Brevibacillus themoruber: a promising microbial cell factory for exopolysaccharide production.

AIMS: This study aims to identify a high level exopolysaccharide (EPS) producer thermophile that in turn could be used as a model organism to study the biological mechanisms and whole genome organization of EPS-producing thermophilic bacteria. METHODS AND RESULTS: Thermophilic isolates were screened, and then growth and EPS production of the best producer Brevibacillus thermoruber strain 423 were investigated under different carbon and nitrogen sources, temperature, pH and agitation rates. Rheological characterization revealed that the EPS behaved like a typical Newtonian fluid and viscosity of the EPS solution increased with increasing Ca(2+) ion concentration. Chemical characterization by TLC, GC-MS, FT-IR and NMR suggested a heteropolymer structure with glucose as major monomer unit. High biocompatibility of pure EPS fractions suggested their potential use in biomedical applications. CONCLUSIONS: This study reports on the comprehensive description of microbial production conditions as well as chemical, rheological and biological characterization of the EPS produced by B. thermoruber strain 423. The bioreactor cultures were found to reach two times higher yields and three times higher productivities when compared with literature. SIGNIFICANCE AND IMPACT OF THE STUDY: Brevibacillus thermoruber strain 423 combined the advantages of its nonpathogenicity with the advantages of fast productivity and hence proved to be a very promising model organism and cell factory for microbial EPS production.

Extremophiles survival to simulated space conditions: an astrobiology model study.

In this work we investigated the ability of four extremophilic bacteria from Archaea and Bacteria domains to resist to space environment by exposing them to extreme conditions of temperature, UV radiation, desiccation coupled to low pressure generated in a Mars' conditions simulator. All the investigated extremophilic strains (namely Sulfolobus solfataricus, Haloterrigena hispanica, Thermotoga neapolitana and Geobacillus thermantarcticus) showed a good resistance to the simulation of the temperature variation in the space; on the other hand irradiation with UV at 254 nm affected only slightly the growth of H. hispanica, G. thermantarcticus and S. solfataricus; finally exposition to Mars simulated condition showed that H. hispanica and G. thermantarcticus were resistant to desiccation and low pressure.

Factors associated with climacteric symptoms in women around menopause attending menopause clinics in Italy.

OBJECTIVE: To obtain data on correlates of climacteric symptoms in women around menopause attending menopause clinics in Italy. METHODS: Since 1997 a large cross sectional study has been conducted on the characteristics of women around menopause attending a network of first level menopause outpatient's clinics in Italy. A total of 66,501 (mean age 54.4 years) women are considered in the present paper. RESULTS: The odds ratios of moderate and severe hot flashes/night sweats were lower in more educated women and (for severe symptoms only) in women reporting regular physical activity. Depression, difficulty to sleep, forgetfulness and irritability tended to be less frequent in more educated women and (depression only) in women reporting regular physical activity. Parous women reported more frequently these symptoms. CONCLUSIONS: This large study confirms in Southern European population that low education, body mass index and low physical activity are associated with climacteric symptoms. Parous women are at greater risk of psychological symptoms.

Risk factors for type 2 diabetes in women attending menopause clinics in Italy: a cross-sectional study.

OBJECTIVE: To analyze risk factors for type 2 diabetes among women attending menopause clinics in Italy for counselling about the menopause. SUBJECTS: Women attending a network of first-level outpatient menopause clinics in Italy for general counselling about menopause or treatment of menopausal symptoms. METHODS: Cross-sectional study with no exclusion criteria. Type 2 diabetes was defined according to National Diabetes Data Groups Indications and the fasting blood glucose at an oral glucose tolerance test within the previous year. RESULTS: Out of the 44 694 considered in this analysis, 808 had a diagnosis of diabetes type 2 (1.8%). In comparison with women aged < 50 years, the multivariate odds ratios (OR) of type 2 diabetes were 1.31 (95% confidence interval (CI), 0.99-1.74) for women aged 50-52 years, 1.66 (95% CI, 1.27-2.17) at 53-56 years and 2.84 (95% CI, 2.20-3.67) in women aged > or = 57 years. Type 2 diabetes was less frequently reported in more educated women (OR high school/university vs. primary school = 0.44 (95% CI, 0.36-0.55)). Being overweight was associated with an increased risk of type 2 diabetes. In comparison with women reporting a low level of physical activity, the multivariate OR of type 2 diabetes was 0.67 (95% CI, 0.54-0.84) for women reporting regular physical activity. In comparison with premenopausal women, the multivariate OR of type 2 diabetes was 1.38 (95% CI, 1.03-1.84) in women with natural menopause. This finding was present also after allowing for the potential confounding effect of age. The multivariate OR of diabetes for users of hormonal replacement therapy was 0.58 (95% CI, 0.46-0.73). CONCLUSIONS: This large cross-sectional study suggests that postmenopausal women are at higher risk of type 2 diabetes after allowance for the effect of age. Other main determinants of risk of type 2 diabetes in women around menopause were low socioeconomic status and being overweight. Diabetes was found less frequently in those taking hormone replacement therapy.

Zinc-catalyzed oxidation of 5-S-cysteinyldopa to 2,2'-bi(2H-1,4-benzothiazine): tracking the biosynthetic pathway of trichochromes, the characteristic pigments of red hair.

Trichochromes, the peculiar pigments of red human hair, featuring the Delta(2,2)(')-bi(2H-1,4-benzothiazine) skeleton, are known to arise from cysteinyldopas, mainly the 5-S-isomer (5). However, the mode of formation and the direct precursors have remained largely undefined. To fill this gap, we investigated the oxidation of 5 in air or with chemical and enzymatic agents under biomimetic conditions. In the presence of zinc ions, which occur in epidermal tissues at significant concentrations, the reaction course is diverted toward the formation of a labile 3-carboxy-2H-1,4-benzothiazine intermediate (11), which was identified by direct NMR analysis. Structural formulation was supported by characterization of the analogous compound 13 isolated from oxidation of the model 5-methyl-3-S-cysteinylcatechol (12) after methylation. In the further stages of the oxidation, diastereomeric 2,2'-bi(2H-1,4-benzothiazine) 15 and 14 were obtained from 5 and 12, respectively, the reaction proceeding at a higher rate and to a greater extent in the presence of acids. The dimers were shown to readily convert to each other in the presence of acids. In the case of the methylated dimers 14, a 2,2'-bi(4H-1,4-benzothiazine) intermediate (16) was isolated and characterized. In acidic media, trichochrome C (1a), the most abundant in red human hair, was smoothly formed from aerial oxidation of 15, and under similar conditions, trichochrome-related products (17 and 18) were obtained from 14 prior to or after methylation. The presence of 1a and precursors 5 and 15 was investigated by HPLC analysis of red hair samples following mild proteolytic digestion. On the basis of these data, a likely biosynthetic route to trichochrome pigments of red human hair is depicted.

Microanalysis of melanins in mammalian hair by alkaline hydrogen peroxide degradation: identification of a new structural marker of pheomelanins.

A highly sensitive, easy-to-perform method for melanin analysis in pigmented tissues based on alkaline hydrogen peroxide degradation has been developed and accomplishes simultaneous determination of eumelanins and pheomelanins. Pyrrole-2,3,5- tricarboxylic acid, the typical eumelanin marker, was obtained in higher yields than in previous procedures. A benzothiazole acid, 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole, characterized in our previous studies as a specific marker of pheomelanins, and the newly identified 1,3-thiazole-2,4, 5-tricarboxylic acid were also used for pigment analysis. Optimal yields of the pigment markers were obtained at 24 h reaction time. Pyrrole-2,3,5-tricarboxylic acid, 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole, and 1, 3-thiazole-2,4,5- tricarboxylic acid were quantified in a single chromatographic analysis without fractionation or work up of the degradation mixture. The linearity (linearity coefficient from 0.997 to 0.999) was excellent and the inter-assay (percentage coefficient of variation values in the range 0.2-2, n = 6) and intra-assay (percentage coefficient of variation values

New regulatory mechanisms in the biosynthesis of pheomelanins: rearrangement vs. redox exchange reaction routes of a transient 2H-1, 4-benzothiazine-o-quinonimine intermediate.

Pheomelanins, the typical epidermal pigments of red haired, Celtic-type Caucasians, arise from oxidative cyclization of cysteinyldopas, mainly the 5-S-isomer CD, via 1,4-benzothiazines. However, the mechanism and the relative yields of formation of these intermediates have remained poorly defined. We have now examined the course of the oxidation of CD at physiological pHs, under different reaction conditions. Surprisingly, a consumption of CD far exceeding the stoichiometry of the oxidant was observed at low oxidant-to-substrate ratios, low temperatures and high substrate concentrations. The yields of the 3,4-dihydro-1,4-benzothiazine-3-carboxylic acid DHBCA vs. the non-carboxylated analogue DHB in the oxidation mixture, after NaBH4 reduction, were also found to depend markedly on the reaction conditions. Based on these and other results, a reaction scheme is proposed involving a transient o-quinonimine generated by oxidative cyclization of CD to which three different paths are offered, namely redox exchange with CD to give DHBCA (path A) or intramolecular rearrangement with (path B) or without (path C) decarboxylation, leading to the benzothiazine BTZ and the 3-carboxy analogue BTZCA, respectively. The relative operation of path A vs. path C was assessed by deuterium labeling experiments. These findings point to new mechanisms of regulation of the initial steps of pheomelanogenesis, bearing significant implications on the structure of the final pigment.

Transient quinonimines and 1,4-benzothiazines of pheomelanogenesis: new pulse radiolytic and spectrophotometric evidence.

Biosynthetic and model in vitro studies have shown that pheomelanins, the distinctive pigments of red human hair, arise by oxidative cyclization of cysteinyldopas mainly 5-S-cysteinyldopa (1) via a critical o-quinonimine intermediate, which rearranges to unstable 1,4-benzothiazines. To get new evidence for these labile species, fast time resolution pulse radiolytic oxidation by dibromide radical anion of a suitable precursor, the dihydro-1,4-benzothiazine-3-carboxylic acid 7 was performed in comparison with that of 1. In the case of 7, dibromide radical anion oxidation leads over a few microseconds (k = 2.1 x 10(9) M(-1) s(-1)) to a phenoxyl radical (lambda(max) 330 nm, epsilon = 6300 M(-1) cm(-1)) which within tens of milliseconds gives rise with second-order kinetics (2k = 2.7 x 10(7) M(-1) s(-1)) to a species exhibiting an absorption maximum at 540 nm (epsilon = 2200 M(-1) cm(-1)). This was formulated as the o-quinonimine 3 arising from disproportionation of the initial radical. The quinonimine chromophore is converted over hundreds of milliseconds (k = 6.0 s(-1)) to a broad maximum at around 330 nm interpreted as due to a 1,4-benzothiazine or a mixture of 1,4-benzothiazines, which as expected are unstable and subsequently decay over a few seconds (k = 0.5 s(-1)). Interestingly, the quinonimine is observed as a labile intermediate also in the alternative reaction route examined, involving cyclization of the o-quinone (lambda(max) 390 nm, epsilon = 6900 M(-1) cm(-1)) arising by disproportionation (2k = 1.7 x 10(8) M(-1) s(-1)) of an o-semiquinone (lambda(max) 320 nm, epsilon = 4700 M(-1) cm(-1)) directly generated by dibromide radical anion oxidation of 1. Structural formulation of the 540 nm species as an o-quinonimine was further supported by rapid scanning diode array spectrophotometric monitoring of the ferricyanide oxidation of a series of model dihydrobenzothiazines.

Adult granulosa cell tumor of the ovary: a clinico pathologic study of 35 cases.

AIMS AND BACKGROUND: Adult granulosa cell tumor has a low malignant potential but requires an extensive follow-up of more than 5 years to accurately assess tumor activity. The aim of the present study was to evaluate the clinical characteristics, the treatment and the outcome of this rare ovarian tumor. STUDY DESIGN: A retrospective review of 35 cases treated at primary onset of disease during a 23-year period from 1971 to 1993. RESULTS: The disease-free survival rate for stages IA-B-C at 5 and 10 years was 90% and 84%, respectively; for stages III-IV the 5-year freedom from progression rate was 16%. CONCLUSIONS: The most important prognostic factor appears to be the extent of tumor involvement outside of the ovary.

Transvaginal ultrasound diagnosis of twin reversed arterial perfusion syndrome at 9 weeks' gestation.

Diagnosis of twin reversed arterial perfusion syndrome was made using transvaginal sonography at 9 weeks' gestation. The morphological evaluation of the abnormal twin revealed a dysmorphic cephalic pole and absence of the upper extremities. There was diffuse subcutaneous edema and no detectable cardiac activity. The other twin had a normal appearance and a size compatible with the gestational age. The fetuses were divided by a thin amniotic membrane with normal amniotic fluid in both sacs. Diagnosis was confirmed by demonstrating fetal growth and lower limb activity in the abnormal twin 10 days later.

Antioxidant vitamins or lactulose for the prevention of the recurrence of colorectal adenomas. Colorectal Cancer Study Group of the University of Modena and the Health Care District 16.

Colonic adenomas represent the natural precursor lesions of most colorectal cancers. The treatment of choice is endoscopic polypectomy. However, after endoscopic removal, polyps recur in a large fraction of cases. Thus, we evaluated the effect of antioxidant vitamins or lactulose on the recurrence rate of adenomatous polyps. After polypectomy, 255 individuals were randomized into three groups. Group 1 was given vitamin A (30,000 IU/day), vitamin C (1 g/day), and vitamin E (70 mg/day); Group 2 was given lactulose (20 g/day); Group 3 received no treatment. Forty-six subjects had to be excluded because the histologic diagnosis was not consistent with adenoma. The remaining 209 individuals were included in the analysis according to the "intention to treat" criterion, though 34 did not adhere to the scheduled treatment or were lost during the follow-up. Subjects were followed at regular intervals for an average of 18 months. Polyps recurring before one year from index colonoscopy were considered missed by the endoscopist. In the 209 evaluable subjects, the percentages of recurrence of adenomas were 5.7 percent, 14.7 percent, and 35.9 percent in the vitamins, lactulose, and untreated groups, respectively. The fraction of subjects remaining free of adenomas, estimated by Kaplan-Meier survival curves, was significantly different among the three groups (log-rank chi-squared = 17.138; P < 0.001). Using Cox's regression analysis, treatment was the only variable that significantly contributed to the model (regression coefficient = 0.905; P < 0.001). In conclusion, either antioxidant vitamins or, to a lesser extent, lactulose lower the recurrence rate of adenomas of the large bowel and can be proposed as chemopreventive agents, at least in high-risk individuals.

Pattern of epithelial cell proliferation in colorectal mucosa of normal subjects and of patients with adenomatous polyps or cancer of the large bowel.

Microautoradiography has been largely used to characterize the proliferative activity of colorectal mucosa. We used this technique in a large series of patients with polyps or cancer of the large bowel and in normal controls with the following objectives: (a) to define the normal pattern of cell replication in different tracts of the large bowel; (b) to compare the proliferative activity of colonic crypts in patients with colorectal cancer or polyps with that of controls; (c) to evaluate replicative activity of colorectal mucosa in the close vicinity and at distance from a neoplastic mass. Specimens of colorectal mucosa were taken during endoscopy (controls and polyps) or at surgery (cancer). During histological examination each intestinal hemicrypt was divided into five equal longitudinal compartments from the base to the surface and the labeled cells in each compartment were counted. In controls, total labeling index (ratio of labeled to total cells) and labeling index per crypt compartment showed only minor differences between the various large bowel tracts. Total labeling index tended to be higher in patients with polyps or cancer than in controls (13.5 +/- 0.4 and 12.5 +/- 0.4, respectively, versus 11.3 +/- 0.5). Labeling index per crypt compartment in the most superficial portions of the crypt (compartments 3 to 5) was significantly higher in the two groups of patients with tumors than in controls. This was particularly evident in the fifth compartment (the most superficial), in which labeled cells were observed in 15.8% (three subjects out of 19) of controls but in 71% (15 out of 21) and 87.5% (14 out of 16) of polyp and cancer patients, respectively. In patients with colorectal cancer there were not significant differences of cell proliferation between mucosal samples taken at various distances from the tumor margin; however, increased cell replication, especially in the most superficial portions of the crypt, has been observed. In conclusion, a significant upwards expansion of the proliferative zone of intestinal glands has been observed in patients with either polyps or cancer of the large bowel. In particular, labeling of the fifth compartment seems to possess the highest discriminatory power between subjects with or without intestinal neoplasms. Hyperproliferation of the entire colonic mucosa seems to be a common feature in patients with colorectal cancer.

Fecal neutral steroids in normal conditions and in patients with polyps or cancer of the large bowel.

There is evidence suggesting that the excretion and conversion of neutral sterols in the human large bowel might be somewhat related to the development of colorectal cancer. Therefore, our objectives were: to characterize the excretion and the major pattern of sterol degradation in normal conditions, both in children and in adults; and to investigate if abnormalities of these parameters are frequent in patients with colorectal cancer or polyps. The study has been carried out in: 38 adult volunteers; 29 children divided into 4 age groups; 22 patients with colorectal cancer; 16 members of 6 families with adenomatosis coli; 15 members of 2 families with a high prevalence of multiple polyps or cancer of the large bowel; 12 subjects with colorectal polyps without familiality. With the subjects kept under metabolic control, fecal samples were collected for at least 3 days and analyzed by thin layer chromatography and gas-liquid chromatography. Total neutral steroid excretion was lower in children than in adult volunteers; in contrast, there was no significant difference between the latter and the other investigated group of patients with cancer or polyps, with values ranging between 230 and 680 mg/day. All the adult volunteers were "high converters" of cholesterol to its intestinal metabolites coprostanol and coprostanone [89 +/- 10% (SE) of degradation]. Children less than 1 year old degraded little or no cholesterol (10.4 +/- 6% of total neutral sterols), whereas with increasing age the fraction of conversion became more similar to that of adults. In patients with colorectal tumors 2 populations could be defined, one characterized by a large degradation of cholesterol and the other by little or no conversion. Low degradation of cholesterol was found in 3 of 6 families with adenomatosis coli. In conclusion, we did not find any significant difference in total neutral sterol excretion among controls, colorectal cancer patients, or subjects at risk. In adult volunteers the normal pattern of cholesterol degradation is characterized by a large conversion of cholesterol to its intestinal metabolites. In children this process changes with increasing age from an absolute "nonconverter" state (after birth) to the pattern typical of adults. Finally, in a minority of patients with either polyps or cancer of the large bowel and of their first-degree relatives, cholesterol is poorly degraded and represents the most abundant fecal sterol.

Effect of small doses of deoxycholic acid on bile cholesterol saturation in patients with liver cirrhosis.

To test the hypothesis that the detergent power of each individual bile acid--that is, its separate capacity to solubilize cholesterol and to induce biliary cholesterol secretion, present in the biliary bile acid mixture might be one of the determinant factors of biliary cholesterol saturation, we studied the effect of feeding small doses of deoxycholic acid on biliary cholesterol saturation in patients with liver cirrhosis and low deoxycholic acid pool. Eleven hospitalised patients with cirrhosis of various degree of severity were put on a standard solid diet. Fasting bile rich duodenal fluid was obtained at the beginning of the study, after a three to four weeks treatment with deoxycholic acid (3 mg/kg/day, in two doses) and one month after discontinuing bile acid ingestion. Before treatment the fraction of deoxycholic acid was 5.3 +/- 4.9% (mean +/- SD); after treatment the fraction rose to 43.9 +/- 12.0 of total bile acids, but returned to the basal values after stopping bile acids. Bile cholesterol saturation increased significantly from a mean of 0.92 +/- 0.26 (before treatment) to a mean of 1.34 +/- 0.34 after deoxycholic acid feeding (p less than 0.005). One month after treatment, bile saturation was not significantly different from the basal values (0.91 +/- 0.44). We conclude that feeding low doses of deoxycholic acid to patients with liver cirrhosis induces a significant increase of the fraction of this bile acid in the total pool and this is followed by a sharp increase of bile cholesterol saturation. These data are compatible with the hypothesis that the detergent capacity of individual bile acids is one of the main determinants of bile cholesterol saturation.

Cholesterol esterase activity of human intestinal mucosa.

It has been suggested that cholesterol absorption in humans is dependent on bile acid pool composition and that expansion of the cholic acid pool size is followed by an increase of the absorption values. Similar observations were reported in rats, where the increase of cholesterol absorption, after trihydroxy bile acid feeding, seems to be due to the stimulatory effect of cholic acid on the intestinal cholesterol esterase. In the present study, therefore, we investigated some general properties of human intestinal cholesterol esterase, with particular emphasis to the effect of bile acids on this enzymatic activity. Twenty-nine segments of small intestine were taken during operations; the enzymatic activity was studied by using mucosal homogenate as a source of enzyme and oleic acid, cholesterol, and 14C-labeled cholesterol as substrates. The time-activity relationship was linear within the first two hours; optimal pH for esterification ranged between 5 and 6.2. There was little difference between the esterifying activity of the jejunal and ileal mucosa. Esterification of cholesterol was observed with all the investigated fatty acids but was maximal with oleic acid. Bile acids did not affect cholesterol esterase activity when present in the incubation mixture at 0.1 and 1.0 mM; the enzymatic activity, however, was significantly inhibited when bile acids were added at 20 mM. In conclusion, this study has shown that the human intestinal mucosa possesses a cholesterol esterase activity; at variance with the rat, however, the human enzyme does not seem to be stimulated by trihydroxy bile acids. Thus, the stimulatory effect of cholic acid on cholesterol absorption induced by the administration of this bile acid does not seem to be simply due to changes of cholesterol esterase activity of the small bowel mucosa.