Susceptibility to db gene and streptozotocin-induced diabetes in C57BL mice: control by gender-associated,MHC-unlinked traits

H-2 haplotype differences distinguish the related C57BL/KsJ (BKs) and C57BL/6J (B6) inbred strains. BKs mice are more susceptible to diabetes induction by a recessive obesity gene, diabetes (db), or by multi-dose streptozotocin (MSZ) administration. The purpose of this study was to evaluate whether the H-2 differences were the important genetic background modifiers determining inbred strain susceptibility or resistance to these diabetogenic stresses. Diabetes susceptibility of BKs.B6-H-2 ^b congenic mice was compared with that of the parental BKs and B6 stocks. In addition, diabetes severity was studied in (B6 × BKs)F₁ and F₂ db/db mice and an H-2 segregation analysis was performed. BKs susceptibility genes expressed in a dominant fashion in the F₁ generation, and were transmitted to F₂ db/db males without apparent segregation. No association between H-2 ^b haplotype and B6-type diabetes resistance was found in response to either the db mutation or to MSZ. Insulitis, associated with development of hyperglycemia in BKs males, also occurred in the H-2 ^b congenic stock. However, an apparent interaction between H-2 ^b haplotype, the db mutation (on chromosome 4), and male gender (Y chromosome?) was indicated by a segregation ratio distortion in recovery of this genotype. A more moderate diabetes in some F₂ db/db females suggested that non-MHC-linked genes controlling sex steroid metabolism were the important determinants of diabetogenic sensitivities in the C57BL stocks. In support of the latter, strain differences were demonstrated in activity levels of steroid sulfatase, which is regulated by a sex-linked gene likely expressed on both the X and Y chromosome, and which may control tissue levels of active androgens and estrogens. We show that the diabetes-susceptible F₁ hybrids exhibit the higher activity associated with the BKs strain.     

Expression of genes in the 111.35–116.16 million bp fragment of chromosome 13 in brain of mice with different predisposition to hereditary catalepsy

Catalepsy is a pathological animal behavior that is usually associated with dysfunctions in the striatal pallidal system of the brain and can be caused by different reasons. It was previously demonstrated that hereditary catalepsy is linked to the 111.35–116.16 million bp fragment of chromosome 13 in mice. The level of mRNA content in 42 genes localized in this fragment was determined in the study. Two brain departments that are functionally associated with catalepsy (striatum and substantia nigra) were studied in mice from AKR line (resistant to catalepsy), cataleptic CBA line, and recombinant cataleptic AKR.CBA-D13Mit76 (D13) line. The latter was obtained by the transfer of indicated fragment of chromosome 13 from the CBA line to the genome of the AKR line. It was found that two genes (Ndufs4 and Ppap2a) in the striatum and ten genes (Esm1, Fst, Gm10735, Gm15322, Gm15323, Gm15324, Gm15325, Il6st, Il31ra, and Itga1) in the substantia nigra differ in the level of mRNA expression in AKR and D13 lines. The Mcidas gene mRNA level is lower in both structures in D13 line mice than in the AKR line. The expression of the Hspb3 and Mocs2 genes (that encode heat shock protein and molybdenum cofactor synthesis, respectively) is lower in the substantia nigra of CBA and D13 cataleptic line mice than in the AKR line resistant to catalepsy. These genes are considered to be the most likely candidate genes of the catalepsy. The coexpression of a large amount of genes in these brain structures in sick animals indicates the existence of a complex gene network that regulates hereditary catalepsy.     

Is AKR2A an essential molecular chaperone for a class of membrane-bound proteins in plants?

The Arabidopsis ankyrin-repeat containing protein 2A (AKR2A) was shown to be an essential molecular chaperone for the peroxisomal membrane-bound ascorbate peroxidase 3 (APX3), because the biogenesis of APX3 depends on the function of AKR2A in plant cells. AKR2A binds specifically to a sequence in APX3 that is made up of a transmembrane domain followed by a few positively charged amino acid residues; this sequence is named as AKR2A-binding sequence or ABS. Interestingly, a sequence in the chloroplast outer envelope protein 7 (OEP7) shares similar features to ABS and is able to bind specifically to AKR2A, suggesting a possibility that proteins with a sequence similar to ABS could bind to AKR2A and they are all likely ligand proteins of AKR2A. This hypothesis was supported by analyzing five additional proteins that contain sequences similar to ABS using the yeast two-hybrid technique. A preliminary survey in the Arabidopsis genome indicates that there are at least 500 genes encoding proteins that contain sequences similar to ABS, which raises interesting questions: are these proteins AKR2A''s ligand proteins and does AKR2A play a critical role in the biogenesis of these proteins in plants?Key words: Arabidopsis, membrane protein, molecular chaperone, protein targeting, transmembrane domainThe Arabidopsis ankyrin-repeat containing protein 2A (AKR2A) is an essential molecular chaperone for the peroxisomal membrane-bound ascorbate peroxidase 3 (APX3).¹ Both AKR2A and APX3 were identified as GF14λ-interacting proteins^2,3 when the mode of action of a 14-3-3 protein, GF14λ⁴ was studied. In characterizing the enzymatic property of APX3, there was some initial difficulty in purifying the expressed APX3 from a bacterial expression system. Although APX3 could be expressed in E. coli cells in large quantities, as evidenced by directly boiling the bacterial cells and analyzing the bacterial cells by SDS-PAGE and Western blot analysis (Fig. 1), APX3 enzymatic activity in the supernatant fraction was not detectable after cells were broken by sonication (Fig. 1). The reason that APX3 activity was not detectable in the supernatant fraction was likely caused by the transmembrane domain that occurs at the C-terminal end of APX3; because these hydrophobic domains could interact with one another, forming insoluble aggregates in bacterial cells. When a truncated APX3 was expressed, i.e., APX3 without the transmembrane domain (APX3Δ in Fig. 1), APX3 activity was then detectable in the supernatant fraction of bacterial cellular extracts. If a protein is able to bind to APX3''s transmembrane domain immediately after or during translation of APX3, this protein could prevent APX3 from forming insoluble aggregates among themselves. APX3 activity would then be detectable in the supernatant fraction. Because some 14-3-3-interacting proteins were shown to interact with one another,⁵ the best candidate that could interact with APX3 should be AKR2A (because they are both GF14λ-interacting proteins). This possibility was tested by simultaneously expressing both APX3 and AKR2A in the same bacterial cell; APX3 activity was indeed detectable in the supernatant fraction of bacterial cellular extracts (Fig. 1).Open in a separate windowFigure 1Protein-protein interaction between AKR2A and APX3 in bacterial cells. (A) Analysis of APX3 activity in supernatant fractions of various bacterial cells. In lanes, APX3, supernatant from cells that express full-length APX3; APX3 + OMT 1, supernatant from cells that express both full-length APX3 and OMT 1 (O-methyltransferase1,⁷); APX3 + AKR2A, supernatant from cells that express both full-length APX3 and AKR2A; APX3Δ, supernatant from cells that express a partial APX3 (i.e., lacking the transmembrane domain and the last seven amino acid residues); APX3Δ + OMT 1, supernatant from cells that express both APX3Δ and OMT 1; APX3Δ + AKR2A, supernatant from cells that express both APX3Δ and AKR2A; OMT 1, supernatant from cells that express OMT1; AKR2A, supernatant from cells that express AKR2A. The white bands in the gel represent APX3 activities as assayed by using the method of Mittler and Zilinskas.⁸ (B) Bacterial cells expressing various target proteins were analyzed directly by using SDS-PAGE method and the positions of the expressed target proteins are marked on the right. (C) Bacterial cells expressing various target proteins were analyzed by western blot. The antibodies used are listed on the right.This was the first evidence that AKR2A interacts with APX3 and the interaction site involves the C-terminal transmembrane domain of APX3. To further define the amino acid residues involved in the AKR2A-APX3 interaction, yeast two-hybrid experiments were conducted with various deletion fragments of AKR2A and APX3.1 It was found that in addition to the transmembrane domain, the positively charged amino acid residues following the transmembrane domain also play a role in the AKR2A-APX3 interaction.¹ This sequence in APX3 was designated as AKR2A-binding sequence (ABS). In order to understand the biological function of the AKR2A-APX3 interaction, several akr2a mutants that displayed reduced or altered interaction with APX3 were created and analyzed. Results indicated that reduced AKR2A activity leads to severe developmental, phenotypic, and physiological abnormalities including reduced steady-state level of APX3 and reduced targeting of APX3 to peroxisomal membranes in Arabidopsis.¹ The pleiotropic nature of akr2a mutants indicated that AKR2A plays more roles in addition to chaperoning APX3. Indeed this work was corroborated by a finding that AKR2A is also required for the biogenesis of the chloroplast outer envelope protein 7 (OEP7).⁶ More importantly, the interaction between AKR2A and OEP7 also involves a sequence in OEP7 that is similar to the ABS found in APX3.There is no apparent similarity, at the amino acid level, between the sequences of the AKR2A-binding site found in APX3 and OEP7; it appears that what AKR2A recognizes in its ligand proteins is the structural feature: single transmembrane domain followed by one or a few positively charged amino acid residues. Therefore, these AKR2A-binding sequences should all be designated as ABS, and it was predicted that any protein with an ABS could be AKR2A''s interacting protein. Five such proteins, APX5, TOC34, TOM20, cytochrome b5 (CB5) and cytochrome b₅ reductase (CB5R) were tested, and indeed all five proteins interacted with AKR2A in the yeast two-hybrid system.¹ More importantly, the interaction sites of these proteins are their ABS in every case tested.¹ Based on these discoveries, it is proposed that AKR2A is a molecular chaperone for this group of ABS-containing proteins.Among the seven AKR2A-interacting proteins that were characterized, the ABS is found at C-terminal end of four proteins (APX3, APX5, CB5 and TOM20), near N-terminal end of two proteins (OEP7 and CB5R), and near C-terminal end of one protein (TOC34), suggesting that the position of ABS in these membrane proteins does not affect its interaction with AKR2A. Furthermore, in all cases, AKR2A binds to its ligand proteins that contain only one ABS. AKR2A does not appear to bind to proteins that contain multiple transmembrane domains such as PMP22,^1,6 even though these transmembrane domains are followed by a few positively charged amino acid residues.APX3 and APX5 are peroxisomal membrane-bound, OEP7 and TOC34 are chloroplast outer envelope proteins, TOM20 is a mitochondrion outer membrane protein and CB5 and CB5R are microsomal membrane (ER-membrane) proteins. Therefore, AKR2A is clearly not responsible for targeting these proteins to their specific membranes; instead AKR2A serves as a molecular chaperone to prevent these proteins from forming aggregates through their hydrophobic domain in ABS after translation (Fig. 2). Perhaps, AKR2A''s binding to the ABS of these membrane proteins also keeps these proteins in insertion competent state before they are sent to their specific destinations. It is clear that other factors, such as organellar membrane-specific receptors, must be required for sending these proteins to their specific membranes (Fig. 2).Open in a separate windowFigure 2Model on how AKR2A chaperones its ligand proteins. (1) AKR2A binds to ABS of a nascent protein that is being synthesized from a free ribosome. (2) AKR2A keeps its ligand protein (L) in the cytoplasm. (3) With the help of membrane-specific receptors, AKR2A''s ligand proteins are sent to their specific membranes.The Arabidopsis proteome was analyzed and it was found that there are at least 500 proteins that contain sequences similar to ABS (http://bio.scu.edu.cn/list.xls). Would these proteins be AKR2A''s ligand proteins? Some of them, if not all, might be, but it will be a challenging task to experimentally test these proteins one by one. A better bioinformatics tool that can provide clues on the mode of action of the protein-protein interactions between AKR2A and its known ligand proteins should help us designing next set of experiments in order to answer the above question in an efficient way.     

Genetic polymorphisms of glutathione S-transferases and cytochrome P450 enzymes as susceptibility factors to systemic lupus erythematosus in southern Brazilian patients

Systemic lupus erythematosus (SLE) is an autoimmune chronic inflammatory disease that presents several clinical manifestations, affecting multiple organs and systems. Immunological, environmental, hormonal and genetic factors may contribute to disease. Genes and proteins involved in metabolism and detoxification of xenobiotics are often used as susceptibility markers to diseases with environmental risk factors. Cytochrome P450 (CYP) enzymes activate the xenobiotic making it more reactive, while the Glutathione S-transferases (GST) enzymes conjugate the reduced glutathione with electrophilic compounds, facilitating the toxic products excretion. CYP and GST polymorphisms can alter the expression and catalytic activity of enzymes. This study aimed to investigate the role of genetic variants of CYP and GST in susceptibility and clinical expression of SLE, through the analysis of GSTM1 null, GSTT1 null, GSTP1*Ile105Val, CYP1A1*2C and CYP2E1*5B polymorphisms. 371 SLE patients from Hospital de Clínicas de Porto Alegre and 522 healthy blood donors from southern Brazil were evaluated. GSTP1 and CYP variants were genotyped using PCR–RFLP and GSTT1 and GSTM1 variants were analyzed by multiplex PCR. Among European-derived individuals, a lower frequency of GSTP1*Val heterozygous genotypes was found in SLE patients when compared to controls (p = 0.005). In African-derived SLE patients, the CYP2E1*5B allelic frequency was higher in relation to controls (p = 0.054). We did not observe any clinical implication of the CYP and GST polymorphisms in patients with SLE. Our data suggest a protective role of the GSTP1*Ile/Val heterozygous genotype against the SLE in European-derived and a possible influence of the CYP2E1*5B allele in SLE susceptibility among African-derived individuals.     

A comparison of type 2 diabetes risk allele load between African Americans and European Americans

The prevalence of type 2 diabetes (T2D) is greater in populations of African descent compared to European-descent populations. Genetic risk factors may underlie the disparity in disease prevalence. Genome-wide association studies (GWAS) have identified >60 common genetic variants that contribute to T2D risk in populations of European, Asian, African and Hispanic descent. These studies have not comprehensively examined population differences in cumulative risk allele load. To investigate the relationship between risk allele load and T2D risk, 46 T2D single nucleotide polymorphisms (SNPs) in 43 loci from GWAS in European, Asian, and African-derived populations were genotyped in 1,990 African Americans (n = 963 T2D cases, n = 1,027 controls) and 1,644 European Americans (n = 719 T2D cases, n = 925 controls) ascertained and recruited using a common protocol in the southeast United States. A genetic risk score (GRS) was constructed from the cumulative risk alleles for each individual. In African American subjects, risk allele frequencies ranged from 0.024 to 0.964. Risk alleles from 26 SNPs demonstrated directional consistency with previous studies, and 3 SNPs from ADAMTS9, TCF7L2, and ZFAND6 showed nominal evidence of association (p < 0.05). African American individuals carried 38–67 (53.7 ± 4.0, mean ± SD) risk alleles. In European American subjects, risk allele frequencies ranged from 0.084 to 0.996. Risk alleles from 36 SNPs demonstrated directional consistency, and 10 SNPs from BCL11A, PSMD6, ADAMTS9, ZFAND3, ANK1, CDKN2A/B, TCF7L2, PRC1, FTO, and BCAR1 showed evidence of association (p < 0.05). European American individuals carried 38–65 (50.9 ± 4.4) risk alleles. African Americans have a significantly greater burden of 2.8 risk alleles (p = 3.97 × 10^?89) compared to European Americans. However, GRS modeling showed that cumulative risk allele load was associated with risk of T2D in European Americans, but only marginally in African Americans. This result suggests that there are ethnic-specific differences in genetic architecture underlying T2D, and that these differences complicate our understanding of how risk allele load impacts disease susceptibility.     

Serum vaspin concentration in elderly patients with type 2 diabetes mellitus and macrovascular complications

Background

Adipose tissue, an endocrine organ of the body, is involved in some obesity-related disease states such as insulin resistance, diabetes mellitus, and atherosclerosis. Vaspin is a novel adipocyte with insulin sensitizing effects. In this study, we planned to estimate serum vaspin concentrations as related to glycemic status and the presence of macrovascular complications among elderly patients with type-2 diabetes mellitus (T2DM).

Methods

A total of 230 elderly patients with T2DM were evaluated. These patients were divided into two groups: patients without complications (T2DM group, n?=?110), and patients with macrovascular complications (T2DM + MC group, n?=?120). In addition, 60 healthy elderly subjects were enrolled and assigned into the control group (NC group). Relevant parameters were matched for age and gender ratio. Serum vaspin concentrations were measured by Enzyme-linked immunosorbent assay (ELISA). Anthropometric measurements, plasma glucose and HbA_1C levels, insulin concentration, liver and kidney functions, and lipid profile were measured for each participant.

Results

Serum vaspin concentrations were significantly higher in the T2DM group than in the T2DM + MC group (F?=?13.122, P?<?0.01). These concentrations were also significantly higher among females, compared to males (T?=?3.567, P?<?0.05). Logistic regression analysis revealed that serum vaspin concentration, systolic blood pressure, HDL-C and T2DM duration were independent influencing factors for diabetic macrovascular complications.

Conclusion

Serum vaspin may be considered as a potential marker to assess the status of elderly patients with T2DM and the risk of developing serious macrovascular complications. Further prospective studies are warranted.

Trial registration

ChiCTR-OPC-14005698, retrospectively registered on 20 Dec. 2014.

     

A genetic association study detects haplotypes associated with obstructive heart defects

The development of congenital heart defects (CHDs) involves a complex interplay between genetic variants, epigenetic variants, and environmental exposures. Previous studies have suggested that susceptibility to CHDs is associated with maternal genotypes, fetal genotypes, and maternal–fetal genotype (MFG) interactions. We conducted a haplotype-based genetic association study of obstructive heart defects (OHDs), aiming to detect the genetic effects of 877 SNPs involved in the homocysteine, folate, and transsulfuration pathways. Genotypes were available for 285 mother-offspring pairs with OHD-affected pregnancies and 868 mother-offspring pairs with unaffected pregnancies. A penalized logistic regression model was applied with an adaptive least absolute shrinkage and selection operator (lasso), which dissects the maternal effect, fetal effect, and MFG interaction effects associated with OHDs. By examining the association between 140 haplotype blocks, we identified 9 blocks that are potentially associated with OHD occurrence. Four haplotype blocks, located in genes MGMT, MTHFS, CBS, and DNMT3L, were statistically significant using a Bayesian false-discovery probability threshold of 0.8. Two blocks in MGMT and MTHFS appear to have significant fetal effects, while the CBS and DNMT3L genes may have significant MFG interaction effects.     

Vascular complications in patients with type 2 diabetes: prevalence and associated factors in 38 countries (the DISCOVER study program)

Background

The global prevalence of type 2 diabetes-related complications is not well described. We assessed prevalence of vascular complications at baseline in DISCOVER (NCT02322762; NCT02226822), a global, prospective, observational study program of 15,992 patients with type 2 diabetes initiating second-line therapy, conducted across 38 countries.

Methods

Patients were recruited from primary and specialist healthcare settings. Data were collected using a standardized case report form. Prevalence estimates of microvascular and macrovascular complications at baseline were assessed overall and by country and region, and were standardized for age and sex. Modified Poisson regression was used to assess factors associated with the prevalence of complications.

Results

The median duration of type 2 diabetes was 4.1 years (interquartile range [IQR]: 1.9–7.9 years), and the median glycated hemoglobin (HbA_1c) level was 8.0% (IQR: 7.2–9.1%). The crude prevalences of microvascular and macrovascular complications were 18.8% and 12.7%, respectively. Common microvascular complications were peripheral neuropathy (7.7%), chronic kidney disease (5.0%), and albuminuria (4.3%). Common macrovascular complications were coronary artery disease (8.2%), heart failure (3.3%) and stroke (2.2%). The age- and sex-standardized prevalence of microvascular complications was 17.9% (95% confidence interval [CI] 17.3–18.6%), ranging from 14.2% in the Americas to 20.4% in Europe. The age- and sex-standardized prevalence of macrovascular complications was 9.2% (95% CI 8.7–9.7%), ranging from 4.1% in South-East Asia to 18.8% in Europe. Factors positively associated with vascular complications included age (per 10-year increment), male sex, diabetes duration (per 1-year increment), and history of hypoglycemia, with rate ratios (95% CIs) for microvascular complications of 1.14 (1.09–1.19), 1.30 (1.20–1.42), 1.03 (1.02–1.04) and 1.45 (1.25–1.69), respectively, and for macrovascular complications of 1.41 (1.34–1.48), 1.29 (1.16–1.45), 1.02 (1.01–1.02) and 1.24 (1.04–1.48), respectively. HbA_1c levels (per 1.0% increment) were positively associated with microvascular (1.05 [1.02–1.08]) but not macrovascular (1.00 [0.97–1.04]) complications.

Conclusions

The global burden of microvascular and macrovascular complications is substantial in these patients with type 2 diabetes who are relatively early in the disease process. These findings highlight an opportunity for aggressive early risk factor modification, particularly in regions with a high prevalence of complications.Trial registration ClinicalTrials.gov; NCT02322762. Registered 23 December 2014. https://clinicaltrials.gov/ct2/show/NCT02322762. ClinicalTrials.gov; NCT02226822. Registered 27 August 2014. https://clinicaltrials.gov/ct2/show/NCT02226822

     

NOD2 and CCDC122-LACC1 genes are associated with leprosy susceptibility in Brazilians

Leprosy is a complex disease with phenotypes strongly influenced by genetic variation. A Chinese genome-wide association study (GWAS) depicted novel genes and pathways associated with leprosy susceptibility, only partially replicated by independent studies in different ethnicities. Here, we describe the results of a validation and replication study of the Chinese GWAS in Brazilians, using a stepwise strategy that involved two family-based and three independent case–control samples, resulting in 3,614 individuals enrolled. First, we genotyped a family-based sample for 36 tag single-nucleotide polymorphisms (SNPs) of five genes located in four different candidate loci: CCDC122-LACC1, NOD2, TNFSF15 and RIPK2. Association between leprosy and tag SNPs at NOD2 (rs8057431) and CCDC122-LACC1 (rs4942254) was then replicated in three additional, independent samples (combined OR_AA = 0.49, P = 1.39e?06; OR_CC = 0.72, P = 0.003, respectively). These results clearly implicate the NOD2 pathway in the regulation of leprosy susceptibility across diverse populations.     

Generation of CD44 gene-deficient mouse derived induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) show good promise for the treatment of defects caused by numerous genetic diseases. Herein, we successfully generated CD44 gene-deficient iPSCs using Oct4, Sox2, Klf4, and vitamin C. The generated iPSCs displayed a characteristic morphology similar to the well-characterized embryonic stem cells. Alkaline phosphatase, cell surface (SSEA1, NANOG, and OCT4), and pluripotency markers were expressed at high levels in these cells. The iPSCs formed teratomas in vivo and supported full-term development of constructed porcine embryos by inter-species nuclear transplantation. Importantly, incubation with trichostatin A increased the efficiency of iPSCs generation by increasing the histone acetylation levels. Moreover, more iPSCs colonies appeared following cell passaging during colony picking, thus increasing the effectiveness of iPSCs selection. Thus, our work provides essential stem cell materials for the treatment of genetic diseases and proposes a novel strategy to enhance the efficiency of induced reprogramming.     

A Mixed Mirror-image DNA/RNA Aptamer Inhibits Glucagon and Acutely Improves Glucose Tolerance in Models of Type 1 and Type 2 Diabetes

Excessive secretion of glucagon, a functional insulin antagonist, significantly contributes to hyperglycemia in type 1 and type 2 diabetes. Accordingly, immunoneutralization of glucagon or genetic deletion of the glucagon receptor improved glucose homeostasis in animal models of diabetes. Despite this strong evidence, agents that selectively interfere with endogenous glucagon have not been implemented in clinical practice yet. We report the discovery of mirror-image DNA-aptamers (Spiegelmer®) that bind and inhibit glucagon. The affinity of the best binding DNA oligonucleotide was remarkably increased (>25-fold) by the introduction of oxygen atoms at selected 2′-positions through deoxyribo- to ribonucleotide exchanges resulting in a mixed DNA/RNA-Spiegelmer (NOX-G15) that binds glucagon with a K_d of 3 nm. NOX-G15 shows no cross-reactivity with related peptides such as glucagon-like peptide-1, glucagon-like peptide-2, gastric-inhibitory peptide, and prepro-vasoactive intestinal peptide. In vitro, NOX-G15 inhibits glucagon-stimulated cAMP production in CHO cells overexpressing the human glucagon receptor with an IC₅₀ of 3.4 nm. A single injection of NOX-G15 ameliorated glucose excursions in intraperitoneal glucose tolerance tests in mice with streptozotocin-induced (type 1) diabetes and in a non-genetic mouse model of type 2 diabetes. In conclusion, the data suggest NOX-G15 as a therapeutic candidate with the potential to acutely attenuate hyperglycemia in type 1 and type 2 diabetes.     

Immune response of mice to Thy-1.1 antigen: Studies on congenic lines

The primary response to Thy-1.1 antigen was measured by a plaque assay that detected cells producing antibodies lytic for AKR thymocytes (PFC). TheH-2 congenic mice (B10.K and B10.BR) carryingH-2 complexes of high responders (CBA and C57BR) on the low-responder background (B10) were found to produce significantly fewer PFC than the corresponding donor of theH-2 complex. On the other hand, C3H.B10 mice carrying theH-2 complex of a low responder on the high-responder background produced significantly more PFC than the donor of theH-2 complex. These findings were interpreted as evidence that alleles at previously described loci believed to be components of theI region of theH-2 complex and controlling immune response to Thy-1.1 are influenced by alleles at another locus. Studies of segregating populations of theH-2 congenic lines supplied evidence that this locus, tentatively calledIr-5, is in chromosome 17 (linkage group IX).     

Characterization of Remitting and Relapsing Hyperglycemia in Post-Renal-Transplant Recipients

Background

Hyperglycemia following solid organ transplant is common among patients without pre-existing diabetes mellitus (DM). Post-transplant hyperglycemia can occur once or multiple times, which if continued, causes new-onset diabetes after transplantation (NODAT).

Objective

To study if the first and recurrent incidence of hyperglycemia are affected differently by immunosuppressive regimens, demographic and medical-related risk factors, and inpatient hyperglycemic conditions (i.e., an emphasis on the time course of post-transplant complications).

Methods

We conducted a retrospective analysis of 407 patients who underwent kidney transplantation at Mayo Clinic Arizona. Among these, there were 292 patients with no signs of DM prior to transplant. For this category of patients, we evaluated the impact of (1) immunosuppressive drugs (e.g., tacrolimus, sirolimus, and steroid), (2) demographic and medical-related risk factors, and (3) inpatient hyperglycemic conditions on the first and recurrent incidence of hyperglycemia in one year post-transplant. We employed two versions of Cox regression analyses: (1) a time-dependent model to analyze the recurrent cases of hyperglycemia and (2) a time-independent model to analyze the first incidence of hyperglycemia.

Results

Age (P = 0.018), HDL cholesterol (P = 0.010), and the average trough level of tacrolimus (P<0.0001) are significant risk factors associated with the first incidence of hyperglycemia, while age (P<0.0001), non-White race (P = 0.002), BMI (P = 0.002), HDL cholesterol (P = 0.003), uric acid (P = 0.012), and using steroid (P = 0.007) are the significant risk factors for the recurrent cases of hyperglycemia.

Discussion

This study draws attention to the importance of analyzing the risk factors associated with a disease (specially a chronic one) with respect to both its first and recurrent incidence, as well as carefully differentiating these two perspectives: a fact that is currently overlooked in the literature.     

Copper tolerance in Frankia sp. strain EuI1c involves surface binding and copper transport

Several Frankia strains have been shown to be copper-tolerant. The mechanism of their copper tolerance was investigated for Frankia sp. strain EuI1c. Copper binding was shown by binding studies. Unusual globular structures were observed on the surface of the bacterium. These globular structures were composed of aggregates containing many relatively smaller “leaf-like” structures. Scanning electron microscopy with energy-dispersive X-ray (SEM-EDAX) analysis of these structures indicated elevated copper and phosphate levels compared to the control cells. Fourier transform infrared spectroscopy (FTIR) analysis indicated an increase in extracellular phosphate on the cell surface of copper-stressed cells. Bioinformatics’ analysis of the Frankia sp. strain EuI1c genome revealed five potential cop genes: copA, copZ, copC, copCD, and copD. Experiments with Frankia sp. strain EuI1c using qRT-PCR indicated an increase in messenger RNA (mRNA) levels of the five cop genes upon Cu²⁺ stress. After 5 days of Cu²⁺ stress, the copA, copZ, copC, copCD, and copD mRNA levels increased 25-, 8-, 18-, 18-, and 25-fold, respectively. The protein profile of Cu²⁺-stressed Frankia sp. strain EuI1c cells revealed the upregulation of a 36.7 kDa protein that was identified as FraEuI1c_1092 (sulfate-binding periplasmic transport protein). Homologues of this gene were only present in the genomes of the Cu²⁺-resistant Frankia strains (EuI1c, DC12, and CN3). These data indicate that copper tolerance by Frankia sp. strain EuI1c involved the binding of copper to the cell surface and transport proteins.     

SNP-SNP Interactions Discovered by Logic Regression Explain Crohn's Disease Genetics

In genome-wide association studies (GWAS), the association between each single nucleotide polymorphism (SNP) and a phenotype is assessed statistically. To further explore genetic associations in GWAS, we considered two specific forms of biologically plausible SNP-SNP interactions, ‘SNP intersection’ and ‘SNP union,’ and analyzed the Crohn''s Disease (CD) GWAS data of the Wellcome Trust Case Control Consortium for these interactions using a limited form of logic regression. We found strong evidence of CD-association for 195 genes, identifying novel susceptibility genes (e.g., ISX, SLCO6A1, TMEM183A) as well as confirming many previously identified susceptibility genes in CD GWAS (e.g., IL23R, NOD2, CYLD, NKX2-3, IL12RB2, ATG16L1). Notably, 37 of the 59 chromosomal locations indicated for CD-association by a meta-analysis of CD GWAS, involving over 22,000 cases and 29,000 controls, were represented in the 195 genes, as well as some chromosomal locations previously indicated only in linkage studies, but not in GWAS. We repeated the analysis with two smaller GWASs from the Database of Genotype and Phenotype (dbGaP): in spite of differences of populations and study power across the three datasets, we observed some consistencies across the three datasets. Notable examples included TMEM183A and SLCO6A1 which exhibited strong evidence consistently in our WTCCC and both of the dbGaP SNP-SNP interaction analyses. Examining these specific forms of SNP interactions could identify additional genetic associations from GWAS. R codes, data examples, and a ReadMe file are available for download from our website: http://www.ualberta.ca/~yyasui/homepage.html.     

A Single Nucleotide Polymorphism within DUSP9 Is Associated with Susceptibility to Type 2 Diabetes in a Japanese Population

Aims

The DUSP9 locus on chromosome X was identified as a susceptibility locus for type 2 diabetes in a meta-analysis of European genome-wide association studies (GWAS), and GWAS in South Asian populations identified 6 additional single nucleotide polymorphism (SNP) loci for type 2 diabetes. However, the association of these loci with type 2 diabetes have not been examined in the Japanese. We performed a replication study to investigate the association of these 7 susceptibility loci with type 2 diabetes in the Japanese population.

Methods

We genotyped 11,319 Japanese participants (8,318 with type 2 diabetes and 3,001 controls) for each of the 7 SNPs–rs5945326 near DUSP9, rs3923113 near GRB14, rs16861329 in ST6GAL1, rs1802295 in VPS26A, rs7178572 in HMG20A, rs2028299 near AP3S2, and rs4812829 in HNF4A–and examined the association of each of these 7 SNPs with type 2 diabetes by using logistic regression analysis.

Results

All SNPs had the same direction of effect (odds ratio [OR]>1.0) as in the original reports. One SNP, rs5945326 near DUSP9, was significantly associated with type 2 diabetes at a genome-wide significance level (p = 2.21×10⁻⁸; OR 1.39, 95% confidence interval [CI]: 1.24−1.56). The 6 SNPs derived from South Asian GWAS were not significantly associated with type 2 diabetes in the Japanese population by themselves (p≥0.007). However, a genetic risk score constructed from 6 South Asian GWAS derived SNPs was significantly associated with Japanese type 2 diabetes (p = 8.69×10⁻⁴, OR  = 1.06. 95% CI; 1.03−1.10).

Conclusions/interpretation

These results indicate that the DUSP9 locus is a common susceptibility locus for type 2 diabetes across different ethnicities, and 6 loci identified in South Asian GWAS also have significant effect on susceptibility to Japanese type 2 diabetes.     

Aldo-keto Reductase Family 1 Member B10 Promotes Cell Survival by Regulating Lipid Synthesis and Eliminating Carbonyls

Aldo-keto reductase family 1 member B10 (AKR1B10) is primarily expressed in the normal human colon and small intestine but overexpressed in liver and lung cancer. Our previous studies have shown that AKR1B10 mediates the ubiquitin-dependent degradation of acetyl-CoA carboxylase-α. In this study, we demonstrate that AKR1B10 is critical to cell survival. In human colon carcinoma cells (HCT-8) and lung carcinoma cells (NCI-H460), small-interfering RNA-induced AKR1B10 silencing resulted in caspase-3-mediated apoptosis. In these cells, the total and subspecies of cellular lipids, particularly of phospholipids, were decreased by more than 50%, concomitant with 2–3-fold increase in reactive oxygen species, mitochondrial cytochrome c efflux, and caspase-3 cleavage. AKR1B10 silencing also increased the levels of α,β-unsaturated carbonyls, leading to the 2–3-fold increase of cellular lipid peroxides. Supplementing the HCT-8 cells with palmitic acid (80 μm), the end product of fatty acid synthesis, partially rescued the apoptosis induced by AKR1B10 silencing, whereas exposing the HCT-8 cells to epalrestat, an AKR1B10 inhibitor, led to more than 2-fold elevation of the intracellular lipid peroxides, resulting in apoptosis. These data suggest that AKR1B10 affects cell survival through modulating lipid synthesis, mitochondrial function, and oxidative status, as well as carbonyl levels, being an important cell survival protein.Aldo-keto reductase family 1 member B10 (AKR1B10,² also designated aldose reductase-like-1, ARL-1) is primarily expressed in the human colon, small intestine, and adrenal gland, with a low level in the liver (1–3). However, this protein is overexpressed in hepatocellular carcinoma, cervical cancer, lung squamous cell carcinoma, and lung adenocarcinoma in smokers, being a potential diagnostic and/or prognostic marker (1, 2, 4–6).The biological function of AKR1B10 in the intestine and adrenal gland, as well as its role in tumor development and progression, remains unclear. AKR1B10 is a monomeric enzyme that efficiently catalyzes the reduction to corresponding alcohols of a range of aromatic and aliphatic aldehydes and ketones, including highly electrophilic α,β-unsaturated carbonyls and antitumor drugs containing carbonyl groups, with NADPH as a co-enzyme (1, 7–12). The electrophilic carbonyls are constantly produced by lipid peroxidation, particularly in oxidative conditions, and are highly cytotoxic; through interaction with proteins, peptides, and DNA, the carbonyls cause protein dysfunction and DNA damage (breaks and mutations), resulting in mutagenesis, carcinogenesis, or apoptosis (10, 13–19). AKR1B10 also shows strong enzymatic activity toward all-trans-retinal, 9-cis-retinal, and 13-cis-retinal, reducing them to the corresponding retinols, which may regulate the intracellular retinoic acid, a signaling molecule modulating cell proliferation and differentiation (6, 20–23). In lung cancer, AKR1B10 expression is correlated with the patient smoking history and activates procarcinogens in cigarette smoke, such as polycyclic aromatic hydrocarbons, thus involved in lung tumorigenesis (24–26).Recent studies have shown that in breast cancer cells, AKR1B10 associates with acetyl-CoA carboxylase-α (ACCA) and blocks its ubiquitination and proteasome degradation (27). ACCA is a rate-limiting enzyme of de novo synthesis of long chain fatty acids, catalyzing the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA (28). Long chain fatty acids are the building blocks of biomembranes and the precursor of lipid second messengers, playing a critical role in cell growth and proliferation (29, 30). Therefore, ACCA activity is tightly regulated by both metabolite-mediated allosteric mechanisms and phosphorylation-dependent mechanisms; the latter are controlled by multiple hormones, such as insulin, glucagon, and growth factors (31–33). ACCA activity is also regulated through physical protein-protein interaction. For instance, breast cancer 1 (BRCA1) protein associates with the ACCA and blocks its Ser⁷⁹ residue from dephosphorylation (34, 35). The AKR1B10-mediated regulation on ACCA stability represents a novel regulatory mechanism, and this current study elucidated the biological significance of this regulation. The results show that AKR1B10 promotes cell survival via modulating lipid synthesis, mitochondrial function and oxidative stress, and carbonyl levels.     

The effects of galectin-1 on the gene expression of the transcription factors TBX21, GATA-3, FOXP3 and RORC

CD4⁺ T cells orchestrate the immune response by differentiating into T helper (Th) or regulatory (Treg) cell subsets that secrete distinct sets of cytokines. They also play a critical role in the pathogenesis of autoimmunity, asthma, allergy and, likely, cancer. The mechanisms involved in the regulation of CD4⁺ T cell homeostasis by galectin-1 remain poorly characterized. To investigate whether galectin-1 modulates the differentiation of CD4⁺ T cells, the effects of galectin-1 on the mRNA expression levels of TBX21, GATA-3, FOXP3 and RORC in activated peripheral blood mononuclear cells were examined. The expression levels of GATA-3 and FOXP3 mRNA were up-regulated after treatment with 1.0 μg/ml galectin-1 and were unchanged (for GATA-3) or slightly elevated (for FOXP3) compared with untreated cells when 2.0 μg/ml galectin-1 was added. At the same time, at both concentrations of galectin-1, we observed reduced TBX21 and RORC mRNA expression levels. These findings support the concept that galectin-1 skews the differentiation of CD4⁺ T cells towards Th2 and Treg cells.