A new physiological method for heart rate correction of the QT interval

AIM: To reassess QT interval rate correction. BACKGROUND: The QT interval is strongly and inversely related to heart rate. To compare QT intervals between different subjects with different heart rates requires the application of a QT interval rate correction formula. To date these formulae have inappropriately assumed a fixed relation between QT interval and heart rate. An alternative method of QT interval rate correction that makes no assumptions about the QT interval-heart rate relation is needed. PROPOSAL: A QT heart rate correction method should maintain or accentuate biological QT interval variability, should totally remove the dependence of the rate corrected QT interval on heart rate, and should be applicable over a wide range of conditions with a wide range of differing autonomic states. METHODS: QT intervals were obtained at rest and during exercise from subjects expected to have different QT intervals and different QT interval-heart rate relations. A linear regression line was obtained from the exercise test data, and the QT interval at a notional heart rate of 60 and 0 beats/min, termed the QT(60) interval, and the QT y intercept obtained by back calculation. RESULTS: QT(60) and QT y intercept values were prolonged in heart failure compared with either left ventricular hypertrophy or controls. There was no relation between heart rate and either QT(60) or QT y intercept CONCLUSIONS: This new physiologically based method of correcting QT interval for heart rate removes the dependence of the corrected QT interval on heart rate, and maintains biological differences.     

From the Cover: PNAS Plus: Changes in genetic selection differentials and generation intervals in US Holstein dairy cattle as a result of genomic selection

Seven years after the introduction of genomic selection in the United States, it is now possible to evaluate the impact of this technology on the population. Selection differential(s) (SD) and generation interval(s) (GI) were characterized in a four-path selection model that included sire(s) of bulls (SB), sire(s) of cows (SC), dam(s) of bulls (DB), and dam(s) of cows (DC). Changes in SD over time were estimated for milk, fat, and protein yield; somatic cell score (SCS); productive life (PL); and daughter pregnancy rate (DPR) for the Holstein breed. In the period following implementation of genomic selection, dramatic reductions were seen in GI, especially the SB and SC paths. The SB GI reduced from ∼7 y to less than 2.5 y, and the DB GI fell from about 4 y to nearly 2.5 y. SD were relatively stable for yield traits, although modest gains were noted in recent years. The most dramatic response to genomic selection was observed for the lowly heritable traits DPR, PL, and SCS. Genetic trends changed from close to zero to large and favorable, resulting in rapid genetic improvement in fertility, lifespan, and health in a breed where these traits eroded over time. These results clearly demonstrate the positive impact of genomic selection in US dairy cattle, even though this technology has only been in use for a short time. Based on the four-path selection model, rates of genetic gain per year increased from ∼50–100% for yield traits and from threefold to fourfold for lowly heritable traits.Genetic improvement of livestock during the second half of the 20th century using pedigree and performance data has been very successful, particularly in dairy cattle populations (e.g., ref. 1). The improvement of dairy cattle has depended heavily on the use of artificial insemination (AI) to maximize the impact of elite bulls globally. Historically, progeny testing (2), or the characterization of these AI bulls by measuring and comparing performance of daughters, has been a critical step in identifying the very best bulls for widespread use. However, traditional genetic improvement schemes in dairy cattle have been limited by time required and expense of the progeny test paradigm. This process remained relatively slow because of the substantial time needed to accumulate sufficient daughter phenotypes to compute genetic evaluations with high accuracy. The recent development of genomic selection (3) programs based on single-nucleotide polymorphism genotypes was expected to increase rates of genetic gain (3, 4) in several ways, including shortened generation interval(s) (GI) (5, 6) and increased reliability of predicted breeding value(s) (PBV) (7). A doubling of rates of genetic gain was predicted when comparing genomic evaluations and traditional progeny testing schemes (5, 6, 8, 9). These advantages have been demonstrated in simulations (10, 11), and increased accuracies have been documented in the US Holstein population (12), but response to the incorporation of genomic data into dairy cattle evaluations has not been characterized.In April 2008, the United States released its first unofficial genomic PBV, and official evaluations for Holsteins and Jerseys were published in January 2009 (9). Genomic selection was rapidly adopted by the industry, and more than half of all AI matings in the United States are now made to genomically tested young bulls (13). Genomic breeding values have been available for 6 y, so a characterization was conducted of the dynamic changes in rates of genetic gain associated with alterations in GI and selection differential(s) (SD). Rendel and Robertson (14) described a four-path model of genetic improvement in which genetic progress occurs with differing selection dynamics, partitioned into improvement due to genetic changes in sire(s) of bulls (SB), sire(s) of cows (SC), dam(s) of bulls (DB), and dam(s) of cows (DC). The objective of this study was to measure the impact of genomic selection on SD and GI in US Holstein cattle using this four-path model, and to compare these observed results with those results predicted by theory.     

Comparison of the anticoagulant effect of a direct thrombin inhibitor and a low molecular weight heparin in an acquired antithrombin deficiency in children with acute lymphoblastic leukaemia treated with L-asparaginase: an in vitro study

Thrombosis occurs in 37% of children with acute lymphoblastic leukaemia (ALL) and is related to an L-asparaginase-induced acquired antithrombin (AT) deficiency. The incidence dictates the need for anticoagulant prophylaxis. Direct thrombin inhibitors (DTI) are independent of AT for effect and may thus have advantages in this population. The objective of this study was to determine the interaction of an AT deficiency with the anticoagulant effects of a DTI and a low molecular weight heparin (LMWH). Plasma samples from children with ALL were pooled (mean AT 0.53 U/ml). LMWH 0.3 and 0.7 U/ml or melagatran 0.3 and 0.5 micromol/l were added to the pools, then divided and AT was added back to one aliquot. In additional experiments, AT was added to AT immuno-depleted plasma. Endogenous thrombin generation capacity (ETGC) was assessed by the continuous method. In plasma with LMWH, there was a 66-88% decrease in ETGC in AT-normalised samples compared with neat. Conversely, no significant difference in ETGC with or without AT added for melagatran was seen. Experiments with AT-depleted plasma showed no effect of AT level on anticoagulant activity of DTI, but a significant relationship for LMWH. By contrast to LMWH, DTI provides a consistent anticoagulant response independent of AT levels in children with AT deficiency.     

Natural course of a prolonged PR interval and the relation between PR and incidence of coronary heart disease. A 7-year follow-up study of 1832 apparently healthy men aged 40-59 years

During a baseline cardiovascular survey PR was measured in a strictly standardized way in 1832 men aged 40-59 years, free from coronary heart disease (CHD). Of 1758 men still alive, 1585 underwent an identical follow-up study 7 years later. A total of 1570 were in sinus rhythm. The following findings were made: (1) Baseline and follow-up prevalence of a prolonged PR (greater than or equal to 0.22 s) was identical (5.3 vs. 5.4%). (2) Only 60% of restudied men with a prolonged PR also had prolonged PR at follow-up. (3) Only 1 of 98 with a prolonged baseline PR had a more advanced AV block at follow-up, whereas an additional 4 had conditions which might influence the AV node (1 Bechterew's disease and 3 mild aortic valve stenosis). (4) The incidence of all CHD events found during the follow-up study (CHD deaths, myocardial infarction, angina pectoris, and pathologic exercise ECGs) was moderately but significantly lower in men with a prolonged PR than among men with a PR less than or equal to 0.21 s. Thus a prolonged PR is rarely an indicator of impending, more severe conduction disturbances; it is mostly a benign, functional finding in middle-aged men free from overt heart disease and is not positively associated with CHD. Rather PR may be moderately and inversely associated with latent CHD.     

Targeted disruption of FANCC and FANCG in human cancer provides a preclinical model for specific therapeutic options

BACKGROUND & AIMS: How specifically to treat pancreatic and other cancers harboring Fanconi anemia gene mutations has raised great interest recently, yet preclinical studies have been hampered by the lack of well-controlled human cancer models. METHODS: We endogenously disrupted FANCC and FANCG in a human adenocarcinoma cell line and determined the impact of these genes on drug sensitivity, irradiation sensitivity, and genome maintenance. RESULTS: FANCC and FANCG disruption abrogated FANCD2 monoubiquitination, confirming an impaired Fanconi anemia pathway function. On treatment with DNA interstrand-cross-linking agents, FANCC and FANCG disruption caused increased clastogenic damage, G2/M arrest, and decreased proliferation. The extent of hypersensitivity varied among agents, with ratios of inhibitory concentration 50% ranging from 2-fold for oxaliplatin to 14-fold for melphalan, a drug infrequently used in solid tumors. No hypersensitivity was observed on gemcitabine, etoposide, 3-aminobenzamide, NU1025, or hydrogen peroxide. FANCC and FANCG disruption also resulted in increased clastogenic damage on irradiation, but only FANCG disruption caused a subsequent decrease in relative survival. Finally, FANCC and FANCG disruption increased spontaneous chromosomal breakage, supporting the role of these genes in genome maintenance and likely explaining why they are mutated in sporadic cancer. CONCLUSIONS: Our human cancer cell model provides optimal controls to elucidate fundamental biologic features of individual Fanconi anemia gene defects and facilitates preclinical studies of therapeutic options. The impact of Fanconi gene defects on drug and irradiation sensitivity renders these genes promising targets for a specific, genotype-based therapy for individual cancer patients, providing a strong rationale for clinical trials.     

Establishment of a reference interval for natural killer cell activity through flow cytometry and its clinical application in the diagnosis of hemophagocytic lymphohistiocytosis

Recently, the Histiocyte Society revised the diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH) to include low or absent natural killer (NK) cell activity, according to local laboratory reference. The aim of this study was to establish reference interval for functional NK-cell activity in 63 healthy Korean individuals using a flow-cytometric assay. We used peripheral blood mononuclear cells (PBMCs) as effector cells and Fluorescein isothiocyanate-labeled K562 cells as target cells. NK-cell activity was calculated using the following equation: NK-cell activity (%) = (test lysis − spontaneous lysis) × 100/(maximum lysis − spontaneous lysis). NK-cell activity was analyzed in 13 known HLH patients and 16 suspected non-HLH patients using a flow-cytometric assay. The mean (±SD) cytotoxicity of PBMCs from healthy individuals was 20.9 ± 5.3% and the reference interval was 11.8–31.9%. The mean NK-cell activity of HLH patients (8.3 ± 8.9%) was significantly lower (P = 0.001) than that of non-HLH patients (20.1 ± 7.8%). The sequential changes in NK-cell activity in the HLH group corresponded to clinical and laboratory findings following treatment. We successfully developed a functional NK-cell activity test for use in the clinical laboratory and obtained a reference interval of NK-cell activity from healthy donors. This assay, and associated reference interval, was used to analyze 30 clinically relevant specimens and the results were shown to be well correlated.     

Major surgery in a severe haemophilia A patient with high titre inhibitor: use of the thrombin generation test in the therapeutic decision

Summary. In haemophilia patients with inhibitor, elective orthopaedic surgery is usually performed under recombinant activated factor VII (rFVIIa). We report here the case of a severe haemophilia A patient with a high inhibitor who needed a bilateral total knee arthroplasty. Recombinant FVIIa was previously shown to be ineffective for the treatment of muscle and joint bleedings, and he had a history of excessive postoperative bleeding under activated prothrombin complex concentrate (APCC). Thrombin generation test (TGT) was used to assess the efficacy of Factor Eight Inhibitor Bypassing Activity (FEIBA). Insufficient correction of thrombin-generating capacity was observed after administration of 75 U kg(-1) FEIBA. In a multidisciplinary environment, a bilateral total knee arthroplasty was performed using a protocol combining immunoadsorption of inhibitors preoperatively associated with FVIII replacement during a first phase followed by FEIBA when the inhibitor reappeared. To our knowledge this is the first direct application of TGT in the management of haemophilia patients with inhibitor, which indicated that a sequential use of immunoadsorption, FVIII and FEIBA was the most appropriate treatment to perform this major elective surgery. This case demonstrates that this combined protocol can be safely used to cover major surgery in inhibitor patients. In addition, it also suggests that TGT may have a major contribution in the decision-making process of the most adapted therapy for the treatment of such high-risk patients.     

分泌性重组人干扰素α-2a治疗慢性乙型肝炎患者疗效和安全性的研究

目的 观察国产分泌性干扰素治疗慢性乙型肝炎的疗效与安全性。方法 采用随机、开放、对照、多中心的研究方法，以国产普通干扰素作为对照药物，观察分泌性干扰素治疗慢性乙型肝炎6个月，停药随访6个月的疗效与安全性。结果 分泌性干扰素在治疗24周时，丙氨酸氨基转移酶(ALT)复常率为48．3％，优于对照组(35．7％)，但随访结束时两组差异无显著性。治疗后分泌性干扰素组的HBVDNA下降幅度优于对照组，但转阴率两组间差异无显著性。治疗结束时分泌性干扰素组与对照组的乙型肝炎e抗原(HBeAg)转阴率分别为26．5％和l9．4％，HBeAg血清转换率分别为13．5％和12．0％；随访结束时两组的HBeAg转阴率继续增加，分别为32．5％与27．2％，HBeAg血清转换率分别为19．0％和18．4％。两种干扰素的安全性良好。结论 分泌性干扰素治疗结束时在ALT复常率、HBVDNA下降幅度方面优于普通干扰素，随访结束时两组间差异无显著性。在HBeAg转阴率和HBeAg血清转换率方面两种干扰素差异无显著性。两种干扰素安全性良好。     

A simple method for the routine detection of somatic quantitative genetic alterations in colorectal cancer

BACKGROUND & AIMS: Several quantitative genetic alterations have been suggested to have in colorectal cancer (CRC) either a prognostic or a therapeutic predictive value. Routine detection of these alterations is limited by the absence of simple methods. METHODS: The somatic quantitative multiplex polymerase chain reaction of short fluorescent fragments (QMPSF) is based on the simultaneous amplification under quantitative conditions of several dye-labeled targets both from tumor and nonmalignant tissues. For each patient, the resulting QMPSF fluorescent profiles are superimposed, and quantitative changes are simply detected by an increase or decrease of the corresponding fluorescent peaks. Two assays were developed and applied to 57 CRC: a "bar code" exploring several loci with known prognostic value and a "kinogram" studying the copy number change of kinase genes, against which inhibitors have been developed. RESULTS: The bar code revealed that the most frequent alterations were the gain of AURKA/20q13 (53%) and MYC/8q24 (39%) and heterozygous deletion of DCC/18q21.3 (39%) and TP53/17p13 (23%). The kinogram detected a gene copy number increase for AURKA, PTK2, MET, and EGFR in 53%, 37%, 33%, and 28% of the tumors, respectively. QMPSF results were validated by comparative genomic hybridization and multiplex real-time polymerase chain reaction on genomic DNA. CONCLUSIONS: The somatic QMPSF is a simple method able to detect simultaneously on a routine basis several quantitative changes in tumors. Its flexibility will allow the integration of clinically relevant genes. This high throughput method should be a valuable complementary tool of fluorescent in situ hybridization and comparative genomic hybridization.     

Comparison of the performance of two spontaneous sedimentation techniques for the diagnosis of human intestinal parasites in the absence of a gold standard

Performance evaluation of diagnostic tests is critical in the search for accurate diagnoses. A gold standard test is usually absent in parasitology, thus rendering satisfactory assessment of diagnostic accuracy difficult. Moreover, reliability (assessed by the study of repeatability) is a rarely studied characteristic of diagnostic tests. This study compared and evaluated the performance (repeatability, concordance and accuracy) of the spontaneous sedimentation technique (SST) and the Paratest for the diagnosis of Giardia lamblia, Entamoeba histolytica complex, Blastocystis spp., Ascaris lumbricoides, hookworm, Trichuris trichiura and Calodium hepaticum. Fecal samples of 143 individuals were separated into three replicates for each test. Concordance and homogeneity of the results between replicates of each test and between tests were evaluated. Proportions of positives, sensitivity and specificity were estimated using a Bayesian Latent Class Model. High repeatability of both tests was found for the detection of intestinal parasites, except for Blastocystis spp. and hookworm. Concordance between tests was generally high (concordance correlation coefficient, 0.72–0.88), except for Blastocystis spp., hookworm and T. trichiura. The Paratest detected more cases of Blastocystis spp. and fewer of hookworm than the SST. The tests were quite discordant in the detection of T. trichiura. A low sensitivity (39.4–49.2% for SST, 35.8–53.8% for Paratest) and a high specificity (93.2–97.2%) were found for both tests. The Paratest presented a slightly higher sensitivity for the diagnosis of Blastocystis spp. (53.8%), and SST did so for hookworm (49.2%). This is the first study on repeatability and accuracy (using a Bayesian approach) of two spontaneous sedimentation techniques. These results suggest underdiagnosis of little dense parasitic forms due to technical limitations in both tests. We conclude that the combined study of repeatability, concordance and accuracy is a key strategy for better evaluation of the performance of tests and is also useful for the identification of technical limitations.     

The Past as a Lens for Biodiversity Conservation on a Dynamically Changing Planet: Changes in the functional diversity of modern bird species over the last million years

Despite evidence of declining biosphere integrity, we currently lack understanding of how the functional diversity associated with changes in abundance among ecological communities has varied over time and before widespread human disturbances. We combine morphological, ecological, and life-history trait data for >260 extant bird species with genomic-based estimates of changing effective population size (N_e) to quantify demographic-based shifts in avian functional diversity over the past million years and under pre-anthropogenic climate warming. We show that functional diversity was relatively stable over this period, but underwent significant changes in some key areas of trait space due to changing species abundances. Our results suggest that patterns of population decline over the Pleistocene have been concentrated in particular regions of trait space associated with extreme reproductive strategies and low dispersal ability, consistent with an overall erosion of functional diversity. Further, species most sensitive to climate warming occupied a relatively narrow region of functional space, indicating that the largest potential population increases and decreases under climate change will occur among species with relatively similar trait sets. Overall, our results identify fluctuations in functional space of extant species over evolutionary timescales and represent the demographic-based vulnerability of different regions of functional space among these taxa. The integration of paleodemographic dynamics with functional trait data enhances our ability to quantify losses of biosphere integrity before anthropogenic disturbances and attribute contemporary biodiversity loss to different drivers over time.

The safeguarding of biological diversity is one of the society’s most pressing challenges (1), but our understanding of its decline and consequences is still limited and often constrained by the spatial and temporal scales at which natural history studies are conducted. Despite such constraints, novel opportunities exist for using information from the Quaternary period (~2.6 Mya–present) to inform conservation practices and policies in the context of climate change, including providing natural baselines of biotic responses and quantifying the vulnerability of the natural world to varying rates and magnitudes of climate change (2). Exploring biodiversity responses during pre-human periods allows us to more clearly infer the impact of climate change without the additional effects of other anthropogenic pressures and thus tackle some of the challenges posed by the difficulty in accurately attributing observed changes to different drivers of biodiversity change (3–5). This “attribution conundrum” exists in part because collinearity, scale dependencies, or interactions among the different potential drivers of biodiversity change (e.g., ongoing climate change, habitat loss, over-exploitation) may obscure their individual effects on species declines across taxa.In addition to their overall effects on species declines across taxa, drivers of biodiversity change can also cause changes in the relative abundance of different species, shifting representation of key functional traits (i.e., the morphological, behavioral, or physiological features of organisms that can affect their fitness) in communities and leading to overall erosion of functional diversity (i.e., the variation among such features) over time (6–8). The loss of functional diversity associated with both species extinctions and overall declines in abundance can lead to declines in the functioning and stability of ecosystems over time, particularly during periods of broad-scale environmental variation such as climate change (9–11). An overrepresentation of species exhibiting similar traits can buffer these effects of declining abundances, allowing key ecosystem functions to be maintained during periods of biodiversity loss. Indeed, over the past century, changes in species abundances are more prevalent than global extinctions (12–15), emphasizing the need to incorporate estimates of species abundances in analyses of global functional diversity change (16). By identifying changes in representation across avian functional space (i.e., multi-dimensional space where species are grouped based on their functional traits) during the Pleistocene epoch (~2.6 Mya – 11.7 thousand years ago [kya]), we aim to provide baseline estimates of functional diversity within avian communities before the widespread effects of humans on global wildlife. Doing so will identify the regions of functional space that are more resilient or more sensitive to climate warming, thus helping provide more plausible scenarios for future changes in avian functional diversity.Current understanding of global and regional dynamics in functional diversity is limited due to missing information about the past abundances of species (2, 17). This missing information hampers our ability to estimate baselines before human impacts and to quantify the magnitude and rates of ongoing losses of functional diversity and associated ecosystem functioning during periods of rapid environmental change. The modern genomic revolution offers a unique opportunity to investigate how demographic changes may have affected functional diversity over the Earth’s history. In particular, species-specific estimates of changing effective population size (N_e) over thousands-to-millions of years, inferred from whole-genome sequence data (18), can provide key estimates of fluctuations in abundance over evolutionary time and across biogeographic realms. Coupling such information with morphological, life-history, and ecological trait data for a broad selection of species permits reconstruction of the representation (in terms of abundance) of different functional traits over different periods of the distant past, revealing trait sets associated with functional diversity across a dynamically changing global environment. For instance, the last million years on Earth have been punctuated by periods of intense climate warming and cooling (19, 20). A recent analysis of genomic data in birds revealed that while overall bird abundance has steadily declined over the past million years, certain sets of morphological/life-history traits were associated with either species-specific population increases or decreases during discrete periods of climate warming and cooling (21). These observations suggest that the functional diversity of avian communities also fluctuates over time and during periods of climate variation. Identifying regions of functional space that are less occupied in more recent time periods due to population declines of certain species can provide baseline information on the diversity and composition of long-term ecological strategies leading up to the modern Holocene epoch (~11 kya–present). Such information will also illuminate regions of the global functional trait space which may be less resilient to future environmental challenges, beyond those documented under contemporary studies of population responses to current climate change.Here, we combine morphological, ecological, and life-history trait data for >260 extant bird species with estimates of changing N_e to quantify shifts in avian functional diversity over the past million years and explore patterns of change over time and space. We reconstruct the abundance-weighted functional trait space of these bird species in three time periods to quantify how functional diversity has changed 1) over time, 2) across the Earth’s major zoogeographic realms, and 3) how abundances have shifted across the avian functional space to potentially influence these patterns. We further quantify changes in functional diversity during the most recent period of abrupt climate warming over the past million years to identify the areas of the functional space most sensitive to broad-scale climate warming.     

A novel three-dimensional computer-assisted method for a quantitative study of microvascular networks of the human cerebral cortex

OBJECTIVE: Detailed information on microvascular network anatomy is a requirement for understanding several aspects of microcirculation, including oxygen transport, distributions of pressure, and wall shear stress in microvessels, regulation of blood flow, and interpretation of hemodynamically based functional imaging methods, but very few quantitative data on the human brain microcirculation are available. The main objective of this study is to propose a new method to analyze this microcirculation. METHODS: From thick sections of india ink-injected human brain, using confocal laser microscopy, the authors developed algorithms adapted to very large data sets to automatically extract and analyze center lines together with diameters of thousands of brain microvessels within a large cortex area. RESULTS: Direct comparison between the original data and the processed vascular skeletons demonstrated the high reliability of this method and its capability to manage a large amount of data, from which morphometry and topology of the cerebral microcirculation could be derived. CONCLUSIONS: Among the many parameters that can be analyzed by this method, the capillary size, the frequency distributions of diameters and lengths, the fractal nature of these networks, and the depth-related density of vessels are all vital features for an adequate model of cerebral microcirculation.     

Evidence for the aldo-keto reductase pathway of polycyclic aromatic trans-dihydrodiol activation in human lung A549 cells

Polycyclic aromatic hydrocarbons (PAHs) are tobacco carcinogens implicated in the causation of human lung cancer. Metabolic activation is a key prerequisite for PAHs to cause their deleterious effects. Using human lung adenocarcinoma (A549) cells, we provide evidence for the metabolic activation of (+/-)-trans-7,8dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-trans-dihydrodiol) by aldo-keto reductases (AKRs) to yield benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione), a redox-active o-quinone. We show that B[a]P-7,8-trans-dihydrodiol (AKR substrate) and B[a]P-7,8-dione (AKR product) lead to the production of intracellular reactive oxygen species (ROS) (measured as an increase in dichlorofluorescin diacetate fluores-cence) and that similar changes were not observed with the regioisomer (+/-)-trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene or the diol-epoxide, (+/-)-anti-7,8-dihydroxy-9alpha,10beta-epoxy-7,8,9,10-tetrahydro-B[a]P. B[a]P-7,8-trans-dihydrodiol and B[a]P-7,8-dione also caused a decrease in glutathione levels and an increase in NADP(+)/NADPH ratios, with a concomitant increase in single-strand breaks (as measured by the comet assay) and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo). The specificity of the comet assay was validated by coupling it to human 8-oxo-guanine glycosylase (hOGG1), which excises 8-oxo-Gua to yield single-strand breaks. The levels of 8-oxo-dGuo observed were confirmed by an immunoaffinity purification stable isotope dilution ([(15)N(5)]-8-oxo-dGuo) liquid chromatography-electrospray ionization/multiple reaction monitoring/mass spectrometry (LC-ESI/MRM/MS) assay. B[a]P-7,8-trans-dihydrodiol produced DNA strand breaks in the hOGG1-coupled comet assay as well as 8-oxo-dGuo (as measured by LC-ESI/MRM/MS) and was enhanced by a catechol O-methyl transferase (COMT) inhibitor, suggesting that COMT protects against o-quinone-mediated redox cycling. We conclude that activation of PAH-trans-dihydrodiols by AKRs in lung cells leads to ROS-mediated genotoxicity and contributes to lung carcinogenesis.     

The molecular genetic analysis of hemophilia A: a directed search strategy for the detection of point mutations in the human factor VIII gene

A directed-search strategy for point mutations in the factor VIII gene causing hemophilia A was used to screen eight potentially hypermutable CpG dinucleotides occurring at sites deemed to be of functional importance. Polymerase chain reaction-amplified DNA samples from 793 unrelated individuals with hemophilia A were screened by discriminant oligonucleotide hybridization. Point mutations were identified in 16 patients that were consistent with a model of 5-methylcytosine (5mC) deamination. Four new examples of recurrent mutation were demonstrated at the following codons: 336 (CGA----TGA), 372 (CGC----TGC), 372 (CGC----CAC), and 1689 (CGC----TGC). These are functionally important cleavage sites for either activated protein C or thrombin. Further novel C----T transitions were identified in the remaining arginine codons screened (-5, 427, 583, 795, and 1696), resulting in the creation of TGA termination codons. Differences in mutation frequency were found both within and between the CpG sites and between ethnic groups. These differences are assumed to be due to differences in the level of cytosine methylation at these sites, although direct evidence for this inference is lacking.     

A novel Y231del mutation of HFE in hereditary haemochromatosis provides in vivo evidence that the Huh-7 is a human haemochromatotic cell line

Hereditary haemochromatosis (HH), which is mainly associated with a C282Y polymorphism in HFE, is common among Caucasians of north European descent, but is very rare among Asians. Herein, we report a 43-year-old Japanese man who was diagnosed as having HH. A laboratory examination revealed an elevated serum iron level (280 μg/dl), hyperferritinemia (1698 ng/ml) and a low serum level of hepcidin-25 (4.0 ng/ml). Abdominal magnetic resonance imaging revealed findings suggestive of iron accumulation in the liver and pancreas. HFE gene sequencing in the patient revealed a novel homozygous TAC nucleotide deletion (c. 691_693del) responsible for the loss of a tyrosine at position 231 (p. Y231del) of the HFE protein. This homozygous Y231del mutation was recently found in the Huh-7 hepatoma cell line and was shown to prevent the translocation of HFE to the cell surface. This clinical case provides in vivo evidence suggesting that Huh-7 is undoubtedly a human haemochromatotic cell line and, as such, is a valuable tool for investigating the pathogenesis of HFE-related HH in humans.