Uterus didelphys demonstrated with echo-planar magnetic resonance imaging.
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Adult↗
Biomedical subjects
V Adams
Publications and source records attributed to V Adams.
Evaluation of methods for hepatitis C virus detection in archival liver biopsies. Comparison of histology, immunohistochemistry, in-situ hybridization, reverse transcriptase polymerase chain reaction (RT-PCR) and in-situ RT-PCR.
To evaluate reliable methods for detection of hepatitis C virus (HCV) infection in routinely processed liver biopsies we analyzed formaldehyde-fixed and paraffin-embedded liver specimens of 10 patients with serological confirmed HCV infection. We compared (1) conventional histology; (2) indirect immunofluorescence using the mAb TORDJI-22 (Clonatec, Paris, France); (3) RT-PCR using total RNA and Southern blotting with chemiluminescent detection; (4) non-radioactive in-situ hybridization (ISH) with digoxigenin-labeled oligo- and cRNA probes; (5) direct in-situ RT-PCR with incorporation of labeled nucleotides into PCR-products, and (6) indirect in-situ RT-PCR using subsequent ISH for the visualization of intracellular PCR-products. Our results indicate that: (1) using the histological criteria described by Lefkowitch et al. [Gastroenerology 1993;104:595] together with clinical data, most chronic HCV infections can be diagnosed by conventional histology, if liver biopsies specimens are adequate; (2) the commercially available mAb TORDJI-22 appears to crossreact with non-HCV epitopes, resulting in false positives; (3) molecular methods performed on routinely fixed and processed liver biopsies frequently yield false negative results due to sampling problems, low viral copy number and RNA degradation in infected cells; (4) analysis of HCV-RNA by RT-PCR of extracted total RNA is more sensitive than indirect in-situ RT-PCR or ISH; and (5) direct in-situ RT-PCR is not reliable despite the use of modifications such as DNase pretreatment and hot-start procedures. It is concluded, that several molecular methods for HCV detection must await further improvements of protocols to be suitable for routine diagnostics on paraffin-embedded liver biopsies.
Base Sequence↗
Estimation of fetal lung volume using echo-planar magnetic resonance imaging.
OBJECTIVE: To measure fetal lung volume by echo-planar magnetic resonance imaging (MRI). METHODS: Twenty singleton pregnancies were scanned on one occasion each using echo-planar imaging, a form of MRI. Cases of fetal growth restriction and macrosomia were excluded from the study. RESULTS: Lung volumes increased with gestational age from 21 mL at 23 weeks' gestation to a maximum of 94 mL at term. The relation between lung volume and gestational age was exponential. The ratio of lung volume to total fetal volume decreased with gestational age. CONCLUSION: The use of echo-planar imaging allows estimation of normal fetal growth; it has clear applications for the diagnosis of lung hypoplasia and potential use for the study of lung maturity.
Echo-Planar Imaging↗
[Comparison of histology, immunohistochemistry, RT-PCR, in situ hybridization, and in situ RT-PCR for demonstration of hepatitis C virus in paraffin-embedded liver biopsies].
To compare immunohistochemical and molecular methods for the detection of hepatitis C virus (HCV) infection in archival liver biopsies we analyzed formalin-fixed and paraffin-embedded liver specimens of 10 patients with serologically confirmed HCV infection. Methods employed included indirect FITC-immunofluorescence, reverse-transcriptase polymerase chain reaction (RT-PCR) using extracted RNA and Southern blotting with chemiluminescence-based detection, non-radioactive in situ hybridization (ISH) with digoxigenin-labeled oligo- and cRNA probes, direct in situ RT-PCR with incorporation of labeled nucleotides into PCR-products, and indirect in situ RT-PCR using subsequent ISH for the visualization of intracellular PCR-products. Our results indicate that: (1) using the histological criteria described by Lefkowitch et al. (Gastroenterology 1993; 104-595) together with clinical data, most chronic HCV infections can be diagnosed by conventional histology, if liver biopsies are representative; (2) the commercially available mAB TORDJI-22 appears to cross-react with non-HCV epitopes; (3) molecular methods performed on routinely fixed and processed liver biopsies frequently yield false negative results due to sampling problems, low viral copy number and RNA degradation in infected cells; (4) analysis of HCV-RNA by RT-PCR of extracted total RNA is more sensitive than indirect in situ RT-PCR or ISH; and (5) direct in situ RT-PCR is not reliable despite the use of modifications such as DNase pretreatment and hot-start procedures. Further studies are required to define both optimal methods for sample processing and improvements of protocols, in order to increase detection sensitivity and specificity of HCV infection by immunohistochemical and molecular methods.
Biopsy↗
[Detection of human herpesvirus type 6, human herpesvirus type 7, cytomegalovirus and human papillomavirus in cutaneous AIDS-associated Kaposi's sarcoma].
In order to evaluate a possible role of viral infections in the pathogenesis of AIDS-associated Kaposi's sarcoma (KS), we investigated 26 cutaneous AIDS-associated KS by polymerase chain reaction (PCR), in situ hybridization, and immunohistochemistry. By PCR we detected human papilloma viruses (HPV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), and for the first time human herpesvirus 7 (HHV-7) in the KS. The prevalence of HPV, HHV-6, and HHV-7 was similar to or lower in KS than in normal skin tissues of AIDS patients without KS, but higher than in normal skin of HIV-seronegative patients. All HHV-6 found in KS were identified as HHV-6 variant B. In addition to the known HPV types 16 and 18 described in KS, we also found HPV types 6 and 33 in KS specimen. By immunohistochemistry HHV-6 could be localized in macrophages in KS, in the adjacent stroma as well as in normal skin of control cases. In situ hybridization for CMV and HPV gave negative results in KS and controls.
Acquired Immunodeficiency Syndrome↗
Cloning and functional expression in yeast of two human isoforms of the outer mitochondrial membrane channel, the voltage-dependent anion channel.
The voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane is a small abundant protein found in all eukaryotic kingdoms which forms a voltage-gated pore when incorporated into planar lipid bilayers. VDAC is also the site of binding of the metabolic enzymes hexokinase and glycerol kinase to the mitochondrion in what may be a significant metabolic regulatory interaction. Recently, there has been speculation that there may be multiple forms of VDAC in mammals which differ in their localization in the outer mitochondrial membrane and in their physiological function. In this report, we describe the identification and characterization of two human cDNAs encoding VDAC homologs (HVDAC1 and HVDAC2). To confirm VDAC function, each human protein has been expressed in yeast lacking the endogenous VDAC gene. Human proteins isolated from yeast mitochondria formed channels with the characteristics expected of VDAC when incorporated into planar lipid bilayers. In addition, expression of the human proteins in such strains can complement phenotypic defects associated with elimination of the endogenous yeast VDAC gene. Since VDAC is the site of binding of hexokinase to the outer mitochondrial membrane, the binding capacity of each VDAC isoform expressed in yeast mitochondria was assessed. When compared with the binding of hexokinase to mitochondria lacking VDAC, the results show that mitochondria expressing HVDAC1 are capable of specifically binding hexokinase, whereas mitochondria expressing HVDAC2 only bind hexokinase at background levels. The expression of each human cDNA has been assessed by Northern blot and polymerase chain reaction techniques. With one exception, each is expressed in all human cell lines and tissues examined.
Amino Acid Sequence↗
Yeast artificial chromosome cloning in the glycerol kinase and adrenal hypoplasia congenita region of Xp21.
The adrenal hypoplasia congenita (AHC) and glycerol kinase (GK) loci are telomeric to the Duchenne muscular dystrophy locus in Xp21. We developed a pair of yeast artificial chromosome (YAC) contigs spanning at least 1.2 Mb and encompassing the region from the telomeric end of the Duchenne muscular dystrophy (DMD) locus to beyond YHX39 (DXS727), including the genes for AHC and GK. The centromeric contig consists of 13 YACs reaching more than 600 kb from DMD through GK. The telomeric contig group consists of 8 YACs containing more than 600 kb including the markers YHX39 (DXS727) and QST-59 (DXS319). Patient deletion breakpoints in the region of the two YAC contigs define at least eight intervals, and seven deletion breakpoints are contained within these contigs. In addition to the probes developed from YAC ends, we have mapped eight Alu-PCR probes amplified from a radiation-reduced somatic cell hybrid, two anonymous DNA probes, and one Alu-PCR product amplified from a cosmid end, for a total of 26 new markers within this region of 2 Mb or less. One YAC in the centromeric contig contains an insert encompassing the minimum interval for GK deficiency defined by patient deletion breakpoints, and this clone includes all or part of the GK gene.
Adrenal Insufficiency↗
Genomic scanning for expressed sequences in Xp21 identifies the glycerol kinase gene.
Rapid genomic scanning methods are required to identify expressed sequences and we report an efficient, sensitive and specific approach which relies upon hybridization of an amplified, labeled cDNA library to digested cosmid DNA. We identified expressed sequences within a cosmid in the glycerol kinase (GK) "critical region" of Xp21 that had impressive similarity to prokaryotic GKs. We used this genomic sequence information to clone the human hepatic GK cDNA. Independent confirmation of the identity of this gene was obtained by functional complementation of GK deficient E. coli mutants with a construct containing the complete human X-linked GK coding sequence.
Amino Acid Sequence↗
Effect of chronic pre- and post-natal low-dose ethanol exposure on brain enolase isoenzyme activities.
Sprague-Dawley dams were treated with 3 v/v % ethanol in liquid diet from the 8th day gestation through 3 weeks nursing period. Offsprings, ages 1, 2 and 3 weeks, and their dams were studied. Brain weights and their total proteins were not affected by this ethanol treatment. Total enolase activity/mg protein and its isoenzymes, i.e. non-neuron-specific, hybrid and neuron-specific, (mumol/min/mg protein) in 100,000 g supernate, were significantly lower in the treated. Additionally, enolase isoenzyme transformation was delayed.
Animals↗
Developmental expression of hexokinase 1 in the rat.
Hexokinase 1 (HK1) activity varies in a developmental stage- and tissue-specific manner and is a key step in glucose homeostasis and energy metabolism. We conducted studies to determine if HK1 expression is regulated at the transcriptional level. Expression of HK1 was examined in selected pre- and postnatal rat tissues using Northern blot analyses and RNAase protection assays. We found that brain and kidney exhibited significantly higher expression than heart, lung, spleen and skeletal muscle. Brain HK1 expression was constant prenatally, peaked at 7 days of age and reached a constant level after weaning. In kidney, HK1 expression was essentially constant or perhaps gradually decreased after weaning. HK1 transcription in heart, skeletal muscle and liver was higher during fetal stages than postnatally. Lung expression was essentially constant except in the adult. HK1 mRNA levels were compared to phosphoglycerate kinase (PGK) mRNA. PGK steady state mRNA levels were relatively constant in all tissues and developmental stages, except in skeletal muscle where there was a postnatal rise. The developmental profiles of HK1 and PGK mRNA expression are not consistent with enzyme activity measurements in all the tissues examined. We conclude that regulation of HK1 expression involves both transcriptional and posttranscriptional mechanisms that are tissue and stage specific.
Animals↗
Targeting of hexokinase 1 to liver and hepatoma mitochondria.
The proportion of hexokinase (HK; EC 2.7.1.1) isozyme 1 (HK1) that is bound to the outer mitochondrial membrane is tissue specific and developmentally regulated. HK activity is known to be markedly elevated in many cancer cells and a significant fraction is mitochondrial bound. This study examined the role of the 15-amino acid N-terminal domain of HK1 in binding to liver and hepatoma mitochondria. A chimeric reporter construct, pCMVHKCAT, encoding this HK1 domain coupled to the chloramphenicol acetyltransferase (CAT) gene was electroporated into mouse Hepa 1-6 hepatoma cells. After digitonin treatment, cell fractions were assayed for HK, lactate dehydrogenase, and CAT activities. Digitonin (75 micrograms/mg of protein) caused cytosolic leak but 70% of HK remained with the pellet. HKCAT, like HK, remained predominantly with the pellet; CAT form the control, pCMVCAT, remained mostly unbound. Binding of membrane-free cell extracts to rat liver mitochondria in vitro showed 91% of the HKCAT bound, whereas only 12% of CAT bound. Specificity of HKCAT binding to mitochondria was demonstrated by competition of HK1 for HKCAT binding sites on rat liver mitochondria as well as by blockage of HKCAT binding by N,N'-dicyclohexylcarbodiimide, which covalently binds to porin and blocks HK1 binding. Deletional mutant constructs of HKCAT showed reduced binding with increasing deletion size. In summary, these studies demonstrate that the 15-amino acid N-terminal domain of HK1 is necessary and sufficient to confer mitochondrial binding properties to CAT and that there is specificity for this binding to the mitochondria.
Animals↗
Characterization of human genomic yeast artificial chromosome inserts containing hexokinase 1 coding information on chromosome 10.
Hexokinase 1 (HK1) is one of four mammalian HK isoenzymes and maps to human chromosome 10. Two yeast artificial chromosomes (YACs) were identified in the Washington University human YAC library using polymerase chain reaction (PCR) primers designed with knowledge of the human HK1 cDNA sequence. YAC B129B12 is 120 kb in length and maps entirely to chromosome 10. YAC A159D5 is 400 kb in length and appears to have resulted from a recombination of chromosome 10 with non-chromosome 10 material. We report these YACs as potential resources for those interested in HK1 gene organization and mapping, as well as those desiring additional genomic information and markers on chromosome 10.
Animals↗
Protein kinase activity of rat brain hexokinase.
Hexokinase 1 (HK1) purified from rat brain exhibits protein kinase activity, including autophosphorylation and phosphorylation of histone H2A. This protein kinase activity is observed only in the absence of the HK1 carbohydrate substrate, glucose. Analysis of the ATP-binding domains of the mammalian HK1 protein sequences shows significant homology with other mammalian protein kinases.
Amino Acid Sequence↗
Localization of the ATP/ADP translocator in the inner membrane and regulation of contact sites between mitochondrial envelope membranes by ADP. A study on freeze-fractured isolated liver mitochondria.
The frequency of contacts between the mitochondrial envelope membranes was determined in freeze-fractured samples of isolated mitochondria by means of quantifying the frequency of fracture plane deflections between the two membranes. It was observed that the formation of contacts correlated with the concentration of free ADP despite of inhibition of electron transport by antimycin A. The activity of ATPase partially inhibited by oligomycin or depletion of membrane potential by K+ and valinomycin had no effect on the induction of the contacts by ADP. ATP was ineffective in creating contacts irrespective of the presence or absence of a membrane potential, whereas carboxyatractyloside induced the contacts under all conditions in a manner similar to ADP. These results suggest the involvement of the ATP/ADP translocator in regulation of contact sites. As a consequence, we analyzed its distribution in the inner membrane of kidney and liver mitochondria by binding of [3H]atractyloside to subfractions of this membrane. The experiments demonstrated that the translocator was located in the peripheral part of the inner membrane as well as in the portion which formed the cristae.
Adenosine Diphosphate↗
Location and regulation of octameric mitochondrial creatine kinase in the contact sites.
Differential extraction of creatine kinase activity (CK, EC 2.7.3.2) from rat brain mitochondria by graded concentrations of digitonin all yielded supernates varying in CK activity. As analyzed by isozyme electrophoresis and gel permeation chromatography the extracts contained different species of creatine kinase: (i) one third of the total CK activity consisting of contaminating cytosolic brain-type CK (B-CK) was liberated by 100 micrograms digitonin/mg of mitochondrial protein, (ii) approx. 20% more CK activity consisting of B-CK, as above, plus dimeric and octameric mitochondrial CK (Mi-CK), was extracted by 300 micrograms/mg digitonin, whereas (iii) all CK activity, consisting of B-CK and mainly octameric Mi-CK, were liberated by 700 micrograms/mg digitonin. In contrast to Mi-CK, B-CK associated with contaminating synaptic vesicles was readily extracted even by low concentrations of digitonin, but on the other hand octameric Mi-CK was significantly more resistant to digitonin extraction than the dimeric enzyme species. It appeared that the Mi-CK resistant to treatment with 300 micrograms/mg digitonin consisted to a large percentage of octamers and was organized as a complex between the two envelope membranes, for its activity was latent and still remained regulated by the outer membrane pore, that is: (i) the Mi-CK activity in such mitoplasts could be inhibited reversibly by cessation of the adenine nucleotide transport through the outer membrane pore with a polyanion, (ii) the ADP produced by Mi-CK in mitoplasts was not available to external pyruvate kinase, (iii) approx. 50% of total CK activity was not susceptible to inhibition by iodo acetate and phosphocreatine. In agreement with these findings a preferential association of octameric Mi-CK was also found in isolated contact site fractions indicating a physiological role of Mi-CK in energy transfer and a structure-function relationship of Mi-CK octamers at these sites. In addition some evidence for an interaction of Mi-CK with the adenylate translocator is presented.
Adenosine Diphosphate↗
Porin interaction with hexokinase and glycerol kinase: metabolic microcompartmentation at the outer mitochondrial membrane.
Porin is the pore-forming protein involved in the movement of adenine nucleotides across the outer mitochondrial membrane (OMM). Hexokinase and glycerol kinase interact with porin on the outer surface of the OMM in a manner which provides these enzymes with preferred access to the ATP generated in the mitochondrion. We review recent evidence which permits refinement of our knowledge of these proteins and their interactions at the OMM. The involvement of this system in metabolic microcompartmentation is discussed, as well as possible pathological consequences of its disruption in malignancy and genetic deficiencies of hexokinase, glycerol kinase, and porin.
Adenine Nucleotides↗
Mammalian hexokinase 1: evolutionary conservation and structure to function analysis.
We have amplified and sequenced the complete coding region of bovine hexokinase isoenzyme 1 (HK1) from brain RNA with PCR primers selected for sequence conservation. The sequence information was analyzed to evaluate the evolutionary and structure-function relationships among the mammalian and yeast HK isoenzymes. Structure to function analysis identified an unduplicated, invariant N-terminal domain involved in HK1 outer mitochondrial membrane targeting, as well as putative carbohydrate and nucleotide-binding sites in the regulatory and catalytic halves of HK1 essential to enzyme function. The ATP-binding site in the catalytic half of the HK1 protein resembles nucleotide-binding regions from protein kinases, with the single amino acid replacement (lysine to glutamate) in the ATP-binding site of the amino half explaining the loss of HK1 catalytic function in the regulatory domain. Sequence comparisons suggest that the 50-kDa mammalian and yeast glucokinases arose separately in evolution. In addition to providing valuable phylogenetic and structure-function insights, this work provides an efficient strategy for rapid cloning and sequencing of the coding regions for other HKs and related proteins.
Amino Acid Sequence↗
Characterization and metabolic function of mitochondrial contact sites.
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Adenosine Diphosphate↗