Flow injection analysis with diode array absorbance detection and dynamic surface tension detection for studying denaturation and surface activity of globular proteins

In this article, a multidimensional dynamic surface tension detector (DSTD), in a parallel configuration with a UV-visible diode array absorbance detector, is presented in a novel flow injection analysis (FIA) application to study the effects of chemical denaturants urea, guanidinium hydrochloride (GdmHCl), and guanidinium thyocyanate (GdmSCN) on the surface activity of globular proteins at the liquid-air interface. The DSTD signal is obtained by measuring the changing pressure across the liquid-air interface of 4-mul drops repeatedly forming at the end of a capillary using FIA. The sensitivity and selectivity of the DSTD signal is related to the surface-active protein concentration in aqueous solution combined with the thermodynamics and kinetics of protein interaction at a liquid-air drop interface. Rapid on-line calibration and measurement of dynamic surface tension is applied, with the surface tension converted into surface pressure results. Continuous surface tension measurement throughout the entire drop growth is achieved, providing insight into kinetic behavior of protein interactive processes at the liquid-air drop interface. Specifically, chemical denaturation of 12 commercial globular proteins-chicken egg albumin, bovine serum albumin, human serum albumin, alpha-lactalbumin (alpha-Lac), myoglobin, cytochrome c, hemoglobin, carbonic anhydrase, alpha-chymotrypsinogen A, beta-lactoglobulin (beta-LG), lysozyme, and glyceraldehyde-3-phosphate-dehydrogenase-is studied in terms of surface pressure (i.e., surface activity) after treatment with increasing concentrations of urea, GdmHCl, and GdmSCN in the 0-8, 0-6, and 0-5 M ranges, respectively. For several of these proteins, the spectroscopic absorbance changes are monitored simultaneously to provide additional information prior to drop formation. Results show that surface pressure of proteins generally increases as the denaturant concentration increases and that effectiveness is GdmSCN > GdmHCl > urea. Protein unfolding curves obtained by plotting surface pressure as a function of denaturant concentration are presented and compared with respect to unfolding curves obtained by using UV absorbance and literature data. Kinetic information relative to the protein adsorption to the air-liquid interface of two proteins, alpha-Lac and beta-LG (chosen as representative proteins for comparison), denatured by the three denaturants is also studied and discussed.     

Analysis of neuron-like differentiation of human bone marrow mesenchymal stem cells

The objective of the study was to evaluate differentiation of human bone marrow mesenchymal stem cells into true or pseudo neurons after treating with chemical induction medium in vitro. The morphological changes were assessed using interference contrast microscopy. Immunocytochemistry and Western blotting were performed using neuronal markers. Further evaluation was conducted with proteomic profiling, DNA microarray analysis and the whole-cell patch clamp test. After three hours of treatment with chemical induction medium, nearly three-fourths of the hMSCs changed to cells with a neuronal phenotype. The results of immunocytochemistry and Western blotting showed a high expression of neuronal markers in these cells at 3 h which decreased at 24 h. The proteomics analysis showed no change of proteins related to neuronal differentiation. DNA microarray showed downregulation of neuron related genes. The patch clamp test was unable to demonstrate any similarity to true neurons. Our findings suggest that neuron-like cells derived from chemical induction of hMSCs are not the genuine neurons as they resemble true neurons phenotypically but are different in genotypic and electrophysiological characteristics.     

Morphological and chromosomal variation of the Dryopteris varia (L.) Kuntze complex (Dryopteridaceae) in Korea

The Dryopteris varia complex is highly variable in morphology, resulting in taxonomic confusion in delimiting taxon boundaries and determining relationships. We have examined the variation in morphology, chromosome number and mode of reproduction of the Korean members of the D. varia complex to clarify their taxonomic identities. Landmark analysis of the leaf blades and pinnae and the principal components analysis of 31 morphological characters revealed seven entities within the D. varia complex in Korea; these comprise D. varia s. str., D. pacifica, D. sacrosancta, D. bissetiana, D. saxifraga, D. saxifragivaria, and the Suak population which is considered to be a new taxon. Mitotic chromosome counts and examination of reproduction modes indicated that D. bissetiana in Korea appears to be agamosporous with diploid (2n = 82) or triploid (2n = 123) chromosome numbers. However, D. saxifraga is sexual diploid or agamosporous triploid, and the other taxa are agamosporous triploid. Dryopteris bissetiana, D. saxifraga and D. saxifragivaria are similar in major morphological characteristics, but show differences in attachment and shape of rachis scales. The results also suggest that agamosporous triploid D. saxifragivaria was probably derived from hybridization between sexual diploid D. saxifraga and D. bissetiana.     

大头金蝇营养成分分析

分析大头金蝇Chrysomya megacephala(Fabricius)的营养成分。结果表明,大头金蝇幼虫、蛹、成虫干粉水分含量分别是6.83,2.07和3.70%,干体粗蛋白含量分别为63.72,76.81和70.54%,粗脂肪分别为16.43,12.82和7.23%。蛋白质中氨基酸总量百分比分别为56.39,55.61和59.12%,其中必需氨基酸占氨基酸总量(E%)的48.34,53.08和43.91%,必需氨基酸与非必需氨基酸之比(E/N)分别为0.94,1.13,0.81,E/T为3.22,3.54,2.93。大头金蝇幼虫、蛹、成虫不饱和脂肪酸占总脂肪酸含量依次是68.1,61.9和65.9%。含有人和动物所必需的常量和微量元素,但没有检测到有毒重金属镉(Cd)、铅(Pb)。大头金蝇与鱼粉、舍蝇幼虫、柞蚕蛹相比,营养成分指标较高,属于理想的昆虫蛋白资源。     

从华蟹甲草中分离的两种活性成分对家蝇的杀虫活性

研究了从我国特有植物华蟹甲草Sinacalia tangutica (Maxim.) B. Nord中经生物活性跟踪实验得出的杀虫活性成分Z,Z,Z-9,12,15-十八碳三烯酸和Z,Z-9,12-十八碳二烯酸对家蝇Musca domestica vicina的生物活性及对其生理生化指标的影响。结果表明,家蝇经该2种化合物1 mg/mL处理后表现出类似神经毒剂中毒的兴奋症状。这2种化合物对家蝇表现出毒杀作用,24 h时,Z,Z,Z-9,12,15-十八碳三烯酸和Z,Z-9,12-十八碳二烯酸对家蝇的LC₅₀值分别为0.26 mg/mL和0.43 mg/mL; 对家蝇触杀作用极低,以1 mg/mL分别点滴处理家蝇后48 h,死亡率分别为21.54%和4.08%。这2种化合物对家蝇的AChE影响不明显,但可引起家蝇的过氧化物酶的活性发生紊乱,对家蝇总糖含量也有一定的影响。Z,Z-9,12-十八碳二烯酸可引起家蝇的Mg-ATPase的比活力降低,而Z,Z,Z-9,12,15-十八碳三烯酸引起家蝇的Na-K-Mg-ATPase和Ca-ATPase的比活力较对照上升。     

Physiological aspects. Part 1 in a series of papers devoted to surfactants in microbiology and biotechnology

Surfactants, both chemical and biological, are amphiphilic compounds which can reduce surface and interfacial tensions by accumulating at the interface of immiscible fluids and increase the solubility, mobility, bioavailability and subsequent biodegradation of hydrophobic or insoluble organic compounds. Investigations on their impacts on microbial activity have generally been limited in scope to the most common and best characterized surfactants. Recently a number of new biosurfactants have been described and accelerated advances in molecular and cellular biology are expected to expand our insights into the diversity of structures and applications of biosurfactants. Biosurfactants play an essential natural role in the swarming motility of microorganisms and participate in cellular physiological processes of signaling and differentiation as well as in biofilm formation. Biosurfactants also exhibit natural physiological roles in increasing bioavailability of hydrophobic molecules and can complex with heavy metals, and some also possess antimicrobial activity. Chemical- and indeed bio-surfactants may also be added exogenously to microbial systems to influence behaviour and/or activity, mimicking the latter effects of biosurfactants. They have been exploited in this way, for example as antimicrobial agents in disease control and to improve degradation of chemical contaminants. Chemical surfactants can interact with microbial proteins and can be manipulated to modify enzyme conformation in a manner that alters enzyme activity, stability and/or specificity. Both chemical- and bio-surfactants are potentially toxic to specific microbes and may be exploited as antimicrobial agents against plant, animal and human microbial pathogens. Because of the widespread use of chemical surfactants, their potential impacts on microbial communities in the environment are receiving considerable attention.     

Biomass functions applicable to Scots pine

This study describes parameterization of biomass functions applicable to Scots pine (Pinus sylvestris, L.) in the conditions of Central Europe. Fifty-two sample trees from seven sites in different regions of the Czech Republic were used for destructive measurements. The observed aboveground biomass (B _AB) and its individual components were examined by different types of non-linear regression models using one to five independent variables including stem diameter (D), tree height (H), tree age (A), crown length (C) and altitude (Z). The best single-equation approximation of B _AB was a three-parameter model using D, H and Z, which was also best suited for bark. Including altitude into classical multiplicative exponential function with three independent variables (DHZ) and four parameters yielded the best model for stem over or under bark. Age was important for assessment of dead branches within DHA model, whereas living branches were best approximated using four variables including C (DHAC model). The most complex model (DHACZ) worked best for needle biomass. The comparison of B _AB assessment by single regression equation and from sum of individual components showed a negligible difference for full-grown trees, whereas the additive assessment yielded considerably higher B _AB for small or young trees. This corresponds to the assessed confidence intervals for individual trees that were larger for smaller trees. The paper also discusses the issue of commonly used linearization of biomass equations and application of linear regression, and provides comparative examples with nonlinear approach used here. The paper presents the parameter sets for the tested equations for B _AB and individual biomass components.     

Chemical composition and biochemical changes in needles of Scots pine (Pinus sylvestris L.) stands at different stages of decline in Bulgaria

Water content was decreased in the needles of Scots pines (Pinus sylvestris L.) with moderate and slight levels of stress-induced decline. In pines with moderate decline a misbalance of foliar nutritional elements was observed. In 1-year-old needles of afflicted pines, the content of potassium and calcium was decreased, but the content of phosphorus, magnesium, iron, manganese and of the heavy metals cadmium and lead was increased. In 2-year-old needles of damaged pines, the content of calcium, iron and of the heavy metal cadmium was decreased, but the content of phosphorus, magnesium, manganese and zinc was increased. Potassium deficiency and zinc levels above toxicity tolerance characterized the nutrient status of declined Scots pines. In the same pines chlorophyll a/b ratio was diminished in 2-year-old needles and the protein content was increased in 1-year-old needles but was decreased in 2-year-old needles.In the second phase of the study 25- and 40-year-old pines with slight decline were examined. An increase in the protein content of 2-year-old needles of 25-year-old declined pines and of both 1- and 2-year-old needles of older 40-year-old pines was observed. A 280% increase of peroxidase activity in 1-year-old and a 178% increase in 2-year-old needles of 25-year-old damaged pines were shown. In older 40-year-old damaged pines a slight increase only in 1-year-old needles was seen. The observed age-related decrease in peroxidase activity in older 40-year-old symptomatic pines might be responsible for the lower resistance to decline in older pines. Our results show that clear but complex chemical and biochemical changes are observed in the needles of Scots pines in Bulgaria and that these changes hold promise for use as indicators of stress.     

Identifying ontogenetic, environmental and individual components of forest tree growth

Background and Aims

This study aimed to identify and characterize the ontogenetic, environmental and individual components of forest tree growth. In the proposed approach, the tree growth data typically correspond to the retrospective measurement of annual shoot characteristics (e.g. length) along the trunk.

Methods

Dedicated statistical models (semi-Markov switching linear mixed models) were applied to data sets of Corsican pine and sessile oak. In the semi-Markov switching linear mixed models estimated from these data sets, the underlying semi-Markov chain represents both the succession of growth phases and their lengths, while the linear mixed models represent both the influence of climatic factors and the inter-individual heterogeneity within each growth phase.

Key Results

On the basis of these integrative statistical models, it is shown that growth phases are not only defined by average growth level but also by growth fluctuation amplitudes in response to climatic factors and inter-individual heterogeneity and that the individual tree status within the population may change between phases. Species plasticity affected the response to climatic factors while tree origin, sampling strategy and silvicultural interventions impacted inter-individual heterogeneity.

Conclusions

The transposition of the proposed integrative statistical modelling approach to cambial growth in relation to climatic factors and the study of the relationship between apical growth and cambial growth constitute the next steps in this research.     

Male hamsters discriminate estrous state from vaginal secretions and individuals from flank marks

It is clear that male hamsters discriminate between the odors of individual, conspecific females, as shown by using habituation-dishabituation methods. However, it is not clear from past research whether male hamsters are able to discriminate between the odors of estrous and non-estrous females. A series of habituation-dishabituation experiments was conducted to determine whether males discriminated between different estrous cycle states using two female secretions, those from flank-glands and vaginal secretions. We found that, when habituated to a female flank-gland secretion, males discriminated between this female and a second female on the test trial, whether both were in estrus, both were in diestrus, or one was in estrus and the other in diestrus. There was no difference, however, in the magnitude of their dishabituation response toward flank-gland odors of females in estrus and diestrus. These results suggest that males use flank-gland odors to gain information primarily about individuals. When tested with vaginal secretions in habituation-dishabituation tests, males only showed differences in investigation when the second female was in estrus, indicating that males use vaginal secretions to gain information primarily about reproductive state.