Formation of Extracellular Enzyme Systems during the Growth of Geotrichum candidum3C on Cell Walls Isolated from Cereal Grain Coats

The activities of extracellular systems of hemicellulases, pectinases, and cellulases was studied during a 72-h cultivation of Geotrichum candidum3C. The culture was grown on a medium containing 3% cell walls isolated from wheat grain coats, which served as the sole carbon source. Enzymes catalyzing the degradation of pectin substances (beet pectin, -L-arabinan, and 1,4--D-galactan), as well as -D-galactosidase and -L-arabinofuranosidase involved in their hydrolysis, were formed first (4 h after the beginning of cultivation). Enzymes hydrolyzing 4-O-methyl--D-glucurono--D-xylan and sodium carboxymethyl xylan were also found in the culture liquid after 4 h of fungal growth. The contents of pectin-degrading and xylanolytic enzymes reached their maximum levels after 52–56 and 72 h of growth, respectively. Cellulolytic enzymes were detected after 8–28 h of cultivation. Enzymes degrading -D-galacto--D-mannan were found 24 h after the beginning of growth; their content was maximum after 72 h of cultivation.     

Invariant amino acids essential for decoding function of polypeptide release factor eRF1

In eukaryotic ribosome, the N domain of polypeptide release factor eRF1 is involved in decoding stop signals in mRNAs. However, structure of the decoding site remains obscure. Here, we specifically altered the stop codon recognition pattern of human eRF1 by point mutagenesis of the invariant Glu55 and Tyr125 residues in the N domain. The 3D structure of generated eRF1 mutants was not destabilized as demonstrated by calorimetric measurements and calculated free energy perturbations. In mutants, the UAG response was most profoundly and selectively affected. Surprisingly, Glu55Arg mutant completely retained its release activity. Substitution of the aromatic ring in position 125 reduced response toward all stop codons. This result demonstrates the critical importance of Tyr125 for maintenance of the intact structure of the eRF1 decoding site. The results also suggest that Tyr125 is implicated in recognition of the 3d stop codon position and probably forms an H-bond with Glu55. The data point to a pivotal role played by the YxCxxxF motif (positions 125–131) in purine discrimination of the stop codons. We speculate that eRF1 decoding site is formed by a 3D network of amino acids side chains.     

DNA-Protein Complex in Circular DNA from Phage ϕ29

THE DNA of the B. subtilis phage ?29 has been described as unpermuted linear duplex molecules¹ of molecular weight 11 × 10⁶, but the formation of circular molecules has also been indicated, suggesting the existence of cohesive ends^1,2.     

Glutamic acid as enhancer of protein synthesis kinetics in hepatocytes from old rats

Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell–cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell–cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.     

Purification and Characterization of endo-(1→4)-β-xylanase fromGeotrichum candidum 3C

A method of purification of endo-( 1 → 4)-β-xylanase (endoxylanase; EC 3.2.1.8) from the culture liquid ofGeotrichum candidum 3C, grown for three days, is described. The enzyme, purified 23-fold, had a specific activity of 32.6 U per mg protein (yield, 14.4%). Endoxylanase was shown to be homogeneous by SDS-PAGE (molecular weight, 60 to 67 kDa). With carboxymethyl xylan as the substrate, the optimum activity (determined viscosimetrically) was recorded at pH 4.0 (pI 3.4). The enzyme retained stability at pH 3.0-4.5 and 30–45°C for 1 h. With xylan from birch wood, the hydrolytic activity of the enzyme (ability to saccharify the substrate) was maximum at 50°C. In 72 h of exposure to 0.2 mg/ml endoxylanase, the extent of saccharification of xylans from birch wood, rye grain, and wheat straw amounted to 10,12, and 7.7%, respectively. At 0.4 mg/ml, the extent of saccharification of birch wood xylan was as high as 20%. In the case of birch wood xylan, the initial hydrolysis products were xylooligosaccharides with degrees of polymerization in excess of four; the end products were represented by xylobiose, xylotriose, xylose, and acid xylooligosaccharides.     

两个苜蓿品种营养器官解剖结构特征比较

为了明确具有极强抗虫特性的‘草原4号紫花苜蓿’(Medicago sativa L.‘Caoyuan No.4’) 营养器官的解剖特征,该研究选择具有抗蓟马特性较强的‘草原2号杂花苜蓿’(Medicago varia Martin.‘Caoyuan No.2’)为对照,采用显微镜观察比较两品种的根、茎、叶解剖结构特征,为揭示‘草原4号紫花苜蓿’ 抗蓟马特性提供理论依据。结果显示：(1)‘草原4号紫花苜蓿’根部解剖结构的皮层薄壁细胞厚度、内皮层厚度、形成层厚度、木质部厚度和木射线宽度等5个指标均极显著高于(P＜0.01)‘草原2号杂花苜蓿’,其中木射线宽度(159.37 μm)是‘草原2号杂花苜蓿’的1.82倍。(2)‘草原4号紫花苜蓿’的茎部厚角组织厚度(21.4 μm)极显著高于‘草原2号杂花苜蓿’(P＜0.01),而韧皮部宽度、髓直径却均极显著低于‘草原2号杂花苜蓿’(P＜0.01)。(3)‘草原4号紫花苜蓿’叶片解剖构造的7个指标均极显著高于‘草原2号杂花苜蓿’(P＜0.01),其中栅栏组织层数(2～3层)极明显地高于‘草原2号杂花苜蓿’(1~2层)。研究表明,‘草原4号紫花苜蓿’的组织结构特征具有明显的抗虫特征,且其组织的抗虫特征比‘草原2号杂花苜蓿’更为突出。     

GTP-dependent structural rearrangement of the eRF1:eRF3 complex and eRF3 sequence motifs essential for PABP binding

Translation termination in eukaryotes is governed by the concerted action of eRF1 and eRF3 factors. eRF1 recognizes the stop codon in the A site of the ribosome and promotes nascent peptide chain release, and the GTPase eRF3 facilitates this peptide release via its interaction with eRF1. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay through its association with cytoplasmic poly(A)-binding protein (PABP) via PAM2-1 and PAM2-2 motifs in the N-terminal domain of eRF3. We have studied complex formation between full-length eRF3 and its ligands (GDP, GTP, eRF1 and PABP) using isothermal titration calorimetry, demonstrating formation of the eRF1:eRF3:PABP:GTP complex. Analysis of the temperature dependence of eRF3 interactions with G nucleotides reveals major structural rearrangements accompanying formation of the eRF1:eRF3:GTP complex. This is in contrast to eRF1:eRF3:GDP complex formation, where no such rearrangements were detected. Thus, our results agree with the established active role of GTP in promoting translation termination. Through point mutagenesis of PAM2-1 and PAM2-2 motifs in eRF3, we demonstrate that PAM2-2, but not PAM2-1 is indispensible for eRF3:PABP complex formation.     

Activation of reconstructive processes in rat tissues under the action of radiofrequency current with a periodic impulse mode of modulation

The action of a current in the radio frequency range with a periodic impulse mode of modulation on the activation of recovery processes in the skin and skeletal muscles has been studied. The action of a radio frequency current with a power of 1 W, as opposed to that of the weaker action (0.1 W) and stronger (4 W) action, leads to the activation of recovery processes in the skin and skeletal muscles. Recovery processes are manifested in the increase in proliferation and activation of angiogenesis in the skin, and also in formation of new muscle fibers. Recovery processes in muscles are accompanied by activation and migration of satellite cells of muscle tissue in the zone of action of the radio frequency current.