Surface structure of the two porcine low-density lipoprotein subclasses — a spin labelling study

Porcine low-density lipoprotein (LDL) subclasses were spin-labelled at both protein and lipid sites. The surface structures of the two subclasses were compared. E.s.r. spectra of stearic acid spin-labels [I(m/n)] and of androstanol spin-label indicated more restricted motion of the labels in LDL₂ (d = 1.063?1.090 g/ml) than in LDL₁ (d = 1.020?1.063 g/ml). Thermotropic change in the surface structure was found in both subclasses by both protein and lipid spin-labels, but at lower temperature in LDl₁, than in LDL₂. These results indicate the relationship between the size and the dynamics of the lipid components in the surface layer of the LDL subclasses.     

Studies on the Lp(a)-lipoprotein of human serum

Summary Lp(a)-lipoprotein of human serum, when isolated by ultracentrifugation and gelfiltration on Sepharose 4 B, may disaggregate into several components either spontaneously or by mild treatment with detergents:1. A lipoprotein with characteristics of LDL₂; 2. a lipid-free protein complex, which reacts immunologically with anti-Lp(a)-sera; 3. albumin and 4. two other protein components, which were also observed in aged LDL₂-preparations and have not been characterized further. The Lp(a)-positive protein moiety could be separated from all other components by chromatography on hydroxyl-apatite.It is concluded that the Lp(a)-lipoprotein represents a complex protein composed of LDL₂-lipoprotein, the Lp(a)-protein moiety, and possibly albumin.

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Zusammenfassung Das durch Ultrazentrifugation und Gelfiltration and Sepharose 4 B isolierte Lp(a)-Lipoprotein des menschlichen Serums zerfällt spontan oder nach milder Behandlung mit Detergentien in mehrere Komponenten:1. Ein Lipoprotein mit den Eigenschaften der LDL₂; 2. einen lipidfreien Proteinkomplex, der immunologisch mit Anti-Lp(a)-Antiseren reagiert; 3. Albumin und 4. zwei weitere Proteinkomponenten, die auch in gealterten LDL₂-Präparationen beobachtet wurden, die aber hier nicht weiter charakterisiert wurden. Der Lp(a)-positive Proteinanteil konnte von allen anderen Bestandteilen durch Chromatographie an Hydroxylapatit abgetrennt werden.Es wird geschlossen, daß das Lp(a)-Lipoprotein ein komplexes Protein darstellt, das aus LDL₂-Lipoprotein, dem Lp(a)-Protein und möglicherweise Albumin zusammengesetzt ist.

     

LDL protein nitration: implication for LDL protein unfolding

Oxidatively- or enzymatically-modified low-density lipoprotein (LDL) is intimately involved in the initiation and progression of atherosclerosis. The in vivo modified LDL is electro-negative (LDL⁻) and consists of peroxidized lipid and unfolded apoB-100 protein. This study was aimed at establishing specific protein modifications and conformational changes in LDL⁻ assessed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and circular dichroism analyses, respectively. The functional significance of these chemical modifications and structural changes were validated with binding and uptake experiments to- and by bovine aortic endothelial cells (BAEC).The plasma LDL⁻ fraction showed increased nitrotyrosine and lipid peroxide content as well as a greater cysteine oxidation as compared with native- and total-LDL. LC/MS/MS analyses of LDL⁻ revealed specific modifications in the apoB-100 moiety, largely involving nitration of tyrosines in the α-helical structures and β₂ sheet as well as cysteine oxidation to cysteic acid in β₁ sheet. Circular dichroism analyses showed that the α-helical content of LDL⁻ was substantially lower (∼25%) than that of native LDL (∼90%); conversely, LDL⁻ showed greater content of β-sheet and random coil structure, in agreement with unfolding of the protein. These results were mimicked by treatment of LDL subfractions with peroxynitrite (ONOO⁻) or SIN-1: similar amino acid modifications as well as conformational changes (loss of α-helical structure and gain in β-sheet structure) were observed. Both LDL⁻ and ONOO⁻-treated LDL showed a statistically significant increase in binding and uptake to- and by BAEC compared to native LDL. We further found that most binding and uptake in control-LDL was through LDL-R with minimal oxLDL-R-dependent uptake. ONOO⁻-treated LDL was significantly bound and endocytosed by LOX-1, CD36, and SR-A with minimal contribution from LDL-R.It is suggested that lipid peroxidation and protein nitration may account for the mechanisms leading to apoB-100 protein unfolding and consequential increase in modified LDL binding and uptake to and by endothelial cells that is dependent on oxLDL scavenger receptors.     

Plasma lipoproteins,postheparin plasma lipoprotein lipase activity intralipid half-life,triglyceride secretion rate and liver lipids in the mouse fed different cholesterol diets

Mice (SC), fed a semipurified diet containing cholesterol, cholic acid and sucrose, exhibited, in comparison to control animals (S), an increase in cholesterol, phospholipid and protein of VLDL, LDL₁ and LDL₂, but triglyceride of the same lipoproteins decreased, as did total plasma triglycerides. Postheparin plasma lipoprotein lipase activity of SC animals was 1.72 times that of S mice. At the same time Intralipid half-life in SC mice was decreased by 52%. Triglyceride secretion rate, after Triton WR 1339 treatment, and liver triglyceride content were reduced in SC animals. HDL mass was decreased in SC mice. Mice (AC) fed a standard diet containing cholesterol showed, in comparison to normal fed animals (A), an increase in cholesterol of VLDL, LDL₁ and LDL₂ but triglyceride of the same lipoproteins decreased as did total plasma triglycerides. Postheparin plasma lipoprotein lipase activity of AC animals was unmodified as was Intralipid half-life. In AC animals triglyceride secretion rate, after Triton WR 1339 treatment, was reduced but in a less extent than in SC mice. Liver triglyceride was unmodified. HDL mass was decreased in AC mice.     

A rapid and simple method for the isolation of S100a0 (αα) protein by high-performance liquid chromatography

A rapid and simple method to isolate S100a₀ protein from the mixture of bovine S100 protein (S100a₀, S100a and S100b) is described. The S100 mixture purified from bovine brain was applied to an anion-exchange column, equilibrated with 50 mM Tris HC1 buffer (pH 8.0) in a high-performance liquid chromatography (HPLC) system. S100a₀, S100a and S100b proteins could be eluted separately from the column, which were identified by the immunoassay method, by the Tris-HC1 buffer containing a linear concentration gradient (0.25–0.4) M of NaCl. Immunoreactive S100a₀ protein was found in two peak fractions, and each S100a₀ fraction could be isolated (S100a₀-1 and S100a₀-2). Both fractions of S100a₀ protein showed a single band at the same position on acrylamide gel electrophoresis, and eluted in a single peak in the same fractions upon gel-filtration column chromatography. There was no significant difference in the amino acid composition between the two S100a₀ fractions. Since the S100a₀-1 fraction aged for several months at 4°C in the presence of 0.1% NaN₃ was found to contain four protein peaks including the fraction corresponding to the S100a₀-2 fraction, the difference between the two S100a₀ fractions is probably due to some modification of amino acid residues in the molecule, which may occur both in vivo and in vitro.     

The Uptake and Metabolism of Sulphate in Scenedesmus as Influenced by Citrate, Carbon Dioxide, and Metabolic Inhibitors

The incorporation of sulphur from external sulphate into different fractions was studied in cells with a P content of 0.6 to 1 mg/g fresh weight as against 3 to 5 mg/g fresh weight in normal cells. — There is a flat and broad optimum for the action of pH in the region between 5.7 and 7. — In most cases, citrate inhibits the uptake of sulphur. — Five per cent CO₂ in the air enhances the incorporation of S. It is suggested that the effects of CO₂ may well be exerted by way of the carboxylation and decarboxylation mechanisms in the cells. — Selenate inhibits the formation of DNA-S and protein S more than that of lipid S, but the main effect in both cases seems to be in an early step of the assimilation of sulphate. In the absence of external phosphate, the inhibition is counteracted by CO₂, especially in darkness. Phosphate enhances the action of selenate on the organic S fractions, and in its presence CO₂ and darkness make the inhibition more pronounced. — Without P in the medium, the effects of selenate on uptake into the inorganic sulphate fraction are smaller than when the organic fractions are concerned. In the presence of CO₂ even stimulations due to selenate have been observed. External phosphate brings the inhibition of uptake into cellular SO₄^2- to the same level as found in the case of organic S. More than one pathway for the uptake as SO₄^2-seem possible. — Anaerobiosis and menadione affect the organic S fractions more than the sulphate; 2,4-dinitrophenol has a more uniform action all over the field.     

Probing of the porcine serum lipoprotein surfaces by Mn(II) binding: an e.s.r. study

Mn(II) ions were used for probing the surfaces of porcine LDL₁, LDL₂ and HDL. From the intensity of the e.p.r. lines corresponding to the unbound Mn(II) the percentage of the ions bound to the lipoprotein surface is determined. From the titration curves the binding parameters, dissociation constant. K_d, and the number of binding sites, n, in all the three lipoproteins studied have been derived. There are at least two types of binding sites in each lipoprotein class. The ”weak’ binding sites are charaterized by approximately the same value of $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{(≈ 6.2 × 10}{}^{\mathrm{?3}}\text{mol l}{}^{\mathrm{?1}}$ and different values for n (n = 114 for LDL₁, n = 135 for LDL₂ and n = 28 for HDL). Similarly, for the ”strong’ binding sites K_d ≈ 1.6 × 10^?4 mol l^?1 and the number of binding sites is 15, 20 and 5 for LDL₁, LDL₂ and HDL respectively. It is concluded that the binding sites are probably located in the protein part of the lipoproteins and that they are mainly associated with the negatively charged amino acids.     

Long term delayed luminescence: A possible fast and convenient assay for nutrition deficiencies and environmental pollution damages in plants

Long Term Delayed Luminescence (LDL) of green plants ranging from 0.3 s up to several minutes after irradiation has been found to exhibit complex decay kinetics which are highly dependent on nutritional deficiencies and environmental pollutive components. As a model organism we utilized the unicellular green algaScenedesmus obliquus with fairly well understood properties ofLDL probably involving both photosynthetic reaction centers PS I and PS II. It is shown thatLDL is strongly affected both by depletion of the growth medium of various essential elements such as N, Fe, Ca, Mg or K, as well as by growth in the presence of environmental stress factors such as Cu, Hg, Cd or NO ₂ ^– . Therefore,LDL offers itself as a convenient, highly sensitive and specific assay for a number of stress factors in photosynthesizing plantsin vivo and in the field.     

The Effect of Light, Carhon Dioxide, and Nitrogen Nutrition on the Incorporation of S from External Sulphate into Different S-Containing Fractions in Scenedesmus, with Special Reference to Lipid S

The experiments showed that incorporation of S from the sulphate in the medium into normal cells of Scenedesmus was enhanced by light, relatively most in the case of lipid S and least in the inorganic sulphate fraction. The effects of light were, generally, increased by the presence of CO₂ and nitrogen salts. CO₂ did not significantly alter the proportions between the fractions, but the presence of nitrogen increased the formation of protein S more than the synthesis of S-containing lipids.—It is suggested that lipid S is formed as a “sink”, when a step between sulpbite and -SH becomes increasingly rate-limiting in the overall reduction of sulphate. Furthermore, incorporation as SO₄^2? and as lipid S may be regulated by more or less independent processes.     

Cholinergic Binding to the Receptor Proteolipid from Torpedo Electroplax Separated by Ion Exchange Chromatography

Abstract

Proteolipids (i.e., hydrophobic proteins) have been extracted with chloroform-methanol (2:1) from lyophilized Torpedo electroplax, and fractionated on a DEAE-cellulose column. The elution system consisted of the same solvent and a gradient of the hydrophobic ion ptoluene sulfonate (0.1–100mM). The three fractions obtained (I, II and III) have different content of protein, lipid P, and reducing sugars. The amino acid composition shows a higher proportion of hydrophobic residues in I and more charged ones in fractions II and III. Polyacryl amide gel electrophoresis of fraction I shows a single major band of 39 kdaltons; in fractions II and III a major band of 42 kdaltons and fainter bands in the range 62–68 kdaltons are observed.

Fraction I has the highest specific binding for [³H]-acetylcholine (7.1 nmol/mg protein) and [³H]α-bungarotoxin (5.2 nmol/mg protein). The nicotinic nature of this proteolipid was demonstrated by blocking experiments. The Scatchard plot showed two affinity sites for [³H]-acetylcholine (Kd₁ = 3nM and Kd₂ = 1.1 μm). 4-(N-maleimido) pheny1 [³H]trimethylammonium specifically labeled the 39 kdaltons band.

The possible factors involved in the fractionation of proteolipids are discussed. The findings suggest that the 39 kdaltons polypeptide contains the receptor site for acetylcholine and that the other proteolipid components may play a different function in the membrane.     

Fractionation of L-cell chromatin into DNA, RNA, and protein fractions on Cs2SO4 equilibruim density gradients

A new procedure is described for fractionation of chromatin into DNA, RNA, and total chromatin protein. By isopycnic gradient centrifugation of chromatin preparations in Cs₂SO₄ solutions containing dimethylsulfoxide and sodium sarcosyl it is possible to obtain highly purified fractions of these components. The method gives a very high yield of these chromatin fractions unlike some other methods, where irreversible binding to columns occurs. Also with this method it is possible to obtain highly concentrated fractions, which after a simple dialysis step, can be conveniently analyzed by polyacrylamide gel electrophoresis.Nuclei from L-929 cells were isolated by a method involving citric acid or by a method using a nonionic detergent. The yields of DNA obtained by both methods was compared. Chromatin was isolated from purified nuclei (prepared in either of the above ways) in two different ways also. In one method, chromatin was extracted from nuclei with 1 m NaCl. A second method involving fractionation of lysed nuclei in sucrose and metrizamide solutions was also used. The yields of DNA obtained by both methods was compared. There appears to be little nuclear membrane contamination of any of these chromatin preparations.A preliminary analysis of L-929 cell chromatin total RNA and protein fractions on polyacrylamide and agarose gels has been made. Both fractions appear to be quite complex with a wide spectrum of subcomponents of differing S values.     

Hydrogen sulfide guards myoblasts from ferroptosis by inhibiting ALOX12 acetylation

Recognized as a novel and important gasotransmitter, hydrogen sulfide (H₂S) is widely present in various tissues and organs. Cystathionine gamma-lyase (CSE)-derived H₂S has been shown to regulate oxidative stress and lipid metabolism. The aim of the present study is to examine the role of H₂S in ferroptosis and lipid peroxidation in mouse myoblasts and skeletal muscles. Ferroptosis agonist RSL3 inhibited the expressions of Gpx4 and reduced CSE/H₂S signaling, which lead to increased oxidative stress, lipid peroxidation, and ferroptotic cell death. In addition, ferroptosis antagonist ferrostatin-1 (Fer-1) up-regulated the expression of CSE, scavenged the generation of reactive oxygen species (ROS) and lipid peroxidation, and improved cell viability. Exogenously applied NaHS was also able to block RSL3-induced ferroptotic cell death. Neither RSL3 nor H₂S affected cell apoptosis. Furthermore, H₂S reversed RSL3-induced Drp1 expression and mitochondrial damage, which lead to abnormal lipid metabolism as evidenced by altered expressions of ACSL4, FAS, ACC and CPT1 as well as higher acetyl-CoA contents in both cytoplasm and mitochondria. RSL3 promoted the protein expression and acetylation of ALOX12, a key protein in initiating membrane phospholipid oxidation, while the addition of NaHS attenuated ALOX12 acetylation and protected from membrane lipid peroxidation. Moreover, we observed that CSE deficiency alters the expressions of ferroptosis and lipid peroxidation-related proteins and enhances global protein acetylation in mouse skeletal muscles under aging or injury conditions. These results indicate that downregulation of CSE/H₂S signaling would contribute to mitochondrial damage, abnormal lipid metabolism, membrane lipid peroxidation, and ferroptotic cell death. CSE/H₂S system can be a target for preventing ferroptosis in skeletal muscle.     

Production of reactive oxygen species by monocyte-derived macrophages from blood of healthy donors and patients with ischemic heart disease

Production of reactive oxygen species (ROS) by macrophages derived from blood monocytes of healthy donors (MP_N) and patients with ischemic heart disease (IHD) (MP_IHD) before, during, and after their incubation with low-density lipoprotein (LDL) isolated from blood plasma of healthy donors (LDL_N) and patients with a high cholesterol level (LDL_H) was investigated by the method of luminol-dependent (spontaneous) and stimulated chemiluminescence (CL) using opsonized zymosan (OZ) or phorbol-12-myristate-13-acetate (PMA) as the CL stimulators. It was shown that proper, luminol-dependent, and zymosan-or PMA-stimulated chemiluminescence of MP_IHD was 1.4-, 1.8-, 2.7-, and 1.6-fold higher than the same types of chemiluminescence of MP_N, respectively, (p<0.05–0.01). Although the effect of OZ on MP_N and MP_IHD was more potent than that of PMA (by 4.3- and 3.2-fold, respectively), but it appeared in 2.5–3.0 times slower than that of PMA. LDL_N and LDL_H incubated with MP_N for the first 15 and 60 min caused the 1.4- and 2.5-increase of the luminol-dependent CL of MP_N; the same treatment of MP_IHD did not influence ROS production by these cells. Repeated increase in the OZ-stimulated CL of MP_N was also observed after preincubation for 15–180 min with LDL_N and LDL_H followed by LDL removal, subsequent MP_N washing and addition of Hanks solution and OZ; the repeated increase in OZ-stimulated CL of MP_N was only observed after incubation with LDL_H than with LDL_N. No increase of CL was observed in experiments with MP_IHD. Thus, more intensive chemiluminescence of macrophages obtained from blood of patients with IHD suggests their in vivo stimulation. LDL_N and LDL_H may cause both primary and secondary (after preincubation) stimulating effect on CL of MP_N but not of MP_IHD. Thus, the analysis of macrophage chemiluminescence is a sensitive test for evaluation the degree of macrophage stimulation; it may be effectively used for monitoring of effectiveness of medical treatment of patients.     

Unit gravity sedimentation analysis of basic nuclear protein synthesis during the HeLa cell cycle

Summary Exponentially growing HeLa cells have been separated according to their cell cycle age by sedimenting at unit gravity for 3 hr on a phosphate-buffered sucrose density gradient. Measurements of cell size, cell number, DNA content, and tritiated thymidine incorporation in consecutive portions of the gradient showed that cells in upper fractions were in G₁, cells in middle fractions were in S, and cells in lower fractions were in G₂. Basic amino acids were rapidly incorporated into nuclear protein during late G₁ and S; some incorporation also took place during G₂. This work is supported by grant A-3458 from the National Research Council of Canada.     

Oxidant and angiotensin II-induced subcellular translocation of protein kinase C in pulmonary artery endothelial cells

We recently reported that nitrogen dioxide (NO₂), an environmental oxidant, alters the dynamics of the plasma membrane lipid bilayer structure, resulting in increased phosphatidylserine content and angiotensin II (Ang II) receptor binding. Angiotensin II is known to elicit receptor-mediated stimulation of diacylglycerol (DAG) production in pulmonary artery endothelial cells. Because protein kinase C (PKC) is a phosphatidylserine-dependent enzyme and is activated by DAG, we examined whether NO₂ resulted in activation and/or translocation of PKC from predominantly cytosolic to membrane fractions of these cells. We also evaluated whether NO₂ exposure resulted in increased production of DAG in pulmonary artery endothelial cells. Exposure to 5 ppm NO₂ for 1–24 hr resulted in significant increases in PKC activity in the cytosolic and membrane fractions (p < 0.05 for both fractions) compared to activities in control fractions. Exposure to Ang II resulted in translocation of PKC activity from cytosol to membrane fractions of both control and NO₂-exposed cells. This translocation of PKC from cytosolic to membrane fraction was prevented by the specific receptor antagonist [Sar¹ Ile⁸] Ang II. Exposure of 5 ppm NO₂ for 1–24 hr provoked rapid increases in [³H]glycerol labeling of DAG in pulmonary artery endothelial cells. These results demonstrate that exposure to NO₂ increases the production of second messenger DAG and activates PKC in both the cytosolic and membrane fractions, whereas Ang II stimulates the redistribution of PKC from cytosolic to membrane fractions of pulmonary artery endothelial cells.     

Antioxidant activities of sulfated polysaccharide fractions from Porphyra haitanesis

Three sulfated polysaccharide fractions (F1, F2, and F3) were isolated from Porphyra haitanesis, an important economic alga in China, through anion-exchange column chromatography and their in vitro antioxidant activities were investigated in this study. Galactose was the main sugar unit of the three fractions. The analytical results indicated that polysaccharide fractions from P. haitanesis had similar chemical components to porphyran from other species, but differed in their high sulfate content. The sulfate content of F1, F2 and F3 was 17.4%, 20.5% and 33.5% respectively. All three polysaccharide fractions showed antioxidant activities. They had strong scavenging effect on superoxide radical, and much weaker effect on hydroxyl free radical. Lipid peroxide in rat liver microsome was significantly inhibited, and H₂O₂ induced hemolysis of rat erythrocyte was partly inhibited by F1, F2 and F3. Among them, F3 showed strongest scavenging effect on superoxide radical; F2 had strongest effect on hydroxyl radical and lipid peroxide.     

Lipid class and molecular species interrelationships among plasma lipoproteins of type III and type IV hyperlipemic subjects

As a further appraisal of lipoprotein interconversion and equilibration of lipid components a detailed examination was made of the chemical class and molecular species interrelationships among the major fasting plasma lipoprotein fractions within each of six male Type III and Type IV hyperlipemic subjects subsisting on free choice diets. The lipoprotein fractions were prepared by conventional ultracentrifugation and the lipid class and molecular species composition of the corresponding lipoprotein fractions were determined by gas chromatography of the intact glycerol esters and ceramides. In general, each lipoprotein fraction possessed a well defined lipid class composition, which was characterized by a dramatically decreasing triacylglycerol and increasing phospholipid and cholesteryl ester content, when progressing from the very low (VLDL) to the low (LDL) and high (HDL) density lipoproteins, as already established for normolipemic subjects. Likewise, the LDL, and LDL₂ of the hyperlipemic subjects contained about two times higher proportion of total phospholipid as sphingomyelin than VLDL and HDL. Furthermore, the sphingomyelins of the HDL fraction contained about 30% more of the higher and 30% less of the lower molecular weight species than the sphingomyelins of the VLDL. Smaller differences were seen in the molecular species composition of the phosphatidylcholines, cholesteryl esters and triacylglycerols among the corresponding lipoproteins. In comparison to normolipemic subjects analyzed previously, the hyperlipemic subjects showed greater individual variability. Despite this variability the lipid class and molecular species composition in the hyperlipemic subjects was again incompatible with the hypothesis which postulates direct VLDL conversion into LDL and HDL under the influence of lipoprotein lipase and lecithin: cholesterol acyltransferase. The main differences between normolipemic and hyperlipemic plasma were found to reside in the number of the VLDL and LDL, lipoprotein particles and not in their chemical composition or physical structure, or in the apparent mechanism of their metabolic interconversion.     

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.     

Comparative study of storage compound breakdown in germinating seeds of three lupine species

Storage lipid and protein breakdown in germinating seeds of yellow (Lupinus luteus L.), white (L. albus L.), and Andean lupine (L. mutabilis Sweet) and regulatory function of sucrose were investigated. Less oil bodies were detected in organs of yellow lupine seeds, whereas the highest content of oil bodies was noticed in the Andean lupine seeds. Mature, air-dried yellow, white and Andean lupine seeds do not contain starch. Starch grains appear the earliest in white lupine seeds during imbibition. Sucrose deficiency in tissues enhances breakdown of storage lipid, protein and temporary starch in cotyledons. In sucrose starved embryo axes of all investigated lupine species, an increased level of vacuolization was noted. Interconnections between catabolism of storage protein and storage lipid in germinating lupine seeds were identified by applying ¹⁴C-acetate. To assess the importance of key processes in storage lipid breakdown NaF (inhibitor of glycolysis and gluconeogenesis), KCN, NaN₃ and SHAM (inhibitors of mitochondrial electron transport chain) and MSO (inhibitor of glutamine synthetase) were used. Radioactivity coming from ¹⁴C-acetate was released as ¹⁴CO₂ but mostly was incorporated into ethanol-soluble fraction of embryo axes and cotyledons. Respiratory inhibitors caused a significant decrease in ¹⁴CO₂ and ethanol fractions in all three lupine species studied. MSO stimulated release of ¹⁴CO₂ and radioactivity of ethanol fractions in yellow lupine organs fed with sucrose, but in Andean lupine MSO enhanced the production of ¹⁴CO₂ and radioactivity of ethanol fractions both in organs fed and not fed with sucrose. Different strategies of storage compound breakdown are proposed, depending on relative proportion in storage protein and lipid content in lupine seeds.     

Proliferation of germ cells and somatic cells in first trimester human embryonic gonads as indicated by S and S+G2+M phase fractions

Objectives: The number of germ cells and somatic cells in human embryonic and foetal gonads has previously been estimated by stereological methods, which are time‐ and labour‐consuming with little information concerning cell proliferation. Here, we studied whether flow cytometry could be applied as an easier method, also enabling estimation of the fraction of cells in S or S+G₂+M (SG₂M) cell‐cycle phases as indicators of cell proliferation. Methods: Cell suspensions from 35 human embryonic gonads at days 37 to 68 post‐conception (pc) were immunomagnetically sorted into C‐KIT positive (germ) cells and negative (somatic) cells. They were stained for DNA content and analysed by flow cytometry. S and SG₂M fractions could be measured for 13 of the female and 20 of the male gonads. The number of cells was estimated using fluorescent reference beads. Results: During the period from 37 to 68 days pc, female germ and somatic cells had a stable S and SG₂M fractions indicating steady growth of both subpopulations, whereas they decreased in both male germ and somatic cells. The number of germ and somatic cells estimated by flow cytometry was significantly lower than in stereological estimates, suggesting loss of cells during preparation. Conclusions: Cell proliferation as indicated by S and SG₂M fractions could be estimated specifically for primordial germ and somatic cells. Estimation of total number of germ and somatic cells was not feasible.