Polyacrylamide Gradient Gel Electrophoresis Research Articles

An Automatic Fast High Resolution HDL Subfractioning Assay Using Microfluidic Electrophoretic System

Objectives: Studies in recent decade demonstrated the importance of HDL subfractionation in precision medicine. It is notable that not all HDL subfractions are “good” lipoprotein. The lack of a reliable technique that can precisely identify each particle of HDL subfractions remains the major obstacle in clinical application. Based on the principle of microfluidic electrophoresis, we have developed an automatic system (MFE) that meets the need of clinical applications. The objectives of this paper is to test the system for its application, precision, and also the normal range in healthy population using one of major subfraction, HDL-2b, as a target. Methods: The MFE was compared with traditional non-denaturing polyacrylamide gradient gel electrophoresis (GGE). Intra-chip, inter-chip, inter-instrument, inter-lab, and inter-operator precisions were all tested by using the sample in various conditions. An instruction was provided with the instrument and HDL subfractioning kit. Data were analyzed with SPSS for correlation of variation, regressions, etc. Results: The new system exhibited a linear correlation with the traditional GGE system. The intra-chip %CVs, inter-chip %CVs, inter-instrument %CVs of HDL-2b were less than 10%, which was the set acceptance criteria. With 243 samples from healthy individuals demonstrated that the mean value of HDL-2b% is about 25% in both male and female. Conclusions: Our data demonstrated that the MFE system is a fast, automatic, and reliable system that can obtain the percentage of HDL-2b with high precision

Expeditious Molecular Weight Determination of Native Proteins by Transverse Pore Gradient Polyacrylamide Gel Electrophoresis Using the PhastGel System

Expeditious Molecular Weight Determination of Native Proteins by Transverse Pore Gradient Polyacrylamide Gel Electrophoresis Using the PhastGel System

Altered high-density lipoprotein particle structure and antioxidant capacity in preeclampsia

Background/Aim. One of the complications that can occur during pregnancy is the development of preeclampsia (PE). The main characteristics of this condition are high blood pressure and very often signs of kidney damage or other organ damage. The condition affects 5?7% of all pregnant women and is one of the main factors of maternal and perinatal morbidity and mortality worldwide. The aim of this study was to investigate the structural and functional modifications of high-density lipoprotein (HDL) particles during high-risk pregnancies (HRP) for PE development. Methods. The longitudinal prospective study included a total of 91 pregnant women with a HRP for developing PE. Out of this total number, 71 women did not develop PE until delivery, and this group was designated as the group without PE (WPE). The rest of the 20 HRP women developed PE before delivery and were designated as the PE group. The blood was sampled toward the end of each trimester and before the delivery. The distribution of HDL particles was determined by the vertical 3?31% polyacrylamide gradient gel electrophoresis method. The antioxidative capacity of HDL particles was measured by the activity of the HDL-associated enzyme ? paraoxonase 1 (PON1). PON1 activity was determined by the method of kinetic spectrophotometry from serum samples. Results. The results have shown that the proportions of HDL2b particles significantly increased in the 2nd trimester (p ? 0.05) and remained increased until the end of pregnancy in the WPE group. PON1 activity was significantly higher in the 3rd trimester (p < 0.05) of the WPE group. In the PE group, we found that the proportions of HDL3a particles significantly decreased in the 2nd trimester (p < 0.05) and remained decreased until the end of pregnancy. PON1 activity has not changed in the PE group during pregnancy. Conclusion. Dyslipidemia in pregnancy could be associated with different modifications of HDL particles. The adaptive pregnancy mechanisms expressed as a functional modification of HDL particles in pregnant women who develop PE seem inadequate and, therefore, lose their atheroprotective role.

Low-density lipoprotein cholesterol and non-high-density lipoprotein cholesterol measurement in Familial Dysbetalipoproteinemia

AimTo compare LDL-C concentrations using the Friedewald formula, the Martin-Hopkins formula, a direct assay and polyacrylamide gradient gel electrophoresis (PGGE) to the reference standard density gradient ultracentrifugation in patients with Familial Dysbetalipoproteinemia (FD) patients. We also compared non-HDL-cholesterol concentrations by two methods. MethodsFor this study data from 28 patients with genetically confirmed FD from the placebo arm of the EVOLVE-FD trial were used. Four different methods for determining LDL-C were compared with ultracentrifugation. Non-HDL-C was measured with standard assays and compared to ultracentrifugation. Correlation coefficients and Bland-Altman plots were used to compare the methods. ResultsMean age of the 28 FD patients was 62 ± 9 years, 43 % were female and 93 % had an ɛ2ɛ2 genotype. LDL-C determined by Friedewald (R2 = 0.62, p <0.01), Martin-Hopkins (R2 = 0.50, p = 0.01) and the direct assay (R2 = 0.41, p = 0.03) correlated with density gradient ultracentrifugation. However, Bland-Altman plots showed considerable over- or underestimation by the four methods compared to ultracentrifugation. Non-HDL-C showed good correlation and agreement. ConclusionIn patients with FD, all four methods investigated over- or underestimated LDL-C concentrations compared with ultracentrifugation. In contrast, standard non-HDL-C assays performed well, emphasizing the use of non-HDL-C in patients with FD.

Plasma HDL pattern, cholesterol efflux and cholesterol loading capacity of serum in carriers of a novel missense variant (Gly176Trp) of endothelial lipase

Loss of function variants of LIPG gene encoding endothelial lipase (EL) are associated with primary hyperalphalipoproteinemia (HALP), a lipid disorder characterized by elevated plasma levels of high density lipoprotein cholesterol (HDL-C). Aim of the study was the phenotypic and genotypic characterization of a family with primary HALP. HDL subclasses distribution was determined by polyacrylamide gradient gel electrophoresis. Serum content of preβ-HDL was assessed by (2D)-electrophoresis. Cholesterol efflux capacity (CEC) of serum mediated by ABCA1, ABCG1 or SR-BI was assessed using cells expressing these proteins. Cholesterol loading capacity (CLC) of serum was assayed using cultured human macrophages. Next generation sequencing was used for DNA analysis. Plasma EL mass was determined by ELISA. Three family members had elevated plasma HDL-C, apoA-I and total phospholipids, as well as a reduced content of preβ-HDL. These subjects were heterozygous carriers of a novel variant of LIPG gene [c.526 G>T, p.(Gly176Trp)] found to be deleterious in silico. Plasma EL mass in carriers was lower than in controls. CEC of sera mediated by ABCA1 and ABCG1 transporters was substantially reduced in the carriers. This effect was maintained after correction for serum HDL concentration. The sera of carriers were found to have a higher CLC in cultured human macrophages than control sera. The novel p.(Gly176Trp) variant of endothelial lipase is associated with changes in HDL composition and subclass distribution as well as with functional changes affecting cholesterol efflux capacity of serum which suggest a defect in the early steps of revere cholesterol transport.

Changes in the size and electrophoretic mobility of HDL subpopulation particles in chronic kidney disease

BackgroundHigh-density lipoprotein (HDL) is a heterogeneous group of particles with anti-atherogenic properties whose metabolism is alterated in chronic kidney disease (CKD). The aim of this study was to evaluate the particle size and mobility of HDL subpopulations in non-dialysis CKD patients.MethodsThe study involved 42 non-dialysis CKD patients (stages 3a–4) and 18 control subjects. HDL was separated by non-denaturing two-dimensional polyacrylamide gradient gel electrophoresis (2D-PAGGE) and eight HDL subpopulations; preβ1, preβ2a-c, and α1-4 were distinguished. The size and electrophoretic mobility of HDL subpopulation particles were compared between the groups, and a regression analysis was conducted.ResultsIn CKD patients, the mean sizes of α-HDL and preβ2-HDL particles were significantly lower compared to the control group (8.42 ± 0.32 nm vs. 8.64 ± 0.26 nm, p = 0.014; 11.45 ± 0.51 vs. 12.34 ± 0.78 nm, p = 0.003, respectively). The electrophoretic mobility of preβ2-HDL relative to α-HDL was significantly higher in CKD patients compared to the control group (Rf 0.65 ± 0.06 vs. 0.53 ± 0.10, p = 0.002). The size and mobility of HDL subpopulations correlated with eGFR values (p < 0.01). These relationships remained statistically significant after adjusting for age, gender, statin treatment, apolipoprotein AI, total cholesterol, and triglyceride levels.DiscussionCKD affects the size and mobility of HDL particles, which can be related to HDL dysfunction. The magnitude of HDL size and mobility changes depended on CKD stage and differed for individual HDL subpopulations, which indicates that some stages of HDL metabolism may be more affected by the presence of chronic kidney disease.Graphical abstract

Lipoproteins and cholesterol homeostasis in paediatric nephrotic syndrome patients.

IntroductionThe aim of this study was to investigate lipoprotein particle distributions and the likelihood of achieving cholesterol homeostasis in the remission phase of nephrotic syndrome (NS) in paediatric patients. We hypothesized that lipoprotein particle distributions moved toward less atherogenic profile and that cholesterol homeostasis was achieved.Materials and methodsThirty-three children, 2 to 9 years old with NS were recruited. Blood sampling took place both in the acute phase and during remission. Serum low-density lipoprotein particles (LDL) and high-density lipoprotein particles (HDL) were separated using non-denaturing polyacrylamide gradient gel (3-31%) electrophoresis. Serum non-cholesterols sterols (NCSs), desmosterol, lathosterol, 7-dehydrocholesterol (7-DHC), campesterol and β-sitosterol were measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).ResultsAll patients had desirable serum HDL cholesterol concentrations during remission. The dominant lipoprotein diameters and LDL subclass distribution did not change significantly during follow-up. In contrast, HDL lipoprotein particle distribution shifted towards larger particles. The absolute concentration of desmosterol was significantly lower during remission (P = 0.023). β-sitosterol concentration markedly increased during remission (P = 0.005). Desmosterol/β-sitosterol (P < 0.001) and 7-DHC/β-sitosterol (P = 0.005) ratios significantly declined during disease remission.ConclusionsFavourable changes in the serum lipid profiles, HDL particle subclass distribution and cholesterol metabolism in paediatric patients with NS during remission took place. For the first time, we found that cholesterol homeostasis changed in favour of increased cholesterol absorption during disease remission. Nevertheless, complete cholesterol homeostasis was not achieved during disease remission.

Comparison of the modified polyacrylamide gradient gel electrophoresis and high-performance liquid chromatography methods in determining LDL size

Abstract Objectives In this study, we aimed to compare modified Krauss polyacrylamide gradient gel electrophoresis (PAGGE) and high-performance liquid chromatography (HPLC) methods in classification, quantification, and separation of lipoproteins and determining low-density lipoprotein (LDL) size. Methods Blood specimens were obtained from eighty-seven volunteers. We measured LDL size using the PAGGE method and HPLC method with total cholesterol (TC) and triglyceride (TG) peaks. In the PAGGE method, Coomassie Brilliant Blue (CBB) staining was used instead of Sudan black staining, unlike the original method. The relationship between PAGGE and HPLC methods was evaluated by Pearson correlation test and Passing-Bablok regression analysis. Agreement between them was evaluated by Kappa analysis and Bland-Altman plots. Results Statistically significant correlation was found between the LDL size with PAGGE and HPLC methods under the cholesterol curve (HPLC-TC) (r=0.924, p&lt;0.001). Similarly, there was a statistically significant correlation between PAGGE and HPLC methods under the TG curve (HPLC-TG) (r=0.910, p&lt;0.001). In the PAGGE method, within-day precision was found as 2% and between-day precision as 3%. It was determined agreement between HPLC-TC vs. HPLC-TG methods and HPLC-TG vs. PAGGE methods was higher than HPLC-TC vs. PAGGE (Kappa values; 0.68, 0.71, and 0.44, respectively). Conclusions The PAGGE method can be a reliable method for measuring LDL size. HPLC method under cholesterol and triglyceride peaks may be used in clinical practice interchangeably, but clinical decision limits should be different. In addition, our study demonstrated that measurement methods for LDL size could be simplified with several modifications.

Uticaj metaboličkih parametara na veličinu i subklasu LDL i HDL kod adolescenata sa dijabetesom tip 1

Alterations in the serum of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) size and subclass contribute to the atherogenesis in coronary artery disease in diabetic patients. The purpose of this study is to evaluate the effect of metabolic parameters on LDL and HDL size and subclasses in adolescents with type 1 diabetes. The cross-sectional study included 43 adolescents (23 females, 20 males) with type 1 diabetes of mean age 15.09±1.94 years, with mean disease duration of 5.86 ± 3.08 years. LDL and HDL particles were separated by polyacrylamide gradient gel electrophoresis, while serum lipid parameters were determined by routine laboratory methods. Patients with inadequate metabolic control (HbA1c ≥ 7.5%) had a higher mean value of triglycerides (TG) (p = 0.041), higher proportions of small, dense LDL particles (p = 0.045), higher proportions of LDL IIA subclasses (p=0.03) and smaller LDL diameter (p = 0.02) and HDL diameter (p = 0.04) than patients with optimal metabolic control (HbA1c &lt; 7.5%). Higher HbA1c and higher TG levels were statistically significantly related to small, dense LDL (r=0.341, p=0.025; r= 0.394, p= 0.009) and HDL particles (r=0.684, p=0.000; r=0.421, p=0.005). Predictors of small, dense LDL and HDL particles, which contribute to atherogenesis, were high HbA1c (HR = 1.52, 95% CI: 0.97-2.40; HR 3.87, 95% CI: 2.11-7.10) and elevated TG (HR= 1.10, 95% CI: 1.00-1.20; HR 1.85, 95% CI: 1.07-3.21). Diabetic adolescents require particular attention in order to minimize factors such as high HbA1c and elevated TGs in the development of future cardiovascular events.

Antioksidativna, antiinflamatorna i anti-hiperlipidemična svojstva speltinog soka

Spelt is a grain rich in bioactive compounds - different phytochemicals, so it is often mentioned in the context of being beneficial to human health. We aimed to investigate the antioxidant, anti-inflammatory, and anti-hyperlipidemic properties of the spelt grass juice. Twenty volunteers consumed a beverage once a day, by dissolving one dose in 200 ml of still water, for 2 months. All individuals were clinically and biochemically examined. The individual profiles of LDL and HDL particles were determined by polyacrylamide gradient gel electrophoresis, while the concentrations of non-cholesterol sterols were measured using the gas chromatography - flame ionization detector. High-sensitivity Creactive protein (hsCRP) was measured using the commercially available kit. Concentrations of oxidative stress status parameters were measured spectrophotometrically. Though no change was observed in lipid profile parameters, values of coronary risk (CRI) (p &lt; 0.05) and the atherogenic indexes (AI) (p &lt; 0.05) after the supplementation were lower compared with values before the dietary intervention. After analyzing the markers of cholesterol synthesis and absorption, we only noticed a significant increase in campesterol concentrations (p &lt; 0.05). Moreover, we found a statistically significant reduction in hsCRP concentrations in subjects who underwent spelt juice supplementation (p &lt; 0.001). A significant decrease in PAB (p &lt; 0.001), AOPP (p &lt; 0.05) and PON1 (p &lt; 0.001) was recorded after supplementation. On the other side, TAS values and concentration of SH groups were significantly higher after the dietary intervention (p &lt; 0.001, both). We suggested a possible beneficial effect of the spelt grass supplementation on the overall lipid profile, the prooxidant-antioxidant balance, and inflammatory parameters.

Association of acute Babesia canis infection and serum lipid, lipoprotein, and apoprotein concentrations in dogs.

Background Babesia canis infection induces a marked acute phase response (APR) that might be associated with alteration in lipid and lipoprotein metabolism and disease prognosis.HypothesisDogs with B. canis‐induced APR develop dyslipidemia with altered lipoprotein concentration and morphology.AnimalsTwenty‐nine client‐owned dogs with acute B. canis infection and 10 clinically healthy control dogs.MethodsObservational cross‐sectional study. Serum amyloid A (SAA) was measured using ELISA. Cholesterol, phospholipids, and triglycerides were determined biochemically. Lipoproteins were separated using agarose gel electrophoresis. Lipoprotein diameter was assessed by polyacrylamide gradient gel electrophoresis; correlation with ApoA‐1 (radioimmunoassay) and SAA was determined.ResultsDogs with B. canis infection had a marked APR (median SAA, 168.3 μg/mL; range, 98.1‐716.2 μg/mL) compared with controls (3.2 μg/mL, 2.0‐4.2 μg/mL) (P < .001). Dogs with B. canis infection had significantly lower median cholesterol (4.79 mmol/L, 1.89‐7.64 mmol/L versus 6.15 mmol/L, 4.2‐7.4 mmol/L) (P = .02), phospholipid (4.64 mmol/L, 2.6‐6.6 mmol/L versus 5.72 mmol/L, 4.68‐7.0 mmol/L) (P = .02), and α‐lipoproteins (77.5%, 27.7%‐93.5% versus 89.2%, 75.1%‐93.5%) (P = .04), and higher ApoA‐1 (1.36 U, 0.8‐2.56 U versus 0.95 U, 0.73‐1.54 U) concentrations (P = .02). Serum amyloid A correlated with high‐density lipoproteins (HDLs) diameter (rho = .43; P = .03) and ApoA‐1 (rho = .63, P < .001).Conclusions and Clinical ImportanceMajor changes associated with B. canis‐induced APR in dogs are related to concentration, composition, and morphology of HDL particles pointing to an altered reverse cholesterol transport. Parallel ApoA‐1 and SAA concentration increase is a unique still unexplained pathophysiological finding.

The Effect of Biochanin A as PPAR γ agonist on LDL Particles Diameter and Type 2 Diabetic Dyslipidemia

Background and Aims: Small dense low-density lipoproteins (sd-LDL) particles are smaller and heavier than typical LDL ones. They can penetrate into the endothelium of coronary arteries more easily because of their small size. Diabetes mellitus is accompanied by dyslipidemia such as increasing concentration of plasma very low density lipoprotein and sd-LDL. Peroxisome proliferator activated receptor γ (PPARγ ) can decrease the level of sd-LDL in plasma. Biochanin A (BCA), a natural compound, is a PPARγ agonist. The present study was designed to investigate the effect of BCA on sd-LDL-Clolesterol level in diabetic animals. Materials and Methods: Adult male rats (Wistar strain) were used as the animal models in this study. Animals were made diabetic by single intraperitoneal injection of Streptozotocin- Nicotinamide and then treated by 1 and 5 mg/kg of BCA for 28 days. Body weight and fasting blood glucose were also tested before and at the end of treatment. Furthermore, the size of LDL particles were measured by nondenaturing polyacrylamide gradient gel electrophoresis assay. Results: Results of the present study indicated that BCA administration at dose of 5mg/kg decreased fasting blood glucose level and increased body weight and diameter of LDL particles in diabetic animals significantly. Conclusions: BCA seems to be an appropriate agent in diabetes mellitus, because it improves the diabetic dyslipidemia, which is the most important complication in diabetic patients.

Molecular basis for the protective effects of low-density lipoprotein receptor-related protein 1 (LRP1)-derived peptides against LDL aggregation

Aggregated LDL is the first ligand reported to interact with the cluster II CR9 domain of low-density lipoprotein receptor-related protein 1 (LRP1). In particular, the C-terminal half of domain CR9, comprising the region Gly1127-Cys1140 exclusively recognizes aggregated LDL and it is crucial for aggregated LDL binding. Our aim was to study the effect of the sequence Gly1127-Cys1140 (named peptide LP3 and its retro-enantio version, named peptide DP3) on the structural characteristics of sphingomyelinase- (SMase) and phospholipase 2 (PLA2)-modified LDL particles. Turbidimetry, gel filtration chromatography (GFC) and transmission electronic microscopy (TEM) analysis showed that LP3 and DP3 peptides strongly inhibited SMase- and PLA2-induced LDL aggregation. Nondenaturing polyacrylamide gradient gel electrophoresis (GGE), agarose gel electrophoresis and high-performance thin-layer chromatography (HPTLC) indicated that LP3 and DP3 prevented SMase-induced alterations in LDL particle size, electric charge and phospholipid content, respectively, but not those induced by PLA2. Western blot analysis showed that LP3 and DP3 counteracted changes in ApoB-100 conformation induced by the two enzymes. LDL proteomics (LDL trypsin digestion followed by mass spectroscopy) and computational modeling methods evidenced that peptides preserve ApoB-100 conformation due to their electrostatic interactions with a basic region of ApoB-100. These results demonstrate that LRP1-derived peptides are protective against LDL aggregation, even in conditions of extreme lipolysis, through their capacity to bind to ApoB-100 regions critical for ApoB-100 conformational preservation. These results suggests that these LRP1(CR9) derived peptides could be promising tools to prevent LDL aggregation induced by the main proteolytic enzymes acting in the arterial intima.

Viroids as Companions of a Professional Career.

Since the early 1970s when “virus-like” agents were considered as the cause of two diseases (potato spindle tuber and citrus exocortis), their study and further characterization have been linked to the development and use of molecular biology tools. Sucrose density gradient centrifugation and polyacrylamide gel electrophoresis (PAGE) played a critical role in the pioneering studies of PSTVd and citrus exocortis viroid (CEVd). This was later modified by using other PAGEs (sequential PAGE, return PAGE, two-dimensional PAGE), and/or different staining methods (ethidium bromide, silver nitrate, etc.). Since then, disease-causing agents suspected to be viroids were usually subjected to a number of tests to define their: (i) Molecular nature (RNA or DNA; single stranded or double stranded; circular or linear RNA); (ii) molecular weight; (iii) secondary and tertiary structure. Further biological assays are also essential to establish the relationship of a viroid with plant disease and to fulfill Koch’s postulates.

Protein profiling of water and alkali soluble cottonseed protein isolates

Currently, there is only limited knowledge on the protein types and structures of the cottonseed proteins. In this work, water-soluble cottonseed proteins (CSPw) and alkali-soluble cottonseed proteins (CSPa) were sequentially extracted from defatted cottonseed meal. Proteins of the two fractions were separated by 4–20% gradient polyacrylamide gel electrophoresis (SDS-PAGE); There were 7 and 12 polypeptide bands on SDS-PAGE of CSPa and CSPw, respectively. These individual bands were then excised from the gel and subjected to mass spectrometric analysis. There were total 70 polypeptides identified from the proteins of the two cottonseed preparations, with molecular weights ranging from 10 to 381 kDa. While many proteins or their fragments were found in multiple bands, 18 proteins appeared only in one SDS-PAGE band (6 in CSPa, 12 in CSPw). Putative functions of these proteins include storage, transcription/translation, synthesis, energy metabolism, antimicrobial activity, and embryogenesis. Among the most abundant are legumin A (58 kDa), legumin B (59 kDa), vicilin C72 (70 kDa), vicilin GC72-A (71 kDa), and vicilin-like antimicrobial peptides (62 kDa). This work enriched the fundamental knowledge on cottonseed protein composition, and would help in better understanding of the functional and physicochemical properties of cottonseed protein and for enhancing its biotechnological utilization.

CETP and LCAT Gene Polymorphisms Are Associated with High-Density Lipoprotein Subclasses and Acute Coronary Syndrome.

We evaluated whether CETP and LCAT gene polymorphisms are statistically associated with the high-density lipoprotein (HDL) size distribution, the cholesterol level of HDL subclasses, and the acute coronary syndrome (ACS) susceptibility. Two CETP gene polymorphisms (rs4783961 and rs708272) and one LCAT polymorphism (rs2292318) were genotyped by 5' exonuclease TaqMan assays in 619 patients with ACS and 607 control individuals. For HDL analysis, a subgroup of 100 healthy individuals was recruited; the HDL subclasses were separated via ultracentrifugation and polyacrylamide gradient gel electrophoresis under native conditions. Under a dominant model, the G allele of the rs708272 polymorphism was associated with an increased risk of ACS (odds ratios [OR] = 1.45, corrected p-value [pCDom ] = 0.036). The linkage disequilibrium analysis showed that one of the eight possible combinations was associated with the risk of developing ACS (OR = 1.52, pC = 0.02), which suggests that it may contribute to coronary atherosclerosis. The rs708272 G allele carriers had a lower concentration of cholesterol associated with the HDL2a and HDL3a subclasses when compared with subjects carrying the A allele. Carriers of LCAT rs2292318 A allele showed a lower concentration of high-density lipoprotein-cholesterol (HDL-C) in comparison to the GG genotype; the cholesterol associated with the each one of the five HDL subclasses was significantly lower in rs2292318 A than in GG subjects. In summary, this study demonstrates that the rs708272 polymorphism is associated with a heightened risk of developing ACS. In addition, we report the association of the rs708272 and rs2292318 polymorphisms with HDL-C levels and HDL subclasses.

Transcoronary gradients of HDL-associated MicroRNAs in unstable coronary artery disease

AimsMicroRNAs (miRNAs) are transported on high-density lipoproteins (HDLs) and HDL-associated miRNAs are involved in intercellular communication. We explored HDL-associated miRNAs concentration gradients across the coronary circulation in stable and unstable coronary artery disease patients and whether changes in the transcoronary gradient were associated with changes in HDL composition and size. MethodsAcute coronary syndrome (ACS, n=17) patients, those with stable coronary artery disease (stable CAD, n=19) and control subjects without CAD (n=6) were studied. HDLs were isolated from plasma obtained from the coronary sinus (CS), aortic root (arterial blood) and right atrium (venous blood). HDL-associated miRNAs (miR-16, miR-20a, miR-92a, miR-126, miR-222 and miR-223) were quantified by TaqMan miRNA assays. HDL particle sizes were determined by non-denaturing polyacrylamide gradient gel electrophoresis. HDL composition was measured immunoturbidometrically or enzymatically. ResultsA concentration gradient across the coronary circulation was observed for all the HDL-associated miRNAs. In ACS patients, there was a significant inverse transcoronary gradient for HDL-associated miR-16, miR-92a and miR-223 (p<0.05) compared to patients with stable CAD. Changes in HDL-miRNA transcoronary gradients were not associated with changes in HDL composition or size. ConclusionHDLs are depleted of miR-16, miR-92a and miR-223 during the transcoronary passage in patients with ACS compared to patients with stable CAD.

Protein Patterns of Silkworm Bombyx mori L. Fed on Leaves of Wild and Cultivated Mulberry Varieties

Sodium dodecyl sulphate polyacrylamide gradient gel electrophoresis (SDS-PAGE) was used to determinethe protein profiles of two mulberry variety leaves: the cultivated Morus alba var. Morittiana and the wild one Moruslaevigata and their effects on haemolymph and silkgland protein patterns in final larval instar of silkworm Bombyx moriL. Results showed some differences in the number and abundance of protein bands in the two studied mulberry varietyleaves as they were 5 bands in Morus alba var. Morittiana ranged between (47.27- 344.47 kDa), while, were 10 bandsin Morus laevigata leaves ranged between (17.79- 350.11 kDa). However there were close similarity in haemolymphand silkgland protein patterns of larvae fed on the both studied mulberry varieties, which registrad 12 protein band inhaemolymph and 15 protein bands in silkgland of larvae fed on the two mulberry varities.

自然発症糖尿病WBN/Kobラットにおけるアルカリホスファターゼについて

The male WBN/Kob rat is an animal model of chronic pancreatitis and spontaneous diabetes. Here, we performed a kinetic study of alkaline phosphatase (ALP) in WBN/Kob rats. The level of serum ALP activities in the WBN/Kob rats was three times higher than that of the control group (p<0.001). Specific ALP activities in bone tissues or the ileum increased significantly (p<0.01 and p<0.05, respectively). By 4-20% gradient polyacrylamide gel electrophoresis, serum ALP isozymes were separated into two bands. One was heat-unstable, showing the same mobility as an ALP preparation from bone tissue, and the other was heat-stable. We considered these isozymes to be bone-type and intestinal-type ALPs, respectively, and both of them were enhanced in the WBN/Kob rats. These results demonstrate the enzyme kinetics of ALP isozymes in WBN/Kob rats, and further studies will be necessary to reveal the association with diabetes.

The New Molecular Entity Evolocumab, One Kind of PCSK9 Inhibitor, Reduce Plasma Small Size LDL-Cholesterol Levels by Using a New Standardized Method of Measuring LDL Size

Aims: There has been no evidence on the effects of evolocumab, protein convertase subtilisin/kexin type 9 (PCSK9) inhibitor, on small size LDL. We observationally investigated the efficacy and side effects of evolocumab on the LDL subfraction particle diameter using PAGE system for lipoprotein analysis. Methods: We defined 30 patients with high-risk hyperlipidemia. As for analysis of LDL subfraction profile, we used polyacrylamide gel electrophoresis three methods: 1) 3% nondenatured poly-acrylamide gel electrophoresis method (3%PAGE), 2) 2% - 16% nondenatured poly-acrylamide gradient gel electro-phoresis method (2% - 16% GGE) and 3) 2.7% - 5% GGE. Evolocumab 140 mg/day administered together with statin significantly improved serum total cholesterol (TC), triglyceride (TG), high-dense lipoprotein-cholesterol (HDL-C), and LDL-C after four-week treatment. Results: TC, TG, HDL-C and LDL-C levels were improved by, respectively, 33%, 20%, 10%, and 54%. The mean LDL size significantly increased from 25.6 ± 0.4 nm to 26.4 ± 0.8 nm. The small dense LDL-cholesterol (sdLDL-C), large buoyant LDL-cholesterol (lbLDL-C), and mid-band lipoprotein-cholesterol were reduced, respectively. Therefore, the preliminary study on this paper can be the first step into a new insight on the world of lipid metabolism. Conclusion: Short-term administration of evolocumab addedons to statin therapy, significantly reduced small size LDL levels.