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Biomedical subjects

S Patton

Publications and source records attributed to S Patton.

At least 109 records · Page 6Linked to original sources
PubMed1984

Intramammary infusion technique for genetic engineering of the mammary gland.

The mammary gland is an appropriate substrate for genetic engineering because of its capacity to synthesize and secrete molecules of biological importance. An approach to mass production of such molecules involves transfer of genes into the lactating cell by infusion via the teat and duct system. We describe an infusion technique with the rat, a useful animal in which to develop such technology. By dye maker, trypan blue, and the ultrastructural marker, ferritin-concanavalin A, infusions by this route can permeate the entire gland and deliver molecules to apical membranes of lactating cells.

Animals↗
PubMed1984

Intramammary infusions of cytochalasin B and dimethyl sulfoxide do not suppress milk secretion in the goat.

Cytochalasin B, an intracellular microfilament antagonist, was evaluated for its capacity to inhibit milk secretion in the goat. Intramammary infusions of the drug via teat canal in amounts to 6 mg had no effects on yields or fat and protein contents and minor, if any, effects on somatic cell counts of consecutive 12-h milkings. Our results suggest that the apical plasma membrane of lactating cells is impermeable to cytochalasin B and that the reduced secretion of lactose and casein caused by the drug in vitro may arise from its interference with glucose uptake at the base of cells. Dimethylsulfoxide, which we used (2 ml) in infusates to solubilize cytochalasin B, also was without effect on the foregoing lactation characteristics.

Animals↗
PubMed1983

In vivo effects of colchicine on milk fat globule membrane.

Milk secretion in lactating goats was suppressed reversibly by infusing colchicine (2.5 to 5 mg) into one half of the udder via the teat canal. Fat globules were isolated from milks before, during and after (96 h post-infusion) this suppression. Protein, phospholipid, cholesterol (free and esterified), 5'-nucleotidase activity and peptide patterns by gel electrophoresis of these globule samples were determined. Association of [14C]colchicine with milk fat globules in vivo and in vitro also was investigated. Amounts of protein, phospholipid and free cholesterol per g of globule and 5'-nucleotidase per mg of globule protein fall following colchicine infusion. The nature of these changes suggests that the supply of membrane for milk secretion is restricted as a result of the drug treatment. Patterns of globule peptides by gel electrophoresis were qualitatively similar during the experimental period. However, a major globule glycoprotein, Mr = 52 000, showed a significant (3-fold) increase relative to the other principal peptide bands during the period of reduced milk flow. Analysis of milks for radioactivity following infusion of [14C]colchicine revealed that a portion of activity returning in milk is associated with fat globules. This activity peaked at 72 h post-infusion. Evaluation of [14C]colchicine binding to milk fat globules in vitro yielded evidence that the drug binds to the cytoplasmic, but not the exterior surface of the globule membrane. Colchicine's inhibition of milk synthesis and secretion is discussed.

Animals↗
PubMed1983

Ultrastructural changes in lactating tissue related to the suppression of milk secretion by concanavalin A.

The plant lectin, concanavalin A (Con A) suppresses milk secretion when infused into the mammary gland or when incubated with lactating tissue in vitro. Toward defining its mode of action, we infused Con A into rat and goat mammary glands via the teats and observed effects on lactating cells. Lectin dosages were 2 and 25 mg per gland for rats and goat, respectively. Tissue samples were taken 1 and 3 h post infusion for rats and at 24 h for the goat. Control and Con A-treated tissues were observed by light microscopy and by both thin section and freeze fracture electron microscopy. In comparison to controls, Con A-treated tissues of both species exhibited alveoli with enlarged cells and relatively empty lumina; cells were distended with secretory vesicles and fat droplets. Apical plasma membranes of lectin-affected cells of the rat displayed a marked reduction in the number of microvilli, and exhibited an atypical branching and folded structure. Morphometry was employed to quantitate changes in cell and secretory product parameters in both rat and goat tissue. Microtubule numbers and distribution did not appear to be altered by Con A but considerable changes were noted in the arrangement of microfilaments associated with the secretory surface of lectin-treated epithelial cells. Various related ultrastructural changes and the role of Con A in perturbing the microfilament system are discussed.

Animals↗
PubMed1983

Evaluation of an enzyme-linked immunosorbent assay for occult dirofilariasis in a population of naturally exposed dogs.

Dogs seen at a veterinary teaching hospital were classified on the basis of their apparent exposure to filarial worms, and their sera were examined in an enzyme-linked immunosorbent assay test, using a Dirofilaria immitis-derived antigen. The geometric mean titers of 103 dogs varied from 30 (61 noninfected dogs) to 2,896 (in 8 dogs without microfilaremia, but with signs of occult dirofilariasis [respiratory insufficiency, radiographic evidence of pulmonary hypertension] and a history of possible exposure to the mosquito vectors of heartworms). The mean titer of 28 dogs with patent filarial infections was 122, slightly higher than the mean titer of 64 in 8 dogs with a history of previous heartworm infection. The titers of dogs with evidence suggestive of occult dirofilariasis were significantly higher (P = 0.05) than the mean titers of those without evidence of filarial infections, with patent filarial infections, or with a history of dirofilariasis.

Animals↗
PubMed1982

Release of remnant plasma membrane from milk fat globules by Triton X-100.

The nonionic detergent, Triton X-100, was investigated as an agent for releasing plasma membrane from milk fat globules. The sedimentable material (50 000 X g, 1 h) derived by treating washed goat globules with the detergent (0.2%) was compared to membrane made by the classical globule churning procedure. Characterization included lipid and protein analyses, gel electrophoresis of peptide components, determination of enzymatic activities, and examination with the electron microscope. The results established that the detergent-releasing material is membrane with similarities to the product by churning. Evaluation of variables revealed that a detergent concentration of 0.1 to 0.2% and reaction temperature of 20-22 degrees C appear optimum with respect to membrane yield when a reaction time of 2 min is employed. At higher detergent concentrations or temperatures removal of phospholipid from the membrane was maximized. Triton X-100 was observed to release membrane from milk fat globules of the goat, human and cow, the latter with a minor procedural modification. The detergent based method is a convenient procedure for obtaining plasma membrane material in good yield for biochemical studies. It also should aid investigations of milk fat globule structure.

Animals↗
PubMed1980

Suppression of milk secretion (exocytosis) by concanavalin a in vitro.

The observation that concanavalin A can inhibit milk secretion was evaluated in an in vitro system employing minced mammary gland or isolated alveoli from lactating rats. Release of milk constituents (casein, lactose and fat globules) into the medium in the presence and absence of concanavalin A was measured during 1 or 2 h incubations. The effect of concanavalin A on glucose uptake and CO2 production of the minced tissue was also studied. Concanavalin A suppressed release of milk components at a concentration as low as 80 micrograms/ml of medium. Respiration of minced mammary tissue in the presence of concanavalin A (100 micrograms/ml of medium) was essentially the same as that of the control. The data are evidence that concanavalin A acts directly on the mammary cell in suppressing milk secretion and that the effect is not due to cytotoxicity.

Animals↗
PubMed1980

Antiserum to the milk fat globule membrane. Preparation and capacity to suppress milk secretion.

A procedure is described for preparing rabbit antiserum to goat milk fat globule membrane. This membrane is derived from the secretory surface of the lactating cell. Immunoelectrophoresis and enzyme-linked immunosorbent assay showed that antibody development reached maximal levels in about 6--8 weeks. Infusion of 5--10 ml of this antiserum into the lactating mammary gland of goats via the teat canal depressed milk yields temporarily on the infused side to 60--80% of normal. Ordinary serum from rabbit, goat or human did not evoke such a response and rabbit complement was not essential for the effect. Fractionation showed that the globulin fraction of the antiserum contained the milk-suppressing principle. Milk from the antiserum-infused side of the udder showed extensive and tenacious clumping of fat globules on standing 12--24 h. The inhibition of milk flow by antibodies to the secretory membrane resembles a previously observed inhibition following infusion of concanavalin A or its succinyl derivative. Binding of antibodies or lectins which recognize specific surface protein components of the lactating cell appears to be involved in the suppression mechanism. The possible relevance of our findings to autoimmune suppression of exocytosis is noted.

Animals↗
PubMed1980

Carotene in bovine milk fat globules: observations on origin and high content in tissue mitochondria.

The location and origin of carotenoids in bovine milk fat globules was investigated using spectral absorption of lipid solutions at 461 nm to quantitate carotene. Release of membrane from globules as a result of churning to butter or by freezing and thawing of the globules yielded membrane preparations which were devoid of carotene. Globule cores from these procedures exhibited carotene concentrations comparable to those in total milk lipids. Fractionation of lactating bovine tissue and analysis of lipid extracts revealed that the intracellular fat droplets have carotene concentrations approximating those of secreted globules. However, intracellular membranes of the tissue, particularly the mitochondria, are much richer in carotenoids than formative or secreted fat globules. The evidence indicates that bovine milk fat globules acquire carotene during their formation in the cell, but that some minor fraction of the total carotene may be extracted from the enveloping secretory membrane. Mean carotene values (microgram/g of lipid) for fractions from three samples of lactating tissue were: whole tissue 47, mitochondria 461, microsomes 69, cytosol 67, fat droplets 8, milk 9. One tissue analysis indicated that Golgi membranes contain somewhat more carotene than do microsomes.

Animals↗
PubMed1980

Origins of the cholesterol in milk.

Studies were conducted to investigate the origin of milk cholesterol in the ruminant. In the first experiment, [1(-14)C]sodium acetate was infused into one side of the udder of a lactating goat via the test canal whereas in the second, (1,2-3H]cholesterol was injected intravenously and concurrently with a [14C]acetate intramammary infusion. In both experiments, blood and milk samples were collected at intervals for 6 days postinjection. Maximum unesterified cholesterol specific activity (sp act) in whole milk appeared at 78 hr after intravenous injections of 3H cholesterol and within 3-7 hr after infusion of [14C]acetate. Virtually all the tritium in milk was associated with unesterified cholesterol. The sp act of 14C-labeled cholesterol was only 20% of gland-synthesized decanoic acid. Decanoic acid is known to be completely synthesized in the mammary gland, and, like cholesterol, acetate is its precursor. The results indicate that, although some milk cholesterol is synthesized in the mammary gland, it is derived principally from serum cholesterol. The data show also that serum cholesterol equilibrates with membrane cholesterol of the lactating cell prior to its secretion in milk.

Acetates↗
PubMed1980

Anesthetic induced changes in endoplasmic reticulum of lactating mammary tissue.

Three anesthetics--Nembutal, Ketaset, and T61--produced extensive large vacuolization of endoplasmic reticulum in lactating goat mammary tissue. This was observable with either light or electron microscopy. No normal looking cells were seen at the electron microscope level. These changes rendered the tissue unusable for morphological and autoradiographic studies. Comparable doses (weight adjusted) of the same anesthetics did not produce cellular distortion in the lactating tissue of rats as observed with light or electron microscopy. This species difference seems to involve susceptibility of the endoplasmic reticulum to swelling. Effects of anesthetics on morphology of lactating tissue from other species is not known but may constitute an important variable.

Anesthesia↗
PubMed1978

Effects of colchicine on ultrastructure of the lactating mammary cell: membrane involvement and stress on the Golgi apparatus.

The effects of colchicine on ultrastructure of the lactating mammary cell in the rat and goat were studied by electron microscopy. Changes in tissue of the rat were examined over time (1, 2 and 4 h). The goat gland was evaluated by comparing ultrastructure of tissue at the time of maximum milk flow suppression induced by the drug with that of untreated tissue. Colchicine produced notable changes in the tissue of both species: 1) the secretion of lipid droplets and Golgi vesicle contents (exocytosis) was inhibited and the droplets and vesicles became randomly distributed throughout the cell, 2) the Golgi apparatus was significantly reduced in size, 3) casein and lipid continued to be synthesized as evidenced by greater numbers of secretory vesicles and increased sizes of casein micelles and lipid droplets, 4) secretory vesicles showed a propensity to cluster around lipid droplets, 5) isolated microtubules were found occasionally in the control tissue, ordinarily in the vicinity of the Golgi apparatus, but rarely in the colchicine-treated tissue. These observations indicate that colchicine has two effects leading to suppression of exocytosis in the mammary cell: one involves early interference with capacity of secretory vesicle membranes to fuse and a further effect, related to higher concentrations of colchicine, causes intracellular disorganization and loss of polarity. Microtubules were not seen as directly involved in the mechanisms of exocytosis. The secretion of milk fat globules is coupled to exocytosis and thereby is also inhibited by colchicine.

Animals↗
PubMed1978

Formation of intracellular fat droplets: interrelation of newly synthesized phosphatidylcholine and triglyceride in milk.

The lipogenic system of fresh goat milk was used to investigate the relationship of lipid synthesis to formation of fat droplets. The skim milk phase was incubated with [1-carbon-14] palmitate and incorporation of activity into the glycerolipids was assayed. In two representative experiments, 44 and 56% of the label was incorporated into the lipids. Most of this activity (78 to 84%) was in triglycerides. Of 12 and 10% activity in the phospholipids, about 60% was contained in phosphatidylcholine with substantially smaller amounts in the other phospholipids. The synthesis of triglyceride and phosphatidylcholine was further investigated in relation to distribution of labeled lipids in a cream layer and a sedimenting fluff (membrane) fraction derived by centrifuging the skim milks following incubation. The correlation coefficient for the amounts of activity in phosphatidylcholine and triglyceride in the cream layer was .97. The data are consistent with synthesis of phosphatidylcholine and triglyceride to meet the requirements of surface and volume expansion, respectively, in formation of fat droplets. Our findings suggest the interesting working hypothesis that phosphatidylcholine synthesis regulates development of fat droplets.

Animals↗
PubMed1978

Milk secretion at the cellular level: a unique approach to the mechanism of exocytosis.

Drugs which interfere with the mechanism of exocytosis such as colchicine and vincristine, so-called microtubule antagonists, are providing a fruitful approach to the study of milk secretion at the cellular level. Intramammary infusions of a milligram or less of these substances into lactating goats produce dramatic drops in milk yields in 24 to 36 h. These depressions are reversed substantially by 48 h. In vitro experiments and tissue observations confirm that these drugs are blocking secretion at the level of the lactating cell and that secretion of all the major milk components (fat globules, casein micelles, lactose, and water) is restrained. Mammary infusion of the plant lectin concanavalin A, a protein which binds to cell surface receptors, produces similar changes in milk flow to those of the microtubule antagonists. This indicates that cell surface membrane components perturbed by concanavalin also must be involved in the secretory mechanism. One of the known receptors for concanavalin A in the apical (secretory) plasma membrane of the lactating cell is the enzyme 5'nucleotidase. The possibility must be considered that this enzyme (glycoprotein), inactivated by concanavalin A, is involved in milk secretion.

Animals↗
PubMed1977

The supression of milk fat globule secretion by clochicine: an effect coupled to inhibition of exocytosis.

The effects of colchicine on release of milk lipids from mammary tissue were evaluated by biochemical analysis of milk and morphological study of the tissue following intramammary infusions of the alkaloid into lactating goats. Colchicine produces a reversible drop in milk yield. As the flow of milk resumes, 36--48 h after infusion, the fat content of the milk increases, phospholipid per g of total globule lipid falls, mean size of milk fat globules increases and diameters of fat droplets (presecretory milk fat globules) within lactating cells approximately double. These observations are consistent with the conclusion that colchicine suppresses milk fat globule secretion but that globules continue to grow in size wihtin cells during the suppression period. These findings indicate that secretion of milk fat globules and the skim milk phase are coupled.

Animals↗