Assay methods and biological roles of labile sulfur in animal tissues

Sulfur is a chemically and biologically active element. Sulfur compounds in animal tissues can be present in two forms, namely stable and labile forms. Compounds such as methionine, cysteine, taurine and sulfuric acid are stable sulfur compounds. On the other hand, acid-labile sulfur and sulfane sulfur compounds are labile sulfur compounds. The sulfur atoms of labile sulfur compounds are liberated as inorganic sulfide by acid treatment or reduction. Therefore, the determination of sulfide is the basis for the determination of labile sulfur. Determination of sulfide has been performed by various methods, including spectrophotometry after derivatization, ion chromatography, high-performance liquid chromatography after derivatization, gas chromatography, and potentiometry with a sulfide ion-specific electrode. These methods were originally developed for the determination of sulfide in air and water samples and were then applied to biological samples. The metabolic origin of labile sulfur in animal tissues is cysteine. The pathways of cysteine metabolism leading to the formation of sulfane sulfur are discussed. Finally, reports on the physiological roles and pathological considerations of labile sulfur are reviewed.     

Biological functions of sphingomyelins

Sphingomyelin (SM) is a dominant sphingolipid in membranes of mammalian cells and this lipid class is specifically enriched in the plasma membrane, the endocytic recycling compartment, and the trans Golgi network. The distribution of SM and cholesterol among cellular compartments correlate. Sphingolipids have extensive hydrogen-bonding capabilities which together with their saturated nature facilitate the formation of sphingolipid and SM-enriched lateral domains in membranes. Cholesterol prefers to interact with SMs and this interaction has many important functional consequences. In this review, the synthesis, regulation, and intracellular distribution of SMs are discussed. The many direct roles played by membrane SM in various cellular functions and processes will also be discussed. These include involvement in the regulation of endocytosis and receptor-mediated ligand uptake, in ion channel and G-protein coupled receptor function, in protein sorting, and functioning as receptor molecules for various bacterial toxins, and for non-bacterial pore-forming toxins. SM is also an important constituent of the eye lens membrane, and is believed to participate in the regulation of various nuclear functions. SM is an independent risk factor in the development of cardiovascular disease, and new studies have shed light on possible mechanism behind its role in atherogenesis.     

Cell-line specific chromatin acetylation at the Sox10-Pax3 enhancer site modulates the RET proto-oncogene expression

IscA homologs are known to be involved in iron-sulfur cluster formation in various organisms. Recombinant proteins of two IscA homologs from the cyanobacterium Synechocystis PCC 6803, designated SLR1417 and SLR1565, were purified. The absorption spectrum of purified SLR1565 was typical for [2Fe-2S] cluster-containing proteins, whereas that of SLR1417 predominantly showed the presence of the iron ion alone. In the cyanobacterial cell extracts, only SLR1565 was found to form a complex with a novel prokaryotic HEAT-repeats-containing protein, SLR1098. Thus, the two cyanobacterial IscA protein homologs exist in distinct molecular states, suggesting different cellular roles for these proteins.     

Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance

Respiratory chains are crucial for cellular energy conversion and consist of multi‐subunit complexes that can assemble into supercomplexes. These structures have been intensively characterized in various organisms, but their physiological roles remain unclear. Here, we elucidate their function by leveraging a high‐resolution structural model of yeast respiratory supercomplexes that allowed us to inhibit supercomplex formation by mutation of key residues in the interaction interface. Analyses of a mutant defective in supercomplex formation, which still contains fully functional individual complexes, show that the lack of supercomplex assembly delays the diffusion of cytochrome c between the separated complexes, thus reducing electron transfer efficiency. Consequently, competitive cellular fitness is severely reduced in the absence of supercomplex formation and can be restored by overexpression of cytochrome c. In sum, our results establish how respiratory supercomplexes increase the efficiency of cellular energy conversion, thereby providing an evolutionary advantage for aerobic organisms.     

Increases in ceramide levels in normal human mesangial cells subjected to different cellular stresses result from changes in distinct enzyme activities and can influence cellular responses to other stimuli

Sphingolipids, ceramide in particular, have come to be regarded as having roles in cellular signaling, most recently being associated with stress and the cellular responses to stress. In the present study we first examined the mechanisms involved in the changes in cellular ceramide levels in normal human mesangial cells (NHMC) in the growth, quiescent, and senescent phases as well as those resulting from environmental stimuli. We found that in NHMC total ceramide levels increase in response to cellular stresses as a result of a combination of enzyme activities. Furthermore, different stresses cause different alterations in various enzyme activities, with age and growth influencing acidic enzymes, but cell density affecting neutral, resulting in final ceramide level increases which most likely are associated with distinct pools of ceramide. Secondly, we examined the influence of changes in ceramide levels on apoptosis induced by sphingosine and its methylated derivative N, N-dimethylsphingosine. We found that increases in cellular ceramide levels prohibited the apoptosis and caused a quiescent state in the cells. The data presented here provide additional insight into the roles of ceramide and related enzymes in cellular responses to stress and suggest a possible relevance to in vivo disease states.     

Sequestered end products and enzyme regulation: the case of ornithine decarboxylase.

The polyamines (putrescine, spermidine, and spermine) are synthesized by almost all organisms and are universally required for normal growth. Ornithine decarboxylase (ODC), an initial enzyme of polyamine synthesis, is one of the most highly regulated enzymes of eucaryotic organisms. Unusual mechanisms have evolved to control ODC, including rapid, polyamine-mediated turnover of the enzyme and control of the synthetic rate of the protein without change of its mRNA level. The high amplitude of regulation and the rapid variation in the level of the protein led biochemists to infer that polyamines had special cellular roles and that cells maintained polyamine concentrations within narrow limits. This view was sustained in part because of our continuing uncertainty about the actual biochemical roles of polyamines. In this article, we challenge the view that ODC regulation is related to precise adjustment of polyamine levels. In no organism does ODC display allosteric feedback inhibition, and in three types of organism, bacteria, fungi, and mammals, the size of polyamine pools may vary radically without having a profound effect on growth. We suggest that the apparent stability of polyamine pools in unstressed cells is due to their being largely bound to cellular polyanions. We further speculate that allosteric feedback inhibition, if it existed, would be inappropriately responsive to changes in the small, freely diffusible polyamine pool. Instead, mechanisms that control the amount of the ODC protein have appeared in most organisms, and even these are triggered inappropriately by variation of the binding of polyamines to ionic binding sites. In fact, feedback inhibition of ODC might be maladaptive during hypoosmotic stress or at the onset of growth, when organisms appear to require rapid increases in the size of their cellular polyamine pools.     

土壤和沉积物中黑碳的环境行为及效应研究进展

土壤和沉积物是全球黑碳排放的主要归宿,土壤和沉积物中黑碳具有复杂的环境行为和环境效应。分析了黑碳的概念,指出应以环境意义为出发点去理解黑碳概念的丰富内涵;描述了黑碳形成过程及其对黑碳理化性质的影响,以及基于此的黑碳分类;总结了黑碳来源辨析的若干种常用方法;讨论了黑碳在土壤/沉积物与其他环境介质之间的迁移循环过程,以及在土壤和沉积物内部的迁移行为;探讨了土壤和沉积物中黑碳的降解行为与稳定性,及其与地-气碳氮温室气体通量、土壤稳定碳库的关系,以及在土壤碳循环模型中的作用;综述了土壤和沉积物中黑碳对有机物、重金属和营养盐的吸附行为及主要机制;提出了今后研究的主要方向,以供相关研究者参考。     

Evidence for a gelsolin-rich, labile F-actin pool in human polymorphonuclear leukocytes.

Filamentous (F) actin is a major cytoskeletal element in polymorphonuclear leukocytes (PMNs) and other non-muscle cells. Exposure of PMNs to agonists causes polymerization of monomeric (G) actin to F-actin and activates motile responses. In vitro, all purified F-actin is identical. However, in vivo, the presence of multiple, diverse actin regulatory and binding proteins suggests that all F-actin within cells may not be identical. Typically, F-actin in cells is measured by either NBDphallacidin binding or as cytoskeletal associated actin in Triton-extracted cells. To determine whether the two measures of F-actin in PMNs, NBDphallacidin binding and cytoskeletal associated actin, are equivalent, a qualitative and quantitative comparison of the F-actin in basal, non-adherent endotoxin-free PMNs measured by both techniques was performed. F-actin as NBDphallacidin binding and cytoskeletal associated actin was measured in cells fixed with formaldehyde prior to cell lysis and fluorescent staining (PreFix), or in cells lysed with Triton prior to fixation (PostFix). By both techniques, F-actin in PreFix cells is higher than in PostFix cells (54.25 +/- 3.77 vs. 23.5 +/- 3.7 measured as mean fluorescent channel by NBDphallacidin binding and 70.3 +/- 3.5% vs. 47.2 +/- 3.6% of total cellular actin measured as cytoskeletal associated actin). These results show that in PMNs, Triton exposure releases a labile F-actin pool from basal cells while a stable F-actin pool is resistant to Triton exposure. Further characterizations of the distinct labile and stable F-actin pools utilizing NBDphallacidin binding, ultracentrifugation, and electron microscopy demonstrate the actin released with the labile pool is lost as filament. The subcellular localization of F-actin in the two pools is documented by fluorescent microscopy, while the distribution of the actin regulatory protein gelsolin is characterized by immunoblots with anti-gelsolin. Our studies show that at least two distinct F-actin pools coexist in endotoxin-free, basal PMNs in suspension: 1) a stable F-actin pool which is a minority of total cellular F-actin, Triton insoluble, resistant to depolymerization at 4 degrees C, gelsolin-poor, and localized to submembranous areas of the cell; and 2) a labile F-actin pool which is the majority of total cellular F-actin, Triton soluble, depolymerizes at 4 degrees C, is gelsolin-rich, and distributed diffusely throughout the cell. The results suggest that the two pools may subserve unique cytoskeletal functions within PMNs, and should be carefully considered in efforts to elucidate the mechanisms which regulate actin polymerization and depolymerization in non-muscle cells.     

Ribosome degradation in growing bacteria

Ribosomes are large ribozymes that synthesize all cellular proteins. As protein synthesis is rate-limiting for bacterial growth and ribosomes can comprise a large portion of the cellular mass, elucidation of ribosomal turnover is important to the understanding of cellular physiology. Although ribosomes are widely believed to be stable in growing cells, this has never been rigorously tested, owing to the lack of a suitable experimental system in commonly used bacterial model organisms. Here, we develop an experimental system to directly measure ribosomal stability in Escherichia coli. We show that (i) ribosomes are stable when cells are grown at a constant rate in the exponential phase; (ii) more than half of the ribosomes made during exponential growth are degraded during slowing of culture growth preceding the entry into stationary phase; and (iii) ribosomes are stable for many hours in the stationary phase. Ribosome degradation occurs in growing cultures that contain almost no dead cells and coincides with a reduction of comparable magnitude in the cellular RNA concentration.     

Compartmentalized ATP pools produced from adenosine are nuclear pools

Incubation of African green monkey kidney (BS-C-1) cells and mouse fibroblasts (3T6) in the presence of adenosine for 4 hours resulted in increases in the nuclear compartment pools of adenosine 5'-triphosphate (ATP) and nuclear ATP/adenosine 5'-diphosphate (ADP) ratios. Adenine and inosine, which yield increases in total cellular ATP pools and ATP/ADP ratios similar to those promoted by adenosine, do not produce similar increases in the nuclear compartment. Adenosine-promoted increases in nuclear ATP pools were higher in the untransformed, serially propagated, BS-C-1 cells than in the spontaneously transformed 3T6 cells. Adenosine-promoted compartmentalized ATP pools in primary chick embryo fibroblasts were reduced upon transformation of these cells with Rous sarcoma virus, resulting in free mixing of all of the ATP pools synthesized from various salvage precursors. The growth regulatory properties of the nuclear compartment pools of adenine nucleotides is suggested by the big increases in nuclear ATPase and adenosine 5'-monophosphate (AMP) deaminase activities upon the entry of 3T6 cells into the S phase of their cycle. These enzymatic activities would tend to lower the nuclear ATP/ADP ratios and reduce the total adenine nucleotide pools in these nuclei respectively--conditions which were shown by earlier in vitro studies to be favorable to DNA replication.     

Compartmentation of NAD+-dependent signalling

NAD(+) plays central roles in energy metabolism as redox carrier. Recent research has identified important signalling functions of NAD(+) that involve its consumption. Although NAD(+) is synthesized mainly in the cytosol, nucleus and mitochondria, it has been detected also in vesicular and extracellular compartments. Three protein families that consume NAD(+) in signalling reactions have been characterized on a molecular level: ADP-ribosyltransferases (ARTs), Sirtuins (SIRTs), and NAD(+) glycohydrolases (NADases). Members of these families serve important regulatory functions in various cellular compartments, e.g., by linking the cellular energy state to gene expression in the nucleus, by regulating nitrogen metabolism in mitochondria, and by sensing tissue damage in the extracellular compartment. Distinct NAD(+) pools may be crucial for these processes. Here, we review the current knowledge about the compartmentation and biochemistry of NAD(+)-converting enzymes that control NAD(+) signalling.     

Intracellular labile iron

Cells maintain organellar pools of "labile iron" (LI), despite its propensity for catalyzing the formation of reactive oxygen species. These pools are identifiable by iron-chelating probes and accessible to pharmacological agents. Cytosolic LI has been assumed to have a dual function: providing a rapidly adjustable source of iron for immediate metabolic utilization, and for sensing by iron-regulatory proteins (IRPs) that regulate iron uptake and compartmentalization via transferrin receptors and ferritin. However, it now appears that IRPs may respond both to fluctuations in LI per se and to secondary signals associated with redox-active species. Recent information also indicates that iron can be delivered to mitochondria via pathways that circumvent cytosolic LI, suggesting possible alternative mechanisms of cell iron mobilization and trafficking. We discuss the changing views of intracellular LI pools in relation to iron homeostasis and cellular distribution in physiological and pathological states.     

Regulation of caspase activation and apoptosis by cellular zinc fluxes and zinc deprivation: A review.

Non-toxic agents that target intracellular signalling pathways in apoptosis may have potential therapeutic use in many diseases. One such agent is the transition metal Zn, a dietary cytoprotectant and anti-oxidant, which stimulates cell proliferation and suppresses apoptosis. Zn is maintained in discrete subcellular pools that are critical for the functional and structural integrity of cells. The present review initially describes the current state of knowledge on the cellular biology of Zn, especially the critical free or loosely bound (labile) pools of Zn, which are thought to regulate apoptosis. We then review the evidence relating Zn to apoptosis, including studies from our laboratory showing potent synergy between intracellular Zn deficiency and the short chain fatty acid butyrate in induction of caspase activation and the downstream events of apoptosis. Our studies have also reported the suppressive effects of micromolar concentrations of Zn on caspase-3 activation in cell-free models. Other key issues that will be discussed include the identification of the putative molecular targets of Zn and the evidence that systemic changes in labile Zn levels are sufficient to alter susceptibility to apoptosis and lead to physiopathological changes in the human body. Finally, we propose that labile Zn may serve as a coordinate regulator of mitosis and apoptosis to regulate tissue growth.     

Lysosomal labilization

The lysosomal compartment is the place for cellular degradation of endocytosed and autophagocytosed material and a center for normal turnover of organelles as well as most long-lived proteins. Lysosomes were long considered stable structures that broke and released their many hydrolytic enzymes only following necrotic cell death. It is now realized that lysosomes instead are quite vulnerable, although in a heterogeneous way. Their exposure to a number of events, such as oxidative stress, lysosomotropic detergents and aldhydes, as well as overexpression of the p53 protein, causes time-and-dose-dependent lysosomal rupture that is followed by apoptosis or necrosis. Partial lysosomal rupture has often been found to be an early upstream event in apoptosis, while necrosis results from fulminant lysosomal rupture. Consequently, factors influencing the stability of lysosomes, for instance their content of labile and redox-active iron, seem to be essential for the survival of cells.     

Degrading and stabilizing effects of Mg ions on microtubule-containing axopodia

Although many divalent cations are known to affect the labile microtubules as their stabilizing agents, it was found here that Mg²⁺ ions resulted in not only stabilization but also degradation of heliozoan axopodia composed mainly of the labile microtubules. The concentration range inducing the cytolysis of organisms (C), axopodial degradation (D), and axopodial stabilization (S) was 2 M C 750 mM, 2 M D 12 mM, and 10 mM S 0.07 mM, respectively. At relatively low Mg²⁺ concentration range inducing the axopodial degradation, three characteristic phenomena were observed: (1) the particles within every axopodia ceased from moving up and downwards as a first sign of axopodial degradation; (2) these particles were then accumulated around the tips of degrading axopodia; and finally (3) the distal parts of axopodia were more labile than the proximal ones. These experimental data suggested that the axopodial microtubules played two important roles not only supporting the elongated axopodial extensions but also in transporting the particles up and downwards the axopodia.     

Plant RABs: Role in Development and in Abiotic and Biotic Stress Responses

Endosomal trafficking plays an integral role in various eukaryotic cellular activities and is vital for higher-order functions in multicellular organisms. RAB GTPases are important proteins that influence various aspects of membrane traffic, which consequently influence many cellular functions and responses. Compared to yeast and mammals, plants have evolved a unique set of plant-specific RABs that play a significant role in their development. RABs form the largest family of small guanosine triphosphate (GTP)-binding proteins, and are divided into eight sub-families named RAB1, RAB2, RAB5, RAB6, RAB7, RAB8, RAB11 and RAB18. Recent studies on different species suggest that RAB proteins play crucial roles in intracellular trafficking and cytokinesis, in autophagy, plant microbe interactions and in biotic and abiotic stress responses. This review recaptures and summarizes the roles of RABs in plant cell functions and in enhancing plant survival under stress conditions.     

Accelerating HIV-1 VLP production using stable High Five insect cell pools

Stable cell pools are receiving a renewed interest as a potential alternative system to clonal cell lines. The shorter development timelines and the capacity to achieve high product yields make them an interesting approach for recombinant protein production. In this study, stable High Five cell pools are assessed for the production of a simple protein, mCherry, and the more complex HIV-1 Gag-eGFP virus-like particles (VLPs). Random integration coupled to fluorescence-activated cell sorting (FACS) in suspension conditions is applied to accelerate the stable cell pool generation process and enrich it with high producer cells. This methodology is successfully transferred to a bioreactor for VLP production, resulting in a 2-fold increase in VLP yields with respect to shake flask cultures. In these conditions, maximum viable cell concentration improves by 1.5-fold, and by-product formation is significantly reduced. Remarkably, a global increase in the uptake of amino acids in the Gag-eGFP stable cell pool is observed when compared with parental High Five cells, reflecting the additional metabolic burden associated with VLP production. These results suggest that stable High Five cell pools are a robust and powerful approach to produce VLPs and other recombinant proteins, and put the basis for future studies aiming to scale up this system.     

The contribution of both extracellular and intracellular calcium to the action of alpha-adrenergic agonists in perfused rat liver

The role of both intracellular and extracellular Ca2+ pools in the expression of alpha-adrenergic-agonist-mediated responses was examined in perfused rat liver. Responses studied included glycogenolysis, respiration, lactate and pyruvate formation, ketone-body production, changes in the cytoplasmic and mitochondrial redox ratio and cellular K+ fluxes. Each of these was shown to be dependent on the mobilization of intracellular Ca2+ and can be grouped into one of two response types. Transient responses (ion fluxes and the redox ratios) are obligatorily dependent on the mobilization of intracellular Ca2+ and occur irrespective of the extracellular Ca2+ concentration. Sustained responses, on the other hand, initially require intracellular Ca2+ and, subsequently, extracellular Ca2+. The data indicate that alpha-adrenergic agonists mobilize extracellular Ca2+ as well as intracellular Ca2+ and that both pools are required for the full expression of hormone-induced responses in rat liver.