Extracellular Mg2+ regulates intracellular Mg2+ and its subcellular compartmentation in fission yeast, Schizosaccharomyces pombe

Effects of extracellular magnesium ions ([Mg²⁺]_o ) on intracellular free Mg²⁺ ([Mg²⁺]_i ) and its subcellular distribution in single fission yeast cells, Schizosaccharomyces pombe, were studied with digital-imaging microscopy and an Mg²⁺ fluorescent probe (mag-fura-2). Using 0.44 mM [Mg²⁺]_o , [Mg²⁺]_i in yeast cells was 0.91±0.08 mM. Elevation of [Mg²⁺]_o to 1.97 mM induced rapid (within 5 min) increments in [Mg²⁺]_i (2.18±0.11 mM). Lowering [Mg²⁺]_o to 0.06 mM, however, exerted no significant effects on [Mg²⁺]_i (0.93±0.14 mM), at least for periods of up to 30 min. Irrespective of the [Mg²⁺]_o used, the subcellular distribution of [Mg²⁺]_i remained hetero geneous, i.e. where the sub-plasma membrane region >cytoplasm >nucleus. [Mg²⁺] in all three subcellular compartments increased significantly, two- to threefold, concomitant with [Mg²⁺]_i when placed in 1.97 mM [Mg²⁺]_o . We conclude that [Mg²⁺]_i in fission yeast is maintained at a physiologic level when [Mg²⁺]_o is low, but intracellular free Mg²⁺ rapidly rises when [Mg²⁺]_o is elevated. Like most eukaryotic cells, yeast may have a Mg²⁺ transport system(s) which functions to maintain gradients of Mg²⁺ from the outside to inside the cell and among its subcellular compartments. Received 18 April 1996; received after revision 4 July 1996; accepted 26 July 1996     

Die katalytische Mutation der Ionen Al3+ und MoO

Summary The catalytic mutation of the ions Al³⁺ and MoO ₄ ^2– on the mixed carrier Cd(OH)₂/Co(OH)₂(Tr) is recognizable by the fact that the combination Tr+Al³⁺+MoO ₄ ^2– is more active in the decomposition of H₂O₂ than the combination Tr+MoO ₄ ^2– +Al³⁺.     

Ionic mechanisms in pancreatic β cell signaling

The function and survival of pancreatic β cells critically rely on complex electrical signaling systems composed of a series of ionic events, namely fluxes of K⁺, Na⁺, Ca²⁺ and Cl^? across the β cell membranes. These electrical signaling systems not only sense events occurring in the extracellular space and intracellular milieu of pancreatic islet cells, but also control different β cell activities, most notably glucose-stimulated insulin secretion. Three major ion fluxes including K⁺ efflux through ATP-sensitive K⁺ (K_ATP) channels, the voltage-gated Ca²⁺ (Ca_V) channel-mediated Ca²⁺ influx and K⁺ efflux through voltage-gated K⁺ (K_V) channels operate in the β cell. These ion fluxes set the resting membrane potential and the shape, rate and pattern of firing of action potentials under different metabolic conditions. The K_ATP channel-mediated K⁺ efflux determines the resting membrane potential and keeps the excitability of the β cell at low levels. Ca²⁺ influx through Ca_V1 channels, a major type of β cell Ca_V channels, causes the upstroke or depolarization phase of the action potential and regulates a wide range of β cell functions including the most elementary β cell function, insulin secretion. K⁺ efflux mediated by K_V2.1 delayed rectifier K⁺ channels, a predominant form of β cell K_V channels, brings about the downstroke or repolarization phase of the action potential, which acts as a brake for insulin secretion owing to shutting down the Ca_V channel-mediated Ca²⁺ entry. These three ion channel-mediated ion fluxes are the most important ionic events in β cell signaling. This review concisely discusses various ionic mechanisms in β cell signaling and highlights K_ATP channel-, Ca_V1 channel- and K_V2.1 channel-mediated ion fluxes.     

Sopra un caso particolare della superficie F 4 (3) di M. Noether

Summary The Noetherian surfaceF ₄ ⁽³⁾ , which is represented on a plane by a linear ³ system ofC ₉(A ₁ ³ A ₂ ³ A ₃ ³ A ₄ ³ A ₅ ³ A ₆ ³ A ₇ ³ A ₈ ³ A ₉ ² A ₁₀), possesses generally only one linear pencil of elliptic cubics. IfA _i (i=1, 2, , 9) are the basis points of aHalphen pencil ofC ₉,A ₁₀ is infinitely near toA ₉, and in this caseF ₄ ⁽³⁾ is a not trivial example of such a surface with two pencils of elliptic cubics.     

Cholinesterases are down-expressed in human colorectal carcinoma

The aberrations of cholinesterase (ChE) genes and the variation of ChE activity in cancerous tissues prompted us to investigate the expression of ChEs in colorectal carcinoma. The study of 55 paired specimens of healthy (HG) and cancerous gut (CG) showed that acetylcholinesterase (AChE) activity fell by 32% and butyrylcholinesterase (BuChE) activity by 58% in CG. Abundant AChE-H, fewer AChE-T, and even fewer AChE-R and BuChE mRNAs were observed in HG, and their content was greatly diminished in CG. The high level of the AChE-H mRNA explains the abundance of AChE-H subunits in HG, which as glycosylphosphatidylinositol (GPI)-anchored amphiphilic AChE dimers (G₂^A) and monomers (G₁^A) account for 69% of AChE activity. The identification of AChE-T and BuChE mRNAs justifies the occurrence in gut of A₁₂, G₄^H and PRiMA-containing G₄^A AChE forms, besides G₄^H, G₄^A and G₁^H BuChE. The down-regulation of ChEs might contribute to gut carcinogenesis by increasing acetylcholine availability and overstimulating muscarinic receptors. Received 19 May 2006; received after revision 5 June 2006; accepted 5 July 2006     

Bicarbonate and chloride transport across rat ileal basolateral membrane

The mechanisms of HCO ₃ ^– and Cl^– transport across basolateral membranes from rat ileum were investigated in isolated vesicles by means of uptake experiments. Neither Cl^–/HCO ₃ ^– exchanger nor Na⁺–(HCO ₃ ^– )_n cotransport seem to be present in ileal basolateral membranes. Moreover Cl^– uptake is unaffected bycis Na⁺ and/or K⁺ gradients, indicating the absence of Na⁺–Cl^–, K⁺–Cl^– and Na⁺–K⁺–2Cl^– symport activity. An electrically conductive pathway seems to be responsible for both HCO ₃ ^– and Cl^– fluxes. Evidence is also given for the presence of a Na⁺/H⁺ exchanger at the basolateral pole of ileal enterocytes.     

Photodynamic sensitizers assay: rapid and sensitive iodometric measurement

A rapid, sensitive and simple spectrophotometric method for the detection of¹O₂ produced by photodynamic photosensitizers in slightly acid and air-saturated aqueous solutions has been developed. The method is based on the reaction of¹O₂ (produced by photodynamic processes) with I^– in the presence of ammonium molybdate as a catalyst. The reaction product I₃ ^–, proportional to¹O₂, is followed spectrophotometrically at 355 nm. Several ways of avoiding interference with other oxidizing compounds, either present before or produced during the irradiation, are described.The method could be used to measure the efficiency of water-soluble photodynamic photosensitizers.     

The superoxide-generating NADPH oxidase: structural aspects and activation mechanism

Flavocytochrome b ₅₅₈ is the catalytic core of the respiratory-burst oxidase, an enzyme complex that catalyzes the NADPH-dependent reduction of O₂ into the superoxide anion O₂ ^- in phagocytic cells. Flavocytochrome b ₅₅₈ is anchored in the plasma membrane. It is a heterodimer that consists of a large glycoprotein gp91phox (phox for phagocyte oxidase) (β subunit) and a small protein p22phox (α subunit). The other components of the respiratory-burst oxidase are water-soluble proteins of cytosolic origin, namely p67phox, p47phox, p40phox and Rac. Upon cell stimulation, they assemble with the membrane-bound flavocytochrome b ₅₅₈ which becomes activated and generates O₂ ^-. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections, illustrating the role of O₂ ^- and the derived metabolites H₂O₂ and HOCl in host defense against invading microorganisms. The electron carriers, FAD and hemes b, and the binding site for NADPH are confined to the gp91phox subunit of flavocytochrome b ₅₅₈ . The p22phox subunit serves as a docking site for the cytosolic phox proteins. This review provides an overview of current knowledge on the structural organization of the O₂ ^--generating flavocytochrome b ₅₅₈ , its kinetics, its mechanism of activation and the regulation of its biosynthesis. Homologues of gp91phox, called Nox and Duox, are present in a large variety of non-phagocytic cells. They exhibit modest O₂ ^--generating oxidase activity, and some act as proton channels. Their role in various aspects of signal transduction is currently under investigation and is briefly discussed. Received 28 May 2002; received after revision 20 June 2002; accepted 24 June 2002     

Proinsulin C-peptide and its analogues induce intracellular Ca2+ increases in human renal tubular cells

Based on the findings that proinsulin C-peptide binds specifically to cell membranes, we investigated the effects of C-peptide and related molecules on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in human renal tubular cells using the indicator fura-2/AM. The results show that human C-peptide and its C-terminal pentapeptide (positions 27–31, EGSLQ), but not the des (27–31) C-peptide or randomly scrambled C-peptide, elicit a transient increase in [Ca²⁺]_i. Rat C-peptide and rat C-terminal pentapeptide also induce a [Ca²⁺]_i response in human tubular cells, while a human pentapeptide analogue with Ala at position 1 gives no [Ca²⁺]_i response, and those with Ala at positions 2–5 induce responses with different amplitudes. These results define a species cross-reactivity for C-peptide and demonstrate the importance of Glu at position 1 of the pentapeptide. Preincubation of cells with pertussis toxin abolishes the effect on [Ca²⁺]_i by both C-peptide and the pentapeptide. These results are compatible with previous data on C-peptide binding to cells and activation of Na⁺,K⁺ATPase. Combined, all data show that C-peptide is a bioactive peptide and suggest that it elicits changes in [Ca²⁺]_i via G-protein-coupled pathways, giving downstream enzyme effects. Received 13 May 2002; accepted 16 May 2002     

Mechanical and biochemical characterization of the contraction elicited by a calcium-independent myosin light chain kinase in chemically skinned smooth muscle

Summary The contraction induced by a Ca²⁺-independent myosin light chain kinase (MLCK-) was characterized in terms of isometric force (F_o), immediate elastic recoil (SE), unloaded shortening velocity (V_us), shortening under a constant load and ATPase activity of chemically skinned smooth muscle preparations. These parameters were compared to those measured in a Ca²⁺-induced contraction to assess the nature of cross bridge interaction in the MLCK-induced contraction. F_o developed in chicken gizzard fibers as well as SE were similar in contractions elicited by either agent. V_us in the contraction induced by MLCK-(0.36 mg/ml) was similar though averaged 39.3±8.9% less than V_us induced by Ca²⁺ (1.6x10^–6M) in the control fibers. Addition of Ca²⁺ (1.6x10^–6M) to a contraction induced by MLCK-resulted in small increases in both F_o and V_us. Shortening under a constant load was similar for both types of contractions. The contraction induced by MLCK-was accompanied by an increased rate of ATP hydrolysis. The MLCK-induced contraction is thus kinetically similar though not identical to a contraction induced by Ca²⁺. We conclude that with respect to actin-myosin interaction, MLCK- and Ca²⁺-induced contractions are similar.     

Differences in luteinizing hormone release stimulated in rat anterior pituitary cells by leukotriene C4 and by gonadotropin-releasing hormone in vitro

Summary Continuous administration of leukotriene C₄ (LTC₄, 10^–10 M) to superfused rat anterior pituitary cells increased LH release for 40 min only, whereas in a parallel experiment gonadotropin-releasing hormone (GnRH, 10^–9 M) evoked a continuous increase in hormone secretion. In contrast to GnRH, LTC₄ did not desensitize rat anterior pituitary cells. The secretory action resulting from the administration of LTC₄ (10^–10 M) was abolished for 40 min after previous stimulation. The results documented the dual action of LTC₄ on LH exocytosis.     

IP<Subscript>3</Subscript> receptor signaling and endothelial barrier function

The endothelium, a monolayer of endothelial cells lining vessel walls, maintains tissue-fluid homeostasis by restricting the passage of the plasma proteins and blood cells into the interstitium. The ion Ca²⁺, a ubiquitous secondary messenger, initiates signal transduction events in endothelial cells that is critical to control of vascular tone and endothelial permeability. The ion Ca²⁺ is stored inside the intracellular organelles and released into the cytosol in response to environmental cues. The inositol 1,4,5-trisphosphate (IP₃) messenger facilitates Ca²⁺ release through IP₃ receptors which are Ca²⁺-selective intracellular channels located within the membrane of the endoplasmic reticulum. Binding of IP₃ to the IP₃Rs initiates assembly of IP₃R clusters, a key event responsible for amplification of Ca²⁺ signals in endothelial cells. This review discusses emerging concepts related to architecture and dynamics of IP₃R clusters, and their specific role in propagation of Ca²⁺ signals in endothelial cells.     

Gitterkonstanten und Raumgruppe von Tetrammincuprisulfat

Summary The space-group of Cu(NH₃)₄SO₄·H₂O isD _2h ¹⁶ orD _2h ¹³ (D _2h ⁵ , D _2h ¹ ) with a=7,07, b=12,12, c=10,66 Å ± 1%.     

Developmental changes of aldehyde dehydrogenase isozymes in human livers: Mitochondrial ALDH2 isozyme is expressed in fetal livers

Summary Previous reports suggested that the major cytosolic aldehyde dehydrogenase (ALDH₁) was present in fetal and infant livers, but the major mitochondrial isozyme (ALDH₂) was absent or severely diminished. Re-examination by means of starch gel electrophoresis followed by enzyme activity staining, and by means of dot blot immuno-hybridization of liver samples with known genotypes of theALDH ₂ locus, indicated that bothALDH ₁ andALDH ₂ genes are expressed in fetal and infant livers. In addition, ALDH₄ isozyme was also observed. The results imply that a fetus with the usual homozygousALDH ₂ ¹ /ALDH ₂ ¹ genotype, but not one with the atypicalALDH ₂ ¹ /ALDH ₂ ² orALDH ₂ ² /ALDH ₂ ² , is capable of detoxifying acetaldehyde transferred from the mother.     

Differences in membrane ion transport between hepatocytes from the periportal and the pericentral areas of the liver lobule

We studied the Na⁺/K⁺ pump, Na⁺/K⁺ ATPase activity, and oxygen consumption (QO₂) in hepatocytes isolated from the periportal (PH) and pericentral (CH) regions of the liver lobule, to provide an insight into the functional properties of these cells. Na⁺/K⁺ pump activity was determined using⁸⁶Rb⁺ (a functional analog of K⁺) and ouabain, a specific inhibitor of this transport system. Our results indicate the the Na⁺/K⁺, pump and Na⁺/K⁺ ATPase activity are significantly lower in CH than in PH, although basal ouabain-sensitive (OS) QO₂ was negligible in both of these cell preparations. However, OSQO₂ was significantly lower in CH than in PH when the Na⁺/K⁺ pump was activated using the ionophore nystatin in a Na⁺-containing medium. These results indicate that the differences in membrane ion transport exist between hepatocytes from different locations of the liver lobule.     

On the synthesis of serine and homoserine samples asymmetrically labelled with tritium and deuterium in the hydroxymethylene group

Summary (1 R) [1-³H,²H₁] 3-Phenylpropanol, the key intermediate in the synthesis of (4 R) [4-³H,²H₁] D, L-homoserine and of the (4 S)-isomer, is obtained from (1 S) [1-²H₁] 3-phenylpropanol and (1 RS) [1-³H] ethanol upon incubation with yeast alcohol dehydrogenase and NAD⁺; under similar conditions 2-phenylethanol undergoes very small exchange with [1-²H₂] ethanol.     

Selective blockade of components of potassium activation inMyxicola axons

Summary The K⁺ conductance inMyxicola giant axons activates in two phases which are pharmacologically separable. The fast phase of K⁺ activation is specifically inhibited by 4-aminopyridine and by the substitution of D₂O for H₂O. We suggestMyxicola giant axons, like the amphibian node of Ranvier, may possess more than one variety of K⁺ channel.     

Effects of pCai and pHi on cell-to-cell coupling

Summary Internal longitudinal resistance (r_i), a determinant of cardiac conduction, is affected by changes in intracellular calcium and protons. However, the role and mechanism by which H⁺ and Ca²⁺ may modulate r_i is uncertain. Cable analysis was performed in cardiac Purkinje fibers to measure r_i during various interventions. In some experiments, intracellular pH (pH_i) was recorded simultaneously to study the pH_i-r_i relation. Both intracellular Ca²⁺ and H⁺ independently modified r_i. However, internal resistance of cardiac fibers was insensitive to pH_i changes compared to other tissues. A latent period preceded the pH_i-related changes in r_i and the amount of change depended upon methodology. The results suggest that direct action of protons on r_i may be subordinate to other regulatory processes. Ionic regulation of internal longitudinal resistance may occur by more than one mechanism: i) direct cationic binding to sites on junctional membrane proteins; and ii) H⁺- or Ca²⁺-dependent phosphorylation of junctional proteins.     

Photoreactivation of UV damage in culturedDrosophila cells

Summary Cell survival and photoreactivation of 254 nm ultraviolet (UV) light damage in a wild typeDrosophila cell line was assayed by colony formation in liquid medium. F_o, F_q, and extrapolation number for the exponential portion of survival curves are 21 J/m², 3.6 J/m², and 1.5 for non-photoreactivated cells and 110 J/m², 11.2 J/m², and 1.3 for those exposed to photoreactivating light. Maximal photoreactivation occurs at the 100 J/m² region of the curve. At 10 and 50% survival, 75–80% of the UV damage was photoreactivable.     

Complex modulation of Cav3.1 T-type calcium channel by nickel

Nickel is considered to be a selective blocker of low-voltage-activated T-type calcium channel. Recently, the Ni²⁺-binding site with critical histidine-191 (H191) within the extracellular IS3–IS4 domain of the most Ni²⁺-sensitive Ca_v3.2 T-channel isoform has been identified. All calcium channels are postulated to also have intrapore-binding site limiting maximal current carried by permeating divalent cations (PDC) and determining the blockade by non-permeating ones. However, the contribution of the two sites to the overall Ni²⁺ effect and its dependence on PDC remain uncertain. Here we compared Ni²⁺ action on the wild-type “Ni²⁺-insensitive” Ca_v3.1_w/t channel and Ca_v3.1_Q172H mutant having glutamine (Q) equivalent to H191 of Ca_v3.2 replaced by histidine. Each channel was expressed in Xenopus oocytes, and Ni²⁺ blockade of Ca²⁺, Sr²⁺, or Ba²⁺ currents was assessed by electrophysiology. Inhibition of Ca_v3.1_w/t by Ni²⁺ conformed to two sites binding. Ni²⁺ binding with high-affinity site (IC₅₀ = 0.03–3 μM depending on PDC) produced maximal inhibition of 20–30 % and was voltage-dependent, consistent with its location within the channel’s pore. Most of the inhibition (70–80 %) was produced by Ni²⁺ binding with low-affinity site (IC₅₀ = 240–700 μM). Q172H-mutation mainly affected low-affinity binding (IC₅₀ = 120–160 μM). The IC₅₀ of Ni²⁺ binding with both sites in the Ca_v3.1_w/t and Ca_v3.1_Q172H was differentially modulated by PDC, suggesting a varying degree of competition of Ca²⁺, Sr²⁺, or Ba²⁺ with Ni²⁺. We conclude that differential Ni²⁺-sensitivity of T-channel subtypes is determined only by H-containing external binding sites, which, in the absence of Ni²⁺, may be occupied by PDC, influencing in turn the channel’s permeation.