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1.
Int J Tuberc Lung Dis ; 17(7): 922-7, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23743311

ABSTRACT

BACKGROUND: Active TB disease can destroy lung parenchyma leading to cavities. Immune responses that predispose or protect individuals from lung damage during TB are poorly defined. OBJECTIVE: To sample lung immune cells and assay bronchoalveolar lavage (BAL) cell cytokine production. DESIGN: Enrolled subjects (n = 73) had bilateral infiltrates and underwent BAL. RESULTS: All had sputum culture demonstrating Mycobacterium tuberculosis and 22/73 (30%) had cavities on their chest radiograph. Those with cavities at presentation had a higher percentage of polymorphonuclear neutrophils (PMN) in BAL as well as lower inducible protein (IP) 10 (P < 0.01) and interleukin (IL) 6 (P = 0.013) in BAL cell supernatants compared to those without cavities. There was no correlation between cavities and other BAL or serum cytokines. IP-10 was negatively associated with BAL PMN. IP-10 and IL-6 expression above median reduces the odds of cavities by 79% and 78% in logistic regression models. IP-10 and IL-6 clustered with interferon-gamma and tumour necrosis factor-alpha in a principal component analysis, while IL-4 clustered with PMN. CONCLUSION: Increasing IP-10 and IL-6 production by BAL cells is associated with non-cavitary TB in patients who present with radiographically advanced TB. IP-10 and IL-6 may reflect an effective T-helper 1 immune control pathway for TB, attenuating tuberculous lung destruction.


Subject(s)
Chemokine CXCL10/metabolism , Interleukin-6/metabolism , Mycobacterium tuberculosis/isolation & purification , Tuberculosis, Pulmonary/physiopathology , Adult , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/microbiology , Case-Control Studies , Cross-Sectional Studies , Female , Humans , Logistic Models , Male , Middle Aged , Neutrophils/microbiology , Principal Component Analysis , Radiography , Sputum/microbiology , Th1 Cells , Tuberculosis, Pulmonary/diagnostic imaging , Tuberculosis, Pulmonary/microbiology , Young Adult
2.
Cases J ; 2: 211, 2009 Nov 19.
Article in English | MEDLINE | ID: mdl-19946455

ABSTRACT

BACKGROUND: Orthodontic management of an ectopically erupted canine requires substantial amount of bodily movement which is difficult to perform and often results in root resorption. CASE PRESENTATION: A 9 year old girl presented with Class I malocclusion with crowding and ectopically positioned upper left canine (23). Treatment involved extraction of all first premolars. The alignment of the upper left canine was achieved by the modified 2 by 1 appliance. CONCLUSION: The design of this modified 2 by 1 appliance allows individualized bodily tooth movement of canine, it provides light and continuous force for physiological orthodontic movement with minimal root resorption.

3.
Int J Tuberc Lung Dis ; 13(6): 731-6, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19460249

ABSTRACT

SETTING: Recent reports indicate a role of chemokine inducible protein 10 (IP-10) in Mycobacterium tuberculosis infection substantiated by the detection of elevated levels in plasma and at infection foci in individuals infected with M. tuberculosis. OBJECTIVE: To evaluate IP-10 as a potential marker for the diagnosis of M. tuberculosis infection in children living in a region of low tuberculosis (TB) prevalence. DESIGN: IP-10 levels were obtained after whole blood stimulation with M. tuberculosis-specific antigens in 127 children. IP-10 results were evaluated upon gradations of exposure risk to M. tuberculosis and correlation with tuberculin skin test and an interferon-gamma release assay (IGRA). RESULTS: IP-10 reactivity correlated well to risk of exposure to M. tuberculosis in children. There was a strong correlation between IP-10 and IGRA results. IP-10 responses, unlike interferon-gamma (IFN-gamma), were not age-dependent and detected more positive results in children aged <5 years. In the children with active disease, the IGRA was more sensitive than IP-10 at detecting M. tuberculosis infection. CONCLUSION: Our findings suggest that IP-10 in combination with IFN-gamma may enhance the diagnostic performance of IGRAs in detecting M. tuberculosis infection, especially in young children.


Subject(s)
Biomarkers/blood , Chemokine CXCL10/blood , Tuberculosis/blood , Tuberculosis/diagnosis , Adolescent , Child , Child, Preschool , Female , Humans , Male , Mycobacterium tuberculosis/immunology , New York City , Reagent Kits, Diagnostic , Risk Factors , Sensitivity and Specificity , Tuberculosis/microbiology
4.
Proc Natl Acad Sci U S A ; 99(21): 13642-6, 2002 Oct 15.
Article in English | MEDLINE | ID: mdl-12370436

ABSTRACT

The exponential expansion of the publicly available human DNA sequence database has increasingly facilitated cloning by homology of genes for biochemically defined, functionally similar proteins. We hypothesized that an as-yet uncloned human alpha-glucosidase (human neutral alpha-glucosidase C or GANC) is a previously uncharacterized member of a paralogous human glycosyl hydrolase gene family 31, sharing sequence homology and related, but not identical, functions with other cloned human alpha-glucosidases. We now report both the in silico and physical cloning of two alleles of human neutral alpha-glucosidase (designated GANC on the human gene map). This cloning and correct identification and annotation as GANC was successful only because of the application of the biochemical and genetic information we had previously developed regarding this gene to the results of the in silico method. Of note, this glucosidase, a member of family 31 glycosyl hydrolases, has multiple alleles, including a "null" allele and is potentially significant because it is involved in glycogen metabolism and localizes to a chromosomal region (15q15) reported to confer susceptibility to diabetes.


Subject(s)
alpha-Glucosidases/genetics , Alleles , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 15/genetics , Cloning, Molecular , Computers , DNA, Complementary/genetics , Humans , Molecular Sequence Data , Multigene Family , RNA, Messenger/genetics , Sequence Homology, Amino Acid
5.
Pediatr Dent ; 23(5): 390-3, 2001.
Article in English | MEDLINE | ID: mdl-11699159

ABSTRACT

PURPOSE: The purpose of this study was to report any differences found among the mean percentages of procedures performed by three types of dental providers for each type of service performed. The study focused on the types of services provided by dentists to Medicaid children in Virginia. METHODS: Medicaid claims field for dental patients younger than age 21 were obtained and analyzed for fiscal years 1994 and 1995. Dental providers were categorized according to their practice: general practice (GP), pediatric dentist (PD) and public health dentist (PH). Each type of practitioner (GP, PD, and PH) was evaluated for percentages of diagnostic, preventive, and corrective services provided to their Medicaid patients. The preventive category was subdivided into preventive services (scaling, prophy, fluoride and oral hygiene instruction) and sealant services. RESULTS: For each type of service, the mean percentages of procedures performed were compared among the three types of dental providers. The evaluation of the diagnostic procedure variable resulted in the finding that GP practitioners performed a significantly greater percentage of diagnostic procedures to their Medicaid patients than do PD and PH dentists (p < 0.0001). The percentage of preventive procedures performed by PD and GP dentists was not significantly different but was significantly lower than those performed by PH dentists (p < 0.0001). Finally, PD dentists performed a significantly greater percentage of corrective procedures than both GP and PH dentists (p > 0.0037). CONCLUSION: Differences were found among the mean percentages of procedures performed by the three types of dental providers for each type of service performed.


Subject(s)
General Practice, Dental/statistics & numerical data , Insurance, Dental/statistics & numerical data , Medicaid/statistics & numerical data , Pediatric Dentistry/statistics & numerical data , Public Health Dentistry/statistics & numerical data , Adolescent , Adult , Analysis of Variance , Child , Child, Preschool , Dental Prophylaxis/statistics & numerical data , Dentistry, Operative/statistics & numerical data , Fluorides, Topical , Humans , Infant , Insurance Claim Review , Oral Surgical Procedures/statistics & numerical data , Pit and Fissure Sealants , Prosthodontics/statistics & numerical data , Root Canal Therapy/statistics & numerical data , Statistics, Nonparametric , Virginia
6.
J Immunol ; 167(4): 2142-50, 2001 Aug 15.
Article in English | MEDLINE | ID: mdl-11489998

ABSTRACT

We recently described the incidence of a SCID disease in a litter of Jack Russell terriers. In this study, we show that the molecular defect in these animals is faulty V(D)J recombination. Furthermore, we document a complete deficit in DNA-dependent protein kinase activity that can be explained by a marked diminution in the expression of the catalytic subunit DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We conclude that as is the case in C.B-17 SCID mice and in Arabian SCID foals, the defective factor in these SCID puppies is DNA-PKcs. In mice, it has been clearly established that DNA-PKcs deficiency produces an incomplete block in V(D)J recombination, resulting in "leaky" coding joint formation and only a modest defect in signal end ligation. In contrast, DNA-PKcs deficiency in horses profoundly blocks both coding and signal end joining. Here, we show that although DNA-PKcs deficiency in canine lymphocytes results in a block in both coding and signal end joining, the deficit in both is intermediate between that seen in SCID mice and SCID foals. These data demonstrate significant species variation in the absolute necessity for DNA-PKcs during V(D)J recombination. Furthermore, the severity of the V(D)J recombination deficits in these three examples of genetic DNA-PKcs deficiency inversely correlates with the relative DNA-PK enzymatic activity expressed in normal fibroblasts derived from these three species.


Subject(s)
Catalytic Domain/genetics , DNA-Binding Proteins , Disease Models, Animal , Dog Diseases/enzymology , Dog Diseases/genetics , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , Severe Combined Immunodeficiency/enzymology , Severe Combined Immunodeficiency/genetics , Alleles , Animals , Base Sequence , Cell Line , DNA-Activated Protein Kinase , Dog Diseases/immunology , Dogs , Fibroblasts/immunology , Fibroblasts/radiation effects , Gene Expression Regulation/immunology , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , Humans , Mice , Mice, Inbred BALB C , Mice, SCID , Molecular Sequence Data , Nuclear Proteins , Phenotype , Polymorphism, Single Nucleotide , Protein Serine-Threonine Kinases/biosynthesis , Purine Nucleotides/genetics , Purine Nucleotides/metabolism , Radiation Tolerance , Receptors, Antigen, T-Cell, alpha-beta/chemistry , Recombination, Genetic/immunology , Recombination, Genetic/radiation effects , Severe Combined Immunodeficiency/veterinary , T-Lymphocyte Subsets/chemistry , T-Lymphocyte Subsets/enzymology
7.
Hum Gene Ther ; 12(5): 527-38, 2001 Mar 20.
Article in English | MEDLINE | ID: mdl-11268285

ABSTRACT

Pompe disease is a lethal cardioskeletal myopathy in infants and results from genetic deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). Genetic replacement of the cDNA for human GAA (hGAA) is one potential therapeutic approach. Three months after a single intramuscular injection of 10(8) plaque-forming units (PFU) of E1-deleted adenovirus encoding human GAA (Ad-hGAA), the activity in whole muscle lysates of immunodeficient mice is increased to 20 times the native level. Direct transduction of a target muscle, however, may not correct all deficient cells. Therefore, the amount of enzyme that can be transferred to deficient cells from virally transduced cells was studied. Fibroblasts from an affected patient were transduced with AdhGAA, washed, and plated on transwell culture dishes to serve as donors of recombinant enzyme. Deficient fibroblasts were plated as acceptor cells, and were separated from the donor monolayer by a 22-microm pore size filter. Enzymatic and Western analyses demonstrate secretion of the 110-kDa precursor form of hGAA from the donor cells into the culture medium. This recombinant, 110-kDa species reaches the acceptor cells, where it can be taken up by mannose 6-phosphate receptor-mediated endocytosis. It then trafficks to lysosomes, where Western analysis shows proteolytic processing to the 76- and 70-kDa lysosomal forms of the enzyme. Patient fibroblasts receiving recombinant hGAA by this transfer mechanism reach levels of enzyme activity that are comparable to normal human fibroblasts. Skeletal muscle cell cultures from an affected patient were also transduced with Ad-hGAA. Recombinant hGAA is identified in a lysosomal location in these muscle cells by immunocytochemistry, and enzyme activity is transferred to deficient skeletal muscle cells grown in coculture. Transfer of the precursor protein between muscle cells again occurs via mannose 6-phosphate receptors, as evidenced by competitive inhibition with 5 mM mannose 6-phosphate. In vivo studies in GAA-knockout mice demonstrate that hepatic transduction with adenovirus encoding either murine or human GAA can provide a depot of recombinant enzyme that is available to heart and skeletal muscle through this mechanism. Taken together, these data show that the mannose 6-phosphate receptor pathway provides a useful strategy for cell-to-cell distribution of virally derived recombinant GAA.


Subject(s)
Gene Transfer Techniques , Genetic Therapy/methods , Glycogen Storage Disease Type II/genetics , Glycogen Storage Disease Type II/therapy , alpha-Glucosidases/genetics , Adenoviridae/genetics , Animals , Blotting, Western , Cells, Cultured , Coculture Techniques , DNA, Complementary/metabolism , Fibroblasts/metabolism , Humans , Immunohistochemistry , Lysosomes/metabolism , Mannosephosphates/metabolism , Mice , Mice, Knockout , Mice, Nude , Muscle, Skeletal/cytology , Myocardium/metabolism , Placenta/metabolism , Receptor, IGF Type 2/metabolism , Recombinant Proteins/metabolism , Time Factors , Transduction, Genetic
8.
Proc Natl Acad Sci U S A ; 98(5): 2682-7, 2001 Feb 27.
Article in English | MEDLINE | ID: mdl-11226299

ABSTRACT

The ability to isolate fetal nucleated red blood cells (NRBCs) from the maternal circulation makes possible prenatal genetic analysis without the need for diagnostic procedures that are invasive for the fetus. Such isolation requires antibodies specific to fetal NRBCs. To generate a panel of antibodies to antigens present on fetal NRBCs, a new type of nonimmune phage antibody library was generated in which multiple copies of antibody fragments are displayed on each phage. Antibody fragments specific for fetal NRBCs were isolated by extensive predepletion of the phage library on adult RBCs and white blood cells (WBCs) followed by positive selection and amplification on fetal liver erythroid cells. After two rounds of selection, 44% of the antibodies analyzed bound fetal NRBCs, with two-thirds of these showing no binding of WBCs. DNA fingerprint analysis revealed the presence of at least 16 unique antibodies. Antibody specificity was confirmed by flow cytometry, immunohistochemistry, and immunofluorescence of total fetal liver and adult RBCs and WBCs. Antibody profiling suggested the generation of antibodies to previously unknown fetal RBC antigens. We conclude that multivalent display of antibodies on phage leads to efficient selection of panels of specific antibodies to cell surface antigens. The antibodies generated to fetal RBC antigens may have clinical utility for isolating fetal NRBCs from maternal circulation for noninvasive prenatal genetic diagnosis. Some of the antibodies may also have possible therapeutic utility for erythroleukemia.


Subject(s)
Antibodies, Monoclonal/immunology , Bacteriophages/genetics , Erythrocytes/immunology , Fetus/cytology , Antibodies, Monoclonal/genetics , Base Sequence , Cell Separation , DNA Primers , Flow Cytometry , Humans , Immunohistochemistry , Liver/cytology , Liver/embryology , Microscopy, Fluorescence
9.
Neuromuscul Disord ; 10(8): 599-603, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11053688

ABSTRACT

We screened 22 Japanese patients with acid maltase deficiency (seven with the infantile type, eight with the juvenile type and seven with the adult type) for three previously described mutations, D645E, S529V and R672Q, and a novel mutation, R600C. Although D645E has been reported to be common in Chinese patients with the infantile type, only three of 44 alleles (two of 14 infantile type alleles) from Japanese patients harbored the D645E mutation. The S529V mutation was identified in six of 14 alleles from adult-onset patients. None of the infantile or juvenile patients harbored the S529V mutation. Therefore, S529V apparently results in the adult type disease and is common in Japanese adult-onset patients. R672Q was identified in two pairs of siblings with the juvenile type. A novel mutation, R600C, was identified in eight of 22 patients (nine of 44 alleles). Therefore, R600C is another common Japanese mutation occurring at a CpG dinucleotide "hot spot". Homozygosity for this mutation apparently results in the infantile phenotype. Genetic diagnosis by detecting these four mutations might be feasible for most Japanese patients with acid maltase deficiency.


Subject(s)
Glycogen Storage Disease Type II/genetics , Mutation/genetics , Adolescent , Adult , Alleles , DNA Mutational Analysis , Genetic Testing , Humans , Infant , Japan
10.
Am J Med Genet ; 85(1): 5-8, 1999 Jul 02.
Article in English | MEDLINE | ID: mdl-10377006

ABSTRACT

Genetic deficiency of lysosomal acid alpha-glucosidase (acid maltase) results in the autosomal recessive disorder glycogen storage disease type II (GSDII) in which intralysosomal accumulation of glycogen primarily affects function of skeletal and cardiac muscle. During an earlier review we noted 3 in 100 cases of GSDII with incidental description of cleft lip. In addition, we identified 2 of 35 GSDII patients referred to us for molecular studies with co-occurence of cleft lip, considerably greater than the estimated frequency of nonsyndromic cleft lip with or without cleft palate of 1 in 700 to 1,000. Because several lines of evidence support a minor cleft lip/palate (Cl/P) locus on chromosome 17q close to the locus for GSDII, we defined the molecular basis for the GSDII in these two patients to determine if they represented a contiguous gene syndrome. Patient I (of Dutch descent) was homozygous and the parents heterozygous for an intragenic deletion of exon 18 (deltaex18), common in Dutch patients. Patient II was heterozygous for delta525T, a mutation also common in Dutch patients and a novel nonsense mutation (172 [corrected] C-->T; Gln58Stop) in exon 2, the first coding exon. The mother was heterozygous for the delta525T and the father for the 172 [corrected] C-->T; Gln58Stop. The finding that both patients carried intragenic mutations eliminates a contiguous gene syndrome. Whereas the presence of cleft lip/cleft palate in a patient with GSDII could be coincidental, these co-occurences could represent a modifying action of acid alpha-glucosidase deficiency on unlinked or linked genes that result in increased susceptibility for cleft lip.


Subject(s)
Cleft Lip/epidemiology , Glycogen Storage Disease Type II/complications , Mutation , Base Sequence , Cleft Lip/complications , Cleft Lip/genetics , Cleft Palate/genetics , Cleft Palate/pathology , DNA Primers , Female , Gene Deletion , Glycogen Storage Disease Type II/genetics , Humans , Infant , Infant, Newborn , Male , Pedigree , Syndrome
11.
Am J Pathol ; 154(4): 1089-96, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10233847

ABSTRACT

Acid alpha-glucosidase (GAA) cleaves the alpha1-4 and alpha1-6 glycosidic linkages of glycogen and related alpha-glucosyl substrates within lysosomes. Its deficiency results in glycogen storage disease type II (GSDII) variants including Pompe disease. To gain insight into the tissue patterns of involvement by glycogen storage in GSDII, GAA mRNA expression in mouse tissues was evaluated by Northern blot and in situ hybridization analyses. Extensive temporal and spatial variation of GAA mRNA was observed. During preterm maturation, GAA mRNA levels of whole mice progressively increased as assessed by Northern analysis. By in situ hybridization with GAA antisense mRNA, low signals were detected in most tissues throughout gestation. However, increased expression in specific cell types of different tissues was observed beginning at 16 days post coitum in developing brain neurons, primitive inner ear cells, and seminiferous tubular epithelium. In adult mice, whole-organ GAA mRNA levels were highest in brain, moderate in heart, liver, and skeletal muscle, and lowest in the series kidney > lung > testis > spleen. By in situ hybridization, the highest-intensity signals were in neurons of the central and peripheral nervous systems whereas neuroglial cells had only low-level signal. Signals of moderate intensity were in cardiomyocytes whereas low signals were in hepatocytes and skeletal muscle myocytes and very low in cells of the lungs, thymus, pancreas, spleen, and adrenal glands. However, testicular Sertoli cells and kidney tubular epithelial cells had significant signals even though surrounding cells had very low signals. The discrete temporal and spatial variations of GAA mRNA during development indicate different physiological roles for this enzyme in various cell types and developmental stages.


Subject(s)
Gene Expression Regulation, Developmental , Glucan 1,4-alpha-Glucosidase/genetics , RNA, Messenger/biosynthesis , alpha-Glucosidases/genetics , Animals , Blotting, Northern , Central Nervous System/enzymology , Embryo, Mammalian , In Situ Hybridization , Liver/enzymology , Male , Mice , Mice, Inbred Strains , Muscle, Skeletal/enzymology , Myocardium/enzymology , Neurons/enzymology , Organ Specificity , RNA, Antisense/metabolism , Time Factors
12.
Hum Genet ; 104(1): 94-8, 1999 Jan.
Article in English | MEDLINE | ID: mdl-10071199

ABSTRACT

Glycogen storage disease type II (GSDII) is an autosomal recessive disorder resulting from inherited deficiency of the enzyme lysosomal acid alpha-glucosidase. Over 40 different mutations have been described but no large deletions have been previously identified. We now describe a homozygous large (9-kb) deletion extending from IVS 15 to 4 kb downstream of the terminal exon (exon 20), detected by polymerase chain reaction (PCR)-based methods. The deletion was initially suspected because of failure to amplify a contiguous group of exons by PCR. We hypothesized an Alu/Alu recombination, based on our prior demonstration by Southern blotting of Alu elements in the regions potentially flanking the deletion. Additional sequence analysis of genomic fragments confirmed the presence of Alu elements and allowed the design of flanking primers for PCR amplification. Amplification resulted in a smaller than normal fragment (0.7 vs. 10 kb) in homozygosity in the proband and in heterozygosity in her parents. Cloning and sequencing of the smaller than normal 0.7-kb deletion fragment revealed an Alu/Alu deletion junction. In heterozygosity this deletion would not be detected by currently standard PCR mutation detection methods. Based on other Alu-mediated deletions, this deletion is likely to be recurrent and should be screened for in all non-consanguineous GSDII patients, particularly when only one mutation has been identified and none of the 12 single-nucleotide polymorphisms in the deleted region are heterozygous. These observations also suggest that initial characterization of genes at disease-causing loci should include a search for Alu and other repetitive elements to facilitate subsequent PCR-based mutation analysis.


Subject(s)
Alu Elements/genetics , Glycogen Storage Disease Type II/genetics , Polymerase Chain Reaction , Sequence Deletion/genetics , Base Sequence , Cloning, Molecular , Exons/genetics , Female , Humans , Infant , Male , Molecular Sequence Data , Pedigree , Sequence Analysis, DNA
14.
J Appl Physiol (1985) ; 85(3): 1175-86, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9729597

ABSTRACT

We examined the hypothesis that glucose flux was directly related to relative exercise intensity both before and after a 12-wk cycle ergometer training program [5 days/wk, 1-h duration, 75% peak O2 consumption (VO2 peak)] in healthy female subjects (n = 17; age 23.8 +/- 2.0 yr). Two pretraining trials (45 and 65% of VO2 peak) and two posttraining trials [same absolute workload (65% of old VO2 peak) and same relative workload (65% of new VO2 peak)] were performed on nine subjects by using a primed-continuous infusion of [1-13C]- and [6,6-2H]glucose. Eight additional subjects were studied by using [6, 6-2H]glucose. Subjects were studied postabsorption for 90 min of rest and 1 h of cycling exercise. After training, subjects increased VO2 peak by 25.2 +/- 2.4%. Pretraining, the intensity effect on glucose kinetics was evident between 45 and 65% of VO2 peak with rates of appearance (Ra: 4.52 +/- 0.25 vs. 5.53 +/- 0.33 mg . kg-1 . min-1), disappearance (Rd: 4.46 +/- 0.25 vs. 5.54 +/- 0.33 mg . kg-1 . min-1), and oxidation (Rox: 2.45 +/- 0.16 vs. 4.35 +/- 0.26 mg . kg-1 . min-1) of glucose being significantly greater (P

Subject(s)
Carbohydrate Metabolism , Physical Fitness/physiology , Adolescent , Adult , Blood Glucose/metabolism , Body Composition/physiology , Exercise/physiology , Female , Hormones/blood , Humans , Lactic Acid/blood , Male , Menstruation/physiology , Oxygen Consumption/physiology , Sex Characteristics
16.
Biochem Biophys Res Commun ; 244(3): 921-7, 1998 Mar 27.
Article in English | MEDLINE | ID: mdl-9535769

ABSTRACT

Glycogen storage disease type II (GSDII), an autosomal recessive myopathic disorder, results from deficiency of lysosomal acid alpha-glucosidase. We searched for mutations in an evolutionarily conserved region in 54 patients of differing phenotype. Four novel mutations (D645N, G448S, R672W, and R672Q) and a previously described mutation (C647W) were identified in five patients and their deleterious effect on enzyme expression demonstrated in vitro. Two novel frame-shifting insertions/deletions (delta nt766-785/insC and +insG@nt2243) were identified in two patients with exon 14 mutations. The remaining three patients were either homozygous for their mutations (D645N/D645 and C647W/C647W) or carried a previously described leaky splice site mutation (IVS1-13T-->G). For all patients "in vivo" enzyme activity was consistent with clinical phenotype. Agreement of genotype with phenotype and in vitro versus in vivo enzyme was seen in three patients (two infantile patients carrying C647W/C647W and D645N/+insG@nt2243 and an adult patient heteroallelic for G648S/IVS1-13T-->G). Relative discordance was found in a juvenile patient homozygous for the non-expressing R672Q and an adult patient heterozygous for the minimally expressing R672W and delta nt766-785/+insC. Possible explanations include differences in in vitro assays vs in vivo enzyme activity, tissue specific expression with diminished enzyme expression/stability in fibroblasts vs muscle, somatic mosaicism, and modifying genes.


Subject(s)
Glucan 1,4-alpha-Glucosidase/genetics , Glycogen Storage Disease Type II/genetics , Mutation , Adult , Child, Preschool , Female , Fibroblasts/enzymology , Frameshift Mutation , Genotype , Glucan 1,4-alpha-Glucosidase/analysis , Glycogen Storage Disease Type II/enzymology , Homozygote , Humans , Male , Middle Aged , Muscles/enzymology , Mutagenesis, Site-Directed , Phenotype , Sequence Analysis, DNA , Sequence Deletion , alpha-Glucosidases
17.
J Clin Invest ; 101(2): 295-300, 1998 Jan 15.
Article in English | MEDLINE | ID: mdl-9435300

ABSTRACT

We and others have shown that an increased extracellular concentration of adenosine mediates the antiinflammatory effects of methotrexate and sulfasalazine both in vitro and in vivo, but the mechanism by which these drugs increase extracellular adenosine remains unclear. The results of the experiments reported here provide three distinct lines of evidence that adenosine results from the ecto-5'-nucleotidase- mediated conversion of adenine nucleotides to adenosine. First, pretreatment of a human microvascular endothelial cell line (HMEC-1) with methotrexate increases extracellular adenosine after exposure of the pretreated cells to activated neutrophils; the ecto-5'-nucleotidase inhibitor alpha, beta-methylene adenosine-5'-diphosphate (APCP) abrogates completely the increase in extracellular adenosine. Second, there is no methotrexate-mediated increase in extracellular adenosine concentration in the supernate of cells deficient in ecto-5'-nucleotidase, but there is a marked increase in extracellular adenosine concentration in the supernates of these cells after transfection and surface expression of the enzyme. Finally, as we have shown previously, adenosine mediates the antiinflammatory effects of methotrexate and sulfasalazine in the murine air pouch model of inflammation, and injection of APCP, the ecto-5'-nucleotidase inhibitor, abrogates completely the increase in adenosine and the decrement in inflammation in this in vivo model. These results not only show that ecto-5'-nucleotidase activity is a critical mediator of methotrexate- and sulfasalazine-induced antiinflammatory activity in vitro and in vivo but also indicate that adenine nucleotides, released from cells, are the source of extracellular adenosine.


Subject(s)
5'-Nucleotidase/physiology , Adenine Nucleotides/metabolism , Adenosine/metabolism , Anti-Inflammatory Agents/pharmacology , Methotrexate/pharmacology , Sulfasalazine/pharmacology , Adenosine Monophosphate/metabolism , Animals , Humans , Mice , Mice, Inbred BALB C , Tumor Cells, Cultured
18.
J Appl Physiol (1985) ; 82(4): 1360-9, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9104876

ABSTRACT

We examined the hypothesis that glucose flux was directly related to relative exercise intensity both before and after a 10-wk cycle ergometer training program in 19 healthy male subjects. Two pretraining trials [45 and 65% of peak O2 consumption (VO2peak)] and two posttraining trials (same absolute and relative intensities as 65% pretraining) were performed for 90 min of rest and 1 h of cycling exercise. After training, subjects increased VO2peak by 9.4 +/- 1.4%. Pretraining, the intensity effect on glucose kinetics was evident with rates of appearance (R(a); 5.84 +/- 0.23 vs. 4.73 +/- 0.19 mg x kg(-1) x min(-1)), disappearance (R(d); 5.78 +/- 0.19 vs. 4.73 +/- 0.19 mg x kg(-1) x min(-1) x min(-1)), oxidation (R(ox); 5.36 +/- 0.15 vs. 3.41 +/- 0.23 mg x kg(-1) x min(-1)), and metabolic clearance (7.03 +/- 0.56 vs. 5.20 +/- 0.28 ml x kg(-1) x min(-1)) of glucose being significantly greater (P < or = 0.05) in the 65% than the 45% VO2peak trial. When R(d) was expressed as a percentage of total energy expended per minute (R(dE)), there was no difference between the 45 and 65% intensities. Training did reduce R(a) (4.63 +/- 0.25), R(d) (4.65 +/- 0.24), R(ox) (3.77 +/- 0.43), and R(dE) (15.30 +/- 0.40 to 12.85 +/- 0.81) when subjects were tested at the same absolute workload (P < or = 0.05). However, when they were tested at the same relative workload, R(a), R(d), and R(dE) were not different, although R(ox) was lower posttraining (5.36 +/- 0.15 vs. 4.41 +/- 0.42, P < or = 0.05). These results show 1) glucose use is directly related to exercise intensity; 2) training decreases glucose flux for a given power output; 3) when expressed as relative exercise intensity, training does not affect the magnitude of blood glucose use during exercise; 4) training alters the pathways of glucose disposal.


Subject(s)
Glucose/metabolism , Physical Fitness , Adolescent , Adult , Blood Glucose/metabolism , Body Composition , Catecholamines/blood , Energy Metabolism/physiology , Exercise Test , Humans , Kinetics , Male , Metabolic Clearance Rate/physiology , Oxidation-Reduction , Physical Endurance/physiology , Pulmonary Gas Exchange/physiology
20.
Ann Hum Genet ; 60(5): 365-8, 1996 09.
Article in English | MEDLINE | ID: mdl-8912788

ABSTRACT

We have identified the molecular basis of the GAA*4 allozyme as a G to A transition at nt2065 which predicts the substitution of glutamic acid by lysine at codon 689 (E689K). The conclusion that this change represents the molecular basis of the GAA*4 allozyme is based on 1) presence of the G2065A in homozygosity in a known GAA*4 homozygote, 2) transient expression studies showing normal enzyme activity expressed by cDNA containing the G2065A transition and 3) isoelectric focusing studies showing a more cathodal pattern for the expressed product as compared to the common GAA*1, analogous to the patterns seen in normal and known GAA*4 lymphoid cells.


Subject(s)
Glutamic Acid/genetics , Mutation , Asian People , Cell Line , Exons , Homozygote , Humans , Japan/ethnology , Lymphoid Tissue/cytology , Sequence Analysis, DNA
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