DECREASED DENSITIES OF INTRAMEMBRANOUS PARTICLES AND CYTOCHEMICALLY DETECTABLE CHOLESTEROL IN MICROVILLI OF STARVED RAT ENTEROCYTES

The densities of intramembranous particles (IMPs) and of sterol complexes induced by treatment of filipin were studied by freeze-fracture replication of intact intestine and/or isolated brush border membranes (BBM) of well-fed and starved rats. The density of IMPs and filipin-sterol complexes (FSCs) decrease considerably during starvation. Biochemical estimations show a decrease in the levels of cholesterol and proteins with respect to phospholipids during starvation which is in agreement with morphological findings. It is suggested that these changes may play a role in regulating membrane fluidity which in turn affects absorption of nutrients through BBM.     

Increased Na(+)-dependent D-glucose transport and altered lipid composition in renal cortical brush-border membrane vesicles from bile duct-ligated rats.

Erythrocyte membranes of patients with liver disease are characteristically enriched in cholesterol, a change known to impair several carrier-mediated membrane transport functions. In the present study we have assessed whether experimental liver disease can affect the membrane lipid composition and transport function of kidney epithelial cells. Small (about 5%) but significant (P less than 0.01) increases were found in the cholesterol-to-phospholipid molar ratio (C/PL) of rat renal cortical brush-border membrane (BBM) vesicles 3, 8, and 15 days after bile duct ligation which correlated closely with increased fluorescence polarization, i.e., decreased membrane fluidity (r = 0.75, P less than 0.001; n = 27). A lipoprotein-mediated pathogenesis was suggested by the close relationship between BBM C/PL and plasma C/PL (r = 0.69, P less than 0.001). The mean high-affinity Na(+)-coupled D-glucose uptake by BBM vesicles was higher 1, 3, 8, and 15 days after ligation than in non-operated rats, significantly so at 3 and 8 days (611 +/- 37 and 593 +/- 22 vs. 507 +/- 21 pmol/mg protein per 4 sec; P less than 0.05), and was positively correlated with BBM C/PL (r = 0.58, P less than 0.01) and fluorescence polarization (r = 0.41, P less than 0.05). Brief incubation of BBM vesicles from normal rats with cholesterol-rich phospholipid liposomes simultaneously increased BBM C/PL and Na(+)-dependent D-glucose uptake. Stimulation of BBM Na(+)-glucose cotransport in ligated rats was not due to delayed dissipation of the Na+ gradient or to a more rapid development of membrane potential. High-affinity Na(+)-dependent D-glucose uptake kinetics in 3-day bile duct-ligated rats showed a lower Kt, without an alteration in maximum velocity, Vmax, compared to sham-operated animals (0.298 +/- 0.015 vs. 0.382 +/- 0.029 mM; P less than 0.05), whilst the binding dissociation constant, Kd of high-affinity phlorizin binding sites was reduced by ligation (0.453 +/- 0.013 vs. 0.560 +/- 0.015 microM; P less than 0.001). We conclude that an early effect of bile duct ligation is to enrich renal cortical brush-border membranes in cholesterol, thereby decreasing membrane fluidity and stimulating Na(+)-dependent D-glucose uptake by increasing the affinity of the carrier.     

Static and dynamic components of renal cortical brush border and basolateral membrane fluidity: Role of cholesterol

Summary Static polarization and differential polarized phase fluorimetry studies on rat renal cortical brush border (BBM) and basolateral membranes (BLM) were undertaken to determine the membrane components responsible for differences in BBM and BLM fluidity, whether these differences were due to the order or dynamic components of membrane fluidity and if a fluidity gradient existed within the bilayer. Surface membrane proteins rigidified both BBM and BLM fluidity. Neutral lipid extraction, on the other hand, caused a larger decrease in BBM than BLM fluorescence polarization (0.104vs. 0.60,P<0.01) using diphenyl hexatriene (DPH). Cholesterol addition to phospholipid fractions restored membrane fluidity to total lipid values in both BBM and BLM phospholipids. The response to cholesterol in the BBM was biphasic, while the BLM response was linear. Lateral mobility, quantitated using dipyrenylpropane, was similar in both BBM and BLM fractions at 35°C. BBM and BLM differed primarily in the order component of membrane fluidity as DPH-limiting anisotropy (r ) (0.212vs. 0.154,P<0.01) differed markedly between the two membrane fractions. The two membrane components also differed with respect to 2 and 12-anthroyloxy stearate (2-AS, 12-AS) probes, indicating a difference in the dynamic component of membrane fluidity may also be present. DPH and 12-As probes were also used to quantitate inner core membrane fluidity and showed the BBM was less fluid than the BLM for intact membranes, total lipid extracts and phospholipids. Results obtained using the surface membrane probes trimethylammonium-DPH (TMA-DPH) and 2-AS suggested a fluidity gradient existed in both BBM and BLM bilayers with the inner core being more fluid in both membranes. These data indicate cholesterol is in large part responsible for fluidity differences between BBM and BLM and that these membranes, while clearly differing in the order component of membrane fluidity, may also difer in the dynamic component as well.     

Thyroid hormones stimulate Na+–Pi transport activity in rat renal brush-border membranes: Role of membrane lipid composition and fluidity

Antecedent studies have suggested that lipid composition and fluidity of cellular membranes of various organs are altered in response to thyroid hormone status. To date, the effects of thyroid hormone status on these parameters have not been examined in rat renal apical membrane in regard to sodium-dependent phosphate transport. In the present study, we determined the potential role of alterations in cortical brush-border membrane lipid composition and fluidity in modulation of Na⁺–P_i transport activity in response to thyroid hormone status. Thyroid hormone status influences the fractional excretion of Pi, which is associated with alteration in renal brush-border membrane phosphate transport. The increment in Na⁺–P_i transport in renal BBMV isolated from Hyper-T rats is manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport. Further, the cholesterol content was significantly increased in renal BBM of Hypo-T rats and decreased in Hyper-T rats as compared to the Eu-T rats. The molar ratio of cholesterol/phospholipids was also higher in renal BBM from hypo-T rats. Subsequently, fluorescence anisotropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the renal BBM of the Hyper-T rats and increased in the Hypo-T rats as compared to Eu-T rats. The result of this study, therefore, suggest that alteration in renal BBM cholesterol, cholesterol/phospholipid molar ratio, and membrane fluidity play an important role in the modulation of renal BBM Na⁺–P_i transport in response to thyroid hormone status of animals. (Mol Cell Biochem 268: 75–82, 2005)     

Molecular basis of renal handling of calcium in response to thyroid hormone status of rat

We investigated the effect of thyroid hormone status on renal handing of Ca2+. Further, like kinetics of Ca2+ transport across brush-border membrane (BBM) and basolateral membrane (BLM) of renal epithelial cells was carried out. FE(Ca) was decreased in hyperthyroid (Hyper-T) rats and increased in hypothyroid (Hypo-T) rats as compared to euthyroid (Eu-T) rats. Ca2+ uptake into renal brush-border membrane vesicles (BBMV) was increased in Hyper-T rats and decreased in Hypo-T rats as compared to Eu-T rats. K(m) was lower in Hyper-T rats and higher in Hypo-T rats as compared to Eu-T rats whereas, V(max) remained unaltered. The transition temperature for calcium uptake varied inversely with the thyroid hormone status. Renal BBM of Hyper-T rats showed decreased anisotropy and polarisation of DPH as compared to EU-T rats whereas these values were increased in Hypo-T rats. Thus, the altered BBM fluidity appears to modulate Ca2+ transport across BBM. Na+/Ca2+ exchange activity of renal cells was increased in Hyper-T and decreased in Hypo-T rats as compared to Eu-T rats. V(max) for Na+/Ca2+ exchange was increased in Hyper-T rats and deceased in Hypo-T rats as compared to Eu-T rats, whereas, [Na+](0.5) was similar in all three groups. The c-AMP levels of renal cortex of Hyper-T rats was increased and that of Hypo-T rats decreased as compared to Eu-T rats. Thus, thyroid hormones increased Ca2+ reabsorption in the kidney of rat. Thyroid hormone-mediated modulation of BBM fluidity appears to stimulate Ca2+ uptake into renal BBMV. Thyroid hormones possibly activated the Na+/Ca2+ exchanger through cAMP-dependent pathway.     

Regional variations in intestinal brush border membrane fluidity and function during diabetes and the role of oxidative stress and non-enzymatic glycation

The physical state (fluidity) of lipids modulates the activities of several membrane bound enzymes and transport proteins. Alteration of brush border membrane (BBM) fluidity is one of the several changes exhibited by the small intestine during diabetes. In the present study, an investigation of the diabetes induced regional changes in fluidity, oxidative damage, non-enzymatic glycation as well as the activities and the kinetic parameters of the enzymes alkaline phosphatase and -glutamyl transpeptidase was carried out on the intestinal BBM. At the end of 6 weeks of diabetes, significant increases in the extent of both oxidative damage and non-enzymatic glycation were observed along the length of the intestine along with a simultaneous decrease in membrane fluidity. A significant correlation between the decrease in BBM fluidity and increase in non-enzymatic glycation was observed in the duodenum and jejunum. Additionally regional variations in the activities and kinetic parameters of both the enzymes were observed.     

Effect of pregnancy, postnatal growth, and gender on renal sulfate transport.

Serum sulfate concentrations are increased in infants, young children, and pregnant women, compared with adult values. The objective of this investigation was to examine the influences of age, gender, and pregnancy on renal sulfate transport using guinea pigs as an animal model. Membrane vesicles were isolated from the kidney cortex of male animals at four different ages, from male and female adult animals, and from pregnant and nonpregnant female animals. There were no significant differences in marker enzymes for the brush-border membrane (BBM) or basolateral membrane (BLM) among all groups examined. Uptake was determined by a rapid filtration method and membrane fluidity by measuring the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. The Vmax values for Na+ /sulfate co-transport in BBM were significantly increased with decreasing age, whereas the Km for this process was unchanged. The Vmax and Km for Na + /sulfate co-transport in BBM of pregnant animals were significantly higher than the values in the nonpregnant group. Bicarbonate-driven anion exchange of sulfate in BLM was not different among the different age groups. The Vmax for the bicarbonate/sulfate exchange process in BLM was not different between pregnant and nonpregnant groups; however, the Km for this process in BLM of pregnant animals was significantly greater than the value in nonpregnant animals. There were no gender-related differences in sulfate transport in BBM or BLM isolated from adult male and female animals. Renal BBM fluidity was increased with decreasing age and in pregnant animals, suggesting that altered membrane fluidity may represent one possible mechanism to explain the increased sodium/sulfate uptake in young and pregnant animals. The higher Vmax for Na+/sulfate co-transport in young and pregnant animals suggests that there is an increased density of co-transporter protein or an increase in the rate of movement of the carrier protein (i.e., turnover) once loaded with sodium and sulfate. This increased conservation of inorganic sulfate in young and pregnant guinea pigs may be related to the increased demand for sulfated substrates, such as sulfated glycosaminoglycans, during growth and development.     

Some physiological observations on the uptake of d-glucose and 2-deoxy-d-glucose by starving and exponentially-growing yeasts

Some methods for measuring the uptake of sugars by yeasts were investigated critically. A study was made of the effects of starvation of Pichia pinus, Candida utilis, Saccharomyces cerevisiae and Rhodosporidium toruloides on their uptake of d-glucose and 2-deoxy-d-glucose. Marked changes in the rates of uptake of these sugars occurred during 10 h of starvation, including (a) an immediate increase of up to 75% above that for growing cells and (b) a continuous decline to as little as 4%. Each yeast behaved differently. The rates did not remain constant during the periods of starvation often used for studies on the transport of sugars into yeasts. For Pichia pinus, there were striking differences, associated with starvation, between the transport of 2-deoxy-d-glucose and d-glucose, despite evidence that the two sugars enter this yeast by means of the same carrier. Some physiological explanations for these findings are discussed.     

Use of phospholipid transfer protein as a probe to study the lipid dynamics and alkaline phosphatase activity in the brush border membrane of human term placenta

Incubation of placental brush border membrane (BBM) along with sonicated vesicles of exogenous lipids (egg yolk PC) in the presence of phospholipid-transfer protein (PL-TP) showed a decrease in the alkaline phosphatase activity due to the change in the membrane micro-environment, such as fluidity. Effect of substrate concentration was tested by Lineweaver-Burk plot, which showed decreased V(max) and K(M). The effect of temperature was probed by the Arrhenius plot, which showed no change in transition temperature, but a decline in the energy of activation both below and above the transition temperature. The protein-catalyzed transfer of phospholipid from the donor unilamellar vesicles resulted in a substantial increase in the BBM phospholipid and a net decrease in cholesterol/phospholipid molar ratio. The change in membrane fluidity was assessed by translational as well as rotational diffusion of membrane extrinsic fluorescent probes, pyrene and diphenyl-hexatriene. An increased lateral mobility was recorded by the increased pyrene excimer formation. A decrease in fluorescent polarization of diphenyl-hexatriene was observed, which led to the decrease in fluorescence anisotropy and order parameter, and therefore, an increase in membrane fluidity (rotational diffusion). Mean anisotropy parameter was also decreased in the presence of PL-TP. Thus, the placental BBM alkaline phosphatase activity showed a distinct lipid dependence which may have important physiological consequences.     

Regulation of chloride transport in parotid secretory granules by membrane fluidity.

Zymogen granule membranes contain Cl- conductance and Cl/anion exchange activities that become important for primary fluid production after fusion with the apical plasma membrane of the acinar cell. We have used steady-state fluorescence anisotropy of diphenylhexatriene derivatives and measurements of Cl- transport in isolated secretory granules to determine the contribution of membrane fluidity to the regulation of transport across the granule membrane. Secretory granules from several unstimulated glands (rat pancreas and parotid, rabbit gastric glands) were shown to have low membrane fluidity compared to plasma membranes. In addition, Cl- transport activity in different granule preparations showed a strong correlation to the membrane fluidity when measured with 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), but not with 3-[p-(6-phenyl)-1,3,5-hexatrienyl)-phenyl]propionic acid (PA-DPH). These data suggest that TMA-DPH preferentially partitions into a specific lipid environment associated with, or which exerts an influence on, the Cl- transport proteins and that increases in the fluidity of this environment are associated with higher transport rates. Data from other types of plasma membranes indicate that TMA-DPH partitions much more than PA-DPH into the cytoplasmic leaflet, suggesting that this part of the granule membrane is involved in the observed fluidity changes. Furthermore, increasing the bulk membrane fluidity with the local anesthetics benzyl alcohol and n-alkanols increased the Cl- transport rates up to 10-fold. This increase was apparently through specific transporters as anion selectivity was maintained in spite of the higher absolute rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyroid hormones increase Na+–Pi co-transport activity in intestinal brush border membrane: Role of membrane lipid composition and fluidity

In the present study, we documented the promising role of thyroid hormones status in animals in modulation of Na⁺–P_i transport activity in intestinal brush border membrane vesicles (BBMV) which was accompanied with alterations in BBM lipid composition and fluidity. Augmentation of net P_i balance in hyperthyroid (Hyper-T) rats was fraternized with accretion of P_i transport across BBMV isolated from intestine of Hyper-T rats as compared to hypothyroid (Hypo-T) and euthyroid (Eu-T) rats while Na⁺–P_i transport across BBMV was decreased in Hypo-T rats relative to Eu-T rats. Increment in Na⁺–P_i transport in intestinal BBMV isolated from Hyper-T rats was manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport system. Furthermore, BBMV lipid composition profile in intestinal BBM from Hyper-T was altered to that of Hypo-T rats and Eu-T rats. The molar ratio of cholesterol/phospholipids was higher in intestinal BBM from Hypo-T rats. Fluorescence anistropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the intestinal BBM of Hyper-T rats and decreased in Hypo-T rats as compared to Eu-T rats which corroborated with the alteration in membrane fluidity in response to thyroid hormone status of animals. Therefore, thyroid hormone mediated change in membrane fluidity might play an important role in modulating Na⁺–P_i transport activity of intestinal BBM. (Mol Cell Biochem 278: 195–202, 2005)     

EPR spin labeling measurements of thylakoid membrane fluidity during barley leaf senescence

Physical properties of thylakoid membranes isolated from barley were investigated by the electron paramagnetic resonance (EPR) spin labeling technique. EPR spectra of stearic acid spin labels 5-SASL and 16-SASL were measured as a function of temperature in secondary barley leaves during natural and dark-induced senescence. Oxygen transport parameter was determined from the power saturation curves of the spin labels obtained in the presence and absence of molecular oxygen at 25 °C. Parameters of EPR spectra of both spin labels showed an increase in the thylakoid membrane fluidity during senescence, in the headgroup area of the membrane, as well as in its interior. The oxygen transport parameter also increased with age of barley, indicating easier diffusion of oxygen within the membrane and its higher fluidity. The data are consistent with age-related changes of the spin label parameters obtained directly by EPR spectroscopy. Similar outcome was also observed when senescence was induced in mature secondary barley leaves by dark incubation. Such leaves showed higher membrane fluidity in comparison with leaves of the same age, grown under light conditions. Changes in the membrane fluidity of barley secondary leaves were compared with changes in the levels of carotenoids (car) and proteins, which are known to modify membrane fluidity. Determination of total car and proteins showed linear decrease in their level with senescence. The results indicate that thylakoid membrane fluidity of barley leaves increases with senescence; the changes are accompanied with a decrease in the content of car and proteins, which could be a contributing factor.     

Effects of Two Highly Monounsaturated Oils on Lipid Composition and Enzyme Activities in Rat Jejunum

The effects of two monounsaturated fatty acid (MUFA) oils, olive oil (OO)and high-oleic sunflower oil (HOSO), with high content in oleic acid butdiffering in their non-fatty acid fraction, on brush-border membrane(BBM) lipid composition and fluidity and on mucosal enzyme activitiesof rat jejunum were studied. Animals were given semipurified diet withlinoleic acid to prevent essential fatty acid deficiency (control group)or semipurified diet containing 10% of either OO or HOSO for 12weeks. There was a significant decrease in the content of jejunalBBM phospholipids together with an increase in the level of freecholesterol in both oil-fed rats, when compared to controlgroup. Although the increase in the BBM free cholesterol levelwas not statistically significant in HOSO-fed rats, a significantdecrease in the phospholipid/free cholesterol ratio was found inboth OO and HOSO-fed animals compared to control group. Rat jejunalBBM had a high level of free fatty acids which was increased in BBMisolated from OO and HOSO-fed animals. There was no statisticalsignificant difference in the phospholipid distribution between thecontrol and the OO group. However, HOSO-fed animals showed the lowestlevel of phosphatidylethanolamine together with the highestphosphatidylcholine content and the phosphatidylcholine/sphingomyelinratio. The fatty acid pattern of jejunal BBM lipids was modifiedaccording to the major fatty acids in the oils. There was a decreasein both stearic acid (18:0) and linoleic acid (18:2 n-6), togetherwith an increase in oleic acid (18:1 n-9) in jenunal BBM isolatedfrom both oil experimental groups. All these results were accompaniedby a significant increase in the BBM fluidity (as assessed bysteady-state fluorescence polarization of diphenylhexatriene) isolatedfrom oil-fed rat, when compared to control group. OO and HOSO-fedanimals had the lowest activities of sucrase and maltase, whilealkaline phosphatase activity only was decreased in HOSO-fedanimals. The specific activity of maltase was not modified in anyexperimental rats. In summary, both MUFA oils induced similar effectson jejunal BBM lipid composition, fluidity, sucrase, maltase andlactase activities. Furthermore, HOSO intake resulted in a lowestalkaline phosphatase activity which was accompanied by changes inindividual phospholipid composition. All these results suggest thateffects of MUFA oils on jejunal BBM lipid composition and hydrolaseactivities are most likely due to the presence of high content ofoleic acid rather than other components contained in the non-fattyacid of olive oil.     

Ectopic expression of alkaline phosphatase in proximal tubular brush border membrane of human renal cell carcinoma

The present study was conducted to find out any alteration in the expression and activity of alkaline phosphatase in the brush border membrane (BBM) from renal cell carcinoma (RCC) in comparison to normal renal BBM. The specific activity of alkaline phosphatase was drastically reduced in homogenate as well as BBM from RCC kidney when compared to ALP activity in BBM of normal kidney. Kinetic studies revealed that diminished activity of alkaline phosphatase in BBM isolated from RCC was fraternized with decrease in maximal velocity (V(max)) and increase in affinity constant (K(m)) of the enzyme. SDS-PAGE studies showed that the BBM proteins having molecular weights ranging from 95 to 170 kDa were poorly expressed in RCC BBM in relative to normal kidney BBM. Incubation of SDS-PAGE gel with BCIP/NBT dye clearly showed that the expression of ALP in tumor renal BBM was markedly reduced as compared to normal kidney. Further, Western blot analysis using anti-alkaline phosphatase antibody also confirmed the reduced expression of ALP in tumor renal BBM. Lipid composition in reference to phospholipids, glycolipids and cholesterol in tumor renal BBM was altered to that of normal renal BBM, indicating alteration in membrane fluidity of tumor renal BBM.     

Horseradish peroxidase binding to intestinal brush-border membranes of Cyprinus carpio. Identification of a putative receptor

Morphologic studies have shown that the classic endocytosis tracer horseradish peroxidase (HRP) is actively internalized by vesicular transport in the carp intestine, suggesting the existence of specific binding sites in the apical membrane of enterocytes. The aim of the present study was to develop an in vitro binding assay using isolated carp intestinal brush-border membranes (BBM) to demonstrate and characterize these specific HRP binding sites. The results obtained show that HRP binding to BBM exhibits a saturable mode and high affinity (K(d) = 22 nM). In addition, HRP binding sites are highly enriched in BBM compared to basolateral membranes. On the other hand, HRP interaction with these sites is apparently of an ionic character because binding increased concomitantly with decreasing NaCl concentrations in the assay, reaching a maximum in the absence of NaCl. Other proteins that are also internalized in carp intestine did not significantly inhibit HRP binding to BBM. A lectin-type of interaction was discarded because neither manan nor ovoalbumin inhibited HRP binding. Proteinase K treatment of BBM reduced HRP binding by 70%, suggesting a proteic nature for this binding site. Finally, ligand blotting assays showed that HRP binds specifically to a 15.3-kDa protein. Taken together, these results are consistent with the existence of a functional receptor for HRP in carp intestinal mucosa that could mediate its internalization.     

Implication of BBM lipid composition and fluidity in mitigated alkaline phosphatase activity in renal cell carcinoma

Previous study has documented reduced alkaline phosphatase (ALP) activity in brush border membrane (BBM) isolated from renal cell carcinoma (RCC). Diminished activity of ALP is associated with alteration in both increased K (m) as well as decreased V (max) of enzyme suggests that there may be a change in the conformation of enzyme as well as decreased number of ALP active molecules. The present study was conducted to find out any role of BBM lipid composition and its fluidity in diminished activity of alkaline phosphatase in renal cell carcinoma. Total phospholipids and glycolipids were significantly augmented in BBM from RCC as compared to control. Fractional analysis of total phospholipids revealed significantly increased phosphatidylethanolamine. Decreased fractions of sphingomyelin and phosphatidylinositol were observed. Cholesterol-to-total phospholipid molar ratios in tumor BBM was a significantly lower in tumor BBM. A significant reduction in polarization and microviscosity was found in BBM from RCC. Therefore, we conclude that alteration in membrane lipid composition and fluidity may play a substantial role in reduced activity of ALP in RCC.     

Biochemical characterization and membrane fluidity of membranous vesicles isolated from boar seminal plasma

Mammalian seminal plasma contains membranous vesicles (MV), which differ in composition and origin. Among these particles, human prostasomes and equine prostasome-like MV have been the most studied. The aim of the present work is to characterize the biochemical composition and membrane fluidity of MV isolated from boar seminal plasma. The MV from boar seminal plasma were isolated by ultracentrifugation and further purification by gel filtration on Sephadex G-200. The MV were examined by electron microscopy (EM), amount of cholesterol, total phospholipid, protein content, and phospholipid composition were analyzed. Membrane fluidity of MV and spermatozoa were estimated from the electron spin resonance (ESR) spectra of the 5-doxilstearic acid incorporated into the vesicle membranes by the order parameter (S). The S parameter gives a measure of degree of structural order in the membrane and is defined as the ratio of the spectral anisotropy in the membranes to the maximum anisotropy obtained in a rigidly oriented system. The S parameter takes into consideration that S = 1 for a rapid spin-label motion of about only one axis and S = 0 for a rapid isotropic motion. Intermediate S values between S = 0 and S = 1 represents the consequence of decreased membrane fluidity. The EM revealed the presence of bilaminar and multilaminar electron-dense vesicles. Cholesterol to phospholipid molar ratio from the isolated MV was 1.8. Phospholipid composition showed a predominance of sphingomyelin. The S parameter for porcine MV and for boar spermatozoa was 0.73 +/- 0.02 and 0.644 +/- 0.008, respectively, with the S for MV being greater (p < 0.001) than the S for spermatozoa. The high order for S found for boar MV was in agreement with the greater cholesterol/phospholipids ratio and the lesser ratio for phosphatidylcholine/sphingomyelin. Results obtained in the present work indicate that MV isolated from boar semen share many biochemical and morphological characteristics with equine prostasome-like MV and human prostasomes. The characteristics of the porcine MV of the seminal plasma, however, differed from those of boar sperm plasma membranes.     

Effect of chronic ethanol and food deprivation on intestinal villus morphology and brush border membrane content of lipid and marker enzymes

Brush border membranes (BBM) were isolated from the jejunum and ileum of control, ad libitum (CAL); control, food-restricted (CFR); control, weight gain (CWG); and ethanol-fed (EF) rabbits. Jejunal alkaline phosphatase activity was similar among control groups, but higher in CAL than EF animals. Sucrase activity was higher in EF and CWG animals than in CAL and CFR. The alkaline phosphatase/sucrase ratio was lower in EF than control animals. Ileal enzyme marker activity was similar among EF and control animals. Sucrase (S) activity was lower in the ileum than in the jejunum. Jejunal free fatty acid and phospholipid/cholesterol (PL/C) were lower in EF than control animals, whereas ileal lipid content was generally similar among all animal groups. Total phospholipid content was similar between sites, but the cholesterol and free fatty acid content were lower in the ileum than the jejunum. The phospholipid/cholesterol ratio was increased only in the ileum of EF animals. The amount of lecithin was decreased in the jejunal BBM of EF animals resulting in a decreased choline/amine phospholipid ratio as compared with control animals. The ileal phospholipid composition was similar among all groups. A large increase in villus height is observed in the jejunum of EF animals. Villus surface area and mucosal surface area are altered with ethanol feeding and food deprivation. Thus, (i) there is a gradient of S and cholesterol between the BBM of jejunum and ileum; (ii) changes in food intake are associated with changes in the morphology as well as the enzyme marker and lipid content of BBM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of phosphatidylserine in carrot cells cultured under carbon-source starvation

When carrot suspension cells were cultured on medium containing no carbon source (starvation), the levels of phosphatidylserine (PS) increased transiently 3-4 d after the initiation of starvation while levels of most other phospholipid (PL) species decreased. We previously reported that fatty acids of these PLs served as an alternative carbon source during starvation. The present study showed that cells possess two different biosynthetic pathways involving phosphatidylcholine (PC)/phosphatidylethanolamine (PE) exchange enzymes and PS synthase to synthesize PS. These activities peaked similarly 4 d after the initiation of starvation and coincided with the peak of PS level. The synthesis of serine was also significantly activated during starvation. The activity of phosphoserine aminotransferase (PSAT) which is involved in serine synthesis increased with a time course similar to that of the increase in the PS level. These observations suggest that the increase in PS level plays an important role in membranes which are degraded during starvation.