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Biomedical subjects

Roger Gaedigk

Publications and source records attributed to Roger Gaedigk.

11 recordsLinked to original sources
PubMed2006

Variability of CYP2J2 expression in human fetal tissues.

CYP2J2 metabolizes arachidonic acid to 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids (EETs), which play a critical role in the regulation of renal, pulmonary, cardiac, and vascular function. However, the contribution of CYP2J2 to EET formation in the liver remains poorly characterized. Likewise, information is sparse regarding the extent and variability of CYP2J2 expression during human development. This investigation was undertaken to characterize the variability of CYP2J2 expression in fetal liver, heart, kidney, lung, intestine, and brain and in postnatal liver samples. CYP2J2 mRNA expression was measured using quantitative polymerase chain reaction, and immunoreactive CYP2J2 was examined using two anti-CYP2J2 antibodies. CYP2J2 mRNA was ubiquitously expressed in pre- and postnatal samples. Fetal hepatic mRNA expression varied 127-fold (1351 +/- 717 transcripts/ng total RNA), but this variation was reduced to 8-fold after exclusion of four samples with extremely low levels of mRNA. Amounts of immunoreactive protein also varied substantially among samples without an apparent relationship with transcript number or genotype. Western blot analysis revealed a different protein pattern between prenatal and postnatal liver samples. DNA resequencing of selected subjects identified a single novel single-nucleotide polymorphism (CYP2J2*10), which was found in only one subject and therefore did not explain the large variability in CYP2J2 protein content. In vitro expression suggests that the protein product of CYP2J2*10 confers reduced enzymatic activity. Aberrant splicing produces three minor transcripts, which were present in all samples tested. Due to premature termination codons, none encodes functional protein. The mechanisms leading to variable amounts of immunoreactive protein and distinct pre- and postnatal CYP2J2 protein patterns warrant further investigation.

Alleles↗
PubMed2006

Improvement in survival and muscle function in an mdx/utrn(-/-) double mutant mouse using a human retinal dystrophin transgene.

Duchenne muscular dystrophy is a progressive muscle disease characterized by increasing muscle weakness and death by the third decade. mdx mice exhibit the underlying muscle disease but appear physically normal with ordinary lifespans, possibly due to compensatory expression of utrophin. In contrast, double mutant mice (mdx/utrn(-/-)), deficient for both dystrophin and utrophin die by approximately 3 months and suffer from severe muscle weakness, growth retardation, and severe spinal curvature. The capacity of human retinal dystrophin (Dp260) to compensate for the missing 427 kDa muscle dystrophin was tested in mdx/utrn(-/-) mice. Functional outcomes were assessed by histology, EMG, MRI, mobility, weight and longevity. MCK-driven transgenic expression of Dp260 in mdx/utrn(-/-) mice converts their disease course from a severe, lethal muscular dystrophy to a viable, mild myopathic phenotype. This finding is relevant to the design of exon-skipping therapeutic strategies since Dp260 lacks dystrophin exons 1-29.

Age Factors↗
PubMed2005

CYP2D7 splice variants in human liver and brain: does CYP2D7 encode functional protein?

A CYP2D7 brain-specific protein metabolizing codeine and encoded by an alternate mRNA has recently been described in Indian subjects. To examine its potential presence in other ethnic backgrounds, CYP2D7 and CYP2D6 full-length splice products were analyzed in liver and brain. CYP2D7 splice variant-specific PCR on 13 subjects revealed the previously reported partial intron 6-containing transcript encoding CYP2D7 in both tissues as a minor variant. This transcript is not predicted to encode functional protein as a frame-shift-reverting deletion in CYP2D7 exon 1 (138delT) was not detected in any transcript or in 285 additional genotyped subjects. The g.14408G > C SNP required for functional transcript also was not observed; all transcripts and individuals genotyped as g.14408G/G that causes a premature stop codon in any splice variants that contain the 57bp intron 6 insertion. Therefore, no evidence for functional CYP2D7 transcripts was observed in Asian, Caucasian or African American individuals.

Alternative Splicing↗
PubMed2005

Nuclear receptor expression in fetal and pediatric liver: correlation with CYP3A expression.

The mechanisms underlying interindividual variation and developmental changes in cytochrome P450 3A (CYP3A) expression and activity are not fully understood. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) methods were used to detect, during human fetal and pediatric development, mRNA expression of nuclear receptors involved in the regulation of CYP3A genes. Quantitative RT-PCR was conducted on RNA extracted from prenatal (n = 60, 76 days to 32 weeks estimated gestational age) and pediatric (n = 20, 4 days to 18 years of age) liver tissue with primers for nuclear receptors implicated in regulating CYP3A gene expression. Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) were expressed at low (and highly variable) levels in pre- and neonatal liver relative to liver tissue derived from older children. CAR was expressed at higher levels relative to PXR in prenatal liver (757 +/- 480 molecules CAR/ng of RNA versus 271 +/- 190 molecules PXR/ng of RNA after correction for 18S rRNA). In contrast, mRNA expression of the heterodimer partner RXRalpha was less variable (33-fold) and did not differ appreciably between pre- and postnatal liver samples (219 +/- 101 molecules/ng of RNA prenatal versus 253 +/- 232 molecules/ng of RNA postnatal). Expression of HNF4alpha1 mRNA was similar to that of RXRalpha. Log CYP3A7 mRNA expression was significantly correlated with PXR (r2 = 0.372) and CAR (r2 = 0.380) mRNA in fetal liver, but associations were weaker than those observed with CYP3A4 mRNA in postnatal liver (r2 = 0.610 and 0.723 for PXR and CAR, respectively). In conclusion, nuclear receptor mRNA expression demonstrates considerable interindividual variability in human fetal and pediatric liver and is significantly correlated with CYP3A expression.

Age Factors↗
PubMed2005

Variability of CYP3A7 expression in human fetal liver.

Fetal liver CYP3A7 plays an important role in placental estriol synthesis during pregnancy, yet little is known concerning the extent or consequences of variability in expression. The purpose of this investigation was to characterize the variability in CYP3A7 expression using several phenotypic measures in a panel of 54 fetal livers ranging in age from 76 days to 32 weeks gestation. CYP3A7 mRNA expression was measured using quantitative polymerase chain reaction, whereas immunoreactive CYP3A7 was determined using an affinity-purified antipeptide antibody. Variability in catalytic activity was evaluated using testosterone and dehydroepiandrosterone (DHEA) as substrates. Across the entire panel, CYP3A7 was the most abundant CYP3A mRNA species present and varied 634-fold from 151 to 95,700 transcripts/ng total RNA, corrected for 18S ribosomal RNA. CYP3A4 expression was minimal based on mRNA expression (1000-fold lower than CYP3A7) and the ratio of testosterone 2alpha- (T2alphaH) to 6beta- (T6betaH) hydroxylation. T2alphaH and T6betaH were highly correlated (r(2) = 0.859), and the correlation increased (r(2) = 0.974) in livers with CYP3A5*3/*3 genotypes implying that the same enzyme (CYP3A7) generated both products. Overall, T2alphaH and DHEA16alphaH activities varied 175- and 250-fold, respectively. A subset of five samples had extremely low mRNA, protein, and catalytic activity, possibly due to pathology affecting fetal viability (anencephaly, porencephaly). In the remaining samples, T2alphaH activity varied 6.7-fold (358 +/- 142, range 97 to 643 pmol/min/mg) and DHEA16alphaH activity varied 6.2-fold (8.07 +/- 2.87, range 2.41 to 14.9 nmol/min/mg). Observed variability in CYP3A7 activity was not related to CYP3A7*2, and alternative regulatory mechanisms require further investigation.

Adult↗
PubMed2005

Characterization of the ALP1 gene locus of Trichophyton tonsurans.

Trichophyton tonsurans is the primary etiologic agent of fungal infections in the pediatric population. Establishing techniques that facilitate strain discrimination offer the opportunity to investigate the relationship between fungal genotype, biochemical phenotype and disease presentation in the host. In the process of expanding efforts to elucidate intra-specific genetic variability in T. tonsurans, we have identified 2 genetic polymorphisms in the ALP1 gene: a fragment length polymorphism in the 5'UTR and a single SNP (G-->A) within the 3'UTR. Full sequence data revealed that the length polymorphism was constituted by a 16 bp repeat element, present in tandem from 3 to 6 times depending on the strain. Quantitative RT-PCR analysis demonstrated a clear association between the length polymorphism and ALP1 mRNA transcript levels. Not only do the sequence variations identified in this study increase our ability to discriminate T. tonsurans strains, but they also reveal the presence of a genetic variation with functional consequences at the transcript level that may play a role in regulating disease severity.

5' Untranslated Regions↗
PubMed2004

Chromosomal localization, hematologic characterization, and iron metabolism of the hereditary erythroblastic anemia (hea) mutant mouse.

Understanding iron metabolism has been enhanced by identification of genes for iron deficiency mouse mutants. We characterized the genetics and iron metabolism of the severe anemia mutant hea (hereditary erythroblastic anemia), which is lethal at 5 to 7 days. The hea mutation results in reduced red blood cell number, hematocrit, and hemoglobin. The hea mice also have elevated Zn protoporphyrin and serum iron. Blood smears from hea mice are abnormal with elevated numbers of smudge cells. Aspects of the hea anemia can be transferred by hematopoietic stem cell transplantation. Neonatal hea mice show a similar hematologic phenotype to the flaky skin (fsn) mutant. We mapped the hea gene near the fsn locus on mouse chromosome 17 and show that the mutants are allelic. Both tissue iron overloading and elevated serum iron are also found in hea and fsn neonates. There is a shift from iron overloading to iron deficiency as fsn mice age. The fsn anemia is cured by an iron-supplemented diet, suggesting an iron utilization defect. When this diet is removed there is reversion to anemia with concomitant loss of overloaded iron stores. We speculate that the hea/fsn gene is required for iron uptake into erythropoietic cells and for kidney iron reabsorption.

Animals↗
PubMed2003

Genetic Heterogeneity in the rRNA Gene Locus of Trichophyton tonsurans.

Trichophyton tonsurans is the major pediatric pathogen in tinea capitis, causing disparate disease presentations. Little is known about genetic variation, which may ultimately be linked to divergent disease status. This investigation was aimed at identifying genetic variants of T. tonsurans by methods that can facilitate strain discrimination in population-based studies. Ninety-two isolates were acquired from six U.S. microbiology laboratories, and genomic DNA was isolated from mature colonies. The nontranscribed spacer (NTS) was amplified by PCR, and products from isolates with various amplicon sizes were fully sequenced. Nested amplification, targeting a variable internal repeat (VIR) region, allowed assignment of variant type by fragment size. Subvariant type was assigned by a combination of PCR-restriction fragment length polymorphism-based assays. Five variants differing in size (348 to 700 bp) and sequence were identified within the VIR region comprised of several large repeats (104, 140, and 194 bp) arranged in tandem. Seven single-nucleotide polymorphisms (SNPs) were detected across the NTS, with five occurring in the constant regions flanking the VIR region and two occurring in the VIR region. Additionally, a 10-bp insertion and a 14-bp deletion were identified upstream of the VIR region. The combination of SNPs revealed seven haplotype patterns which were stable upon serial passage over 1 year. No sequence variations were identified within the internal transcribed spacer regions. Unique NTS sequences were utilized to develop a duplex PCR assay that discriminated T. tonsurans from other dermatophytes. Of the 92 isolates evaluated, this genotyping scheme distinguished 12 distinct strains, providing evidence of genetic heterogeneity in T. tonsurans.

Base Sequence↗
PubMed2002

Primary Sjögren's syndrome and deficiency of ICA69.

BACKGROUND: Sjögren's syndrome is a common (about 1% of the population) autoimmune disease of salivary and lacrimal glands. Its cause and pathogenesis are poorly understood, and treatments are mostly for symptoms of the disease. ICA69 is a self-antigen expressed in brain, pancreas, salivary, and lacrimal glands. NOD-strain mice are an animal model of spontaneous Sjögren's syndrome. We aimed to assess the role of ICA69 in autoimmunity against Sjögren's syndrome. METHODS: We inactivated the genomic ICA69 locus, generated NOD congenic mice that were deficient in ICA69, and assessed development of Sjögren's syndrome. ICA69 autoimmunity was investigated in controls and in patients with primary Sjögren's syndrome or systemic lupus erythematosus, and in various NOD mice, some of which were given an ICA69-directed prototype peptide vaccine. FINDINGS: Disruption of the ICA69 locus prevented lacrimal gland disease and greatly reduced salivary gland disease in NOD mice. In healthy NOD mice, ICA69-specific T cells accumulated in lymph nodes that drain salivary tissue. T-cell and B-cell autoreactivity against ICA69 was much the same in patients with primary Sjögren's syndrome, but not in those with systemic lupus erythematosus or in healthy controls. Immunotherapy with a high-affinity mimicry peptide targeting ICA69-specific T-cells reduced established Sjögren's syndrome in wild-type NOD mice in the long term. INTERPRETATION: ICA69 is a new autoantigen in primary Sjögren's syndrome that has an important role in progression of disease and could be of diagnostic value. Immunotherapy of primary Sjögren's syndrome is promising, since autoimmunity in NOD mice with Sjögren's syndrome seems to be uniquely susceptible to such treatment even late in disease.

Adult↗
PubMed2002

ICA69(null) nonobese diabetic mice develop diabetes, but resist disease acceleration by cyclophosphamide.

ICA69 (islet cell Ag 69 kDa) is a diabetes-associated autoantigen with high expression levels in beta cells and brain. Its function is unknown, but knockout of its Caenorhabditis elegans homologue, ric-19, compromised neurotransmission. We disrupted the murine gene, ica-1, in 129-strain mice. These animals aged normally, but speed-congenic ICA69(null) nonobese diabetic (NOD) mice developed mid-life lethality, reminiscent of NOD-specific, late lethal seizures in glutamic acid decarboxylase 65-deficient mice. In contrast to wild-type and heterozygous animals, ICA69(null) NOD congenics fail to generate, even after immunization, cross-reactive T cells that recognize the dominant Tep69 epitope in ICA69, and its environmental mimicry Ag, the ABBOS epitope in BSA. This antigenic mimicry is thus driven by the endogenous self Ag, and not initiated by the environmental mimic. Insulitis, spontaneous, and adoptively transferred diabetes develop normally in ICA69(null) NOD congenics. Like glutamic acid decarboxylase 65, ICA69 is not an obligate autoantigen in diabetes. Unexpectedly, ICA69(null) NOD mice were resistant to cyclophosphamide (CY)-accelerated diabetes. Transplantation experiments with hemopoietic and islet tissue linked CY resistance to ICA69 deficiency in islets. CY-accelerated diabetes involves not only ablation of lymphoid cells, but ICA69-dependent drug toxicity in beta cells that boosts autoreactivity in the regenerating lymphoid system.

Adoptive Transfer↗