Mammalian Tissue Homogenates Research Articles

Vitex doniana Leaves Extract Ameliorates Alterations Associated with 7, 12-Dimethyl Benz[a]Anthracene-Induced Mammary Damage in Female Wistar Rats

Vitex doniana leaves are used traditionally in West Africa for the treatment of swellings and cancer. We investigated if Vitex doniana leaves extract could ameliorate 7, 12-dimethylbenz[a]anthracene (DMBA)-induced mammary damage. Female Wistar rats aged 52 ± 2 day were administered 80 mg/kg DMBA. After monitoring for 150 day, rats were administered 0, 50, 100, 200 mg/kg Vitex doniana and 20 mg/kg Tamoxifen for 14 day. Serum estrogen receptor-α, IL-1β and TNF -α levels were determined using ELISA kits. Oxidative stress markers in mammary tissue homogenates were determined using standard spectrophotometric methods. Histopathological examination was done using hematoxylin and eosin staining and cyclooxygenase-2 (COX-2) expression using immunohistochemistry. Liquid chromatography-mass spectrometry was used to determine components present in the extract. Although tumors were not observed, significantly (p < 0.05) lower estrogen receptor-α, malondialdehyde, IL-1β and TNF -α levels, significantly (p < 0.05) higher glutathione and catalase activity, attenuation of malignant epithelial hyperplasia and mild COX-2 expression were observed in rats administered Vitex doniana when compared to DMBA-induced untreated control. Liquid chromatography-mass spectrometry analysis of the V. doniana extract revealed the presence of 4,5-dihydroxy-7-methoxy-6-methylflavone and vanillylamine, which are compounds with reported antioxidant and anti-inflammatory effects. Collectively, treatment with Vitex doniana ameliorated some derangement observed in DMBA-induced rats.

ADDITIVE EFFECT OF GENES POLYMORPHISM OF FOLATE CYCLE PROTEINS AND HOMOCYSTEIN LEVEL IN PATIENTS WITH PROLIFERATIVE DISEASES OF THE BREAST AS A POTENTIAL FACTOR OF THE RISK OF THROMBOSSES

Aim.The aim of study was to examine the relationship between serum and mammary gland homocysteine levels with the carrier of separate SNP (single nucleotide polymorphism) genes of the folate metabolism system in patients with proliferative diseases and breast cancer.Methods and results.The study included 182 patients with proliferative diseases of the mammary gland in transbaikalia. The control group included 144 women who did not have oncological diseases. The serum homocysteine level and the supernatant of the mammary tissue homogenate were evaluated by high performance liquid chromatography. Genotyping for the detection of polymorphism MTHFRС677T, MTHFRА1298C, MTRA2756G, MTRRA66G was carried out by polymerase chain reaction with the detection of the amplification product in real time. In the course of molecular genetic testing in patients with proliferative diseases of the mammary gland, there was found: 1) the absence of an explicit association of the carriage of genetic polymorphism MTHFRС677T, MTHFRА1298C, MTRA2756G and MTRRA66G with serum homocysteine concentration, however, comparative hyperhomocysteinemia and, to a lesser extent, in women with the benign breast diseases; 2) the highest homocysteine content in the blood in patients with breast cancer whose genotype was characterized by combinations of polymorphic alleles MTR2756G x MTRR66G; 3) that the MTR2756A allele and genotype MTHFR1298AC, especially their combination of MTHFR1298AC x MTR2756A, increase the risk of developing benign breast formations; 4) the effect of the risk alleles MTR2756G and MTRR66GON the concentration of homocystein in the tumor tissue of the mammary gland.Conclusion. These patterns indicate a certain contribution of the polymorphisms studied, especially their additive effect, both in the development of proliferative diseases of the mammary gland and in the possible potentiation of prothrombotic effects in these patients against the background of tumor progression and homocysteine metabolism disorders.

Assessing problems of organic hydroperoxide-induced chemiluminescence related to biological/medical samples

The supernatants obtained from mammalian tissue homogenates, blood serum or other body fluids, and supplemented with organic hydroperoxide show a multiplicity of light-emitting reactions. The low-level chemiluminescence of biological samples denotes that after hydroperoxide-induction electronically excited species are produced. The light-emission measured by photomultipliers is related to (i) the geometrical location of the induced sample, (ii) light gathering capacity of the phototubes, and (iii) both the quantity and concentration of samples and reactants. The fewest blood contamination of biological samples leads to an increased photoemission. The very high sensitivity of light detectors requires stable measuring and sampling conditions and peculiarly cautious evaluation of the reactions.

Mammary secretion of oestrogens in the cow

Two experiments in vivo and one experiment in vitro were conduced to examine the mechanisms involved, which lead to mammary secretion of oestrogens and its importance for milk production and udder health in cows. In experiment 1 in six cows of the White–Black breed on day 268 of pregnancy catheters were inserted into uterine vein of pregnant horn, the abdominal aorta and the caudal superficial epigastric (milk) vein. Blood samples for estimation of oestrone, oestrone sulphate, oestradiol-17α and -17β by RIA were obtained daily from day 7 pre-partum until day 1 post-partum. Only the concentration of oestradiol-17β was statistically higher ( P≤0.01) in mammary venous plasma than in aortal and uterine plasma. In experiment 2 forty late-pregnant cows were divided into two groups according to their milk production in the previous lactation: group 1 ( n=20) high-yielding cows (>6500 kg milk per lactation), and group 2 ( n=20) low-yielding cows (<3700 kg milk per lactation). Blood samples for measurement of oestradiol-17β by RIA were collected from milk and tail veins every fourth day during a period from day 20 prior to parturition to day 4 post-partum. The concentration of oestradiol-17β was significantly higher ( P≤0.01) in the milk vein than in the peripheral plasma from day 12 pre-partum to parturition. In high-yielding cows the level of oestradiol-17β in mammary venous blood was significantly higher ( P≤0.01) than in low-yielding cows. In six cows with pathological udder oedema ante-partum the concentration of oestradiol-17β in milk vein was significantly higher ( P≤0.05) than in control cows. There were no statistically significant differences in the levels of oestradiol-17β in cows with clinical mastitis ( n=10) during 2 weeks after parturition and without it ( P≥0.05). In an in vitro experiment, homogenates of mammary tissue collected on day 7 pre-partum from two cows were incubated with 3 H -androstendione. After incubation the samples were extracted and 3 H -oestradiol-17β was separated by HPLC. 3 H -oestradiol-17β was formed in a total yield of 37%. These results indicate that oestrone, oestrone sulphate and oestradiol-17α are not secreted by bovine mammary gland. Furthermore, the secretion of oestradiol-17β starts about day 12 pre-partum and is associated with milk yield and udder oedema. Preliminary in vitro study suggests the synthesis of oestradiol-17β by mammary tissue.

10-Hydroxystearic acid--identified after homogenization of tissue--is derived from bacteria.

10-Hydroxystearic acid seems to be widely distributed in nature: Bacteria generate it by hydroxylation of oleic acid, but it was found also as constituent of plants, in cancer cell cultures and in mammalian tissue homogenates. Investigation of 10-hydroxystearic acid, obtained from mammalian tissue homogenates, revealed its identity with that of bacteria. Thus not 10-hydroxystearic acid is widely distributed in nature but its producers: bacteria. When biological material is processed in aqueous media, lipases are activated, these cleave membrane phospholipids. Thus liberated oleic acid is the substrate for widespread bacteria which are introduced into the media when the work up procedure is done in not sterile surrounding. The bacteria transform then oleic acid to 10R-hydroxystearic acid.

Occurrence of steroidogenic enzymes in the bovine mammary gland at different functional stages.

After the incubation of minced mammary tissues from non-lactating/non-pregnant (NL/NP), nonlactating/pregnant (NL/P), fully lactating (FL) and late-lactating (LL) cows with [14C]-labelled pregnenolone or progesterone and dehydroepiandrosterone (DHEA), the following metabolites were identified at all stages: 20alpha-dihydropregnenolone, progesterone (from pregnenolone), 5alpha-pregnanedione, 5alpha-pregnan-3beta-ol-20-one, 20alpha- and 20beta-dihydroprogesterone (from progesterone), 5-androstene-3beta,17beta-diol, 5alpha-androstanedione, 5alpha-androstan-3beta-ol-17-one, androstenedione, testosterone and DHEA acyl ester (from DHEA). These products indicate the occurrence of 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase, 17beta-hydroxysteroid oxidoreductase (17beta-HOR), 20alpha- and 20beta-hydroxysteroid dehydrogenases, steroid 5alpha-reductase and acyl transferase activities. Incubation of mammary tissue homogenates with [1,2,6,7-(3)H]androstenedione and testosterone confirmed the presence of a 17beta-HOR acting prevalently in a reductive way but failed to show evidence of any aromatase activity beyond background level. When total RNA from mammary tissues of NL/NP and LL cows was reverse-transcribed and amplified by polymerase chain reaction (PCR) with three sets of primers specific for bovine P450scc, P450c17 and P450arom cDNAs, no fragment of the expected size could be detected on gel. Southern analysis with corresponding digoxigenin-labelled ovarian probes, however, gave a positive signal for P450arom cDNA in five out of eight samples of LL mammary tissue. These data indicate that the bovine mammary gland has very limited steroidogenic capabilities that are essentially compatible with the terminal activation of circulating steroids from steroidogenic endocrines. It is uncertain, however, whether this conclusion applies to anestrous or ovariectomized lactating cows as well.

Cloning and expression of cystolic phospholipase A2 (cPLA2) and a naturally occurring variant. Phosphorylation of Ser505 of recombinant cPLA2 by p42 mitogen-activated protein kinase results in an increase in specific activity.

Full-length cytosolic phospholipase A2 (cPLA2) was cloned from U937 cells and polymorphonuclear leukocytes (PMNLs) while a naturally occurring variant of cPLA2, which lacks residues Val473-Ala749 but has a C-terminal extension of ILMNLSEYMLWMSKVKRFM (DcPLA2) was cloned from PMNLs and mononuclear leukocytes. We were unable to clone DcPLA2 from U937 cells. When cPLA2 and DcPLA2 were expressed in insect cells, both proteins were detected in cell lysates by SDS/PAGE as single bands of apparent molecular masses 100 kDa and 57 kDa, respectively. Full-length cPLA2 was phosphorylated stoichiometrically by p42 mitogen-activated protein (MAP) kinase in vitro at a similar rate to other physiological substrates of this protein kinase and the major site of phosphorylation was identified by amino acid sequencing as Ser505. [32P]Ser(P)505 in cPLA2 was only dephosphorylated at a slow rate by mammalian tissue homogenates. Protein phosphatases 2A, 2B and 2C all contributed significantly to the overall dephosphorylation of cPLA2. The phosphorylation of cPLA2 by p42 MAP kinase correlated with an approximately 1.5-fold increase in specific enzyme activity which was reversed by dephosphorylation.

HIGH FISH OIL DIET INCREASES OXIDATIVE STRESS POTENTIAL IN MAMMARY GLAND OF SPONTANEOUSLY HYPERTENSIVE RATS

1. The purpose of this study was to determine whether high omega-3 (19% menhaden oil, 1% corn oil) or high omega-6 (20% corn oil) fatty acid diets would decrease expression of hypertension in the female spontaneously hypertensive rat (SHR), promote tumourigenesis in the rat 7,12-dimethylbenz[a]anthracene (DMBA) model of mammary cancer or increase the susceptibility of the mammary gland to lipid peroxidation. A group of rats on a 5% corn oil diet served as the low fat control group. 2. We found that the high omega-3 and high omega-6 fatty acid diets did not significantly decrease mean arterial pressure. Marked differences occurred between the effects of omega-3 and omega-6 high fatty acid diets on baseline oxidation, auto-oxidation and iron-ascorbate catalyzed oxidation. The omega-3 diet showed 675% increase in basal oxidation, a 2624% increase in auto-oxidation and a 4244% increase in iron-ascorbate catalyzed oxidation compared to the omega-6 diet in mammary tissue homogenates. Although all rats were given 5 mg DMBA (i.g.), no mammary tumours were observed in any of the dietary groups. 3. We conclude that: (i) high polyunsaturated fatty acid diets do not decrease blood pressure in the female SHR; (ii) high fish oil diet markedly increases oxidative potential in the mammary gland; and (iii) the female SHR is resistant to DMBA-induced tumourigenesis.

Cleavage of rat prolactin occurs within rat mammary tissue, and the cleaved form is present in serum but not in milk.

The present study was undertaken to determine whether cleavage of rat prolactin (rPRL) occurs within rat mammary tissue and to investigate whether the cleaved form and its 16-kDa amino terminal fragment are present in rat serum and milk. The relative abundance of the different forms of rPRL was determined by SDS-PAGE and immunoblot analysis using an antiserum to the 16-kDa fragment. Explants of mammary glands from lactating rats were incubated with intact rPRL. The tissue was then washed extensively to remove external PRL forms. The ratios of cleaved to intact rPRL in mammary tissue homogenate, in mammary-conditioned medium, and in serum from lactating rats were 4.2, 0.44, and 0.16, respectively. Although intact rPRL was found in milk, the cleaved form was not detectable. The 16-kDa fragment of rPRL was not detected in concentrated extracts of either serum or milk. These results indicate that lactating rat mammary tissue can internalize and cleave rPRL, but the cleaved form does not appear in milk. The cleaved rPRL is present in rat serum, but no evidence was obtained for the presence of the 16-kDa fragment of rPRL in either serum or milk.

Effect of soybean,Vicia faba,and vitamin C on the carcinogenicity of DMBA

A single dose of 10 mg of 7,12-dimethylbenz[a]anthracene (DMBA), administered to rats through intragastric intubation, was sufficient to induce many biochemical and histopathological changes in their mammary tissue. Significant increases were observed in the activity levels of the enzymes acid ribonuclease, 5-nucleotidase, alkaline phosphatase, and beta-glucuronidase in mammary tissue homogenates of DMBA-treated rats after an experimental period of five months. Histopathological studies of the mammary tissue also revealed malignant epithelial tumors (cribriform carcinoma) induced among 85% of the treated rats, with an incidence of 4 tumors in 12 mammary glands. Nevertheless, administration of 30% soybean in the diet of rats or 5,000 ppm ascorbic acid in their drinking water in addition to DMBA revealed a significant chemoprotective effect against the carcinogenesis induced by DMBA alone. This chemoprotective effect was demonstrated by the normalization of the activity levels of the enzymes studied in mammary tissue homogenates, because most of the enzymes were maintained at near the levels in the control animals. The incidence and number of tumors were also decreased. Cribriform carcinoma was observed in 50% of the rats, and the incidence of the affected glands was 2 in 12 mammary glands among both groups. On the other hand, a less chemoprotective effect was observed due to Vicia faba administration.

Mammary-derived growth inhibitor in lactation and involution

Presence of mammary-derived growth inhibitor (MDGI) in mammary tissue of lactating and involuted cows was investigated. Eighteen lactating, non-pregnant high-producing Holstein cows were randomly assigned to 3 experimental groups of 6 cows each. Cows of the first group were slaughtered while in lactation. Cows of the second group were slaughtered at 2–3 d, and the others at 4–8 d following sudden cessation of milking. Cessation of milking occurred at approximately 300 d in lactation. Western blot analysis revealed the presence of MDGI in the cytosolic and microsomal fractions of mammary tissue homogenates. High levels of MDGI were detected in mammary tissue obtained from lactating non-pregnant cows. A dramatic reduction in MDGI was observed in early involution (2–3 or 4–8 d following cessation of milking). These data suggest that a relationship exists between MDGI levels and the physiological status of the gland. Lack of MDGI may play a role during the processes of mammary involution and development prior to parturition.

An immunoassay for qualitative estimation of collagenase activity in mammalian tissues

Collagen, the most abundant protein of the mammalian body, is specifically degraded by collagenase. Collagenase activity and subsequent collagen degradation are the main aspects of essential biological processes such as bone remodeling, tissue repair and wound healing. Measurement of collagenase activity is of interest to a wide variety of investigations using mammalian tissues, including clinical specimens. Most assays for collagenase activity are based on chemical modification of the substrate collagen. A unique feature of the present method is that it allows the rapid, qualitative measurement of collagenase activity without the requirement of substrate modification. It is based upon both substrate digestion by collagenase and reaction of undigested collagen with its antibody. Collagenase activity is measured by quantitation of immunoreactivity of undigested collagen using an enzyme-linked immunosorbent assay (ELISA). The assay is performed in 96-well microtiter plates used for ELISA. The advantages of this method are several: (a) a highly specific reaction between substrate collagen and its antibody that rules out the possibility of nonspecific quantitation; (b) the use of a nonmodified substrate; (c) the ease and rapidity of performance of a microassay. Application of the microassay to mammalian tissue homogenates was demonstrated in rat uterus tissue and ventricular myocardium of normal and hypertensive rats.

Formation of a stable l-ascorbic acid α-glucoside by mammalian α-glucosidase-catalyzed transglucosylation

Enzymatic transglucosylation from maltose to l-ascorbic acid (AA) with mammalian tissue homogenates was determined by a high-performance liquid chromatography method and compared with the reaction catalyzed by α-glucosidase from Aspergillus niger. The homogenates of small intestine and kidney had a high transglucosylase activity to form a new type of glucosylated AA, which was associated with α-glucosidase activity. The new compound was demonstrated to be an equimolar conjugate of AA and glucose by the spectral and quantitative analyses. In particular, it showed a high stability in a neutral solution and no reducing activity toward cytochrome c and a dye. These properties were very different from those of AA and l-ascorbic acid α-glucoside formed with α-glucosidase form A. niger, but they were consistent with those of l-ascorbic acid 2- O-phosphate and l-ascorbic acid 2- O-sulfate. Moreover, it exhibited a reducing power associated with AA after mild acid hydrolysis or treatment with rat intestinal α-glucosidase. These results indicate that it should be assigned the 2- O- α-glucoside structure. Consequently, i should be assigned the 2- O- α-glucoside structure. Consequently, it is concluded that mammalian α-glucosidase is able to form a very stable and nonreducing form of glucosylated AA through a specific transglucosylation reaction distinct from that of microbial α-glucosidase.

Enzymatic Formation of a Nonreducing L-Ascorbic Acid a-Glucoside: Purification and Properties of a-Glucosidases Catalyzing Site-Specific Transglucosylation from Rat Small Intestine

We have previously found that some mammalian tissue homogenates can catalyze a unique transglucosylation from maltose to L-ascorbic acid (AA), resulting in a chemically stable AA derivative, L-ascorbic acid alpha-glucoside (AAG). In the present study, the enzyme responsible for this transglucosylation was isolated from rat intestinal membrane. The formation of AAG was determined by HPLC with an ODS column. The specific activity of AAG-forming enzyme was increased in parallel with that of alpha-glucosidase (maltose hydrolase) during the purification, and two neutral alpha-glucosidases, termed alpha-glucosidases I and II, were purified to apparent homogeneity. Their enzymological properties showed that they corresponded to maltase [EC 3.2.1.20] and sucrase-isomaltase complex [EC 3.2.1.48/10], respectively. Both enzymes could form AAG by splitting only maltose among the disaccharides examined, although alpha-glucosidase I possessed a considerably higher activity than the other enzyme. Both AAG formation and maltose hydrolysis were dependent on incubation temperature with the maximal activity at 60 degrees C, but there was an apparent difference between their pH optima. AAG thus formed could also be hydrolyzed by the purified enzymes. From these results, it is concluded that membrane-bound neutral alpha-glucosidases from rat intestine have site-specific transglucosylase activity to form nonreducing AAG which is distinct from L-ascorbic acid-6-O-alpha-D-glucoside.

An assay for superoxide dismutase activity in mammalian tissue homogenates

During the course of measuring superoxide dismutase (SOD) activity in rat breast tissue, interferences in the nitroblue tetrazolium (NBT) and cytochrome c assay systems were noted. These interferences inhibit accurate measurement of SOD activity in breast tissues, necessitating the development of a new NBT-based assay that includes compounds capable of inhibiting tissue specific interferences. The most effective compounds were metal chelators that were also electron transport chain inhibitors. Bathocuproine sulfonate (BCS) was the most effective of these compounds. The inclusion of BCS in the NBT assay system was shown to make the accurate measurement of SOD activity in tissues with interferences possible.

Rat and human mammary tissue can synthesize choline moiety via the methylation of phosphatidylethanolamine.

The normal mammal requires large amounts of choline for maintenance and growth of tissue mass. Since milk, the only food for neonates, has many-fold higher free choline concentration than does maternal plasma, it is possible that mammary gland can synthesize choline molecules. The only known mammalian pathway for the synthesis de novo of choline molecules is catalysed by phosphatidylethanolamine N-methyltransferase (PeMT), which synthesizes phosphatidylcholine (PtdCho) via sequential methylation of phosphatidylethanolamine (PtdEtn) using S-adenosylmethionine (AdoMet) as a methyl donor. We identified PeMT activity in rat mammary tissue, and differences in affinities for substrate, as well as in activities as a function of pH, suggest that at least two distinct enzyme activities are involved [i.e. one catalysing the methylation of PtdEtn to form phosphatidyl-N-methylethanolamine (PtdMeEtn) and the other catalysing the methylation of PtdMeEtn and phosphatidyl-NN-dimethylethanolamine (PtdMe2Etn) to form PtdMe2Etn and PtdCho, respectively]. The relationships between AdoMet concentrations and PtdCho formation from endogenous PtdEtn in rat mammary homogenate were complex: a sigmoidal component (with a Hill coefficient of 2.2), requiring 55 microM-AdoMet for half saturation (Vmax. = 9 pmol/h per mg of protein), and a high affinity component (Kapparent = 8.7 microM and Vmax. = 3.8 pmol/h per mg of protein) were identified. When exogenous PtdMe2Etn was added as substrate, PtdCho formation exhibited Michaelis-Menten kinetics for AdoMet, and its affinity for AdoMet was high (Kapparent = 9 microM, Vmax. = 85 pmol/h per mg of protein). In the presence of endogenous substrates, the rates of PeMT-catalysed PtdCho formation within homogenates of rat mammary tissue were similar in tissue from lactating and non-lactating animals. When exogenous PtdMe2Etn was added to homogenates of rat mammary tissue, tissue from lactating rats made twice as much PtdCho as did tissue from non-lactating rats. Isolated mammary epithelial cells also exhibited PeMT activity; the rate of formation of PtdCho was much greater in intact versus broken cells. We also identified PeMT activity in homogenates of mammary tissue from non-lactating humans. The rate of PtdCho formation was of similar magnitude to that seen in rat tissue. This evidence supports the hypothesis that some of the choline found in milk could have been synthesized de novo in the mammary gland.

Activity of Lysosomal Enzymes in Murine Mammary Tissue Through Pregnancy, Lactation, and Involution

Murine mammary tissue homogenates obtained during pregnancy, lactation, and involution were assayed for activities of two lysosomal marker enzymes, acid phosphatase, and N-acetyl-β-D-glucosaminidase. Acid phosphatase activity per milligram protein was relatively constant through pregnancy and lactation, although a decrease was detected at parturition. Acid phosphatase activity per milligram deoxyribonucleic acid was also stable through pregnancy and lactation except for a peak of activity during lactogenesis. Acid phosphatase activity per gram wet weight also remained stable during pregnancy and lactation, but activity was significantly higher during lactation than pregnancy. Glucosaminidase activity, whether expressed as milligrams deoxyribonucleic acid of milligrams protein, tended to decrease during pregnancy, decreased further with lactogenesis, and remained significantly lower throughout lactation. Glucosaminidase activity per gram wet weight increased as pregnancy progressed and slowly decreased through lactogenesis to midpregnancy levels, which remained stable throughout the remainder of lactation. Both acid phosphatase and glucosaminidase activities were slightly higher at the end of lactation, and both decreased within 24h of weaning. Significant increases in activities of both enzymes were observed from d 1 to 4 of involution.

Alcohol and acetaldehyde in rat's milk following ethanol administration

Alcohol and acetaldehyde were measured in milk and peripheral blood in chronic alcoholic rats, at 5 and 15 days of lactation. Ethanol in blood increased throughout lactation and the levels of acetaldehyde were much higher than in nonlactating alcoholic rats. The concentration of acetaldehyde in milk was always ca. 50% of that in blood, whereas that of ethanol varied within the range of 44–80% of the blood levels. Blood alcohol levels in the corresponding sucking pups were much lower than in maternal blood and increased thoughout lactation. The time course of ethanol and acetaldehyde concentration in blood and milk were determined in normal lactating rats after cyanamide (40mg/kg) and ethanol administration (2 or 4 g/kg). Milk alcohol reached higher concentrations than in blood within the first hour of ethanol administration, decreasing and remaining constant thereafter at ca. 65% of those in blood. Acetaldehyde levels in milk were always 35–45% lower than in blood. No alcohol dehydrogenase activity was found in homogenates of mammary tissue; however there was some aldehyde dehydrogenase activity. A significant decrease in mammary tissue aldehyde dehydrogenase was found in chronic alcoholic rats. The role of this enzyme is discussed.

Iodoprotein formation by rat mammary glands during pregnancy and early postpartum period.

Iodide organification by rat mammary glands was studied during the trimesters of pregnancy and early postpartum period. Organification was followed by measuring trichloroacetic acid (TCA) precipitation of delipidated tissue homogenates. The radiolabeled material was sensitive to proteolytic cleavage by a bacterial protease indicating that the 125I was protein-bound. Gel filtration column chromatography in the presence of sodium dodecyl sulfate (SDS) of delipidated mammary tissue homogenates of pregnant and postpartum rats reproducibly resolved several iodoproteins from free iodide. The Kav value for each iodoprotein peak was calculated and was used to estimate each subunit molecular weight which averaged 37,500, 25,100, and 8500. Another iodoprotein with a very large subunit molecular weight of greater than 300,000 was also detected in mammary tissue. Incorporation of 125I-iodide into the three smaller iodoproteins increased logarithmically from the start of the second trimester of pregnancy through the early postpartum period when approximately 20% of the total 125I uptake by mammary tissue was incorporated into protein. Hyperplasia, acinar development, and intracytoplasmic vacuolization of mammary tissue correlated with the increased incorporation of 125I-iodide into these iodoproteins. The characterization and quantitation of specific iodoproteins in mammary tissue may be important as organification of iodide is believed to be a marker for normal hormone-responsive cells.

Techniques for Preparation of Bovine Mammary Smooth Membranes

Most procedures for fractionation of lactating mammary tissue membranes have utilized centrifuge tubes of small volume. We report a method for large-scale fractionation of bovine mammary membranes by a zonal rotor. Because of the large volume of the zonal rotor (1350ml), greater amounts of starting material can be fractionated to obtain greater yields of desired fractions. We report results of linear sucrose gradients to fractionate mammary tissue homogenates and step gradient adaptations to accelerate membrane separations. Assay of membrane fractions for marker enzyme activities demonstrated that zonal fractionation and derived techniques were applicable for large scale preparation with higher yields of selected bovine mammary membranes. Application of this technique to small volumes is discussed for laboratories without a zonal rotor.