Gene expression profiling in subcutaneous, visceral and epigastric adipose tissues of patients with extreme obesity.

OBJECTIVE: The goal of the present study was to identify differences in gene expression between SAT, VAT and EAT depots in Class III severely obese individuals. DESIGN: Human subcutaneous (SAT) and visceral (VAT) adipose tissues exhibit differential gene expression profiles. There is little information, however, about the other proximal white adipose tissue, epigastric (EAT), in terms of its function and contribution to metabolism. SUBJECTS AND METHODS: Using RNA from adipose biospecimens obtained from Class III severely obese patients undergoing open Roux-en-Y gastric bypass surgery, we compared gene expression profiles between SAT, VAT and EAT, using microarrays validated by real-time quantitative PCR. RESULTS: The three depots were found to share 1907 genes. VAT had the greatest number of genes (66) expressed exclusively in this depot, followed by SAT (23), and then EAT (14). Moreover, VAT shared more genes with EAT (65) than with SAT (38). Further analyses using ratios of SAT/EAT, VAT/EAT and SAT/VAT identified specific as well as overlapping networks and pathways of genes representing dermatological diseases, inflammation, cell cycle and growth, cancer and development. Targeted analysis of genes, having a role in adipose tissue development and function, revealed that Peroxisome proliferator-activated receptor Gamma Coactivator 1-alpha (PGC1-α) that regulates the precursor of the hormone Irisin (FNCD5) were abundantly expressed in all three fat depots, along with fibroblast growth factors (FGF) FGF1, FGF7 and FGF10, whereas, FGF19 and FGF21 were undetectable. CONCLUSIONS: These data indicate that EAT has more in common with VAT, suggesting similar metabolic potential. The human epigastric adipose depot could have a significant functional role in metabolic diseases and should be further investigated.

Genetic determinants of weight of fast- and slow-twitch skeletal muscle in 500-day-old mice of the C57BL/6J and DBA/2J lineage.

C57BL/6J (B6) and DBA/2J (D2) strains and two derivative populations, BXD recombinant inbred strains (BXD RIs) and B6D2F2, were used to explore genetic basis for variation in muscle weight at 500 days of age. In parallel with findings in 200-day-old mice (Lionikas A, Blizard DA, Vandenbergh DJ, Glover MG, Stout JT, Vogler GP, McClearn GE, and Larsson L. Physiol Genomics 16: 141-152, 2003), weight of slow-twitch soleus, mixed gastrocnemius, and fast-twitch tibialis anterior (TA) and extensor digitorum longus (EDL) muscles was 13-22% greater (P < 0.001) in B6 than in D2. Distribution of BXD RI strain means indicated that genetic influence on muscle weight (strain effect P < 0.001, all muscles) was of polygenic origin, and effect of genetic factors differed between males and females (strain-by-sex interaction: P < 0.01 for soleus, EDL; P < 0.05 for TA, gastrocnemius). Linkage analyses in B6D2F2 population identified QTL affecting muscle weight on Chr 1, 2, 6, and 9. Pleiotropic influences were observed for QTL on Chr 1 (soleus, TA), 2 (TA, EDL, gastrocnemius), and 9 (soleus, TA, EDL) and were not related to muscle type (fast/slow-twitch) or function (flexor/extensor). Effect of QTL on Chr 9 on soleus muscle was male specific. QTL on Chr 2 and 6 were previously observed at 200 days of age, whereas QTL on Chr 1 and 9 are novel muscle weight QTL. In summary, muscle weight in B6/D2 lineage is affected by a polygenic system that has variable influences at different ages, between males and females, and across muscles in a manner independent of muscle type.

Coagulation parameters of ABO group-specific cryosupernatant.

Patients with thrombotic thrombocytopenic purpura that is refractory to conventional fresh frozen plasma (FFP) exchange therapy are sometimes switched to cryosupernatant as the replacement fluid, although its hemostatic properties are not well defined. We performed several key coagulation assays on three pools of four units from each of three ABO groups of cryosupernatant and FFP. Fibrinogen, factor VIII activity, and von Willebrand factor antigen (vWF:Ag) levels were all significantly lower in cryosupernatant compared with FFP, although at levels usually not considered clinically significant. We confirmed that group O FFP contained significantly less factor VIII activity and vWF:Ag compared with groups AB and B. In contrast to FFP, group AB cryosupernatant contained lower levels of fibrinogen, factor V activity, factor VIII activity, and vWF:Ag than groups O or B. Group AB cryosupernatant, with the lowest levels of vWF:Ag and universal ABO compatibility, may be the product of choice for refractory thrombotic thrombocytopenic purpura.

Molecular biology of aging.

Aging is an extremely complex biologic phenomenon of immense importance. Currently we have only a poor and incomplete understanding of the fundamental molecular mechanisms involved. Despite numerous observations and diverse theories, no unifying or proven hypotheses have emerged. It is reasonable to conclude, however, that aging is a multifactorial process composed of both genetic and environmental components. Each physiologic system within an organism, each tissue within a system, and each cell type with a tissue appears to have its own trajectory of aging. Thus, aging must be studied as parts of a whole and understood as the sum of its parts. Cellular "clocks" exist and operate in the absence of higher-order "clocks". However, higher-order clocks are certainly in place in vivo, but their relationship to cellular clocks is not well understood. All aging changes have a cellular basis, and aging is perhaps best studied, fundamentally, at the cellular level under defined and controlled environmental conditions. Aging changes at the cellular level must be viewed, however, as components of a hierarchical, dynamic, and interacting network whose functional integrity progressively deteriorates with time. The powerful tools of molecular biology are now being applied by scientists to evaluate the leading hypotheses. The results of these studies should serve to advance our understanding of aging and to focus future research efforts. This work should provide the scientific foundation to enhance the quality of life for people suffering the failings of age.

Molecular cloning and sequence analysis of the Danio rerio catalase gene.

Catalase is an antioxidant enzyme that plays a central role in the protection against oxidative stress through the metabolism of hydrogen peroxide. Catalase has been well studied in plants, bacteria, and mammals, but little work has been done in other vertebrate species. We have cloned the zebrafish (Danio rerio) catalase cDNA containing the complete coding region and analyzed expression by both reverse transcription polymerase chain reaction and western blot. The deduced amino acid sequence predicts a protein of 526 amino acids with both the primary DNA and amino acid sequences highly conserved among vertebrate species. The major protein-heme contact points in the catalase enzyme complex are also well conserved, although several amino acids associated with the second and third levels of the major substrate channel are not, suggesting potential differences in substrate access or specificity. The 3' flanking region of the cDNA contains a dinucleotide repeat near the termination codon consisting of a near perfect CA array that is polymorphic. The rat and mouse catalase genes also contain a CA repeat sequence in the 3' untranslated region, which, along with an adjacent 5' stem-loop structure, has previously been shown to be a site for mRNA protein binding (Clerch, 1995, Arch. Biochem. Biophys. 317 (1995) 267-274). A stem-loop structure is also predicted adjacent to the zebrafish CA repeat, suggesting a similar role in catalase gene regulation.

Vibrio vulnificus septicemia in a patient with the hemochromatosis HFE C282Y mutation.

Vibrio vulnificus is an extremely invasive gram-negative bacillus found in marine waters that causes overwhelming bacteremia and shock that is associated with high mortality. Impaired iron metabolism has been implicated in the susceptibility to V vulnificus bacterial infections. We report a case of fatal V vulnificus sepsis in a 56-year-old man who died within 1 to 3 days after consuming raw seafood. At autopsy, he was found to have micronodular cirrhosis and iron overload. Postmortem genetic analysis revealed the presence of the hemochromatosis gene (HFE) C282Y mutation. To our knowledge, this is this first documented fatal case of V vulnificus infection in a patient proven to carry the HFE C282Y mutation. Because this patient was heterozygous for the major hereditary hemochromatosis mutation and was not previously diagnosed with clinical iron overload, the spectrum of clinical susceptibilities to V vulnificus infection may include carriers of the C282Y mutation.

Genetic architecture for hole-board behaviors across substantial time intervals in young, middle-aged and old mice.

A quantitative trait locus (QTL) analysis of behaviors across the life span was conducted in F(2) mice from a C57BL/6J x DBA/2J cross and 22 BXD recombinant inbred (RI) strains. Mice of three age groups were tested in a hole-board apparatus for 3 min on three occasions approximately 1 month apart (average age at test 150, 450 and 750 days, approximately 400 mice per group, divided equally by sex). Quantitative trait loci with small effect size were found on 11 chromosomes for hole-board activity (Hbact) and hole-board rearing (Hbrear). Analysis of 22 RI strains tested at 150 and 450 days of age found only suggestive linkage, with four QTL for Hbact overlapping with those from the F(2) analysis. There was a significant phenotypic correlation between Hbact and Hbrear (approximately 0.55-0.69) and substantial commonality among QTL for the two behaviors. QTL analyses of head-pokes (HP) and fecal boli (FB) only identified QTL at the suggestive level of significance. Age accounted for approximately 15% of the phenotypic variance (sex approximately 3%), and there were genotype by age interactions at approximately 25% of the Hbact and Hbrear QTL. Quantitative trait loci for Hbrear were relatively stable across the three measurement occasions (those for Hbact somewhat less so), although mean levels of each index declined markedly comparing the first to subsequent trials. Considered as a whole, the polygenic system influencing exploratory behaviors accounts for approximately the same amount of phenotypic variance as age (within the range studied), is stable across substantial periods of time, and acts, for the most part, independently of age and sex.

Physical determinants of line pressures during apheresis.

Line pressure changes are inherent to apheresis procedures, although little work has been done to determine their physical basis. We developed a mathematical model in which fluid pressure within apheresis tubing is primarily dependent upon viscosity and hematocrit (HCT). Return line flow rates and pressures during plasma collections for peripheral blood stem cell (PBSC) harvest and during plasma exchange procedures using the COBE Spectra were recorded and used to model return line pressures. Pressure estimates were similar to measured values for both PBSC plasma collection (r2 = 0.70) and plasma exchange (r2 = 0.86). HCT and viscosity thus appear to be primary physical determinants that underlie these pressure changes.

EGF- and PDGF-stimulated phosphorylation in young and senescent WI-38 cells.

We have examined the ability of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) to stimulate cultures of young and senescent WI-38 cells to carry out tyrosine-specific phosphorylation of their respective membrane receptors. Previously we reported no reduction in EGF-stimulated phosphorylation in plasma membrane preparations of senescent cells. In this study we found no reduction in PDGF-stimulated phosphorylation in plasma membrane preparations from senescent cells. Furthermore, we found no differences in the EGF- or PDGF-stimulated phosphorylation of their respective receptors in intact cells. These data support the previous findings that although the EGF receptor autokinase activity becomes highly labile during extraction and immunoprecipitation of senescent cells, in situ loss of receptor tyrosine kinase activity is apparently not responsible for the age-associated loss of mitogenic responsiveness.

Mathematical modeling and computer simulation of erythrocytapheresis for SCD.

BACKGROUND: Erythrocytapheresis is used to prevent acute chest syndrome and stroke in patients with sickle cell disease (SCD). However, such regimens are associated with significant risks, such as iron overload and potential exposure to transfusion-transmitted infectious diseases. Computer modeling of erythrocytapheresis procedures may help optimize treatments and minimize risks. STUDY DESIGN AND METHODS: Mathematical models based upon material balance equations and patient-specific statistical analyses were developed to estimate HbS levels immediately after erythrocytapheresis and immediately before the next treatment. The equations were incorporated into a software application that was used to model the effects of various treatment values on four patients treated with 90 erythrocytapheresis procedures. RESULTS: Immediate postprocedure HbS values were accurately estimated with correlations between measured and calculated values ranging from R(2) = 0.83 to 0.96. Estimates of HbS just before the next treatment correlated well in three patients (R(2) = 0.71 to 0.83) but poorly in one (R(2) = 0.28 to 0.46). Varying the treatment values by computer simulation led to a wide variation in the number of RBC units and the net RBC volume transfused. CONCLUSION: Computer modeling of erythrocytapheresis can be used to optimize chronic treatment regimens for SCD patients and potentially to minimize the risks of overtransfusion.

Loss of red cells from the use of blood warmers during apheresis procedures.

In-line blood warmers increase the extracorporeal volume of apheresis procedures. A saline rinse of the blood warmer may be employed to minimize the loss of red blood cells (RBCs) from this extra volume. We measured the amount of RBCs remaining in three different brands of blood warmers at the end of a COBE Spectra rinseback to determine the clinical significance of blood warmer saline rinses. The volume of RBCs removed by a warmer rinse ranged from 15 to 24 ml per procedure and was statistically different among the three brands of warmers. The COBE warmer contained significantly less RBCs than either the Fenwal or Pharmaseal warmers. Patient hematocrit was not highly correlated with the residual RBC volume (r = 0.30). We estimate that 15-20 procedures are required before the equivalent of one unit of packed RBCs is lost to blood warmers. We conclude that the blood warmer saline rinse may be safely omitted for most patients.

Expression and regulation of superoxide dismutase activity in human skin fibroblasts from donors of different ages.

We have determined the activities, protein, and mRNA abundances as well as the level of transcriptional activation of two intracellular forms of the free radical metabolizing enzyme superoxide dismutase in 29 human skin fibroblast lines established from donors of different ages. SOD-1 (a copper and zinc containing form of SOD) and SOD-2 (a manganese containing form of the enzyme) activities were both observed to be significantly lower in cell lines derived from fetal skin than in lines established from postnatal skin (ages 17-94 years). The percent of total activity contributed by SOD-1 decreased in an age-associated manner from approximately 50% in the fetal lines to less than 20% in lines established from old tissue donors. All of the cell lines were screened to exclude the possibility that they contained a polymorphism known to influence SOD-2 activity. Northern blot analysis revealed three SOD-1 mRNA transcripts that were 0.5, 0.7, and 1.9 kb in length. Although SOD-1 protein abundance was lower in fetal lines than in lines derived from postnatal donors, SOD-1 mRNA abundance did not differ between fetal cells and cell lines derived from young donors. SOD-2 protein abundance and mRNA abundance were both significantly lower in fetal lines than in postnatal lines. No postnatal age-dependent differences were observed in any of the SOD-2 parameters examined. Nuclear run-on analysis revealed that fetal cell lines exhibited a lower level of transcriptional initiation for SOD-1 than postnatal lines. The transcription of SOD-2 was readily detected in postnatal lines, but undetectable in fetal lines. These results are consistent with multiple levels of control for SOD-1 expression and with a strong transcriptional influence on SOD-2 expression.

Localization of the hemochromatosis disease gene: linkage disequilibrium analysis using an American patient collection.

The genetic basis of idiopathic hemochromatosis, a common disorder of iron metabolism, has remained an enigma for over two decades. In an attempt to refine the chromosomal localization of this gene, we have conducted a linkage disequilibrium mapping study utilizing a large group of unrelated American patients. The 12 microsatellites used as genetic markers in this analysis include a series of recently described polymorphic dinucleotide (D6S1558, D6S1545 and D6S1554) and tetranucleotide (D6S1016 and D6S1281) repeats which map between D6S105 and D6S299. Haplotype reconstructions indicate that a core genotype, composed of D6S464 allele 3/D6S1260 allele 4/D6S1558 allele 5, exists on a majority of disease chromosomes. Stringent statistical measures of marker-disease disequilibrium suggest that only associations with D6S1260 are significant and furthermore, aid in the assignment of refined centromeric and telomeric limits for the likely location of the hemochromatosis gene. In summary, the genetic data presented in this report predict that the hemochromatosis locus resides between D6S464 and D6S1558, most likely very close to marker D6S1260. Because a single yeast artificial chromosome clone contains all three of the above loci, a thorough search for coding sequences in this region is likely to identify the gene mutated in this common disorder.

Development and age-associated differences in electron transport potential and consequences for oxidant generation.

We determined the activities of NADH dehydrogenase (ND), succinate dehydrogenase, and cytochrome c oxidase (COX) in 29 skin fibroblast lines established from donors ranging in age from 12 gestational weeks to 94 years. The results of this study demonstrate that all three of the enzyme activities examined are greater in adult-derived fibroblasts than in the fetal cell lines. The ratio of enzyme activities that control electron entry into and exit from the electron transport chain varied directly with lucigenin-detected chemiluminescence (an indicator of .O2- generation) and inversely with H2O2 generation. These results indicate a clear difference in the predominant oxidant species generated during fetal and adult stages of life. We also examined the mRNA abundances of different components of the electron transport chain complexes. We observed higher abundances of mitochondrial encoded mRNAs (COX 1 and ND 4) in cell lines established from adults than in fetal cells. No differences in the mRNA abundances of the nuclear encoded sequences (COX 4 and ND 51) were observed in fetal and postnatal-derived lines. Succinate dehydrogenase mRNA abundance was greater in cell lines established from postnatal donors than in fetal cell lines. No significant differences between cell lines established from young and old adults were detected in any of the parameters examined.

Uroporphyria in Hfe mutant mice given 5-aminolevulinate: a new model of Fe-mediated porphyria cutanea tarda.

Porphyria cutanea tarda (PCT), a liver disease with skin lesions caused by excess liver production of uroporphyrin (URO), is associated with consumption of alcoholic beverages or estrogens, and moderate iron overload. Recently, it has been shown that many PCT patients carry mutations in the HFE gene, which is responsible for hereditary hemochromatosis. Mice homozygous for either the null mutation in the Hfe gene or the C282Y missense mutation rapidly accumulate hepatic parenchymal iron similar to patients with hemochromatosis. Here we investigated whether disruption of the murine Hfe gene would result in hepatic uroporphyria. Mice homozygous for the Hfe-null mutation accumulated high levels of hepatic URO when fed 5-aminolevulinate (ALA). Hfe (+/-) mice also accumulated hepatic URO when fed ALA, but at a much slower rate. The amount of accumulated URO in the null mutant mice was similar to that in wild-type mice treated with iron carbonyl in the diet, or injected with iron dextran. Iron in both wild-type and Hfe (+/-) mice was mostly in Kupffer cells. In contrast, Hfe (-/-) mice had considerable parenchymal iron deposition as well, in a pattern similar to that observed in wild-type mice treated with iron carbonyl. URO accumulation was accompanied by 84% and 33% decreases in hepatic uroporphyrinogen decarboxylase activities in Hfe (-/-) and Hfe (+/-) mice, respectively. No increases in CYP1A2 or other cytochrome P450s were detected in the Hfe-null mutant mice. We conclude that this experimental model of uroporphyria is a valid model for further investigations into the mechanism of PCT.

46,XX, inv(6)(p21.1p23) in a pedigree with hereditary haemochromatosis.

Hereditary haemochromatosis (HFE) is a recessive genetic disease of iron overload which has been shown by linkage analysis to reside on the short arm of chromosome 6, close to the major histocompatibility complex (MHC). Positional cloning of the putative HFE locus has been hampered, in part, by the lack of a structural alteration on 6p. In this report, we describe a pedigree with HFE which carries a balanced paracentric inversion of chromosome 6, inv(6)(p21.1p23), a rarely reported chromosomal rearrangement in this region. We have determined the inheritance of the chromosome harbouring the inversion, which segregates as an HFE chromosome. Because the HFE locus has been mapped distal to the HLA-F class I locus at 6p21.3, the breakpoints associated with this chromosomal rearrangement may provide a significant genomic landmark for positional cloning of the HFE gene.

Mutation analysis in hereditary hemochromatosis.

The DNA of 147 patients of European origin clinically diagnosed with idiopathic hemochromatosis and 193 controls was examined for mutations of the HLA-H gene at nt 845 and nt 187. One hundred twenty-one (82.3%) of the hemochromatosis patients were homozygous and 10 (6.8%) heterozygous for the 845A (C282Y) mutation. All of the homozygous patients were also homozygous for nt 187C, and all 845A heterozygotes had at least one copy of 187C. Thus, the nt 845 and nt 187 mutations were in complete linkage disequilibrium; nt 187 was a C on all chromosomes with the 845A mutation. Eight of the 10 heterozygotes for 845A were heterozygous for 187G(H63D). The excess of heterozygotes at both nt 187 and nt 845 suggested either the presence of as yet undiscovered mutations existing in trans with 845A and in linkage disequilibrium with 187G, or that the 187G itself is a deleterious mutation, which in concert with the 845A can give rise to hemochromatosis. None of the 193 normal controls were homozygous for 845A and 29/193 (15%) were heterozygous for 845A. Although 47/193 (24.3%) of normal controls were heterozygous for the 187G mutation only two of these carried the 845A mutation. If the 187G mutation complemented the 845A mutation with high penetrance in causing hemochromatosis, then the population frequency of the two genes would require that a high proportion of patients with hemochromatosis be heterozygous for 845A and 187G. Instead, the frequency of homozygotes for the 845A mutation was much higher than that of the 845A/187G genotype. Based on our data, the penetrance of the 845A/187G genotype is only 1.5% and based on the data of Feder et al. only 0.5%. In contrast, the penetrance of the homozygous 845A/845A genotype seems to be very high. Thus, screening for this genotype should be very useful.