A comparative study of the estrogenic potential of two synthetic estrogens (mestranol and ethinylestradiol)

The purpose of this study was to establish the relative estrogenicity of ethinylestradiol and mestranol and to establish .05 and .1 mg of mestranol as standard reference preparations for the comparative study of other compounds. Patients were selected from a large group of primary amenorrhea, secondary amenorrhea, and postmenopausal women. All required hormonal replacement therapy. Patients with chromosomal abnormalities or those with diminished or abnormal response were excluded. Vaginal cytology was used as the main method for the assessment of the response. Before treatment all had either an atrophic pattern (Maturation Index = 50-50-0) or a hypertrophic pattern (MI=5-95-0) with a mean value of 10-87-3 for patients with primary amenorrhea and 18-79-3 in the other 2 groups. Patients with a proliferative intermediate cell pattern and postmenopausal women thought to have continuing endogenous estrogen activity were omitted. Ethinylestradiol or mestranol were given orally in doses of .025, .05, and .1 mg for a period of 10 days during which frequent vaginal smears were made. When cell patterns showed extremely marked cytolysis of the glycogen-containing intermediate cells and a low percentage of karyopyknotic superficial cells these patients were excluded. 40 women were finally included. The effects of estrogen on the vaginal epithelium in all 40 women after 10 days therapy were quite uniform. In those cases showing delayed regression of estrogenic effects 4 mg of chlormadinone acetate was given daily for 10 days. These patients then showed a reduction in the superficial cell percentage accompanied by a shift to the left in the maturation index such as 0-40-60 before to 0-95-5 after treatment. A few women had an insignificant reduction in the superficial cell percentages. Changes in the percentages of the intermediate type of cells were not significant. The regression was considered to be complete when the smear contained mainly small intermediate cells and parabasal cells with numerous leukocytes and some necrotic debris. Both estrogen preparations produced the same vaginal response at .05 mg doses. Tablets of .05 and .1 mg mestranol were chosen for further study. The increase in the superficial cell index to 30 (SD plus or minus 18.5) following treatment with .05 mg and the increase to 54.1 (SD plus or minus 26.7) following .1 mg have been adopted as standard vaginal responses. The term estrogenic effect used in vaginal cytology is taken to mean the local vaginal effect induced by estrogens.     

Normal functions of the thyroid gland of the pygmy goat.

Normal values were obtained for blood serum PBI, T3 index. T4, and cholesterol in a colony of pygmy goats (n equal to 55) of mixed sex and age. Serum PBI values averaged 8.1 plus or minus 1.2 mug/dl with no significant sex differences. The mean T3 index and T4 value were 1.1 plus or minus 0.1 and 7.2 plus or minus 1.1 mug/dl, respectively, with no sex differences. The mean serum cholesterol value was 90.0 mg/dl, with sex differences apparent. Serum cholesterol averaged 84.9 mg/dl (n equal to 44) for females and was significantly higher in intact males (97.4 mg/dl; n=8) and significantly lower in castrate males 69.2; n=6. There was a consistent and significant increase in cholesterol values with age in females, an unexplained phenomenon also observed in humans. There was no evidence of thyroid malfunction in the animals studied.     

Effects of dehydroepiandrosterone, Premarin and Acolbifene on histomorphology and sex steroid receptors in the rat vagina

To assess the specific estrogenic and/or androgenic effects of a potential novel hormone replacement therapy, we have examined the morphology of the rat vagina 9 months after ovariectomy (OVX) and treatment of OVX animals with dehydroepiandrosterone (DHEA), conjugated estrogens Premarin and the selective estrogen receptor modulator Acolbifene. OVX led to atrophy and inflammatory changes while Acolbifene reduced the inflammation incidence and induced mucification of the vaginal epithelium. Premarin induced a typical keratinized stratified squamous epithelium while DHEA induced stimulation of the vaginal epithelium, with mucous cells typical of an androgenic effect, combined with increased collagen fiber compactness of the lamina propria. On the other hand, after OVX, the vaginal muscle layer decreased by 46%, an effect which was 41 and 100% reversed by DHEA and Premarin, respectively. The present data show particularly interesting effects of DHEA on the three layers of the vaginal wall, namely a highly mucified epithelium, an increased muscularis thickness and increased collagen fiber compactness in the lamina propria. DHEA exerts both androgenic and estrogenic effects on the vaginal mucosa, thus providing a more physiological replacement therapy.     

Structure and synthesis of a lipid-containing bacteriophage. The molecular weight and other physical properties of bacterophage PM2.

Several physical and chemical parameters of bacteriophage PM2 have been measured. The sedimentation constant was determined to be s-20,w=293 S. The buoyant density in sucrose at 20 degrees C was 1.24 g cm+-3 and in CsCl at 25 degrees C was 1.29 g cm-3. The high-speed equilibrium centrifugation method of Yphantis (1964) was used to measure the molecular weight of PM2. The necessary auxiliary parameters were also determined. A value of 0.771 plus or minus 0.005 cm-3 g-1 for the apparent specific volume at constant chemical potential in 1 M sodiium chloride has been obtained by pycnometry; the viral concentration was determined using the absorption coefficient at 260 nm (4.60 plus or minus 0.10 cm-2 mg-1), which in turn was calculated from the phosphorous content of the virus (17.89 plus or minus 0.28 mu-g of P per mg dry weight dry weight of virus). The molecular weight of PM2 determined with these parameters is (44.1 plus or minus 1.2 x 10-6). From the phosphorous content of the virus, the percentage of phosphorous known to be in its DNA (Camerini-Otero and Franklin, 1972), and the molecular weight of the bacteriophage, we have calculated a molecular weight for PM2 DNA of 6.26 x 10-6, which confirms values determined using empirical relationships.     

Cholesterol sulfate. II. Studies on its metabolism and possible function in canine blood.

Previous in vitro studies to evaluate the possible role of cholesterol sulfate in the stabilization of the human erythrocyte membrane have been extended to the dog in vivo. Thus, following the injection of labelled cholesterol sulfate, a large fraction of the administered sterol conjugate is taken up by the membrane of the canine erythrocyte. Peak membrane levels were obtained within 30-60 min. Measurement of radioactivity associated with the plasma and red cell fractions in serial samples allowed the calculation of the half-life of cholesterol sulfate in each fraction. From the data obtained from the plasma of four dogs, the half-life was calculated to 5.8 plus or minus 0.9 h. The half-life of chlesterol sulfate associated with the erythrocyte membrane was calculated to be 6.7 plus or minus 1.2 h. In addition, following the intravenous administration of 0.2-0.7 mg of cholesterol sulfate/kg of body weight and withdrawal of serial blood samples, a significant diminution in the degree of hemolysis was observed when the red cells were exposed to hypotonic saline solutions. Maximal stabilization effects were observed at approx. 6-7 h after the administration of the sterol conjugate. Hemolytic properties returned to normal at approx. 24 h following the injection.     

DNA-bound lipids from eukaryotic (loach spermatozoa, pigeon erythrocytes) and prokaryotic (E. coli B, phage T2) cells

Using thin-layer chromatography, some specific DNA-bound neutral lipids and phospholipids of loach spermatozoa, pigeon erythrocytes, E. coli B and phage T2 cells were studied. These lipids are represented by loosely and firmly bound components. The content of neutral lipids in the above DNAs (per mg of DNA) is 10.6, 4.8, 7.81 and 1.43 micrograms, respectively; that of phospholipids is 4.31, 1.28, 1.14 and 0.54 micrograms, respectively. The eucaryotic DNA-bound lipids are highly deficient of free cholesterol, phosphatidylcholine, phosphatidylinositol and phosphatidylserine but are rich in cardiolipin, phosphatidylethanolamine, cholesterol esters, diglycerides and free fatty acids. The quantitative and qualitative composition of DNA-bound lipids of loach spermatozoa changes during the transition from the superhelical to the relaxed conformation of DNA. Procaryotic DNA-bound neutral lipids are also represented by the free cholesterol, diglyceride and free fatty acid fractions, whereas the DNA-bound phospholipids of procaryotes consist of only two fractions, i.e., cardiolipin and phosphatidylethanolamine. The role of DNA-bound lipids in the structural and functional organization of eucaryotic and procaryotic genomes is discussed.     

Properties of (Na+ plus K+)-activated ATPase in rat liver plasma membranes enriched with bile canaliculi.

Liver plasma membranes enriched in bile canaliculi were isolated from rat liver by a modification of the technique of Song et al. (J. Cell Biol. (1969) 41, 124-132) in order to study the possible role of ATPase in bile secretion. Optimum conditions for assaying (Na+ plus K+)-activated ATPase in this membrane fraction were defined using male rats averaging 220 g in weight. (Na+ plus K+)-activated ATPase activity was documented by demonstrating specific cation requirements for Na+ and K+, while the divalent cation, Ca(2+), and the cardiac glycosides, ouabain and scillaren, were inhibitory. (Na+ plus K+)-activated ATPase activity averaged 10.07 plus or minus 2.80 mumol Pi/mg protein per h compared to 50.03 plus or minus 11.41 for Mg(2+)-activated ATPase and 58.66 plus or minus 10.07 for 5'-nucleotidase. Concentrations of ouabain and scillaren which previously inhibited canalicular bile secretion in the isolated perfused rat liver produced complete inhibition of (Na+ plus K+)-activated ATPase without any effect on Mg(2+)-activated ATPase. Both (Na+ plus K+)-activated ATPase and Mg(2+)-activated ATPase demonstrated temperature dependence but differed in temperature optima. Temperature induced changes in specific activity of (Na+ plus K+)-activated ATPase directly paralleled previously demonstrated temperature optima for bile secretion. These studies indicate that (Na+ plus K+)-activated ATPase is present in fractions of rat liver plasma membranes that are highly enriched in bile canaliculi and provide a model for further study of the effects of various physiological and chemical modifiers of bile secretion and cholestasis.     

Role of prolactin on Leydig, Sertoli and germ cellular neutral lipids in bonnet monkeys, Macaca radiata.

To elucidate the specific influence of prolactin on neutral lipids in Leydig, Sertoli and germ cell compartments of the testis in immature and mature monkeys, the present study was carried out by injecting ovine prolactin (oPRL) (1 mg/kg body weight/twice daily for 10 days ip), to both age groups. Similarly, bromocryptine (an ergot alkaloid which inhibits prolactin secretion) was given to other sets of immature and mature monkeys (1 mg/kg body weight/twice daily for 10 days ip) to induce hypoprolactinemia. It was observed that after oPRL administration the total lipid accumulated in the germ cells of immature and mature monkeys. Total lipid was markedly decreased in the Leydig cells of mature monkeys only. But no such influence of PRL was evident in the Leydig cells of immature monkeys, suggesting an age-dependent effect of PRL on the Leydig cells. The increase in total lipid in the germ cells following PRL treatment was contributed by mono, di- and triacyl glycerols and free cholesterol. However, an opposite effect of PRL was evident in the Leydig cells of mature monkeys, where the cholesterols and glyceride fractions registered a decrease. The reduced cholesterol fractions in the Leydig cells following PRL treatment suggests the utilization of cholesterol for steroidogenesis. Sertoli cells were found to be comparatively resistant to change in PRL status. Bromocryptine treatment brought about the opposite effect of PRL in almost all parameters studied in both immature and mature monkeys. In general, these findings with prolactin suggests that PRL has a specific and definite influence on testicular neutral lipids and the response of different cellular compartments was found to vary.     

Efflux of cholesterol from different cellular pools

Free cholesterol is very efficiently removed from cells by 2-hydroxypropyl-beta-cyclodextrins. The efflux of cholesterol occurs from two distinct kinetic pools: the half-times (t(1/2)) for the two pools in CHO-K1 cells are 15 +/- 5 s and 21 +/- 6 min and they represent 25% +/- 5% and 75% +/- 5% of the readily exchangeable cell cholesterol, respectively. In this study we have determined that the fast pool and the majority of the slow kinetic pool for cholesterol efflux are apparently present in the plasma membrane. Numerous agents that inhibit intracellular cholesterol trafficking are unable to affect either the size or the t(1/2) for efflux of either kinetic pool. In contrast, treatment of the cells with N-ethylmaleimide (NEM), exogenous lipases such as sphingomyelinase and phospholipase C, calcium ionophore A23187, or heat resulted in the dramatic increase in the size of the fast kinetic pool of cholesterol. These changes in the kinetics of cholesterol efflux are not specific to the nature of the extracellular acceptor indicating that they are a consequence of changes in the cell plasma membrane. The above treatments disrupt the normal organization of the lipids in the plasma membrane via either hydrolysis or randomization. The phosphatidylcholine and sphingomyelin present in the plasma membrane are critical for maintaining the two kinetic pools of cholesterol; any alteration in the amount or the location of these phospholipids results in an enhancement of efflux by redistributing cholesterol into the fast kinetic pool.     

Carbonic Anhydrase Activity in Isolated Chloroplasts of Wild-Type and High-CO2-Dependent Mutants of Chlamydomonas reinhardtii as Studied by a New Assay

In an assay of carbonic anhydrase (CA), NAH14CO3 soltution at the bottom of a sealed vessel releases 14CO2, which diffuses to the top of the vessel to be assimilated by photosynthesizing Chlamydomonas reinhardtii cells that have been adapted to a low-CO2 environment. The assay is initiated by illuminating the cells and is stopped by turning the light off and killing the cells with acid. Enzyme activity was estimated from acid-stable radioactivity. With bovine CA, 1.5 Wilbur-Anderson units (WAU) was consistently measured at 5- to 6-fold above background. Sonicated whole cells of air-adapted wild-type C. reinhardtii had 740 [plus or minus] 12.4 WAU/mg chlorophyll (Chl). Sonicated chloroplasts from a mixotrophically grown wall-less strain, cw-15, had 35.5 [plus or minus] 2.6 WAU/mg Chl, whereas chloroplasts from wall-less external CA mutant strain cia5/cw-15 had 33.8 [plus or minus] 1.9 WAU/mg Chl. Sonicated chloroplasts from the wall-less mutant strain cia-3/cw-15, believed to lack an internal CA, had 2.8 [plus or minus] 3.2 WAU/mg Chl. Sonicated whole cells from cia3/cw-15 had 2.8 [plus or minus] 7.8 WAU/mg Chl. Acetazolamide, ethoxyzolamide, and p-aminomethylbenzene sulfonamide (Mafenide) at 100 [mu]M inhibited CA in sonicated chloroplasts from cia-5/cw-15. Treatment at 80[deg]C for 10 min inhibited this CA activity by 90.8 [plus or minus] 3.6%. Thus, a sensitive 14C assay has confirmed the presence of a CA in cw-15 and cia-5/cw-15 chloroplasts and the lack of a CA in cia-3/cw-15 chloroplasts. Our results indicate that HCO3- is the inorganic carbon species that is accumulated by chloroplasts of Chlamydomonas and that chloroplastic CA is responsible for the majority of internal CA activity.     

Effect of different durations of exercise on transferrin-bound iron uptake by rat erythroblast

This study evaluated effects of different durations of exercise on transferrin receptor (TfR) expression on the membrane of rat erythroblasts. Female rats were assigned to six groups: 3, 6 and 12 months of strenuous exercise (swimming 2 h/day, 5 days/wk) groups and their corresponding controls. At the end of experiments, the erythroblasts were isolated for Tf binding assay and transferrin-bound iron (Tf-Fe) uptake. Tissue non-heme iron and hematological iron indices were also measured. The TfR number on the cells was about 603,189 plus minus 107,562, 890,150 plus minus 164,849 and 384,695 plus minus 46,295 molecules/cell in three control groups (3, 6, 12 months) respectively. Exercise groups had significantly higher levels of TfR than those of the control groups, being 1,374,137 plus minus 243,677, 2,175,360 plus minus 462,737 and 1,012,759 plus minus 249,423 molecules/cell in 3, 6 and 12 months of exercise groups respectively (p < 0.05). After 30 min of incubation, cellular Tf approached to levels of 8.28 plus minus 1.94, 10.73 plus minus 3.30 and 6.60 plus minus 0.93 fmole/10(6) cells in 3, 6 and 12 months of exercise groups, while the corresponding control values were 3.09 plus minus 0.36, 5.03 plus minus 1.01 and 2.51 plus minus 0.88 fmole/10(6) cells respectively (all P < 0.05). The rates of cellular iron accumulation were 7.07 (3), 8.79 (6) and 5.96 (12 month) fmole/10(6) cells/min in the exercised rats and 2.91, 3.85, and 2.03 fmole/10(6) cells/min in their corresponding controls (all p < 0.05). However, no significant difference was observed in the ratios (Exercise/Corresponding control) of the increased TfR expression, Tf-Fe uptake and Tf endocytosis as well as of the decreased plasma iron and tissue non-heme iron levels induced by different periods of exercise. Furthermore, the increase in the length of exercise (6 or 12 month) did not induce a remarkable decrease in plasma hemoglobin and hematocrit. These results indicate that a true iron deficiency or 'sport anemia' can not develop even if under longer periods (6 or 12 month) of strenuous exercise.     

Changes induced by sarcolysine in vivo in the composition of DNA-bound lipids in sarcoma 37

The two DNA fractions were isolated from sarcoma 37 by the use of the phenol method: supramolecular complex of DNA (SC DNA, 60%) and "phenol" nuclear matrix DNA (PNM DNA, 40%). The lipids in SC DNA represented of light and tightly bound components, the latter was similar to the lipid composition of PNM DNA. SC DNA contains 20 micrograms of neutral lipids (NL) and 6.5 micrograms of phospholipids (PL), while PNM DNA contains 9.8 micrograms of NL and 3.5 micrograms of PL per mg DNA. SC DNA-bound lipids of sarcoma 37 are deficient in free cholesterol (FC, 13%), but rich in cholesterol esters (CE, 39%) and free fatty acids (FFA, 23%); very rich in cardiolipin (CL, 43%) and phosphatidylethanolamine (PE, 28%), but deficient in phosphatidylcholine (PC, 12%). The tumor contains triglycerides (TG) that is absent in DNA of the normal cells. The injection of sarcolysine (10 micrograms/kg) markedly increased (1.5-3 times) the content of all LN and PL fractions in SC DNA, which was accompanied by both the accumulation of FC, TG, PC and the reduction of the remaining lipid fractions in PNM DNA. It is supposed, that DNA-bound lipids may be the target for the action of sarcolysine.     

Low density lipoprotein metabolism in hypertriglyceridemic and normolipidemic patients with coronary heart disease

The turnover rates of low density lipoprotein-apolipoprotein B (LDL-apoB) were determined in 32 men with coronary heart disease (CHD) and 11 control men with normal plasma lipids. Thirty patients with CHD had normal levels of LDL-cholesterol (LDL-C); of these patients, 9 had hypertriglyceridemia and 21 had normal plasma lipids. Mean concentrations of total cholesterol and LDL-C were similar among the control subjects and CHD patients, although the latter had significantly lower HDL-C. In control subjects, transport rates and fractional catabolic rates (FCR) of LDL-B were 10.6 +/- 0.5 (SEM) mg/kg-day and 0.31 +/- 0.01 pools/day, respectively. In 10 hypertriglyceridemic patients with CHD, transport rates were 21.7 +/- 1.7 mg/kg-day, and FCRs averaged 0.56 +/- 0.06 pools/day; both were significantly higher than normal (P less than 0.05). Six normolipidemic patients also had abnormally high transport rates of LDL-apoB (19.4 +/- 2.8 mg/kg-day) and FCRs (0.51 +/- 0.03 pools/day); again both were higher than normal. The remaining 16 normolipidemic patients with CHD had normal transport rates (9.9 +/- 0.6 mg/kg-day) and FCRs (0.28 +/- 0.01 pools/day). Thus, hypertriglyceridemic patients with CHD and a portion of normolipidemic patients with CHD were characterized by increases in both transport and fractional catabolic rate of LDL-apoB; these abnormalities in LDL metabolism may have contributed to their coronary heart disease. However, the majority of normolipidemic patients with CHD did not show a distinct defect in their LDL metabolism.     

Postcopulatory effects of two antifertility agents on ova transport and implantation

The daily doses which prevented implantation in 50 percent of treated animals (ED50) of 2,3 - bis (4-hydroxyphenol) valeronitrile (SC-3402) and 2,3 - bis (4-methoxyphenyl) pent-2-enenitrile (SC-3296) injected in rats s on Days 1 to 3, or Days 4 to 7, or Days 1 to 7 (Day 1 = pregnancy) were 100, 200, and 40 mcg and 50, 100, and 12 mcg respectively, ED50 doses of estrone were 4,8 and 3.5 mcg. Control animals showed ova in the oviduct only on Days 1, 2 and 3, also in the uterus on Day 4, and only in the uterus on Day 5. Very few ova were found in rats treated with 10 mcg estrone daily Day 1-2 and autopsied on Day 3. The same treatment period with 200 mcg SC-3402 caused similar results. 64 mcg SC-3402 resulted in a smaller reduction of ova. Acceleratory potency of 200 mcg SC-3402 is greater than can be due to its estrogenic activity equivalent, 0.5 mcg estrone; that of 64 mcg SC-3296 (4.8 equivalents estrone) can be so ascribed. Rats receiving daily 4-8 mg 17 alpha-acetoxy-6 alpha-methylprogesterone (MAP) from Day 1 to 9 to delay nidation, and 200 mcg SC-3402, autopsied on Day 10 showed no free blastocysts and a few implantation sites in the process of resorption (Free blastocysts were found in rats similarly treated but with ligation of the uterus at the cervix on Day 5 to prevent expulsion of blastocysts). Control rats on Day 10 showed a few implantation sites and free blastocysts. The normal number of implantations were present in SC-3296 treated rats. The average weight of cornu traumatized by threading one cornu in psuedopregnant rats with a silk thread on Day 5 (Day 1=cervical stimulus) in rats treated with 200 mcg SC-3402 on Days 5-8, 404 plus or minus 50 mg was significantly (P less than .05) lower than mean control weight, 794 plus or minus 48 mg. The difference between the mean weight of non-traumatized cornu of rats given 100 mcg, 284 plus or minus 36 and 232 plus or minus 12 mg respectively was significantly (P less than .05) greater than in controls, 159 plus or minus 5.8 mg. The deciduoma-inhibiting activity of SC-3402 is further evidence that it initiates nidation but impedes early implantation stages.     

Synthesis and coenzyme A-dependent esterification of cholesterol in rat intestinal epithelium. Differences in cellular localization and mechanisms of regulation

The current studies were undertaken to characterize the localization and regulation of cholesterol synthesis and acyl-CoA:cholesterol acyltransferase activity in rat intestinal crypt and villus cells. Both parameters were determined in groups of animals with widely varying sterol fluxes across the intestinal mucosa. In animals on control diet the rates of cholesterol synthesis, measured by the incorporation of [3H]water per mg of protein, were similar along the villus/crypt axis in the jejunum, whereas in the ileum, villus cells were significantly more active than crypt cells. In both areas, however, the majority of total synthetic activity was found in cells from the crypts and lower villi. In contrast, the highest specific and total acyl-CoA:cholesterol acyltransferase activity was recovered in the villus cells of the jejunum and ileum. Dietary cholesterol did not affect sterol synthesis in any of the cell fractions but increased acyl-CoA:cholesterol acyltransferase activity approximately 2-fold in jejunal cell fractions. Inhibition of cholesterol absorption or sequestration of intestinal bile acids stimulated sterol synthetic activity up to 7-fold, and this occurred mainly in the lower villus and crypt cells in both jejunum and ileum. An increased demand for lipoprotein cholesterol, generated by triglyceride feeding, similarly was associated with enhanced synthetic rates. However, unlike cholesterol feeding, these manipulations did not increase acyl-CoA:cholesterol acyltransferase activity in any of the villus cell fractions. These studies suggest, therefore, that the intracellular pools of cholesterol that regulate the rate of cholesterol synthesis and the rate of cholesterol esterification are functionally distinct.     

Effects of thrombin on washed, human platelets: changes in the subcellular fractions.

Pressure homogenization and subcellular fractionation has been performed on washed, human platelets and platelets treated with thrombin to undergo the so-called release reaction. Electron microscopy revealed that the particulate zones obtained from the control sample corresponded to membrane vesicles (B), small storage granules (D) as well as mitochondria and larger storage granules (E). Only a few storage granules could be observed in the particulate zones isolated from thrombin-treated platelets. Visual comparison of the sucrose gradient patterns revealed that one granule fraction (D) had disappeared from the thrombin-treated sample. Sodium dodecysulfate-polyacrylamide gel electrophoresis showed a major protein band (mol. wt 145 500 plus or minus 1000) in the extracellular phase (supernatant after removal of the platelets) of the thrombin-treated sample and in the granule fractions (D and E) of the control (mol. wt 147 000 plus or minus 1000). Incubation of whole, washed platelets with thrombin for 5 min at 37 degrees C followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis of the isolated membrane fraction revealed no reproducible differences in the protein band pattern compared to membranes isolated from control platelets. However, after treatment with thrombin for 30 min, a protein band (mol. wt 183 000 plus or minus 3500) had disappeared. The distribution of protein and beta-N-acetylglucosaminidase activity among the subcellular fractions were measured. Both were mainly recovered in the soluble fraction (greater than 77%). The granule fractions, D and E of the control contained 3.0% plus or minus 0.8% and 6.4% plus or minus 1.3% of the total amount of beta-N-acetylglucosaminidase in the gradient. Fraction E of the thrombin-treated cells contained 3.3% plus or minus 1.0% of total while fraction D was lacking.     

Effect of mercury chloride on the level of lipids and products of their peroxidation in rat vital organs

The influence of mercury chloride on peroxidation processes of lipids and level of common lipids, phospholipids and spectrum of neutral lipids in liver, heart, lung and kidney of rats has been investigated. Administration of mercury chloride in a dose 0.7 mg/100 g of body weight to animals has invoked accumulation of lipids peroxide products in fractions of neutral lipids and phospholipids so it testifies the development of oxidative stress. Decrease of the most sensitive to oxidation fractions in the early stages of oxidative stress development and increase of free cholesterol and its ethers content in kidney and free cholesterol in the heart in more later terms as a result of mercury chloride administration have been revealed.     

Defined protein and animal component-free NS0 fed-batch culture

A chemically defined protein and animal component-free fed-batch process for an NS0 cell line producing a human IgG(1) antibody has been developed. The fed-batch feed profile was optimised in a step-wise manner. Depletion of measurable compounds was determined by direct analysis. The cellular need for non-measurable compounds was tested by continued culturing of cell suspension, removed from the bioreactor, in shake-flasks supplemented with critical substances. In the final fed-batch culture, 8.4 x 10(6) viable cells mL(-1) and 625 mg antibody L(-1) was obtained as compared to 2.3 x 10(6) cells mL(-1) and 70 mg antibody L(-1) in batch. The increase in cell density, in combination with a prolonged declining phase where antibody formation continued, resulted in a 6.2-fold increase in total cell yield, a 10.5-fold increase in viable cell hours and an 11.4-fold increase in product yield. These improvements were obtained by using a feed with glucose, glutamine, amino acids, lipids, sodium selenite, ethanolamine and vitamins. Specifically, supplementation with lipids (cholesterol) had a drastic effect on the maximum viable cell density. Calcium, magnesium and potassium were not depleted and a feed also containing iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield. The growth and death profiles in the final fed-batch indicated that nutrient deprivation was not the main cause of cell death. The ammonium concentration and the osmolality increased to potentially inhibitory levels, but an imbalance in the supply of growth/survival factors may also contribute to termination of the culture.     

Omega-3 fatty acids increase the arachidonic acid content of liver cholesterol ester and plasma triacylglycerol fractions in the rat.

Recent studies have demonstrated that dietary fish oils rich in eicosapentaenoic acid (C20:5,omega 3) lower the content of arachidonic acid and its metabolites in plasma and tissue phospholipids. The present study examined the fatty acid composition of cholesterol ester and triacylglycerol fractions from plasma and livers of rats fed diets enriched with saturated fatty acids (beef tallow), alpha-linolenic acid (linseed oil) or eicosapentaenoic acid (fish oil). Feeding diets containing linseed oil or fish oil for 28 days increased arachidonic acid (C20:4,omega 6) levels in the cholesterol ester fraction of liver and in the triacylglycerol fraction of the plasma lipids. Plasma cholesterol esters were depleted of C20:4,omega 6 after feeding of the diet containing either linseed oil or fish oil. The changes in C20:4,omega 6 content cannot be explained by alterations in cholesterol ester or triacylglycerol pools of plasma and liver. These results suggest that the decrease in phospholipid C20:4,omega 6 content generally observed after fish oil consumption may be partly due to a shift of C20:4,omega 6 from phospholipid to the triacylglycerol and/or cholesterol ester pools in the same tissue. Triacylglycerols and cholesterol esters may therefore play a buffering role in the homeostatic maintenance of tissue phospholipid levels of arachidonic acid.     

Effects of enrichment of fibroblasts with unesterified cholesterol on the efflux of cellular lipids to apolipoprotein A-I.

This study elucidates the factors underlying the enhancement in efflux of human fibroblast unesterified cholesterol and phospholipid (PL) by lipid-free apolipoprotein (apo) A-I that is induced by cholesterol enrichment of the cells. Doubling the unesterified cholesterol content of the plasma membrane by incubation for 24 h with low density lipoprotein and lipid/cholesterol dispersions increases the pools of PL and cholesterol available for removal by apoA-I from about 0.8-5%; the initial rates of mass release of cholesterol and PL are both increased about 6-fold. Expression of the ATP binding cassette transporter A1 (ABCA1) is critical for this increased efflux of lipids, and cholesterol loading of the fibroblasts over 24 h increases ABCA1 mRNA about 12-fold. The presence of more ABCA1 and cholesterol in the plasma membrane results in a 2-fold increase in the level of specific binding of apoA-I to the cells with no change in binding affinity. Characterization of the species released from either control or cholesterol-enriched cells indicates that the plasma membrane domains from which lipids are removed are cholesterol-enriched with respect to the average plasma membrane composition. Cholesterol enrichment of fibroblasts also affects PL synthesis, and this leads to enhanced release of phosphatidylcholine (PC) relative to sphingomyelin (SM); the ratios of PC to SM solubilized from control and cholesterol-enriched fibroblasts are approximately 2/1 and 5/1, respectively. Biosynthesis of PC is critical for this preferential release of PC and the enhanced cholesterol efflux because inhibition of PC synthesis by choline depletion reduces cholesterol efflux from cholesterol-enriched cells. Overall, it is clear that enrichment of fibroblasts with unesterified cholesterol enhances efflux of cholesterol and PL to apoA-I because of three effects, 1) increased PC biosynthesis, 2) increased PC transport via ABCA1, and 3) increased cholesterol in the plasma membrane.