The association between the HLA-G 14-bp insertion/deletion polymorphism and type 1 diabetes.

Type 1 diabetes (T1D) is a multifactorial disease that has a strong genetic component. The HLA-G is a nonclassical HLA class I locus that is associated with immunomodulatory functions, including downregulation of innate and adaptive immune responses and induction of immune tolerance. However, there is currently limited information about the involvement of HLA-G in T1D susceptibility. This case-control study aims to investigate the T1D susceptibility association of alleles and genotypes of a widely investigated 14-bp insertion/deletion polymorphism in the HLA-G and to provide further evidence of the frequency distribution of class II HLA-DR-DQ-risk genotypes in T1D children and adolescents in the Brazilian population. The deletion allele and the homozygous deletion genotype are associated with susceptibility to T1D and the insertion allele and the heterozygous deletion/insertion genotype are associated with protection from T1D. We also confirm that genetic susceptibility to T1D is associated with the DRB1*03:01-DQA1*05:01-DQB1*02:01 and DRB1*04-DQA1*03:01-DQB1*03:02 haplotypes in Brazilian northeast region. The DR3-DQ2/DR4-DQ8 genotype conferred the highest detected risk for T1D. Our results identify a novel association of the 14-bp deletion allele and the homozygous deletion genotype with T1D development and provide additional evidence of the importance of HLA class II heterozygous DR3-DQ2/DR4-DQ8 genotype in T1D susceptibility.

Genetic and non-genetic factors that increase the risk of non-syndromic cleft lip and/or palate development.

OBJECTIVES: We investigated the relationship between non-syndromic cleft lip/palate (NSCLP) and polymorphisms in methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR), and RFC1, as well as the corresponding interactions with environmental factors. SUBJECTS AND METHODS: One hundred and forty NSCLP patients and their mothers, as well as 175 control individuals and their mothers, were recruited. Information regarding smoking and alcohol consumption was recorded. Blood samples were obtained in order to measure serum folate and cobalamin, as well as, plasma total homocysteine concentrations and to extract DNA. Polymorphisms in MTHFR(677C>T and 1298A>C), MTR(2756A>G), MTR(66A>G), and RFC1(80A>G) were analyzed by PCR-restriction fragment length polymorphism. RESULTS: Among the patients, 59.5% had cleft lip and palate, 22.0% had cleft palate, and 18.5% had cleft lip only. Maternal alcohol consumption and reduced folic acid concentrations in both children and mothers (P < 0.001 and P = 0.003, respectively) were risk factors for NSCLP. Patients and their mothers carrying the MTHFR 667T allele showed lower serum folate than CC (P = 0.011 and P = 0.030, respectively). Mothers who carried the MTHFR 1298C allele exhibited increased risk of having a child with NSCLP, after adjusting for alcohol consumption (OR: 1.75, 95% CI: 1.03-2.99, P = 0.038). CONCLUSIONS: Reduced folic acid levels, alcohol consumption, and the MTHFR 677T and 1298C alleles may have contributed to NSCLP development in this sample population from Rio Grande do Norte.

Chlamydophila pneumonia and increased TLR4 gene expression in leukocytes are associated with acute myocardial infarction.

We investigated the relationship of the positivity for Chlamydophila pneumoniae (Cpn) and Mycoplasma pneumonia (Mpn), inflammatory and metabolic markers, and mRNA expression and polymorphisms of the TLR2, TLR4, IL-6 and TNFA genes with acute myocardial infarction (AMI). Two hundred and eighteen individuals (98 AMI and 120 non-AMI) were selected at two Clinical Centers. Blood samples were drawn to extract DNA and RNA and to measure laboratory variables including anti-Cpn IgM and IgG. Cpn and Mpn genomic DNA as well as TLR2, TLR4, IL-6 and TNFA mRNA expression were evaluated by quantitative real-time PCR (qPCR). Gene polymorphisms were detected by PCR-HRM. AMI patients had higher positivity for Cpn-DNA (17.3%) than non-AMI group (6.7%, p=0.018). In addition, Cpn-DNA positivity was an independent predictor of risk for AMI (OR: 2.56, CI: 1.08 - 6.04, p=0.031). Positivity for anti-Cpn IgG and Mpn-DNA was similar between AMI and non-AMI (> 0.05). TLR4 mRNA expression was higher in AMI than non-AMI individuals (p=0.005). CD14 -260C> T, TNFA -308A> G, TLR2 c.2258G> A, TLR4 c.896A> G and TLR4 c.1196> T variants were not associated with increased risk for AMI (p> 0.05). In the AMI group, individuals carrying CD14 -260CC genotype had higher hsCRP levels than CT/TT carriers (p=0.041). These results are suggestive that Cpn-DNA positivity and increased TLR4 mRNA expression in blood leukocytes may be associated with AMI and could be useful markers to evaluate the severity and progression of the atherosclerotic disease in AMI patients.

MSX1 gene polymorphisms in non-syndromic cleft lip and/or palate.

OBJECTIVE: The aim of this study was to investigate the contribution of 6 polymorphic variants of the MSX1 gene in non-syndromic cleft lip and/or palate (NSCL/P). METHODS: Three hundred and fifty-eight individuals (158 NSCL/P cases and 200 controls) were genotyped by TaqMan allelic discrimination using predesigned SNP assays. Statistical analyses were conducted using the software spss 15.0 and the r statistical suite. Haplotype block structure and haplotype frequencies were determined using the Haploview. A P-value of 0.05 and confidence interval of 95% were used for all of statistical tests. RESULTS: The patients with non-syndromic cleft lip and/or palate were characterized by similar distribution of MSX1 genotypes and allele in comparison to subjects without oral clefts (P > 0.05). Two haplotype blocks were constructed with polymorphisms of MSX1 gene and haplotypes formed showed a similar frequency in patients with and without oral clefts. CONCLUSIONS: The present study provides no evidence that MSX1 polymorphisms (rs3775261, rs1042484, rs12532, rs6446693, rs4464513 and rs1907998) play a major role in NSCL/P.

Genetic susceptibility in pharmacodynamic and pharmacokinetic pathways underlying drug-induced arrhythmia and sudden unexplained deaths.

Drug-induced arrhythmia is an adverse drug reaction that can be potentially fatal since it is mostly related to drug-induced QT prolongation, a known risk factor for Torsade de Pointes and sudden cardiac death (SCD). Several risk factors have been described in association to these drug-induced events, such as preexistent cardiac disease and genetic variation. Our objective was to study the genetic susceptibility in pharmacodynamic and pharmacokinetic pathways underlying suspected drug-induced arrhythmias and sudden unexplained deaths in 32 patients. The genetic component in the pharmacodynamic pathway was studied by analysing 96 genes associated with higher risk of SCD through massive parallel sequencing. Pharmacokinetic-mediated genetic susceptibility was investigated by studying the genes encoding cytochrome P450 enzymes using medium-throughput genotyping. Pharmacodynamic analysis showed three probably pathogenic variants and 45 variants of uncertain significance in 28 patients, several of them previously described in relation to mild or late onset cardiomyopathies. These results suggest that genetic variants in cardiomyopathy genes, in addition to those related with channelopathies, could be relevant to drug-induced cardiotoxicity and contribute to the arrhythmogenic phenotype. Pharmacokinetic analysis showed three patients that could have an altered metabolism of the drugs they received involving CYP2C19 and/or CYP2D6, probably contributing to the arrhythmogenic phenotype. The study of genetic variants in both pharmacodynamic and pharmacokinetic pathways may be a useful strategy to understand the multifactorial mechanism of drug-induced events in both clinical practice and forensic field. However, it is necessary to comprehensively study and evaluate the contribution of the genetic susceptibility to drug-induced cardiotoxicity.

Association between decreased vitamin levels and MTHFR, MTR and MTRR gene polymorphisms as determinants for elevated total homocysteine concentrations in pregnant women.

OBJECTIVES: To examine the association between methylenetetrahydrofolate reductase (MTHFR) (C677T and A1298C), methionine synthase (MTR) A2756G and methionine synthase reductase (MTRR) A66G gene polymorphisms and total homocysteine (tHcy), methylmalonic acid (MMA) and S-adenosylmethionine/S-adenosylhomocysteine (SAM/SAH) levels; and to evaluate the potential interactions with folate or cobalamin (Cbl) status. SUBJECTS/METHODS: Two hundred seventy-five healthy women at labor who delivered full-term normal babies. Cbl, folate, tHcy, MMA, SAM and SAH were measured in serum specimens. The genotypes for polymorphisms were determined by PCR-restriction fragment length polymorphism (RFLP). RESULTS: Serum folate, MTHFR 677T allele and MTR 2756AA genotypes were the predictors of tHcy levels in pregnant women. Serum Cbl and creatinine were the predictors of SAM/SAH ratio and MMA levels, respectively. The gene polymorphisms were not determinants for MMA levels and SAM/SAH ratios. Low levels of serum folate were associated with elevated tHcy in pregnant women, independently of the gene polymorphisms. In pregnant women carrying MTHFR 677T allele, or MTHFR 1298AA or MTRR 66AA genotypes, lower Cbl levels were associated with higher levels of tHcy. Lower SAM/SAH ratio was found in MTHFR 677CC or MTRR A2756AA genotypes carriers when Cbl levels were lower than 142 pmol/l. CONCLUSIONS: Serum folate and MTHFR C677T and MTR A2576G gene polymorphisms were the determinants for tHcy levels. The interaction between low levels of serum Cbl and MTHFR (C677T or A1298C) or MTRR A66G gene polymorphisms was associated with increased tHcy.

Lipoprotein lipase PvuII polymorphism is associated with variations in serum lipid levels in non-diabetic pregnant women.

The aim of the present study was to determine if there is an association between the single nucleotide polymorphisms (SNPs) of the lipoprotein lipase (LPL) and apolipoprotein E (apo E) genes and the serum lipid profile in pregnancy and puerperium. Non-diabetic women of European descent in the third semester of pregnancy (N = 120) were selected. Those with diseases or other condition that could modify their lipid profile were excluded from the study (N = 32). Serum lipids were measured by routine laboratory procedures and genomic DNA was extracted by a salting out method. LPL (PvuII and HindIII) and apo E (HhaI) SNPs were detected by the polymerase chain reaction and restriction fragment length polymorphism. Categorical and continuous variables were compared by the chi-square test and Student t-test or ANOVA, respectively. Women carrying the LPL P1P1 genotype had higher serum LDL cholesterol (N = 21; 155 +/- 45 mg/dL) than women carrying the P1P2/P2P2 genotypes (N = 67; 133 +/- 45 mg/dL; P = 0.032). During the puerperium period, serum levels of triglycerides and VLDL cholesterol were significantly reduced in women carrying the P1P1 (73%, P = 0.006) and P1P2 (51%, P = 0.002) genotypes but not in women carrying the P2P2 genotype (23%, P > 0.05). On the other hand, serum concentrations of lipids did not differ between the LPL HindIII and apo E genotypes during pregnancy and after delivery. We conclude that LPL PvuII SNP is associated with variations in serum lipids during pregnancy and the puerperal period in non-diabetic women.

Competition between chylomicrons and their remnants for plasma removal: a study with artificial emulsion models of chylomicrons.

In previous studies, protein-free emulsions of defined lipid composition were shown capable of simulating either the metabolism of chylomicrons (chylomicron-like emulsion) or their remnants (remnant-like emulsion), depending on the content of free, unesterified cholesterol. To validate further the assumption that remnant-like and chylomicron-like emulsion have metabolic pathways in common with their natural counterparts, studies of competition for plasma removal were undertaken: the remnant-like emulsion labeled with [3H]triolein was injected sequentially twice in the carotid arteries of rats to compare the clearance of remnant-like emulsion of the second injection with the first (control). Prior to the second injection, a large bolus of the chylomicron-like emulsion or rat lymph chylomicron was injected, to check the hypothesis that remnant generated from chylomicron-like emulsion or natural chylomicrons could compete with and displace remnant-like emulsion particles from their tissue receptor sites. Experiments were also performed in rats treated with Triton WR-1339, to block the generation of remnants. Results showed that remnants derived from either natural chylomicrons or chylomicron-like emulsion both strongly competed with the remnant-like emulsion. In contrast, when transformation of remnants was prevented by Triton, the undegraded particles of chylomicron-like emulsion or natural chylomicron were unable to compete with or displace remnant-like emulsion from its sites of removal from the plasma. In agreement with plasma clearance data, the hepatic uptake of the remnant-like emulsion was inhibited by the surplus dose of natural chylomicrons. In contrast, the spleen uptake was unaffected by it.

The effects of Triton WR-1339, protamine sulfate and heparin on the plasma removal of emulsion models of chylomicrons and remnants in rats.

Protein-free lipid emulsions with compositions modelling chylomicrons (chylomicron-like emulsion) or chylomicron remnants (remnant-like emulsion) were injected intra-arterially into nonanesthetized rats. Compared with control untreated rats, treatment with Triton WR-1339, protamine sulfate or heparin strongly modified the plasma removal of triacylglycerols and cholesteryl ester moieties of chylomicron-like emulsions, but had little effect on removal rates of triacylglycerols or cholesteryl esters of remnant-like emulsions. The effects on chylomicron-like removal were similar to those on natural lymph chylomicrons. The relative lack of effects on remnant-like emulsion removal provides additional evidence that remnant-like emulsions are a metabolic model for natural chylomicron remnants.

Effects of apolipoprotein B-100 on the metabolism of a lipid microemulsion model in rats.

In previous studies, it was shown that lipid microemulsions resembling LDL (LDE) but not containing protein, acquire apolipoprotein E when injected into the bloodstream and bind to LDL receptors (LDLR) using this protein as ligand. Aiming to evaluate the effects of apolipoprotein (apo) B-100 on the catabolism of these microemulsions, LDE with incorporated apo B-100 (LDE-apoB) and native LDL, all labeled with radioactive lipids were studied after intraarterial injection into Wistar rats. Plasma decay curves of the labels were determined in samples collected over 10 h and tissue uptake was assayed from organs excised from the animals sacrificed 24 h after injection. LDE-apo B had a fractional clearance rate (FCR) similar to native LDL (0.40 and 0.33, respectively) but both had FCR pronouncedly smaller than LDE (0.56, P<0.01). Liver was the main uptake site for LDE, LDE-apoB, and native LDL, but LDE-apoB and native LDL had lower hepatic uptake rates than LDE. Pre-treatment of the rats with 17alpha-ethinylestradiol, known to upregulate LDLR, accelerated the removal from plasma of both LDE and LDE-apoB, but the effect was greater upon LDE than LDE-apoB. These differences in metabolic behavior documented in vivo can be interpreted by the lower affinity of LDLR for apo B-100 than for apo E, demonstrated in in vitro studies. Therefore, our study shows in vivo that, in comparison with apo E, apo B is a less efficient ligand to remove lipid particles such as microemulsions or lipoproteins from the intravascular compartment.

High baseline serum total and LDL cholesterol levels are associated with MDR1 haplotypes in Brazilian hypercholesterolemic individuals of European descent.

The MDR1 gene encodes the P-glycoprotein, an efflux transporter with broad substrate specificity. P-glycoprotein has raised great interest in pharmacogenetics because it transports a variety of structurally divergent drugs, including lipid-lowering drugs. The synonymous single-nucleotide polymorphism C3435T and the nonsynonymous single-nucleotide polymorphism G2677T/A in MDR1 have been indicated as potential determinants of variability in drug disposition and efficacy. In order to evaluate the effect of G2677T/A and C3435T MDR1 polymorphisms on serum levels of lipids before and after atorvastatin administration, 69 unrelated hypercholesterolemic individuals from São Paulo city, Brazil, were selected and treated with 10 mg atorvastatin orally once daily for four weeks. MDR1 polymorphisms were analyzed by PCR-RFLP. C3435T and G2677T polymorphisms were found to be linked. The allelic frequencies for C3435T polymorphism were 0.536 and 0.464 for the 3435C and 3435T alleles, respectively, while for G2677T/A polymorphism allele frequencies were 0.580 for the 2677G allele, 0.384 for the 2677T allele and 0.036 for the 2677A allele. There was no significant relation between atorvastatin response and MDR1 polymorphisms (repeated measures ANOVA; P > 0.05). However, haplotype analysis revealed an association between T/T carriers and higher basal serum total (TC) and LDL cholesterol levels (TC: 303 +/- 56, LDL-C: 216 +/- 57 mg/dl, respectively) compared with non-T/T carriers (TC: 278 +/- 28, LDL-C: 189 +/- 24 mg/dl; repeated measures ANOVA/Tukey test; P < 0.05). These data indicate that MDR1 polymorphism may have an important contribution to the control of basal serum cholesterol levels in Brazilian hypercholesterolemic individuals of European descent.

Human interferon-alpha receptor: identification of the region involved in binding to interferon-alpha B.

Three polypeptides comprising amino acids 1-102, 93-260, and 261-410 of the extracellular domain of the human interferon-alpha receptor HuIFN-alpha R (Uzé, G., Lutfalla, G., and Gresser, I. Cell 1990; 60:225-234) have been expressed in Escherichia coli. The polypeptides were sequestered within bacterial inclusion bodies. Inclusion body material was solubilized by 8 M urea, and the polypeptides were purified by gel filtration or histidine tag-based affinity chromatography. Overall recovery of each purified and refolded polypeptide was approximately 0.5-0.8 mg/liter of cell culture. The polypeptides migrated as homogeneous monomers of 12 kDa, 22 kDa, and 17 kDa, respectively on reduced sodium dodecylsulfate polyacrylamide gel electrophoresis. The polypeptide fragments corresponding to amino acids 1-102, and 93-260 of the extracellular domain of HuIFN-alpha R lacked the ability to bind to IFN-alpha B and to inhibit its biologic activities. The polypeptide fragment corresponding to amino acids 261-410 of the receptor molecule inhibited the antiproliferative activity of IFN-alpha B and competed with the Daudi cell surface receptor for binding to this IFN-alpha species.

Isolation of a biologically active soluble human interferon-alpha receptor-GST fusion protein expressed in Escherichia coli.

The cDNA encoding the extracellular domain of the human interferon-alpha (IFN-alpha) receptor (Uzé, G., Lutfalla, G., and Gresser, I. Cell 1990;60:225-234) lacking the signal peptide has been expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The fusion protein represented 12% of total bacterial proteins and was found exclusively within cytoplasmic inclusion bodies. Inclusion body material was completely solubilized by 8 M urea; 20% solubilization was achieved by cell lysis in the presence of 0.45% cholamidopropyl dimethylammoniol-propane sulfonate and 1% Triton X-100. The soluble fusion protein was purified by gel filtration and affinity chromatography. Overall recovery of affinity purified fusion protein was approximately 100-200 micrograms/liter of cell culture. The affinity purified and refolded fusion protein exhibited the expected amino terminal sequence and M(r) of 68,000 on reduced sodium dodecylsulfate gel electrophoresis. The protein reacted with antibodies specific for the cloned IFN-alpha receptor and inhibited the antiviral and antiproliferative activities of recombinant IFN-alpha B. We have demonstrated that the fusion protein binds to IFN-alpha B and competes with the cell surface receptor for binding to this IFN-alpha species.

Sialic acid and oxidizability of low density lipoprotein subfractions of hyperlipidemic patients.

OBJECTIVES: Low density lipoprotein (LDL) does not constitute an homogenous fraction and it is known that the heavy LDL subfraction is potentially more atherogenic than the light one. Because concentration of LDL subfractions tend to be different in hyperlipidemias, it was verified whether these subfractions can also differ in sialic acid and neutral sugar content, as well as their resistance to oxidation. DESIGN AND METHODS: Two subfractions of low density lipoprotein (light LDL, density 1.019-1.034 g/mL and heavy LDL, density 1.034-1.063 g/mL) were isolated from the plasma of 17 patients with hypercholesterolemia, 11 with combined hyperlipidemia, 7 with hypertriglyceridemia, and 19 normolipidemic subjects. The content of sialic acids and neutral sugars of apo B was determined, respectively, by the periodate-thiobarbituric acid method and by reaction with phenol. The oxidation of LDL subfractions was determined by exposure to 5 microM copper (II) followed by the measurement of lipid hydroperoxides production by high performance liquid chromatography with electrochemical detection. RESULTS: The study groups did not differ in the neutral sugar content of LDL subfractions. However, compared to normolipidemic subjects, the sialic acid concentration of both LDL subfractions was lower in patients with hypercholesterolemia and hypertriglyceridemia and higher in those with combined hyperlipidemia (p < 0.05). In the hypercholesterolemia and combined hyperlipidemia groups, the lipid hydroperoxide content (microM) of heavy LDL was higher than in normolipidemic subjects (p < 0.05). CONCLUSIONS: The heavy LDL subfraction was more susceptible to oxidation in the patients with combined hyperlipidemia compared to controls and the other hyperlipidemic groups. The effect of sialic acids on heavy LDL oxidizability seems to vary according to the type of hyperlipidemia.

Seven DNA polymorphisms at the candidate genes of atherosclerosis in Brazilian women with angiographically documented coronary artery disease.

The possible association of genetic markers at the apolipoprotein E (HhaI polymorphism), apolipoprotein B (XbaI, EcoRI and Ins/Del polymorphisms), and low-density lipoprotein receptor (LDLR) (AvaII, HincII and PvuII polymorphisms) with coronary artery disease (CAD) was evaluated in 50 Brazilian women with CAD diagnosed by angiography and in 100 healthy women (controls). The frequency of E3/E4 genotype for HhaI polymorphism at the Apo E gene was significantly higher in CAD patients than in controls (40% vs. 14%, respectively, P<0.001). Similarly, the X-X- genotype for XbaI polymorphism was more frequent in CAD individuals than controls (42% vs. 12%, P<0.0001). The A+A+ and P1P1 genotypes for AvaII and PvuII polymorphisms at the LDLR locus were also higher in CAD subjects than controls (44% vs. 16%, P<0.001 and 64% vs. 39%, P<0.05, respectively). The estimated relative risks for CAD in women carrying the E3/E4, X-X-, A+A+ and P1P1 genotypes were 4.1 [95% confidence interval (CI), 3.0-5.6], 5.3 (95% CI, 3.8-7.5), 4.1 (95% CI, 3.0-5.5), and 2.8 (95% CI, 2.2-3.6), respectively. This study demonstrates that Apo E, Apo B and LDLR gene polymorphisms are associated with CAD in Brazilian Caucasian women.

Apolipoprotein B gene polymorphisms: prevalence and impact on serum lipid concentrations in hypercholesterolemic individuals from Brazil.

Genetic polymorphisms at the apolipoprotein B (apo B) have been associated with elevated plasma concentrations of low-density lipoprotein (LDL) cholesterol, atherosclerosis and increased risk for coronary artery disease (CAD). In the present study, four apo B gene polymorphisms (MspI, XbaI, Ins/Del and 3'HVR) have been investigated to determine their frequencies and influence on the lipid profile of 177 hypercholesterolemic white Brazilian subjects (HG) and 100 control individuals (CG). The genotype distribution and allele frequency of MspI, XbaI and Ins/Del polymorphisms of apo B gene were similar between HG and CG groups. The frequency of the alleles smaller than 43 repeats (< or =43) of 3'HVR polymorphism in the HG group was higher when compared to controls (16.4 vs. 8.5%, P<0.05). Moreover, these alleles were associated with higher total cholesterol concentrations in serum of hypercholesterolemic individuals (P<0.05). In addition, an association between Ins/Del and 3'HVR polymorphism was observed. The alleles < or =43 and Del were more frequent in the HG when compared to the CG individuals (P<0.05). We concluded that 3'HVR polymorphism at the apo B gene may be an important genetic marker to evaluate atherosclerotic disease risk.

Pvu II intron 15 polymorphism at the LDL receptor gene is associated with differences in serum lipid concentrations in subjects with low and high risk for coronary artery disease from Brazil.

Coronary artery disease (CAD) has a high prevalence in the Brazilian population. Nevertheless, studies of genetic risk factors for CAD in this country have not been sufficiently conducted. We used the Pvu II polymorphism (intron 15) at the low-density lipoprotein receptor (LDLR) gene to study the effect of variation at this locus in determining plasma lipid concentrations in 128 white subjects presenting a lipid profile suggesting high risk for CAD (HRG) and 100 white normolipidemic individuals (controls, CG). The Pvu II polymorphism was detected by PCR-RFLP. The P1P1 genotype for Pvu II polymorphism (homozygous for absence of restriction site) was greater in HRG individuals than in CG subjects (57% vs. 38%, P<0.05). Moreover, the P1P1 genotype was strongly associated with high concentrations of total cholesterol (P=0.0001), triglycerides (P=0. 0295), LDL-C (P=0.0001), and VLDL-C concentrations (P=0.0280) and lower HDL-C concentrations (P=0.0051) in HRG subjects. Similarly, the CG individuals with P1P1 genotype presented high concentrations of total cholesterol and LDL-C compared to other genotypes (P=0. 0001). This study demonstrates the influence of Pvu II polymorphism of the LDLR on serum lipid concentrations of individuals with low and high risk for CAD from Brazil.

Effects of Triton WR 1339 and heparin on the transfer of surface lipids from triglyceride-rich emulsions to high density lipoproteins in rats.

The influence of lipolytic mechanisms on the transfer of phospholipids and unesterified cholesterol from artificial emulsions, serving as chylomicron models to other plasma lipoproteins, mainly high density lipoproteins (HDL) were tested in vivo. The emulsions labeled with radioactive lipids were injected into the bloodstream of rats (controls) and the results were compared with those obtained from rats that had previously been treated with Triton WR 1339 or heparin. Plasma clearance and the distribution of cholesteryl esters, phospholipids and unesterified cholesterol in the different plasma lipoprotein fractions were then determined. Whereas virtually no cholesteryl esters were found in d greater than 1.006 g/mL density fraction of the three experimental groups, 2.8 +/- 1.3% of the injected phospholipids were in the 1.063-1.210 g/L density fraction of the Triton treated rats, and 12.6 +/- 5.4% of the heparin treated rats, as compared to 10.1 +/- 1.7% in controls. This indicates that lipolysis directly influences phospholipid transfer to HDL. In contrast, free-cholesterol transfer to HDL, besides being less pronounced than phospholipid transfer, was enhanced by Triton and diminished by heparin, indicating that lipolytic mechanisms were not important determinants in this process.

Metabolic behavior in rats of a nonprotein microemulsion resembling low-density lipoprotein.

A protein-free microemulsion (LDE) with a lipid composition resembling that of low-density lipoprotein (LDL) was used in metabolic studies in rats to compare LDE with the native lipoprotein. LDE labeled with radioactive lipids was injected into the bloodstream of male Wistar rats, and plasma kinetics of the labeled lipids were followed on plasma samples collected at regular intervals for 12 h after injection. The 24-h LDE uptake by different tissues was also measured in tissue samples excised after the animals had been sacrificed. We found that LDE plasma kinetics were similar to those described for native LDL [fractional clearance rate (FCR) of cholesteryl ester, 0.42 +/- 0.11 h-1]. The major site for LDE uptake was the liver, and the tissue distribution of the LDE injected radioactivity was as one would expect for LDL. To test whether LDE was taken up by the specific LDL receptors, the LDE emulsion was injected into rats treated with 17 alpha-ethinylestradiol, which is known to increase the activity of these receptors; as expected, removal of LDE from the bloodstream increased (FCR = 0.90 +/- 0.35 h-1). On the other hand, saturation of the receptors that remove remnants by prior infusion of massive amounts of lymph chylomicrons did not change LDE plasma kinetics. These results indicate that LDE is cleared from plasma by B,E receptors and not by the E receptors that remove remnants. Incorporation of free cholesterol into LDE increased LDE plasma clearance. Incubation studies also showed that LDE incorporates a variety of apolipoproteins, including apo E, a ligand for recognition of lipoproteins by specific receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-alpha receptor.

Abnormal production of interferon alpha (IFN-alpha) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-alpha. IFN-alpha can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the development of IFN-alpha inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have expressed two polypeptides encoding amino acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of subunit 1 of the interferon-alpha receptor (IFNAR 1-EC) in E. coli. The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays. Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A into E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified by serial sonication steps and by gel filtration chromatography with 8 M urea and refolded by dialysis. Under reducing SDS-PAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively. Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. P1 and P2 and their respective polyclonal antibodies were able to block the antiproliferative activity of 6.25 nM IFN-alphaB on Daudi cells, but did not block IFN-alphaB activity at higher concentrations (>6. 25 nM). On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFN-alphaB on Hep 2/c cells challenged with encephalomyocarditis virus.