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1.
Neurology ; 60(5): 865-8, 2003 Mar 11.
Article in English | MEDLINE | ID: mdl-12629249

ABSTRACT

Leigh syndrome (LS) is a heterogeneous disorder, usually due to a defect in oxidative metabolism. Typically, signs and symptoms commence in infancy or childhood, although rare cases of adult onset have been described. Progressive deterioration is the norm. The authors describe a 22-year-old woman with partial cytochrome c oxidase deficiency who developed fulminant LS following an acute febrile illness and who subsequently showed dramatic clinical and neuroradiologic improvement.


Subject(s)
Cytochrome-c Oxidase Deficiency/drug therapy , Leigh Disease/drug therapy , Leigh Disease/enzymology , Adult , Ascorbic Acid/administration & dosage , Brain/pathology , Drug Therapy, Combination , Female , Humans , Leigh Disease/diagnosis , Magnetic Resonance Imaging , Remission Induction , Riboflavin/administration & dosage , Thiamine/administration & dosage , Ubiquinone/administration & dosage
2.
Laryngoscope ; 111(7): 1275-80, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11568554

ABSTRACT

OBJECTIVES: Previous studies of decreased cochlear DNA binding in autoimmune mice suggested that antibodies against a cochlear cell surface DNA receptor cause autoimmune hearing loss. However, the presence of a cochlear DNA receptor has not been determined. Therefore, immunohistochemistry with an anti-DNA receptor antibody was performed on MRL.MpJ-Fas(lpr) (MRL/lpr) autoimmune mice to determine 1) which inner ear structures contain DNA receptors and 2) whether the receptor staining pattern changes as autoimmune disease progresses and hearing thresholds increase. STUDY DESIGN: A prospective study of the progression of hearing loss in autoimmune mice and correlated alterations in immunostaining for the inner ear DNA receptor. METHODS: One group of MRL/lpr mice (n = 10) was allowed to develop autoimmune disease, and auditory brainstem response (ABR) audiometry was performed at 4, 6, and 9 months of age to measure the progression of hearing loss. A second group (n = 5) was tested for ABR thresholds at 2 months of age and immediately killed to assess receptor staining before the onset of autoimmune disease and hearing loss. The inner ears from all mice were immunohistochemically stained with an anti-DNA receptor antibody, and a qualitative analysis of the staining of cochlear structures was performed. RESULTS: Auditory brainstem response audiometry revealed a significant 20- to 30-dB elevation of thresholds as systemic disease progressed. Anti-DNA receptor staining was heaviest in the spiral ligament and less intense in the spiral ganglion and cochlear nerve. Both groups showed a similar pattern of staining in these structures. The stria vascularis and hair cells also stained in both groups. However, the stria cells of normal-hearing mice showed diffuse intracellular immunoreactivity, whereas older mice displayed less staining that was confined to the cell membranes. CONCLUSIONS: The inner ears of MRL/lpr mice contain DNA receptors. Autoimmune hearing loss was correlated with weaker overall intracellular staining in the stria vascularis and hair cells but increased staining of the cell membranes. This suggested DNA receptors have impaired endocytosis and more receptors remain on the cell membrane, possibly as a result of binding by circulating autoantibodies.


Subject(s)
Autoimmune Diseases , Cochlea/metabolism , DNA/analysis , Hearing Loss, Sensorineural/etiology , Sensory Receptor Cells/metabolism , Age Factors , Animals , Audiometry, Evoked Response , Autoimmune Diseases/complications , Autoimmune Diseases/diagnosis , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , Cochlea/physiology , Hearing Loss, Sensorineural/diagnosis , Immunohistochemistry , Lupus Erythematosus, Systemic/complications , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Mice, Inbred MRL lpr , Prospective Studies , Staining and Labeling
3.
Mol Genet Metab ; 72(1): 67-71, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11161831

ABSTRACT

Smith-Lemli-Opitz syndrome (SLOS) is a multiple congenital anomaly/mental retardation syndrome of variable severity with an incidence previously estimated at 1 in 20,000-60,000 based on case frequency surveys. Identification of the gene defect in SLOS has made it possible to calculate the carrier frequency and estimate disease incidence using molecular methods to identify carriers. Using a previously described PCR-RFLP assay we screened 1503 anonymous blood samples from random newborn screening blood spot cards for the presence of the common SLOS mutation IVS8-1G>C in order to determine the carrier frequency. Sixteen carriers were identified in the 1503 samples. Since the frequency of the IVS8-1G>C mutation among all SLOS gene mutations is known, the overall carrier frequency for all mutations can be calculated. The calculated carrier frequency for all mutations based on this result is 1 in 30, predicting an SLOS incidence of 1 in 1590 to 1 in 13,500. The current incidence estimate may, therefore, significantly underestimate the true incidence of SLOS. This discrepancy between calculated and observed incidence could be due to undiagnosed mild cases, misdiagnosed severe cases, death prior to diagnosis, or fetal loss. More comprehensive incidence studies are needed to determine if SLOS is as common as predicted by the very high (1 in 30) carrier frequency determined in this study.


Subject(s)
Heterozygote , Mutation , Oxidoreductases Acting on CH-CH Group Donors , Oxidoreductases/genetics , Smith-Lemli-Opitz Syndrome/genetics , Cohort Studies , Gene Frequency , Genetic Testing , Humans , Infant, Newborn , Models, Genetic , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length
4.
J Biol Chem ; 275(43): 33655-62, 2000 Oct 27.
Article in English | MEDLINE | ID: mdl-10938276

ABSTRACT

Studies to clone a cell-surface DNA-binding protein involved in the binding and internalization of extracellular DNA have led to the isolation of a gene for a membrane-associated nucleic acid-binding protein (MNAB). The full-length cDNA is 4.3 kilobases with an open reading frame of 3576 base pairs encoding a protein of approximately 130 kDa (GenBank accession numbers and ). The MNAB gene is on human chromosome 9 with wide expression in normal tissues and tumor cells. A C3HC4 RING finger and a CCCH zinc finger have been identified in the amino-terminal half of the protein. MNAB bound DNA (K(D) approximately 4 nm) and mutagenesis of a single conserved amino acid in the zinc finger reduced DNA binding by 50%. A potential transmembrane domain exists near the carboxyl terminus. Antibodies against the amino-terminal half of the protein immunoprecipitated a protein of molecular mass approximately 150 kDa and reacted with cell surfaces. The MNAB protein is membrane-associated and primarily localized to the perinuclear space, probably to the endoplasmic reticulum or trans-Golgi network. Characterization of the MNAB protein as a cell-surface DNA-binding protein, critical in binding and internalization of extracellular DNA, awaits confirmation of its localization to cell surfaces.


Subject(s)
DNA-Binding Proteins/genetics , Membrane Proteins/genetics , Amino Acid Sequence , Animals , COS Cells , Cell Membrane/chemistry , Cloning, Molecular , DNA/metabolism , DNA-Binding Proteins/chemistry , HeLa Cells , Humans , Membrane Proteins/chemistry , Molecular Sequence Data , Recombinant Proteins/metabolism , Zinc Fingers
5.
J Immunol Methods ; 241(1-2): 141-6, 2000 Jul 31.
Article in English | MEDLINE | ID: mdl-10915856

ABSTRACT

DNA binding to cell-surfaces has been documented in several studies. The interaction of DNA with cells has been shown to have therapeutic potential as a non-viral form of gene delivery and DNA vaccination. Recently, bacterial DNA binding and internalization has been demonstrated in some cells to trigger secretion of cytokines and cell activation. Previous studies to quantify DNA binding to cells have used radiolabeled DNA. Here we report a non-radioactive assay for quantification of cell-surface DNA binding based on the isoparametric analysis of flow cytometric data as described by Chatelier et al., Embo J., 5 (1986) 1181. This assay has the advantage over previously used procedures in not employing radioactive material and being able to discriminate viable from non-viable cells that bind DNA. With the importance of understanding the interaction of DNA with cells, this assay may have application for the identification and characterization of reagents designed to either enhance or inhibit DNA binding to cells.


Subject(s)
Cell Membrane/metabolism , Flow Cytometry/methods , Plasmids/metabolism , Cell Line, Transformed , Humans
6.
Biochim Biophys Acta ; 1264(2): 243-8, 1995 Nov 07.
Article in English | MEDLINE | ID: mdl-7495870

ABSTRACT

The gene for Thermus aquaticus (Taq) DNA polymerase enzyme (Taq Pol I) was mutagenized and sixty-two candidate clones were screened for enzyme activity. Two of the clones expressed enzymes (*Taq-3 and *Taq-5) that showed very reduced 5'-3' exonuclease activity and normal DNA polymerase activity. These two enzymes showed heat resitance and storage stability similar to Taq Pol I and had similar effectiveness in PCR. Processivity of the polymerases was compared by measuring the extension of an end-labeled primer annealed to a single stranded DNA, as well as by a PCR method. The processivity of *Taq-3 and *Taq-5 was similar to Taq Pol I (50-80 nucleotides) and more processive than a Taq Pol I deficient in the 5'-3' exonuclease due to absence of the first 290 amino acids (Stoffel fragment). The results indicate two amino acids which are required for normal 5'-3' exonuclease activity in Taq Pol I (Arg-25 and Arg-74).


Subject(s)
DNA-Directed DNA Polymerase/metabolism , Exodeoxyribonucleases/metabolism , Thermus/enzymology , Cloning, Molecular , DNA Primers , DNA-Directed DNA Polymerase/biosynthesis , Enzyme Stability , Exodeoxyribonuclease V , Exodeoxyribonucleases/antagonists & inhibitors , Exodeoxyribonucleases/biosynthesis , Genes, Bacterial , Mutagenesis , Nucleic Acid Synthesis Inhibitors , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Restriction Mapping , Taq Polymerase , Thermus/genetics
7.
Genomics ; 29(1): 285-7, 1995 Sep 01.
Article in English | MEDLINE | ID: mdl-8530088

ABSTRACT

We have cloned a novel human cDNA, INPPL1 (GenBank Accession No. L36818), which maps to 11q23. The corresponding mRNA is 4657 nt in length and is widely expressed in both fetal and adult tissues. An open reading frame of 3441 nt encodes a putative polypeptide that shares several domains with inositol triphosphate phosphatases. Several polymorphisms have been mapped to the 3'-untranslated region, yet the putative coding region showed no polymorphisms in nine independent cDNA samples.


Subject(s)
Chromosomes, Human, Pair 11 , Hominidae/genetics , Phosphoric Monoester Hydrolases/genetics , Adult , Amino Acid Sequence , Animals , Chromosome Mapping , Cloning, Molecular , Conserved Sequence , DNA, Complementary , Fetus , Humans , Molecular Sequence Data , Open Reading Frames , Phosphoric Monoester Hydrolases/biosynthesis , Polymorphism, Genetic , Sequence Homology, Amino Acid
8.
Proc Soc Exp Biol Med ; 184(1): 92-7, 1987 Jan.
Article in English | MEDLINE | ID: mdl-3797429

ABSTRACT

We determined whether addition of human lipoprotein-TG to the perfusate for the isolated rat kidney would increase net Na+ reabsorption or maintain renal tissue K+ content. Rat kidneys (n = 6) were perfused for 75 min with a perfusate containing 6 g% of substrate-free albumin in Krebs-Ringer bicarbonate and a mixture of human chylomicrons and very low density lipoproteins (human lipoprotein-triacylglycerol (HL-TG]. Control kidneys (n = 6) were perfused in the substrate-limited state, i.e., without any exogenous substrates added to the perfusate. Means (n = 6) for function of control kidneys were GFR = 808 +/- 50 microliter g-1 X min-1; %T-Na+ = 63.3 +/- 1.3%. A significant loss of tissue K+ occurred: tissue K+ remaining after 75 min of perfusion = 79.1 +/- 1.9%. Although kidney tissue contains lipoprotein lipase, HL-TG (n = 6) did not increase %Na+ reabsorption (64.3 +/- 2.6%) or maintain tissue K+ content (80.6 +/- 2.0%). Therefore, the TG might have been hydrolyzed and taken up for biosynthesis, the rat kidney lipoprotein lipase might have been inactive, or the rat kidney might not use lipoprotein-TG for biosynthesis or oxidation.


Subject(s)
Chylomicrons/metabolism , Kidney/metabolism , Lipoproteins, VLDL/metabolism , Triglycerides/metabolism , Animals , Biological Transport , Diuresis , Glomerular Filtration Rate , Kidney Tubules/metabolism , Perfusion , Potassium/metabolism , Rats , Sodium/metabolism
10.
Am J Physiol ; 238(5): F415-27, 1980 May.
Article in English | MEDLINE | ID: mdl-7377350

ABSTRACT

When the ratio delta TNa+/delta QO2 is used to estimate the energy requirements for net Na+ reabsorption (TNa+), it is assumed that the entire change in renal O2 uptake (delta QO2) is utilized only for the delta TNa+. However, if increases in renal synthetic work also occur when TNa+ is increased, the energy cost for TNa+ will be overestimated. We perfused the substrate-limited isolated rat kidney at 38 degrees C, pH 7.4, a mean arterial pressure of 120 mmHg, and mean lactate concentrations between 0 and 8.3 mM. We measured QO2, TNa+, net reabsorption of lactate (Tlac), net utilization of lactate (Qlac), lactate decarboxylation rate (Qlacox), as well as the net entry rate of lactate into biosynthetic pathways (Qxslac). When no exogenous substrate was present (rates are means, g wet wt-1 . min-1) GFR was 351 +/- 38 microliter, %TNa+ was 54 +/- 2%, and QO2 was 2.85 +/- 0.31 mumol; there was also a loss of about 20% of renal tissue K+ content. When mean [lactate] greater than or equal to 0.73 mM, the loss of tissue K+ was completely prevented and %TNa+ increased to and remained at about 85%. At mean [lactate] of 8.3 mM, Tlac was 5.1 +/- 0.6 mumol, QO2 was 6.12 +/- 1.24 mumol, and GFR was 709 +/- 83 microliter. Qlac, delta Qlacox and delta TNa+ increased in parallel with each other and approaches maximal rates as [lactate] was raised. By contrast, Tlac increased as a linear function of perfusate [lactate] and was not related to changes in Qlac. The molar increases in TNa+ were 10- to 20-fold greater than the increases in Tlac. It is more probable, therefore, that lactate enhances TNa+ by providing energy from its oxidation rather than by a co-transport phenomenon. At all concentrations of lactate, more lactate was utilized (Km = 1.2 mM; Vmax = 3.4) than was decarboxylated (Km = 1.6 mM; Vmax = 1.7), indicating that as lactate concentration increased, both the synthesis of new products from lactate and Na+ reabsorption increased. We conclude that the ratio delta TNa+/delta QO2, overestimates the energy cost of Na+ reabsorption. In order to obtain an accurate estimate of the energy requirements for TNa+ in kidney, the simultaneous changes in the rate of net biosynthetic work must also be quantified as TNa+ is changed.


Subject(s)
Kidney/metabolism , Oxygen Consumption , Sodium/metabolism , Absorption , Animals , Energy Metabolism , Gluconeogenesis , Glucose/metabolism , Lactates/metabolism , Male , Rats
12.
Curr Probl Clin Biochem ; 8: 418-23, 1977.
Article in English | MEDLINE | ID: mdl-616375

ABSTRACT

The renal oxidation rates of glucose and lactate in the dog in vivo, in the dog cortical slice and in the isolated perfused rat kidney were compared. Lactate decarboxylation rate, on a carbon-atom basis, was from 2 to 10 fold greater than that of glucose. In the substrate-limited perfused kidney, glucose replaced only 30-40% of the substrates oxidized in vivo, while lactate replaced up to 80% of the substrates oxidized in vivo. Insulin lack does not account for these differences in the rates of lactate and glucose oxidation. Glucose and lactate support GFR and Na+ reabsorption to approximately the same extent in spite of their different rates of oxidation. Thus Na+ reabsorptive rate: CO2 production rate is not a constant and depends on the substrate being oxidized. The virtual absence of glucose oxidation by the dog cortical slice suggests either that: 1) glucose oxidation supports primarily medullary Na+ reabsorption while lactate oxidation supports cortical Na+ reabsorption as well of 2) glucose oxidation is more selectively coupled to Na+ reabsorptive work than is lactate oxidation.


Subject(s)
Glucose/metabolism , Kidney Cortex/metabolism , Kidney/metabolism , Lactates/metabolism , Sodium/metabolism , Animals , Biological Transport, Active , Dogs , In Vitro Techniques , Oxidation-Reduction , Perfusion , Rats , Species Specificity
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