Brown adipose tissue function in short-chain acyl-CoA dehydrogenase deficient mice.

Brown adipose tissue is a highly specialized organ that uses mitochondrial fatty acid oxidation to fuel non-shivering thermogenesis. In mice, mutations in the acyl-CoA dehydrogenase family of fatty acid oxidation genes are associated with sensitivity to cold. Brown adipose tissue function has not previously been characterized in these knockout strains. Short-chain acyl-CoA dehydrogenase (SCAD) deficient mice were found to have increased brown adipose tissue mass as well as modest cardiac hypertrophy. Uncoupling protein-1 was reduced by 70% in brown adipose tissue and this was not due to a change in mitochondrial number, nor was it due to decreased signal transduction through protein kinase A which is known to be a major regulator of uncoupling protein-1 expression. PKA activity and in vitro lipolysis were normal in brown adipose tissue, although in white adipose tissue a modest increase in basal lipolysis was seen in SCAD-/- mice. Finally, an in vivo norepinephrine challenge of brown adipose tissue thermogenesis revealed normal heat production in SCAD-/- mice. These results suggest that reduced brown adipose tissue function is not the major factor causing cold sensitivity in acyl-CoA dehydrogenase knockout strains. We speculate that other mechanisms such as shivering capacity, cardiac function, and reduced hepatic glycogen stores are involved.

Tumor necrosis factor-alpha gene polymorphisms are associated with severity of acute graft-versus-host disease following matched unrelated donor bone marrow transplantation in children: a Pediatric Blood and Marrow Transplant Consortium study.

Tumor necrosis factor (TNF)-alpha plays a significant role in conditioning related toxicities and the development of acute graft-versus-host disease (aGVHD). TNF-alpha gene polymorphisms are associated with rejection after organ transplantation and aGVHD in matched related donor blood and marrow transplantation (BMT) recipients. Few studies have been published on unrelated donor BMT in the pediatric age group. In this study, we examined the relationship between specific polymorphisms in TNF pathway genes and the occurrence and severity of aGVHD. Recipient single-nucleotide polymorphisms (SNPs) in TNF-alpha and TNF receptor superfamily members 1A (TNFRSF1A) and 1B (TNFRSF1B) were investigated. In a multi-institutional Pediatric Blood and Marrow Transplant Consortium trial, a total of 180 pediatric patients (mean age, 11.0 years) were prospectively evaluated for clinical outcomes after matched unrelated donor BMT. All patients received myeloablative conditioning and two-drug GVHD prophylaxis with cyclosporine or tacrolimus, with methotrexate in the majority of patients. TNF-alpha genotypes were not correlated with the overall incidence of aGVHD. Significant associations were seen between TNF-alpha variant alleles and the severity of aGVHD (grade II-IV and grade III-IV), especially when analyzed in whites only (n = 165). Grade II-IV aGVHD was correlated with recipient -857T allele (hazard ratio [HR], 0.47; P = .04), -238A allele (HR, 1.76; P = .002), and d3/d3 genotype (HR, 0.64; P = .03). Severe (grade III-IV) aGVHD was associated with TNF-alpha -1031C allele (HR, 2.38; P = .03), -863A allele (HR, 3.18; P = .003), and d4/d4 genotype (HR, 2.82; P = .01). After adjusting for clinical factors, the association of -1031C, -863A, -238A, and d4/d4 genotypes with severity of aGVHD remained statistically significant. No correlation between selected SNPs in TNFRSF1A or TNFRSF1B and the incidence or severity of aGVHD was found. Our findings indicate clinically important relationships between genetic polymorphisms in TNF-alpha and the severity of aGVHD in this cohort. Improved understanding of this relationship may allow for a risk-adjusted approach to GVHD prevention in pediatric BMT.

Effects of exercise on mitochondrial content and function in aging human skeletal muscle.

Skeletal muscle mitochondria are implicated with age-related loss of function and insulin resistance. We examined the effects of exercise on skeletal muscle mitochondria in older (age = 67.3 +/- 0.6 years) men (n = 5) and women (n = 3). Similar increases in (p <.01) cardiolipin (88.2 +/- 9.0 to 130.6 +/- 7.5 microg/mU creatine kinase activity [CK]) and the total mitochondrial DNA (1264 +/- 170 to 1895 +/- 273 copies per diploid of nuclear genome) reflected increased mitochondria content. Succinate oxidase activity, complexes 2-4 of the electron transport chain (ETC), increased from 0.13 +/- 0.02 to 0.20 +/- 0.02 U/mU CK (p <.01). This improvement was more pronounced (p <.05) in subsarcolemmal (127 +/- 48%) compared to intermyofibrillar (56 +/- 12%) mitochondria. NADH oxidase activity, representing total ETC activity, increased from 0.51 +/- 0.09 to 1.00 +/- 0.09 U/mU CK (p <.01). In conclusion, exercise enhances mitochondria ETC activity in older human skeletal muscle, particularly in subsarcolemmal mitochondria, which is likely related to the concomitant increases in mitochondrial biogenesis.

Functional single nucleotide polymorphism haplotypes in the human equilibrative nucleoside transporter 1.

The human equilibrative nucleoside transporter 1 gene (hENT1) is the primary nucleoside transporter for cytosine arabinoside (AraC), a deoxycytidine analog used for treatment of acute leukemias and lymphomas. We screened approximately 1.6 kb upstream of the transcription initiation site of hENT1 for single nucleotide polymorphisms (SNPs) that affect gene expression. We identified one SNP at position -706G>C with a frequency of 21% in whites and 5% in African-Americans. In African-Americans, we observed two SNPs at positions -1345C>G and -1050G>A with allele frequencies of 8% and 19%, respectively. TRANSFAC analysis suggested that -1345C>G and -706G>C may alter transcription factor binding sites. Four naturally occurring haplotypes (CGG, CAG, CGC and GAG) were cloned into a luciferase expression plasmid, transfected into Cos-1 cells, and reporter activity measured at 24 and 48 h. Three haplotypes, CAG, CGC and GAG, respectively, showed average expression that was approximately two-fold (P<0.05), 1.4-fold (P<0.05) and 1.1-fold (P>0.05) higher than lowest expression haplotype CGG at 48 h. When reanalysed as single SNPs, the differences in expression were significant for -1345C>G and -1050G>A genotypes, and not for -706G>C. However, the magnitude of difference was reduced, suggesting that no single SNP completely accounts for the expression differences observed at the haplotype level. By real-time quantitative reverse transcriptase-polymerase chain reaction assay, individuals with CGG/CGC haplotypes showed 1.37-fold higher median expression of hENT1 transcript than those with common CGG/CGG haplotypes. Although not statistically significant (P=0.12), this difference is in the direction predicted by the in vitro data. hENT1 promoter region haplotypes may influence gene expression and alter AraC chemosensitivity.

The PANE1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in B-lymphoid cells and B-CLL.

Minor histocompatibility antigens (mHAg's) are peptides encoded by polymorphic genes that are presented by major histocompatibility complex (MHC) molecules and recognized by T cells in recipients of allogeneic hematopoietic cell transplants. Here we report that an alternative transcript of the proliferation-associated nuclear element 1 (PANE1) gene encodes a novel human leukocyte antigen (HLA)-A(*)0301-restricted mHAg that is selectively expressed in B-lymphoid cells. The antigenic peptide is entirely encoded within a unique exon not present in other PANE1 transcripts. Sequencing of PANE1 alleles in mHAg-positive and mHAg-negative cells demonstrates that differential T-cell recognition is due to a single nucleotide polymorphism within the variant exon that replaces an arginine codon with a translation termination codon. The PANE1 transcript that encodes the mHAg is expressed at high levels in resting CD19(+) B cells and B-lineage chronic lymphocytic leukemia (B-CLL) cells, and at significantly lower levels in activated B cells. Activation of B-CLL cells through CD40 ligand (CD40L) stimulation decreases expression of the mHAg-encoding PANE1 transcript and reciprocally increases expression of PANE1 transcripts lacking the mHAg-encoding exon. These studies suggest distinct roles for different PANE1 isoforms in resting compared with activated CD19(+) cells, and identify PANE1 as a potential therapeutic target in B-CLL.

Comparative gene expression analysis of ovarian carcinoma and normal ovarian epithelium by serial analysis of gene expression.

Despite the poor prognosis of ovarian cancer and the importance of early diagnosis, there are no reliable noninvasive biomarkers for detection in the early stages of disease. Therefore, to identify novel ovarian cancer markers with potential utility in early-stage screening protocols, we have undertaken an unbiased and comprehensive analysis of gene expression in primary ovarian tumors and normal human ovarian surface epithelium (HOSE) using Serial Analysis of Gene Expression (SAGE). Specifically, we have generated SAGE libraries from three serous adenocarcinomas of the ovary and, using novel statistical tools, have compared these to SAGE data derived from two pools of normal HOSE. Significantly, in contrast to previous SAGE-based studies, our normal SAGE libraries are not derived from cultured cell lines. We have also compared our data with publicly available SAGE data obtained from primary tumors and "normal" HOSE-derived cell lines. We have thus identified several known and novel genes whose expressions are elevated in ovarian cancer. These include but are not limited to CLDN3, WFDC2, FOLR1, COL18A1, CCND1, and FLJ12988. Furthermore, we found marked differences in gene expression patterns in primary HOSE tissue compared with cultured HOSE. The use of HOSE tissue as a control for these experiments, along with hierarchical clustering analysis, identified several potentially novel biomarkers of ovarian cancer, including TACC3, CD9, GNAI2, AHCY, CCT3, and HMGA1. In summary, these data identify several genes whose elevated expressions have not been observed previously in ovarian cancer, confirm the validity of several existing markers, and provide a foundation for future studies in the understanding and management of this disease.

Distinct domains of the limbic system-associated membrane protein (LAMP) mediate discrete effects on neurite outgrowth.

The limbic system-associated membrane protein (LAMP) is a glycosylphosphatidylinositol-anchored glycoprotein with three immunoglobulin (Ig) domains that can either enhance or inhibit neurite outgrowth depending upon the neuronal population examined. In the present study, we investigate the domains responsible for these activities. Domain deletion revealed that the N-terminal IgI domain is necessary and sufficient for the neurite-promoting activity observed in hippocampal neurons. In contrast, inhibition of neurite outgrowth in SCG neurons, which is mediated by heterophilic interactions, requires full-length LAMP, although selective inhibition of the second Ig domain, but not the first or third domains, prevented the inhibitory effect. This indicates that the IgII domain of LAMP harbors the neurite-inhibiting activity, but only in the context of the full-length configuration. Covasphere-binding analyses demonstrate IgI/IgI interactions, but no interaction between IgII and any other domain, consistent with the biological activities that each domain mediates. The data suggest that LAMP may serve as a bifunctional guidance molecule, with distinct structural domains contributing to the promotion and inhibition of neurite outgrowth.

Sequence variation in hypoxia-inducible factor 1alpha (HIF1A): association with maximal oxygen consumption.

Hypoxia-inducible factor 1 (HIF1) is a DNA transcription factor composed of two subunits, one of which is regulated by hypoxia (HIF1alpha, encoded by HIF1A). Genes regulated by HIF1 are involved in the processes of angiogenesis, erythropoiesis, and metabolism, making HIF1A a candidate gene in establishing maximal oxygen consumption (VO2 max) before and after aerobic exercise training. The purpose of the present study was to screen HIF1A for sequence variation and determine whether such variation is associated with VO2 max before and after aerobic exercise training. A total of 233 Caucasian and African-American subjects were available for screening of HIF1A and determination of allele frequencies, with 155 of those subjects used to study VO2 max in relation to identified variants. We measured VO2 max before and after 24 wk of aerobic exercise training. Screening revealed several rare and common polymorphisms in HIF1A with race-specific allele frequencies. African Americans with AT or TT genotype at the A-2500T locus exhibited significantly lower baseline VO2 max compared with those of AA genotype (21.9 +/- 0.99 vs. 25.1 +/- 1.0, P = 0.03). An age by P582S (C/T) genotype interaction was observed in Caucasian subjects, such that those of CT or TT genotype exhibited significantly lower change in VO2 max after training than those of CC genotype when compared at ages 65 and 60 yr, but not at age 55 yr. No other significant differences were noted among genotype groups at the A-2500T, P582S, or T+140C sites. Based on these findings, we conclude that HIF1A sequence variation is associated with VO2 max before and after aerobic exercise training in older humans.