Regulation of triglyceride synthesis in the parturient guinea-pig mammary gland.
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ABSTRACT:
SUBMITTER: Kuhn NJ
PROVIDER: S-EPMC1198293 | biostudies-other | 1967 Oct
REPOSITORIES: biostudies-other
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Project description:1. The presence of palmitoyl-CoA-l-glycerol 3-phosphate palmitoyltransferase (EC 2.3.1.15) has been demonstrated in a particulate fraction of mammary tissue from lactating guinea pigs. 2. Cell-free preparations also catalysed the activation of palmitate and oleate, and the conversion of enzymically formed phosphatidic acid into glycerides, in accord with the Kennedy pathway of glyceride formation. 3. The properties of the system that esterifies l-glycerol 3-phosphate were studied with respect to substrates and cofactors, and the reaction product was shown to be phosphatidic acid (1,2-diacyl glycerol 3-phosphate). 4. The extent to which newly formed phosphatidic acid was converted into glyceride in a cell-free system was dependent on the nature of the acyl donor, the concentration of subcellular particles, the time of incubation and the concentration of Mg(2+).
| S-EPMC1198292 | biostudies-other
Project description:1. 32-P-labelled polyribosome preparations were made from the mammary glands of lactating and late-pregnant guinea pigs after injection of (32-P)i into the animals. 2. The RNA of polyribosomes, ribosomal subunits and that released from polyribosomes by EDTA were analysed by zone velocity centrifugation and by polyacrylamide-gel electrophoresis. 3. RNA species which have the physical properties expected for the milk protein mRNA were detected. RNA species of a size which could code for the caseins were present in lactating but not in pre-lactating mammary-gland polyribosomes.
| S-EPMC1165346 | biostudies-other
Project description:Tryptophan uptake by the isolated perfused lactating guinea-pig mammary gland was 46.5+/-4.6 mug/h per g. Results of absorption studies and the use of [methylene-14C]tryptophan suggest that tryptophan is one of the group of amino acids that are transferred almost quantitatively from blood plasma to milk protein.
| S-EPMC1164025 | biostudies-other
Project description:The molecular clock plays key roles in daily physiological functions, development and cancer. Period 2 (PER2) is a repressive element, which inhibits transcription activated by positive clock elements, resulting in diurnal cycling of genes. However, there are gaps in our understanding of the role of the clock in normal development outside of its time-keeping function. Here, we show that PER2 has a noncircadian function that is crucial to mammalian mammary gland development. Virgin Per2-deficient mice, Per2-/- , have underdeveloped glands, containing fewer bifurcations and terminal ducts than glands of wild-type mice. Using a transplantation model, we show that these changes are intrinsic to the gland and further identify changes in cell fate commitment. Per2-/- mouse mammary glands have a dual luminal/basal phenotypic character in cells of the ductal epithelium. We identified colocalization of E-cadherin and keratin 14 in luminal cells. Similar results were demonstrated using MCF10A and shPER2 MCF10A human cell lines. Collectively this study reveals a crucial noncircadian function of PER2 in mammalian mammary gland development, validates the Per2-/- model, and describes a potential role for PER2 in breast cancer.
| S-EPMC5897596 | biostudies-literature
Project description:Golgi and endoplasmic-reticulum fractions were prepared from the lactating guinea-pig mammary gland. The endoplasmic-reticulum fraction was highly active in the processing and sequestration of milk-protein primary translation products. Explants from the lactating gland in organ culture were used to identify milk-protein intermediates present in the secretory pathway, and the timing of the events leading to their post-translational modification. With [35S]methionine, the milk proteins labelled after a short pulse (3 min) were represented by the partially processed (but not phosphorylated) caseins and alpha-lactalbumin sequestered within membrane-bound vesicles. After a 30 min labelling period, higher-Mr caseins with electrophoretic mobilities identical with those of the phosphorylated caseins isolated from milk were identified in the incubation medium, and sequestered within membrane-bound vesicles. Pulse-chase experiments established a precursor-product relationship between these forms. Secretion is apparent approx. 30 min after sequestration. Caseins are highly phosphorylated; removal of the phosphate residues with acid phosphatase results in proteins with increased electrophoretic mobility, similar to those of the partially processed early casein intermediates found sequestered in explants after a 3 min pulse with [35S]methionine, and those sequestered within microsomal membranes after mRNA-directed cell-free protein synthesis. A comparison of the proteins labelled during both short (5 min) and long (30 min) pulses with [35S]methionine and [32P]Pi shows that, in contrast with the 35S-labelled caseins, those labelled with [32P]Pi exhibit only electrophoretic mobilities identical with those of the mature caseins isolated from milk and those identified after long labelling periods with [35S]methionine. No phosphorylated early intermediate forms of caseins were identified. We conclude that the synthesis and post-translational modification of guinea-pig caseins occurs in two stages, (i) an early event involving synthesis and sequestration within the endoplasmic reticulum, an event that involves signal-peptide removal, followed (ii) 10-20 min later by phosphorylation at a different point in the secretory pathway, probably in the Golgi complex. Secretion of the phosphorylated caseins occurs 10-20 min later.
| S-EPMC1144205 | biostudies-other
Project description:The sequence of guinea pig cytomegalovirus (GPCMV) was determined by direct sequencing of salivary gland homogenates obtained following sustained, serial in vivo passage of pathogenic virus in guinea pigs. The 233,501-nucleotide salivary gland (SG) genome was noted to have 11 differences compared to the tissue culture-passaged virus, although no variations were noted in putative protein coding sequences.
| S-EPMC3622957 | biostudies-literature
Project description:Cytosolic malic enzyme was purified from rat mammary gland by L-malate affinity chromatography. The pure enzyme obtained was used to produce a specific antiserum in a rabbit. Relative synthesis of malic enzyme in the mammary gland of mid-lactating rats was 0.097%, measured by labelling the enzyme in isolated acini. When food was removed, malic enzyme synthesis decreased to 35% and 20% of the control value at 4 and 6 h respectively. Incorporation of [3H]leucine into soluble proteins was constant during the first 6 h of starvation. When lactating rats (maintained with their pups) were starved for 24 h and then re-fed, the relative rate of enzyme synthesis increased 2.5-, 4-, and 4.5-fold at 3 h, 6 h and 18 h respectively after initiation of re-feeding. The relative rate of malic enzyme synthesis was about 50% of normal at 15 h after weaning, whereas the rate of synthesis of soluble proteins did not change. Administration of bromocriptine or adrenalectomy of lactating rats decreased the relative rate of synthesis of malic enzyme by 40% or 30% respectively; these effects were counteracted by hormone supplementation. Hormone therapy also caused an increase in the rate of incorporation of [3H]leucine into soluble proteins and in malic enzyme activity.
| S-EPMC1146859 | biostudies-other
Project description:Prolactin receptors present in the particulate fraction of lactating pig mammary gland were solubilized by 7.5mM-3-[(3-cholamidopropyl)dimethylammonio]-1-propane-su lph onic acid (Chaps) and purified by affinity chromatography on prolactin coupled to Affi-Gel 10. Nearly 30% of the particulate receptors were solubilized by the detergent and over a 1000-fold purification from homogenates was achieved. A water-soluble fraction rich in receptors was observed during the preparation of membranes, although this fraction has not yet been purified. Prolactin binding to the receptors was a time-dependent, reversible and saturable reaction in particulate, Chaps-solubilized and purified receptors. In all forms, receptors showed the same specificity to peptide hormones. Prolactin and human growth hormone bound to the same receptors, whereas bovine growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and insulin failed to bind. After solubilization, the dissociation constant (Kd) for prolactin was decreased 5-fold from 9.8 X 10(-11) M in the particulate receptors to 1.8 X 10(-11) M in solubilized and purified receptors, being due principally to an increase in the association rate constant from 1.0 X 10(9)M-1 X h-1 to (3.9-4.6) X 10(9)M-1 X h-1, respectively, with the dissociation rate constant remaining unchanged at (1.1-1.3) X 10(-2)h-1. Isoelectric focusing of the prolactin-receptor complex revealed two peaks, one at a pI of 5.5-5.6 and the other at 5.2-5.3. Microsomal receptors were covalently cross-linked to 125I-labelled ovine prolactin with ethylene glycol bis(succinimidyl succinate) and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Autoradiography of the gel revealed a major subunit of Mr 28 000-35 000 and a minor one of Mr 67 000-69 000. Anti-(prolactin receptor) antibodies raised against rabbit mammary gland prolactin receptors were equally effective in inhibiting prolactin binding to particulate, solubilized and affinity-purified receptors, suggesting that purified prolactin receptors have a structure indistinguishable immunologically from particulate receptors and rabbit mammary gland prolactin receptors. The present demonstration shows that particulate prolactin receptors from a domestic animal can be solubilized and purified without losing the original properties of high affinity and binding specificity for hormones.
| S-EPMC1144528 | biostudies-other
Project description:Background and purposeTo investigate the role of connexin 43 in the maintenance of spontaneous activity in prostate tissue from young and old guinea pigs.Experimental approachConventional intracellular microelectrode and tension recording techniques, coupled with Western blot analysis and immunohistochemistry for connexin 43 (CX43) were used. The effects of three gap junction uncouplers, 18? glycyrrhetinic acid (10?µM, 40?µM), carbenoxolone (10?µM, 50?µM) and octanol (0.5?mM, 1?mM), were studied in cells displaying slow wave activity and on spontaneously contracting tissue from prostate glands of young (2-5 months) and old (9-16 months) guinea pigs.Key results18? Glycyrrhetinic acid (40?µM), carbenoxolone (50?µM) or octanol (0.5?mM) abolished slow wave activity in prostate tissue from young and old guinea pigs and depolarized membrane potential by approximately 5?mV. These treatments also abolished all contractions in both sets of prostate tissue. These effects were reversed upon washout. Western blot analysis and CX43 immunohistochemistry showed that there was no age-related difference in the expression and distribution of CX43 in prostate tissues.Conclusion and implicationsWhen gap junctional communication via CX43 was disrupted, spontaneous activity was abolished at a cellular and whole tissue level; CX43 is therefore essential for the maintenance of spontaneous slow wave activity and subsequent contractile activity in the guinea pig prostate gland.
| S-EPMC3010576 | biostudies-literature
Project description:1. The expression of alpha-lactalbumin and casein genes was examined in guinea-pig mammary tissue taken from animals both pre- and post-partum. 2. Analysis of total RNA by RNA excess hybridization with sequence-specific complementary DNA probes demonstrated that alpha-lactalbumin mRNA was present late in pregnancy, and that maximum concentrations were present at parturition. Casein gene transcripts were absent late in pregnancy (62 days), but by parturition were present at concentrations identical to those found at all time points examined throughout lactation. 3. Studies using mammary explants in organ culture showed that tissue from pregnant animals, or animals at parturition, synthesized and secreted only alpha-lactalbumin. After parturition, at the onset of casein synthesis, differential rates of secretion of alpha-lactalbumin and the caseins were observed. 4. The results are discussed in terms of the multiple intracellular mechanisms involved in the regulation of milk protein gene expression in the guinea-pig mammary gland.
| S-EPMC1162838 | biostudies-other