Electrostatic interactions in leucine zippers: thermodynamic analysis of the contributions of Glu and His residues and the effect of mutating salt bridges

Electrostatic interactions play a complex role in stabilizing proteins. Here, we present a rigorous thermodynamic analysis of the contribution of individual Glu and His residues to the relative pH-dependent stability of the designed disulfide-linked leucine zipper AB(SS). The contribution of an ionized side-chain to the pH-dependent stability is related to the shift of the pK(a) induced by folding of the coiled coil structure. pK(a)(F) values of ten Glu and two His side-chains in folded AB(SS) and the corresponding pK(a)(U) values in unfolded peptides with partial sequences of AB(SS) were determined by 1H NMR spectroscopy: of four Glu residues not involved in ion pairing, two are destabilizing (-5.6 kJ mol(-1)) and two are interacting with the positive alpha-helix dipoles and are thus stabilizing (+3.8 kJ mol(-1)) in charged form. The two His residues positioned in the C-terminal moiety of AB(SS) interact with the negative alpha-helix dipoles resulting in net stabilization of the coiled coil conformation carrying charged His (-2.6 kJ mol(-1)). Of the six Glu residues involved in inter-helical salt bridges, three are destabilizing and three are stabilizing in charged form, the net contribution of salt-bridged Glu side-chains being destabilizing (-1.1 kJ mol(-1)). The sum of the individual contributions of protonated Glu and His to the higher stability of AB(SS) at acidic pH (-5.4 kJ mol(-1)) agrees with the difference in stability determined by thermal unfolding at pH 8 and pH 2 (-5.3 kJ mol(-1)). To confirm salt bridge formation, the positive charge of the basic partner residue of one stabilizing and one destabilizing Glu was removed by isosteric mutations (Lys-->norleucine, Arg-->norvaline). Both mutations destabilize the coiled coil conformation at neutral pH and increase the pK(a) of the formerly ion-paired Glu side-chain, verifying the formation of a salt bridge even in the case where a charged side-chain is destabilizing. Because removing charges by a double mutation cycle mainly discloses the immediate charge-charge effect, mutational analysis tends to overestimate the overall energetic contribution of salt bridges to protein stability.     

Cryopreservation of canine spermatozoa: theoretical prediction of optimal cooling rates in the presence and absence of cryoprotective agents

In the present study a shape independent differential scanning calorimeter (DSC) technique was used to measure the dehydration response during freezing of ejaculated canine sperm cells. Volumetric shrinkage during freezing of canine sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPAs (6 different combinations of freezing media were used, ranging from a media with no CPAs, and those with 0.5%, 3%, and 6% glycerol and with 0.5% and 3% Me(2)SO). Using previously published data, the canine sperm cell was modeled as a cylinder of length 105.7mum and a radius of 0.32mum with an osmotically inactive cell volume, V(b), of 0.6 V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data the best fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The "combined best fit" membrane permeability parameters at 5 and 10 degrees C/min for canine sperm cells in the absence of CPAs are: L(pg)=0.52x10(-15)m(3)/Ns (0.0029mum/min-atm) and E(Lp)=64.0kJ/mol (15.3kcal/mol) (R(2)=0.99); and the corresponding parameters in the presence of CPAs ranged from L(pg)[cpa]=0.46 to 0.53x10(-15) m(3)/Ns (0.0027-0.0031mum/min-atm) and E(Lp)[cpa]=46.4-56.0kJ/mol (11.1-13.4kcal/mol). These parameters are significantly different than previously published parameters for canine and other mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that optimal rates of freezing canine sperm cells ranges from 10 to 30 degrees C/min; these theoretical cooling rates are found to be in close conformity with previously published but empirically determined optimal cooling rates.     

红豆杉抗苯丙氨酸细胞系的筛选及悬浮培养特性

通过胁迫筛选法筛选红豆杉抗－苯丙氨酸细胞变异系,并比较了该细胞系与同步培养的原型细胞系悬浮培养的几项生理指标,结果表明,抗苯丙氨酸细胞系的紫杉醇含量显著高于原型细胞系的3－5倍,并且抗性细胞系的生长速率,细胞活性,pH值,胞内可溶性糖含量及褐变强度均与原型细胞系存在显著差别。     

Feast and famine in plant genomes

Plant genomes vary over several orders of magnitude in size, even among closely related species, yet the origin, genesis and significance of this variation are not clear. Because DNA content varies over a sevenfold range among diploid species in the cotton genus (Gossypium) and its allies, this group offers opportunities for exploring patterns and mechanisms of genome size evolution. For example, the question has been raised whether plant genomes have a one-way ticket to genomic obesity, as a consequence of retroelement accumulation. Few empirical studies directly address this possibility, although it is consistent with recent insights gleaned from evolutionary genomic investigations. We used a phylogenetic approach to evaluate the directionality of genome size evolution among Gossypium species and their relatives in the cotton tribe (Gossypieae, Malvaceae). Our results suggest that both DNA content increase and decrease have occurred repeatedly during evolution. In contrast to a model of unidirectional genome size change, the frequency of inferred genome size contraction exceeded that of expansion. In conjunction with other evidence, this finding highlights the dynamic nature of plant genome size evolution, and suggests that poorly understood genomic contraction mechanisms operate on a more extensive scale that previously recognized. Moreover, the research sets the stage for fine-scale analysis of the evolutionary dynamics and directionality of change for the full spectrum of genomic constituents.     

Effects of Nigella sativa L. and Urtica dioica L. on selected mineral status and hematological values in CCl4-treated rats

This study was designed to investigate the effects of Nigella sativa L. (NS), known as black seed, or/and Urtica dioica L. (UD), known as stinging nettle root, treatments on serum Na, K, Cl, and Ca levels and some hematological values of CCl₄-treated rats. Sixty healthy male Sprague-Dawley rats, weighing 250–300 g, were randomly allotted into 1 of 4 experimental groups: A (CCl₄-only treated), B (CCl₄+UD treated), C (CCl₄+NS treated), and D (CCl₄+UD+NS treated), each containing 15 animals. All groups received CCl₄ (0.8 mL/kg of body weight, subcutaneously, twice a week for 90 d starting d 1). In addition, B, C, and D groups also received the daily ip injection of 0.2 mL/kg NS and/or 2 mL/kg UD oils for 45 d starting d 46. Group A, on the other hand, received only 2 mL/kg normal saline solution for 45 d starting d 46. Blood samples for the biochemical analysis were taken by cardiac puncture from five randomly chosen rats in each treatment group at the beginning, d 45, and d 90 of the experiment. The CCl₄ treatment for 45 d significantly (p<0.05) increased the serum K and Ca and decreased (p<0.05) the red blood cell count (RBC), white blood cell count (WBC), packed cell volume (PCV), and Hb levels without changing (p>0.05) the serum Na and Cl levels. NS or UD treatments (alone or combination) for 45 d starting d 46 significantly (p<0.05) decreased the elevated serum K and Ca levels and also increased (p<0.05) the reduced RBC, WBC, PCV, and Hb levels. It is concluded that NS and/or UD treatments might ameliorate the CCl₄-induced disturbances of anemia, some minerals, and body’s defense mechanism in CCl₄-treated rats.     

Extending the Boundaries,Bridging the Gaps: Crafting Mental Health: Culture,Race, and Ethnicity,a Supplement to the Surgeon General's Report on Mental Health

The August 2001 issuance of Mental Health: Culture, Race, and Ethnicity--A Supplement to Mental Health: A Report of the Surgeon General, represents a landmark in the dialogue--political and scientific--regarding health disparities in the United States. This paper offers a critical appraisal of the process and structure of generating these reports, paying particular attention to issues that marked serious epistemological tensions among the participants. These issues revolved around the relative emphasis placed on (1) mental illness and mental health; (2) risk, etiology, and treatment versus prevention and promotion; (3) large-scale, population-based surveys and randomized clinical trials as the standard bearers of scientific evidence; (4) variation related to gender, social class, and culture; (5) ethnicity and culture as dispositional variables or individual glosses as opposed to dynamic, collective phenomena; and (6) the historical forces that shaped the contemporary context for much of this discussion. It describes the sometimes subtle, other times stark differences in assumptions and experience that sprang from disciplinary orientations, investigative methods, institutional affiliations, and personal histories and agendas.     

Quality Control and Assurance: crucial for the sustainability of the applied phycology industry

The chemical and nutritional properties of microalgae are well known, which has led to an ever expanding industry for foods and dietary supplements both in terms of quantity and products. Little has been done to regulate or control quality and assurance in the applied phycology industry and it is known that it varies considerably. Nutritional aspects of produced biomass and consumption as dietary supplements have become issues of concern, especially since the industry is lucrative and fast growing. Various claims are made regarding dietary and food supplements that include health, nutrition, structure and functioning, many often unsubstantiated. Although quality is a subjective term many organisations are involved in testing, controlling and determining criteria. Today quality is more than just standards where it is an "integrated quality management approach" involving amongst others "hazard analyses and critical control points" (HACCP) practices. Microalgae are not recognised as a food or food supplement and they are also not categorised under herbals or botanicals, but as "other supplements". Produced microalgal biomass is subject to contamination from the entire range of contaminants and pathogens. Contamination of products by algal toxins in mixed culture populations also occurs. The industry has largely regulated itself, but there is considerable scope for improvement. There is a need for support and dissemination of information in the industry.     

Discounting and the environment should current impacts be weighted differently than impacts harming future generations?

Background  In Life-Cycle Assessment (LCA), decision makers are often faced with tradeoffs between current and future impacts. One typical example is waste incineration, where immediate emissions to the air from the incineration process have to be weighted against future emissions of slag landfills. Long-term impacts are either completely taken into account or they are entirely disregarded in case of a temporal cut-off. Temporal cutoffs are a special case of discounting. Objective  In this paper, discounting is defined as valuing damages differently at different points of time using a positive or negative discount rate. Apart from temporal cut-offs, discounting has rarely been applied in LCA so far. It is the goal of this paper to discuss the concept of discounting and its applicability in the context of LCA. Methods  For this purpose, we first review the arguments for discounting and its principles in economic sciences. Discounting in economics can be motivated by pure time preference, productivity of capital, diminishing marginal utility of consumption, and uncertainties. The nominal discount rate additionally includes changes in the price level. These arguments and their justification are discussed in the context of environmental impacts harming future generations. Results and Discussion  It is concluded that discounting across generations because of pure time preference contradicts fundamental ethical values and should therefore not be applied in LCA. However, it has to be acknowledged that in practice decision makers often use positive discount rates because of pure time preference — either because they might profit from imposing environmental damage on others instead of themselves or because people in the far future are not of immediate concern to them. Discounting because of the productivity of capital assumes a relationship between monetary values and environmental impact. If such a relationship is accepted, discounting could be applied. However, future generations should be compensated for the environmental damage. It is likely that they would demand a higher compensation if the real per capita income increases. As both the compensation and the discount rate are related to economic growth, the overall discount rate might be close to zero. It is shown that the overall discount rate might even be negative considering that the required compensation could increase (even to infinite) if natural assets remain scarce, whereas the utility of consumption diminishes with increasing income. Uncertainties could justify both positive and negative discount rates. Since the relationship between uncertainties and the magnitude of damage is generally not exponential, we recommend to model changes in the magnitude of damage in scenario analysis instead of considering it in discounting (which requires an exponential function of time in the case of a constant discount rate). We investigated the influence of discounting in a case study of heavy metal emissions from slag landfills. It could be shown that even small discount rates of less than 1 % lead to a significant reduction of the impact score, whereas negative discount rates inflate the results. Conclusions and Recommendations  Discounting is only applicable when temporally differentiated data is available. In some cases, such a temporal differentiation is necessary to take sound decisions, especially when long emission periods are involved. An example is the disposal of nuclear or heavy metal-containing waste. In these cases, the results might completely depend on the discount rate. This paper helps to structure arguments and thus to support the decision about whether or not discounting should be applied in an LCA.     

Predicted SAR in Sprague-Dawley rat as a function of permittivity values

Specific absorption rate (SAR) value is dependent on permittivity value. However, variability in the published permittivity values for human and animal tissue and the development of sophisticated 3-dimensional digital anatomical models to predict SAR values has resulted in the need to understand how model parameters (permittivity value) affect the predicted whole body and localized SAR values. In this paper, we establish the partial derivative of whole body SARs and localized SAR values (defined as SAR for individual organs with respect to a change in the permittivity values of all tissue types, as well as for those tissues with the most variable permittivity values. Variations in the published permittivity values may substantially influence whole body and localized SAR values, but only under special conditions. Orientation of the exposed object to the incident electromagnetic wave is one of the most crucial factors. Published 2001 Wiley-Liss, Inc.     

人工饲养条件下恒河猴血液学、血液生化正常参考值测定及分析

目的测定人工饲养条件下104只健康恒河猴的血液学、血液生化正常参考值,并分析不同性别、不同年龄阶段(青幼年组∶3～4岁,成年组∶5～10岁,老年组∶11～25岁)恒河猴的血液学、血液生化值的差异及相互关系.方法应用全自动血液细胞分析仪和全自动血液生化分析仪,测定及分析非麻醉状态下健康恒河猴血液学和血液生化正常值.结果血液学值中较明显低 (P<0.05) 的项目为MED.血液生化值中♀猴比♂猴明显低 (P<0.01)的项目有ALT、ALP、GGT、CHOL、APOA、HDL、LDL、P3+、BUN,较明显低 (P<0.05) 的项目为ALB.较明显高 (P<0.05) 的项目为UA.各年龄组之间的分析如下:血液学值中RDW值为青幼年组比成年组及老年组均明显低(P<0.01),MEDIAN值为成年组比青幼年组较明显高 (P<0.05),血液生化值中青幼年组比成年组及老年组均明显高 (P<0.01)的项目有ALT、ALP、GGT、P3+,青幼年组比成年组明显高(P<0.01)的项目为BUN、ALP、AST、,青幼年组比老年组明显高(P<0.01)的项目为CK、TF;成年组比老年组明显低(P<0.01)的项目为CREAT,较明显低 (P<0.05) 的为TRIG.结论本文建立了人工饲养条件下健康恒河猴血液学和血液生化值的正常指标,并讨论了年龄、性别对其正常指标的影响,可供参考.