The relevance of flow cytometry for biochemical analysis

Flow cytometry (FCM) allows the simultaneous measurement of multiple fluorescences and light scatter induced by illumination of single cells or microscopic particles in suspension, as they flow rapidly through a sensing area. In some systems, individual cells or particles may be sorted according to the properties exhibited. By using appropriate fluorescent markers, FCM is unique in that multiple structural and functional parameters can be quantified simultaneously on a single-particle basis, whereas up to thousands of biological particles per second may be examined. FCM is increasingly used for basic, clinical, biotechnological, and environmental studies of biochemical relevance. In this critical review, we summarize the main advantages and limitations of FCM for biochemical studies and discuss briefly the most relevant parameters and analytical strategies. Graphical examples of the biological information provided by multiparametric FCM are presented. Also, this review contains specific sections on flow cytoenzymology, FCM analysis of isolated subcellular organelles, and cell-free FCM.     

Biomarker-enhanced assessment of reproductive disorders in Monoporeia affinis exposed to contaminated sediment in the Baltic Sea

Introducing biomarkers into monitoring programs requires understanding of their responses in relation to higher-level biological effects as well as modulating effects of confounding environmental factors. We evaluated relationships between the general toxicity biomarkers (acetylcholinesterase [AChE], lysosomal membrane stability [LMS], oxygen radical absorbance capacity [ORAC]) and reproductive performance (fecundity and embryo aberrations) in the amphipod Monoporeia affinis in the Baltic Sea. To further link biomarker response to contaminant (PCBs, PAHs and metals) levels in the surrounding sediments as well as environmental factors (salinity, bottom depth and total organic carbon in sediments [TOC]), correlation and partial least square regression (PLSR) analyses were applied. The observed contaminants levels were frequently elevated for heavy metals and PAHs, but not PCBs. In the amphipod populations, female ORAC values were positively related to the occurrence of females carrying malformed or membrane-damaged embryos and to the percentage of such embryos in their broods, but also to the fecundity. Female AChE activity was negatively related to the frequency of the membrane-damaged embryos, and positively to the frequency of embryos with arrested development in the broods. Moreover, higher AChE activity and ORAC values in the females occurred at elevated concentrations of metals and PAHs, while there was a negative correlation between embryo ORAC and some PCB congeners. The PLSR models explained over 80% of the variation in the female ORAC and AChE values by variation in contaminant concentrations in combination with environmental variables. Specifically, CB180 and PAM4,9 were identified as negative predictors for ORAC, whereas many PAHs and some metals were positive predictors. The AChE activity was positively related to some metals and negatively to PCBs. In the PLSR models, environmental factors had significant modulating effects, with positive effect of salinity on female ORAC and AChE, and negative effect of TOC on the AChE. The LMS data were less informative, with no apparent relation to any of the contaminants. Linking subcellular responses to the reproduction effects facilitates environmental stress assessment and understanding of the response mechanisms, but also calls for more experimental and field data providing a mechanistic understanding to these linkages.     

Plant peroxisomes at the crossroad of NO and H_2O_2 metabolism

Plant peroxisomes are subcellular compartments involved in many biochemical pathways during the life cycle of a plant but also in the mechanism of response against adverse environmental conditions. These organelles have an active nitro-oxidative metabolism under physiological conditions but this could be exacerbated under stress situations. Furthermore, peroxisomes have the capacity to proliferateand also undergo biochemical adaptations depending on the surrounding cellular status. An important characteristic of peroxisomes is that they have a dynamic metabolism of reactive nitrogen and oxygen species(RNS and ROS) which generates two key molecules, nitric oxide(NO) and hydrogen peroxide(H_2O_2). These molecules can exert signaling functions by means of post-translational modifications that affect the functionality of target molecules like proteins, peptides or fatty acids. This review provides an overview of the endogenous metabolism of ROS and RNS in peroxisomes with special emphasis on polyamine and uric acid metabolism as well as the possibility that these organelles could be a source of signal molecules involved in the functional interconnection with other subcellular compartments.     

Global organellar proteomics

Cataloging the proteomes of single-celled microorganisms, cells, biological fluids, tissue and whole organisms is being undertaken at a rapid pace as advances are made in protein and peptide separation, detection and identification. For metazoans, subcellular organelles represent attractive targets for global proteome analysis because they represent discrete functional units, their complexity in protein composition is reduced relative to whole cells and, when abundant cytoskeletal proteins are removed, lower abundance proteins specific to the organelle are revealed. Here, we review recent literature on the global analysis of subcellular organelles and briefly discuss how that information is being used to elucidate basic biological processes that range from cellular signaling pathways through protein-protein interactions to differential expression of proteins in response to external stimuli. We assess the relative merits of the different methods used and discuss issues and future directions in the field.     

Hygienic evaluation and regulation of some environmental factors to counteract their potential carcinogenic hazards

The evident growth of tumour morbidity makes analysis of the fundamental problems of neoplasm prophylaxis in man particularly urgent. The establishment of maximum permissible concentrations for carcinogenic agents calls for further exploration into the problem of threshold action of carcinogens and elaboration of a methodology for quantitative assessment of the biological effects of certain environmental factors. In particular, the available data on the ability of low-intensity environmental factors to modify the organism's general resistance and thereby produce in it conditions for materialization of the untoward effects of carcinogens invite further work in this direction. The study of environmental factors as possible modifiers of blastomatosis and investigation of the modification mechanisms involved are an immediate and topical task. Quantitative analysis of these modifying effects would allow establishment of well-grounded hygienic norms as the next step. Thus, study of the regularities involved, development of a fundamental methodology and accumulation of facts on the influence of low-intensity environmental factors on carcinogenesis and the cancer effect should be helpful in devising scientifically based measures of lowering the growth of tumour morbidity.     

Theoretical and methodological bases of experimental simulation of the prepathological state under membrane-damaging effects of environmental factors

The paper theoretically substantiates the importance for detecting the prepathological state of experimental simulation of those situations in the organism which adequately reflect changes in human metabolism exposed to multiple environmental factors. A putative experimental model has been proposed in the shape of sexually immature and mature generations of animals of differing age with a "metabolic burden" whose characteristics, as regards impairments in biochemical, immunological and other functional reactions in biological fluids, are to some extent similar to those occurring in certain groups of humans. It has been suggested that the further investigation of the mechanisms and biological significance of systemic changes of the body's internal milieu may hold promise for studies of environmental hygiene in evaluating populational health and detecting the early signs of metabolic alterations with the aim of their timely prevention.     

高山植物叶片δ13C的海拔响应及其机理

植物 1 3C的分辨研究已成为植物生态学和全球碳循环研究的核心问题之一。植物 1 3C的分辨是环境和生物因子共同作用的综合结果 ,海拔梯度变化不仅可以造成植物生存环境的变化 ,而且还可以造成植物形态和生理特征的变化 ,因此 ,高山植物 1 3C分辨随海拔的变化为深入揭示植物 1 3C分辨的环境和生物因子的作用机理提供了非常理想的研究条件。在简单介绍植物 1 3C分辨基本理论的基础上 ,对目前国际上高山植物 1 3C分辨的海拔响应研究进行了述评。重点介绍了随海拔变化的大气 1 3C组成、温度、气压、水分等环境因子和植物叶片的气孔导度、羧化效率、氮含量和叶肉细胞导度等生物因子对高山 C3植物 1 3C分辨的影响 ,指出高山植物 1 3C分辨的海拔响应机理仍存在一些不确定性 ,为国内相关研究的开展提供了一定参考     

Importance of the functional state of alveolar macrophages of the lungs for hygienic evaluation of protective reactions and cell damage due to atmospheric pollution

Total number of cells, their viability and ability to adhesion were examined in surface alveolar macrophages isolated from rat livers after exposure to sulphur dioxide during 2, 4 and 6 weeks (0.05, 0.5, 1.0 and 5.0 mg/m3); to nitrogen oxide during 5, 8 and 15 hours, 28 and 56 days (19 mg/m3) and to carbon monoxide during 2, 28 and 56 days (0.01% or 10 MAC). In the experiment with exposure to sulphur dioxide, the activity of enzymes of varying localization in the macrophages - soluble in the cytoplasm (lactate dehydrogenase) and connected with subcellular structures - lysosomes (beta-galactosidase, beta-glucosidase and acid phosphatase) was tested by means of biochemical methods in parallel with cytological examinations. Low concentrations of various chemical contaminants of the atmospheric air (sulphur dioxide, nitrogen oxides, carbon monoxide) have an unfavourable biological effect on rats, manifest in the impairment of local immunity, i.e., decreased number of alveolar macrophages, disturbance of their viability and reduced ability of the macrophages to adhesion. At the same time, sulphur dioxide induces enzyme disorganization in lactate dehydrogenase and in a number of lysosomal enzymes of the macrophages. These results serve as a basis for the recommendation of cytobiochemical methods of elaborating methodological approaches to the regulation of environmental factors. Alveolar macrophages as a constituent part of the mononuclear phagocytic system ensuring local non-specific and specific resistance of the organism form one of the most important cellular mechanisms of protection of the organism against the harmful effect of environmental factors including chemical contaminants of the atmospheric air (1, 2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacterial microcompartments: widespread prokaryotic organelles for isolation and optimization of metabolic pathways

Prokaryotes use subcellular compartments for a variety of purposes. An intriguing example is a family of complex subcellular organelles known as bacterial microcompartments (MCPs). MCPs are widely distributed among bacteria and impact processes ranging from global carbon fixation to enteric pathogenesis. Overall, MCPs consist of metabolic enzymes encased within a protein shell, and their function is to optimize biochemical pathways by confining toxic or volatile metabolic intermediates. MCPs are fundamentally different from other organelles in having a complex protein shell rather than a lipid‐based membrane as an outer barrier. This unusual feature raises basic questions about organelle assembly, protein targeting and metabolite transport. In this review, we discuss the three best‐studied MCPs highlighting atomic‐level models for shell assembly, targeting sequences that direct enzyme encapsulation, multivalent proteins that organize the lumen enzymes, the principles of metabolite movement across the shell, internal cofactor recycling, a potential system of allosteric regulation of metabolite transport and the mechanism and rationale behind the functional diversification of the proteins that form the shell. We also touch on some potential biotechnology applications of an unusual compartment designed by nature to optimize metabolic processes within a cellular context.     

Recent developments in cryo-electron microscopy reconstruction of single particles

Cryo-electron microscopy and single-particle 3D image reconstruction techniques have been used to examine a broad spectrum of samples ranging from 500 kDa protein complexes to large subcellular organelles. The attainable resolution has improved rapidly over the past few years. Structures of both symmetric and asymmetric assemblies at approximately 7.5 A have been reported. Together with X-ray crystallography, three-dimensional cryo-electron microscopy reconstruction has provided important insights into the function of many biological systems in their native biochemical contexts.     

Subcellular Localization of Catalase in Sea Urchin (Tetrapigus niger) Gametes: Implications for Peroxisome Biogenesis

Peroxisomes are essential subcellular organelles that appear to be derived from pre-existing organelles. To test the presence of peroxisomes in sea urchin (Tetrapigus niger) sperm and eggs, we performed biochemical and morphological experiments to evaluate the subcellular distribution of catalase as the typical peroxisomal marker. In sea urchin sperm, we found that catalase is localized in the cell cytosol. In contrast, sea urchin eggs contain sedimentable catalase, presumably contained in peroxisome-like structures detected by immunomicroscopy and by cytochemistry. Our results show, for the first time, evidence for the presence of peroxisome-like structures in sea urchin eggs and provide evidence for the peroxisome biogenesis hypothesis by division of pre-existing organelles.     

Analytical tools for characterizing heterogeneity in organelle content

Heterogeneity in the content and function of subcellular organelles on the intercellular and intracellular level plays an important role in determining cell fate. These variations extend to normal-state and disease-state cellular functions and responses to environmental stimuli, such as oxidative stress and therapeutic drugs. Analytical tools to characterize variation in all types of organelles are essential to provide insights that can lead to advances in medicine, such as therapies targeted to specific subcellular regions. In this review, we discuss analytical techniques for interrogating individual intact organelles (e.g. mitochondria and synaptic vesicles) and lysates in a high-throughput manner, including a recently developed nanoscale fluorescence-activated subcellular sorter and techniques based on capillary electrophoresis with laser-induced fluorescence detection. We then highlight the advantages that droplet microfluidics offers for probing subcellular heterogeneity.     

Acid hydrolases in the suckling rat small intestine I. Fractionation of mucosal homogenates

Synopsis Small intestine mucosal homogenates of suckling rats have been fractionated by centrifugation and analyzed for acid hydrolases and for biochemical markers of subcellular organelles. The results indicate that the acid hydrolases are associated with particles having sedimentation properties similar to those of mitochrondria. The acid hydrolases exhibited latent activity. Subfractionation on a continuous density gradient of sucrose in deuterium oxide demonstrated that these enzymes are associated with particles distinct from other subcellular organelles. Electron micrographs of the acid hydrolase-rich region of the gradient show the presence of numerous small electron dense bodies bounded by a unit membrane.     

A Conceptual Framework for the Interpretation of Biological Markers for Environmental Exposure Assessment

Human exposure to environmental contaminants occurs via air, water, soil, dust, food, and other environmental media. Given this multitude of sources, environmental exposure assessment is moving away from single route exposure assessment to more integrated measures of exposure. Biological markers are frequently advocated as appropriate exposure assessment tools since they provide a measure of internal dose integrated over all routes of exposure. However, contributing sources may be difficult to identify through use of biological markers, and thus, have had limited utility in the regulatory community. To explore the different perspectives on the use and application of biological markers for exposure assessors, epidemiologists, and regulatory personnel, we have developed a biological marker conceptual framework. This framework is developed as a paradigm for the interpretation of biological markers for environmental exposure assessment linking the exposure assessment and the health effects assessment perspectives regarding biological markers. Further, it incorporates issues of source-specific exposures, aggregate exposure assessment, route-specific contributions, and biological variation in response to exposure. This structure provides an approach to explore the current constraints in using biological markers to evaluate source-specific exposures. This framework is discussed in the context of currently available biological markers for lead, carbon monoxide, and toluene. Biological markers represent a complex tool to assess human exposures to environmental contaminants; the biological marker framework presents a structure for their interpretation recognizing that many of the determinants of exposure, bioavailablity, and toxicokinetics are still being evaluated. The conceptual framework presented here provides another tool for the researcher in assessing the utility of biological markers in exposure assessment and epidemiology.     

Comparative biochemical and morphological investigation of the liver of experimental animals in the process of hepatotropic effect of atmospheric pollution (on the model of carbon tetrachloride)

The authors effected a comparative examination of morphologic and biochemical investigation of structural and enzymic reactions of the rat liver during different periods of permanent inhalation influence of carbon tetrachloride. Three stages of morphologico-biochemical parllels in the development of hepatotropic effect characterized by certain regularities in the enzymic desorganization of lysosyme, mitochondrias and cytasol by derangement of the exchange of biopolymers containing carbohydrate (of glycoproteids, glycosaminoglycans) and DNK-synthetizing function in liver in comparison with control animals were revealed. Mechanisms of the hepatotropic effect and hygienic importance of structural function changes of hepatocytes due to action of factors polluting the atmosphere are appreciated.     

Generation of transgenic Arabidopsis plants expressing mcherry-fused organelle marker proteins

In eukaryotic cells, a major proportion of the cellular proteins localize to various subcellular organelles where they are involved in organelle-specific cellular processes. Thus, the localization of a particular protein in the cell is an important part of understanding the physiological role of the protein in the cell. Various approaches such as subcellular fractionation, immunolocalization and live imaging have been used to define the localization of organellar proteins. Of these various approaches, the most powerful one is the live imaging because it can show in vivo dynamics of protein localization depending on cellular and environmental conditions without disturbing cellular structures. However, the live imaging requires the ability to detect the organelles in live cells. In this study, we report generation of a new set of transgenic Arabidopsis plants using various organelle marker proteins fused to a fluorescence protein, monomeric Cherry (mCherry). All these markers representing different subcellular organelles such as chloroplasts, mitochondria, peroxisomes, endoplasmic reticulum (ER) and lytic vacuole showed clear and specific signals regardless of the cell types and tissues. These marker lines can be used to determine localization of organellar proteins by colocalization and also to study the dynamics of organelles under various developmental and environmental conditions.     

Binding of cytochrome b5 to membranes of isolated subcellular organelles from rat liver

The in vitro incorporation of a well-characterized integral protein cytochrome b5 into membranes of various subcellular organelles was investigated by biochemical and immunochemical methods. Microsomes, peroxisomes, and outer mitochondrial membranes, all containing endogenous cytochrome b5, incorporated large amounts of the hemoprotein in such a way that it was reducible by an inherent NADH cytochrome b5 reductase. Lysosomal membranes did not incorporate cytochrome b5. Inner mitochondrial and Golgi membranes, which do not naturally contain cytochrome b5, bound it in vitro but it was not reduced in the presence of NADH. These results show some discrepancies between the natural localization and the in vitro binding of cytochrome b5. They confirm one aspect of the fluid membrane theory and bring new elements to our understanding of the maintenance of the specific features of the membranes of subcellular organelles with respect to the cell dynamism.     

Exosomes: Extracellular organelles important in intercellular communication

In addition to intracellular organelles, eukaryotic cells also contain extracellular organelles that are released, or shed, into the microenvironment. These membranous extracellular organelles include exosomes, shedding microvesicles (SMVs) and apoptotic blebs (ABs), many of which exhibit pleiotropic biological functions. Because extracellular organelle terminology is often confounding, with many preparations reported in the literature being mixtures of extracellular vesicles, there is a growing need to clarify nomenclature and to improve purification strategies in order to discriminate the biochemical and functional activities of these moieties. Exosomes are formed by the inward budding of multivesicular bodies (MVBs) and are released from the cell into the microenvironment following the fusion of MVBs with the plasma membrane (PM). In this review we focus on various strategies for purifying exosomes and discuss their biophysical and biochemical properties. An update on proteomic analysis of exosomes from various cell types and body fluids is provided and host-cell specific proteomic signatures are also discussed. Because the ectodomain of ~ 42% of exosomal integral membrane proteins are also found in the secretome, these vesicles provide a potential source of serum-based membrane protein biomarkers that are reflective of the host cell. ExoCarta, an exosomal protein and RNA database (http://exocarta.ludwig.edu.au), is described.     

Purification of autophagosomes from rat hepatocytes

To facilitate the purification of rat liver autophagosomes, isolated rat hepatocytes are first incubated for 2 h at 37°C with vinblastine, which induces autophagosome accumulation by blocking the fusion of these organelles with endosomes and lysosomes. The hepatocytes are then electrodisrupted and homogenized, and the various cellular organelles sequentially removed by subcellular fractionation. A brief incubation of the homogenate with the cathepsin C substrate, glycyl-phenylalanine-naphthylamide (GPN), causes rapid osmotic disruption of the lysosomes due to intralysosomal accumulation of GPN cleavage products. Nuclei are removed by differential centrifugation, and the postnuclear supernatant subsequently fractionated on a two-step Nycodenz density gradient. Autophagosomes are recovered in an intermediate density fraction, free from cytosol and mitochondria. The autophagosomes are finally separated from the membranes and vesicles of the endoplasmic reticulum, Golgi, endosomes, etc. by sieving through a density gradient of colloidal silica particles (Percoll). The final preparation contains about 95% autophagosomes and 5% amphisomes according to morphological and biochemical criteria.     

Hygienic behavior of the honey bee (Apis mellifera) is independent of sucrose responsiveness and foraging ontogeny

Hygienic behavior in honey bees is a behavioral mechanism of disease resistance. Bees bred for hygienic behavior exhibit an increased olfactory sensitivity to odors of diseased brood, which is most likely differentially enhanced in the hygienic line by the modulatory effects of octopamine (OA), a noradrenaline-like neuromodulator. Here, we addressed whether the hygienic behavioral state is linked to other behavioral activities known to be modulated by OA. We specifically asked if, during learning trials, bees from hygienic colonies discriminate better between odors of diseased and healthy brood because of differences in sucrose (reward) response thresholds. This determination had to be tested because sucrose response thresholds are susceptible to OA modulation and may have influenced the honey bee's association of the conditioned stimulus (odor) with the unconditioned stimulus (i.e., the sucrose reward). Because the onset of first foraging is also modulated by OA, we also examined whether bees from hygienic colonies differentially forage at an earlier age compared to bees from non-hygienic colonies. Our study revealed that 1-day- and 15- to 20-day-old bees from the hygienic line do not have lower sucrose response thresholds compared to bees from the non-hygienic lines. In addition, hygienic bees did not forage at an earlier age or forage preferentially for pollen as compared to non-hygienic bees. These results support the idea that OA does not function in honey bees simply to enhance the detection of all chemical cues non-selectively or control related behaviors regardless of their environmental milieu. Our results indicate that the behavioral profile of the hygienic bee is sculpted by multiple factors including genetic, neural, social and environmental systems.