Prior placental bed disorders and later dementia: a retrospective Swedish register-based cohort study.

OBJECTIVE: To investigate the association between a history of placental bed disorders and later dementia. DESIGN: Retrospective population-based cohort study. SETTING: Sweden. SAMPLE: All women giving birth in Sweden between 1973 and 1993 (1 128 709). METHODS: Women with and without placental bed disorders (hypertensive disorders of pregnancy including pre-eclampsia, fetal growth restriction, spontaneous preterm labour and birth, preterm premature rupture of membranes, abruptio placenta, late miscarriages) and other pregnancy complications were identified by means of the Swedish Medical Birth Register. International classification of disease was used. Data were linked to other National Registers. Participants were followed up until 2013. The Cox proportional hazards model was used to calculate hazard ratios for women with and without pregnancy complications and were adjusted for possible confounders. MAIN OUTCOME MEASURES: Diagnosis of vascular dementia and non-vascular dementia. RESULTS: Adjusted for cardiovascular disease and socio-demographic factors, an increased risk of vascular dementia was shown in women with previous pregnancy-induced hypertension (Hazard ratio [HR] 1.88, 95% CI 1.32-2.69), pre-eclampsia (HR 1.63, 95% CI 1.23-2.16), spontaneous preterm labour and birth (HR 1.65, 95% CI 1.12-2.42) or preterm premature rupture of membranes (HR 1.60, 95% CI 1.08-2.37). No statistically significant increased risk was seen for other pregnancy complications or non-vascular dementia even though many of the point estimates indicated increased risks. CONCLUSIONS: Women with placental bed disorders have a higher risk for vascular disease. Mechanisms behind the abnormal placentation remain elusive, although maternal constitutional factors, abnormal implantation as well as impaired angiogenesis have been suggested. TWEETABLE ABSTRACT: Placental bed syndromes associated with vascular dementia even after adjusting for cardiovascular disease.

A 37-year-old Menkes disease patient-Residual ATP7A activity and early copper administration as key factors in beneficial treatment.

Menkes disease (MD) is a lethal disorder characterized by severe neurological symptoms and connective tissue abnormalities; and results from malfunctioning of cuproenzymes, which cannot receive copper due to a defective intracellular copper transporting protein, ATP7A. Early parenteral copper-histidine supplementation may modify disease progression substantially but beneficial effects of long-term treatment have been recorded in only a few patients. Here we report on the eldest surviving MD patient (37 years) receiving early-onset and long-term copper treatment. He has few neurological symptoms without connective tissue disturbances; and a missense ATP7A variant, p.(Pro852Leu), which results in impaired protein trafficking while the copper transport function is spared. These findings suggest that some cuproenzymes maintain their function when sufficient copper is provided to the cells; and underline the importance of early initiated copper treatment, efficiency of which is likely to be dependent on the mutant ATP7A function.

Psychiatric illness in women requesting caesarean section.

OBJECTIVE: To compare psychiatric in- and outpatient care during the 5 years before first delivery in primiparae delivered by caesarean section on maternal request with all other primiparae women who had given birth during the same time period. DESIGN: Prospective, population-based register study. SETTING: Sweden. SAMPLE: Women giving birth for the first time between 2002 and 2004 (n = 64 834). METHODS: Women giving birth by caesarean section on maternal request (n = 1009) were compared with all other women giving birth (n = 63 825). The exposure of interest was any psychiatric diagnosis according to the International Statistical Classification of Diseases and Related Health Problems (ninth revision, ICD-9, 290-319; tenth revision, ICD-10, F00-F99) in The Swedish national patient register during the 5 years before first delivery. MAIN OUTCOME MEASURES: Psychiatric diagnoses and delivery data. RESULTS: The burden of psychiatric illnesses was significantly higher in women giving birth by caesarean section on maternal request (10 versus 3.5%, P < 0.001). The most common diagnoses were 'Neurotic disorders, stress-related disorders and somatoform disorders' (5.9%, aOR 3.1, 95% CI 1.1-2.9), and 'Mood disorders' (3.4%, aOR 2.4, 95% CI 1.7-3.6). The adjusted odds ratio for caesarean section on maternal request was 2.5 (95% CI 2.0-3.2) for any psychiatric disorder. Women giving birth by caesarean section on maternal request were older, used tobacco more often, had a lower educational level, higher body mass index, were more often married, unemployed, and their parents were more often born outside of Scandinavia (P < 0.05). CONCLUSIONS: Women giving birth by caesarean section on maternal request more often have a severe psychiatric disease burden. This finding points to the need for psychological support for these women as well as the need to screen and treat psychiatric illness in pregnant women.

Expression patterns of DLK1 and INSL3 identify stages of Leydig cell differentiation during normal development and in testicular pathologies, including testicular cancer and Klinefelter syndrome.

STUDY QUESTION: What is the differentiation stage of human testicular interstitial cells, in particular Leydig cells (LC), within micronodules found in patients with infertility, testicular cancer and Klinefelter syndrome? SUMMARY ANSWER: The Leydig- and peritubular-cell populations in testes with dysgenesis contain an increased proportion of undifferentiated cells when compared with control samples, as demonstrated by increased delta-like homolog 1 (DLK1) and decreased insulin-like peptide 3 (INSL3) expression. WHAT IS KNOWN ALREADY: Normal LC function is essential for male development and reproduction. Signs of LC failure, including LC micronodules, are often observed in patients with reproductive disorders. STUDY DESIGN, SIZE, PARTICIPANTS: In this retrospective study, a panel of markers and factors linked to the differentiation of LCs was investigated in 33 fetal and prepubertal human specimens and in 58 adult testis samples from patients with testicular germ cell tumours, including precursor carcinoma in situ (CIS), infertility or Klinefelter syndrome. PARTICIPANTS/MATERIALS, SETTING, METHODS: The expression patterns of DLK1, INSL3, chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII), cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) and smooth muscle actin (SMA) were investigated by immunohistochemistry and quantitative RT-PCR. The percentage of positive LCs was estimated and correlated to total LC numbers and serum levels of reproductive hormones. MAIN RESULTS AND THE ROLE OF CHANCE: DLK1, INSL3 and COUP-TFII expression changed during normal development and was linked to different stages of LC differentiation: DLK1 was expressed in all fetal LCs, but only in spindle-shaped progenitor cells and in a small subset of polygonal LCs in the normal adult testis; INSL3 was expressed in a subset of fetal LCs, but in the majority of adult LCs; and COUP-TFII was expressed in peritubular and mesenchymal stroma cells at all ages, in fetal LCs early in gestation and in a subset of adult LCs. CYP11A1 was expressed in the majority of LCs regardless of age and pathology and was the best general LC marker examined here. SMA was weakly expressed in peritubular cells in the fetal and infantile testis, but strongly expressed in the adult testis. In pathological testes, the numbers of DLK1-positive interstitial cells were increased. The proportion of DLK1-positive LCs correlated with total LC numbers (R = 0.53; P < 0.001) and was higher in testis with enlargement of the peritubular layers (P < 0.01), which was also highly associated with DLK1 expression in the peritubular compartment (P < 0.001). INSL3 expression was absent in some, but not all LC micronodules, and in the majority of LCs, it was mutually exclusive of DLK1. LIMITATIONS, REASONS FOR CAUTION: The number of samples was relatively small and no true normal adult controls were available. True stereology was not used for LC counting, instead LCs were counted in three fields of 0.5 µm(2) surface for each sample. WIDER IMPLICATIONS OF THE FINDINGS: The population of LCs, especially those clustered in large nodules, are heterogeneous and comprise cells at different stages of differentiation. The study demonstrated that the differentiation and function of LCs, and possibly also peritubular cells, are impaired in adult men with testicular pathologies including testis cancer and Klinefelter syndrome. STUDY FUNDING/COMPETING INTERESTS: This work was funded by Rigshospitalet's research funds, the Danish Cancer Society and Kirsten and Freddy Johansen's foundation. The authors have no conflicts of interest.

Hemolytic properties of synthetic nano- and porous silica particles: the effect of surface properties and the protection by the plasma corona.

Novel silica materials incorporating nanotechnology are promising materials for biomedical applications, but their novel properties may also bring unforeseen behavior in biological systems. Micro-size silica is well documented to induce hemolysis, but little is known about the hemolytic activities of nanostructured silica materials. In this study, the hemolytic properties of synthetic amorphous silica nanoparticles with primary sizes of 7-14 nm (hydrophilic vs. hydrophobic), 5-15 nm, 20 nm and 50 nm, and model meso/macroporous silica particles with pore diameters of 40 nm and 170 nm are investigated. A crystalline silica sample (0.5-10 µm) is included for benchmarking purposes. Special emphasis is given to investigations of how the temperature and solution complexity (solvent, plasma), as well as the physicochemical properties (such as size, surface charge, hydrophobicity and other surface properties), link to the hemolytic activities of these particles. Results suggests the potential importance of small size and large external surface area, as well as surface charge/structure, in the hemolysis of silica particles. Furthermore, a significant correlation is observed between the hemolytic profile of red blood cells and the cytotoxicity profile of human promyelocytic leukemia cells (HL-60) induced by nano- and porous silica particles, suggesting a potential universal mechanism of action. Importantly, the results generated suggest that the protective effect of plasma towards silica nanoparticle-induced hemolysis as well as cytotoxicity is primarily due to the protein/lipid layer shielding the silica particle surface. These results will assist the rational design of hemocompatible silica particles for biomedical applications.

Insulin resistance after a 72-h fast is associated with impaired AS160 phosphorylation and accumulation of lipid and glycogen in human skeletal muscle.

During fasting, human skeletal muscle depends on lipid oxidation for its energy substrate metabolism. This is associated with the development of insulin resistance and a subsequent reduction of insulin-stimulated glucose uptake. The underlying mechanisms controlling insulin action on skeletal muscle under these conditions are unresolved. In a randomized design, we investigated eight healthy subjects after a 72-h fast compared with a 10-h overnight fast. Insulin action on skeletal muscle was assessed by a hyperinsulinemic euglycemic clamp and by determining insulin signaling to glucose transport. In addition, substrate oxidation, skeletal muscle lipid content, regulation of glycogen synthesis, and AMPK signaling were assessed. Skeletal muscle insulin sensitivity was reduced profoundly in response to a 72-h fast and substrate oxidation shifted to predominantly lipid oxidation. This was associated with accumulation of both lipid and glycogen in skeletal muscle. Intracellular insulin signaling to glucose transport was impaired by regulation of phosphorylation at specific sites on AS160 but not TBC1D1, both key regulators of glucose uptake. In contrast, fasting did not impact phosphorylation of AMPK or insulin regulation of Akt, both of which are established upstream kinases of AS160. These findings show that insulin resistance in muscles from healthy individuals is associated with suppression of site-specific phosphorylation of AS160, without Akt or AMPK being affected. This impairment of AS160 phosphorylation, in combination with glycogen accumulation and increased intramuscular lipid content, may provide the underlying mechanisms for resistance to insulin in skeletal muscle after a prolonged fast.

Circulating soluble urokinase plasminogen activator receptor predicts cancer, cardiovascular disease, diabetes and mortality in the general population.

BACKGROUND: Low-grade inflammation is thought to contribute to the development of cardiovascular disease (CVD), type-2 diabetes mellitus (T2D), cancer and mortality. Biomarkers of inflammation may aid in risk prediction and enable early intervention and prevention of disease. OBJECTIVE: The aim of this study was to investigate whether plasma levels of the inflammatory biomarker soluble urokinase plasminogen activator receptor (suPAR) are predictive of disease and mortality in the general population. DESIGN: This was an observational prospective cohort study. Cohort participants were included from June 1993 to December 1994 and followed until the end of 2006. SETTING: General adult Caucasian population. PARTICIPANTS: The MONICA10 study, a population-based cohort recruited from Copenhagen, Denmark, included 2602 individuals aged 41, 51, 61 or 71 years. MEASUREMENTS: Blood samples were analysed for suPAR levels using a commercially available enzyme-linked immunosorbent assay. Risk of cancer (n = 308), CVD (n = 301), T2D (n = 59) and mortality (n = 411) was assessed with a multivariate proportional hazards model using Cox regression. RESULTS: Elevated baseline suPAR level was associated with an increased risk of cancer, CVD, T2D and mortality during follow-up. suPAR was more strongly associated with cancer, CVD and mortality in men than in women, and in younger compared with older individuals. suPAR remained significantly associated with the risk of negative outcome after adjustment for a number of relevant risk factors including C-reactive protein levels. LIMITATION: Further validation in ethnic populations other than Caucasians is needed. CONCLUSION: The stable plasma protein suPAR may be a promising biomarker because of its independent association with incident cancer, CVD, T2D and mortality in the general population.

[Update hyperthyreoidism]. / Update Hyperthyreose.

Hyperthyroidism is mainly caused by Graves' disease and toxic adenoma or multinodular goiter. In Europe, treatment of both disorders is usually started with antithyroidal drugs such as methimazole. Complications include agranulocytosis and the risk is dose-dependent. The starting dose of methimazole should not exceed 15-20 mg/d. Propylthiouracil can cause severe liver failure, leading to liver transplantation or death. Propylthiouracil, therefore, should not be used as first line agent and is only recommended when an antithyroid drug is to be started during the first trimester of pregnancy or in individuals who have experienced adverse responses to methimazole. Toxic adenoma is finally treated with radioioidine. To reduce the risk of treatment failure, antithyroidal drugs should be stopped at least one week prior to radioiodine. For Graves' disease, remission is unlikely if antibodies against the TSH-receptor remain above 10 mU/l after 6 months of antithyroidal treatment and radioiodine or thyroidectomy can be recommended. Thyroidectomy should be performed as (near) total thyreoidectomy.

Gender-related differences in response to mutagens and carcinogens.

The incidences of many cancers can be very different in men and women. Besides differences in exposures to putative causative agents, it is plausible that both genetic and epigenetic effects play roles in these differences. In addition, gender-specific lifestyle and behavioural factors may modulate the effects of exposure to genotoxins. This commentary focuses on several aspects of gender-related differences in responses to mutagens and carcinogens, including sensitivity to chromosome damage, the contribution of genotypic variation and the role of DNA methylation. It is concluded that the reasons for gender differences in cancer susceptibility remain largely unknown in many cases, and the subject deserves more attention and study.

Mortality of life-insured Swedish cats during 1999-2006: age, breed, sex, and diagnosis.

BACKGROUND: A cat life insurance database can potentially be used to study feline mortality. HYPOTHESIS: The aim was to describe patterns of mortality in life-insured Swedish cats. Cats: All cats (<13 years of age) with life insurance during the period 1999-2006 were included. METHODS: Age-standardized mortality rates (MR) were calculated with respect to sex (males and females), age, breed, and diagnosis. Survival to various ages is presented by time period and breed. RESULTS: The total number of cats insured was 49,450 and the number of cat-years at risk (CYAR) was 142,049. During the period, 6,491 cats died and of these 4,591 cats (71%) had a diagnosis, ie, were claimed for life insurance. The average annual MR was 462 deaths per 10,000 CYAR (95% confidence interval, 431-493). Sex-specific rates did not differ significantly. The overall mortality of the Persian and the Siamese groups was higher than that of several other breeds. Overall and breed-specific (for most breeds) survival increased with time when analyzed by 2-year periods. The 6 most common diagnostic categories (ignoring cats recorded as dead with no diagnosis) were urinary, traumatic, neoplastic, infectious, cardiovascular, and gastrointestinal. The MR within diagnostic categories varied by age and breed. CONCLUSIONS AND CLINICAL IMPORTANCE: In this mainly purebred, insured cat population, the overall mortality varied with age and breed but not with sex. The increase in survival over time is likely a reflection of willingness to keep pet cats longer and increased access to and sophistication of veterinary care.

Major hepatectomy for colorectal metastases: is preoperative portal occlusion an oncological risk factor?

BACKGROUND: This study investigates oncological risks and benefits of portal occlusion (PO) in major resection for colorectal liver metastases (CLM). METHODS: Between 1995 and 2004, 107 patients were scheduled for major hepatectomy for CLM. Of these, 53 patients were selected for PO due to insufficient future liver remnant (FLR), and 54 patients had straightforward hepatectomy. Associations of clinicopathologic factors with resectability, and outcome after PO were analyzed. RESULTS: 21 of 53 patients (39.6%) after PO were unresectable. These patients had a significant smaller volume of the FLR than the 32 resected patients after PO (P = .029). In total, 17 patients (80.9%) did not undergo resection due to cancer progression. Among these, 11 patients (52.4%) exhibited either a progression of known metastases located in the occluded lobes, or new metastases in the nonoccluded portion of the liver. In another 4 individuals (19%), the decision against resection resulted from insufficient hypertrophy of the FLR. Following major hepatectomy, the 5-year survival was 43.66%. Although there was a significantly higher rate of extended hepatectomies versus formal hepatectomies (P < .001), more bilobar distributed metastases versus unilobar manifestations (P = .015), and a smaller resection margin (P = .01) in patients who had PO, no adverse effect on mortality, morbidity, recurrence and survival was observed. CONCLUSION: Unresectability after PO is a major problem that warrants multidisciplinary improvements, and randomization to resection with or without PO remains ethically problematic. However, following adequate patient selection, PO may provide a significant survival benefit for patients with prior unresectable CLM.

Protective effects of organosulfur compounds towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay.

The aim of this study was to investigate the protective effect of organosulfur compounds towards N-nitrosamine-induced DNA damage in the single-cell gel electrophoresis (SCGE)/HepG2 assay. N-Nitrosopyrrolidine (NPYR) and N-nitrosodimethylamine (NDMA) incubated with formamidopyrimidine-DNA glycosylase (Fpg), caused a significant increase in oxidative DNA damage in comparison to the solvent control, the lowest effective concentrations, being 5 and 27 mM, respectively. NPYR exerted greater genotoxic effects than NDMA. None of the organosulfur compounds (OSCs) concentrations tested in presence or absence of Fpg enzyme, caused DNA damage per se. OSCs (diallyl sulfide, DAS and dipropyl sulfide, DPS, 1-50 microM; diallyl disulfide, DADS and dipropyl disulfide, DPDS, 1-5 microM) reduced the genotoxic effects of the N-nitrosamines in a dose-dependent manner when HepG2 cells were simultaneously treated with OSCs and N-nitrosamines. The effect of NPYR was attenuated by about 61-67%, respectively, with the highest concentration of DAS (50 microM) and DADS (5 microM). The protective effect of DADS (5 microM, 66%) was higher than DAS (50 microM, 53%) towards NDMA-induced oxidative DNA damage. A concentration of 50 microM DPS and 5 microM DPDS led to a 65-63% and 59-65% reduction in NPYR/NDMA-induced oxidative DNA damage, respectively. Our results indicate that OSCs protect human-derived cells against the oxidative DNA damaging effect of NPYR and NDMA, two carcinogenic compounds which occur in the environment.

Congenital cataract, muscular hypotonia, developmental delay and sensorineural hearing loss associated with a defect in copper metabolism.

Deficiencies of different proteins involved in copper metabolism have been reported to cause human diseases. Well-known syndromes, for example, are Menkes and Wilson diseases. Here we report a patient presenting with congenital cataract, severe muscular hypotonia, developmental delay, sensorineural hearing loss and cytochrome-c oxidase deficiency with repeatedly low copper and ceruloplasmin levels. These findings were suggestive of a copper metabolism disorder. In support of this, the patient's fibroblasts showed an increased copper uptake with normal retention. Detailed follow-up examinations were performed. Immunoblotting for several proteins including ATP7A (MNK or Menkes protein), ATP7B (Wilson protein) and SOD1 showed normal results, implying a copper metabolism defect other than Wilson or Menkes disease. Sequence analysis of ATOX1 and genes coding for proteins that are known to play a role in the mitochondrial copper metabolism (COI-III, SCO1, SCO2, COX11, COX17, COX19) revealed no mutations. Additional disease genes that have been associated with cytochrome-c oxidase deficiency were negative for mutations as well. As beneficial effects of copper histidinate supplementation have been reported in selected disorders of copper metabolism presenting with low serum copper and ceruloplasmin levels, we initiated a copper histidinate supplementation. Remarkable improvement of clinical symptoms was observed, with complete restoration of cytochrome-c oxidase activity in skeletal muscle.

Hospital volume and outcome of rectal cancer surgery in Denmark 1994-99.

OBJECTIVE: The association between hospital volumen and outcome of major cancer surgery is being debated at present. We analysed the outcome of rectal cancer surgery in Denmark during the period 1994-99. METHODS: All patients with a first-time rectal cancer were registered in a national database during the 5-year period. In this observational cohort study, the influence of hospital case volume on resectional procedure, complications, 30-day mortality and 5-year mortality was analysed. RESULTS: The register comprised 5021 patients. Surgery was performed in 27 hospitals with <15 operations per year, 15 hospitals with 15-30 operations per year and 11 hospitals with >30 operations per year. In a multivariate model, the risk of permanent colostomy was significantly increased in the group of low-volume hospitals. On the contrary, volume did not influence the risk of anastomotic leakage, 30-day mortality and 5-year mortality. However, a large variation in 5-year mortality was observed particularly within the low-volume group of hospitals. CONCLUSIONS: In this study, only risk of having a permanent colostomy during surgery for rectal cancer was significantly related to hospital case volume. When individual hospitals were analysed, a large variation in 5-year mortality was observed within the low-volume group of hospitals.

Survival of rectal cancer patients in Denmark during 1994-99.

OBJECTIVE: As survival from rectal cancer in Denmark is below the European average, we analysed survival during the period of 1994-99 focusing upon improvement strategies. METHOD: All patients with a first-time rectal cancer were registered in a national database during this 5-year period. In the observational cohort study, data on patient age and gender, tumour stage, surgical procedures, adjuvant radiotherapy, anastomotic leakage, 30-day mortality and long-term survival were evaluated. RESULTS: The database comprised 5021 patients. Sixty-four percent had a localized tumour. Less than a third of patients with fixed tumours had pre-operative radiotherapy and curative surgery was achieved in 70%. Anastomotic leakage occurred in 13%, and 30-day mortality was 4% following abdominoperineal or anterior resection and 11% following a Hartmann's procedure. The relative 5-year survival in the entire series was 39% in males and 47% in females, respectively. Following curative surgery the relative 5-year survival was 55% in males and 63% in females, respectively. Survival was 71% in the subset of patients receiving curative total mesorectal excision. CONCLUSION: The average tumour stage upon diagnosis was probably more advanced compared to the other Nordic countries and pre-operative radiotherapy was administered to a minority of patients with fixed tumours. The anastomotic leakage rate was relatively high, whereas the 30-day mortality was comparable to other studies. Survival from rectal cancer in Denmark is still less favourable compared to the other Nordic and several European countries but improved from 1996 and onwards.

DNA adduct formation of 14 heterocyclic aromatic amines in mouse tissue after oral administration and characterization of the DNA adduct formed by 2-amino-9H-pyrido[2,3-b]indole (AalphaC), analysed by 32P_HPLC.

Heterocyclic aromatic amines (HAAs) are produced during cooking of proteinaceous food such as meat and fish. Humans eating a normal diet are regularly exposed to these food-borne substances. HAAs have proved to be carcinogenic in animals and to induce early lesions in the development of cancer. DNA adduct levels in mouse liver have been measured by 32P-HPLC after oral administration each of 14 different HAAs. The highest DNA adduct levels were detected for 3-amino-1-methyl-5H-pyrido[4,3-b]-indole (Trp-P-2), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-9H-pyrido[2,3-b]indole (AalphaC), respectively. To assess a relative risk in a human population, a relative risk index was calculated by combining the DNA adduct levels in mouse liver with human daily intake of heterocyclic amines in a US and in a Swedish population. Such calculations suggest that AalphaC presents the highest risk for humans, e.g. nine-fold higher compared with the most abundant amines in food, 2-amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP). Therefore, the distribution of DNA adducts in different tissues of mouse was investigated after oral administration of AalphaC. The highest AalphaC-DNA adduct levels were found in liver (137 adducts/10(8) normal nucleotides) followed by heart, kidney, lung, large intestine, small intestine, stomach and spleen, in descending order. To characterize the chemical structure of the major DNA adduct, chemical synthesis was performed. The major DNA adduct from the in vivo experiments was characterized by five different methods. On the basis of these results, the adduct was characterized as N2-(deoxyguanin-8-yl)-2-amino-9H-pyrido [2,3-b]indole. Considering the abundance of AalphaC not only in grilled meat, but also in other products like grilled chicken, vegetables and cigarette smoke and in light of the results of the present study, it is suggested that the human cancer risk for AalphaC might be underestimated.

Probiotic mixture decreases DNA adduct formation in colonic epithelium induced by the food mutagen 2-amino-9H-pyrido[2,3-b]indole in a human-flora associated mouse model.

Consumption of probiotic bacteria such as bifidobacteria has been shown to reduce the risk of colon cancer in animal models. However, the composition and metabolic activities of the intestinal flora of experimental animals are significantly different from those of humans. The aim of the study was to examine whether the probiotic mixture, which consisted of Streptococcus faecalis, Clostridium butyricum and Bacillus mesentericus, could decrease DNA adduct formation induced by 2-amino-9H-pyrido[2,3-b]indole (2-amino-alpha-carboline; AAC) in the colonic epithelium of a human-flora-associated (HFA) mouse model. Ten HFA mice were divided into a control group (n=4) and a probiotic group (n=6). The control group was administered AAC for 3 days and sacrificed 24 h after the last dose. The probiotic group was administered the probiotic mixture for 2 weeks prior to the administration of AAC. Analysis of DNA adducts with the 32P-high-performance liquid chromatography method was performed on stomach, jejunum and colonic epithelium, representing direct exposure sites of AAC, and colon wall, liver and kidney, representing indirect exposure sites. The mean level of the DNA adducts in the colonic epithelium of the probiotic group was significantly lower than that of control group, while the mean levels at the other sites did not differ significantly between the groups. The results indicated that the probiotic mixture could decrease the DNA adduct formation in the colonic epithelium induced by AAC.

X-linked recessive Menkes disease: identification of partial gene deletions in affected males.

Menkes disease is an X-linked recessive lethal disorder of copper metabolism, caused by defects in the ATP7A gene. Partial gene deletions comprise about 15% of the mutations causing Menkes disease. We have previously demonstrated identification of partial ATP7A deletions in patients by Southern blot analysis. In the present study, we report the use of three fast and reliable polymerase chain reaction (PCR)-based methods for the identification of partial ATP7A deletions in Menkes disease patients. First we demonstrate the use of multiplex PCR, a fast method for identification and rough localization of partial gene deletions, in which two exons of ATP7A are coamplified. Second, we present PCR amplification of genomic DNA across the deletion junctions, a method enabling identification of the deletion breakpoints and hence the exact size of the deletion. Finally, application of reverse transcription PCR (RT-PCR) for identification and localization of gene deletions at the cDNA level is demonstrated. By studying the mutation at the cDNA level the predicted effect of the mutation on the amino acid sequence and consequently the protein structure and function can be inferred. We demonstrate characterization of partial gene deletions in five patients, and in three of these we were able to determine the breakpoint sequences.