Foundations of Vectorial Metabolism and Osmochemistry

Chemical transformations, like osmotic translocations, are transport processes when looked at in detail. In chemiosmotic systems, the pathways of specific ligand conduction are spatially orientated through osmoenzymes and porters in which the actions of chemical group, electron and solute transfer occur as vectorial (or higher tensorial order) diffusion processes down gradients of total potential energy that represent real spatially directed fields of force. Thus, it has been possible to describe classical bag-of-enzymes biochemistry as well as membrane biochemistry in terms of transport. But it would not have been possible to explain biological transport in terms of classical transformational biochemistry or chemistry. The recognition of this conceptual asymmetry in favour of transport has seemed to be upsetting to some biochemists and chemists; and they have resisted the shift towards thinking primarily in terms of the vectorial forces and co-linear displacements of ligands in place of their much less informative scalar products that correspond to the conventional scalar energies. Nevertheless, considerable progress has been made in establishing vectorial metabolism and osmochemistry as acceptable biochemical disciplines embracing transport and metabolism, and bioenergetics has been fundamentally transformed as a result.     

Biochemical topology: From vectorial metabolism to morphogenesis

In living cells, many biochemical processes are spatially organized: they have a location, and often a direction, in cellular space. In the hands of Peter Mitchell and Jennifer Moyle, the chemiosmotic formulation of this principle proved to be the key to understanding biological energy transduction and related aspects of cellular physiology. For H. E. Huxley and A. F. Huxley, it provided the basis for unravelling the mechanism of muscle contraction; and vectorial biochemistry continues to reverberate through research on cytoplasmic transport, motility and organization. The spatial deployment of biochemical processes serves here as a point of departure for an inquiry into morphogenesis and self-organization during the apical growth of fungal hyphae.     

Effects of DDT on carbohydrate metabolism and translocation in secale species

An inhibition of photosynthetic electron transport in susceptible rye following treatment with DDT is accompanied by an increase in dry weight of leaves contacting the pesticide due to an accumulation of fructose, glucose, and to lesser extent, sucrose. Several days after treatment over 40% of the dry weight is due to these sugars. The assimilation of ¹⁴CO₂ by leaf segments was decreased as a consequence of DDT treatment, but labelling patterns were similar to those for leaf segments from untreated plants. However, if given a prolonged period in darkness before extraction of assimilates the leaf segments from treated seedlings retained ¹⁴C in sugars and did not show the substantial decrease in extractable soluble material which was characteristic of untreated controls. In DDT-treated seedlings the translocation of metabolites from leaves to roots was severely impaired.     

Algorithms for computational solvent mapping of proteins

Computational mapping methods place molecular probes (small molecules or functional groups) on a protein surface to identify the most favorable binding positions by calculating an interaction potential. We have developed a novel computational mapping program called CS-Map (computational solvent mapping of proteins), which differs from earlier mapping methods in three respects: (i) it initially moves the ligands on the protein surface toward regions with favorable electrostatics and desolvation, (ii) the final scoring potential accounts for desolvation, and (iii) the docked ligand positions are clustered, and the clusters are ranked on the basis of their average free energies. To understand the relative importance of these factors, we developed alternative algorithms that use the DOCK and GRAMM programs for the initial search. Because of the availability of experimental solvent mapping data, lysozyme and thermolysin are considered as test proteins. Both DOCK and GRAMM speed up the initial search, and the combined algorithms yield acceptable mapping results. However, the DOCK-based approaches place the consensus site farther from its experimentally determined position than CS-Map, primarily because of the lack of a solvation term in the initial search. The GRAMM-based program also finds the correct consensus site for thermolysin. We conclude that good sampling is the most important requirement for successful mapping, but accounting for desolvation and clustering of ligand positions also help to reduce the number of false positives.     

Auxin metabolism and transport during gravitropism

Auxins are endogenous, growth-regulating compounds in plants: for decades investigators have hypothesized that plants change their growth rates and patterns in response to environmental signals by changing their transport of, metabolism of, or sensitivity to their endogenous auxins. The Cholodny-Went hypothesis, for example, postulates that plants respond to tropic signals by changing the distribution of free indoleacetic acid within their tissues. This hypothesis was based on data from experiments investigating phototropism and gravitropism in oat ( Avena sativa L.) and maize ( Zea mays L.) coleoptiles. The results of recent experiments support the Cholodny-Went hypothesis for maize coleoptile gravitropism. Recent experiments conducted on the gravitropisms of other developmental stages of grasses, and other species of plants, however, indicate that the Cholodny-Went hypothesis may not adequately describe how all plants respond to gravity.     

Selenium metabolism and bioavailability

Selenium (Se) is at once an essential and toxic nutrient that occurs in both inorganic and organic forms. The biological functions of Se are mediated through at least 13 selenoproteins that contain Se as selenocysteine (Se-cyst). The endogenous synthesis of this amino acid from inorganic Se (selenide Se⁻²) and serine is encoded by a stop codon UGA in mRNA and involves a unique tRNA. Selenium can also substitute for sulfur in methionine to form an analog, selenomethionine (Se-meth), which is the main form of Se found in food. Animals cannot synthesize Se-meth or distinguish it from methionine and as a result it is nonspecifically incorporated into a wide range of Se-containing proteins. The metabolic fate of Se varies according to the form ingested and the overall Se status of an individual. This paper reviews the bioavailability, including absorption, transport, metabolism, storage, and excretion, of the different forms of exogenous and endogenous Se.     

Regulation of redox states of plasma proteins by metabolism and transport of glutathione and related compounds

Glutathione is one of the most abundant naturally occurring thiols in living organisms and is synthesized in its reduced from (GSH). GSH has been known to play a fundamental role in cellular events in different cells and tissues, including protection of organisms against oxidative stress. The two peptide linkages of GSH are sequentially degraded by -glutamyltransferase and peptidases that hydrolyze the cysteinylglycine bond; all these enzymes are localized on the outer surface of cell membranes. The turnover of GSH in animals can be understood on the basis of the following three factors: (1) synthesis of GSH occurs exclusively intracellularly, while its degradation occurs predominantly extracellularly; (2) plasma membranes of many tissues and cells have secretory transport systems for GSH and its derivatives; (3) levels of the transferase, a key enzyme for GSH degradation, differ from one tissue to another. Thus, GSH released from tissues with low transferase activity (such as the liver) must be transferred for its rapid turnover to tissues with high enzyme activity (such as the kidney). Further studies on the states of thiol compounds transported via the circulation should be relevant to the understanding of the full scope and physiological significance of the interorgan cooperation of GSH metabolism. Many enzymes and proteins have free SH and disulfide groups within molecules. Function, stability, and in vivo fate of these macromolecules could be affected significantly by their redox state. Although cells and tissues have enzymic defense mechanisms against oxidative stress, the mechanism by which the homeostasis of the redox state of extracellular compartments (such as plasma, urine, bile, etc.) is maintained remains obscure. Plasma mercaptoalbumin (M-Alb) has 17 disulfide bonds and one free cysteinyl residue (Cys-34). This free thiol group can form mixed disulfides with low-molecular weight compounds, such as GSH and cysteine, to generate nonmercaptoalbumin (NM-Alb). Thus, when titrated by several different thiol reagents, less than 1 mole of free SH group (0.4–0.7) was usually detected per mole albumin. The ratio of M-Alb to NM-Alb in plasma samples varies significantly from one sample to another. Many plasma proteins in nonalbumin fractions also formed mixed disulfides with GSH and cysteine. The extent of mixed disulfide formation and the ratio of M-Alb to NM-Alb appeared to change markedly, depending on the redox state of the organisms. The present paper describes the mode of interorgan metabolism and transport of GSH and related compounds, the mechanism by which the redox state of albumin and other plasma proteins is controlled, and their biological significance in healthy and diseased conditions in normal and analbuminemic mutant rats.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

病毒侵染对转SCMV-CP基因甘蔗光合速率和活性氧代谢的影响

对转SCMV-CP基因甘蔗叶绿素含量、光合参数、活性氧代谢有关的酶活性进行了测定分析,结果表明转基因甘蔗由于外源CP基因的导入,病毒在植株体内无法正常复制繁殖,从而保护了植株细胞免受因病毒侵染造成的伤害。未转基因甘蔗接种后病毒迅速繁殖蔓延,受害叶片对活性氧清除能力的下降,促使有毒物质活性氧的积累,启动膜脂过氧化,造成膜的损伤。     

Glucose transport and metabolism in Gymnodinium breve

Florida's red tide organism, Gymnodinium breve, utilized exogenous glucose in the light for the synthesis of cellular components. Glucose was not taken up in the dark. Kinetic parameters for glucose uptake include a K_FD of 11 μM and a V_max of 1 × 10^?10 mol of glucose taken up/mg cellular protein/hr. Glucose uptake was competitively inhibited by phloridzin (K_i = 40 μM), mannose (K_i = 12O μM), and 2-deoxy-d-glucose (K_i = 190 μM) and non-competitively inhibited by galactose (K_i = 125 μM). Kinetics and inhibition of glucose uptake are consistent with a facilitated diffusion transport system.     

Experimental pitfalls in evaluating vectorial protein secretion in vitro; Sertoli cell secretion of androgen-binding protein and transferrin in two-compartment culture chambers

Summary We examined the influence of various Millipore filter pretreatments on the amounts of androgen-binding protein (ABP) and transferrin (Trf) found in the outer (OC) and inner (IC) compartment of two-compartment Sertoli cell (Sc) cultures. When Sc were cultured on untreated Millipore filters, less than 10% of ABP was found in OC during 3 initial culture days compared to similar cultures on pretreated filters. Most of the glycoprotein was shown to be bound by the filters. Pretreatment of Millipore filters with 5% bovine serum albumin (BSA) or 2% fetal bovine serum (FBS) maximally saturated the nonspecific protein-binding sites resulting in OC:IC ratio of ABP similar to that found in cultures on polycarbonate membranes, which exhibit very low protein-binding capacity. In contrast to ABP, about 40% of Trf was bound by the Millipore filter on Day 1, with only trace amounts bound thereafer. These differences were due to much higher secretion rate of Trf than ABP, resulting in a relatively smaller fraction of Trf bound to the filter. Again, the nonspecific binding of Trf was greatly reduced by filter pretreatment with 5% BSA or 2% FBS. It is concluded that complete saturation of protein-binding sites of cellulose ester supports is necessary for reliable evaluation of vectorial protein secretion by Sc and other polarized epithelial cells maintained in this type of culture. The implications of partial saturation of protein-binding sites of culture support in interpreting experimental results are discussed. This work was supported in part by grant HD-17802 (A.S.) from the National Institutes of Health, Bethesda, MD.     

Differential expression of genes related to glucose metabolism in domesticated pigs and wild boar

Glucose metabolism is a basic biological process that shows substantial variation within and between species. Using pig as a model organism, we investigated differences in glucose metabolic genes in seven tissues from domesticated pigs (Rongchang pig and Tibetan pig, meanwhile, the Tibetan pig just as a special case of the domesticated pig under plateau condition) and wild boar. We found large differences in the expression of genes involved in multiple aspects of glucose metabolism, including genes associated with glucose transport, gluconeogenesis, and glycolysis. In addition, we identified microRNAs (miRNAs) that may be involved in the divergence of glucose metabolism in pig. A combined analysis of mRNA and miRNA expression indicated that some miRNA:mRNA pairs showed ab facto function in it. Our results provide a valuable resource for further determination of miRNA regulatory roles in pig glucose metabolism and reveal the divergence of glucose metabolism in pigs under domestication.     

砷胁迫对小麦根系生长及活性氧代谢的影响

在水培和盆载条件下研究了砷对小麦根系生长及活性氧代谢的影响。结果表明：随砷浓度的提高,小麦胚根、次生根条数减少,总根长度、胚芽长度缩短、根体积、干重也较对照冯少超氧物歧化酶（ＳＯＤ）活性下降趋势,质脂过氧化产物（ＭＤＡ）含理增加,质膜相对透性增大、泪科越冬期,拔节期、灌浆期表现出同样的趋势。     

Transport,metabolism and distribution space of octanoate in the perfused rat liver

The scope of the present work was to investigate the metabolism and the passage of octanoate from albumin into the phospholipid bilayer of the plasma membrane and from thence into the cell space. The experiments were done in the isolated perfused rat liver with infusions of albumin and octanoate at various concentrations. Once steady-state conditions were attained, trace amounts of [1-¹⁴C]-octanoate, [¹³¹I]-albumin and [³H]-water were injected simultaneously and the effluent perfusate was fractionated. The normalized dilution curves were used for model analysis. The model which gives the best fit to the experimental results and which also produces the most consistent parameters is one that presupposes a rapid distribution of octanoate into the cell membrane and a slow transfer from the cell membrane into the cytosol. The concentration dependence of the distribution between the membrane and the extracellular space is parabolic, suggesting that octanoate changes the properties of the cell membrane when present at higher concentrations. The passage from the cell membrane into the cell space is relatively slow and limits metabolic transformation partly or totally, depending on the octanoate concentration in the plasma membrane. The rapid transfer of octanoate from the albumin space into the plasma membrane corroborates previous measurements of the dissociation of the albumin–octanoate complex. © 1997 John Wiley & Sons, Ltd.     

Alanine metabolism, transport, and cycling in the brain

Brain glutamate/glutamine cycling is incomplete without return of ammonia to glial cells. Previous studies suggest that alanine is an important carrier for ammonia transfer. In this study, we investigated alanine transport and metabolism in Guinea pig brain cortical tissue slices and prisms, in primary cultures of neurons and astrocytes, and in synaptosomes. Alanine uptake into astrocytes was largely mediated by system L isoform LAT2, whereas alanine uptake into neurons was mediated by Na⁺-dependent transporters with properties similar to system B⁰ isoform B⁰AT2. To investigate the role of alanine transport in metabolism, its uptake was inhibited in cortical tissue slices under depolarizing conditions using the system L transport inhibitors 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and cycloleucine (1-aminocyclopentanecarboxylic acid; cLeu). The results indicated that alanine cycling occurs subsequent to glutamate/glutamine cycling and that a significant proportion of cycling occurs via amino acid transport system L. Our results show that system L isoform LAT2 is critical for alanine uptake into astrocytes. However, alanine does not provide any significant carbon for energy or neurotransmitter metabolism under the conditions studied.     

铜污染对天蓝苜蓿幼苗生长及活性氧代谢的影响

通过盆栽实验研究了重金属铜(Cu)污染对天蓝苜蓿(Medicago lupulinaL.)幼苗的生长及活性氧代谢系统的影响。结果表明,低Cu污染(<500 mg.kg-1)对天蓝苜蓿幼苗生长无明显抑制现象,甚至还具有一定的促进作用,电导率略微升高,而植株鲜重、干重、叶片可溶性蛋白质含量及叶片色素含量均在500 mg.kg-1处理浓度时达到峰值。同时,丙二醛(MDA)水平降低,活性氧清除系统内超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性均略微升高,保护酶系统仍保持平衡。但随Cu浓度继续增加(500—3 000 mg.kg-1)则显示对幼苗生长的一定的负效应,与对照组相比,植株鲜重、干重和可溶性蛋白质含量均显著下降,叶片电导率明显增大,MDA水平上升,且SOD和CAT活性显著下降(分别下降了19.14%和20.81%),而POD活性却明显上升(比对照上升了2.01倍),表明活性氧清除系统遭到破坏,保护酶系统失衡。     

Complexity of vitamin E metabolism

Bioavailability of vitamin E is influenced by several factors, most are highlighted in this review. While gender, age and genetic constitution influence vitamin E bioavailability but cannot be modified, life-style and intake of vitamin E can be. Numerous factors must be taken into account however, i.e., when vitamin E is orally administrated, the food matrix may contain competing nutrients. The complex metabolic processes comprise intestinal absorption, vascular transport, hepatic sorting by intracellular binding proteins, such as the significant α-tocopherol-transfer protein, and hepatic metabolism. The coordinated changes involved in the hepatic metabolism of vitamin E provide an effective physiological pathway to protect tissues against the excessive accumulation of, in particular, non-α-tocopherol forms. Metabolism of vitamin E begins with one cycle of CYP4F2/CYP3A4-dependent ω-hydroxylation followed by five cycles of subsequent β-oxidation, and forms the water-soluble end-product carboxyethylhydroxychroman. All known hepatic metabolites can be conjugated and are excreted, depending on the length of their sidechain, either via urine or feces. The physiological handling of vitamin E underlies kinetics which vary between the different vitamin E forms. Here, saturation of the side-chain and also substitution of the chromanol ring system are important. Most of the metabolic reactions and processes that are involved with vitamin E are also shared by other fat soluble vitamins. Influencing interactions with other nutrients such as vitamin K or pharmaceuticals are also covered by this review. All these processes modulate the formation of vitamin E metabolites and their concentrations in tissues and body fluids. Differences in metabolism might be responsible for the discrepancies that have been observed in studies performed in vivo and in vitro using vitamin E as a supplement or nutrient. To evaluate individual vitamin E status, the analytical procedures used for detecting and quantifying vitamin E and its metabolites are crucial. The latest methods in analytics are presented.     

The mechanism of coupling chemical and physical reactions by the calcium ATPase of sarcoplasmic reticulum and other coupled vectorial systems

The coupling of the chemical reaction of ATP hydrolysis to the transport of calcium from the cytoplasm into the lumen of sarcoplasmic reticulum vesicles can be defined by a set of rules that define alternating changes in the specificities of the enzyme for catalysis of chemical and physical reactions.     

Movement to bark and metabolism of xylem cytokinins in stems of Lupinus angustifolius

Following uptake of [(3)H]zeatin riboside and [(3)H]dihydrozeatin riboside by girdled lupin (Lupinus angustifolius L.) stems via the transpiration stream, rapid lateral movement of the radioactivity from xylem to bark was observed. Short-term studies with intact stems, and other studies with excised stem tissues, revealed that the ribosides and/or the corresponding nucleotides were the cytokinin forms which actually moved into the bark tissues. Relative to cytokinin metabolism in xylem plus pith, metabolism in bark was both more rapid and more complex. Riboside cleavage and formation of the O-acetylzeatin and O-acetyldihydrozeatin ribosides and nucleotides were almost completely confined to bark tissues. Exogenous (3)H-labelled O-acetylzeatin riboside was converted to zeatin riboside in bark tissue, but the presence of the acetyl group suppressed degradation to adenine metabolites. The sequestration and modification of xylem cytokinins by stem tissues probably contributes significantly to the cytokinin status of the shoot. New cytokinins identified by mass spectrometry in lupin were: O-acetyldihydrozeatin 9-riboside, a metabolite of exogenous dihydrozeatin riboside in stem bark; O-methylzeatin nucleotide and O-methyldihydrozeatin 9-riboside, metabolites of endogenous cytokinins in stem bark; O-methylzeatin nucleotide and O-methylzeatin 9-riboside, metabolites of exogenous zeatin riboside in excised pod walls.     

Tracking metabolism and imaging transport in arbuscular mycorrhizal fungi. Metabolism and transport in AM fungi

In the last few years the application of modern techniques to the study of arbuscular mycorrhizas has greatly increased our understanding of the mechanisms underlying carbon metabolism in these mutualistic symbioses. Arbuscular mycorrhizal (AM) monoxenic cultures, nuclear magnetic resonance spectroscopy together with isotopic labeling, and analyses of expressed sequence tags (ESTs) have shed light on the metabolic processes taking place in these interactions, particularly in the case of the mycobiont. More recently, in vivo multiphoton microscopy has provided us with some new insights in the allocation and translocation processes which play crucial roles in the distribution of host plant-derived C throughout the fungal colony. In this mini-review we highlight recent advances in these fields, with special attention to the visualization of oleosomes (i.e., lipid bodies) as they move along the long, coenocytic AM fungal hyphae. Volumetric measurements of such oleosomes have allowed us to estimate the flux of triacylglycerides from the intraradical to the extraradical phase of the AM fungal colony. We raise questions and postulate regulatory mechanisms for C metabolism and translocation within the arbuscular mycorrhizal fungal colony.     

Evaluation of cellular purine transport and metabolism in the Caco-2 cell using comprehensive high-performance liquid chromatography method for analysis of purines

ABSTRACT

Using Caco-2 cells and our previously developed high-performance liquid chromatography method for quantification of purine bases, nucleosides, and nucleotides, we evaluated cellular purine transport and uptake. The analytes were separated using YMC-Triart C18 column with gradient elution. We used Caco-2 cells as intestinal model cells and monitored purine transport across a monolayer for 2 h. The degree of change of purine concentrations in the permeate was very slight; however, it was possible to simultaneously determine these parameters for all purines because of our method's high sensitivity. In the present study, the purine bases (adenine, guanine, hypoxanthine, and xanthine) showed a relatively high permeability as compared with the nucleosides (adenosine, guanosine, inosine, and xanthosine). Increased concentration of metabolites in the permeate was also observed following the addition of purines. In a cell uptake assay, both the cell culture medium (extracellular) and the cells extracted from Caco-2 with acetonitrile:water (7:3) (intracellular) were measured. The additional nucleoside did not increase significantly within the cells. On the other hand, we observed that nucleotide, such as ATP, increased in the cell in a time-dependent manner following the addition of nucleoside. The additional nucleosides were considered to be rather recycled via the salvage pathway than metabolized to purine bases and/or uric acid in the cell. Such differences might have affected the increase in the serum uric acid levels depending on purine form.