Functional properties of a manganese-activated exo-polygalacturonase produced by a thermotolerant fungus Aspergillus niveus

A thermotolerant fungus identified as Aspergillus niveus was isolated from decomposing materials and it has produced excellent levels of hydrolytic enzymes that degrade plant cell walls. A. niveus germinated faster at 40 °C, presenting protein levels almost twofold higher than at 25 °C. The crude extract of the A. niveus culture was purified by diethylaminoethyl (DEAE)-cellulose, followed by Biogel P-100 column. Polygalacturonase (PG) is a glycoprotein with 37.7 % carbohydrate, molecular mass of 102.6 kDa, and isoelectric point of 5.4. The optimum temperature and pH were 50 °C and 4.0–6.5, respectively. The enzyme was stable at pH 3.0 to 9.0 for 24 h. The DEAE-cellulose derivative was about sixfold more stable at 60 °C than the free enzyme. Moreover, the monoaminoethyl-N-aminoethyl-agarose derivative was tenfold more stable than the free enzyme. PG was 232 % activated by Mn²⁺. The hydrolysis product of sodium polypectate corresponded at monogalacturonic acid, which classifies the enzyme as an exo-PG. The K _m, V _max, K _cat, and K _cat/K _m values were 6.7 mg/ml, 230 U/mg, 393.3/s, and 58.7 mg/ml/s, respectively. The N-terminal amino acid sequence presented 80 % identity with PglB1, PglA2, and PglA3 putative exo-PG of Aspergillus fumigatus and an exo-PG Neosartorya fischeri.     

Regulation of dihydropyridine receptor gene expression in mouse skeletal muscles by stretch and disuse

This study examined dihydropyridine receptor (DHPR) gene expression in mouse skeletal muscles during physiological adaptations to disuse. Disuse was produced by three in vivo models—denervation, tenotomy, and immobilization—and DHPR _1s mRNA was measured by quantitative Northern blot. After 14-day simultaneous denervation of the soleus (Sol), tibialis anterior (TA), extensor digitorum longus (EDL), and gastrocnemius (Gastr) muscles by sciatic nerve section, DHPR mRNA increased preferentially in the Sol and TA (+1.6-fold), whereas it increased in the EDL (+1.6-fold) and TA (+1.8-fold) after selective denervation of these muscles by peroneal nerve section. It declined in all muscles (–1.3- to –2.6-fold) after 14-day tenotomy, which preserves nerve input but removes mechanical tension. Atrophy was comparable in denervated and tenotomized muscles. These results suggest that factor(s) in addition to inactivity per se, muscle phenotype, or associated atrophy can regulate DHPR gene expression. To test the contribution of passive tension to this regulation, we subjected the same muscles to disuse by limb immobilization in a maximally dorsiflexed position. DHPR _1s mRNA increased in the stretched muscles (Sol, +2.3-fold; Gastr, +1.5-fold) and decreased in the shortened muscles (TA, –1.4-fold; EDL, –1.3-fold). The effect of stretch was confirmed in vitro. DHPR protein did not change significantly after 4-day immobilization, suggesting that additional levels of regulation may exist. These results demonstrate that DHPR _1s gene expression is regulated as an integral part of the adaptive response of skeletal muscles to disuse in both slow- and fast-twitch muscles and identify passive tension as an important signal for its regulation in vivo. dihydropyridine receptor mRNA; decreased use; passive tension; denervation; tenotomy; hindlimb immobilization     

Beta-glucosidase activity from the thermophilic fungus Scytalidium thermophilum is stimulated by glucose and xylose

An inducible mycelial beta-glucosidase from Scytalidum thermophilum was characterized. The enzyme exhibited a pI of 6.5, a carbohydrate content of 15%, and an apparent molecular mass of about 40 kDa. Optima of temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable up to 1 h at 50 degrees C and exhibited a half-life of 20 min at 55 degrees C. The enzyme hydrolyzed p-nitrophenyl-beta-d-glucopyranoside, p-nitrophenyl-beta-d-xylopyranoside, o-nitrophenyl-beta-d-galactopyranoside, p-nitrophenyl-alpha-arabinopyranoside, cellobiose, laminaribiose and lactose. Kinetic studies indicated that the same enzyme hydrolyzed these substrates. Beta-Glucosidase was activated by glucose or xylose at concentration varying from 50 to 200 mM. The apparent affinity constants (K0.5) for glucose and xylose were 36.69 and 43.24 mM, respectively. The stimulatory effect of glucose and xylose on the S. thermophilum beta-glucosidase is a novel characteristic which distinguish this enzyme from all other beta-glucosidases so far described.     

Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+

The Na,K-ATPase α2 isoform is the predominant Na,K-ATPase in adult skeletal muscle and the sole Na,K-ATPase in the transverse tubules (T-tubules). In quiescent muscles, the α2 isozyme operates substantially below its maximal transport capacity. Unlike the α1 isoform, the α2 isoform is not required for maintaining resting ion gradients or the resting membrane potential, canonical roles of the Na,K-ATPase in most other cells. However, α2 activity is stimulated immediately upon the start of contraction and, in working muscles, its contribution is crucial to maintaining excitation and resisting fatigue. Here, we show that α2 activity is determined in part by the K⁺ concentration in the T-tubules, through its K⁺ substrate affinity. Apparent K⁺ affinity was determined from measurements of the K_1/2 for K⁺ activation of pump current in intact, voltage-clamped mouse flexor digitorum brevis muscle fibers. Pump current generated by the α2 Na,K-ATPase, Ip, was identified as the outward current activated by K⁺ and inhibited by micromolar ouabain. Ip was outward at all potentials studied (−90 to −30 mV) and increased with depolarization in the subthreshold range, −90 to −50 mV. The Q₁₀ was 2.1 over the range of 22–37°C. The K_1/2,K of Ip was 4.3 ± 0.3 mM at −90 mV and was relatively voltage independent. This K⁺ affinity is lower than that reported for other cell types but closely matches the dynamic range of extracellular K⁺ concentrations in the T-tubules. During muscle contraction, T-tubule luminal K⁺ increases in proportion to the frequency and duration of action potential firing. This K_1/2,K predicts a low fractional occupancy of K⁺ substrate sites at the resting extracellular K⁺ concentration, with occupancy increasing in proportion to the frequency of membrane excitation. The stimulation of preexisting pumps by greater K⁺ site occupancy thus provides a rapid mechanism for increasing α2 activity in working muscles.     

Blood pressure in night shift workers: circadian rhythms and levels and their seasonal differences

The average diurnal blood pressure profiles (DBPPs) were studied in subjects working at different hours of the day, including nighttime. The DBPPs and the curve levels in winter and in summer were compared. The study material included more than 497 000 prework blood pressure (BP) measurement points in 30 566 locomotive drivers. It was found that the average DBPP of the subjects working at different hours of the day is of a markedly nondipper type; i.e., despite night wakefulness, the BP was lower at night but not as low as that of those sleeping at night. In the cohort studied, the shapes of the DBPP curve did not differ in winter and in summer; however, the BP levels in winter were significantly higher.     

Effects of salicylic acid and cold on freezing tolerance in winter wheat leaves

The effects of salicylic acid (SA) (0.01, 0.1 and 1 mM) and cold on freezing tolerance (freezing injury and ice nucleation activity) were investigated in winter wheat (Triticum aestivum cv. Dogu-88) grown under control (20/18 °C for 15, 30 and 45-day) and cold (15/10 °C for 15-day, 10/5 °C for 30-day and 5/3 °C for 45-day) conditions. Cold acclimatisation caused a decrease of injury to leaf segments removed from the plants and subjected to freezing conditions. Exogenous SA also decreased freezing injury in the leaves grown under cold (15/10 °C) and control (15 and 30-day) conditions. Cold conditions (10/5 and 5/3 °C) caused an increase in ice nucleation activity by apoplastic proteins, which were isolated from the leaves. For the first time, it was shown that exogenous SA caused an increase in ice nucleation activity under cold (15/10 and 10/5 °C) and control conditions. These results show that salicylic acid can increase freezing tolerance in winter wheat leaves by affecting apoplastic proteins.