Confomational and molecular responses to pH variation of the purified membrane adenosine triphospate of Micrococcus lysodeikticus

A preparation of ATPase from the membranes of Micrococcus lysodeikticus, solubilized and more than 95 %. pure, showed two main bands in analytical polyacrylamide gel electrophoresis. They did not correspond to isoenzymes because one band could be converted into the other by exposure to a mildly alkaline pH value. The conversion was paralleled by changes in molecular weight, circular dichroism and catalytic properties. Denaturation by pH at 25 °C was followed by means of circular dichroism, ultracentrifugation and polyacrylamide gel electrophoresis. A large conformational transition took place in the acid range with midpoints at about pH = 3.6 (I = 10^?4 M), 4.3 (I = 0.03 M) and 5.3 (I = 0.1 M). The transition was irreversible. Strong aggregation of the protein occurred in this range of pH. The final product was largely random coil, but even at pH 1.5 dissociation into individual subunits was not complete. However, partial dissociation took place at pH 5 (I = 0.028 M). At this pH value the enzyme was inactive, but 20–30 % of the activity could be recovered when the pH was returned to 7.5.In the alkaline region the midpoint of the transition occurred near pH = 11 (I = 0.028 M). The pK of most of the tyrosine residues of the protein was about 10.9. The unfolding was irreversible and the protein was soon converted into peptide species with molecular weights lower than those determined for the subunits by gel clectrophoresis in the presence of sodium dodecyl sulphate. Conventional proteolysis did not account for the transformation.     

Photoreceptor Pigment for Blue Light in Neurospora crassa

Irradiating the mycelium of Neurospora crassa with moderate intensities of blue light causes a reversible photoreduction of a b-type cytochrome. The action spectrum for the photoreduction of cytochrome b is very similar to the absorption spectrum of flavin pigments. Prolonged irradiation of the mycelium with strong blue light irreversibly bleaches flavin-like pigments and as these pigments are bleached the photoresponse of cytochrome b is lost. We conclude from these and other data that a flavin is the photoreceptor pigment for the photoreduction of cytochrome b. The close similarity between the action spectrum for the photoreduction of cytochrome b and action spectra for a number of physiological photoresponses suggests that this photoreceptor pigment controls a wide variety of photobiological processes in a wide diversity of organisms.     

Determination of plasma and tissue levels of tyramine by radioimmunoassay.

Antibodies were prepared against tyramine. The antigen was prepared as follows: p-Aminohippuric acid was coupled to mBSA using a carbodiimide reagent. The amino group was diazotized an attached to the aromatif ring of TYR. The immunogen in Freund's complete adjuvant was injected into rabbits. The specificity of the resulting antibody was determined by radioimmunoassay. Using random-labeled TYR-3H, TYR, its metabolites, phenethylamine analogs, catecholamines, and certain amino acids were evaluated by a competitive binding assay method. With this technique 4 ng of TYR inhibited the binding of TYR-3H by 50%. The radioimmunoassay of TYR was used to measure the plasma, urine, and tissue levels of TYR in rabbits. The plasma disappearance curve of TYR revealed a biphasic pattern with t1/2 of 2 min and 54 min. The highest concentration of TYR was found in adrenals and spleen. The factthat the major metabolites of TYR and a series of pharmacologically important sympathomimetics and catecholamines did not interfere, makes the radioimmunoassay of TYR a useful, simple, sensitive, and spedific method for the direct analysis of TYR in biological meterials.     

西藏接合菌物种多样性初探

【背景】接合菌在自然界广泛分布,在工业、食品、医药、生物防治等方面扮演着重要的角色,部分接合菌为有害菌。西藏地区接合菌只有少数零星的报道,其系统调查研究几乎还是空白,大量潜在的物种需要分离、鉴定、认识、保藏和开发。【目的】了解西藏地区的接合菌物种多样性和生物资源现状,为该地区有害接合菌的控制和有益接合菌的开发利用奠定基础。【方法】对西藏全境7个地区19个代表县的701个样品采用平板稀释分离法得到单菌落,然后进行形态和分子(SSU、ITS和LSU r DNA)鉴定,并在此基础上进行生物多样性以及优势和稀有属种分析。【结果】得到西藏接合菌10属26种,包括5个西藏已知种和21个西藏新记录种;其中4个为中国新记录种,分别是:类变形被孢霉(Mortierella amoeboidea)、球孢高山被孢霉(M.globalpina)、灰褐毛霉(Mucor brunneogriseus)、暗色毛霉(M.fuscus)。西藏接合菌的多样性指数分析表明,不同地区间的物种数量和组成存在显著差异,排在前4位的分别是波密县、米林县、当雄县和八宿县。属种分析显示,西藏接合菌的2个优势属为被孢霉属(Mortierella)和毛霉属(Mucor);3个优势种为高山被孢霉(Mortierella alpina)、冻土毛霉(Mucor hiemalis)、匍枝根霉(Rhizopus stolonifer);常见属8个,分别是犁头霉属(Absidia)、放射毛霉属(Actinomucor)、小克银汉霉属(Cunninghamella)、根毛霉属(Rhizomucor)、根霉属(Rhizopus)、共头霉属(Syncephalastrum)、伞形霉属(Umbelopsis)和接霉属(Zygorhynchus);常见种9个,稀有种14个。【结论】西藏地区接合菌资源丰富,各地区生物多样性差异显著,稀有物种占一半以上的比例提示西藏自然环境保护的重要性。     

Hemin with Peroxidase Activity Can Inhibit the Oxidative Damage Induced by Ultraviolet A

Excessive reactive oxygen species (ROS), a highly reactive substance that contains oxygen, induced by ultraviolet A (UVA) cause oxidative damage to skin. We confirmed that hemin can catalyze the reaction of tyrosine (Tyr) and hydrogen peroxide (H₂O₂). Catalysis was found to effectively reduce or eliminate oxidative damage to cells induced by H₂O₂ or UVA. The scavenging effects of hemin for other free-radical ROS were also evaluated through pyrogallol autoxidation, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·)-scavenging assays, and phenanthroline–Fe²⁺ assays. The results show that a mixture of hemin and tyrosine exhibits strong scavenging activities for H₂O₂, superoxide anion (O₂⁻·), DPPH·, and the hydroxyl radical (·OH). Furthermore, the inhibition of oxidative damage to human skin keratinocyte (HaCaT) cells induced by H₂O₂ or UVA was evaluated. The results show that catalysis can significantly reduce the ratio of cell apoptosis and death and inhibit the release of lactate dehydrogenase (LDH), as well as accumulation of malondialdehyde (MDA). Furthermore, the resistance to apoptosis was found to be enhanced. These results show that the mixture of hemin and tyrosine has a significantly protective effect against oxidative damage to HaCaT cells caused by UVA, suggesting it as a protective agent for combating UVA damage.     

The type of competition modulates the ecophysiological response of grassland species to elevated CO2 and drought

The effects of elevated CO₂ and drought on ecophysiological parameters in grassland species have been examined, but few studies have investigated the effect of competition on those parameters under climate change conditions. The objective of this study was to determine the effect of elevated CO₂ and drought on the response of plant water relations, gas exchange, chlorophyll a fluorescence and aboveground biomass in four grassland species, as well as to assess whether the type of competition modulates that response. Elevated CO₂ in well‐watered conditions increased aboveground biomass by augmenting CO₂ assimilation. Drought reduced biomass by reducing CO₂ assimilation rate via stomatal limitation and, when drought was more severe, also non‐stomatal limitation. When plants were grown under the combined conditions of elevated CO₂ and drought, drought limitation observed under ambient CO₂ was reduced, permitting higher CO₂ assimilation and consequently reducing the observed decrease in aboveground biomass. The response to climate change was species‐specific and dependent on the type of competition. Thus, the response to elevated CO₂ in well‐watered grasses was higher in monoculture than in mixture, while it was higher in mixture compared to monoculture for forbs. On the other hand, forbs were more affected than grasses by drought in monoculture, while in mixture the negative effect of drought was higher in grasses than in forbs, due to a lower capacity to acquire water and mineral nutrients. These differences in species‐level growth responses to CO₂ and drought may lead to changes in the composition and biodiversity of the grassland plant community in future climate conditions.     

Single chain antibody displays glutathione S-transferase activity

Substrate binding and the subsequent reaction are the two principal phenomena that underlie the activity of enzymes, and many enzyme-like catalysts were generated based on the phenomena. The single chain variable region fragment of antibody 2F3 (scFv2F3) was elicited against hapten GSH-S-DN2phBu, a conjugate of glutathione (GSH), butyl alcohol, and 1-chloro-2,4-dinitrobenzene (CDNB); it can therefore bind both GSH and CDNB, the substrates of native glutathione S-transferases (GSTs). It was shown previously that there is a serine residue that is the catalytic group of GST in the CDR regions of scFv2F3 close to the sulfhydryl of GSH. Thus, we anticipated that scFv2F3 will display GST activity. The experimental results showed that scFv2F3 indeed displayed GST activity that is equivalent to the rat-class GST T-2-2 and exhibited pH- and temperature-dependent catalytic activity. Steady-state kinetic studies showed that the Km values for the substrates are close to those of native GSTs, indicating that scFv2F3 has strong affinities for the substrates. Compared with some other GSTs, its kcat value was found to be low, which could be caused by the similarity between the GSH-S-DN2phBu and the reaction product of GSH and CDNB. These results showed that our approach to imitating enzymes is correct, which is that an active site may catalyze a chemical reaction when a catalytic group locates beside a substrate-binding site of a receptor. It is important to consider product inhibition in hapten design in order to obtain a mimic with a high catalytic efficiency.     

Analysis of temperature‐mediated changes in the wine yeast Saccharomyces bayanus var uvarum. An oenological study of how the protein content influences wine quality

Saccharomyces bayanus var. uvarum plays an important role in the fermentation of red wine from the D.O. Ribera del Duero. This is due to the special organoleptic taste that this yeast gives the wines and their ability to ferment at low temperature. To determine the molecular factors involved in the fermentation process at low temperature, a differential proteomic approach was performed by using 2D‐DIGE, comparing, qualitatively and quantitatively, the profiles obtained at 13 and 25°C. A total of 152 protein spots were identified. We detected proteins upregulated at 13°C that were shown to be related to temperature stress, the production of aromatic compounds involved in the metabolism of amino acids, and the production of fusel alcohols and their derivatives, each of which is directly related to the quality of the wines. To check the temperature effects, an aromatic analysis by GC–MS was performed. The proteomic and “aromatomic” results are discussed in relation to the oenological properties of S. bayanus var. uvarum.