Liver fatty acid binding protein enhances sterol transfer by membrane interaction

Among the large family of fatty acid binding proteins, the liver L-FABP is unique in that it not only binds fatty acids but also interacts with sterols to enhance sterol transfer between membranes. Nevertheless, the mechanism whereby L-FABP potentiates intermembrane sterol transfer is unknown. Both fluorescence and dialysis data indicate L-FABP mediated sterol transfer between L-cell fibroblast plasma membranes occurs by a direct membrane effect: First, dansylated-L-FABP (DNS-L-FABP) is bound to L-cell fibroblast plasma membranes as indicated by increased DNS-L-FABP steady state polarization and phase resolved limiting anisotropy. Second, coumarin-L-FABP (CPM-L-FABP) fluorescence lifetimes were significantly increased upon interaction with plasma membranes. Third, dialysis studies with³H-cholesterol loaded plasma membranes showed that L-FABP added to the donor compartment of the dialysis cell stimulated³H-cholesterol transfer whether or not the dialysis membrane was permeable to L-FABP. However, L-FABP mediated intermembrane sterol transfer did require a sterol binding site on L-FABP. Chemically blocking the ligand binding site also inhibited L-FABP activity in intermembrane sterol transfer. Finally, L-FABP did not act either as an aqueous carrier or in membrane fusion. The fact that L-FABP interacted with plasma membrane vesicles and required a sterol binding site was consistent with a mode of action whereby L-FABP binds to the membrane prior to releasing sterol from the bilayer.Abbreviations ³H-CHO [1,2-³H(N)]-cholesterol - ANTS 8-aminonaphthalene-1,3,6-trisulfonic acid - CF carboxyfluorescein - CHO cholesterol - CPM (coumarin maleimide) 7-diethylamino-3-(4-maleimidylphenyl)-4-methylcoumarin - cPNA cisparinaric acid - DHE (dehydroergosterol) ^5,7,9(11),22-ergostatetraen-3-ol - DMF dimethyl formamide - DMPOPOP 1,4-bis[4-methyl-5-phenyl-2-oxazolyl]benzene - DNS (dansyl chloride) 5-dimethylaminonaphthalene-1-sulfonylchloride - DPX p-xylene-bis-pyridinium bromide - FBS fetal bovine serum - fluorescamine 4-phenylspiro[furan-2(3H), 1 phthalan]-3,3-dione - L-FABP liver fatty acid binding protein - NPG p-nitrophenylglyoxal - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine - SUV small unilamellar vesicle(s) - TNM tetranitromethane This work was supported in part by the National Institutes of Health United States Public Health Service (GM31651 and DK41402) and the American Heart Association (Postdoctoral Fellowship to JKW). The helpful assistance of Dr. Scott M. Colles and Mr. Daniel R. Prows in isolating L-FABP was much appreciated.     

Lack of Correlation between Activation of Jun–NH2-terminal Kinase and Induction of Apoptosis after Detachment of Epithelial Cells

Detachment of epithelial cells from the extracellular matrix leads to induction of programmed cell death, a process that has been termed “anoikis.” It has been reported recently that detachment of MDCK cells from matrix results in activation of Jun–NH₂-terminal kinases (JNKs) and speculated that these stress activated protein kinases play a causal role in the induction of anoikis (Frisch, S.M., K. Vuori, D. Kelaita, and S. Sicks. 1996. J. Cell Biol. 135:1377–1382). We report here that although JNK is activated by detachment of normal MDCK cells, study of cell lines expressing activated signaling proteins usually controlled by Ras shows that stimulation of JNK fails to correlate with induction of anoikis. Activated phosphoinositide 3-OH kinase and activated PKB/Akt protect MDCK cells from detachment-induced apoptosis without suppressing JNK activation. Conversely, activated Raf and dominant negative SEK1, a JNK kinase, attenuate detachment-induced JNK activation without protecting from apoptosis. zVAD-fmk, a peptide inhibitor of caspases, prevents MDCK cell anoikis without affecting JNK activation. p38, a related stress-activated kinase, is also stimulated by detachment from matrix, but inhibition of this kinase with SB 203580 does not protect from anoikis. It is therefore unlikely that either JNK or p38 play a direct role in detachment-induced programmed cell death in epithelial cells.     

Partial purification of a phosphoethanolamine methyltransferase from rat brain cytosol

The conversion of phosphoethanolamine to phosphocholine requires 3 separate N-methyltransferases. We had previously purified the enzyme catalyzing the last methylation, phosphodimethylethanolamine N-methyltransferase. We have successfully purified the enzyme catalyzing the initial methylation of phosphoethanolamine. A 434 fold purified enzyme from rat brain was obtained by the sequential use of ammonium sulfate fractionation, Q-Sepharose fast flow column chromatography and a -aminoethyl agarose column chromatography. The pH optimum was 11 or greater, the Km value for phosphoethanolamine was 167.8±41.7 M and the Vmax was 487.3±85 mmoles/mg/hr. The kinetics for S-adenosyl-methionine, the methyldonor, has characteristics of cooperative binding with a Km of 1.805±0.59 mM and a Vmax of 16.9±3.6 moles/mg/hr. The activity was stimulated 6 fold by 2.5 mM MnCl₂ and inhibited by DZA and S-adenosylhomocysteine. These results reinforce the early in vivo observations which had provided suggestive evidence for the existence of a pathway for the methylation of phosphoethanolamine to phosphocholine in rat brain.Abbreviations used Adomet S-adenosylmethionine - AdoHcy S-adenosyl-homocysteine - CAPS 3-(cyclohexyl)amino-1-propanesulphonic acid - Cho choline - 3-DZA 3-deazaadenosine - Etn ethanolamine - N-MT N-methyltransferase - PEG polyethyleneglycol - PMSF phenylmethanesulphonyl fluoride - PEtn phosphoethanolamine - PCho phosphocholine - PMe₂Etn phosphodimethylethanolamine - PtdCho phosphatidylcholine - PtdEtn phosphatidylethanolamine     

Effects of cytosolic calcium and limited, possible dual, effects of G protein modulators on guard cell inward potassium channels

The cellular mechanisms that regulate potassium (K⁺) channels in guard cells have been the subject of recent research, as K⁺ channel modulation has been suggested to contribute to stomatal movements. Patch clamp studies have been pursued on guard cell protoplasts of Vicia faba to analyze the effects of physiological cytosolic free Ca²⁺ concentrations, Ca²⁺ buffers and GTP-binding protein modulators on inward-rectifying K⁺ channels. Ca²⁺ inhibition of inward-rectifying K⁺ currents depended strongly on the concentration and effectiveness of the Ca²⁺ buffer used, indicating a large Ca²⁺ buffering capacity and pH increases in guard calls. When the cytosolic Ca²⁺ concentration was buffered to micromolar levels using BAPTA, inward-rectifying K⁺ channels were strongly inhibited. However, when EGTA was used as the Ca²⁺ buffer, much less inhibition was observed, even when pipette solutions contained 1 µM free Ca²⁺. Under the imposed conditions, GTPγS did not significantly inhibit inward-rectifying K⁺ channel currents when cytosolic Ca²⁺ was buffered to low levels or when using EGTA as the Ca²⁺ buffer. Furthermore, GDPβS reduced inward K⁺ currents at low cytosolic Ca²⁺, indicating a novel mode of inward K⁺ channel regulation by G-protein modulators, which is opposite in effect to that from previous reports. On the other hand, when Ca²⁺ was effectively elevated in the cytosol to 1 µM using BAPTA, GTPγS produced an additional inhibition of the inward-rectifying K⁺ channel currents in a population of cells, indicating possible Ca²⁺-dependent action of GTP-binding protein modulators in K⁺ channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K⁺ currents does not prohibit, but slows, stomatal opening and reduces stomatal apertures by only 34% after 2 h light exposure. These data suggest that limited K⁺ channel down-regulation alone may not be rate-limiting, and it is proposed that the concerted action of proton-pump inhibition and additional anion channel activation is likely required for inhibition of stomatal opening. Furthermore, G-protein modulators regulate inward K⁺ channels in a more complex and limited, possibly Ca²⁺-dependent, manner than previously proposed.     

Correlation of tumor metastasis with sterol carrier protein and plasma membrane sterol levels

Squalene and sterol carrier protein (SCP) levels and sterol/phospholipid molar ratios of whole cells and plasma membranes were measured in cultured primary tumor and metastatic cell lines. SCP is abundant in all cell lines. However, metastatic lines have significantly lower SCP levels and plasma membrane sterol/phospholipid ratios than do primary lines. The results indicate that extremely malignant, metastatic cells are unable to produce or maintain adequate levels of both SCP and plasma membrane sterols when grown in lipoprotein deficient media. This defect, in vivo, probably causes excess uptake of SCP and lipid.     

Comparison of gravimetry and planimetry in determining dry matter budgets for three species of phytophagous lepidopteran larvae

Gravimetric and a combination areal-gravimetric methods for determining dry matter budgets for leaf eating Lepidoptera were compared. The gravimetric method is based on dry weight/live weight ratios of the leaves fed to the larvae. In the areal-gravimetric method, the quantity of food offered to the larvae is determined from the area of leaf tracings and the dry matter content per unit area of the leaves. The areal-gravimetric method permits the use of larger leaf sprays and an open, gauze enclosed rearing chamber. There were no consistent differences in budget factors (growth, ingestion or egestion), nor were there any differences in the observed variability of the data attributable to the method used. However, the expected variability based on instrument precision for the gravimetric method is less than for the areal-gravimetric method. Experimental factors inherent in the gravimetric method introduce variability to the measurements that are not present in the areal method. Thirty to 60% of the variability in budget factors was attributed to intrinsic properties of the larvae, even though the larvae were taken from the same egg masses.

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Résumé Les budgets en matière sèche consommée par des lépidoptères ont été comparés par les méthodes gravimétrique et planimétrique. La méthode gravimétrique est basée sur le rapport poids sec/poids frais de feuilles consommées par les chenilles. Avec la méthode planimétrique, la quantité d'aliment proposée aux chenilles est déterminée par les tracés de la surface des feuilles et le contenu de matière sèche par unité de surface des feuilles. La méthode de planimétrie permet l'utilisation de plus grands rameaux de feuilles et de cages d'élevage extérieures en gaze. Il n'y avait pas de différence appréciable dans les éléments du budget (croissance, ingestion et déjection), ni aucune différence dans la variabilité observée des données attribuable à la méthode utilisée. Cependant, la variabilité attendue d'après la précision des mesures avec la méthode gravimétrique est inférieure à celle de la méthode planimétrique. est inférieure à celle de la méthode planimétrique. Des éléments expérimentaux, inhérents à la méthode gravimétrique, introduisent une variabilité dans les mesures que l'on n'a pas avec la méthode planimétrique. 30–60% de la variabilité dans la consommation ont été attribués à des paramètres internes à la chenille, même quand elles provenaient toutes de la même ooplaque.

     

Microtubule arrays in the cortex and near the germinal vesicle of immature starfish oocytes

An extensive array of long, crisscrossing microtubules has been discovered in the cortex of oocytes of the starfish Pisaster ochraceus. The microtubules were visualized in cortex preparations by indirect immunofluorescence microscopy using antibodies to tubulin. The cortical array of microtubules is present in all oocytes before and for about 30 min after the application of 1-methyladenine, the hormone that induces oocyte maturation. The presence of microtubules was confirmed by electron microscopy. The microtubules in this array are depolymerized when oocytes are treated with colchicine or nocodozole and are augmented when oocytes are treated with taxol. Dihydrocytochalasin B treatment of the oocytes causes the microtubules to aggregate, presumably by altering a microfilament network also found in the cortex. The distribution of microtubules was also explored in whole oocytes stained with antitubulin. One or two aster-like structures were observed adjacent to the germinal vesicle of each oocyte.     

Organization of microtubules in stabilized meristematic plant cells revealed by a rat monoclonal antibody reacting only with the tyrosinated form of alpha-tubulin

A rat monoclonal antibody against yeast tubulin (clone YL 1/2; Kilmartin et al., 1982) that reacts specifically with mammalian alpha-tubulin carrying a carboxyterminal tyrosine residue (Wehland et al., 1983) was used to localize microtubules in plant cells derived from onion root apices (Allium cepa L.). YL 1/2 reacted with all types of microtubular arrays known to occur in higher plant meristematic cells such as interphase cortical microtubules, pre-prophase bands, the mitotic spindle and phragmoplast microtubules. The specific labeling of microtubules in isolated cells from onion root tips by YL 1/2 indicates that plant cells like animal cells contain tubulin tyrosine ligase, the enzyme which posttranslationally modifies alpha-tubulin. This enzyme could be involved in the dynamic regulation of microtubular arrays in all eukaryotic cells.     

Role of exercise testing early after myocardial infarction in identifying candidates for coronary surgery.

It has been suggested that ST depression in lead V5 or equivalent on early exercise testing after acute myocardial infarction predicts a high risk of death. To evaluate exercise testing and radionuclide ventriculography in this context 103 consecutive patients with myocardial infarction who were able to undertake a limited exercise test before discharge from hospital were exercised and underwent gated blood pool scanning. No serious complications resulted from exercise testing. Twenty nine patients developed ST depression in lead V5, 19 had exertional hypotension, 31 developed a heart rate of greater than or equal to 130 beats/min, and 15 had complex ventricular arrhythmias. Death during the first year after discharge from hospital was associated with exertional hypotension (p less than 0.001) and a heart rate on exercise testing of greater than or equal to 130 beats/min (p less than 0.05); these two variables identified all nine deaths. Inability to complete the exercise protocol for any reason was also predictive of death (p less than 0.01). Ventricular arrhythmias and ST depression in lead V5 induced by exercise were not significantly associated with an increased risk of death. The mean (SD) radionuclide ejection fraction in the patients who died was 29 (16%) compared with 43 (11)% in the patients who survived (p less than 0.001). ST changes on exercise testing after myocardial infarction appear to be less predictive of later complications than haemodynamic signs, which may indicate left ventricular damage rather than ischaemia.     

Carbohydrate composition of purified serum glycoproteins in Mucolipidosis II and Mucolipidosis III

Summary Mucolipidosis II (I-cell disease) and Mucolipidosis III (ML III) are inherited disorders in which the molecular defect may involve an abnormality in a common post-translational modification step (possibly glycosylation) shared by lysosomal hydrolases. We tested whether such an alteration might be a generalized defect in glycoprotein biosynthesis and, thus, be reflected in an abnormal carbohydrate composition of non-lysosomal glycoproteins. The apoprotein of low density lipoprotein (apo-LDL) and immunoglobulin G (IgG) were purified to apparent homogeneity. Gas liquid chromatographic (glc) analysis of the carbohydrate content of these glycoproteins from ML II, ML III and normal sera revealed no differences in the relative ratios and total amounts of mannose, galactose, N-acetylglucosamine and sialic acid. These results suggest that if the postulated post-translational defect in these disorders involves changes in carbohydrate composition, it is not a general defect in glycosylation and may be specific for lysosomal hydrolases.