Effects of Dietary Copper Loading on Livers of Rats: I. Changes in Subcellular Acid Phosphatases and Detection of an Additional Acid p-Nitrophenylphosphatase in the Cellular Supernatant During Copper Loading

Significant changes occurred in lysosomal structure and function as copper was metabolized by rat livers. Hepatic total acid p-nitrophenylphosphatase (pNPase) activity was markedly increased in copper-loaded rats, and this increase was almost completely accounted for by heat- and formalin-stable (HFS) acid pNPase. Heat- and formalin-labile acid pNPase was essentially unchanged. On a subcellular level, the microsomal and supernatant fractions reflected the greatest relative increase in HFS acid pNPase. Increases in lysosomal, HFS acid pNPase in the large-granule fractions correlated with increase in solubilized large-granule enzymes, LG I, with mol wt > 200,000. LG II, representng solubilized large-granule enzymes with mol wt < 200,000, remained unchanged. Marked increases in supernatant acid pNPase were principally accounted for by a sevenfold increase in a supernatant lysosomal-like enzyme, DEAE Pk 1, separated by DEAE cellulose chromatography. An additional enzyme, DEAE Pk 2A′, that was hardly or not detectable in normal rats, was consistently demonstrated and increased in copper-loaded rats. Serum HFS acid pNPase increased in copper-loaded rats, suggesting that the increased hepatic supernatant acid pNPase in part escaped into the circulating fluid. Copper was principally associated with cytoplasmic organelles and was highest in mitochondrial and lysosomal fractions.     

Possible role for Toll-like receptors in interaction of Fasciola hepatica excretory/secretory products with bovine macrophages

Alternative activation of macrophages (M) during helminth infection is a characteristic feature of the host immune response. Alternatively activated macrophages (AAM) are distinguished from others by high arginase 1 (Arg-1) activity, low nitric oxide (NO), and high interleukin 10 (IL-10) production. In murine models, these cells have been shown to possess anti-inflammatory properties. They have also been implicated in exacerbating a subsequent infection with a secondary pathogen. In this study we used cattle experimentally infected with Fasciola hepatica to monitor the kinetics of IL-4 and IL-10 over the course of infection. Using naïve M in vitro, we examined the effects of exposure to F. hepatica excretory/secretory products (FhepES) alone or in combination with IL-4. Our results suggest that FhepES may work in combination with IL-4 to produce AAM. The effects of FhepES on the subsequent responses to lipopolysaccharide (LPS) and purified protein derivative from Mycobacterium bovis (PPD-B), which are bovine Toll-like receptor 4 (TLR4) and TLR2 antagonists, respectively, were also examined. We found that M stimulated with FhepES together with LPS or PPD-B have reduced NO or gamma interferon production, respectively. The ability of FhepES to produce AAM was found to be heat labile and partially dependent on glycan residues. A possible role for TLR recognition is discussed.     

Vibrio cholerae Hemagglutinin/Protease Inactivates CTXφ

Pathogenic strains of Vibrio cholerae are lysogens of the filamentous phage CTX, which carries the genes for cholera toxin (ctxAB). We found that the titers of infective CTX in culture supernatants of El Tor CTX lysogens increased rapidly during exponential growth but dropped to undetectable levels late in stationary-phase growth. When CTX transducing particles were mixed with stationary-phase culture supernatants of El Tor strains, CTX infectivity was destroyed. Our data indicate that this growth phase-regulated factor, designated CDF (CTX-destroying factor), is the secreted hemagglutinin/protease (HA/P) of V. cholerae. A strain containing a disrupted hap gene, which encodes HA/P of V. cholerae, did not produce CDF activity in culture supernatants. Introduction of the HA/P-expressing plasmid pCH2 restored CDF activity. Also, CDF activity in culture supernatants of a variety of pathogenic V. cholerae isolates varied widely but correlated with the levels of secreted HA/P, as measured by immunoblotting with anti-HA/P antibody. CDF was purified from V. cholerae culture supernatants and shown to contain a 45-kDa polypeptide which bound anti-HA/P antibodies and which comigrated with HA/P in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The production of high levels of secreted HA/P by certain V. cholerae strains may be a factor in preventing CTX reinfection in natural environments and in the human host.     

A Low Concentration of Ethanol Reduces the Chemiluminescence of Human Granulocytes and Monocytes but Not the Tumor Necrosis Factor Alpha Production by Monocytes after Endotoxin Stimulation

The ability of polymorphonuclear neutrophils (PMNs) and monocytes (M) to produce reactive oxygen species (ROS) has been related closely to their potential in the killing of microorganisms. Ethanol has been shown to impair the generation of ROS in these phagocytes after stimulation with some immunogens and to increase the susceptibility of alcohol abusers to infectious diseases. As endotoxemia is common in alcohol abusers, we investigated the effect of ethanol (21.7 mmol/liter) on the luminol-amplified chemiluminescence of PMNs and M after endotoxin stimulation and the release of tumor necrosis factor alpha (TNF-α) from M. Further, the efficiency of ethanol to inactivate chemically generated ROS was tested. Significant stimulation of ROS release occurred at endotoxin concentrations of 1 ng/ml or higher in both PMNs and M. Ethanol significantly suppressed the formation of ROS in both cell types, the decrease being more pronounced in M (−73.8%) than in PMNs (−45.7%). The correlations between endotoxin concentration and the amount of released ROS showed a dose-dependent, sigmoidal course. Concentrations of endotoxin necessary for half-maximum stimulation were nearly identical (6 to 8 ng/ml) in both PMNs and M, independent of the presence of ethanol. In contrast to ROS formation, ethanol had no effect on the amount of TNF-α produced by endotoxin-stimulated M. Ethanol was shown to be unable to decrease the levels of chemically generated ROS under physiological conditions. Therefore, ethanol cannot be assumed to be an “antioxidative” compound but rather seems to modify processes of endotoxin recognition, intracellular signal transduction, or metabolism.     

Lutzomyia longipalpis salivary gland homogenate impairs cytokine production and costimulatory molecule expression on human monocytes and dendritic cells

In this report, we describe an investigation of the effects of Lutzomyia longipalpis sand fly salivary gland homogenates (SGH) on cytokine production and expression of costimulatory molecules on human monocytes, macrophages (Ms), and dendritic cells (DCs). SGH of L. longipalpis induced an increase in interleukin-6 (IL-6), IL-8 and IL-12p40 production but a decrease in tumor necrosis factor alpha and IL-10 production by lipopolysaccharida (LPS)-stimulated monocytes. We also examined the expression of costimulatory molecules on the surface of monocytes, Ms, and DCs. Whereas SGH affected the expression of these molecules on monocytes and Ms, it had little effect on these molecules on DCs. However, when DCs were generated from human monocytes in the presence of SGH, SGH inhibited the expression of costimulatory molecules. In addition, a decrease in the maturation of DCs induced by CD40L was observed in the presence of SGH. Finally, preincubating SGH with human sera containing anti-SGH-specific antibodies abolished the effects of SGH on cytokine production by LPS-stimulated monocytes.     

Activation of human monocytes by both human β1H and C3b

We tested whether highly purified human β1H and C3b, two proteins of the alternative pathway of complement activation, could exert an influence on the activity of human monocytes (M). The activation process of M was assessed by measurements of the respiratory burst in terms of nitro blue tetrazolium (NBT) reduction and by chemiluminescence (CL) tests. In NBT reduction experiments, we found a tendency for β1H to increase NBT reduction, while C3b was found to be rather inhibitory. In CL measurements, both β1H and C3b displayed a stimulatory effect on M, showing different time- and dose-dependency. For β1H, the maximum stimulation occurred after 15 min, whereas for C3b after 45 min. Zymosan particles which served as a positive control also showed the highest stimulation after 45 min. In dose—response experiments, β1H reached a plateau ranging from 30 to 80 μg/ml. In contrast, using C3b up to 170 μg/ml, no plateau was reached. M-depletion and enrichment studies suggested at M as being responsible for the stimulatory effects found.

The differences between NBT reduction and CL could possibly be explained by the measurement of only cell-bound reductive potentials by NBT reduction, while in CL measurements, products of the extracellular space are also assessed. Our results suggest that both human β1H and C3b are appropriate stimuli for human monocytes.

     

Validation of an In-House Assay for Cytomegalovirus Immunoglobulin G (CMV IgG) Avidity and Relationship of Avidity to CMV IgM Levels

Measurement of cytomegalovirus (CMV)-specific immunoglobulin G (IgG) avidity has proven to be a powerful tool for distinguishing primary from nonprimary CMV infection. An in-house enzyme-linked immunosorbent assay (ELISA) for measuring CMV IgG avidity was validated using 84 sera from pregnant women who had recently seroconverted following primary CMV infection and 74 sera from individuals with past CMV infection (IgG-positive and IgM-negative profile). Of the 84 sera from pregnant women, 73 sera were collected within 120 days of the last IgG-negative sample, and 72 of these 73 sera (99%) exhibited an avidity index (AI) of <50%. In contrast, 71 of 74 (96%) sera from individuals with past CMV infection exhibited CMV AI values of >60%. Thus, low avidity in the in-house ELISA was defined as an AI of 50%, whereas high avidity was defined as an AI of 60%. In additional studies, the relationship between CMV IgG avidity and CMV IgM levels was examined using 64 CMV IgG-positive sera (time since seroconversion unknown) exhibiting equivocal or positive results in a CMV IgM capture ELISA (Diamedix). Of these 64 sera, 29 exhibited IgM index values of 3.0, and 27 of these 29 (93%) exhibited low IgG avidity. A similar trend was observed when a subset of these 64 sera (n = 48) was tested in another CMV IgM capture ELISA (Trinity); of 18 sera with IgM index values of 3.0, 17 (94%) exhibited low IgG avidity. These findings demonstrate the validity of an in-house ELISA for CMV IgG avidity and further show that strong reactivity of CMV IgG-positive sera in either of two CMV IgM capture assays is a reliable indicator of low CMV IgG avidity, and thus, recent CMV infection.     

Studies on IgA of the guinea-pig

The secretory immunoglobulin of the guinea-pig, IgA, was antigenically unique when compared with the 7Sγ₁-, 7Sγ₂- and γM-globulins, although it did share Fab determinants in common with the 7Sγ₂ globulin. Specific antigen binding capacity was detected in serum IgA after sequential oral and parenteral sensitization with purified protein antigens. IgA was prominent in saliva and succus entericus; in colostrum its concentration was 4–8 times that in normal serum. IgA in serum and secretory fluids sedimented at similar rates (12S), although colostral IgA contained an additional 16S population. Serum and secretory IgA appeared antigenically identical, however, serum IgA was especially sensitive to mild reductive procedures and became 7S after treatment. Serum IgA migrated as a β-protein on electrophoresis and was faster than IgA of colostrum. Antisera to IgA were produced by a procedure which involved simple precipitation of secretory IgA with an antiserum containing antibodies to common determinants of guinea-pig Ig.     

Comparative Sequence of Human and Mouse BAC Clones from the mnd2 Region of Chromosome 2p13

The mnd2 mutation on mouse chromosome 6 produces a progressive neuromuscular disorder. To determine the gene content of the 400-kb mnd2 nonrecombinant region, we sequenced 108 kb of mouse genomic DNA and 92 kb of human genomic sequence from the corresponding region of chromosome 2p13.3. Three genes with the indicated sizes and intergenic distances were identified: D6Mm5e (81 kb)–787 bp–DOK (2 kb)–845 bp–LOR2 (6 kb). D6Mm5e is expressed in many tissues at very low abundance and the predicted 526-residue protein contains no known functional domains. DOK encodes the p62^dok rasGAP binding protein involved in signal transduction. LOR2 encodes a novel lysyl oxidase-related protein of 757 amino acid residues. We describe a simple search protocol for identification of conserved internal exons in genomic sequence. Evolutionary conservation proved to be a useful criterion for distinguishing between authentic exons and artifactual products obtained by exon amplification, RT–PCR, and 5′ RACE. Conserved noncoding sequence elements longer than 80 bp with 75% nucleotide sequence identity comprise ~1% of the genomic sequence in this region. Comparative analysis of this human and mouse genomic DNA sequence was an efficient method for gene identification and is independent of developmental stage or quantitative level of gene expression.     

Development of a mammalian fast muscle: dynamic and biochemical properties correlated

1. The isometric properties and related biochemical properties of a developing rat fast muscle (extensor digitorum longus, EDL) have been determined during the 21 days after birth.

2. At birth the maximum rate of rise of tension in a tetanus, a measure of speed of contraction, is slow; it begins to increase only after the 5th day and reaches adult values by 21 days.

3. The increase in actomyosin ATPase activity of the rat EDL correlates closely (P 0·001) with the changes in the maximum rate of rise during the same period of development.

4. The relaxation phase of a tetanic response begins to increase in speed promptly after birth, and reaches adult values by the 21st day. The rate of calcium accumulation by the isolated sarcoplasmic reticulum (SR) increases with a similar time course.

5. The separate contributions of contraction and relaxation mechanisms to the changes in the more conventional isometric properties of maturing muscles have been analysed.

     

Analysis of the Humoral Immune Response to Chlamydia Outer Membrane Protein 2

The humoral immune response to Chlamydia outer membrane protein 2 (Omp2) was studied. Omp2 is a highly genus-conserved structural protein of all Chlamydia species, containing a variable N-terminal fragment. To analyze where the immunogenic parts were localized, seven highly purified truncated fusion proteins constituting different regions of the protein were produced (Chlamydia pneumoniae-Omp2_aa23-aa93, Chlamydia psittaci-Omp2_aa23-aa94, and Chlamydia trachomatis-Omp2_{aa23-aa84, aa87-aa547, aa23-aa182, aa167-aa434, aa420-aa547}). By an enzyme-linked immunosorbent assay with serologically defined patient sera, Omp2 was found to be a major immunogen of both C. pneumoniae and C. trachomatis infections (P 0.0001). The humoral immune responses were not confined to any particular region of the Omp2 protein, and no species-specific anti-Omp2 immunoglobulins were detected.     

Increased Levels of ɛ and γ Isoforms of 14-3-3 Proteins in Cerebrospinal Fluid in Patients with Creutzfeldt-Jakob Disease

We established four hybridoma cell lines producing monoclonal antibodies (MAbs) against 14-3-3 proteins. Immunoblot analysis revealed that and γ isoforms were specifically increased in premortem cerebrospinal fluid samples from patients with sporadic Creutzfeldt-Jakob disease. Furthermore, dot immunoblot analysis showed that MAbs were more specific for native antigen than polyclonal antibodies were.     

Hemoglobin Toxicity in Experimental Bacterial Peritonitis Is Due to Production of Reactive Oxygen Species

Hemoglobin (Hb) is a toxic molecule responsible for the extreme lethality associated with experimental Escherichia coli peritonitis, but the mechanism has yet to be elucidated. Hb, but not globin, showed toxic effects in a live E. coli model but not in a model using killed E. coli. Methemoglobin, hematin, and the well-known Fenton reagents iron and iron-EDTA demonstrated the same lethal effect in E. coli peritonitis as Hb, while the addition of the Fenton inhibitors desferrioxamine (DF) and diethylenetriamine pentaacetate removed most of the cytotoxic activity of iron. Administration of a combined dose of superoxide dismutase and catalase minimized the action of Hb and iron-EDTA, suggesting that both O₂⁻ and H₂O₂ are involved in the toxic action of Hb in this rat model. The combination of the antioxidative enzymes and DF further suppressed iron-mediated lethality. An electron spin resonance technique with the spin-trapping reagent 5,5-dimethyl-1-pyroline-N-oxide (DMPO) showed O₂⁻ generation in the peritoneal fluid of rats injected with E. coli alone or E. coli plus iron-DF, and OH generation was detected in the peritoneal fluid of the rats injected with iron-EDTA. Hb did not show any spin adduct of oxygen radicals, suggesting that Hb produces non-spin-trapping radical ferryl ion, which decayed the spin adduct of DMPO. In the presence of Hb or iron-EDTA, O₂⁻-generating activity and viability of phagocytes decreased, whereas lipid peroxidation of peritoneal phagocytes increased. Generation of oxygen radicals and lipid peroxidation did not differ in the live and dead bacterial models. Bacterial numbers in the peritoneal cavity and blood were markedly increased in the live bacterial model with Hb and iron-EDTA. The Fenton inhibitor iron-DF prevented the loss of phagocyte function, lipid peroxidation, and bacterial proliferation. These results led us to conclude that the lethal toxicity of Hb in bacterial peritonitis is associated with a Fenton-type reaction, the products of which decrease phagocyte viability, through the induction of lipid peroxidation, allowing bacterial proliferation and resulting in mortality.     

Evaluation of an Immunochromatographic Assay Kit for Rapid Identification of Mycobacterium tuberculosis Complex in Clinical Isolates

We evaluated a new immunochromatographic assay (ICA) using mouse monoclonal anti-MPT64 antibody for rapid discrimination between Mycobacterium tuberculosis and nontuberculous mycobacteria in clinical isolates. A study with mycobacteria and other organisms showed excellent sensitivity (99%) and specificity (100%) and an appropriate detection limit (10⁵ CFU/ml) when tested with M. tuberculosis H37Rv. This ICA can simplify the identification of M. tuberculosis in clinical laboratories.     

Growth in North American white children with neurofibromatosis 1 (NF1)

OBJECTIVE—To analyse the distributions of and generate growth charts for stature and occipitofrontal circumference (OFC) in neurofibromatosis 1 (NF1) patients.
DESIGN—Cross sectional database survey.
SETTING—The National Neurofibromatosis Foundation International Database (NFDB) includes clinical information on NF1 patients from 14 participating centres in North America.
SUBJECTS—A total of 569 white, North American, NF1 patients, 55% female and 45% male.
MAIN OUTCOME MEASURES—Stature and OFC measurements of NF1 patients were compared to age and sex matched population norms using z score standardisation and centile curves.
RESULTS—The distributions of stature and OFC are shifted and unimodal among NF1 patients; 13% of patients have short stature (2 standard deviations below the population mean) and 24% have macrocephaly (OFC 2 standard deviations above the population mean).
CONCLUSIONS—Alterations of stature and OFC are not limited to NF1 patients with frank short stature or macrocephaly.


Keywords: neurofibromatosis 1; stature; occipitofrontal circumference; macrocephaly     

Effects of Porcine Circovirus Type 2 (PCV2) Maternal Antibodies on Experimental Infection of Piglets with PCV2

To determine the effects of porcine circovirus type 2 (PCV2) maternal antibodies on and response to experimental PCV2 infection, 24 piglets were divided into four groups on the basis of the enzyme-linked immunosorbent assay titers of PCV2 maternal antibodies: group A (n = 6; sample/positive [S/P] ratio, <0.2), group B (n = 5; S/P ratio, >0.2 to <0.5), and groups C (n = 8) and D (n = 5) (S/P ratio, >0.5). Piglets in groups A, B, and C were inoculated with PCV2 at day 0 and challenged with PCV2 at day 42. Group D piglets were not exposed to PCV2 at day 0 but were challenged at day 42. Before challenge, seroconversion to PCV2 antibodies occurred in fiveofsix group A piglets, and the antibody level rose above the cutoff level in oneoffive group B piglets. Viremia was detected in fiveofsix, fouroffive, and twoofeight pigs in groups A, B, and C, respectively. After challenge, PCV2 DNA was detectable from 7 to 21 days postchallenge in the sera from sixofsix, fouroffive, threeofeight, and fiveoffive pigs in groups A, B, C, and D, respectively. The results indicated that protection against PCV2 infection conferred by maternal antibodies is titer dependent: higher titers are generally protective, but low titers are not.     

Different modulation by histamine of IL-4 and interferon-gamma (IFN-γ) release according to the phenotype of human Th0, Th1 and Th2 clones

Histamine, an important inflammatory mediator in allergic diseases and asthma, has been reported to have modulator effects on T cells, suggesting that the bronchial microenvironment may regulate the function of resident T cells. We examined the effect of histamine on the release of the Th2-associated cytokines IL-4 and IL-5 and the Th1-associated cytokine IFN-γ by 30 CD4⁺ T cell clones from peripheral blood or bronchial biopsy of one atopic subject. Based on the IL-4/IFN-γ ratio, the clones were ascribed to the Th2 (ratio >1), Th0 (ratio 0.1 and 1) or Th1 (ratio <0.1) phenotype. Histamine inhibited IFN-γ production by Th1-like cells (P<0.02, Kruskall–Wallis), especially from bronchial biopsy, but had no effect on IL-4 release. Regarding Th0 clones, histamine inhibited IL-4 production (P<0.02) in a dose-dependent manner and slightly inhibited IFN-γ production, but had no effect on Th2-like cells. Histamine had a heterogeneous and insignificant effect on IL-5 production. The H₂-receptor antagonist ranitidine completely reversed the inhibition of IL-4 and IFN-γ production, whereas the agonist dimaprit mimicked this effect. In contrast, H₁- and H₃-receptor agonists and antagonists had no significant effect. These data demonstrate that histamine has different effects on IL-4 and IFN-γ release by T helper cells according to their phenotype via H₂-receptors. This study extends the immunomodulatory effects of histamine which may contribute to the perpetuation of airway inflammation in asthma.     

Association of TPH1 with suicidal behaviour and psychiatric disorders in the Chinese population

Tryptophan hydroxylase (TPH), the rate limiting enzyme in serotonin biosynthesis, is one of the most important regulating factors in the serotonergic system. Recently, polymorphisms of the TPH gene have been identified as being associated with suicide, but the evidence is inconsistent. To investigate the role in suicide of one of the isoforms, TPH1, we examined the association of five single nucleotide polymorphisms (SNPs) in the promoter region and in intron 7 of the TPH1 gene based on a sample from the Chinese population of 810 subjects, of whom 329 had made no suicide attempts (NSA), 297 had made suicide attempts (SA), and 184 were healthy subjects (HS). In this study, we observed statistically significant differences between NSA and HS subjects in allele distributions on one marker, −6526A (p=0.0329; odds ratio (OR) 1.36; 95% confidence interval (CI) 1.01 to 1.81). No significant difference in genotype distribution or allele frequencies of other polymorphisms was found between the suicide victims and the controls. The overall haplotype frequency was significantly different between cases and healthy controls (p=0.000024 NSA v HS; p<0.000001, SA v HS; p<0.000001, cases v HS). We found the haplotype TCAAA of −7180/−7065/−6526/218/779 to be strongly associated with suicidal behaviour and psychiatric disorders (p=0.00243; OR=1.62; 95% CI 1.17 to 2.24 and p=0.018; OR=1.41; 95% CI 1.05 to 1.91), which suggests an association of TPH1 with suicidal behaviour and indicates that TPH1 may play a significant role in the aetiology of psychiatric disorders in the Han Chinese population.     

Sour Cherry (Prunus cerasus L) Anthocyanins as Ingredients for Functional Foods

In the recent years many studies on anthocyaninshave revealed their strong antioxidant activity and their possibleuse as chemotherapeutics. The finding that sour cherries(Prunus cerasus L) (also called tart cherries) containhigh levels of anthocyanins that possess strong antioxidant andanti-inflammatory properties has attracted muchattention to this species. Here we report the preliminary resultsof the induction of anthocyanin biosynthesis in sour cherrycallus cell cultures. The evaluation and characterization of thein vitro produced pigments are compared to those of theanthocyanins found in vivo in fruits of several sour cherrycultivars. Interestingly, the anthocyanin profiles found in wholefruit extracts were similar in all tested genotypes but weredifferent with respect to the callus extract. The evaluation ofantioxidant activity, performed by ORAC and TEAC assays, revealeda relatively high antioxidant capacity for the fruitextracts (from 1145 to 2592μmol TE/100g FW) and alower one for the callus extract (688μmol TE/100g FW).     

Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence

The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10⁻⁶ must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or “PCR noise”. Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5×10⁻⁷, and five of its more frequent mutations (hot spots) consisted of three transversions (GC→TA, AT→TA, and AT→CG), one transition (AT→GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be reduced.