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Glenn S Gerhard

Publications and source records attributed to Glenn S Gerhard.

17 recordsLinked to original sources
PubMed2004

High-throughput non-heme iron assay for animal tissues.

Iron has been widely studied in nearly every realm of biology. However, current methodologies, such as genetic mapping or mutation screening, have been difficult to apply due to the lack of robust high-throughput methods for quantifying iron levels from cells or tissues. The measurement of total iron levels in tissues, usually done with atomic absorption spectroscopy, is impractical for large numbers of samples and includes the contribution of heme iron from hemoglobin contained in red blood cells. The measurement of non-heme iron by reaction with a bathophenanthroline reagent, a commonly used assay reported more than 30 years ago, is also not feasible for large-scale analyses because it is cuvette-based. We therefore have modified this method to a microplate format that will facilitate large-scale analysis. The microplate assay is highly sensitive and specific, and is a simple and effective method for the measurement of non-heme iron for animal tissues that will enable the application of high-throughput of genetic methodologies.

Animals↗
PubMed2004

Genetic factors influence ethanol-induced uroporphyria in Hfe(-/-) mice.

Two major risk factors for porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). We recently described an animal model for alcohol-induced uroporphyria, using Hfe(-/-) mice. In the present study we show that this effect is dependent on genetic background and ethanol dose. In the 129S6/SvEvTac (129) strain, treatment with 15% ethanol in the drinking water for 6.5 months produced an accumulation of hepatic uroporphyrin (URO) 4-fold higher than that observed with 10% ethanol, a 90% decrease in uroporphyrinogen decarboxylase activity (UROD), and further increased the activities of hepatic 5-aminolevulinate synthase (ALAS) and CYP1A2. Hepatic nonheme iron (NHFe) and hepatocyte iron staining were not further increased by 15% compared to 10% ethanol. Treatment of C57BL/6 Hfe(-/-) mice with 15% ethanol for 6.5 months did not increase hepatic URO. Although NHFe was increased by ethanol, the resulting level was only half that of ethanol-treated 129 Hfe(-/-) mice. ALAS induction was similar in both Hfe(-/-) strains. In wild-type 129 mice treated with ethanol for 6 to 7 months, administration of iron dextran increased hepatic URO accumulation and decreased UROD activity. In conclusion, this study demonstrates a strong effect of genetic background on ethanol-induced uroporphyria, which is probably due to a greater effect of ethanol on iron metabolism in the susceptible strain.

5-Aminolevulinate Synthetase↗
PubMed2004

The effects of temperature reduction on gene expression and oxidative stress in skeletal muscle from adult zebrafish.

Longevity is inversely proportional to ambient temperature in ectothermic organisms such as fish. However, the mechanism by which reducing temperature over a physiological range increases life span is not known and available data are derived primarily from invertebrates. With a rodent-like longevity and abundant biological resources, the zebrafish is an ideal vertebrate ectothermic model in which to investigate this phenomenon. As an initial approach, the effects of a year-long 10 degrees C reduction in water temperature on global gene expression in tail skeletal muscle from adult zebrafish were determined using an oligonucleotide microarray representing 15,512 genes. Expression levels for approximately 600 genes were up-regulated by 1.7-fold or greater by the reduction in temperature, while a similar number of transcripts were down regulated by more than 1.7-fold. Using gene ontology (GO) classifications for molecular function, two functional groups, "oxygen and reactive oxygen species metabolism" and "response to oxidative stress," were found to be overrepresented among up-regulated genes. Transcripts levels for the genes in these two categories were increased by temperature reduction (TR). However, temperature reduction did not suppress lipid peroxidation potential, protein carbonyl content, or 8-oxoguanine level. Additional studies will be required to further delineate the role of altered gene expression and oxidative stress on the longevity-promoting effects of temperature reduction.

Aging↗
PubMed2003

The enigmatic role of the hemochromatosis protein (HFE) in iron absorption.

The HFE gene, a member of the class-I transplantation antigen gene family, is responsible for hereditary hemochromatosis, one of the most common inherited diseases in individuals of European descent. Patients exhibit predictable changes in iron homeostasis, including elevations in both transferrin saturation and serum ferritin levels. A subset of patients progress to overt clinical sequelae, resulting from iron overload. A hallmark of the disease is increased absorption of iron by the intestine. Although the HFE protein appears to modulate the function of the transferrin receptor in vitro, its precise role in vivo remains obscure. With multiple cell types involved in iron metabolism, the function of HFE is likely to be complex.

Animals↗
PubMed2003

Uroporphyria caused by ethanol in Hfe(-/-) mice as a model for porphyria cutanea tarda.

Two major risk factors for the development of porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). To develop an animal model, Hfe knockout mice were treated continuously with 10% ethanol in drinking water. By 4 months, uroporphyrin (URO) was detected in the urine. At 6 to 7 months, hepatic URO was increased and hepatic uroporphyrinogen decarboxylase (UROD) activity was decreased. Untreated Hfe(-/-) mice or wild-type mice treated with or without ethanol did not show any of these biochemical changes. Treatment with ethanol increased hepatic nonheme iron and hepatic 5-aminolevulinate synthase activity in Hfe(-/-) but not wild-type mice. The increases in nonheme iron in Hfe(-/-) mice were associated with diffuse increases in iron staining of parenchymal cells but without evidence of significant liver injury. In conclusion, the results of this study suggest that the uroporphyrinogenic effect of ethanol is mediated by its effects on hepatic iron metabolism. Ethanol-treated Hfe(-/-) mice seem to be an excellent model for studies of alcohol-mediated PCT.

5-Aminolevulinate Synthetase↗
PubMed2002

Mitochondrial DNA mutation analysis in human skin fibroblasts from fetal, young, and old donors.

Multibase deletions in mitochondrial DNA (mtDNA) have been shown to accumulate with age in several tissues, including skin, whereas point mutations have only recently been demonstrated to increase during aging, with several specific mutations occurring at high levels (up to 50%) in skin fibroblasts obtained from old donors [Science 286(1999)774]. We have conducted a survey for a specific deletion and for point mutations in several regions of mtDNA from cultured skin fibroblasts derived from eight fetal (12-20 weeks gestational age), ten young (17-33 years of age) and 11 old (78-92 years of age) human donors. Using PCR analysis, detectable levels of the 4977 basepair (bp) 'common deletion' were present in all three age groups, with the highest deletion levels of up to 0.3% of total mtDNA found in several cell lines from old donors, although other old donor cell lines had much lower levels. Single strand conformation polymorphism (SSCP) analysis for point mutations in the non-coding D-loop region and two regions of the cytochrome oxidase 2 gene failed to reveal the presence of any single base mutations. We infer that age-related high level mutational damage in mtDNA from human skin fibroblasts may manifest both sequence and inter-individual specificity.

Adolescent↗
PubMed2002

Genetic differences in hepatic lipid peroxidation potential and iron levels in mice.

Oxidative damage to macromolecules, including lipids, has been hypothesized as a mechanism of aging. One end product of lipid peroxidation, malondialdehyde (MDA), is often quantified as a measure of oxidative damage to lipids. We used a commercial colorimetric assay for MDA (Bioxytech LPO-586, Oxis International, Portland, OR) to measure lipid peroxidation potential in liver tissue from young (2 month) male mice from recombinant inbred (RI) mouse strains from the C57BL/6J (B6)xDBA/2J (D2) series (BXD). The LPO-586 assay (LPO) reliably detected significant differences (P<0.0001) in lipid peroxidation potential between the B6 and D2 parental strains, and yielded a more than two-fold variation across the BXD RI strains. In both B6 and D2 mice, LPO results were greater in old (23 month) mice, with a larger age-related increase in the D2 strain. As the level of iron can influence lipid peroxidation, we also measured hepatic non-heme iron levels in the same strains. Although iron level exhibited a slightly negative overall correlation (r(2)=0.119) with LPO results among the entire group of BXD RI strains, a sub-group with lower LPO values were highly correlated (r(2)=0.704). LPO results were also positively correlated with iron levels from a group of 8 other inbred mouse strains (r(2)=0.563). The BXD RI LPO data were statistically analyzed to nominate quantitaive trait loci (QTL). A single marker, Zfp4, which maps to 55.2 cM on chromosome 8, achieved a significance level of P<0.0006. At least two potentially relevant candidate genes reside close to this chromosomal position. Hepatic lipid peroxidation potential appears to be a strain related trait in mice that is amenable to QTL analysis.

Aging↗
PubMed2002

A call to fins! Zebrafish as a gerontological model.

Among the wide variety of model organisms commonly used for studies on aging, such as worms, flies and rodents, a wide research gap exists between the invertebrate and vertebrate model systems. In developmental biology, a similar gap has been filled by the zebrafish (Danio rerio). We propose that the zebrafish is uniquely suited to serve as a bridge model for gerontology. With high fecundity and economical husbandry requirements, large populations of zebrafish may be generated quickly and cheaply, facilitating large-scale approaches including demographic studies and mutagenesis screens. A variety of mutants identified in such screens have led to modelling of human disease, including cardiac disorders and cancer. While zebrafish longevity is at least 50% longer than in commonly used mouse strains, as an ectothermic fish species, its life span may be readily modulated by caloric intake, ambient temperature and reproductive activity. These features, coupled with a growing abundance of biological resources, including an ongoing genome sequencing project, make the zebrafish a compelling model organism for studies on aging.

Aging↗
PubMed2002

Uroporphyria in mice: thresholds for hepatic CYP1A2 and iron.

In mice treated with 5-aminolevulinic acid (ALA) and polyhalogenated aromatic compounds, the levels of both hepatic cytochrome P450 (CYP)1A2 and iron-which can be quite different among inbred strains-are critical in causing experimental uroporphyria. Here we investigate the development of uroporphyria as a function of CYP1A2 and iron levels in the liver of mice having a common C57BL/6 genetic background. We compared Cyp1a2(-/-) knockout mice, Cyp1a2(+/-) heterozygotes, Cyp1a2(+/+) wild type, and Cyp1a2(+/+) mice pretreated with a low dose of 3,3',4,4',5-pentachlorobiphenyl (PCB126) (4 microg/kg). Cyp1a2(+/-) mice contain about 60% of the hepatic CYP1A2 content of Cyp1a2(+/+) mice, and the PCB126-pretreated Cyp1a2(+/+) mice have about twice the wild-type levels of CYP1A2. ALA- and iron-treated Cyp1a2(+/+) mice are known to accumulate hepatic uroporphyrin; this accumulation was increased 7-fold by pretreatment with the low dose of PCB126. ALA- and iron-treated Cyp1a2(+/-) heterozygote mice accumulated no uroporphyrin in 4 weeks, but by 8 weeks accumulated significant amounts of uroporphyrin. As previously reported, the ALA- and iron-treated Cyp1a2(-/-) knockout mouse has no CYP1A2 and exhibits no detectable uroporphyrin accumulation. Iron dose-response curves in ALA- and PCB126-treated Cyp1a2(+/+) mice showed that hepatic iron levels greater than 850 microg/g liver were required to produce significant uroporphyrin accumulation in the liver. Other measures of hepatic effects of iron (iron-response element-binding protein [IRP]-iron response element [IRE] binding activity and accumulation of protoporphyrin from ALA) decreased when the level of iron was considerably lower than 850 microg/g liver. At low iron doses, accumulation of iron was principally in Kupffer cells, whereas at the higher doses (required to stimulate uroporphyrin accumulation), more iron was found in parenchymal cells. We conclude that small changes in hepatic CYP1A2 levels can dramatically affect uroporphyria in C57BL/6 mice, providing the animals have been sufficiently loaded with iron; these data might be clinically relevant to acquired (sporadic) porphyria cutanea tarda, because humans show greater than 60-fold genetic differences in hepatic basal CYP1A2.

Aminolevulinic Acid↗
PubMedDate not supplied

Hemochromatosis protein (HFE) and tumor necrosis factor receptor 2 (TNFR2) influence tissue iron levels: elements of a common gut pathway?

Quantitative genetic analysis of hepatic and splenic iron levels in recombinant inbred mice yielded a quantitative trait locus that was found to coincide with the genomic locale encompassing the tumor necrosis factor receptor 2 gene (Tnfr2). When fed an iron-enriched diet, mice nullizygous with respect to Tnfr2, but not the Tnfr1 gene, showed a significant increase in splenic non-heme iron levels. This result contrasted with mice deficient in the hemochromatosis protein, HFE, which demonstrated a significant increase in normally high hepatic iron levels, but no change in splenic iron, when fed an iron-enriched chow. Both Tnfr2 knockout and HFE knockout mice fed an iron-enriched diet failed to demonstrate intestinal epithelial cell iron following the application of the Perls' stain, as compared to both Tnfr1 knockout and normal control mice. Moreover, intestinal intraepithelial lymphocytes (IELs) isolated from HFE knockout mice did not show an increase in TNF expression following challenge with the iron-enriched diet, in contrast to normal controls. These results suggest that HFE and TNFR2 are both involved in regulating iron deposition in tissues and that the regulation occurs at the level of the intestine through IEL-orchestrated production of TNF following the binding to TNFR2. These data suggest that HFE and TNFR2 may contribute to a common pathway of the iron stores regulator insuring the controlled efflux of gut iron.

Animals↗
PubMedDate not supplied

Comparative aspects of zebrafish (Danio rerio) as a model for aging research.

The zebrafish has emerged over the past decade as a major model system for the study of development due to its invertebrate-like advantages coupled with its vertebrate biology. These features also make it a potentially valuable organism for gerontological research. The main advantages of zebrafish include its economical husbandry, small yet accessible size, high reproductive capacity, genetic tractability, and a large and growing biological database. Although zebrafish life span is longer than rodents, it shares the feasibility of large-scale mutational analysis with the extremely short-lived invertebrate models. This review compares zebrafish with the more widely used model organisms used for aging research, including yeast, worms, flies, mice, and humans.

Aging↗
PubMedDate not supplied

Life spans and senescent phenotypes in two strains of Zebrafish (Danio rerio).

Zebrafish have become a widely used model organism in developmental biology research. In order to initiate an experimental foundation for aging studies, we have determined some basic gerontological parameters for populations of outbred zebrafish, and the golden sparse strain. Outbred zebrafish manifested a mean life span of about 42 months, with the longest living individual surviving for 66 months. The golden sparse populations had a mean life span of 36 months and a maximum longevity of 58 months. Skeletal length at death increased with age, suggestive of indeterminate growth. A common age-related phenotype was spinal curvature. Radiographic analysis excluded bony changes as the cause of the spinal curvature, suggesting muscle abnormalities as a primary mechanism. These data and a growing abundance of related biological resources suggest that the zebrafish may be a compelling model organism for studies on aging.

Aging↗
PubMedDate not supplied

Separation of IgG and IgM from albumin in citrated human plasma using electrodialysis and metal ion affinity precipitation.

Plasmapheresis is the process by which plasma is separated from whole blood for therapeutic purposes. The primary reason to perform plasmapheresis is to remove immunoglobulins and not to necessarily remove the remainder of the plasma proteins. Nevertheless, most plasmapheresis is performed by centrifugal separation to remove bulk plasma. We have investigated whether electrodialysis and metal ion affinity precipitation can be used to selectively remove immunoglobulins from citrated plasma and potentially serve as an adjunctive technique to centrifugal plasmapheresis. Using a commercial electrodialysis device, we have desalted citrated plasma to separate immunoglobulins from albumin, and have determined the effects of pH adjustment and addition of zinc acetate for concomitant metal ion affinity precipitation. With desalted pH adjusted citrated plasma containing 1.5 mM zinc acetate, we achieved more than 80% recovery of albumin with removal of almost 60% of the immunoglobulin (Ig)G. Almost 80% of polyclonal IgM and 90% of monoclonal IgM was also removed. IgA was not effectively removed under any of the conditions tested. Selective precipitation with electrodialysis and zinc acetate precipitation appears to be an effective technique for the separation of IgG and IgM from albumin in citrated plasma.

Antibodies, Monoclonal↗