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Biomedical subjects

W J Craigen

Publications and source records attributed to W J Craigen.

At least 19 recordsLinked to original sources
PubMed2004

Serum leptin level is a regulator of bone mass.

Leptin is a powerful inhibitor of bone formation in vivo. This antiosteogenic function involves leptin binding to its receptors on ventromedial hypothalamic neurons, the autonomous nervous system and beta-adrenergic receptors on osteoblasts. However, the mechanisms whereby leptin controls the function of ventromedial hypothalamic antiosteogenic neurons remain unclear. In this study, we compared the ability of leptin to regulate body weight and bone mass and show that leptin antiosteogenic and anorexigenic functions are affected by similar amounts of leptin. Using a knock-in of LacZ in the leptin locus, we failed to detect any leptin synthesis in the central nervous system. However, increasing serum leptin level, even dramatically, reduced bone mass. Conversely, reducing serum-free leptin level by overexpressing a soluble receptor for leptin increased bone mass. Congruent with these results, the high bone mass of lipodystrophic mice could be corrected by restoring serum leptin level, suggesting that leptin is an adipocyte product both necessary and sufficient to control bone mass. Consistent with the high bone mass phenotype of lipodystrophic mice, we observed an advanced bone age, an indirect reflection of premature bone formation, in lipodystrophic patients. Taken together, these results indicate that adipocyte-derived circulating leptin is a determinant of bone formation and suggests that leptin antiosteogenic function is conserved in vertebrates.

Animals↗
PubMed2004

Interstitial deletion of 10p and atrial septal defect in DiGeorge 2 syndrome.

We present molecular genetic investigations of a 4-year-old boy with craniofacial dysmorphism and developmental delay. Trivial mitral and tricuspid regurgitation without gross structural abnormality was diagnosed by echocardiography. High-resolution chromosome analysis revealed an interstitial deletion, del(10)(p12.1p12.32). To characterize the deletion size and breakpoints, we performed fluorescence in situ hybridization analysis using 27 BAC clones. Our data demonstrate an approximately 5.5 Mb deletion del(10)(p12.1p12.31). Surprisingly, the BAC clone RP11-56H7 that contains NEBL, an apparent downstream gene of the cardiogenic transcription factor HAND2 previously shown to be deleted in the patients with DiGeorge 2 syndrome and 10p13 deletion, was deleted in our patient with 10p12.1-p12.31 deletion. In addition, we provide clinical data and results of molecular analysis for a patient with multiple congenital anomalies including Ebstein's anomaly, kidney malformations, and 10p13-p14 deletion. We also reviewed 19 patients with congenital heart defects and deletions involving 10p and propose that atrial septal defect (ASD) is a common cardiac anomaly associated with DiGeorge 2 syndrome. Based on genotype-phenotype analysis of published patients and those reported herein, we propose an approximately 1.0 Mb critical region between loci D10S547 and D10S2176 in 10p14 to be associated with ASD. Considering that septal defects are the most frequent congenital heart anomaly, we suggest that further investigations in the 10p critical region are important to identify gene(s) responsible for this common birth defect.

Child, Preschool↗
PubMed2002

PTPN11 mutations in Noonan syndrome type I: detection of recurrent mutations in exons 3 and 13.

We surveyed 16 subjects with the clinical diagnosis of Noonan Syndrome (NS1) from 12 families and their relevant family members for mutations in PTPN11/SHP2 using direct DNA sequencing. We found three different mutations among five families. Two unrelated subjects shared the same de novo missense substitution in exon 13 (S502T); an additional two unrelated families had a mutation in exon 3 (Y63C); and one subject had the amino acid substitution Y62D, also in exon 3. None of the three mutations were present in ethnically matched controls. In the mature protein model, the exon 3 mutants and the exon 13 mutant amino acids cluster at the interface between the N' SH2 domain and the phosphatase catalytic domain. Six of eight subjects with PTPN11/SHP2 mutations had pulmonary valve stenosis while no mutations were identified in those subjects (N = 4) with hypertrophic cardiomyopathy. An additional four subjects with possible Noonan syndrome were evaluated, but no mutations in PTPN11/SHP2 were identified. These results confirm that mutations in PTPN11/SHP2 underlie a common form of Noonan syndrome, and that the disease exhibits both allelic and locus heterogeneity. The observation of recurrent mutations supports the hypothesis that a special class of gain-of-function mutations in SHP2 give rise to Noonan syndrome.

Catalytic Domain↗
PubMed2001

Immotile sperm and infertility in mice lacking mitochondrial voltage-dependent anion channel type 3.

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are small channel proteins involved in the translocation of metabolites across the mitochondrial outer membrane. A single channel-forming protein is found in yeast, whereas higher eukaryotes express multiple VDACs, with humans and mice each harboring three distinct channels (VDAC1-3) encoded by separate genes. To begin to assess the functions of each of the three isoforms, the VDAC3 gene was inactivated by targeted disruption in embryonic stem cells. Here we show that mice lacking VDAC3 are healthy, but males are infertile. Although there are normal sperm numbers, the sperm exhibit markedly reduced motility. Structural defects were found in two-thirds of epididymal axonemes, with the most common abnormality being loss of a single microtubule doublet at a conserved position within the axoneme. In testicular sperm, the defect was only rarely observed, suggesting that instability of a normally formed axoneme occurs with sperm maturation. In contrast, tracheal epithelial cilia showed no structural abnormalities. In addition, skeletal muscle mitochondria were abnormally shaped, and activities of the respiratory chain complexes were reduced. These results demonstrate that axonemal defects may be caused by associated nonaxonemal components such as mitochondrial channels and illustrate that normal mitochondrial function is required for stability of the axoneme.

Animals↗
PubMed2000

Altered mitochondrial sensitivity for ADP and maintenance of creatine-stimulated respiration in oxidative striated muscles from VDAC1-deficient mice.

Voltage-dependent anion channels (VDACs) form the main pathway for metabolites across the mitochondrial outer membrane. The mouse vdac1 gene has been disrupted by gene targeting, and the resulting mutant mice have been examined for defects in muscle physiology. To test the hypothesis that VDAC1 constitutes a pathway for ADP translocation into mitochondria, the apparent mitochondrial sensitivity for ADP (Km(ADP)) and the calculated rate of respiration in the presence of the maximal ADP concentration (Vmax) have been assessed using skinned fibers prepared from two oxidative muscles (ventricle and soleus) and a glycolytic muscle (gastrocnemius) in control and vdac1(-/-) mice. We observed a significant increase in the apparent Km((ADP)) in heart and gastrocnemius, whereas the V(max) remained unchanged in both muscles. In contrast, a significant decrease in both the apparent Km((ADP)) and V(max) was observed in soleus. To test whether VDAC1 is required for creatine stimulation of mitochondrial respiration in oxidative muscles, the apparent Km((ADP)) and Vmax were determined in the presence of 25 mm creatine. The creatine effect on mitochondrial respiration was unchanged in both heart and soleus. These data, together with the significant increase in citrate synthase activity in heart, but not in soleus and gastrocnemius, suggest that distinct metabolic responses to altered mitochondrial outer membrane permeability occur in these different striated muscle types.

Adenosine Diphosphate↗
PubMed2000

Cloning and characterization of a putative human d-2-hydroxyacid dehydrogenase in chromosome 9q.

There is little information on d-isomer-specific dehydrogenases in humans. Identification of d-2-hydroxyglutaric aciduria, an inherited metabolic disorder associated with severe neurological dysfunction, highlights the role of d-isomers in human metabolism. The possibility of a defect in d-2-hydroxyglutarate dehydrogenation prompted us to employ E. coli d-2-hydroxyacid dehydrogenase cDNA to search the human expressed sequence tags database. Two human EST homologues were retrieved and sequenced. Analysis showed the two clones were identical with 1258 nucleotides encoding 248 amino acids of the putative human d-2-hydroxyacid dehydrogenase. It was highly homologous to bacterial d-2-hydroxyacid dehydrogenases (46%), d-phosphoglycerate dehydrogenase (38%), and formate dehydrogenase (36%) at the amino acid level. The gene is expressed ubiquitously in tissue, most abundantly in liver, and was mapped to chromosome 9q between markers WI-3028 and WI-93330. To our knowledge this is the first cloning and characterization of the cDNA for a human d-isomer specific NAD(+)-dependent 2-hydroxyacid dehydrogenase.

Alcohol Oxidoreductases↗
PubMed2000

The tissue-specific, alternatively spliced single ATG exon of the type 3 voltage-dependent anion channel gene does not create a truncated protein isoform in vivo.

Voltage-dependent anion channels (VDACs) are small, integral membrane proteins that traverse the outer mitochondrial membrane and conduct ATP and other small metabolites. They are known to bind several kinases of intermediary metabolism in a tissue-specific fashion, have been found in close association with the adenine nucleotide translocator of the inner mitochondrial membrane, and are hypothesized to form part of the mitochondrial permeability transition pore, which results in the release of cytochrome c at the onset of apoptotic cell death. VDACs are found throughout all strata of eukaryotic evolution and exhibit biophysical characteristics that are well conserved from yeast to mammals. The mammalian VDAC gene family consists of three isoforms, each of which shares approximately 70% sequence identity with the other two family members. Recently, we reported that a single codon (ATG) exon is alternatively spliced into the transcript of the type 3 voltage-dependent anion channel (VDAC3) in a tissue-specific fashion. This unusual splicing event was shown to be conserved between mouse and human, and we theorized that the spliced exon could lead to the creation of an alternative translational initiation site. Here we report that a highly specific polyclonal VDAC3 antibody was unable to detect the truncated protein isoform indicative of this putative downstream start site. From these in vivo studies, we conclude that the alternatively spliced exon results in the insertion of a single methionine residue at amino acid position 39 of the mature VDAC3 protein. Additionally, we have used a cross-species genomic sequence comparison to identify conserved regions that may be involved in the regulation of small exon splicing.

Alternative Splicing↗
PubMed2000

Partial COL1A2 gene duplication produces features of osteogenesis imperfecta and Ehlers-Danlos syndrome type VII.

Type I collagen is the most abundant structural protein in the mammalian body. It exists as a heterotrimer of two subunits in the form [alpha1(I)]2alpha2(I). Pathogenic mutations in COL1A1 and COL1A2, the genes that encode the two subunits, cause a range of phenotypes including mild to lethal forms of osteogenesis imperfecta and a restricted set of Ehlers-Danlos syndrome phenotypes. Lethal mutations usually result from missense mutations that disrupt the normal triple helical structure of the molecule. Multi-exon duplication or deletion in type I collagen genes has rarely been observed and has generally resulted in a lethal or severe phenotype. We report a partial duplication in the COLIA2 gene that causes a relatively mild phenotype, despite the addition of 477 amino acids to the triple helical domain of the proalpha2(I) chain. The abnormal molecule is synthesized and secreted by cultured dermal fibroblasts in a normal fashion. Electron microscopy of dermal tissue reveals small but otherwise near normal collagen fibrils. The gene duplication occurred by mitotic sister chromatid exchange in the mother who is mosaic for the duplication allele. Examination of the abnormal sequence suggests a means by which the duplicated molecule could be processed and properly incorporated into mature collagen fibrils.

Base Sequence↗
PubMed1999

Each mammalian mitochondrial outer membrane porin protein is dispensable: effects on cellular respiration.

Voltage-dependent anion channels (VDACs, also known as mitochondrial porins) are small pore-forming proteins of the mitochondrial outer membrane found in all eukaryotes. Mammals harbor three distinct VDAC isoforms, with each protein sharing 65-70% sequence identity. Deletion of the yeast VDAC1 gene leads to conditional lethality that can be partially or completely complemented by the mammalian VDAC genes. In vitro, VDACs conduct a variety of small metabolites and in vivo they serve as a binding site for several cytosolic kinases involved in intermediary metabolism, yet the specific physiologic role of each isoform is unknown. Here we show that mouse embryonic stem cells lacking each isoform are viable but exhibit a 30% reduction in oxygen consumption. VDAC1 and VDAC2 deficient cells exhibit reduced cytochrome c oxidase activity, whereas VDAC3 deficient cells have normal activity. These results indicate that VDACs are not essential for cell viability and we speculate that reduced respiration in part reflects decreased outer membrane permeability for small metabolites necessary for oxidative phosphorylation.

Animals↗
PubMed1999

Retrotransposition of glycerol kinase-related genes from the X chromosome to autosomes: functional and evolutionary aspects.

Glycerol kinase catalyzes the metabolism of endogenously derived and dietary glycerol. GyK is a member of a small group of kinases termed ambiquitous enzymes, which are found either in the cytosol or as membrane-bound complexes associated with the voltage-dependent anion channel of the mitochondrial outer membrane. In Homo sapiens, the GyK gene family consists of an X-encoded locus and several X-linked and autosomal intronless retroposons, which, apparently, comprise both functional genes and processed pseudogenes. To study the role of the autosomal genes in mammalian physiology, we have isolated two murine GyK-like genes, determined their structures and chromosomal locations, and examined their functions. These sequences are intronless retroposons, which appear to be paralogues of the X-encoded, brain-specific GyK isoform and are expressed only in the testes. Though both retrotransposition events appear to have occurred prior to the primate-rodent divergence of some 65-80 million years ago, only one of the retrotransposed murine gene sequences, based upon its chromosomal location, is conserved with modern H. sapiens. To test the hypothesis that the murine GyK-like genes encode functional GyK activity, transient transfection of the gene sequences into COS7 cells was carried out. While in vitro translation confirmed that the transcripts could direct the synthesis of proteins of the appropriate size, no GyK activity was detected. Such data suggest that the autosomal GyK-like genes have evolved novel, testis-specific functions. A comparison of the human and mouse GyK-like gene sequences demonstrates the evolutionary relationships between each autosomal isoform and its corresponding X-linked ancestral locus.

Amino Acid Sequence↗
PubMed1999

Mouse VDAC isoforms expressed in yeast: channel properties and their roles in mitochondrial outer membrane permeability.

The channel-forming protein called VDAC forms the major pathway in the mitochondrial outer membrane and controls metabolite flux across that membrane. The different VDAC isoforms of a species may play different roles in the regulation of mitochondrial functions. The mouse has three VDAC isoforms (VDAC1, VDAC2 and VDAC3). These proteins and different versions of VDAC3 were expressed in yeast cells (S. cerevisiae) missing the major yeast VDAC gene and studied using different approaches. When reconstituted into liposomes, each isoform induced a permeability in the liposomes with a similar molecular weight cutoff (between 3,400 and 6,800 daltons based on permeability to polyethylene glycol). In contrast, electrophysiological studies on purified proteins showed very different channel properties. VDAC1 is the prototypic version whose properties are highly conserved among other species. VDAC2 also has normal gating activity but may exist in 2 forms, one with a lower conductance and selectivity. VDAC3 can also form channels in planar phospholipid membranes. It does not insert readily into membranes and generally does not gate well even at high membrane potentials (up to 80 mV). Isolated mitochondria exhibit large differences in their outer membrane permeability to NADH depending on which of the mouse VDAC proteins was expressed. These differences in permeability could not simply be attributed to different amounts of each protein present in the isolated mitochondria. The roles of these different VDAC proteins are discussed.

Animals↗
PubMed1999

D-2-Hydroxyglutaric aciduria: biochemical marker or clinical disease entity?

D-2-Hydroxyglutaric aciduria has been observed in patients with extremely variable clinical symptoms, creating doubt about the existence of a disease entity related to the biochemical finding. An international survey of patients with D-2-hydroxyglutaric aciduria was initiated to solve this issue. The clinical history, neuroimaging, and biochemical findings of 17 patients were studied. Ten of the patients had a severe early-infantile-onset encephalopathy characterized by epilepsy, hypotonia, cerebral visual failure, and little development. Five of these patients had a cardiomyopathy. In neuroimaging, all patients had a mild ventriculomegaly, often enlarged frontal subarachnoid spaces and subdural effusions, and always signs of delayed cerebral maturation. In all patients who underwent neuroimaging before 6 months, subependymal cysts over the head or corpus of the caudate nucleus were noted. Seven patients had a much milder and variable clinical picture, most often characterized by mental retardation, hypotonia, and macrocephaly, but sometimes no related clinical problems. Neuroimaging findings in 3 patients variably showed delayed cerebral maturation, ventriculomegaly, or subependymal cysts. Biochemical findings included elevations of D-2-hydroxyglutaric acid in urine, plasma, and cerebrospinal fluid in both groups. Cerebrospinal fluid gamma-aminobutyric acid was elevated in almost all patients investigated. Urinary citric acid cycle intermediates were variably elevated. The conclusion of the study is that D-2-hydroxyglutaric aciduria is a distinct neurometabolic disorder with at least two phenotypes.

Biomarkers↗
PubMed1998

A novel isoform of the mitochondrial outer membrane protein VDAC3 via alternative splicing of a 3-base exon. Functional characteristics and subcellular localization.

Voltage-dependent anion channels (VDACs) are pore-forming proteins found in the outer mitochondrial membrane of all eucaryotes. VDACs are the major pathway for metabolites through the outer mitochondrial membrane and, in mammals, bind several cytosolic carbohydrate kinases. Whereas yeast contain a single VDAC (YVDAC), to date three isoforms have been described in the mouse that constitute a gene family. We have observed an additional isoform of VDAC3 that appears to be generated via the tissue-specific alternative splicing of a 3-base exon (ATG). The exon is predicted to introduce a methionine 39 amino acids downstream of the amino terminus of the polypeptide. Between exons 3 and 4 is an intronic sequence that potentially encodes the exon, with flanking splice enhancer elements. Expression of this alternative form in the mouse is limited to brain, heart, and skeletal muscle. Complementation of YVDAC-deficient yeast by the two isoforms and with other sequence variants of VDAC3 suggests this residue is an important modulator of VDAC3 function. In transfected mammalian cells both isoforms localize to mitochondria. A similar variant is present in humans.

3T3 Cells↗