Highly thermostable xylanase purified from Rhizomucor miehei NRL 3169

A thermostable xylanase was purified and characterized from the thermophilic fungus Rhizomucor miehei (Cooney & Emerson) Schipper. The enzyme was purified to homogeneity by ammonium sulfate precipitation, sephadex G-100 gel filtration and diethylaminoethyl cellulose anion exchange chromatography with a 29.1-fold. The enzyme was highly active within a range of pH from 5.0 to 6.5. The optimum temperature of the purified enzyme was 75°C. The enzyme showed high thermal stability at 70°C and 75°C and the half-life of the xylanase at 90°C was 30 min. Km and Vmax values at 50°C of the purified enzyme were 0.055 mg/ml and 113.5 μmol min?1 mg?1 respectively. The enzyme was activated by Ca2+, Cu2+, K+ and Na+. On the other hand, Ag2+, Hg2+, Ba2+, and Zn2+ inhibited the enzyme. The molecular weight of the xylanase was estimated to be 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present study is among the first works to examine and describe a secreted highly thermostable endoxylanase from the Rhizomucor miehei fungus. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for industrial and commercial application in pulp bleaching.     

Serum level of prostate-specific antigen (PSA) in women with breast cancer

ObjectiveTo identify the diagnostic role of total and free prostate-specific antigen (TPSA and FPSA) in breast cancer in women.MethodsBlood samples of 55 women with breast cancer were prospectively analyzed for PSA before and after breast surgery, with a control group of 82 healthy women.ResultsTotal and free PSA levels were significantly higher in women with breast cancer (preoperatively) than in healthy women (P < 0.001). Both serum TPSA and FPSA showed a significant decline in their pre-surgical values after surgical removal of the tumor (P < 0.001). A significant proportion of breast cancer patients (83.6%) had free PSA as the predominant molecular form in serum as compared to 0% of controls and 1.8% of postoperative groups (P < 0.001). TPSA and FPSA levels were significantly associated with younger age and earlier cancer stage, whereas no significant association was found between these two variables and FPSA as a predominant molecular form.ConclusionsThis study indicated a clinical significance of preoperative measurement of serum TPSA and FPSA in the diagnosis of women with breast cancer, and may be a useful marker for monitoring the response to treatment.     

Thermostable alkaline protease from Thermomyces lanuginosus: optimization,purification and characterization

A serine alkaline protease (EC.3.4.21) was isolated, purified and characterized from culture filtrate of the thermophilic fungus Thermomyces lanuginosus Tsiklinsky. Fructose (1.5 %) and gelatin (0.5 %) proved to be the best carbon and nitrogen sources, giving a maximum enzyme yield of 9.2 U/mL. Dates waste was utilized as a sole organic source to improve enzyme productivity, and the yield was calculated to be 11.56 U/mL. This yield was expressed also as 231.2 U/g of assimilated waste. The alkaline protease produced was precipitated by iso-propanol and further purified by gel filtration through Sephadex G-₁₀₀ and ion exchange column chromatography on diethyl amino ethyl (DEAE)-cellulose with a yield of 30.12 % and 13.87-fold purification. The enzyme acted optimally at pH 9 and 60 °C and had good stability at alkaline pH and high temperatures. The enzyme possessed a high degree of thermostability and retained full activity even at the end of 1 h of incubation at 60 °C. Michaelis–Menten constant (K _m), maximal reaction velocity (V _max) and turnover number (K _cat) of the purified enzyme on gelatin as a substrate were calculated to be 4.0 mg/mL, 18.5 U/mL and 1.8 s^?1, respectively. The best enzyme activators were K⁺, Ca²⁺ and Mn², respectively, while phenylmethylsulfonyl fluoride (PMSF) was the strongest inhibitory agent, thus suggesting that the enzyme is a serine type protease. The enzyme is a glycoprotein with molecular mass of 33 kDa as determined by SDS-PAGE. It retained full activity after 15 min incubation at 60 °C in the presence of the detergent Ariel, thus indicating its suitability for application in the detergent industry.     

The protective effects of 17beta-estradiol against ischemia–reperfusion injury and its effect on pacing postconditioning protection to the heart

The role of pacing postconditioning (PPC) in the heart protection against ischemia–reperfusion injury is not completely understood. The aim of this study was to investigated if 17-β-estradiol (estrogen, E2), endogenous atrial natriuretic peptide (ANP), endogenous brain natriuretic peptide (BNP), and tumor necrosis factor-alpha (TNF-α) are involved in PPC-mediated protection. Langendorff perfused female Wistar rat hearts were used for this study. Hearts challenged with regional ischemia for 30 min subjected to no further treatment served as a control. The PPC protocol was 3 cycles of 30 s pacing alternated between the right atrium and left ventricle (LV). Protection was assessed by recovery of LV contractility and coronary vascular–hemodynamics. Ischemia induced a significant (P?<?0.05) deterioration in the heart function compared with baseline data. PPC alone or in combination with short-term E2 treatment (E2 infusion at the beginning of reperfusion) significantly (P?<?0.05) improved the heart functions. Short-term E2 treatment post-ischemically afforded protection similar to that of PPC. However, long-term E2 substitution for 6 weeks completely attenuated the protective effects of PPC. Although no changes were noted in endogenous ANP levels, PPC significantly increased BNP expression level and decreased TNF-α in the cardiomyocyte lysate and coronary effluent compared to ischemia and controls. Our data suggested a protective role for short-term E2 treatment similar to that of PPC mediated by a pathway recruiting BNP and downregulating TNF-α. Our study further suggested a bad influence for long-term E2 substitution on the heart as it completely abrogated the protective effects of PPC.     

Multivitamin and Iron Supplementation to Prevent Periconceptional Anemia in Rural Tanzanian Women: A Randomized,Controlled Trial

Objective

Women’s nutritional status during conception and early pregnancy can influence maternal and infant outcomes. This study examined the efficacy of pre-pregnancy supplementation with iron and multivitamins to reduce the prevalence of anemia during the periconceptional period among rural Tanzanian women and adolescent girls.

Design

A double-blind, randomized controlled trial was conducted in which participants were individually randomized to receive daily oral supplements of folic acid alone, folic acid and iron, or folic acid, iron, and vitamins A, B-complex, C, and E at approximately single recommended dietary allowance (RDA) doses for six months.

Setting

Rural Rufiji District, Tanzania.

Subjects

Non-pregnant women and adolescent girls aged 15–29 years (n = 802).

Results

The study arms were comparable in demographic and socioeconomic characteristics, food security, nutritional status, pregnancy history, and compliance with the regimen (p>0.05). In total, 561 participants (70%) completed the study and were included in the intention-to-treat analysis. Hemoglobin levels were not different across treatments (median: 11.1 g/dL, Q1-Q3: 10.0–12.4 g/dL, p = 0.65). However, compared with the folic acid arm (28%), there was a significant reduction in the risk of hypochromic microcytic anemia in the folic acid and iron arm (17%, RR: 0.61, 95% CI: 0.42–0.90, p = 0.01) and the folic acid, iron, and multivitamin arm (19%, RR: 0.66, 95% CI: 0.45–0.96, p = 0.03). Inverse probability of treatment weighting (IPTW) to adjust for potential selection bias due to loss to follow-up did not materially change these results. The effect of the regimens was not modified by frequency of household meat consumption, baseline underweight status, parity, breastfeeding status, or level of compliance (in all cases, p for interaction>0.2).

Conclusions

Daily oral supplementation with iron and folic acid among women and adolescents prior to pregnancy reduces risk of anemia. The potential benefits of supplementation on the risk of periconceptional anemia and adverse pregnancy outcomes warrant investigation in larger studies.

Trial Registration

ClinicalTrials.gov NCT01183572     

α2β1 integrin promotes chemoresistance against doxorubicin in cancer cells through extracellular signal-regulated kinase (ERK)

The role and the mechanisms by which β1 integrins regulate the survival and chemoresistance of T cell acute lymphoblastic leukemia (T-ALL) still are poorly addressed. In this study, we demonstrate in T-ALL cell lines and primary blasts, that engagement of α2β1 integrin with its ligand collagen I (ColI), reduces doxorubicin-induced apoptosis, whereas fibronectin (Fn) had no effect. ColI but not Fn inhibited doxorubicin-induced mitochondrial depolarization, cytochrome c release, and activation of caspase-9 and -3. ColI but not Fn also prevented doxorubicin from down-regulating the levels of the prosurvival Bcl-2 protein family member Mcl-1. The effect of ColI on Mcl-1 occurred through the inhibition of doxorubicin-induced activation of c-Jun N-terminal kinase (JNK). Mcl-1 knockdown experiments showed that the maintenance of Mcl-1 levels is essential for ColI-mediated T-ALL cell survival. Furthermore, activation of MAPK/ERK, but not PI3K/AKT, is required for ColI-mediated inhibition of doxorubicin-induced JNK activation and apoptosis and for ColI-mediated maintenance of Mcl-1 levels. Thus, our study identifies α2β1 integrin as an important survival pathway in drug-induced apoptosis of T-ALL cells and suggests that its activation can contribute to the generation of drug resistance.     

Surface electromyography and peak torque of repetitive maximum isokinetic plantar flexions in relation to aspects of muscle morphology.

This study investigates the relationships between surface electromyography (EMG [Mean frequency of the power spectrum (MNF)]) and peak torque variables obtained during 100 maximum concentric plantar flexions with the right limb at 60 degrees s(-1) and different muscle morphological variables. Surface EMG was recorded from the right gastrocnemius lateralis and muscle biopsies were taken from the same site as the EMG electrodes were positioned. Muscle fibre area and fibre type composition were determined on serial muscle cross sections using both histochemistry (myofibrillar adenosine triphosphatase) and immunohistochemistry (monoclonal antibodies against specific myosin heavy chain isoforms). Forty-three female and nine male students participated in the study. Gastrocnemius lateralis contained predominantly type I fibres (50%) and type IIA fibres (40%) in both sexes and large individual differences were found. Principal component analysis (PCA) was used for the intercorrelation analyses, and projection to latent structures (PLS) was used for the multivariate regression analysis. MNF correlated positively with different fibre areas and with the proportion of type I fibres. Fibre areas and sex were the most important factors in the regression of maximum peak torque. High proportion of type I fibres and sex were the most important regressors of peak torque endurance normalised for lean body mass. More studies are needed to understand the complex interrelationships between intrinsic muscle properties and the frequency content of the surface EMG before theoretical models can be formulated that incorporate both fibre areas and fibre type proportions.     

The role of ACE2, angiotensin-(1–7) and Mas1 receptor axis in glucocorticoid-induced intrauterine growth restriction

Background

Plasma and urine levels of the potent vasodilator Ang-(1–7) are elevated in mid and late pregnancy and are correlated with elevated placental angiogenesis, fetal blood flow, and rapid fetal growth. We hypothesized that Ang-(1–7), its receptor (Mas1) and the enzymes involved in Ang-(1–7) production (ACE2 and Membrane metallo-endopeptidase; MME) are down regulated in response to glucocorticoid administration contributing to IUGR.

Methods

Pregnant female Sprague–Dawley rats were injected with dexamethasone (DEX; 0.4 mg/kg/day) starting from 14 day gestation (dg) till sacrifice at 19 or 21 dg while control groups were injected with saline (n?=?6/group). The gene and protein expression of ACE2, MME, Ang-(1–7) and Mas1 receptor in the placental labyrinth (LZ) and basal zones (BZ) were studied.

Results

DEX administration caused a reduction in LZ weight at 19 and 21 dg (p?<?0.001). IUGR, as shown by decreased fetal weights, was evident in DEX treated rats at 21 dg (p?<?0.01). ACE2 gene expression was elevated in the LZ of control placentas at 21 dg (p?<?0.01) compared to 19 dg and DEX prevented this rise at both gene (p?<?0.01) and protein levels (p?<?0.05). In addition, Ang-(1–7) protein expression in LZ was significantly reduced in DEX treated rats at 21 dg (p?<?0.05). On the other hand, Mas1 and MME were upregulated in LZ at 21 dg in both groups (p?<?0.05 and p?<?0.001, respectively).

Conclusion

The results of this study indicate that a reduced expression of ACE2 and Ang-(1–7) in the placenta by DEX treatment may be responsible for IUGR and consequent disease programming later in life.