Purification of alpha-galactosidase by affinity precipitation with alginate

Affinity precipitation is a technique which is known for over 20 years, but has recently received more attention due to the development of new materials for its implementation. It is a relatively simple, convenient, and reproducible technique that results in high target molecule recovery at high specificity. We describe, here, an efficient and rapid purification procedure for Vicia faba alpha-galactosidase (EC 3.2.1.22) by using affinity precipitation with alginate. The enzyme was purified with 43% activity yield and 40-fold purification. SDS-PAGE of the purified enzyme showed a single band and a subunit weight of 44 kDa. The properties of the enzyme were also searched. The results showed that the general properties of the enzyme offer potential for use of this alpha-galactosidase in several production processes.     

Chemo- and baroresponses differ in African-Americans and Caucasians in sleep

To determine sleep effects on baro- andventilatory responses to transient chemo- and barostimulation inAfrican-Americans and Caucasians, 26 nonobese normotensive youngsubjects (13 African-Americans and 13 Caucasians) were studied awakeand in non-rapid-eye movement (NREM) and rapid-eye-movement sleepduring induced transient hypoxemia (N₂), hypertension(phenylephrine, PE), and concomitant hypoxemia and hypertension(N₂ + PE). Arterial blood pressurewas recorded by plethysmographic volume clamp, minute ventilation bypneumotachograph, and arterial O₂saturation by pulse oximeter. For all subjects, chronotropicbaroresponse (pulse interval/systolic blood pressure, where  is change) increased with NREM sleep(P = 0.007). Baroresponse slope wasgreater in Caucasians than in African-Americans (ANOVA, P = 0.02). Hypoxemic ventilatoryresponse (minute ventilation/arterial O₂ saturation) was greater inAfrican-Americans than in Caucasians in NREM sleep(P = 0.01), as was hypoxemicattenuation of baroresponse (N₂ + PE, P = 0.03). These data suggestsleep-related differences in arterial chemo- and baroreceptor responsesin normal young African-Americans and Caucasians, which may haveimplications concerning development of systemic hypertension.

     

Breath-by-breath interactions between inspiratory and expiratory duration in occlusive sleep apnea

We examined interactions between inspiratory duration (TI), expiratory duration (TE), and inspiratory (esophageal) pressure (Pes) generation in seven subjects with confirmed occlusive sleep apnea. Breath-by-breath values of TI, TE, and Pes were identified by digital computer during 21 260-s epochs of repetitive occlusive apnea during non-rapid-eye-movement sleep. The control theory of interacting nonlinear oscillators was used to categorize the interaction between TI and TE for each epoch as either 1) synchronization, the strongest possible interaction between biological oscillators; 2) relative entrainment, a moderate interaction between oscillators; or 3) relative coordination, a weak interaction. The latter two interactions were characterized by systemic oscillations in the moving cross-correlation between TI and TE. The relationship between TI and Pes was analyzed in a similar fashion. Significant oscillations were present in all three parameters (P less than 0.0001 for each). We observed significant negative correlations between TI and TE and between TI and Pes (P less than 0.001 for each) when all breaths for all epochs were pooled. In no epoch was there a significant positive correlation between TI and TE or Pes. All three interactions were observed between TI and TE: five epochs of synchronization, nine of relative entrainment, and seven of relative coordination. In contrast, 19 of 21 epochs exhibited synchronization between TI and Pes, with 2 epochs of relative entrainment. The relative frequency of TI vs. Pes synchronization was significantly greater than TI vs. TE synchronization (P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Gamma-radiolysis of poly(A) in aqueous solution: efficiency of strand break formation by primary water radicals

gamma-Radiation-induced single-strand break formation (ssb) in polyadenylic acid (poly(A] has been determined in Ar and N2O-saturated aqueous solution in the presence of different concentrations of t-butanol. Strand breaks were monitored by a low-angle laser light-scattering technique. The efficiencies for strand breakage caused by solvated electrons, hydrogen atoms and OH radicals have been found to be 0.25, 0.20 and 7.8 per cent, respectively. The efficiency of OH radicals depends only slightly on pH (pH 5.0, 7.5 and 9.0) and is independent of the presence of salt (0.01 mol dm-3 NaC1O4) and of the irradiation temperature (20 degrees C and 70 degrees C). The efficiency of OH for ssb formation obtained in this work with poly(A) is much smaller than that of poly(dA). This is explained by the different molecular conformations of the sugar moiety of poly(A) (3'-endo) and poly(dA) (2'-endo). With increasing t-butanol concentration more strand breaks are formed than expected from simple homogeneous competition kinetics of poly(A) and t-butanol for OH radicals. This result is considered to be due to nonhomogeneous reaction kinetics in the above-mentioned competition. The rate constants for the reaction of OH and H with poly(A) have been determined.     

Comparative Evaluation of Polyphenol Contents and Biological Activities of Five Cistus L. Species Native to Turkey

In this study phytochemical compounds and antioxidant capacity, cytotoxic, antimicrobial and anti-biofilm activities of hydroethanolic extracts of five Cistus species (C. creticus L., C. laurifolius L., C. monspeliensis L., C. parviflorus Lam. and C. salviifolius L.) distributed in Turkey were investigated. (+)-catechin, epigallocatechin gallate, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, luteolin were detected in different amounts. Strongest antioxidant capacities were observed with C. creticus, and C. parvifolius (0.476 and 0.452, respectively). Minimum inhibitory concentrations (MIC) of the extracts were determined between 32 and 128 μg/mL against different bacteria and Candida strains. C. monspeliensis and C. laurifolius extracts were inhibited the biofilm production levels of three Gram-negative bacteria (E. coli, S. enterica, P. aeruginosa), two Gram-positive bacteria (S. aureus, B. subtilis) and three Candida strains (C. albicans, C. parapsilosis, C. krusei). C. creticus extract showed strongest cytotoxic activity against human breast adenocarcinoma (MCF-7) and prostate cell lines (PC-3) (IC₅₀: 14.04±2.78 μg/mL and 34.04±2.74 μg/mL, respectively) among all plants tested.     

Respiratory muscle function during CO2 rebreathing with inspiratory flow-resistive loading

We investigated the respiratory muscle contribution to inspiratory load compensation by measuring diaphragmatic and intercostal electromyograms (EMGdi and EMGic), transdiaphragmatic pressure (Pdi), and thoracoabdominal motion during CO2 rebreathing with and without 15 cmH2O X l-1 X s inspiratory flow resistance (IRL) in normal sitting volunteers. During IRL compared with control, Pdi measured during airflow and during airway occlusion increased for a given change in CO2 partial pressure and EMGdi, and there was a greater decrease in abdominal (AB) end expiratory anteroposterior dimensions with increased expiratory gastric pressure (Pga), this leading to an inspiratory decline in Pga with outward AB movement, indicating a passive component to the descent of the abdomen-diaphragm. The response of EMGic to IRL was similar to that of EMGdi, though rib cage (RC)-Pga plots did infer intercostal muscle contribution. We conclude that during CO2 rebreathing with IRL there is improved diaphragmatic neuromuscular coupling, the prolongation of inspiration promoting a force-velocity advantage, and increased AB action serving to optimize diaphragm length and configuration, as well as to provide its own passive inspiratory action. Intercostal action provides increased assistance also. Therefore, compensation for inspiratory resistive loads results from the combined and integrated effort of all respiratory muscle groups.     

Effects of subacute treatment of ethylene glycol on serum marker enzymes and erythrocyte and tissue antioxidant defense systems and lipid peroxidation in rats

The present study was aimed to investigate the effects of ethylene glycol (EG) on serum marker enzymes, antioxidant defense systems and lipid peroxidation concentration (malondialdehyde=MDA) in various tissues of rats exposed to ethylene glycol. EG (1.25% or 2.5%) in drinking water was administered orally to rats (Sprague-Dawley albino) ad libitum for 21 days continuously. EG treatments caused different effects on the serum marker enzymes, antioxidant defense system and MDA content in various tissues of the treatment groups as compared with the controls. EG also caused a significant increase in the serum marker enzyme activities with 2.5% dosage whereas, no changes were not observed with 1.25% dosage of EG treatment. Lipid peroxidation significantly increased in all the tissues except for in the heart and stomach of rats treated with both dosages of EG. Also, the antioxidative systems were also seriously affected by EG. For example, SOD significantly decreased in the liver treated with both dosages whereas, SOD activity in the erythrocytes, kidney, heart and stomach were significantly increased and not changed in the brain with two dosages of EG. Also, while CAT activity significantly decreased in the erythrocytes, liver and kidney, the activity in the stomach significantly increased, but did not change in the brain and heart with two doses of EG. GR activity significantly decreased in the erythrocytes treated with both dosages of EG whereas GR was not affected in other tissues by EG treatment. GST activity significantly elevated in the heart and brain but did not change in the other tissues of rats treated with both dosages of EG. Meanwhile, GSH depletion in the erythrocytes of rats treated with 2.5% dosage of EG was found to be significant whereas, the level of GSH in the brain was significantly increased treated with both the dosages of EG. The observations presented led us to conclude that the administration of subacute EG promotes lipid peroxidatin content, elevates tissue damage serum marker enzymes and changes in the antioxidative systems in rats. These data, along with the determined changes suggest that EG produced substantial systemic organ toxicity in the erythrocyte, liver, brain, heart kidney and stomach during the period of a 21-day subacute exposure.     

Immobilization and stabilization of α-galactosidase on Sepabeads EC-EA and EC-HA

α-Galactosidase from tomato has been immobilized on Sepabead EC-EA and Sepabead EC-HA, which were activated with ethylendiamino and hexamethylenediamino groups, respectively. Two strategy was used for the covalent immobilization of α-galactosidase on the aminated Sepabeads: covalent immobilization of enzyme on glutaraldehyde activated support and cross-linking of the adsorbed enzymes on to the support with glutaraldehyde. By using these two methods, all the immobilized enzymes retained very high activity and the stability of the enzyme was also improved. The obtained results showed that, the most stable immobilized α-galactosidase was obtained with the second strategy. The immobilized enzymes were characterized with respect to free counterpart. Some parameters effecting to the enzyme activity and stability were also analyzed. The optimum temperature and pH were found as 60 °C and pH 5.5 for all immobilized enzymes, respectively. All the immobilized α-galactosidases were more thermostable than the free enzyme at 50 °C. The stabilities of the Sepabead EC-EA and EC-HA adsorbed enzymes treated with glutaraldehyde compared to the stability of the free enzyme were a factor of 6 for Sepabead EC-EA and 5.3 for Sepabead EC-HA. Both the free and immobilized enzymes were very stable between pH 3.0 and 6.0 and more than 85% of the initial activities were recovered. Under the identical storage conditions the free enzyme lost its initial activity more quickly than the immobilized enzymes at the same period of time. The immobilized α-galactosidase seems to fulfill the requirements for different industrial applications.     

Effect of organic loading rate on the performance of an up-flow anaerobic sludge blanket reactor treating olive mill effluent

The primary objective of this study was to evaluate the effects of the organic loading rate on the performance of an up-flow anaerobic sludge blanket (UASB) reactor treating olive mill effluent (OME), based on the following indicators: (i) chemical oxygen demand (COD) removal efficiency; and (ii) effluent variability (phenol, suspended solids, volatile fatty acids, and pH stability). The UASB reactor was operated under different operational conditions (OLRs between 0.45 and 32 kg COD/m³·day) for 477 days. The results demonstrated that the UASB reactor could tolerate high influent COD concentrations. Removal efficiencies for the studied pollution parameters were found to be as follows: COD, 47∼92%; total phenol, 34∼75%; color, 6∼46%; suspended solids, 34∼76%. The levels of VFAs in the influent varied between 310 and 1,750 mg/L. Our measurements of the VFA levels indicated that some of the effluent COD could be attributed to VFAs (principally acetate, butyrate, iso-butyrate, and propionate) in the effluent, which occurred at levels between 345 and 2,420 mg/L. As the OLRs were increased, more VFAs were measured in the effluent. A COD removal efficiency of 90% could be achieved as long as OLR was kept at a level of less than 10 kg COD/m³·day. However, a secondary treatment unit for polishing purposes is necessary to comply with discharge standards.