DypB peroxidase for aflatoxin removal: New insights into the toxin degradation process.

Aflatoxin B1 (AFB1) is one of the most potent carcinogens and a widespread food and feed contaminant. As for other toxins, many efforts are devoted to find efficient and environmentally-friendly methods to degrade AFB1, such as enzymatic treatments, thus improving the safety of food and feed products. In this regard, the dye decolorizing peroxidase of type B (DypB) can efficiently degrade AFB1. The molecular mechanism, which is required to drive protein optimization in view of the usage of DypB as a mycotoxin reduction agent in large scale application, is unknown. Here, we focused on the role of four DypB residues in the degradation of AFB1 by alanine-scanning (residues 156, 215, 239 and 246), which were identified from biochemical assays to be kinetically relevant for the degradation. As a result of DypB degradation, AFB1 is converted into four products. Interestingly, the relative abundancy of these products depends on the replaced residues. Molecular dynamics simulations were used to investigate the role of these residues in the binding step between protein and manganese, a metal ion which is expected to be involved in the degradation process. We found that the size of the haem pocket as well as conformational changes in the protein structure could play a role in determining the kinetics of AFB1 removal and, consequently, guide the process towards specific degradation products.

Copper binding to naturally occurring, lactam form of angiogenin differs from that to recombinant protein, affecting their activity.

Angiogenin is a member of the ribonuclease family and a normal constituent of human plasma. It is one of the most potent angiogenic factors known and is overexpressed in different types of cancers. Copper is also an essential cofactor in angiogenesis and, during this process, it is mobilized from inside to outside of the cell. To date, contrasting results have been reported about copper(ii) influencing angiogenin activity. However, in these studies, the recombinant form of the protein was used. Unlike recombinant angiogenin, that contains an extra methionine with a free terminal amino group, the naturally occurring protein present in human plasma starts with a glutamine residue that spontaneously cyclizes to pyroglutamate, a lactam derivative. Herein, we report spectroscopic evidence indicating that copper(ii) experiences different coordination environments in the two protein isoforms, and affects their RNase and angiogenic activity differently. These results show how relatively small differences between recombinant and wild type proteins can result in markedly different behaviours.

Risk factors for smoking abuse after heart transplantation.

Patients (n = 103) were studied before heart transplantation with regard to smoking habits by means of a clinical interview, and 81 were submitted to Minnesota Multiphasic Personality Inventory (MMPI). After a mean time of 50.8 +/- 24.2 months from transplant, they were once again interviewed to ascertain their smoking habits after intervention. Nonsmokers (35 of 103) were still nonabusers. Of the remaining 68 patients who ceased smoking before heart transplant, 12 (17.6%) had returned to tobacco abuse. Dividing these 68 patients into two groups based upon the length of smoking cessation before heart transplant (less than 1 year: short term [ST] more than 1 year: long term [LT]), we noticed that the ST group showed a much greater rate of reabuse (8 of 20, 40%) than the LT group (4 of 48, 8.3%, P =.006). Analyzing six scales of MMPI, we found a statistically different score for self-control ability (scale K) in ST and LT smokers compared to nonsmokers (45.5 and 45.5 vs 51.2, P =.026), and for difficult adaptation (scale Ma) in ST compared both to LT smokers and nonsmokers (ST 57, LT 50.5, NS 47.6; P =.042 LT vs ST, P =.0005 ST vs NS). We concluded that patients who have recently decided to stop smoking and show after MMPI compilation a score of >50 for K and <50 for Ma scale have a higher risk of reabuse and need a greater effort by the transplant team to reinforce their will to stop smoking.

Epidemiology of cholelithiasis in southern Italy. Part II: Risk factors.

OBJECTIVE: To determine behavioural, dietary and other common factors associated with new cases of gallstones, diagnosed by ultrasonography, in a prospective cohort study conducted in southern Italy. SUBJECTS AND METHODS: Between May 1985 and June 1986, systematic sampling from the electoral register of Castellana, a small town in southern Italy, yielded 2472 subjects who had had their gallbladder checked for gallstones by ultrasonography. Between May 1992 and June 1993, 1962 out of the 2235 (87.7%) subjects without gallstones at baseline agreed to a further ultrasound examination. At the first survey a standardized questionnaire was administered, inquiring about medical history, diet, cigarette smoking and other behavioural characteristics. Height and weight were also measured, and blood levels of glucose, cholesterol, HDL-cholesterol and triglycerides were determined by standard methods. The same variables were measured at the second survey. The diagnosis of gallstones was made with the same echograph by echographists working in the same department. Multiple logistic regression was used to determine which factors measured at the first survey were associated with the incident cases of gallstones. RESULTS: One hundred and four subjects had developed gallstones, an incidence of 9.7 per 1000 persons per year. Age, body mass index (BMI), weight change, a history of diabetes, constipation (shown by use of laxatives), cigarette smoking, years of schooling, consumption of fried foods and excessive oil, and pregnancy in females, were positively associated with the incidence of gallstones. Consumption of wine, coffee, fish and wholemeal bread was inversely associated. Sex, family history of cholelithiasis, use of oral contraceptives and serum lipids were not independent risk factors for gallstones. CONCLUSION: The results of this study confirm many gallstone-associated factors reported in previous cross-sectional and case-control studies, as well as in other cohort studies based on the clinical diagnosis of gallstones, such as BMI, ageing and wine consumption. Furthermore, use of laxatives, considered a proxy of constipation, appears to be another important independent risk factor for gallstones.

Endogenous sex hormones and cholesterol gallstones: a case-control study in an echographic survey of gallstones.

In a population survey of gallstones, the serum levels of hormones of the pituitary-gonadal axis and the sex hormone-binding globulin (SHBG) were compared in subjects with cholesterol gallstones and in a control group. In 84 subjects who entered the survey, echographic gallstones that had been identified at the survey, turned out to be radiolucent or mixed (predominantly of cholesterol) at subsequent x-ray. The controls were without gallstones at echography, matched to the cases for potential confounders of the association sex hormones-cholelithiasis. Testosterone (T) 17-beta-estradiol (E2), 17-OH progesterone (P), and SHBG were dosed by radioimmunoassay; follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (Prl) by dissociation-enhanced lanthanide fluoro immunoassay (DELFIA). Men with gallstones had lower LH than controls (n = 34, median difference = -0.62 mU/ml, 95% confidence interval (CI) -1.20 to -0.26 mU/ml, paired sign test, p = 0.003). Premenopausal women in the luteal phase of the menstrual cycle with gallstones had higher E2 than controls (n = 7, median difference: +117, pg/ml, 95% CI: +10 to +218 pg/ml, p = 0.008). Postmenopausal women had lower LH than controls (n = 35, median difference = -4.57 mU/ml, 95% CI -9.5 to -1.0 mU/ml, p = 0.04). No other hormones showed statistically significant differences between cases and controls, in either males or females. The findings of this exploratory study in subjects with radiolucent and mixed gallstones suggest that men and postmenopausal women have lower LH, and premenopausal women in the luteal phase of the cycle have higher E2, than controls.

Prevalence of pigment gallstones in sheep.

In a survey of 666 sheep at a slaughterhouse, gallstones (concretions with a diameter greater than or equal to 1 mm) were found in the gallbladder of 50 sheep (7.5%), sludge (concretions with a diameter less than 1 mm) was found in 9 sheep (1.4%), and sludge plus gallstones were found in 7 sheep (1.1%). Gallstones and sludge were associated, and were more frequent in lambs and females, compared with adults and males. Qualitative analysis of the stones revealed all to be pigment (bilirubin) stones. There was a statistically significant increase of biliary bilirubin (total and indirect quota) only in sheep with gallstones plus sludge, compared with control sheep without sludge or gallstones. Concentrations of bilirubin, cholesterol, phospholipids, total and single bile aids, and total and ionized calcium were similar in the bile of sheep with gallstones, sludge, or both and control sheep. Bacteriologic analysis of the bile in 10 sheep with gallstones and 10 controls revealed bacteria in 50% of the first group and in 75% of the second group (Escherichia coli in all sheep and Salmonella spp also in 1 sheep with gallstones). These findings confirm our earlier findings of a high prevalence of black pigment gallstones in sheep. On that basis, we suggest that gallstones are associated with high total bilirubin concentration in the bile, and deconjugating bacteria are common in the biliary tract of these animals.

Duodenogastric reflux of bile acids, gastrin and parietal cells, and gastric acid secretion before and 6 months after cholecystectomy.

In order to evaluate the effect of cholecystectomy on the gastric mucosa, the duodenogastric reflux of total and single bile acids, the number of parietal and gastrin cells, and the volume of gastric acid secretion were examined in 15 patients with gallstones and functioning gallbladders before and 6 months after cholecystectomy. The duodenogastric reflux of the total bile acids increased from a mean preoperative value of 1.9 mumol/hour to a mean postoperative value of 21 mumol/hour (p = 0.008). The duodenogastric reflux of all single bile acids increased after cholecystectomy, with a higher increase in glycoconjugated compared with tauroconjugated bile acids. The parietal cells decreased from a mean preoperative value of 82.8 to a mean postoperative value of 68.7 (p = 0.05), whereas there was only a mild increase in the number of gastrin cells; the output of gastric acid remained unchanged. The variation of the gastrin cells before and after cholecystectomy was negatively correlated only with the variation of taurocholic acid (r = -0.50, p = 0.05), while the variation of the parietal cells was mildly correlated with all single bile acids (r = 0.35-0.50, 0.05 less than p less than 0.02). These findings show an increased duodenogastric reflux of bile acids 6 months after cholecystectomy with a mild morphologic alteration of the gastric mucosa.

Serum and bile lipid levels in a postmenopausal woman after percutaneous and oral natural estrogens.

17 beta-Estradiol was administered for 4 weeks percutaneously (5 mg/day) and, after a 6-week period to allow the drug to wash out, orally (2 mg/day) to a postmenopausal woman in whom a Kerr tube had been placed because of an iatrogenic lesion of the common bile duct. After both methods of administration, there was an increase in the biliary cholesterol concentration, but after oral administration, bile flow also increased and cholesterol crystals appeared in the bile. The percutaneous method of estradiol administration in postmenopausal women seems to be less dangerous for the biliary tract.

Spontaneous cholelithiasis in sheep: prevalence survey and analysis of gallstones and bile.

In an abattoir survey of the consecutive series of 206 sheep at a local slaughterhouse, gallstones (concretions with a diameter equal to or greater than 1 mm) were found in the gall bladder of 24 animals. There were stones in 19 out of 146 adult sheep and in 5 out of 60 lambs, with the higher frequency in the male sex in both groups. Qualitative analysis of the stones showed that all of them were pigment (bilirubin) stones. There was no statistically significant difference (paired t test, P greater than 0.05) between the amounts of biliary cholesterol, phospholipids, bile acids and total and indirect bilirubin in 12 sheep with gallstones and 12 control sheep without gallstones, paired for age, sex and day of slaughter.

Serum and bile lipids in young women with radiolucent gallstones.

To investigate the relationship between blood and bile lipids, serum cholesterol, high density lipoprotein cholesterol, and triglycerides were correlated with cholesterol saturation index of bile in 21 women-10 with radiolucent gallstones and 11 without stones. All of the women had regular menstrual cycles, were normolipidemic, and on a hospital diet. On the same morning, blood and the darkest duodenal bile were taken after cholecystokinin (CCK) stimulation. Standard laboratory procedures were used to analyze serum and bile lipids. We found: 1) statistically significant (t test, p less than 0.05) but only slight hypercholesterolemia (+ 12%) in patients with gallstones; 2) a negative correlation of serum cholesterol with cholesterol saturation index of bile, both in the control group (r = -0.654, p less than 0.05) and in gallstone patients (r = -0.665, p less than 0.05); 3) a correlation of high density lipoprotein cholesterol with cholesterol saturation index only in normal women (r = -0.619, p less than 0.05); 4) conversely, a correlation of triglycerides with the same index in only gallstone patients (r = 0.641, p less than 0.05). With the stepwise multiple regression analysis (independent variables: diagnosis of gallstones, serum cholesterol, HDL cholesterol, triglycerides; dependent variable: biliary cholesterol saturation index), only gallstone diagnosis and serum cholesterol influenced significantly (F test, p less than 0.05) the biliary cholesterol saturation index. These findings suggest that young women with radiolucent gallstones are slightly hypercholesterolemic, that in women both with and without gallstones there is a negative correlation between serum cholesterol and biliary cholesterol saturation, but women with gallstones have a higher cholesterol saturation index of the bile than women without gallstones with the same level of cholesterol in the blood.

Gallstones and uterine fibroids.

In a series published in 1961, an unusual frequency of hysterectomies for uterine leiomyomas (fibroids) was reported in women with gallstones. The purpose of this study was to confirm the association between gallstones and uterine leiomyomas with a patient control study and to investigate its physiopathologic basis comparing the cholesterol saturation of bile in women with gallstones, in women with leiomyomas but no gallstones and in those in the control group with no gallstones or leiomyomas. Patients admitted to the surgical department have, routinely, echography of the gallbladder before and manual exploration of the pelvic floor during surgical intervention. For the first part of the study, we collected information concerning the diagnosis of leiomyomas from the operating room registers and about the diagnosis of gallstones from the clinical records. In 1982, 42 of 139 women operated upon consecutively for gallstones and five of 69 operated upon for other diseases of the gastrointestinal tract had leiomyomas, a statistically significant difference (chi-square test, p less than 0.001). This difference persisted stratifying women with gallstones and those in the control group for age. In the second part of the study, we examined the bile collected at duodenal drainage after gallbladder stimulation with cholecystokinin, in 11 young women with radiolucent gallstones (echography and cholecystography), in ten women with leiomyomas (gynecologic examination and pelvic echography) but no gallstones (echography) and in 11 women with no leiomyomas (gynecologic examination or pelvic echography) or gallstones (echography). Cholesterol, phospholipids and total bile acids in the biliary tract were analyzed with standardized enzymatic methods. The cholesterol saturation index of the biliary tract was higher in patients with leiomyomas than in those in the control group (Wilcoxon rank sum test, p less than 0.01) and similar to that of women with radiolucent gallstones. These data suggest that gallstones and leiomyomas are associated diseases, probably sharing a common cause.