Isolated cardiac myocytes A new cellular model for studying insulin modulation of monosaccharide transport

The usefulness of isolated Ca²⁺-tolerant myocytes as a cellular model system for investigating modulation of monosaccharide transport by insulin was investigated. We have found that the isolation technique described by Haworth et al. (Haworth, R.A., Hunter, D.R. and Berkoff, H.A. (1980) J. Mol. Cell. Cardiol. 12, 715–724), with some minor modifications, consistently gave the highest yield of quiescent, rod-shaped myocytes which maintained their integrity in the presence of 2 mM calcium. Using 3-O-methylglucose, a non-metabolized sugar, transport was shown to possess saturability, substrate stereospecificity, competition and countertransport; all of which have been thoroughly established for d-glucose transport in other systems. The apparent K_m of transport ranged from 2.3 to 3.5 mM. Insulin (10 nM) caused a small but significant increase in K_m and a 2–3-fold increase in V_max. These results suggest that this myocyte preparation will provide a useful model for studying the transport-related effects of insulin as well as current hypotheses regarding the mechanism of insulin modulation of transport at the cellular level.     

水稻器官干物质运转特性的因子分析

本文对33个水稻品种（组合）5个器官的干物质转运率和移动率（共10个性状）进行了因子分析,结果表明,5个器官的干物质运转特性均可自成主因子,均具有重要作用,主因子1为茎杆运转因子,主因子2为叶片运转因子,主因子3为功能叶片运转因子,主因子4为功能叶外其它叶片运转因子,主因子5为叶鞘运转因子．除主因子1具有较大的方差贡献外,其余主因子方差贡献接近．杂交F1比常规品种具有更大的主因子l得分,部分常规品种也具有较高的主因子l得分,可作为亲本加以利用．     

Tubulin and Neurofilament Proteins Are Transported Differently in Axons of Chicken Motoneurons

SUMMARY 1. We previously showed that actin is transported in an unassembled form with its associated proteins actin depolymerizing factor, cofilin, and profilin. Here we examine the specific activities of radioactively labeled tubulin and neurofilament proteins in subcellular fractions of the chicken sciatic nerve following injection of L-[³⁵S]methionine into the lumbar spinal cord.2. At intervals of 12 and 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Analysis of the fractions by high-resolution two-dimensional gel electrophoresis, immunoblotting, fluorography, and computer densitometry showed that tubulin was transported as a unimodal wave at a slower average rate (2–2.5 mm/day) than actin (4–5 mm/day). Moreover, the specific activity of soluble tubulin was five times that of its particulate form, indicating that tubulin is transported in a dimeric or small oligomeric form and is assembled into stationary microtubules.3. Neurofilament triplet proteins were detected only in the particulate fractions and transported at a slower average rate (1 mm/day) than either actin or tubulin.4. Our results indicate that the tubulin was transported in an unpolymerized form and that the neurofilament proteins were transported in an insoluble, presumably polymerized form.     

The mycorrhizal fungus Paxillus involutus transports magnesium to Norway spruce seedlings. Evidence from stable isotope labeling

Although it is well established that ectomycorrhizas improve the mineral nutrition of forest trees, there has been little evidence that they mediate uptake of divalent cations such as Mg. We grew nonmycorrhizal seedlings and seedlings mycorrhizal with Paxillus involutus Batsch in a sand culture system with two compartments separated by a 45-μm Nylon mesh. Hyphae, but not roots, can penetrate this net. Labeling the compartment only accessible to hyphae with ²⁵Mg showed that hyphae of the ectomycorrhizal fungus Paxillus involutus transported Mg to their host plant. No label was found in nonmycorrhizal control plants. Our data support the idea that ectomycorrhizas are important for the Mg nutrition of forest trees. This revised version was published online in June 2006 with corrections to the Cover Date.     

Muscle palmitate uptake and binding are saturable and inhibited by antibodies to FABPPM

Studies show that uptake of long-chain fatty acids (LCFA) across the plasma membranes (PM) may occur partly via a carrier-mediated process and that the plasma membrane fatty acid-binding protein (FABP_PM) may be a component of this system. To test the hypothesis that FABP_PM is involved in transsarcolemmal transport of LCFA in muscle, we measured palmitate uptake in giant sarcolemmal vesicles and palmitate binding to PM proteins in rat muscles, (1) in the presence of increasing amounts of unbound palmitate and (2) in the absence or presence of antibody to FABP_PM. Both palmitate uptake and binding were found to be saturable functions of the unbound palmitate concentration with calculated V_max values of 10.5 ± 1.2 pmol/mg protein/15 sec and 45.6 ± 2.9 nmol/mg protein/15 min and K_m values of 12.8 ± 3.8 and 18.4 ± 1.8 nmol/L, respectively. The V_max values for both palmitate uptake and binding were significantly decreased by 75-79% in the presence of a polyclonal antibody to the rat hepatic FABP_PM. Antibody inhibition was found to be dose-dependent and specific to LCFA. Glucose uptake was not affected by the presence of the antibody to FABP_PM. Palmitate uptake and binding were also inhibited in the presence of trypsin and phloretin. These results support the hypothesis that transsarcolemmal LCFA transport occurs in part by a carrier-mediated process and that FABP_PM is a component of this process in muscle.     

Basolateral and canalicular transport of xenobiotics in the hepatocyte: A review

The molecular and functional characterization of severalproteins involved in the uptake and excretion of xenobioticsand endogenous compounds in the hepatocyte has been achievedthrough intensive research conducted in the past few years.These studies have lead to the identification of specificmembrane transporters located in the basolateral andcanalicular membrane domains of the hepatocyte. The organicanion-transporting polypeptide (OATP), present in thebasolateral membrane of the hepatocyte, is responsible for thetranslocation of xenobiotics from the sinusoidal space into thehepatocyte. Once inside the cell, unconjugated neutral, anionicand cationic xenobiotics can be secreted into bile by themultidrug-resistance P-glycoprotein 1 (MDR1). Conjugatedxenobiotics (e.g. glucuronides and glutathione conjugates) aresecreted into bile by the canalicular multispecific organicanion transporter (cMOAT). Other transporters play keyphysiological roles, including the basolateral uptake of bilesalts (sodium-taurocholate cotransporter, NTCP) and thesecretion into bile of conjugated and unconjugated bile salts(bile salt export pump, BSEP) and phospholipids (MDR2).Experimental approaches used to investigate the role of thebasolateral and canalicular transporters in the hepatocyte haveincluded both in vivo and in vitro models. Animalmodels lacking canalicular transporters include the`hyperbilirubinemic' rats (Groningen-Yellow (GY), Eisaihyperbilirubinemic (EHB) and TR<sup>-</sup> rats), which aredeficient in the cMOAT protein, and `knock-out' mice, lackingeither the MDR1 or MDR2 transporter. Although no animal modelsare currently available for the study of basolateraltransporters, their function has been conveniently investigatedthrough heterologous expression in Xenopus laevis oocytesand also with basolateral membrane vesicles isolated fromhepatocytes. The total number of basolateral and canaliculartransport proteins present in the hepatocyte is still unknown,but current knowledge indicates that there are at least fourpresent in the basolateral membrane and five in the canaliculardomain. The present review focuses on the current knowledgeabout the most relevant hepatocyte transporters involved in theuptake of foreign and endogenous compounds from the sinusoidalspace and in their active secretion into bile. The first partof the review deals with the basolateral (sinusoidal) transportof organic anions, and the major basolateral transporters (e.g.NTCP, OATP) are described here, both in terms of their knownbiochemistry and physiology. In the second part of the review,the canalicular (apical) transport of organic anions isdiscussed and the biochemistry and physiological role of MDR1,MDR2, cMOAT and BSEP is described in detail. The concludingremarks point out areas of research that need to be addressedin order to answer important questions that still remainunanswered in this important field of study.     

Membrane Transport Generated by the Osmotic and Hydrostatic Pressure. Correlation Relation for Parameters L p, σ, and ω

Standard approach to membrane transport generated by osmotic andhydrostatic pressures, developed by Kedem and Katchalsky, is based onprinciples of thermodynamics of irreversible processes. In this paper wepropose an alternative technique. We derive transport equations from fewfairly natural assumptions and a mechanistic interpretation of the flows.In particular we postulate that a sieve-type membrane permeability isdetermined by the pore sizes and these are random within certain range.Assuming that an individual pore is either permeable or impermeable tosolute molecules, the membrane reflection coefficient depends on the ratioof permeable and impermeable pores. Considering flows through permeableand impermeable pores separately, we derive equations for the total volumeflux, solute flux and the solvent flux across the membrane. Comparing themechanistic equations to the Kedem-Katchalsky equations we find the formereasier to interpret physically. Based on the mechanistic equations we alsoderive a correlation relation for the membrane transport parameters L _p,, and . This relation eliminates the need for experimentaldetermination of all three phenomenological parameters, which in somecases met with considerable difficulties.     

Effect of cystine loading and cystine dimethylester on renal brushborder membrane transport

The effect of loading renal tubule cells with cystine was studied by incubating them with cystine dimethylester. Proline uptake into brushborder membrane vesicles isolated from the cystine loaded cells was not different from that observed into brushborder vesicles isolated from tubules incubated in buffer alone. Incubating brushborder membranes with 2 mM cystine dimethylester for 10 minutes reduced the uptake of proline by 27% after 15 seconds of incubation and by 21% after 60 seconds of incubation. There was no effect after 20 minutes of incubation. Pre-incubating brushborder membrane vesicles with cystine dimethylester had no statistically significant effect on the affinity of priline for the carrier, but did reduce the maximal rate of proline uptake by 49%.     

Anion exchange reactions in bacteria

Bacterial anion exchange now includes both carboxylate-linked reactions, in which there is an antiport of mono- and dicarboxylic acids, and Pi-linked reactions that build on phosphate (Pi) and organic phosphates. To illustrate the general features of this expanding class, this article discussed the biochemistry, physiology, and molecular biology of Pi-linked antiporters that accept glucose 6-phosphate (G6P) as their primary substrate. Kinetic and biochemical analysis suggsts that Pi-linked exchangers have a bifunctional active site that accepts a pair of negative charges. For this reason, exchange stoichiometry moves between the limits of 2:1 and 2:2 to reflect the ratio of mono- and divalent substrates at either membrane surface. This results in a particularly interesting reaction sequencein vivo, where, because cytosolic pH is relatively alkaline, one can expect the asymmetric exchange of two monovalent G6P anions against a single divalent G6P. In this way, an otherwise futile self-exchange of G6P gives a net flux driven (indirectly) by the pH gradient. Despite this biochemical and physiological complexity, Pi-linked carriers resemble all other secondary carriers at a molecular level. Indeed, sequence analysis leads one to infer a common (albeit low resolution) structural theme in which each functional unit has two sets of six trans-membrane helices separated by a central hydrophilic loop. Present examples show that this topology can derive from either a single protein, as is typical in bacteria, or from pairs of identical subunits, as found in mitochondria and chloroplasts. The finding of this common structure should make it possible to build detailed structural models that have implications for all membrane carrier proteins.     

Involvement of the donor tyrosine-D1 (Yd) in Photosystem II electron transport in the green alga, Chlamydobotrys stellata

The light-induced oxidation of the accessory donor tyrosine-D (Y_D) has been studied by measurements of the EPR Signal II_slow at room temperature in the autotrophically and photoheterotrophically cultivated alga Chlamydobotrys stellata. After illumination and dark adaptation, Y_D Signal II_slow was observed only in autotrophic algae, i.e. under conditions of a linear photosynthetic electron transfer from water to NADP⁺. The addition of artificial electron acceptors phenyl-p-benzoquinone (PPQ) or dichloro-p-benzoquinone (DCQ) to the autotrophic cells caused an almost negligible increase of this signal. When photosynthetic electron flow and oxygen evolution were diminished by removal of the carbon source CO₂ and addition of acetate (photoheterotrophy), a pronounced Y_D Signal II_slow was seen only in presence of DCQ or PPQ. Several possibilities are discussed to explain the absence of Y_D Signal II_slow in photoheterotrophic Chl. stellata such as the existence of a cyclic PS II electron flow very effectively reducing P₆₈₀ and thereby preventing the possibility of Y_D oxidation. Artificial electron acceptors withdraw electrons from this cycle thus keeping the primary quinone acceptor, Q_A, oxidized and thereby diminishing the reduction of P₆₈₀ ⁺ by cyclic PSII. This leads to the appearance of the Y_D Signal II_slow also in the photoheterotrophically grown algae.Abbreviations A-band- thermoluminescence band associated with S₂Q_A ^- charge recombination - DCQ- 2,5-dichlorobenzoquinone - D₂- structure protein of Photosystem II - EPR- electron paramagnetic resonance - OEC- oxygen evolving complex - PPQ- phenyl-p-benzoquinone - PS II- Photosystem II - P₆₈₀- reaction center of Photosystem II - Q-band- thermoluminescence band associated with S₂Q_A ^- charge recombination - S_i- oxidation levels of the OEC - Y_D- tyrosine-D accessory donor to P₆₈₀ - Y_Z- tyrosine-Z electron donor to P₆₈₀ Dedicated to Prof. Dr E. Schnepf/Heidelberg.