Modelling of parameters for optimization of maturity in composting trimming residues

In this paper, changes in physico-chemical parameters during trimmings residue composting (cation exchange capacity, germination index, self-heated, NH₄/NO₃ ratio and C_FA/C_HA ratio) in relation to environmental composting parameters (time, aeration, moisture and particle size) of the composting process were studied. A central composite experimental design was used to obtain the polynomial model for each dependent variable. Results of the modelling showed that among the studied range, moisture was the highest influenced parameter in maturity evaluation, with respect to aeration and particle size. An exception was found for CEC evolution. In this parameter, the highest influence was found for particle size. Moreover, a product with acceptable chemical properties entails operating at medium moisture content (55%) and medium-to-high particle size (3–5 cm). Moderate to low aeration (0.2 m³ air kg⁻¹ d⁻¹) would be the best compromise to composting this residue, due to the scarce statistical influence of this independent variable.     

Derivatives of rhodamine 19 as mild mitochondria-targeted cationic uncouplers

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C(12)R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H(+) ions was generated in the presence of C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C(12)R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.     

Keeping It Simple,Transport Mechanism and pH Regulation in Na+/H+ Exchangers

Na⁺/H⁺ exchangers are essential for regulation of intracellular proton and sodium concentrations in all living organisms. We examined and experimentally verified a kinetic model for Na⁺/H⁺ exchangers, where a single binding site is alternatively occupied by Na⁺ or one or two H⁺ ions. The proposed transport mechanism inherently down-regulates Na⁺/H⁺ exchangers at extreme pH, preventing excessive cytoplasmic acidification or alkalinization. As an experimental test system we present the first electrophysiological investigation of an electroneutral Na⁺/H⁺ exchanger, NhaP1 from Methanocaldococcus jannaschii (MjNhaP1), a close homologue of the medically important eukaryotic NHE Na⁺/H⁺ exchangers. The kinetic model describes the experimentally observed substrate dependences of MjNhaP1, and the transport mechanism explains alkaline down-regulation of MjNhaP1. Because this model also accounts for acidic down-regulation of the electrogenic NhaA Na⁺/H⁺ exchanger from Escherichia coli (EcNhaA, shown in a previous publication) we conclude that it applies generally to all Na⁺/H⁺ exchangers, electrogenic as well as electroneutral, and elegantly explains their pH regulation. Furthermore, the electrophysiological analysis allows insight into the electrostatic structure of the translocation complex in electroneutral and electrogenic Na⁺/H⁺ exchangers.     

A Simple, Sensitive, and Economic Assay for Choline and Acetylcholine Using HPLC, an Enzyme Reactor, and an Electrochemical Detector

A simple, efficient, economic, and sensitive method is presented for the detection of choline and acetylcholine in neuronal tissue using HPLC, a postcolumn enzyme reactor with immobilized enzyme, and electrochemical detection. The method is based on a separation of choline and acetylcholine by cation exchange HPLC followed by passage of the effluent through a postcolumn reactor containing a mixture of acetylcholinesterase and choline oxidase; the latter enzyme converts choline to betaine and hydrogen peroxide, the former enzyme hydrolyzes acetylcholine to acetate and choline. The hydrogen peroxide produced is electrochemically detected. A simple and efficient preparation of neuronal tissue is described using an optional prepurification step on Sephadex G-10 columns, offering the possibility to detect choline and acetylcholine as well as catecholamines and their related metabolites in the same tissue sample. The sensitivity of the assay system is 250 fmol for choline and 500 fmol for acetylcholine.     

Structural features of cation transport ATPases

Several cation transport ATPases, sharing the common feature of a phosphorylated intermediate in the process of ATP utilization, are compared with respect to their subunit composition and amino acid sequence. The main component of these enzymes is a polypeptide chain of MW slightly exceeding 100,000, comprising an extramembranous globular head which is connected through a stalk to a membrane-bound region. With reference to the Ca²⁺ ATPase of sarcoplasmic reticulum, it is proposed that the catalytic (ATP binding and phosphorylation) domain resides in the extramembranous globular head, while cation binding occurs in the membrane region. Therefore, these two functional domains are separated by a distance of approximately 50 Å. Alignment of amino acid sequences reveals extensive homology in the isoforms of the same ATPases, but relatively little homology in different cation ATPases. On the other hand, all cation ATPases considered in this analysis retain a consensus sequence of high homology, spanning the distance between the phosphorylation site and the preceding transmembrane helix. It is proposed that this sequence provides long-range functional linkage between catalytic and cation-binding domains. Thereby, translocation of bound cation occurs through a channel formed by transmembrane helices linked to the phosphorylation site. Additional sequences at the carboxyl terminal provide regulatory domains in certain ATPases.     

Ion Channel Permeable for Divalent and Monovalent Cations in Native Spinach Thylakoid Membranes

A cation-selective channel was characterized in isolated patches from osmotically swollen thylakoids of spinach (Spinacea oleracea). This channel was permeable for K⁺ as well as for Mg²⁺ and Ca²⁺ but not for Cl⁻. When K⁺ was the main permeant ion (symmetrical 105 mm KCl) the conductance of the channel was about 60 pS. The single channel conductance for different cations followed a sequence K⁺ > Mg²⁺≥ Ca²⁺. The permeabilities determined by reversal potential measurements were comparable for K⁺, Ca²⁺, and Mg²⁺. The cation channel displayed bursting behavior. The total open probability of the channel increased at more positive membrane potentials. Kinetic analysis demonstrated that voltage dependence of the total open probability was determined by the probability of bursts formation while the probability to find the channel in open state within a burst of activity was hardly voltage-dependent. The cation permeability of intact spinach thylakoids can be explained on the single channel level by the data presented here. Received: 26 December 1995/Revised: 17 April 1996     

Theczc operon ofAlcaligenes eutrophus CH34: from resistance mechanism to the removal of heavy metals

Summary The plasmid-borneczc operon ensures for resistance to Cd²⁺, Zn²⁺ and Co²⁺ ions through a tricomponent export pathway and is associated to various conjugative plasmids ofA. eutrophus strains isolated from metal-contaminated industrial areas. Theczc region of pMOL30 was reassessed especially for the segments located upstream and downstream the structural genesczc CBA. In cultures grown with high concentrations of heavy metals,czc-mediated efflux of cations is followed by a process of metal bioprecipitation. These observations led to the development of bioreactors designed for the removal of heavy metals from polluted effluents.     

Large-Conductance Cation Channels in the Envelope of Nuclei from Rat Cerebral Cortex

Eucaryotic nuclei are surrounded by a double-membrane system enclosing a central cisterna which is continuous with the endoplasmic reticulum and serves as a calcium store for intracellular signaling. The envelope regulates protein and nucleic acid traffic between the nucleus and the cytoplasm via nuclear pores. These protein tunnels cross through both nuclear membranes and are permeable for large molecules. Surprisingly, patch clamp recordings from isolated nuclei of different cell species have revealed a high resistance of the envelope, enabling tight seals and the resolution of single ion channel activity. Here we present for the first time single-channel recordings from nuclei prepared from neuronal tissue. Nuclei isolated from rat cerebral cortex displayed spontaneous long-lasting large conductances in the nucleus-attached mode as well as in excised patches. The open times are in the range of seconds and channel activity increases with depolarization. The single-channel conductance in symmetrical K⁺ is 166 pS. The channels are selective for cations with P _K/P _Na= 2. They are neither permeable to, nor gated by Ca²⁺. Thus, neuronal tissue nuclei contain a large conductance ion channel selective for monovalent cations which may contribute to ionic homeostasis in the complex compartments surrounding these organelles. Received: 12 November 1996/Revised: 18 February 1997     

Red Blood Cells of a Transgenic Mouse Expressing High Levels of Human Hemoglobin S Exhibit Deoxy-stimulated Cation Flux

+ and Na⁺ transport in RBCs from control mice (C57Bl/6J) and a transgenic (α^Hβ^S[β^MDD]) mouse line that expresses high levels of human α^H and β^S-chains and has a small percent dense cells but does not exhibit anemia. In transgenic mouse RBCs (n= 5) under oxygenated conditions, K⁺ efflux was 0.22 ± 0.01 mmol/L cell × min and Na⁺ influx was 0.17 ± 0.02 mmol/L cell × min. Both fluxes were stimulated by 10 min deoxygenation in transgenic but not in control mice. The deoxy-stimulated K⁺ efflux from transgenic mouse RBCs was about 55% inhibited by 5 nm charybdotoxin (CTX), a blocker of the calcium activated K⁺-channel. To compare the fluxes between human and mouse RBCs, we measured the area of mouse RBCs and normalized values to area per liter of cells. The deoxy-simulated CTX-sensitive K⁺ efflux was larger than the CTX-sensitive K⁺ efflux observed in RBCs from SS patients. These results suggest that in transgenic mice, deoxygenation increases cytosolic Ca²⁺ to levels which open Ca²⁺-activated K⁺ channels. The presence of these channels was confirmed in both control and transgenic mice by clamping intracellular Ca²⁺ at 10 μm with the ionophore A23187 and measuring Ca²⁺-activated K⁺ efflux. Both types of mouse had similar maximal rates of CTX-sensitive, Ca²⁺-activated K⁺ efflux that were similar to those in human SS cells. The capacity of the mouse red cell membrane to regulate cytosolic Ca²⁺ levels was examined by measurements of the maximal rate of calmodulin activated Ca²⁺-ATPase activity. This activity was 3-fold greater than that observed in human RBCs thus indicating that mouse RBC membranes have more capacity to regulate cytosolic Ca²⁺ levels. In summary, transgenic mouse RBCs exhibit larger values of deoxy-stimulated K⁺ efflux and Na⁺ influx when compared to human SS cells. They have a similar Ca²⁺-activated K⁺ channel activity to human SS cells while expressing a very high Ca²⁺ pump activity. These properties may contribute to the smaller percent of very dense cells and to the lack of adult anemia in this animal model. Received: 23 October/Revised: 15 May 1997