Polyacrylamide Gradient Gel Electrophoresis Research Articles

Lipoproteins containing apolipoprotein A-IV but not apolipoprotein A-I take up and esterify cell-derived cholesterol in plasma.

Two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-PAGGE) identifies distinct apoA-I-or apoE-containing subclasses of high-density lipoproteins (HDLs), each of which plays a different role in reverse cholesterol transport. In this study we used 2D-PAGGE to investigate the role of apoA-IV-containing lipoproteins in reverse cholesterol transport in native plasma. Incubation of 2D electrophoretograms with anti-apoA-IV antibodies identified up to three subclasses of particles. The smaller particle subclasses, LpA-IV-1 and LpA-IV-2, were found in every plasma sample. The largest particle subclass, LpA-IV-3, was observed in fewer than 10% of the plasmas analyzed. 2D-PAGGE of apoA-I-deficient plasma and apoA-I-depleted plasma and anti-apoA-I immunosubtracting 2D-PAGGE of normal plasma revealed that LpA-IV-1 and LpA-IV-2 do not contain apoA-I. The importance of LpA-IV-1 and LpA-IV-2 for uptake and esterification of cell-derived cholesterol was investigated using pulse-chase incubations of plasma with [3H]cholesterol-labeled fibroblasts followed by anti-apoA-I immunosubtracting 2D-PAGGE. During 1-minute pulse incubation with cells, [3H]cholesterol was taken up by gamma-LpE > LpA-IV-1 > pre-beta 1-LpA-I > LpA-IV-2 (">" denotes "more than"). During subsequent chase incubation without cells, proportionately less radioactivity disappeared from LpA-IV-1 and LpA-IV-2 than from pre-beta 1-LpA-I and gamma-LpE. During 5-minute pulse incubations, radioactive cholesteryl esters were formed in pre-beta 3-LpA-I > alpha-LpA-I > LpA-IV-1 > LpA-IV-2. The fractional estertification rate was highest in pre-beta 2-LpA-I and lowest in alpha-LpA-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of Pre-β 1 -HDL and Conversion Into α-HDL

HDL encompasses several apoA-I-containing particles that differ by size and show pre-beta- or alpha-mobility on agarose gel electrophoresis: pre-beta 1-LpA-I, pre-beta 2-LpA-I, pre-beta 3-LpA-I, alpha-LpA-I2, and alpha-LpA-I3. The quantitatively minor subclass pre-beta 1-LpA-I serves as an initial acceptor of cell-derived cholesterol. In this study, we generated a pre-beta 1-LpA-I-like particle in vitro by the incubation of biotinylated apoA-I with cholesterol-loaded macrophages. Both native pre-beta 1-LpA-I and in vitro-generated pre-beta 1-LpA-I were indistinguishable from lipid-free apoA-I by two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis but exhibited a different size upon gel filtration. In vitro-generated biotin-pre-beta 1-LpA-I took up twofold to threefold more [3H]cholesterol from labeled fibroblasts during a 1-minute pulse incubation than lipid-free apoA-I. The in vitro conversion of biotin-pre-beta 1- LpA-I was investigated in the presence of plasmas of healthy probands and patients with Tangier disease, with apoA-I deficiency, and with lecithin-cholesterol acyltransferase (LCAT) deficiency. Incubation of biotin-pre-beta 1-LpA-I with plasmas either from normoalphalipoproteinemic probands or from a patient with apoA-I deficiency generated a biotinylated particle with the size and electrophoretic mobility of alpha-LpA-I2. This conversion was sensitive to heating at 56 degrees C but not to the removal of calcium. Inhibition of LCAT by dithiobisnitrobenzoic acid led to the formation of alpha-LpA-I3 instead of alpha-LpA-I2.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol efflux, cholesterol esterification, and cholesteryl ester transfer by LpA-I and LpA-I/A-II in native plasma.

HDLs encompass structurally heterogeneous particles that fulfill specific functions in reverse cholesterol transport. Two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-PAGGE) of normal plasma and subsequent immunoblotting with anti-apolipoprotein (apo) A-I antibodies differentiates an abundant particle with electrophoretic alpha-mobility and less abundant particles with electrophoretic pre-beta-mobility (pre beta 1-LpA-I, pre beta 2-LpA-I, pre beta 3-LpA-I). Immunodetection with anti-apoA-II antibodies identifies a single particle with alpha-mobility. To differentiate alpha-migrating HDL without apo A-II (alpha-LpA-I) from those with apoA-II (alpha-LpA-I/A-II), we combined 2D-PAGGE with immunoadsorption of apoA-II. Incubation of plasma with [3H]cholesterol-labeled fibroblasts in combination with immunosubtracting 2D-PAGGE allowed us to analyze the role of alpha-LpA-I and alpha-LpA-I/A-II in the uptake and esterification of cell-derived cholesterol in native plasma. Depending on the duration of incubations with cells, alpha-LpA-I took up two to four times more [3H]cholesterol than alpha-LpA-I/A-II. Irrespective of the duration of incubation, two to three times more [3H]cholesteryl esters accumulated in alpha-LpA-I than in alpha-LpA-I/A-II. Subsequent incubations in the presence of an inhibitor of lecithin:cholesterol acyltransferase led to preferential accumulation of [3H]cholesteryl esters in alpha-LpA-I/A-II. In conclusion, our data indicate that alpha-LpA-I is more effective than alpha-LpA-I/A-II in both uptake and esterification of cell-derived cholesterol. Moreover, alpha-LpA-I/A-II appears to accumulate cholesteryl esters, at least partially, from alpha-LpA-I.

Presence and formation of 'free apolipoprotein A-I-like' particles in human plasma.

The influence of dilution on apolipoprotein (apo) A-I-containing subpopulations was studied in human plasma. Agarose electrophoresis and two-dimensional agarose nondenaturing gradient polyacrylamide gel electrophoresis were used. Both in one- and two-dimensional electrophoresis, an increase of charge was observed that resulted in an increase of subpopulations with pre-alpha mobility. Dilution of plasma also resulted in a decrease in the size of apo A-I-containing pre-beta 1 subpopulations. The existence of smaller pre-beta 1 particles was confirmed by subjecting undiluted and 8x diluted plasma to 3% to 16% nondenaturing gradient gel electrophoresis for 4 hours. In addition to the generally observed pre-beta 1 subpopulations, smaller particles similar in size to the free apo A-I were detected even in the undiluted plasma. During dilution, the proportion of larger pre-beta 1 particles decreased while the smaller ones increased, and in 8x diluted plasma, almost all the pre-beta 1 was present in smaller sizes. Using 3% to 35% nondenaturing polyacrylamide gels run for 24 hours, no pre-beta 1 particles could be detected in 8x diluted plasma because the small pre-beta 1 electrophoresed out. These studies show that pre-beta 1 particles can be converted to smaller ones during dilution. It also was demonstrated that "free apo A-I-like" pre-beta 1 particles are present in undiluted plasma. The presence of these particles may have important physiological and pathophysiological functions.

Isolation and partial characterization of β-galactosidase activity produced by a thermotolerant strain of Kluyveromyces marxianus during growth on lactose-containing media

A thermotolerant yeast strain, Kluyveromyces marxianus IMB3, has previously been shown to be capable of ethanol production following growth on lactose-containing media at 45°C. Here, we demonstrate that the organism is capable of producing inducible β-galactosidase activity in the presence of lactose. The majority of the activity appears to be cell associated. Enzyme, isolated following disruption of the cells, has been partially characterized and shown to have a K m of 5 m m for the substrate o-nitrophenyl-β- d-galactoside, whereas that for lactose was found to be 40 m m. The activity was shown to have an optimal operating temperature of 50°C, although the half-life of the enzyme was shown to be <5 min at that temperature. The half-life of the isolated enzyme at 45°C, the optimal fermenting temperature of the yeast, was shown to be 15 min. The enzyme was stable at 30°C for at least 8 h. The optimum pH was 7.5. When we analyzed the result using nondenaturing polyacrylamide gradient gel electrophoresis together with zymogram staining with methylumbelliferyl-β- d-galactoside as substrate, two distinct forms of activity were observed following prolonged incubation with substrate. The results suggest that although this yeast is capable of ethanol production at 45°C, thermotolerance is not reflected in the enzyme activity associated with assimilation of lactose.

Evidence That Meizothrombin Is an Intermediate Product in the Clotting of Whole Blood

Meizothrombin is an intermediate that is produced during the conversion of prothrombin to thrombin in systems composed of purified factor Xa and factor Va that are quantitatively assembled on an anionic phospholipid surface. The biological significance of this intermediate has recently been challenged by the apparent absence of meizothrombin during clotting of sodium citrate-anticoagulated plasma. We analyzed the formation of thrombin during coagulation of nonanticoagulated, unchilled, minimally manipulated whole blood in glass tubes. The process was stopped at 0, 3, 5, and 7 minutes by the addition of biotinylated peptidyl chloromethyl-ketone active-site labeling reagents. Plasma/serum was separated by centrifugation, and labeled species were extracted by immunoadsorption with a polyclonal anti-prothrombin antibody. The purified prothrombin-derived species were separated by SDS-polyacrylamide gradient gel electrophoresis and visualized on a chemiluminescent avidin blot. Meizothrombin appeared as an intermediate product of this reaction and persisted with some increase through the 7-minute time point. We also observed incorporation of the active-site label into a species of lower molecular weight consistent with the B1 chain of beta- and/or gamma-thrombin. These degraded forms of thrombin have not been previously demonstrated in a biologically relevant preparation. Our data clearly establish the generation of meizothrombin as an intermediate product of thrombin generation during whole-blood clotting. The data also represent the first experimental evidence for the generation of beta- and gamma-thrombin in a biologically relevant environment and time scale.

LDL particle size in mildly hypertriglyceridemic subjects: no relation to insulin resistance or diabetes

We examined 18 Type 2 diabetic and 19 non-diabetic subjects in order to determine the association between insulin resistance and LDL particle size distribution in mildly hypertriglyceridemic and hyperinsulinemic subjects with and without Type 2 diabetes. Insulin sensitivity of the patients was characterized by their insulin-stimulated glucose uptake rate determined by euglycemic clamp technique. LDL particle size distribution was determined by nondenaturing polyacrylamide gradient gel electrophoresis. Type 2 diabetic and non-diabetic subjects had closely similar serum lipid and lipoprotein concentrations as well as the mean particle diameters of the major LDL peak (246 ± 6 Å and 244 ± 6 Å, respectively). To evaluate the effect of insulin resistance on LDL particle size the participants were categorized into two subgroups using the median of their insulin-stimulated glucose uptake rate (14.67 μmol/kg/min) as a cut-off point. Neither lipid and lipoprotein concentrations nor the LDL particle size distributions differed between the more insulin resistant group (nine diabetic and nine non-diabetic subjects) and less insulin resistant group (nine diabetic and ten non-diabetic subjects). LDL particle size was not associated with the insulin-stimulated glucose uptake rate or with the mean 24-h concentration of serum insulin. Mean 24-h concentration of serum triglycerides was the strongest discriminator for LDL particle size ( r = −0.44, P < 0.01). In conclusion, neither Type 2 diabetes nor insulin resistance seem to have any direct effect on LDL particle size in mildly hypertriglyceridemic subjects. The fact that LDL particle size was associated with serum triglycerides indicates that the effect of diabetes and insulin resistance on LDL particle size could be explained by the effects of insulin resistance and/or hyperinsulinism on VLDL metabolism.

Preparation and structural characterization of large heparin-derived oligosaccharides.

Porcine mucosal heparin was partially depolymerized with heparin lyase I and then fractionated into low-molecular-weight (< 5000) and high-molecular-weight (> 5000) oligosaccharides by pressure filtration. The high-molecular-weight oligosaccharide mixture (approximately 50 wt% of the starting heparin) also contained intact heparin. This intact polymer complicates oligosaccharide purification. Thus, the low-molecular-weight fraction was used to prepare homogeneous oligosaccharides for structural characterization. The low-molecular-weight oligosaccharide mixture was first fractionated by low-pressure gel permeation chromatography into size-uniform mixtures of disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, dodecasaccharides, tetradecasaccharides and higher oligosaccharides. Each size-fractionated mixture was then purified on the basis of charge by repetitive semi-preparative strong-anion-exchange high-performance liquid chromatography. This approach has led to the isolation of 14 homogeneous oligosaccharides from disaccharide to tetradecasaccharide. The purity of these heparin-derived oligosaccharides was determined by gradient polyacrylamide gel electrophoresis, analytical strong-anion-exchange high-performance liquid chromatography, capillary electrophoresis and one-dimensional nuclear resonance spectroscopy. The structure of these oligosaccharides was established using 600 MHz two-dimensional nuclear resonance spectroscopy. The spectral methods used included homonuclear correlation spectroscopy, nuclear Overhauser effect spectroscopy and heteronuclear multiple quantum coherence spectroscopy. The 1H/1H connectivities of the protons of each sugar residue in an oligosaccharide were established by two-dimensional homonuclear correlation spectroscopy, while 1H/13C assignments were made using 1H inverse detection. One- and two-dimensional nuclear resonance spectroscopic analysis of these heparin oligosaccharides showed two closely related groups of heparin-oligosaccharides are afforded by enzymatic depolymerization of heparin. One group is fully sulphated, having the structures delta UAp2S(1[-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S( 1]n-->4)-alpha- D-GlcNpS6S, where delta UAp is 4-deoxy-alpha-L-threo-hex-4-eno-pyranosyluronic acid, GlcNp is 2-deoxy-2-aminoglucopyranose, IdoAp is idopyranosyluronic acid, S is sulphate and n = 0-6. The other group of oligosaccharides differ in that they contain beta-D-glucuronic acid in place of the alpha-L-iduronic acid residue nearest to the reducing end. The present study describes the isolation and structural elucidation of seven new oligosaccharides: an octasaccharide, two decasaccharides, two dodecasaccharides and two tetradecasaccharides. The utility of two-dimensional nuclear resonance spectroscopy to determine the structure of complex heparin oligosaccharides is also illustrated.

The associations of high-density lipoprotein subclasses with insulin and glucose levels, physical activity, resting heart rate, and regional adiposity in men with coronary artery disease: The stanford coronary risk intervention project baseline survey

We used nondenaturing polyacrylamide gradient gel electrophoresis to examine the associations of high-density lipoprotein (HDL) subclasses with adiposity, physical activity, resting heart rate (an indicator of sympathetic drive), and plasma insulin and glucose levels in 97 men with angiographically documented coronary artery disease. These men neither smoked nor used medications known to affect lipoproteins. The absorbency of protein stain was used as an index of mass concentrations at intervals of 0.01 nm within five HDL subclasses: HDL 3c (7.2 to 7.8 nm), HDL 3b (7.8 to 8.2 nm), HDL 3a (8.2 to 8.8 nm), HDL 2a (8.8 to 9.7 nm), and HDL 2b (9.7 to 12 nm). HDL peak diameter was determined from the predominant peak of the HDL particle distribution when plotted against particle diameter. Four men who were non—insulin-dependent diabetics as defined by a fasting glucose exceeding 140 mg/dL had significantly higher plasma HDL 3b levels and significantly smaller HDL peak diameters than nondiabetic men, and were therefore excluded from further analyses. In the remaining 93 nondiabetic men, plasma HDL 3b levels correlated positively with indices of truncal obesity (waist to hip ratio and subscapular skinfold), whereas plasma HDL 2b levels correlated negatively with indices of total adiposity (body mass index [BMI]) and truncal obesity (subscapular and abdominal skinfold). Fasting plasma insulin levels correlated negatively with HDL 3a, HDL 2a, and HDL 2b. Obesity significantly affected the relationships of resting heart rate with insulin and HDL subclasses. In heavier men (BMI > 25.8 kg/m 2) but not in the less-obese men (BMI < 25.8 kg/m 2), resting heart rate was negatively correlated with HDL 3a, HDL 2a, and HDL 2b levels and HDL peak diameter and positively correlated with fasting plasma insulin concentrations. Although the reported physical activity in heavier men also correlated with HDL 3a, HDL 2a, and resting heart rate, the associations of resting heart rate with HDL 3a, HDL 2a, and HDL 2bcould not be attributed to activity level. These analyses suggest that the influences of plasma insulin, regional adiposity, physical activity, and resting heart rate on HDL involve specific HDL subclasses. In the presence of increased adiposity, sympathetic drive and physical inactivity may reduce levels of larger HDL and the peak diameter of the major HDL subspecies.

Characterisation of D‐Arabinono‐1,4‐Lactone Oxidase from Candida albicans ATCC 10231

D-Erythroascorbic acid was detected from the cell extracts of a dimorphic fungus, Candida albicans. Its concentration in yeast cells grown at 25 degrees C was estimated to be about 0.45 mumol/ml cell water. D-Arabinono-1,4-lactone oxidase, which catalyses the final step in the biosynthesis of D-erythroascorbic acid, was purified 639-fold from the mitochondrial fraction of C. albicans to apparent homogeneity, with an overall yield of 21.2%, by a purification procedure consisting of Triton X-100 solubilisation, ammonium sulphate precipitation, anion-exchange, hydrophobic-interaction, gel-filtration and dye-ligand chromatographies. Gel-filtration chromatography and polyacrylamide-gradient gel electrophoresis in the presence of deoxycholate gave apparent molecular masses of 110 kDa and 84.4 kDa, respectively. SDS/PAGE showed only one protein band corresponding to a molecular mass of 66.7 kDa. Considering the binding of detergents, the enzyme is suggested to be a single polypeptide. The enzyme showed a typical fluorescence excitation spectrum of a flavin-containing enzyme. The flavin was not released by treatment with SDS, CCl3CO2H or boiling, indicating that it may be covalently bound to the enzyme protein. The enzyme was optimally active at 40 degrees C and at pH 6.1. The enzyme was stable in the range pH 7.5-10. An apparent Km value for D-arabinono-1,4-lactone was 44.1 mM. L-Galactono-1,4-lactone, L-gulono-1,4-lactone and L-xylono-1,4-lactone could also serve as substrates. Competitive inhibition was demonstrated with D-glucono-1,5-lactone, L-arabinono-1,4-lactone, D-galactono-1,4-lactone and D-gulono-1,4-lactone. p-Chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, iodoacetamide and divalent metal ions such as Cd2+, Hg2+, Mn2+ and Zn2+ exhibited inhibitory effects on the enzyme.

Purification and characterization of a human erythrocyte-derived growth factor with a wide target cell spectrum.

A cell extract from human erythrocytes promoted the growth of a wide variety of cell types, namely human and mouse myeloid cells, human and mouse T cells, human B cells, human melanoma cells, mouse transformed fibroblast cells, mouse mastocytoma cells, human lung fibroblast cells, and mouse bone marrow fibroblast/stroma-like cells. The growth-promoting activity was acid- and heat-labile and sensitive to proteases, indicating the proteinaceous nature of the molecule. The activity was also lost upon exposure to 2-mercaptoethanol. The novel growth-promoting factor, termed basic growth factor because of its fundamental effect and a wide target cell spectrum, was purified by sequential anion-exchange, hydrophobic, gel filtration, hydroxylapatite, and reverse-phase high performance liquid chromatographies. The purified factor has an apparent molecular mass of 53 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The factor migrated at 270 kDa on native gradient polyacrylamide gel electrophoresis. Therefore, the factor consists of a homopolymer of a single polypeptide chain. The purified factor promoted the growth of the same cell types as the cell extract, except for bone marrow cells.

Glycogen phosphorylase from flight muscle of the hawk moth, Manduca sexta: purification and properties of three interconvertible forms and the effect of flight on their interconversion

Glycogen phosphorylase (EC 2.4.1.1) of Manduca sexta flight muscle was separated into three distinct peaks of activity on diethylaminoethyl-Sephacel. The three fractions of phosphorylase activity were further purified by affinity chromatography on AMP-Sepharose and shown to have the same relative molecular mass (=178000) on polyacrylamide gradient gel electrophoresis under non-denaturating conditions and to produce subunits of molecular mass =92000 on SDS gelelectrophoresis. On the basis of their kinetic properties with respect to the activator AMP and the inhibitor caffeine, the three fractions of phosphorylase activity were assigned as follows: peak 1=phosphorylase b (unphosphorylated form), peak 3=phosphorylase a (phosphorylated form); peak 2 represented a phospho-dephospho hybrid in which only one subunit of the dimeric enzyme was phosphorylated. This hypothesis was corroborated as the various forms could be interconverted in vitro by either dephosphorylation by an endogenous protein phosphatase producing the b form, or by phosphorylation catalyzed by purified phosphorylase kinase from rabbit muscle producing phosphorylase ab and a. From muscle of resting moths more phosphorylase was isolated in the b form (41%) than in the forms ab (28%) and a (31%), respectively. This proportion was changed in favour of the fully phosphorylated a form after a brief interval of flight when 68% of the phosphorylase activity was represented by the a form and only 13% by the b form. Unlike the phosphorylated forms a and ab of phosphorylase, the b form had low affinities for the substrates and for the activator AMP, and was virtually inactive if near-physiological concentrations of substrates and effectors were employed in the assays. The results demonstrate that in Manduca flight muscle three forms of phosphorylase coexist and that their interconversion is a mechanism for regulating phosphorylase activity in vivo.

Purification and characterization of two serine collagenolytic proteases from crab Paralithodes camtschatica

Two enzymes possessing collagenolytic activity were isolated from the hepatopancreas of crab Paralithodes camtschatica by ammonium sulfate fractionation and DEAE-Sepharose chromatography. It was shown that the specific activities of proteases A and C toward insoluble collagen were equal to 400 and 300 Mandl units/mg protein, respectively. The mol. wt of homogenous proteases A and C determined by gradient polyacrylamide gel electrophoresis in the presence of SDS and 2-mercaptoethanol were equal to 30 and 24 kDa, respectively. The isoelectric point values for the enzymes were determined as 2.5 and 2.9. Both enzymes lack carbohydrates. The amino acid compositions of two crab proteases were measured. The optimal conditions for the enzyme catalysis and the catalytic constants for collagenolytic proteases A and C with respect to Bz-Arg-pNA and Bz-Tyr-OEt have been determined. Inhibition data led to classification of the purified enzymes as serine proteases.

Large and Cholesteryl Ester-Rich High-Density Lipoproteins in Cholesteryl Ester Transfer Protein (CETP) Deficiency Can Not Protect Macrophages from Cholesterol Accumulation Induced by Acetylated Low-Density Lipoproteins1

High-density lipoprotein (HDL) has been speculated to have an anti-atherogenic function. Many in vitro studies have demonstrated that HDL has the ability to remove cholesteryl ester (CE) from lipid-laden macrophages. However, the effect of alteration in chemical composition and particle diameter on the in vivo function of HDL is unknown. In the study described here, we have isolated the HDL from patients homozygous for cholesteryl ester transfer protein (CETP) deficiency and examined its function in vitro, in order to clarify the anti-atherogenic property of HDL in CETP-deficient subjects. Apolipoprotein (apo) E-free HDL2 from the patients, separated by heparin-Sepharose column chromatography, was rich in CE, poor in triglycerides (TG), and enlarged in size on 4-30% nondenaturing polyacrylamide gradient gel electrophoresis. In contrast, HDL3 from the patients was normal in size and in its chemical composition. First, we examined the effect of HDL on CE accumulation in macrophages. After mouse peritoneal macrophages had been incubated with both acetylated low-density lipoproteins (Ac-LDL) and HDL, cellular CE content was determined by an enzymatic, fluorometric method. Ac-LDL alone induced a 9-fold accumulation of CE. The addition of apo E-free HDL2 and HDL3 from controls and patients' HDL3 prevented CE accumulation in macrophages, while patients' HDL2 had no preventive effect. We next investigated the in vitro ability of HDL to remove cellular CE from lipid-laden macrophages after incubation with Ac-LDL. After loading of macrophages with cholesterol by Ac-LDL, HDL was added to the culture medium and the cellular CE content was measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Transgenic mice expressing human lipoprotein lipase driven by the mouse metallothionein promoter. A phenotype associated with increased perinatal mortality and reduced plasma very low density lipoprotein of normal size.

We have produced transgenic mice expressing human lipoprotein lipase (LPL) driven by the mouse metallothionein I promoter. We found that integration of the LPL gene construct was associated with a high perinatal mortality. Animals that survived the first 2 weeks of life grew normally afterwards. Compared with controls, transgenic animals had higher post-heparin plasma LPL and tissue LPL activities. Immunoreactive human LPL was detected in their post-heparin plasma but not in controls. Transgenic animals had significantly lower plasma very low density lipoprotein (VLDL) while on a regular laboratory chow. By electron microscopic analysis and nondenaturing polyacrylamide gradient gel electrophoresis, the size and morphology of the plasma VLDL were very similar in transgenic and control animals, which suggests that VLDL particles acted on by the increased tissue LPL in the transgenic animals were mostly taken up by the cell without being released back into circulation. The hypertriglyceridemia and elevated VLDL in response to sucrose feeding were completely abolished in transgenic animals. They also had lower VLDL lipids compared with control animals when they were fed a high-fat, high-cholesterol diet. Feeding the mother of transgenic mice a high-fat diet during pregnancy completely reversed the high perinatal mortality associated with the integrated transgene, which suggests that the deleterious effect of LPL overexpression may be related to the depletion of some essential lipid nutrient.

Hypertriglyceridemia during late pregnancy is associated with the formation of small dense low-density lipoproteins and the presence of large buoyant high-density lipoproteins

Late pregnancy is a unique metabolic state where there are transient increases in the concentrations of plasma triglyceride (TG), cholesterol, and apolipoprotein (apo) B. Despite the hypertriglyceridemic environment, we recently reported that there is an unusual shift in high-density lipoprotein (HDL) subclass distribution from smaller HDL subclasses to the largest, most buoyant HDL 2b subclass. In the present investigation, we determined whether the subclasses of low-density lipoprotein (LDL) also change during this transient hyperlipidemic state and whether such changes were associated with plasma TG and apolipoprotein concentrations. Thirty-six Hispanic subjects at 35 to 36 weeks' gestation and at 6 weeks' postpartum were studied. At 35 to 36 weeks of gestation, plasma concentrations of TG, cholesterol, and apo B were increased (218 ± 62, 234 ± 48, and 130 ± 35 mg/dL, respectively) over levels at 6 weeks' pospartum (112 ± 69, 197 ± 36, and 97 ± 25 mg/dL respectively). However, lipoprotein(a) [Lp(a)] concentrations were not changed during pregnancy compared with postpartum. LDL subclass patterns (A, B, or I) were determined by nondenaturing polyacrylamide gradient gel electrophoresis in our group of 36 pregnant women. During late pregnancy, 97% of subjects were categorized as LDL subclass patterns B or I, indicating that small, dense LDL particles predominated. This predominance of small, dense LDL was associated with plasma TG concentration, where there was a significant inverse relationship ( r = −.45, P < .01) between the LDL peak particle diameter and plasma TG concentration. In an apparent anomaly, there were significant increases in the concentrations of HDL cholesterol (HDL-C) and HDL 2 mass, even though small, dense LDL particles predominated. We speculate that this unusual late-pregnancy lipid/lipoprotein profile is probably the result of insulin resistance combined with the action of the hormone estrogen.

Purification and characterization of an early-expressed polydnavirus-induced protein from the hemolymph of Manduca sexta larvae parasitized by Cotesia congregata

Parasitism of Manduca sexta by Cotesia congregata causes major qualitative and quantitative changes in the host's hemolymph proteins, with several novel parasitism-specific proteins being expressed in newly infected larvae. This report describes evidence for the production of several early-induced proteins (EP's) all of which have subunit molecular weights in the range of 30,000–40,000 and appear in the hemolymph of M. sexta within a few hours following parasitization. Injection of purified polydnavirus into non-parasitized larvae induced production of several EP proteins, suggesting that onset of their synthesis is virally mediated. Following analysis of hemolymph proteins on 2-dimensional gels, N-terminal sequence data were obtained for three EP proteins and one was selected for further study, which was designated EP1. This protein was purified to homogeneity using gel filtration and chromatofocusing; treatment of native purified protein with glycosidases showed that EP1 was heavily glycosylated with polysaccharides of the high-mannose type and the denatured, deglycosylated protein had a subunit molecular weight of c. 33,000. The native molecular weight of EP1 was estimated by gel filtration and native gradient polyacrylamide gel electrophoresis (PAGE) c. 190,000. Western blots of hemolymph from parasitized larvae showed that the EP1 antisera detected proteins only in parasitized larvae; the antibodies showed no cross-reactivity with other proteins present in the hemolymph of non-parasitized larvae. The Western blots indicated the early protein(s) were immunologically detectable in the hemolymph from 2 to 144 h following parasitization. Slot blots using a labeled internal restriction fragment from an early protein-specific cDNA (believed to be EP1 cDNA) as probe showed that transcripts from fat body of parasitized larvae were detectable from 0.5 to 96 h post-oviposition, suggesting that synthesis of transcripts rapidly occurs. At 24 h post-oviposition, these transcripts were detected primarily in fat body and hemocytes; lower amounts were also detected in Malpighian tubules and midgut tissues.

Action pattern of polysaccharide lyases on glycosaminoglycans

The action pattern of polysaccharide lyases on glycosaminoglycan substrates was examined using viscosimetric measurements and gradient polyacrylamide gel electrophoresis (PAGE). Heparin lyase I (heparinase, EC 4.2.2.7) and heparin lyase II (no EC number) both acted on heparin in a random endolytic fashion. Heparin lyase II showed an ideal endolytic action pattern on heparan sulphate, while heparin lyase I decreased the molecular weight of heparan sulphate more slowly. Heparin lyase III (heparitinase, EC 4.2.2.8) acted endolytically only on heparan sulphate and did not cleave heparin. Chondroitin ABC lyase (chondroitinase ABC, EC 4.2.2.4) from Proteus vulgaris acted endolytically on chondroitin-6-sulphate (chondroitin sulphate C) and dermatan sulphate at nearly identical initial rates, but acted on chondroitin-4-sulphate (chondroitin sulphate A) at a reduced rate, decreasing its molecular weight much more slowly. Two chondroitin AC lyases (chondroitinase AC, both EC 4.2.2.5) were examined towards chondroitin-4- and -6-sulphates. The exolytic action of chondroitin AC lyase A from Arthrobacter aurescens on both chondroitin-4- and -6-sulphates was demonstrated viscosimetrically and confirmed using both gradient PAGE and gel permeation chromatography. Chondroitin AC lyase F from Flavobacterium heparinum (Cytophagia heparinia) acted endolytically on the same substrates. Chondroitin B lyase (chondroitinase B, no EC number) from F.heparinum acted endolytically on dermatan sulphate giving a nearly identical action pattern as observed for chondroitin ABC lyase acting on dermatan sulphate.

Lipoprotein abnormalities in non-insulin-dependent diabetic patients with impaired extrahepatic insulin sensitivity, hypertension, and microalbuminuria.

We investigated whether specific lipoprotein abnormalities are present in non-insulin-dependent diabetes mellitus (NIDDM) patients with hypertension and/or microalbuminuria. Fifteen normotensive normoalbuminuric (H-M-), 32 hypertensive normoalbuminuric (H+M-), and 22 hypertensive microalbuminuric (H+M+) NIDDM patients and 20 sex-, age-, and weight-matched nondiabetic control subjects were studied. Lipoprotein size was measured by nondenaturing polyacrylamide gradient gel electrophoresis; insulin sensitivity was assessed by using a euglycemic hyperinsulinemic clamp and [6,6(2)H]glucose tracer infusion for simultaneous measurement of hepatic glucose output and whole-body glucose utilization. Total plasma and very-low-density lipoprotein cholesterol were higher in H+M+ than in control subjects (5.84 +/- 0.98 versus 4.97 +/- 0.98 and 0.57 +/- 0.54 versus 0.26 +/- 0.21 mmol/L, mean +/- SD, P < .05). Plasma triglycerides were higher in H+M+ than in either control or H-M- subjects (2.17 +/- 1.32 versus 1.18 +/- 0.67 and 1.30 +/- 0.59 mmol/L, respectively; P < .05). The mean low-density lipoprotein diameter was 27.2 +/- 0.8 in control, 26.7 +/- 0.8 in H-M-, 26.5 +/- 0.8 nm in H+M- (P < .05 versus control subjects), and 26.0 +/- 0.8 nm in H+M+ subjects (P < .05 versus control subjects). The mean cholesterol level of the large high-density lipoprotein particles was lower in H+M- and H+M+ (0.37 +/- 0.14 and 0.36 +/- 0.16 mmol/L) than in control and H-M- (0.54 +/- 0.41 and 0.54 +/- 0.27 mmol/L, P < .05) subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of bezafibrate therapy on subfractions of plasma low-density lipoprotein and high-density lipoprotein, and on activities of lecithin: cholesterol acyltransferase and cholesteryl ester transfer protein in patients with hyperlipoproteinemia

We investigated the effects of 12 weeks of bezafibrate treatment on plasma lipoprotein subfraction levels and on activities of LCAT and CETP in 25 patients with hyperlipoproteinemia. Bezafibrate reduced plasma levels of VLDL-TC and VLDL-TG by 69% and 66% ( P < 0.001) and plasma levels of IDL-TC and IDL-TG were decreased by 37% and 31% ( P < 0.01). Bezafibrate had no significant effects on plasma levels of LDL 1(1.019 < d < 1.045)-TC and LDL 1-TG in the study population as a whole but significantly increased the plasma level of LDL 1-TC in the subgroup of 9 patients with type IV hyperlipoproteinemia. Bezafibrate reduced plasma levels of LDL 2 (1.045 < d < 1.063)-TC, LDL 2-TG by 48% and 44% ( P < 0.001) in both type II and type IV hyperlipoproteinemic patients. Gradient polyacrylamide gel electrophoresis revealed a decrease in small LDL particles. Bezafibrate did not affect the plasma level of HDL 2-TC but reduced the HDL 2-TG concentration significantly ( P < 0.001). Bezafibrate increased the plasma level of HDL 3-TC by 37% and reduced the HDL 3-TG level significantly by 20% ( P < 0.001). Gradient polyacrylamide gel electrophoresis revealed an increase in HDL 3a and a decrease in HDL 2a. Bezafibrate suppressed the activities of LCAT and CETP by 21% ( P < 0.001) and 17% ( P < 0.01), respectively. The bezafibrate-induced decrease in plasma levels of small, heavy LDL might be related to its inhibition of LCAT and CETP activities which resulted in suppression of heteroexchange of HDL-EC with triglyceride in large, light LDL. The bezafibrate-induced increase in large HDL 3 (HDL 3a) could not be explained solely by its suppression of LCAT and CETP activities. The decrease of plasma small, heavy LDL as well as TG-rich lipoproteins by bezafibrate seems to be beneficial for prevention of atherosclerotic diseases.