The objectives of this study were to evaluate all the non-synonymous single nucleotide polymorphisms (SNPs) in the DNase I and DNase I-like 3 (1L3) genes potentially implicated in autoimmune diseases as a functional SNP in terms of alteration of the activity levels. We examined the genotype distributions of the 32 and 20 non-synonymous SNPs in DNASE1 and DNASE1L3, respectively, in three ethnic groups, and the effect of these SNPs on the DNase activities. Among a total of 44 and 25 SNPs including those characterized in our previous studies [Yasuda et al., Int J Biochem Cell Biol42 (2010) 1216-1225; Ueki et al. Electrophoresis32 (2012) 1465-1472], only four and one, respectively, exhibited genetic heterozygosity in one or all of the ethnic groups examined. On the basis of alterations in the activity levels resulting from the corresponding amino acid substitutions, 11 activity-abolishing and 11 activity-reducing SNPs in DNASE1 and two activity-abolishing and five activity-reducing SNPs in DNASE1L3 were confirmed as a functional SNP. Phylogenetic analysis showed that all of the amino acid residues in activity-abolishing SNPs were completely or well conserved in animal DNase I and 1L3 proteins. Although almost all non-synonymous SNPs in both genes that affected the catalytic activity showed extremely low genetic heterogeneity, it seems plausible that a minor allele of 13 activity-abolishing SNPs producing a loss-of-function variant in both the DNase genes would be a direct genetic risk factor for autoimmune diseases. These findings may have clinical implications in relation to the prevalence of autoimmune diseases.
DNA fragmentation factor beta (DFFB) polypeptide, endonuclease G (EndoG), and Flap endonuclease-1 (FEN-1) are responsible for DNA fragmentation, a hallmark of apoptosis. Although the human homologs of these genes show three, four, and six nonsynonymous single-nucleotide polymorphisms (SNPs), respectively, data on their genotype distributions in populations worldwide are limited. In this context, the objectives of this study were to elucidate the genetic heterogeneity of all these SNPs in wide-ranging populations, and thereby to clarify the genetic background of these apoptosis-related endonucleases in human populations. We investigated the genotype distribution of their SNPs in 13 different populations of healthy Asians, Africans, and Caucasians using novel genotyping methods. Among the 13 SNPs in the 3 genes, only 3 were found to be polymorphic: R196K and K277R in the DFFB gene, and S12L in the EndoG gene. All 6 SNPs in the FEN-1 gene were entirely monoallelic. Although it remains unclear whether each SNP would exert any effect on endonuclease functions, these genes appear to exhibit low degree of genetic heterogeneity with regard to nonsynonymous SNPs. These findings allow us to conclude that human apoptosis-related endonucleases, similarly to other human DNase genes, revealed previously, are well conserved at the protein level during the course of human evolution.
An inherited deficiency of low density lipoprotein (LDL) receptors in familial hypercholesterolemia (FH) is assumed to be the cause of the decreased catabolism of LDL. In contrast, in hyperthyroidism there is an increased rate of LDL catabolism. A 58-year-old female patient with heterozygous FH revealed a normal level of serum cholesterol (193mg/dl) in coexistence of hyperthyroidism. After the therapy of hyperthyroidism with radioiodine the patient's lipid profile revealed an increased concentrations of cholesterol (Whole serum 338, VLDL 68, LDL 199, HDL 42mg/dl). There was a significant inverse correlation between her serum cholesterol levels and her serum thyroxine levels (r=-0.815, p<0.01).Since LDL catabolism in vivo is thought to be mediated largely through the LDL receptor, the role of thyroid hormone in this receptor-mediated degradation of LDL was studied in human skin fibroblasts. Confluent cells were exposed to a medium containing 5% lipoprotein deficient serum prepared from a myxedematous patient by ultracentrifugation for 2 days with or without added triiodothyronine (T3). After 125I-LDL was added, the cells were incubated for 6 hours at 37°C for the determinations of 125I-LDL uptake and degradation. In the cells from 2 normal subjects, LDL uptake and degradation was enhanced 29% by preincubation with T3 (1.0μg/ml). In the cells from 2 heterozygous patients with FH, LDL uptake and degradation was enhanced 23% with T3(1.0μg/ml). While, in the cells from 2 homozygous patients with FH, no effect of T3 was observed. These results suggest that normal serum cholesterol levels in a heterozygous patient with FH result in part from an enhanced degradation of LDL by extrahepatic cells exposed to excess thyroid hormone.
Abstract Deoxyribonucleases (DNases) have been suggested to be implicated in the pathophysiology of autoimmune diseases. In the DNASE1L3 gene encoding human DNase I‐like 3 (DNase 1L3), a member of the DNase I family, only two non‐synonymous (R178 H and R206C) single nucleotide polymorphisms (SNPs) have been examined [Ueki et al., Clin. Chim. Acta 2009, 407 , 20–24]. Three other non‐synonymous (G82R, K96N, and I243M) and four synonymous (S17S, T84T, R92R, and A181A) SNPs, in addition to R206C and R178H, have been identified in DNASE1L3 . We investigated the distribution of all these SNPs in exons of the gene in eight Asian, three African, and three Caucasian populations worldwide using newly devised genotyping methods. SNP T84T showed polymorphism in all the populations, and R92R was polymorphic in the three African and three Caucasian populations; R206C was distributed only in Caucasian populations. In contrast, no minor allele was found in five SNPs (S17S, G82R, K96N, A181A, and I243M) in DNASE1L3. Generally, the DNase 1L3 gene shows relatively low genetic diversity with regard to exonic SNPs. When the effect of amino acid/nucleotide substitutions resulting from the SNPs on DNase 1L3 activity was examined, none of the synonymous SNPs had any effect on the DNase 1L3 activity, whereas among non‐synonymous SNPs, SNP G82R diminished the activity of the enzyme, being similar to R206C. These findings permit us to assume that, although only R206 exhibits polymorphisms in a Caucasian‐specific manner, at least SNPs G82R and R206C in DNASE1L3 might be potential risk factors for autoimmune disease.
Chlorhexidine (CHX) is a cationic biguanide compound that has been widely used for disinfection of skin, mucous membranes, and medical instruments. Poisoning has been occurred occasionally due to its easy accessibility. Some fatal cases developed acute respiratory distress syndrome (ARDS) from aspiration of CHX directly into the lung. There is no preclinical information about the pulmonary toxicity of CHX available since the products of CHX are usually developed for disinfection by topical use. In this study, the acute pulmonary toxic effects of CHX following an intratracheal instillation in rats were investigated. Rats were exposed either to CHX at concentrations of 0.02% and 0.2% or to distilled water at a volume of 500 μl/kg b.w. CHX at concentration of 0.2% caused changes in hematological and biochemical values including white blood cell count (WBC), total protein (TP), albumin (ALB), lactate dehydrogenase (LDH), blood urea nitrogen (BUN) and creatinine (CRE), and induced inflammatory reactions including intra-alveolar edema and hemorrhages, as well as resulted in the target organ concentration in lungs at the level of about 1.0 μg/g and maintained for more than 1 week, when administered intratracheally in rats. The cytotoxic action of CHX might induce those detrimental reactions in rats.
