Purification and properties of arylmannosidases from mung bean seedlings and soybean cells

Two arylmannosidases (signified as A and B) were purified to homogeneity from soluble and microsomal fractions of mung bean seedlings. Arylmannosidase A from the microsomes appeared the same on native gels and on SDS gels as soluble arylmannosidase A, the same was true for arylmannosidase B. Sedimentation velocity studies indicated that both enzymes were homogeneous, and that arylmannosidase A had a molecular mass of 237 kd while B had a molecular mass of 243 kd. Arylmannosidase A showed two major protein bands on SDS gels with molecular masses of 60 and 55 kd, and minor bands of 79, 39 and 35 kd. All of these bands were N-linked since they were susceptible to digestion by endo-glucosaminidase H. In addition, at least the major bands could be detected by Western blots with antibody raised against the xylose moiety of N-linked plant oligosaccharides, and they could also be labeled in soybean suspension cells with [2-3H]mannose. Arylmannosidase B showed three major bands with molecular masses of 72, 55 and 45 kd, and minor bands of 42 and 39 kd. With the possible exception of the 45 and 42 kd bands, all of these bands are glycoproteins. Arylmannosidases A and B showed somewhat different kinetics in terms of mannose release from high-mannose oligosaccharides, but they were equally susceptible to inhibition by swainsonine and mannostatin A. Polyclonal antibody raised against the arylmannosidase B cross-reacted equally well with arylmannosidase A from mung bean seedlings and with arylmannosidase from soybean cells. However, monoclonal antibody against mung bean arylmannosidase A was much less effective against arylmannosidase B. Antibody was used to examine the biosynthesis and structure of the carbohydrate chains of arylmannosidase in soybean cells grown in [2−3H]mannose. Treatment of the purified enzyme with Endo H released ∼50% of the radioactivity, and these labeled oligosaccharides were of the high-mannose type, i.e. mostly Man9GlcNAc. The precipitated protein isolated from the Endo H treatment still contained 50% of the radioactivity, and this was present in modified structures that probably contain xylose residue

Genetic Risk Factors for Type 2 Diabetes: A Trans-Regulatory Genetic Architecture?

To date, 68 loci have been associated with type 2 diabetes (T2D) or glucose homeostasis traits. We report here the results of experiments aimed at functionally characterizing the SNPs replicated for T2D and glucose traits. We sought to determine whether these loci were associated with transcript levels in adipose, muscle, liver, lymphocytes, and pancreatic β-cells. We found an excess of trans, rather than cis, associations among these SNPs in comparison to what was expected in adipose and muscle. Among transcripts differentially expressed (FDR \u3c 0.05) between muscle or adipose cells of insulin-sensitive individuals and those of insulin-resistant individuals (matched on BMI), trans-regulated transcripts, in contrast to the cis-regulated ones, were enriched. The paucity of cis associations with transcripts was confirmed in a study of liver transcriptome and was further supported by an analysis of the most detailed transcriptome map of pancreatic β-cells. Relative to location- and allele-frequency-matched random SNPs, both the 68 loci and top T2D-associated SNPs from two large-scale genome-wide studies were enriched for trans eQTLs in adipose and muscle but not in lymphocytes. Our study suggests that T2D SNPs have broad-reaching and tissue-specific effects that often extend beyond local transcripts and raises the question of whether patterns of cis or trans transcript regulation are a key feature of the architecture of complex traits

Global Gene Expression Profiles of Subcutaneous Adipose and Muscle from Glucose-Tolerant, Insulin-Sensitive, and Insulin-Resistant Individuals Matched for BMI

OBJECTIVE - To determine altered gene expression profiles in subcutaneous adipose and skeletal muscle from nondiabetic, insulin-resistant individuals compared with insulin-sensitive individuals matched for BMI. RESEARCH DESIGN AND METHODS - A total of 62 nondiabetic individuals were chosen for extremes of insulin sensitivity (31 insulin-resistant and 31 insulin-sensitive subjects; 40 were European American and 22 were African American) and matched for age and obesity measures. Global gene expression profiles were determined and compared between ethnic groups and between insulin-resistant and insulin-sensitive participants individually and using gene-set enrichment analysis. RESULTS - African American and European American subjects differed in 58 muscle and 140 adipose genes, including many inflammatory and metabolically important genes. Peroxisome proliferator-activated receptor γ cofactor 1A (PPARGC1A) was 1.75-fold reduced with insulin resistance in muscle, and fatty acid and lipid metabolism and oxidoreductase activity also were down-regulated. Unexpected categories included ubiquitination, citrullination, and protein degradation. In adipose, highly represented categories included lipid and fatty acid metabolism, insulin action, and cell-cycle regulation. Inflammatory genes were increased in European American subjects and were among the top Kyoto Encyclopedia of Genes and Genomes pathways on gene-set enrichment analysis. FADS1, VEGFA, PTPN3, KLF15, PER3, STEAP4, and AGTR1 were among genes expressed differentially in both adipose and muscle. CONCLUSIONS - Adipose tissue gene expression showed more differences between insulin-resistant versus insulin-sensitive groups than the expression of genes in muscle. We confirm the role of PPARGC1A in muscle and show some support for inflammation in adipose from European American subjects but find prominent roles for lipid metabolism in insulin sensitivity independent of obesity in both tissues. Diabetes 60:1019–1029, 201

A controlled trial of Partners in Dementia Care: veteran outcomes after six and twelve months

Introduction: “Partners in Dementia Care” (PDC) tested the effectiveness of a care-coordination program integrating healthcare and community services and supporting veterans with dementia and their caregivers. Delivered via partnerships between Veterans Affairs medical centers and Alzheimer’s Association chapters, PDC targeted both patients and caregivers, distinguishing it from many non-pharmacological interventions. Hypotheses posited PDC would improve five veteran self-reported outcomes: 1) unmet need, 2) embarrassment about memory problems, 3) isolation, 4) relationship strain and 5) depression. Greater impact was expected for more impaired veterans. A unique feature was self-reported research data collected from veterans with dementia. Methods and Findings: Five matched communities were study sites. Two randomly selected sites received PDC for 12 months; comparison sites received usual care. Three structured telephone interviews were completed every 6 months with veterans who could participate. Results: Of 508 consenting veterans, 333 (65.6%) completed baseline interviews. Among those who completed baseline interviews, 263 (79.0%) completed 6-month follow-ups and 194 (58.3%) completed 12-month follow-ups. Regression analyses showed PDC veterans had significantly less adverse outcomes than those receiving usual care, particularly for more impaired veterans after 6 months, including reduced relationship strain (B = −0.09; p = 0.05), depression (B = −0.10; p = 0.03), and unmet need (B = −0.28; p = 0.02; and B = −0.52; p = 0.08). PDC veterans also had less embarrassment about memory problems (B = −0.24; p = 0.08). At 12 months, more impaired veterans had further reductions in unmet need (B = −0.96; p < 0.01) and embarrassment (B = −0.05; p = 0.02). Limitations included use of matched comparison sites rather than within-site randomization and lack of consideration for variation within the PDC group in amounts and types of assistance provided. Conclusions: Partnerships between community and health organizations have the potential to meet the dementia-related needs and improve the psychosocial functioning of persons with dementia. Trial Registry NCT0029116

Analysis of coding variants in the betacellulin gene in type 2 diabetes and insulin secretion in African American subjects

BACKGROUND: Betacellulin is a member of the epidermal growth factor family, expressed at the highest levels predominantly in the pancreas and thought to be involved in islet neogenesis and regeneration. Nonsynonymous coding variants were reported to be associated with type 2 diabetes in African American subjects. We tested the hypotheses that these previously identified variants were associated with type 2 diabetes in African Americans ascertained in Arkansas and that they altered insulin secretion in glucose tolerant African American subjects. METHODS: We typed three variants, exon1 Cys7Gly (C7G), exon 2 Leu44Phe (L44F), and exon 4 Leu124Met (L124M), in 188 control subjects and 364 subjects with type 2 diabetes. We tested for altered insulin secretion in 107 subjects who had undergone intravenous glucose tolerance tests to assess insulin sensitivity and insulin secretion. RESULTS: No variant was associated with type 2 diabetes, and no variant altered insulin secretion or insulin sensitivity. However, an effect on lipids was observed for all 3 variants, and variant L124M was associated with obesity measures. CONCLUSION: We were unable to confirm a role for nonsynonymous variants of betacellulin in the propensity to type 2 diabetes or to impaired insulin secretion

The TCF7L2 locus and type 1 diabetes

<p>Abstract</p> <p>Background</p> <p><it>TCF7L2 </it>belongs to a subfamily of TCF7-like HMG box-containing transcription factors, and maps to human chromosome 10q25.3. A recent study identified genetic association of type 2 diabetes (T2D) with this gene, correlated with diminished insulin secretion. This study aimed to investigate the possibility of genetic association between <it>TCF7L2 </it>and type 1 diabetes (T1D).</p> <p>Methods</p> <p>The SNP most significantly associated with T2D, rs7903146, was genotyped in 886 T1D nuclear family trios with ethnic backgrounds of mixed European descent.</p> <p>Results</p> <p>This study found no T1D association with, and no age-of-onset effect from rs7903146.</p> <p>Conclusion</p> <p>This study suggests that a T2D mechanism mediated by <it>TCF7L2 </it>does not participate in the etiology of T1D.</p