Biomedical subjects

M M Blanchard

Publications and source records attributed to M M Blanchard.

10 recordsLinked to original sources

PubMed1994

High-throughput rapid yeast DNA extraction. Application to yeast artificial chromosomes as polymerase chain reaction templates.

An automation-assisted method enables DNA extraction of over a thousand yeast colonies in a day by one person. Yeast DNA is extracted essentially quantitatively in three steps: (a) cells are converted into spheroplasts with yeast lytic enzyme, (b) cells are lysed with proteinase K, and (c) DNA is collected by ethanol precipitation. The DNA stored at 4 degrees C is active in polymerase chain reaction experiments for more than a year.

Chromosomes, Artificial, Yeast↗

PubMed1993

Screening yeast artificial chromosome libraries with robot-aided automation.

Screening of collections of yeast artificial chromosomes utilizing the polymerase chain reaction (PCR) requires large numbers of reactions in parallel. Four steps were implemented to reduce the labor involved: (a) The number of initial samples for DNA extractions was decreased by compressing libraries up to 12-fold. (b) DNA extraction from yeast clones was robot assisted. (c) A BIOMEK 1000 station was adapted to pipette samples for PCR assays. (d) Sample preparation was integrated with a temperature cycler constructed to carry out up to 576 reactions in six 8 x 12-well trays. The implementation of these steps increases the number of reactions per person per day by an order of magnitude. In tests with X-chromosome-specific probes, the robot-aided screening recovered all of the clones detected by slower manual methods.

Chromosomes, Artificial, Yeast↗

PubMed1993

PCR buffer optimization with uniform temperature regimen to facilitate automation.

To facilitate PCR(1,2) reactions in large numbers with uniform conditions, the annealing temperature was fixed and the stringency of the reactions was adjusted by optimizing the ion conditions of the reaction. The buffer system is based primarily on Tris (T), ammonium (N), and potassium (K) to adapt assay conditions to different primer pairs. The TNK buffers have permitted successful screening of a 60,000-clone yeast artificial chromosome (YAC) library with more than 200 primer pairs.

Automation↗

PubMed1991

GRF treatment of late pregnant ewes alters maternal and fetal somatotropic axis activity.

To examine the effects of anabolic agents given during late gestation on the maternal and fetal somatotropic axes, we injected pregnant ewes twice daily with 0.15 mg somatocrinin (GRF)-(1-29) for 10 days beginning on day 130 of gestation. Maternal and fetal endocrine changes were compared with control animals using both in vivo and in vitro approaches. Treatment with GRF increased maternal plasma levels of growth hormone (GH) and insulin-like growth factor I (IGF-I;P less than 0.05) but not IGF-II. Under in vitro test conditions, maternal pituitary cells showed a greater maximal response (P less than 0.001) to GRF. In the fetuses of treated ewes, cord plasma GH levels were not significantly increased compared with controls. These animals had similar IGF-I but higher IGF-II (P less than 0.05) plasma levels. The maximal response of fetal pituitary cells to GRF was increased (P less than 0.001). GRF treatment had no influence on maternal and fetal pituitary cell responses to somatostatin under either basal or GRF-stimulated conditions. In addition, these treatments did not affect plasma levels of placental lactogen, glucose, or free fatty acids in the maternal and fetal sheep. These data are compatible with the hypothesis that treatment of pregnant ewes in the last days of gestation with GRF could support accelerated fetal growth.

Animals↗

PubMed1989

Effects of a chronic GRF treatment on lambs having low or normal birth weight.

The effects of a long term treatment with human GRF(1-29)NH2 on plasma growth hormone (GH), somatomedin C (Sm-C), histomorphometric parameters of bone growth and body composition were investigated in normal and low birthweight male lambs. The animals were divided into two groups according to their birthweight: 24 normal birthweight (NBW) lambs weighing more than 4 kg and 22 low birthweight (LBW) lambs weighing less than 2.5 kg at birth. Half of the animals in each group received two daily subcutaneous injections (8 micrograms/kg body weight) of hGRF(1-29) NH2 (GRF) from birth to slaughter at 45 or 90 days of age. The other animals received the solvent only. At the beginning and at the end of the treatment, plasma GH and serum Sm-C concentrations were measured in all groups. After slaughter, a histomorphometric study was performed on undecalcified sections of metacarpal growth plates, and the remaining of the carcass was pulverized to study the chemical body composition. GRF induced GH release in both GRF-treated groups. However, plasma GH reached higher (P less than .001) concentrations and the GRF-induced GH peak lasted longer in LBW than in NBW lambs. At day 45, the GRF treatment increased (P less than .05) serum Sm-C concentrations in LBW. Most of histomorphometric parameters reflecting the metacarpal growth in length, were not statistically modified under GRF treatment. However, the size of degenerative cells was smaller (P less than .05) in LBW treated lambs as compared to controls. Consequently, the cell production in the growth plate was increased (P less than .05) under GRF treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Characterization of the insulin-like growth factor (IGF) receptor in K562 erythroleukemia cells; evidence for a biological function for the type II IGF receptor.

Erythroleukemia cells (K562) were found to bind insulin-like growth factor-II (IGF-II) about 10 times as much as IGF-I, insulin and human growth hormone. The specific binding of IGF-II increased as a function of cell number in a range of 0.5-6 X 10(6) cell/ml. Kinetic studies revealed that binding was time and temperature dependent and showed a broad pH optimum. Specificity studies showed no inhibition of 125I-IGF-II binding by insulin or unrelated peptide hormones. The half-maximal inhibition of 125I-IGF-II binding to K562 cells was achieved at 87 and 28 ng/ml of IGF-I and IGF-II respectively. However, the addition of 7.5 and 1.7 ng/ml of unlabeled IGF-I and IGF-II respectively increased the binding of 125I-IGF-II by 55%. This 'hook' effect was greatly reduced when K562 cell membranes were used. Scatchard analysis of IGF-II binding showed a comparable equilibrium constant with either intact cells (Ka = 8.9 X 10(8) M-1) or microsomal membranes (Ka = 5.4 X 10(8) M-1). Cross-linking studies indicated that both 125I-IGF-II and 125I-IGF-I bound to an entity of 215 kDa which increased to 260 kDa under reducing conditions. Both IGF-I and II stimulated 3H-thymidine incorporation into K562 cells whereas insulin was without effect. These data show that both IGF-I and II bind predominantly to a type II-IGF receptor in K562 cells. Since both peptides stimulate 3H-thymidine incorporation in these cells it is possible that the type II-IGF receptor is mediating an anabolic biological response in these cells.

Cell Line↗

PubMed1988

In vitro regulation of growth hormone (GH) release from ovine pituitary cells during fetal and neonatal development: effects of GH-releasing factor, somatostatin, and insulin-like growth factor I.

Using a monolayer approach, we have examined the acute (3 h) effects of GRF, somatostatin (SRIF), and insulin-like growth factor I (IGF-I) on GH release from pituitary cells of male and female 70-, 100-, and 130-day-old fetuses and newborn lambs and of prepubertal male lambs. GRF stimulated basal GH release in a dose-dependent (10(-12)-10(-8) M) manner at each stage in development. There was no linear relationship between maximal response and increasing age of the donor animals. The ED50 values for GRF were similar in all groups, except in the pituitaries from male and female 130-day-old fetuses, where the ED50 values were significantly higher. SRIF elicited a dose-related (10(-10)-10(-6) M) inhibition of basal GH secretion at each stage of fetal life and in the prepubertal period; although the response was lower in the youngest fetal pituitaries, there was no significant change in maximal response during the fetal or prepubertal period. No effect of SRIF on basal GH secretion was observed in newborn lambs. However, SRIF (10(-7) M) was able to block GRF (10(-8) M)-stimulated GH release in 100- and 130-day-old fetal and prepubertal as well as newborn lamb pituitary cells. Plasma IGF-I concentrations increased from 15.0 +/- 0.7 (mean +/- SE) and 13.8 +/- 0.9 ng/ml for male and female animals, respectively, at 70 days gestation to 55.8 +/- 3.2 and 51.8 +/- 11.1 ng/ml at the time of birth. The increase was much more pronounced in prepubertal lambs, especially in male animals, where IGF-I levels reached 300.8 +/- 37.7 ng/ml. IGF-I (100 ng/ml) had no effect on basal GH release in 70- and 100-day-old fetal, newborn, and prepubertal lamb pituitary cultures, but significantly inhibited basal GH secretion from 130-day-old fetal cells. This dose of IGF-I had no effect on GRF (10(-9) M)-stimulated GH release at 70 days gestation. It significantly inhibited this effect at 100 days and in prepubertal lamb cells. In 130-day-old fetal and newborn lamb pituitary cultures, IGF-I completely blocked the GH response to GRF.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗

PubMed1987

Effects of hypothalamic hormones (GRF, TRH, somatostatin) and insulin-like growth factor I on growth hormone secretion from prepubertal male lamb pituitary cultures.

We have examined the regulation of GH secretion from monolayer cultures of prepubertal male lamb anterior pituitary cells. Growth hormone-releasing factor (GRF 1-44) stimulated GH release in a dose-related manner: the maximal effective dose was 10(-10) M, which caused a 500% increase in basal GH secretion, while the half-maximal effect was reached with a dose of 2.5 x 10(-11) M (ED50). Thyrotropin-releasing hormone (TRH) also elicited a dose-dependent stimulation of GH secretion, although it was approximately 1000 times less potent than GRF. GRF and TRH did not have additive or synergistic effects on GH secretion. Somatostatin (SRIF) at a concentration of 10(-7) M maximally inhibited basal GH release to 40% of that of the control; the ED50 was 2.0 x 10(-9) M. Moreover, 10(-7) M SRIF blocked the stimulation of GH secretion induced by 10(-8) M GRF. However, when the cells were incubated with these two peptides at an identical concentration (10(-8) M), GH secretion was stimulated significantly above control values. When added at the same concentration (10(-7) M, TRH ans SRIF nullified their respective effects. A dose of 100 ng/ml of synthetic IGF-I was without effect on basal GH release, but significantly decreased 10(-9) M GRF-induced stimulation of GH secretion. these data indicate that in prepubertal male lambs: the stimulatory effect of GRF is predominant over the inhibitory effect of SRIF, somatostatin inhibits TRH stimulation of GH secretion in vitro, and IGF-I may control GH secretion by modulating GRF effects at the pituitary level.

Animals↗

PubMed1987

Insulin-like growth factor (IGF) binding in hypophysectomized rat liver microsomes: alteration by a soluble binding moiety.

In this study, we compared the binding of IGF-I and IGF-II to liver microsomes of normal and hypophysectomized (Hypox) rats. The binding of [125I]-IGF-II, measured by centrifugation of membrane-bound ligand, was lower in hypox than in normal rats (15 +/- 2 vs 26 +/- 1%, p less than 0.001) but binding was increased (46 +/- 1.5 vs 31 +/- 1%, p less than 0.001) when bound and free hormones were separated using polyethyleneglycol (PEG) precipitation. This was due to the presence of soluble binding activity which dissociated from membranes to compete for IGF binding. When soluble binding activity was first removed from microsomal membranes by a washing procedure no difference was found in [125I]-IGF-II binding to microsomes of Hypox and normal animals (33 +/- 1 s 32 +/- 1%). However, in the microsomal washing supernatant from Hypox (containing soluble binding activity) IGF-II binding was much higher than in that from normals (17 +/- 2 vs 6 +/- 0.5%, p less than 0.001). The binding of [125I]-IGF-I was lower than that of [125I]-IGF-II but was comparably changed. By contrast, [125I]-insulin binding was similar in Hypox and normal rats and was not influenced by PEG precipitation or prewashing of the membranes. Inhibition dose-response curves showed a paradoxical increase in [125I]-IGF-II binding to unwashed microsomes of Hypox rats in the range of 0.5-5 ng/ml cold IGF-II. In normal animals [125I]-IGF-II binding to microscomes was progressively inhibited by IGF-II in a range of 0.5-500 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1983

Analysis of the covariance structure of digital ridge counts in the offspring of monozygotic twins.

Improved methods for analysis of covariance structures now permit the rigorous testing of multivariate genetic hypotheses. Using Jöreskog's Lisrel IV computer program we have conducted a confirmatory factor analysis of dermal ridge counts on the individual fingers of 509 offspring of 107 monozygotic twin pairs. Prior to the initiation of the model-fitting procedure, the sex-adjusted ridge counts for the offspring of male and female twins were partitioned by a multivariate nested analysis of variance yielding five 10 X 10 variance-covariance matrices containing a total of 275 distinctly observed parameters with which to estimate latent sources of genetic and environmental variation and test hypotheses about the factor structure of those latent causes. To provide an adequate explanation for the observed patterns of covariation, it was necessary to include additive genetic, random environmental, epistatic and maternal effects in the model and a structure for the additive genetic effects which included a general factor and allowed for hand asymmetry and finger symmetry. The results illustrate the value of these methods for the analysis of interrelated metric traits.

Analysis of Variance↗