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Biomedical subjects

R D Bremel

Publications and source records attributed to R D Bremel.

At least 37 records · Page 2Linked to original sources
PubMed1988

Prolactin, estradiol, and progesterone changes in paretic and nonparetic cows during the periparturient period.

We studied the relationship of serum prolactin, estradiol-17 beta, and progesterone concentrations to plasma calcium, phosphorus, and free hydroxyproline concentrations, as well as to dry matter intake, in 14 aged dairy cows (mean of 4.5 parities), 7 of which became paretic, from 28 days before to 4 days after calving. Plasma calcium and phosphorus concentrations and dry matter intake decreased more at parturition in paretic cows than in nonparetic cows. Prolactin concentrations were not different between paretic and nonparetic cows, but were variable. Concentrations of estradiol were higher in paretic cows from 15 to 5 days before parturition, whereas hydroxyproline concentration was lower in paretic cows on days 10 through 3 before parturition. Progesterone concentration was lower in paretic cows and decreased earlier at parturition, compared with that in nonparetic cows. The findings suggested that high estradiol concentrations in late pregnancy inhibit bone resorption and predispose aged cows to parturient paresis. The earlier decrease in progesterone concentration at parturition and lower concentrations throughout late pregnancy might have contributed to the greater inappetence in paretic cows at parturition. The importance of prolactin in the pathogenesis of parturient paresis is not clear.

Animals↗
PubMed1987

The concentration of bovine placental lactogen and the incidence of different forms in fetal cotyledons and in fetal serum.

The concentration of bovine placental lactogen (bPL) was determined in fetal placentomes, allantoic fluid, amniotic fluid, maternal and fetal plasma throughout pregnancy. In addition, chromatofocusing chromatography was used to separate the different forms of bPL found both in fetal serum and in placental homogenates in order to determine whether the different forms that have been reported to exist in the cotyledon are also found in the fetal circulation. Reproductive tracts were collected from cows between 109 and 247 days of pregnancy. The concentration of bPL in the fetal cotyledonary tissue was measured by both radioreceptor assay and radioimmunoassay, both assays showed that the concentration of bPL in the fetal portion of the placentomes remained constant throughout the period of pregnancy tested. The mass of the placenta increased approximately 10-fold during the period of study but the concentration of bPL in the maternal plasma was low (0.9 +/- 0.1 ng/ml) at all stages of pregnancy tested. The mean concentration of bPL (Mean +/- S.E.M.) in amniotic and allantoic fluid was 0.4 +/- 0.1 and 1.2 +/- 0.2 ng/ml respectively. Fetal blood contained the highest concentrations of bPL, from 11.6 to 18.4 ng/ml, and the concentration tended to decrease with advancing gestation (slope = 0.07, P = 0.001). Several forms of bPL were found in the fetal circulation; however, a higher percentage of forms with more acidic isoelectric points were found in the fetal serum than in placental homogenates. These results suggest that either some forms of bPL are more stable or that the hormone isolated from placental tissue is not representative of the final secreted product.

Allantois↗
PubMed1986

Isolation and characterization of multiple forms of bovine placental lactogen from secretory granules of the fetal cotyledon.

Previous work has shown that, unlike other species, placental lactogen (PL) in the bovine (bPL) has a mol wt of approximately 32,000 and exists in several different forms with different isoelectric points. This study was carried out to develop a more rapid purification scheme, whereby the yield of bPL obtained was increased while at the same time the possibility of artifacts from a prolonged purification protocol was decreased. A procedure was developed in which a fraction enriched in bPL-containing granules was obtained after gentle disruption of the binucleate cells of the fetal cotyledon. The fetal portion of the placentomes from midpregnant cows was minced with scissors and vigorously stirred in order to remove and disrupt binucleate cells within the fetal villi. The supernatant from this step was fractionated by differential centrifugation followed by a four-step discontinuous Percoll gradient of 1.03-1.08 g/ml. A granule-enriched fraction was isolated from the 1.04 g/ml zone from which membrane-enclosed protein was released by freezing and thawing. Membranes and insoluble proteins were sedimented by high speed centrifugation to yield an extract which contained approximately 20% of the hormone initially in the tissue. Two subsequent chromatographic steps, gel filtration on Sephadex G-75 and high performance reversed phase chromatography with a C-4 column, resulted in a preparation of greater than 98% homogeneity. Two-dimensional gel electrophoresis of purified bPL revealed at least nine protein spots in the 31,000-33,000 mol wt range with isoelectric points ranging from 4.85-6.3. All forms exhibited parallel dilution curves in a RIA for bPL. It would appear, therefore, that multiple forms of bPL exist and that they are not artifacts of the prolonged purification protocol previously used.

Animals↗
PubMed1986

Immunohistochemical localization of placental lactogen in binucleate cells of bovine placentomes.

Bovine placental lactogen (bPL) has been isolated from bovine trophoblast and characterized as a 32 K mol wt protein which exists in three different forms which differ in their isoelectric point values and their amino acid compositions. Two of the three forms have been shown to have both bovine GH (bGH)- and bovine PRL (bPRL)-like activities equal on a molar basis to bGH and BPRL in radioreceptor assays. It has been postulated that, in sheep, PL is delivered to the maternal circulation by the migration of fetal binucleate cells from the trophoblast across the fetal-maternal boundary into the uterine epithelium. To determine whether an analogous situation exists in the cow, antibodies to bPL were used to localize bPL in bovine placentomes and to measure its concentration in fetal and maternal sera. For cytology, bPL was localized on sections of placentomes from midgestation and term bovine placentas using an indirect immunoperoxidase technique. Stained binucleate cells were demonstrated throughout the trophoblast, often in close association with the microvillous boundary which separates the trophoblast from the maternal epithelium. In cross-sections of fetal villi, binucleate cells with cytoplasmic processes extending into and through the uterine epithelium were immunostained as well as cells within the plane of the uterine epithelium in close approximation or apposition to the maternal basement membrane. RIA demonstrated bPL to be present in maternal sera in concentrations of 1-2 ng/ml and in fetal sera at 5-12 ng/ml. These data are consistent with the hypothesis that binucleate cell migration accomplishes the delivery of bPL to the maternal circulation.

Animals↗
PubMed1986

Lactogenic effect of bovine placental lactogen on pregnant rabbit but not pregnant heifer mammary gland explants.

The biological function of bovine placental lactogen is unknown. However, bovine placental lactogen is known to bind prolactin receptors in rabbit mammary gland. This hormone was therefore cultured with rabbit mammary gland explants to confirm that it is lactogenic in this species. Mammary explants from pregnant heifers were also cultured with bovine placental lactogen to determine if the same preparation of hormone possessed similar lactogenic potential in homologous species. Bovine placental lactogen was tested over a range of concentrations from 10 to 500 ng/ml and was about 50 to 80% as potent as bovine prolactin when cultured with mammary explants from 19-d pregnant virgin rabbits. Lactogenic response was assessed by both the incorporation of [14C]acetate into lipid and by the synthesis of casein. Bovine placental lactogen displayed negligible lactogenic activity when cultured with mammary explants from 6 to 7-mo pregnant heifers. Lactogenic response was assessed using the same criteria as used with the rabbit mammary explants; in addition, accumulation of alpha-lactalbumin in the explants was also measured. Although bovine placental lactogen was lactogenic in the rabbit, the same hormone preparation was apparently not lactogenic in cattle. It is therefore vital to test for the biological activity of a hormone in a homologous system, because inappropriate conclusions may be drawn from the response obtained in a heterologous system.

Animals↗
PubMed1984

Oxytocin-stimulated myosin phosphorylation in mammary myoepithelial cells: roles of calcium ions and cyclic nucleotides.

Oxytocin (10 nM) stimulated the phosphorylation of the 20,000 mol wt myosin light chain in rat mammary myoepithelial cells from a basal level of 0.17 to 0.85 mol phosphate/mol light chain within 30 sec. Of the smooth muscle stimulants tested, oxytocin appears to be the only normal physiological stimulus for myosin phosphorylation in these cells. The roles of cAMP, cGMP, and calcium ions were investigated in the mode of action of oxytocin and the regulation of myosin phosphorylation. Although oxytocin had no effect on cGMP metabolism, there was an increase in the cAMP content of the treated myoepithelial cells. Further investigation suggested that the increase in cAMP levels in response to oxytocin was not directly involved in the regulation of myosin phosphorylation. Various agents known to interfere with calcium ion transport were used to study the role of calcium ions in the action of oxytocin and the regulation of myosin phosphorylation. The results indicate that the duration of the cellular response to oxytocin depends on an influx of extracellular calcium through calcium-specific channels in the plasma membrane.

Animals↗
PubMed1983

Oxytocin binding by myoepithelial cell membranes from involuted mammary tissue.

Oxytocin binding activity of myoepithelial cell membranes from mammary tissue was measured under a variety of different experimental conditions. Mammary tissue from non-lactating rats bound oxytocin with a Kd of 9.2 +/- 1.6 nM (+/- S.E.) and indicates that receptors are retained by the myoepithelial cells in a non-lactating state. Ovariectomy of non-lactating rats did not depress the binding activity of the membranes. Administration of the estrogenic compounds estradiol-17 beta and diethylstibestrol at doses which affect uterine weight and are known to increase uterine oxytocin binding did not influence the binding activity of the myoepithelial cells. This indicates that the oxytocin receptors of the mammary gland are not under the same endocrine control as the uterine receptors.

Animals↗
PubMed1983

Determination of myosin light chain phosphorylation using astronomical image analysis programs on autoradiographs of electrophoretic gels.

Computer image analysis programs developed by astronomers for celestial photometry were used for the analysis of autoradiographs of electrophoretic slab gels. Oxytocin-stimulated myosin light chain phosphorylation in mammary myoepithelial cells was studied by incubating cells with [32P]orthophosphate followed by oxytocin addition, polyacrylamide gel electrophoresis of cellular protein, and autoradiography of electrophoretic slab gels. After scanning and digitization of autoradiographs, [32P]phosphate incorporation into the myosin light chain was measured by the determination of radiation flux recorded on the X-ray film. Within seconds after oxytocin addition, the myosin light chain was fully phosphorylated. The large library of astronomical computer programs has applications for the quantitative analysis of both one- and two-dimensional electrophoretic gels.

Autoradiography↗
PubMed1983

Purification and characterization of bovine placental lactogen.

Bovine placental lactogen (bPL) was purified 4,200-fold from cotyledon homogenates by 1) salt precipitation and 2) ultrafiltration, followed by 3) gel filtration, 4) anion exchange, 5) hydroxylapatite, 6) chromatofocusing, and 7) final gel filtration chromatography. Purification was monitored by radioreceptor assay (RRA) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. During chromatofocusing, three distinct peaks of bPL were eluted: a small peak at pH 5.34 (bPL-1), a larger peak at pH 5.09 (bPL-2), and the largest peak at pH 4.84 (bPL-3). These three were further purified separately by the final gel filtration. The purified bPLs were compared with bovine (b) PRL (0.81 IU/mg) and bGH (30 IU/mg) by RRA. The bPRL-like activities were 67.1, 750, and 1,090 micrograms/mg, and the bGH-like activities were 74.4, 787, and 715 micrograms/mg for bPL-1, bPL-2, and bPL-3, respectively. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weights of all bPLs were the same, 31,100. In gel filtration under nondenaturing conditions, all bPLs had a 40,000 mol wt, with a Stokes radius of 2.70 nm. The isoelectric point (pI) of bPL-1 was higher than 5.85, the pI of bPL-2 was 5.52, and that of bPL-3 was 5.39. The amino acid compositions of bPL-2 and bPL-3 were virtually identical, except that bPL-3 had a few more acidic amino acids. Both bPL-2 and bPL-3 had an estimated 277 amino acid residues. Thus, bPL appeared to have both PRL- and GH-like activities equivalent to bPRL and bGH in the RRA on a mass basis, to be 50% larger than bPRL, bGH, and PLs in other species, and to occur in at least three different forms differing in their pI values and in their acidic amino acid compositions.

Amino Acids↗
PubMed1982

A 32,000-molecular weight protein from bovine placenta with placental lactogen-like activity in radioreceptor assays.

Considerable discrepancies exist in the literature concerning the size and activity of bovine placental lactogen. Our bovine placental lactogen purification preparations were 20% as active as bovine PRL (bPRL) on a per weight basis when compared to bPRL in a lactogenic radioreceptor assay. To identify the active component in these preparations, the proteins were radioiodinated and bound to membrane receptors in the presence and absence of competing hormones, bPRL, and bovine GH (bGH). After centrifugation, membrane pellets were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gels were autoradiographed. Only one radioiodinated protein band was present. This protein was displaced in the presence of competing hormone and comigrated which a major component of the purification preparations. In radioreceptor assays the active component was as active as bPRL and bGH. From the migration of protein standards included with the radioiodinated purification preparations in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we estimate that the protein is 32,000 mol wt--about 10,000 mol wt larger than placental lactogens isolated in other species. The possibility that the active molecule was a precursor protein was investigated by examining proteins secreted by bovine placenta tissue cultures. The binding activity in these secretions, as well as in the purification preparations, eluted between ovalbumin (43,000 mol wt) and bPRL (22,000 mol wt) under nondenaturing conditions using high performance gel filtration chromatography. Analysis of this secreted protein, also by binding to membrane receptors, showed that the protein had the same molecular weight as that isolated from the purification preparations and was specifically displaced by the same hormones.

Animals↗
PubMed1982

Phosphorylation of myosin in mammary myoepithelial cells in response to oxytocin.

The response of mammary myoepithelial cells to oxytocin was studied by monitoring the level of phosphorylation of the 20,000 mol wt light chain of myosin. Myoepithelial cells were obtained by collagenase dispersion of involuted rat mammary tissue. The cells were equilibrated with [32P]orthophosphate, and then stimulated with oxytocin. Phosphorylated proteins were separated by polyacrylamide gel electrophoresis, and incorporation of 32P into the proteins was detected by autoradiography. Nanomolar concentrations of oxytocin caused a 3-fold increase in the level of phosphorylation of the myosin light chain within 0.5 min. When the cells were incubated with oxytocin in a calcium-free medium, there was only a transient phosphorylation of myosin. However, readdition of calcium to these cells resulted in phosphorylation of the myosin light chain. The results suggest that calcium is involved in the intracellular events following stimulation of the cells with oxytocin.

Animals↗
PubMed1980

Membrane filter-deoxyribonucleic acid method of somatic cell counting: collaborative study.

In the membrane filter-deoxyribonucleic acid test, somatic cells are trapped on a special filter. A chemical determination of the DNA of the trapped material gives an estimate of the number of cells. The MF-DNA was evaluated and compared with the direct microscopic somatic cell counts (DMSCCs) in 10 laboratories. Cell levels in the 10 bulk milk samples analyzed ranged from 380,000 to 1,700,000/mL. Regression analysis gave a relation of 0.181 absorbance at 490 nm, when 2.5 mL of milk was analyzed and 5.0 mL reagent was used for color development. The 95% confidence limits on the slope were 0.158-0.204. Analysis of variance showed that both procedures had significant (P less than 0.001) systematic errors, but that only the DMSCC method showed a significant laboratory X sample interaction (P less than 0.001). The repeatability standard deviation of the MD-DNA procedure was 117,000 cells/mL (14%), and the reproducibility standard deviation was 198,000/mL (23%). This compared favorably with the DMSCC method which has a repeatability of 162,000/mL (19%) and a reproducibility of 187,000/mL (34%). The error of the MF-DNA was constant at all cell counts, whereas there was a tendency for the error to increase in the DMSCC method at high cell counts. This method has been adopted as official first action.

Animals↗
PubMed1978

Actomyosin from mammary myoepithelial cells and phosphorylation by myosin light chain kinase.

The oxytocin-sensitive myoepithelial cells of the mammary gland form a system with characteristics of a potentially useful model for studying the mechanism of action of oxytocin and coupling phenomena of excitation-contraction. Our objectives were to develop a method for isolating mammary actomyosin, to determine the amount of actomyosin in the glands of lactating and nonlactating animals, and to investigate control of contractile protein interaction. Actomyosin in mammary glands represented a substantial portion of the soluble protein in the gland ranging from 9% of the total in lactating to 17% in weaned rats. The isolated actomyosin had a molecular composition like that of actomyosin of smooth muscle and the isolated actomyosin contained a light chain kinase that phosphorylated the 20,000 dalton light chain of myosin (L20). The kinase isolated as a component of actomyosin preparations did not show calcium control, but it did when isolated from mammary cytosol. Strips of involuted mammary tissue from rats developed tension when oxytocin was added to the bathing medium; thus, the myoepithelial cells appeared to retain their sensitivity to oxytocin even in nonlactating animals and may be a useful model for studying the action of oxytocin. We suggest that one of the final steps in the milk-ejection reflex is phosphorylation of myosin causing a contraction of the myoepithelial cells of the mammary gland.

Actomyosin↗
PubMed1975

Preparation and properties of vertebrate smooth-muscle myofibrils and actomyosin.

A new technique for obtaining a myofibril-like preparation from vertebrate smooth muscle has been developed. An actomyosin can be readily extracted from these myofibrils at low ionic strength and in yields 20 times as high as previously reported. The protein composition of all preparations has been monitored using dodecylsulfate-gel electrophoresis. By this method smooth muscle actomyosin showed primarily only the major proteins, myosin, actin and tropomyosin, while the myofibrils contained, additionally, three new proteins not previously described with polypeptide chain weights of 60000, 110000 and 130000. The ATPase activities of both the myofibrils and actomyosin preparations are considerably higher than previously described for vertebrate smooth muscle. They are sensitive to micromolar Ca2+ ion concentrations to the same degree as comparable skeletal and cardiac muscle preparations, even though troponin-like proteins could not be identified in these smooth muscle preparations. From the latter observation and the presence of Ca2+-sensitivity in tropomyosin-free actomyosin it is suggested that this calcium sensitivity is, as in some invertebrate muscles, a property of the myosin molecule.

Actomyosin↗
PubMed1975

Calcium binding to rabbit skeletal myosin under physiological conditions.

At a free Mg2+ concentration of 1.0 mM, myosin binds one Ca2+ per molecule when the Ca2+ concentration is 20 muM, a value in the concentration range expected during contraction of skeletal muscle. Mg2+ alters Ca2+ binding in a complex manner, not by simple competition. In the range from 20 to 100 muM Mg2+ it produces positive cooperativity between the high-affinity Ca2+ binding sites, in addition to shifting binding to higher Ca2+ concentrations. High-affinity Ca2+ binding is not significantly affected by the addition of ATP, increase in ionic strength to 0.1 and changes in temperature. Ca2+ binding did not increase actin-activated ATPase activity in the absence of regulatory proteins, but rather inhibited it.

Actins↗