Rapid detection of sequence polymorphisms in the human mitochondrial DNA control region by polymerase chain reaction and single-strand conformation analysis in mutation detection enhancement gels

The article describes a rapid approach for the detection of sequence polymorphisms in the mitochondrial (mt)DNA control region that involves enzymatic amplification of each entire mtDNA control region (HV1 and HV2) and the subsequent analysis of the PCR products by single-strand conformation analysis (SSCA) in mutation detection enhancement (MDE) gels, followed by silver stain detection. HV1 and HV2 SSC reference ladders were developed to standardize the classification of the different mtDNA types. Twenty-five mtDNA types were observed among the 45 Spanish individuals analyzed: 11 types were observed in the HV1 region as compared with 10 types in the HV2 region. This mutation scanning strategy could be a promising method of potential use not only in forensic genetics but also in population and evolutionary studies.     

Sensorineural hearing loss caused by mitochondrial DNA mutations: special reference to the A1555G mutation

Mutations in mitochondrial DNA, which are maternally inherited, have been thought to be one of the causes of sensorineural hearing loss. Two mitochondrial mutational sites (A1555G, A7445G) have been reported to be responsible for non-syndromic hearing impairments. The A1555G mutation causes increased susceptibility to aminoglycoside antibiotic-induced hearing loss as well as non-syndromic sensorineural hearing loss. Our wide screening study showed that there may be a great number of subjects within the Japanese population who have the A1555G mutation. Recent reports suggest that high-risk populations may exist throughout the world. The aminoglycoside-induced hearing loss associated with a mitochondrial mutation is commonly bilateral, symmetric, high frequency involved, and is sometimes associated with progressive sensorineural hearing loss.     

A quality control algorithm for DNA sequencing projects

Heterologous DNA sequences from rearrangements with the genomes of host cells, genomic fragments from hybrid cells, or impure tissue sources can threaten the purity of libraries that are derived from RNA or DNA. Hybridization methods can only detect contaminants from known or suspected heterologous sources, and whole library screening is technically very difficult. Detection of contaminating heterologous clones by sequence alignment is only possible when related sequences are present in a known database. We have developed a statistical test to identify heterologous sequences that is based on the differences in hexamer composition of DNA from different organisms. This test does not require that sequences similar to potential heterologous contaminants are present in the database, and can in principle detect contamination by previously unknown organisms. We have applied this test to the major public expressed sequence tag (EST) data sets to evaluate its utility as a quality control measure and a peer evaluation tool. There is detectable heterogeneity in most human and C.elegans EST data sets but it is not apparently associated with cross-species contamination. However, there is direct evidence for both yeast and bacterial sequence contamination in some public database sequences annotated as human. Results obtained with the hexamer test have been confirmed with similarity searches using sequences from the relevant data sets.     

A PCR-RFLP method for the detection and species identification of human microsporidia

Amputation in a growing child should be performed through a joint and not above or below a joint, as is commonly the case in an adult. We describe a technique for performing a through-knee amputation in a situation in which a soft-tissue sarcoma involved a leg, reaching up to the knee joint. Soft-tissue dissection was performed above the knee, leaving a safe zone from the tumor. The distal femur was dissected free of all attachments to the tibia and leg, leaving it intact but protruding from the soft-tissue sleeve of the thigh. To be able to close the stump over the protruding distal femur, the femur was shortened in the metaphysodiaphyseal area. Follow-up over 3 years shows a good through-knee stump, tumor free, and a normal distal growth plate.     

Polymorphic sites in human mitochondrial DNA control region sequences: population data and maternal inheritance

Short-circuit current (Isc), transepithelial conductance (Gt), electrical capacitance (CT) and the fluctuation in Isc were analyzed in polarized epithelial cells from the distal nephron of Xenopus laevis (A6 cell line). Tissues were incubated with Na+- and Cl--free solutions on the apical surface. Basolateral perfusate was NaCl-Ringer. Agents that increase cellular cAMP evoked increases in Gt, CT, Isc and generated a Lorentzian Isc-noise. The responses could be related to active, electrogenic secretion of Cl-. Arginine-vasotocin and oxytocin caused a typical peak-plateau response pattern. Stimulation with a membrane-permeant nonhydrolyzable cAMP analogue or forskolin showed stable increases in Gt with only moderate peaking of Isc. Phosphodiesterase inhibitors also stimulated Cl- secretion with peaking responses in Gt and Isc. All stimulants elicited a spontaneous Lorentzian noise, originating from the activated apical Cl- channel, with almost identical corner frequency (40-50 Hz). Repetitive challenge with the hormones led to a refractory behavior of all parameters. Activation of the cAMP route could overcome this refractoriness. All agents caused CT, a measure of apical membrane area, to increase in a manner roughly synchronous with Gt. These results suggest that activation of the cAMP-messenger route may, at least partly, involve exocytosis of a vesicular Cl- channel pool. Apical flufenamate depressed Cl- current and conductance and apparently generated blocker-noise. However, blocking kinetics extracted from noise experiments could not be reconciled with those obtained from current inhibition, suggesting the drug does not act as simple open-channel inhibitor.     

Oxidative stress and mitochondrial DNA mutations in human aging

The effects of chemical sympathectomy on the mucosal compartments of the immune system were examined in adult rats. Ablation of the sympathetic nervous system using 6-hydroxydopamine in recipient animals reduced the migration into Peyer's patches and mesenteric lymph nodes (MLN) of adoptively transferred cells from MLN of normal donors. The mucosal immune response to ovalbumin (OVA), assessed by enumeration of anti-OVA antibody containing cells (AOCC) in the lamina propria after intestinal immunisation, was reduced in animals sympathectomized prior to immunization. In order to identify whether this reduction in AOCC response in intestinally immunized sympathectomized animals was due to a defect in migration of AOCC precursors to the intestinal lamina propria, the effect of chemical sympathectomy on the appearance of AOCC in the gut of immunized animals after adoptive transfer of AOCC precursors was investigated. The IgA-specific AOCC response was significantly reduced in sympathectomized recipients compared to the control group. Taken together these results demonstrate that the peripheral sympathetic nervous system influences the migration and accumulation in vivo of both naive and memory/effector lymphocytes in mucosal lymphoid tissues.     

Identification of war victims from mass graves in Croatia, Bosnia, and Herzegovina by use of standard forensic methods and DNA typing

The postmortem remains of sixty-one war victims were excavated from 6 mass graves in Bosnia and Herzegovina one and a half years after interment Using standard identification methods, including the matching of medical and dental records, the recognition of distinguishing characteristics such as the use of clothing and belongings, and video superimposition, 35 persons were identified. For the remaining 26 persons identification efforts continue. DNA typing was performed at the HLA DQA1 locus and five PM system loci. Results from DNA typing were confirmed by other methods. DNA profiles of family members of 150 missing persons are now being developed using the 6 loci. These DNA profiles will then be compared with those generated from the bone and teeth remains of the unidentified victims.     

A mutation in a novel yeast proteasomal gene, RPN11/MPR1, produces a cell cycle arrest, overreplication of nuclear and mitochondrial DNA, and an altered mitochondrial morphology

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24 degreesC growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36 degreesC on either carbon source. Microscopic observation of cells growing on glucose at 24 degreesC shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho degrees] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.     

A canine ex vivo shunt for isotopic hemocompatibility evaluation of a NHLBI DTB primary reference material and of a IUPAC reference material

Factors determining the thrombogenic response to particular artificial surfaces were investigated ex vivo in a canine shunt model. Methods using radioisotopic tracers made it possible to dynamically monitor the deposition of labelled blood cells and proteins on a NHLBI.DTB primary reference material polydimethylsiloxane (PRM.PDMS) and on a IUPAC reference material polyvinyl chloride (IUPAC.PVC). On the one hand, leukocyte affinity tau s(leu) (number of deposited leukocytes mm-2s-1) was not significantly different between IUPAC.PVC (tau s(leu) = 1.2-2.5) and PRM.PDMS (tau s(leu) = 1.5-3.4) and the fibrinogen adsorption rate varied from 33 to 48.10(-5) micrograms mm-2s-1 for both these materials. On the other hand, platelet affinity tau s(plat) (number of deposited platelets mm-2s-1) was significantly different (p < 0.05) for IUPAC.PVC and PRM.PDMS (tau s(plat)PVC = 683 +/- 200 > tau s(plat)PDMS = 327 +/- 80). Scanning electron micrographs of adherent platelets, red cells and leukocytes after blood contact ex vivo were performed after each experiment. This preliminary work contributes not only to quantify the adsorption of different radiotracers, but also to evaluate the superficial distribution of the labelled biological species on the inner surface of the tested biomaterials.     

Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts

Techniques have been developed for extracting, amplifying and directly sequencing mitochondrial DNA (mtDNA) from human hair shafts. The hair shaft is ground in a glass micro-tissue grinder, and the DNA is extracted with organic solvent and purified by filtration. The filtrate subsequently provides the mtDNA template for the PCR. The two hypervariable segments of the mtDNA control region are amplified in four separate reactions. After a purification step to remove unincorporated PCR primers, amplified products are quantitated by capillary electrophoresis and subjected to cycle sequencing. The products are separated and analyzed on an automated DNA sequencer. The mtDNA sequences from the hair shaft match the mtDNA sequences from blood samples taken from the same donor.     

Smoking-associated mitochondrial DNA mutations and lipid peroxidation in human lung tissues

BACKGROUND/AIMS: The objective of the present study was to analyze the expression and regulation of intercellular adhesion molecule-1 (ICAM-1) in organotypic cultures of rat liver slices, which preserve the normal microenvironment of liver cells. METHODS: Rat liver slices were maintained in culture for 15 min to 24 h and examined for ICAM-1 expression by immunohistochemistry and Western blotting in basal conditions and after stimulation with 1000 IU/ml interferon-gamma (IFNgamma), 1000 IU/ml tumor necrosis factor-alpha (TNF alpha) and 50 microg/ml endotoxin. Immunohistochemical results were evaluated using a semiquantitative scoring system. RESULTS: In uncultured slices, ICAM-1 was not detected on hepatocytes. In unstimulated liver slices maintained in organotypic culture, ICAM-1 was induced at the surface of scattered hepatocytes (score at 15 min, 0.33+/-0.47 and at 24 h, 1.17+/-0.69). After 4 h of stimulation, a significant increase in ICAM-1 expression by hepatocytes and adjacent sinusoidal cells, but not by intra-hepatic biliary epithelial cells, was observed for IFNgamma (score: 2.35+/-0.47) and endotoxin (score: 2.67+/-0.47), but not with TNF alpha (score: 0.66+/-0.47). After 24 h of stimulation, a further increase in the extent of ICAM-1 expression by hepatocytes was observed for IFNgamma (score: 3.67+/-0.47) and endotoxin (score: 4.0+/-0.0), and a significant overexpression of ICAM-1 by hepatocytes was detectable after treatment with TNF alpha (score: 3.67+/-0.47). CONCLUSIONS: In rat liver organotypic cultures, TNF alpha, IFNgamma and endotoxin induce the expression of ICAM-1 in hepatocytes and adjacent sinusoidal endothelial cells, but not in portal tracts.     

Development of sera reference panels for quality control of ELISA diagnostic kits in Russia

The clinico-immunological examination of 57 patients with chronic bacterial prostatitis was carried out. The clinical analysis made it possible to divide the patients into 3 groups characterized by the presence of chronic inflammatory diseases of other organs which had appeared before (group 1) or after (group 2) the manifestation of the symptoms of prostatitis, aw well as by the absence of concomitant inflammatory diseases (group 3). At the same time these patients were found to have changes in their immune status, most pronounced in patients of groups 1 and 2. The clinico-immunological analysis of the patients with chronic bacterial prostatitis revealed the fact that chronic bacterial prostatitis was a chronic inflammatory process linked with changes in the immune system; these changes had the signs of secondary immunodeficiency and required immunocorrective therapy.     

Comprehensive, rapid and sensitive detection of sequence variants of human mitochondrial tRNA genes

In the present study, a comprehensive, rapid and sensitive method for screening sequence variation of the human mitochondrial tRNA genes has been developed. For this purpose, the denaturing gradient gel electrophoresis (DGGE) technique has been appropriately modified for simultaneous mutation analysis of a large number of samples and adapted so as to circumvent the problems caused by the anomalous electrophoretic behavior of DNA fragments encoding tRNA genes. Eighteen segments of mitochondrial DNA (mtDNA), each containing a single uniform melting domain, were selected to cover all tRNA-encoding regions using the computer program MELT94. All 18 segments were simultaneously analyzed by electrophoresis through a single broad range denaturing gradient gel under rigorously defined conditions, which prevent band broadening and other migration abnormalities from interfering with detection of sequence variants. All base substitutions tested, which include six natural mutations and 14 artificially introduced ones, have been detected successfully in the present study. Several types of evidence strongly suggest that the anomalous behavior in DGGE of tRNA gene-containing mtDNA fragments reflects their tendency to form temporary or stable alternative secondary structures under semi-denaturing conditions. The high sensitivity of the method, which can detect as low as 10% of mutant mtDNA visually, makes it valuable for the analysis of heteroplasmic mutations.     

Multiplex fluorescence-based primer extension method for quantitative mutation analysis of mitochondrial DNA and its diagnostic application for Alzheimer's disease

A sensitive and highly reproducible multiplexed primer extension assay is described for quantitative mutation analysis of heterogeneous DNA populations. Wild-type and mutant target DNA are simultaneously probed in competitive primer extension reactions using fluorophor-labeled primers and high fidelity, thermostable DNA polymerases in the presence of defined mixtures of deoxy- and dideoxynucleotides. Primers are differentially extended and the resulting products are distinguished by size and dye label. Wild-type:mutant DNA ratios are determined from the fluorescence intensities associated with electrophoretically resolved reaction products. Multiple nucleotide sites can be simultaneously interrogated with uniquely labeled primers of different lengths. The application of this quantitative technique is shown in the analysis of heteroplasmic point mutations in mitochondrial DNA that are associated with Alzheimer's disease.     

A mitochondrial tRNA(Val) gene mutation (G1642A) in a patient with mitochondrial myopathy, lactic acidosis, and stroke-like episodes

We studied a patient with the diagnosis of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) for mitochondrial DNA mutations in muscle. Established MELAS mutations were excluded. Mitochondrial DNA was further analyzed for mutations in the 22 tRNA genes by single-strand conformation polymorphism (SSCP) analysis; a tRNA(Val) mutation (G1642A) was found. The structure of the altered tRNA, the heteroplasmy, and the absence of the mutation in the mother and in 100 control subjects suggests that the tRNA(Val) mutation is associated with the MELAS syndrome.     

Expression, characterization, and detection of human uridine phosphorylase and identification of variant uridine phosphorolytic activity in selected human tumors

Uridine phosphorylase (UPase) catalyzes the reversible phosphorolysis of uridine to uracil. We purified the enzyme from the murine colon 26 tumor using a two-step procedure through 5-amino-benzylacyclouridine affinity chromatography. Antibodies raised in rabbits against the purified protein revealed single bands in Western blots of normal human tissue and tumor extracts. The polyclonal antibody used to screen a human liver expression library allowed the isolation of a 1.2-kb clone that contained the entire open reading frame of the human UPase. The UPase cDNA has been expressed as a fusion protein in Escherichia coli using the pMal-C2 vector. The kinetic analysis demonstrated that the recombinant UPase preferentially uses uridine, 5-fluorouracil, and uracil as substrates, although lower levels of activity were observed with 2-deoxyuridine and thymidine. Clinical samples of human tumors and adjacent normal tissues were assayed for phosphorolytic activity and sensitivity to 5-benzylacyclouridine (BAU), a potent inhibitor of the enzyme presently in Phase I-II clinical trial. Activity in normal tissues appeared to be low but very sensitive to BAU (approximately 90% inhibition at 10 microM). Tumors had generally 2-3-fold greater activity compared with adjacent normal tissues. In breast cancer specimens and head-neck squamous carcinomas, however, uridine cleavage was only partially inhibited (40-60%) by 10 or 100 microM BAU. The BAU-insensitive activity requires phosphate and pH conditions similar to the normal enzyme, and the new phosphorolytic activity was independent from thymidine phosphorylase. The BAU-insensitive phosphorolytic activity in selected tumors, coupled with the potent inhibitory activity of BAU against the "classical" uridine phosphorylase in normal human tissues, provides the rationale for combining BAU with 5-fluorouracil in the treatment of breast and head-neck tumors.     

Absence of association between a mitochondrial DNA mutation at nucleotide position 3243 and schizophrenia in Japanese

PURPOSE: To evaluate the effect that increased numbers of women medical school graduates have had on the composition of orthopedic surgery residencies, and to evaluate trends over time in the likelihood of women medical students to select orthopedic residencies. METHOD: The author analyzed JAMA's "Reports on Graduate and Undergraduate Medical Education" for the years 1977 to 1996, calculating the numbers of women and men in orthopedic surgery and other surgery residencies, and medical school composition. RESULTS: Although there have been modest gains in the number of women in orthopedic surgery training programs in the United States, women continue to choose orthopedics only one-seventh as often as do men. CONCLUSION: Orthopedics remains an unattractive career choice for women medical students compared with their men counterparts. Biases and stereotypes about women and about orthopedic surgery may account for this difference.