Heterologous expression of an Aspergillus niveus xylanase GH11 in Aspergillus nidulans and its characterization and application

A xylanase was cloned from Aspergillus niveus and successfully expressed in Aspergillus nidulans (XAN). The full-length gene consisted of 890 bp and encoded 275 mature amino acids with a calculated mass of 31.3 kDa. The deduced amino acid sequence was highly homologous with the xylanase belonging to family 11 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic homogeneity by anion-exchange chromatography and gel filtration. The optima of pH and temperature for the recombinant enzyme were 5.0 and 65 degrees C, respectively. The thermal stability of the recombinant xylanase was extremely improved by covalent immobilization on glyoxyl agarose with 91.4% of residual activity after 180 min at 60 degrees C, on the other hand, the free xylanase showed a half-life of 9.9 min at the same temperature. Affinity chromatography on Concanavalin A- and Jacalin-agarose columns followed by SDS-PAGE analyses showed that the XAN has O- and N-glycans. XAN promotes hydrolysis of xylan resulting in xylobiose, xylotriose and xylotetraose. Intermediate degradation of xylan resulting in xylo-oligomers is appealing for functional foods as the beneficial effect of oligosaccharides on gastrointestinal micro flora includes preventing proliferation of pathogenic intestinal bacteria and facilitates digestion and absorption of nutrients. (C) 2011 Elsevier Ltd. All rights reserved.

Removal from the Membrane Affects the Interaction of Rat Osseous Plate Ecto-Nucleosidetriphosphate Diphosphohydrolase-1 with Substrates and Ions

We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1) from rat osseous plate membranes. A novel finding of the present study is that the solubilized enzyme shows high- and low-affinity sites for the substrate in contrast with a single substrate site for the membrane-bound enzyme. In addition, contrary to the Michaelian chraracteristics of the membrane-bound enzyme, the site-site interactions after solubilization with 0.5% digitonin plus 0.1% lysolecithin resulted in a less active ectonucleoside triphosphate diphosphohydrolase, showing activity of about 398.3 nmol Pi min(-1) mg(-1). The solubilized enzyme has M(r) of 66-72 kDa, and its catalytic efficiency was significantly increased by magnesium and calcium ions; but the ATP/ADP activity ratio was always < 2.0. Partial purification and kinetic characterization of the rat osseous plate E-NTPDase1 in a solubilized form may lead to a better understanding of a possible function of the enzyme as a modulator of nucleotidase activity or purinergic signaling in matrix vesicle membranes. The simple procedure to obtain the enzyme in a solubilized form may also be attractive for comparative studies of particular features of the active sites from this and other ATPases.

Hemolymph ionic regulation and adjustments in gill (Na(+), K(+))-ATPase activity during salinity acclimation in the swimming crab Callinectes ornatus (Decapoda, Brachyura)

We evaluate hemolymph osmotic and ionic regulatory abilities and characterize a posterior gill microsomal (Na(+), K(+))-ATPase from the marine swimming crab, Callinectes ornatus, acclimated to 21 parts per thousand or 33 parts per thousand salinity. C ornatus is isosmotic after acclimation to 21 parts per thousand but is hyposmotic at 33 parts per thousand salinity; hemolymph ions do not recover initial levels on acclimation to 21 parts per thousand salinity but are anisoionic compared to ambient concentrations, revealing modest regulatory ability. NH(4)(+) modulates enzyme affinity for K(+), which increases 187-fold in crabs acclimated to 33%. salinity. The (Na(+), K(+))-ATPase redistributes into membrane fractions of different densities, suggesting that altered membrane composition results from salinity acclimation. ATP was hydrolyzed at maximum rates of 182.6 +/- 7.1 nmol Pi min(-1) mg(-1) (21 parts per thousand) and 76.2 +/- 3.5 nmol Pi min(-1) mg(-1) (33 parts per thousand), with little change in K(M) values (approximate to 50 mu mol L(-1)). K(+) together with NH(4)(+) synergistically stimulated activity to maximum rates of approximate to 240 nmol Pi min(-1) mg(-1). K, values for ouabain inhibition (approximate to 110 mu mol L(-1)) decreased to 44.9 +/- 1.0 mu mol L(-1) (21 parts per thousand) and 28.8 +/- 1.3 mu mol L(-1) (33 parts per thousand) in the presence of both K(+) and NH(4)(+). Assays employing various inhibitors suggest the presence of mitochondrial F(0)F(1)- and K(+)- and V-ATPase activities in the gill microsomes. (C) 2009 Elsevier Inc. All rights reserved.

Seasonal dynamics of a drosophilid (Diptera) assemblage and its potencial as bioindicator in open environments

Drosophila Fallen, 1823 (Diptera, Drosophilidae) is for long a well-established model organism for genetics and evolutionary research. The ecology of these flies, however, has only recently been better studied. Recent papers show that Drosophila assemblies can be used as bioindicators of forested environment degradation. In this work the bioindicator potential of drosophilids was evaluated in a naturally opened environment, a coastal strand-forest (restinga). Data from nine consecutive seasonal collections revealed strong temporal fluctuation pattern of the majority of Drosophila species groups. Drosophila willistoni group was more abundant at autumns, whereas D. cardini and D. tripunctata groups were, respectively, expressive at winters and springs, and D. repleta group at both seasons. The exotic species D. simulans Sturtevant, 1919 (from D. melanogaster group) and Zaprionus indianus Gupta, 1970 were most abundant at summers. Overall, the assemblage structure did not show the same characteristics of forested or urban environments, but was similar to the forests at winters and to cities at summers. This raises the question that this locality may already been under urbanization impact. Also, this can be interpreted as an easily invaded site for exotic species, what might lead to biotic homogenization and therefore can put in check the usage of drosophilid assemblages as bioindicators at open environments.

Serum Ferritin Level Remains a Reliable Marker of Bone Marrow Iron Stores Evaluated by Histomorphometry in Hemodialysis Patients

Background and objectives: As well as being a marker of body iron stores, serum ferritin (sFerritin) has also been shown to be a marker of inflammation in hemodialysis (HD) patients. The aim of this study was to analyze whether sFerritin is a reliable marker of the iron stores present in bone marrow of HD patients. Design: Histomorphometric analysis of stored transiliac bone biopsies was used to assess iron stores by determining the number of iron-stained cells per square millimeter of bone marrow. Results: In 96 patients, the laboratory parameters were hemoglobin = 11.3 +/- 1.6 g/dl, hematocrit = 34.3 +/- 5%, sFerritin 609 +/- 305 ng/ml, transferrin saturation = 32.7 +/- 22.5%, and C-reactive protein (CRP) = 0.9 +/- 1.4 mg/dl. sFerritin correlated significantly with CRP, bone marrow iron, and time on HD treatment W = 0.006, 0.001, and 0.048, respectively). The independent determinants of sFerritin were CRP (beta-coef = 0.26; 95% CI = 24.6 to 132.3) and bone marrow iron (beta-coef = 0.32; 95% CI = 0.54 to 2.09). Bone marrow iron was higher in patients with sFerritin >500 ng/ml than in those with sFerritin :5500 ng/ml. In the group of patients with sFerritin :5500 ng/ml, the independent determinant of sFerritin was bone marrow iron (beta-coef = 0.48, 95% CI = 0.48 to 1.78), but in the group of patients with sFerritin >500 ng/ml, no independent determinant of sFerritin was found. Conclusions: sFerritin adequately reflects iron stores in bone marrow of HD patients.

Association of Changes in Bone Remodeling and Coronary Calcification in Hemodialysis Patients: A Prospective Study

Background: Vascular calcification is common and constitutes a prognostic marker of mortality in the hemodialysis population. Derangements of mineral metabolism may influence its development. The aim of this study is to prospectively evaluate the association between bone remodeling disorders and progression of coronary artery calcification (CAC) in hemodialysis patients. Study Design: Cohort study nested within a randomized controlled trial. Setting & Participants: 64 stable hemodialysis patients. Predictor: Bone-related laboratory parameters and bone histomorphometric characteristics at baseline and after 1 year of follow-up. Outcomes: Progression of CAC assessed by means of coronary multislice tomography at baseline and after 1 year of follow-up. Baseline calcification score of 30 Agatston units or greater was defined as calcification. Change in calcification score of 15% or greater was defined as progression. Results: Of 64 patients, 26 (40%) had CAC at baseline and 38 (60%) did not. Participants without CAC at baseline were younger (P < 0.001), mainly men (P = 0.03) and nonwhite (P = 0.003), and had lower serum osteoprotegerin levels (P = 0.003) and higher trabecular bone volume (P = 0.001). Age (P 0.003; beta coefficient = 1.107; 95% confidence interval [Cl], 1.036 to 1.183) and trabecular bone volume (P = 0.006; beta coefficient = 0.828; 95% Cl, 0.723 to 0.948) were predictors for CAC development. Of 38 participants who had calcification at baseline, 26 (68%) had CAC progression in 1 year. Progressors had lower bone-specific alkaline phosphatase (P = 0.03) and deoxypyridinoline levels (P = 0.02) on follow-up, and low turnover was mainly diagnosed at the 12-month bone biopsy (P = 0.04). Low-turnover bone status at the 12-month bone biopsy was the only independent predictor for CAC progression (P = 0.04; beta coefficient = 4.5; 95% Cl, 1.04 to 19.39). According to bone histological examination, nonprogressors with initially high turnover (n = 5) subsequently had decreased bone formation rate (P = 0.03), and those initially with low turnover (n = 7) subsequently had increased bone formation rate (P = 0.003) and osteoid volume (P = 0.001). Limitations: Relatively small population, absence of patients with severe hyperparathyroidism, short observational period. Conclusions: Lower trabecular bone volume was associated with CAC development, whereas improvement in bone turnover was associated with lower CAC progression in patients with high- and low-turnover bone disorders. Because CAC is implicated in cardiovascular mortality, bone derangements may constitute a modifiable mortality risk factor in hemodialysis patients.

Phosphate Binder Impact on Bone Remodeling and Coronary Calcification - Results from the BRiC Study

Background and Aims: Calcium-containing phosphate binders have been shown to increase the progression of vascular calcification in hemodialysis patients. This is a prospective study that compares the effects of calcium acetate and sevelamer on coronary calcification (CAC) and bone histology. Methods: 101 hemodialysis patients were randomized for each phosphate binder and submitted to multislice coronary tomographies and bone biopsies at entry and 12 months. Results: The 71 patients who concluded the study had similar baseline characteristics. On follow-up, the sevelamer group had higher levels of intact parathyroid hormone (498 +/- 352 vs. 326 +/- 236 pg/ml, p = 0.017), bone alkaline phosphatase (38 +/- 24 vs. 28 +/- 15 U/l, p = 0.03) and deoxypyridinoline (135 +/- 107 vs. 89 +/- 71 nmol/l, p = 0.03) and lower LDL cholesterol (74 +/- 21 vs. 91 +/- 28 mg/dl, p = 0.015). Phosphorus (5.8 +/- 1.0 vs. 6 +/- 1.0 mg/dl, p = 0.47) and calcium (1.27 +/- 0.07 vs. 1.23 +/- 0.08 mmol/l, p = 0.68) levels did not differ between groups. CAC progression (35 vs. 24%, p = 0.94) and bone histological diagnosis at baseline and 12 months were similar in both groups. Patients of the sevelamer group with a high turnover at baseline had an increase in bone resorption (eroded surface, ES/BS = 9.0 +/- 5.9 vs. 13.1 +/- 9.5%, p = 0.05), whereas patients of both groups with low turnover at baseline had an improvement in bone formation rate (BFR/BS = 0.015 +/- 0.016 vs. 0.062 +/- 0.078, p = 0.003 for calcium and 0.017 +/- 0.016 vs. 0.071 +/- 0.084 mu m(3)/mu m(2)/day, p = 0.010 for sevelamer). Conclusions: There was no difference in CAC progression or changes in bone remodeling between the calcium and the sevelamer groups. Copyright (C) 2008 S. Karger AG, Basel

Fibroblast Growth Factor 23 in Hemodialysis Patients: Effects of Phosphate Binder, Calcitriol and Calcium Concentration in the Dialysate

Background: Fibroblast growth factor 23 (FGF23) concentrations increase early in chronic kidney disease (CKD), and the influence of current CKD-mineral and bone disorder (MBD) therapies on serum FGF23 levels is still under investigation. Methods: In this post-hoc analysis of a randomized clinical trial, phosphate binders and calcitriol were washed out of 72 hemodialysis patients who were then submitted to bone biopsy, coronary tomography and biochemical measures, including FGF23. They were randomized to receive sevelamer or calcium acetate for 1 year and the prescription of calcitriol and the calcium concentration in the dialysate were adjusted according to serum calcium, phosphate and PTH and bone biopsy diagnosis. Results: At baseline, bone biopsy showed that 58.3% had low-turnover bone disease, whereas 38.9% had high-turnover bone disease, with no significant differences between them with regard to FGF23. Median baseline FGF23 serum levels were elevated and correlated positively with serum phosphate. After 1 year, serum FGF23 decreased significantly. Repeated measures ANOVA analysis showed that the use of a 3.5-mEq/l calcium concentration in the dialysate, as well as the administration of calcitriol and a calcium-based phosphate binder were associated with higher final serum FGF23 levels. Conclusions: Taken together, our results confirm that the current CKD-MBD therapies have an effect on serum levels of FGF23. Since FGF23 is emerging as a potential treatment target, our findings should be taken into account in the decision on how to manage CKD-MBD therapy. Copyright (C) 2010 S. Karger AG, Basel

Quality of life and neuropsychological changes in mild head trauma - Late analysis and correlation with S100B protein and cranial CT scan performed at hospital admission

Introduction: mild head trauma (MHT) is defined as a transient neurological deficit after trauma with a history of impairment or loss of consciousness lasting less than 15 min and/or posttraumatic amnesia, and a Glasgow Coma Scale between 13 and 15 on hospital admission. We evaluated 50 MHT patients 18 months after the trauma, addressing signs and symptoms of post-concussion syndrome, quality of life and the presence of anxiety and depression. We correlate those findings with the S100B protein levels and cranial CT scan performed at hospital admission after the trauma. Method: patients were asked to fill out questionnaires to assess quality of life (SF36), anxiety and depression (HADS), and signs and symptoms of post-concussion syndrome. For the control group, we asked the patient`s household members, who had no history of head trauma of any type, to answer the same questionnaires for comparison. Results: total quality of life index for patients with MHT was 58.16 (+/-5), lower than the 73.47 (+/-4) presented by the control group. Twenty patients (55.2%) and four (11.1%) controls were depressed. Seventeen patients (47.2%) presented anxiety, whereas only eight (22.2%) controls were considered anxious. Victims of MHT complained more frequently of loss of balance, dry mouth, pain in the arms, loss of memory and dizziness than their respective controls (p < 0.05). We found no correlation between the presence of these signs and symptoms, quality of life, presence of anxiety and depression with S100B protein levels or with presence of injury in the cranial CT performed at hospital admission. Conclusion: MHT is associated with a higher incidence of post-concussion syndrome symptoms, lower quality of life and anxiety than their respective controls even 18 months after the trauma. (C) 2007 Elsevier Ltd. All rights reserved.

Genetic Contribution for Non-Syndromic Cleft Lip With or Without Cleft Palate (NS CL/P) in Different Regions of Brazil and Implications for Association Studies

Non-syndromic cleft lip with or without cleft palate (NS CL/P) is a complex disease in which heritability estimates vary widely depending on the population studied. To evaluate the importance of genetic contribution to NS CL/P in the Brazilian population, we conducted a study with 1,042 families from five different locations (Santarem, Fortaleza, Barbalha, Maceio, and Rio de Janeiro). We also evaluated the role of consanguinity and ethnic background. The proportion of familial cases varied significantly across locations, with the highest values found in Santarem (44%) and the lowest in Maceio (23%). Heritability estimates showed a higher genetic contribution to NS CL/P in Barbalha (85%), followed by Santarem (71%), Rio de Janeiro (70%), Fortaleza (64%), and Maceio (45%). Ancestry was not correlated with the occurrence of NS CL/P or with the variability in heritability. Only in Rio de Janeiro was the coefficient of inbreeding significantly larger in NS CL/P families than in the local population. Recurrence risk for the total sample was approximately 1.5-1.6%, varying according to the location studied (0.6-0.7% in Maceio to 2.2-2.8% in Barbalha). Our findings show that the degree of genetic contribution to NS CL/P varies according to the geographic region studied, and this difference cannot be attributed to consanguinity or ancestry. These findings suggest that Barbalha is a promising region for genetic studies. The data presented here will be useful in interpreting results from molecular analyses and show that care must be taken when pooling samples from different populations for association studies. (C) 2011 Wiley-Liss, Inc.

Liver transplantation and quality of life: relevance of a specific liver disease questionnaire

Aim: A positive effect of liver transplantation on health-related quality of life (HRQOL) has been well documented in previous studies using generic instruments. Our aim was to re-evaluate different aspects of HRQOL before and after liver transplantation with a relatively new questionnaire the `liver disease quality of life` (LDQOL). Methods: The LDQOL and the Short Form 36 (SF-36) questionnaires were applied to ambulatory patients, either in the transplant list (n=65) or after 6 months to 5 years of liver transplant (n=61). The aetiology of cirrhosis, comorbidities, model for end-stage liver disease (MELD) Child-Pugh scores and recurrence of liver disease after liver transplantation were analysed using the Mann-Whitney and Kruskall-Wallis tests. Results: In patients awaiting liver transplantation, MELD scores >= 15 and Child-Pugh class C showed statistically significant worse HRQOL, using both the SF-36 and the LDQOL questionnaires. HRQOL in pretransplant patients was found to be significantly worse in those with cirrhosis owing to hepatitis C (n=30) when compared with other aetiologies (n=35) in 2/7 domains of the SF-36 and in 7/12 domains of the LDQOL. Significant deterioration of HRQOL after recurrence of hepatitis C post-transplant was detected with the LDQOL questionnaire although not demonstrated with the SF-36. The statistically significant differences were in the LDQOL domains: symptoms of liver disease, concentration, memory and health distress. Conclusions: The LDQOL, a specific instrument for measuring HRQOL, has shown a greater accuracy in relation to liver symptoms and could demonstrate, with better reliability, impairments before and after liver transplantation.

Reconstruction of large cranial defects in nonimmunosuppressed experimental design with human dental pulp stem cells

The main aim of this study is to evaluate the capacity of human dental pulp stem cells (hDPSC), isolated from deciduous teeth, to reconstruct large-sized cranial bone defects in nonimmunosuppressed (NIS) rats. To our knowledge, these cells were not used before in similar experiments. We performed two symmetric full-thickness cranial defects (5 x 8 mm) on each parietal region of eight NIS rats. In six of them, the left side was supplied with collagen membrane only and the right side (RS) with collagen membrane and hDPSC. In two rats, the RS had collagen membrane only and nothing was added at the left side (controls). Cells were used after in vitro characterization as mesenchymal cells. Animals were euthanized at 7, 20, 30, 60, and 120 days postoperatively and cranial tissue samples were taken from the defects for histologic analysis. Analysis of the presence of human cells in the new bone was confirmed by molecular analysis. The hDPSC lineage was positive for the four mesenchymal cell markers tested and showed osteogenic, adipogenic, and myogenic in vitro differentiation. We observed bone formation 1 month after surgery in both sides, but a more mature bone was present in the RS. Human DNA was polymerase chain reaction-amplified only at the RS, indicating that this new bone had human cells. The us e of hDPSC in NIS rats did not cause any graft. rejection. Our findings suggest that hDPSC is an additional cell resource for correcting large cranial defects in rats and constitutes a promising model for reconstruction of human large cranial defects in craniofacial surgery.

Evaluation of Maxillary Alveolar Reconstruction Using a Resorbable Collagen Sponge with Recombinant Human Bone Morphogenetic Protein-2 in Cleft Lip and Palate Patients

Introduction: A resorbable collagen matrix with recombinant human bone morphogenetic protein (rhBMP-2) was compared with traditional iliac crest bone graft for the closure of alveolar defects during secondary dental eruption. Methods: Sixteen patients with unilateral cleft lip and palate, aged 8 to 12 years, were selected and randomly assigned to group 1 (rhBMP-2) or group 2 (iliac crest bone graft). Computed tomography was performed to assess both groups preoperatively and at months 6 and 12 postoperatively. Bone height and defect volume were calculated through Osirix Dicom Viewer (Pixmeo, Apple Inc.). Overall morbidity was recorded. Results: Preoperative and follow-up examinations revealed progressive alveolar bone union in all patients. For group 1, final completion of the defect with a 65.0% mean bone height was detected 12 months postoperatively. For group 2, final completion of the defect with an 83.8% mean bone height was detected 6 months postoperatively. Dental eruption routinely occurred in both groups. Clinical complications included significant swelling in three group 1 patients (37.5%) and significant donor-site pain in seven group 2 patients (87.5%). Conclusions: For this select group of patients with immature skeleton, rhBMP-2 therapy resulted in satisfactory bone healing and reduced morbidity compared with traditional iliac crest bone grafting.

Evaluation of Primary Cleft Nose Repair: Severity of the Cleft Versus Final Position of the Nose

Background: Since 1957, when the concept of rotation-advancement repair was introduced by Millard, this technique has become the procedure of choice for unilateral cleft lip worldwide. More recently, modifications described by Noordhoof, Mohler, Skoog, and McComb started being jointly performed so that better results could be obtained. In this study, the nasal position was evaluated and related to the size of the cleft. The primary unilateral cleft lip repair was performed through a modified technique. Methods: Forty-five patients with unilateral cleft lip underwent primary surgical repair through this technique. To analyze aesthetic results, a severity classification of deformities and a scoring system for evaluation of the results were established based on nasal alar lateralization, dome position, alignment of bone segments, and deviation of the columella. Results: By means of the established system, 26.6% of mild forms, 13.4% of moderate forms, and 60% of severe forms were observed. Among aesthetic results, 17.8% were found to be good, and 82.2% were considered excellent. Among aspects considered negative, late deformity of the lower lateral cartilage prevailed. Conclusions: Through the presented evaluation, the authors observed that there was no relation between severity of the cleft and final position of the nose. Among the 27 patients considered to have had severe forms of cleft deformity, 22 were classified as excellent results (81.5%). To obtain better results along time, technical refinements and the critical analysis of results must be performed on a routinely basis.

Novel mutations of the BSCL2 and AGPAT2 genes in 10 families with Berardinelli-Seip congenital generalized lipodystrophy syndrome

P>Context Congenital generalized lipodystrophy, or Berardinelli-Seip syndrome, is a rare autosomal recessive disease caused by mutations in either the BSCL2 or AGPAT2 genes. This syndrome is characterized by an almost complete loss of adipose tissue usually diagnosed at birth or early infancy resulting in apparent muscle hypertrophy. Common clinical features are acanthosis nigricans, hepatomegaly with or without splenomegaly and high stature. Acromegaloid features, cardiomyopathy and mental retardation can also be present. Design We investigated 11 kindreds from different geographical areas of Brazil (northeast and southeast). All coding regions as well as flanking intronic regions of both genes were examined. Polymerase chain reaction (PCR) amplifications were performed using primers described previously and PCR products were sequenced directly. Results Four AGPAT2 and two BSCL2 families harboured the same set of mutations. BSCL2 gene mutations were found in the homozygous form in four kindreds (c.412C > T c.464T > C, c.518-519insA, IVS5-2A > G), and in two kindreds compound mutations were found (c.1363C > T, c.424A > G). In the other four families, one mutation of the AGPAT2 gene was found (IVS3-1G > C and c.299G > A). Conclusions We have demonstrated four novel mutations of the BSCL2 and AGPAT2 genes responsible for Berardinelli-Seip syndrome and Brunzell syndrome (AGPAT2-related syndrome).