Characterization of acetylcholine receptor isolated from Torpedo californica electroplax through the use of an easily removable detergent, β-d-octylglucopyranoside

Non-ionic detergents used for the solubilization and purification of acetylcholine receptor from Torpedo californica electroplax may remain tightly bound to this protein. The presence of detergent greatly hinders spectrophotometric and hydrodynamic studies of the receptor protein. β-d-Octylglucopyranoside, however, is found to be effective in solubilizing the receptor from electroplax membranes with minimal interference in the characterization of the protein. The acetylcholine receptor purified from either octylglucopyranoside or Triton X-100-solubilized extracts exhibits identical amino acid compositions, α-Bungarotoxin and (+)-tubocurarine binding parameters, and subunit distributions in SDS-polyacrylamide gels. The use of octylglucopyranoside allows for the assignment of a molar absorptivity for the purified receptor at 280 nm of approx. 530 000 $\text{M}{}^{\mathrm{?1}}\text{·}\text{cm}{}^{\mathrm{?1}}$. Additionally, successful reconstitution of octylglucopyranoside-extracted acetylcholine receptor into functional membrane vesicles has recently been achieved (Gonzales-Ros, J.M., Paraschos, A. and Martinez-Carrion, M. (1980) Proc.Natl. Acad. Sci. U.S.A. 77, 1796–1799).Removal of octylglucopyranoside by dialysis does not alter the specific toxin and antagonist binding ability of the receptor or its solubility at low protein concentrations. Sedimentation profiles of the purified acetylcholine receptor in sucrose density gradients reveal several components. Sedimentation coefficients obtained for the slowest sedimenting species agree with previously reported molecular weight values. Additionally, the different sedimenting forms exhibit distinctive behavior in isoelectric focusing gels. Our results suggest that both the concentration and type of detergent greatly influence the physicochemical behavior of the receptor protein.     

Intracellular phosphate pools show compartmentalization of intracellular pH in turtle urinary bladder

The urinary bladder of the fresh water turtle is capable of acidification and Na transport, in vitro, and it has been extensively used as a model of distal nephron of the kidney. In the course of measuring intracellular pH of stripped turtle bladder mucosa with phosphorus nuclear magnetic resonance, we observed the consistent presence of two inorganic phosphorus resonances under aerobic conditions, indicating the existence of a pH gradient possibly between cytosol and mitochondrion. This pH gradient was collapsed by addition of N₂ and could be restored by reintroduction of oxygen. These observations demonstrate the existence of a spontaneous pH gradient between cytosol and mitochondria of turtle bladder epithelial cells.     

Structural investigation of the capsular polysaccharide of klebsiella serotype K26

The structure of the capsular polysaccharide from Klebsiella K26 has been determined by using the techniques of methylation, periodate oxidation, partial hydrolysis, and β-elimination. N.m.r. spectroscopy (¹H and ¹³C) was used to establish the nature of the anomeric linkages and to identify oligosaccharides obtained by the different degradative techniques employed.The polysaccharide is comprised of repeating units of the heptasaccharide shown.

     

Subcellular distribution of DNA-binding proteins from cultured hamster fibroblasts

The distribution between nuclei and cytoplasm of DNA-binding proteins from growing NIL cells was studied. To obtain the subcellular fractions, cell monolayers or cells previously detached from the culture dish were treated with the non-ionic detergent Nonidet P-40. Proteins with affinity for DNA were isolated from nuclear or cytoplasmic fractions by chromatography on DNA-cellulose columns and were further analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results show that P8, one of the major components in the 0.15 M NaCl-eluted proteins, is found predominantly in the cytoplasmic fractions, whereas P6, the other main protein peak in this eluate, is more prominent in the nuclear fraction. Among the other proteins eluted at 0.15 M NaCl from the DNA-cellulose column, P5 and P5′ are detected in both nuclear and cytoplasmic fractions. All the other proteins in the 0.15 M NaCl eluate are present almost exclusively in the cytoplasmic fraction. On the other hand, most of the proteins with higher affinity for DNA, eluted from the column at 2 M NaCl, are present in the nuclear fraction, although they are also detected in the cytoplasm in amounts similar to those observed in the nuclei.     

Effects of pair migratory behavior on breeding phenology and success in a partially migratory shorebird population

In migratory systems, variation in individual phenology can arise through differences in individual migratory behaviors, and this may be particularly apparent in partial migrant systems, where migrant and resident individuals are present within the same population. Links between breeding phenology and migratory behavior or success are generally investigated at the individual level. However, for breeding phenology in particular, the migratory behaviors of each member of the pair may need to be considered simultaneously, as breeding phenology will likely be constrained by timing of the pair member that arrives last, and carryover effects on breeding success may vary depending on whether pair members share the same migratory behavior or not. We used tracking of marked individuals and monitoring of breeding success from a partially migrant population of Eurasian oystercatchers (Haematopus ostralegus) breeding in Iceland to test whether (a) breeding phenology varied with pair migratory behavior; (b) within‐pair consistency in timing of laying differed among pair migratory behaviors; and (c) reproductive performance varied with pair migratory behavior, timing of laying, and year. We found that annual variation in timing of laying differed among pair migratory behaviors, with resident pairs being more consistent than migrant and mixed pairs, and migrant/mixed pairs breeding earlier than residents in most years but later in one (unusually cold) year. Pairs that laid early were more likely to replace their clutch after nest loss, had higher productivity and higher fledging success, independent of pair migratory behavior. Our study suggests that the links between individual migratory behavior and reproductive success can vary over time and, to a much lesser extent, with mate migratory behavior and can be mediated by differences in laying dates. Understanding these cascading effects of pair phenology on breeding success is likely to be key to predicting the impact of changing environmental conditions on migratory species.     

Lifelong cytomegalovirus and early‐LIFE irradiation synergistically potentiate age‐related defects in response to vaccination and infection

While whole‐body irradiation (WBI) can induce some hallmarks of immune aging, (re)activation of persistent microbial infection also occurs following WBI and may contribute to immune effects of WBI over the lifespan. To test this hypothesis in a model relevant to human immune aging, we examined separate and joint effects of lifelong latent murine cytomegalovirus (MCMV) and of early‐life WBI over the course of the lifespan. In late life, we then measured the response to a West Nile virus (WNV) live attenuated vaccine, and lethal WNV challenge subsequent to vaccination. We recently published that a single dose of non‐lethal WBI in youth, on its own, was not sufficient to accelerate aging of the murine immune system, despite widespread DNA damage and repopulation stress in hematopoietic cells. However, 4Gy sub‐lethal WBI caused manifest reactivation of MCMV. Following vaccination and challenge with WNV in the old age, MCMV‐infected animals experiencing 4Gy, but not lower, dose of sub‐lethal WBI in youth had reduced survival. By contrast, old irradiated mice lacking MCMV and MCMV‐infected, but not irradiated, mice were both protected to the same high level as the old non‐irradiated, uninfected controls. Analysis of the quality and quantity of anti‐WNV immunity showed that higher mortality in MCMV‐positive WBI mice correlated with increased levels of MCMV‐specific immune activation during WNV challenge. Moreover, we demonstrate that infection, including that by WNV, led to MCMV reactivation. Our data suggest that MCMV reactivation may be an important determinant of increased late‐life mortality following early‐life irradiation and late‐life acute infection.     

Biochemical functionality and recovery of hepatocytes after deep freezing storage

Summary The present study was undertaken to define the conditions for optimal cryopreservation of hepatocytes. Two different freezing procedures were analyzed: a slow freezing rate (SFR) (−2° C/min down to −30°C and then quick freezing to −196° C) and a fast freezing rate (FFR) (direct freezing of tubes to −196° C: −39° C/min). Cells were frozen in fetal bovine serum containing 10% Dimethyl sulfoxide (DMSO). After rapid thawing at 37° C, followed by dilution and removal of the cryoprotectant, cells were plated and several parameters were followed as criteria for optimal cryopreservation of cells. The FFR cells showed no apparent ultrastructural damage after 24 h of culture. Plating efficiency and spreading were similar as controls. Gluconeogenesis from pyruvate and fructose, tyrosine amino transferase induction by glucagon and dexamethasone, urea production, and plasma protein synthesis of FFR cells were similar to those found in control cultures. The FFR procedure, in comparison to the SFR method, seemed to render the best preserved hepatocytes. The financial support for this work was from Fondo de Investigaciones Sanitarias de la Seguridad Social, Grants 41/82 and 48/82.     

Environmental Profile of Brazilian Green Coffee (6 pp)

Goal, Scope and Background

Brazil is the world's biggest producer of coffee beans with approx. a 30% market share. Depending on climate conditions, approx. 30 million bags of coffee beans are exported annually from Brazil, while domestic consumption is around 10 million bags, which makes Brazil the world's third largest coffee-consuming country. Therefore, the goal of this paper is to present the LCA of green coffee produced in Brazil for the reference crops 2001/02 and 2002/03 in order to generate detailed production inventory data as well as to identify the potential environmental impacts of its tillage in order to realize how to reduce those impacts and increase the environmental sustainability of this product. Only the inputs and outputs relative to the coffee tillage were considered. The production of fertilizers, correctives and pesticides were not included in the boundary, but only their amounts. The functional unit selected for this study was 1,000 kg of green coffee destined for exportation.

Methods

The LCI was performed according to the ISO 14040 standard series. All information considered in this study (use of water, fossil based energy, fertilizers and chemicals) were taken up in in-depth data collection and evaluation by questionnaires applied on a farm level and/or received by mail. Four Brazilian coffee producer regions were evaluated: Cerrado Mineiro, South of Minas Gerais State, the Marília and Alta Mogiana regions in São Paulo State. These regions have the following geographic coordinates: 44 to 50° W longitude and 18 to 24° S latitude. The data refer to a production of 420,000 coffee bean bags and a productive area of approx. 14,300 ha. The varieties of coffee beans considered in this study were Mundo Novo, Catuaí (yellow and red), Icatu (yellow and red), Catucaí (yellow and red) and Obatã. Farm specific data along with agricultural production data have been combined to elaborate a coffee cultivation inventory, which will be applied in an emissions estimation.

Results and Conclusion

The production of 1,000 kg of green coffee in Brazil requires approx. 11,400 kg of water, 94 kg of diesel, 270 kg of fertilizers as NPK, 900 kg of total fertilizers, 620 kg of correctives, 10 kg of pesticides and 0.05 hectare of annual land use. Outputs related to these functional units are approx. 3,000 kg of waste water from coffee washing, 8,500 kg of waste water from the wet method and 750 kg of organic residue that is reincorporated to the tillage as fertilizer. The publication of an LCI of agricultural products is a fundamental step for understanding the potential environmental impacts of each tillage and then establishes the basis for product sustainability. In this way, this work is the first Brazilian initiative for applying LCA to coffee cultivation.

Recommendation and Perspective

Different agricultural practices demonstrate different environmental profiles. The amount of agricultural pesticide is directly related to agricultural practices as tillage rotation, density of plants, etc. This study supplied important results for a better correlation of the agricultural practices and potential environmental impacts of coffee. Future updates of this study will show the evolution of the natural resource management such as land use, new agricultural practices, lower fertilizers and chemicals use.