGP30 indicated that the glycoprotein is present in the endometrial epithelium, with the surface epithelium demonstrating the strongest reaction product. Discrete changes in staining and cellular localization were observed during the early stages of the estrous cycle (Days 0-5) and the midluteal (Day 10) phase. A similar response was achieved with administration of steroids to ovariectomized gilts. Data indicate that discrete changes in epithelial synthesis of the endometrial glycoprotein occur at the time of conceptus trophoblastic elongation and placental attachment in the pig.
Animals↗
A functional test for maternally inherited cadherin in Xenopus shows its importance in cell adhesion at the blastula stage.
We report here on the consequences of reducing the expression of EP-cadherin at the earliest stages of Xenopus development. Injection of oligodeoxynucleotides antisense to maternal EP-cadherin mRNA into full-grown oocytes reduced the mRNA level in oocytes, and the protein level in blastulae. Adhesion between blastomeres was significantly reduced, as seen in whole embryos, and in assays of the ability of blastomeres to reaggregate in culture. This effect was especially conspicuous in the inner cells of the blastula and included the disruption of the blastocoel. The severity of the EP-cadherin mRNA depletion and of the disaggregation phenotype was dose dependent. This phenotype was rescued by the injection into EP-cadherin mRNA-depleted oocytes of the mRNA coding for a related cadherin, E-cadherin, that is normally expressed at the gastrula stage in the embryonic ectoderm.
Animals↗
Sequence and magnitude of ventricular volume changes in painful and painless myocardial ischemia.
Stimulation of left ventricular stretch receptors has been proposed as a possible mechanism for the occurrence of cardiac pain. Changes in left ventricular volume were continuously assessed in 12 patients during 11 spontaneous (two painful) and 12 ergometrine-induced (nine painful) ischemic attacks with a precordial scintillation probe and blood pool labeling with technetium-99m. In all ischemic episodes, spontaneous or induced, painful or painless, severe dilatation of the left ventricle was consistently observed. These changes always preceded the onset of ST segment shifts and occurred long before pain, when present. The maximum increase in end-diastolic volume was slightly greater in painful than in painless episodes, 38 +/- 8.0% versus 28 +/- 12.4%, but no significant difference was observed in the rate of volume change or in the maximum increase of end-systolic volume (133 +/- 50% and 110 +/- 27.3%), stroke volume (-28 +/- 15% and -25 +/- 12.4%), or ejection fraction (-32 +/- 8.7% and -26 +/- 6.0%). Although the maximum end-diastolic volume achieved is greater in painful episodes, this effect cannot be separated from that of duration, and, furthermore, there was no significant difference in end-diastolic volume at the moment chest pain began. Thus, in patients with angina at rest, transient asymptomatic ST segment shifts are consistently associated with large changes in left ventricular volume, similar to those observed during painful episodes. The rate and extent of acute left ventricular dilatation do not appear to be factors directly causing anginal pain.
Angina Pectoris↗
The fractional distribution of the cardiac output in man using microspheres labelled with technetium 99m.
To investigate the distribution of organ blood flow in patients we have developed a method of quantitating the whole-body fractional distribution of 99Tcm-labelled microspheres. The microspheres were injected into the left ventricle in nine patients with normal cardiac indices (greater than 3 1/min/m2; Group A) and 11 patients with low cardiac indices (less than 2.51 l/min/m2; Group B). The fractional organ content of the total injected dose was estimated following correction for geometry and transmission using a gamma camera. Cerebral blood flow was 579 +/- 163 ml/min (mean +/- SD) in Group A and 593 +/- 158 ml/min in Group B (p not significant (NS)). Myocardial flow in Group A was 266 +/- 82 ml/min and in Group B was 237 +/- 57 ml/min (p, NS). Total renal blood flow was 749 +/- 161 ml/min in Group A and 614 +/- 181 ml/min in Group B (p less than 0.01). There was a negative correlation between cardiac index and the percentage of the cardiac output distributed to brain (r = -0.70, p less than 0.01), heart (r = -0.67, p less than 0.01) and kidneys (r = -0.47), p less than 0.05). Low output cardiac failure is, therefore, associated with relative preservation of cerebral and myocardial blood flow and, to a lesser extent, of renal flow. A similar technique using dual labelling would allow an accurate estimation in individual patients, of the change in organ blood flow associated with transient alterations in cardiac output states.
Adult↗
Sialyltransferase of the 13762 rat mammary ascites tumor cells.
The MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma differ in morphology, agglutinability with concanavalin A, and xenotransplantability. Both cell lines contain a major mucin-type glycoprotein, but the MAT-C1 (xenotransplantable) subline contains a 3-fold-greater content of sialic acid on the glycoprotein than does the MAT-B1 (nonxenotransplantable) subline. The present work indicates that whole cells of both lines incorporate radioactivity from labeled CMP-sialic acid into a component which comigrates with the major glycoprotein by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and that label incorporated by MAT-B1 cells is released by alkaline-borohydride treatment. Sialyltransferase can be purified from 250- to 400-fold by chromatography of a Triton X-100 extract of microsomes on CDP-agarose. The purified fraction of both cell lines has a Km for CMP-sialic acid of 0.40 +/- 0.10 mM with asialofetuin as the acceptor, and gives 35 to 40% of the activity with the acceptor asialotransferrin as with asialofetuin. When assayed with a variety of acceptors, the MAT-C1 extract showed higher sialyltransferase activity at a pH below 6.5 than did the MAT-B1 extract. Analysis of the products following incubation with lactose yields only 3'-sialyllactose for both cell lines. The results indicate that the differences in MAT-B1 and MAT-C1 sialyltransferase when assayed with glycoprotein acceptors are not large enough to account for the differences in sialic acid content of the two cell lines.
Animals↗
An investigation in patients with previous myocardial infarction who present with chest pain.
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Adult↗
Where National Board Examinations pass and fail in evaluating knowledge of surgical clerks.
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Certification↗
The relationship of interest in surgery to learning styles, grades and residency choice.
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Achievement↗
The undergraduate surgical clerkship. A cutting edge which separates the clinical from the nonclinical medical specialists.
The role of the surgical clerkship in the curriculum has often been debated. This study highlights its function in the students' decision making process. Junior students (N = 164) were measured on interest in surgery before and after the clerkship and divided into groups that changed significantly in a positive or negative direction, or that remained essentially unchanged. Background factors, learning styles of the students, and their evaluations of the clerkship showed that students who changed negatively (12%), compared with those who changed positively (44%), did not learn as well from clinical experiences and evaluated certain aspects of the clerkship, related to time and clinical demands, less positively. The unchanged group resembled the positive change students. Performance in the clerkship did not differ significantly among the three groups. The group that changed positively had none interested in surgery as a career before the clerkship, 15% who chose surgery afterward, and who later entered a surgical residency. The negative change group entered with high, perhaps unrealistic, levels of interest, left with none selecting surgery, and a third entering nonclinical type residencies. The surgical clerkship appears to have a unique role to help students make decisions about future careers based on their underlying personalities and learning styles.
Adult↗
The effects of nifedipine on acute experimental myocardial ischemia and infarction in dogs.
We studied 25 anesthetized and thoracotamized dogs before and during 5 hours of acute regional myocardial ischemia. Krypton-81m (81mKr) was infused constantly into the aortic sinuses. The myocardial equilibrium of this tracer was used to image and assess the distribution of regional myocardial perfusion using a gamma camera and digital computer. The epicardial ECG was recorded, S-T segment elevation and the loss of R and appearance of Q waves were measured, and the plasma activity of creatine kinase (CK) was determined in aortic and coronary venous blood throughout these experiments. Ten dogs underwent left anterior descending coronary artery (LAD) narrowing for 5 hours and received no drugs. Five dogs received nifedipine 13 microgram/kg, and another five received 1.0 microgram/kg intravenously 30 minutes after LAD narrowing. Those dogs receiving nifedipine, 13 microgram/kg, showed a 30% fall in aortic pressure, a 12% rise in heart rate, and an extension of regional ischemia. The ECG showed an extension of infarct size, and CK release into the coronary vein appeared earlier than in the controls. Dogs receiving nifedipine, 1 microgram/kg, showed a 12% fall in blood pressure, no rise in heart rate, an improvement in regional perfusion, and ECG signs that suggested limitation of infarct size. There also was delayed release of coronary venous CK. The effects of nifedipine on the natural history of regional myocardial perfusion, the electrocardiogram, and enzyme release from the heart were dose related and cannot be generalized. These observations warrant further clinical investigation to improve the use of this agent in man.
Animals↗