[Hindfoot valgus. Diagnosis and therapy of flatfoot]. / Rückfußvalgus. Diagnose und Therapie des Knick-Senkfußes.

The clinical finding of flatfoot is characterized by a flattening of the medial longitudinal arch and valgus deformity of the hindfoot. The differential diagnosis of flatfoot is the physiological, flexible, contracted flatfoot, which occurs as a congenital or acquired deformity. Congenital flatfoot deformity requires early intensive therapy, while a flexible flatfoot in children has a good prognosis and conservative treatment usually leads to a stable and sufficient load-bearing foot. Severe flatfoot in children can be corrected successfully by simple, minimally invasive procedures. In adults with symptomatic flatfoot, which usually occurs due to an insufficiency of the tendon of the tibialis posterior, conservative therapy with insoles, shoe modifications and physiotherapeutic measures can lead to significant improvement, otherwise surgical correction is recommended. Early, stage-appropriate therapy helps to prevent an impending decompensation of the hindfoot.

Mouse germline restriction of Oct4 expression by germ cell nuclear factor.

The POU-domain transcription factor Oct4 is essential for the maintenance of the mammalian germline. In this study, we show that the germ cell nuclear factor (GCNF), an orphan nuclear receptor, represses Oct4 gene activity by specifically binding within the proximal promoter. GCNF expression inversely correlates with Oct4 expression in differentiating embryonal cells. GCNF overexpression in embryonal cells represses Oct4 gene and transgene activities, and we establish a link to transcriptional corepressors mediating repression by GCNF. In GCNF-deficient mouse embryos, Oct4 expression is no longer restricted to the germ cell lineage after gastrulation. Our studies suggest that GCNF is critical in repressing Oct4 gene activity as pluripotent stem cells differentiate and in confining Oct4 expression to the germline.

Luteolin protects rat PC12 and C6 cells against MPP+ induced toxicity via an ERK dependent Keap1-Nrf2-ARE pathway.

Oxidative stress is central to neuronal damage in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. In consequence, activation of the cerebral oxidative stress defence is considered as a promising strategy of therapeutic intervention. Here we demonstrate that the flavone luteolin confers neuroprotection against oxidative stress via activation of the nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor central to the maintenance of the cellular redox homeostasis. Luteolin protects rat neural PC12 and glial C6 cells from N-methyl-4-phenyl-pyridinium (MPP+) induced toxicity in vitro and effectively activates Nrf2 as shown by ARE-reporter gene assays. This protection critically depends on the activation of Nrf2 since downregulation of Nrf2 by shRNA completely abrogates the protection of luteolin in vitro. Furthermore, the neuroprotective effect of luteolin is abolished by the inhibition of the luteolin-induced ERK1/2-activation. Our results highlight the relevance of Nrf2 for neural cell survival conferred by flavones. In particular, we identified luteolin as a promising lead for the search of orally available, blood brain barrier permeable compounds to support the therapy of neurodegenerative disorders.

Cdc25A phosphatase suppresses apoptosis induced by serum deprivation.

The phosphatase Cdc25A was shown to be a target of the transcription factor c-Myc. Myc-induced apoptosis appeared dependent on Cdc25A expression and Cdc25A over-expression could substitute for Myc-triggered apoptosis. These findings suggested that an important downstream component of Myc-mediated apoptosis was identified. However and in contrast, we recently reported that during TNFalpha-induced apoptosis, which required c-Myc function, Cdc25A was down-regulated in a human carcinoma cell line. We now provide evidence that Cdc25A rendered the non-transformed rat embryonic cell line 423 refractory to apoptosis, which was induced by serum deprivation and in absence of detectable c-myc levels. The survival promoting activity of cdc25A was abolished upon infection of cells with a full-length cdc25A antisense construct. To identify the signaling proteins mediating the survival function of the phosphatase, cdc25A- and akt- over-expressing pooled clones were exposed to selected chemicals, which inhibit or activate key proteins in signaling pathways. Inhibition of apoptosis by SU4984, NF023 and Rapamycin placed Cdc25A and Akt function downstream of FGF.R, PDGF.R, and compensated G-protein- and PP2A- activity. Interestingly, upon treatment with LY-294002, cdc25A- and akt- over-expressing clones exhibited similar apoptotic patterns as control cells, which indicates that neither Akt- nor Cdc25A-mediated survival functions are dependent on PI.3 kinase activity in rat 423 cells. In cdc25A-overexpressing cells increased levels of serine 473 phosphorylated Akt were found, which co-precipitated with Cdc25A and Raf1. Since activation of proteins requires dephosphorylation of particular residues in addition to site-specific phosphorylation, the anti-apoptotic effect of Cdc25A might derive from its participation in a multimeric protein complex with phosphoAkt and Raf1, two prominent components of survival pathways.

Distributed and partially separate pools of neurons are correlated with two different components of the gill-withdrawal reflex in Aplysia.

We compared the spike activity of individual neurons in the Aplysia abdominal ganglion with the movement of the gill during the gill-withdrawal reflex. We discriminated four populations that collectively encompass approximately half of the active neurons in the ganglion: (1) second-order sensory neurons that respond to the onset and offset of stimulation of the gill and are active before the movement starts; (2) neurons whose activity is correlated with the position of the gill and typically have a tonic output during gill withdrawal; (3) neurons whose activity is correlated with the velocity of the movement and typically fire in a phasic manner; and (4) neurons whose activity is correlated with both position and velocity. A reliable prediction of the position of the gill is achieved only with the combined output of 15-20 neurons, whereas a reliable prediction of the velocity depends on the combined output of 40 or more cells.

Misuse of graphical analysis in nonlinear sugar transport kinetics by Eadie-Hofstee plots.

It has become common practice to analyse the sugar transport kinetics from initial uptake rates in Saccharomyces cerevisiae cells with Eadie-Hofstee plots. These plots often demonstrate a nonlinear behaviour. They have been resolved incorrectly into two quasilinear components indicating the presence of (at least) two uptake systems or components, with Km values differing by a factor of about 10. This graphical analysis neglects the obvious additivity of the two hypothetical systems and is therefore in error. A more efficient way to determine kinetic parameters from initial uptake experiments is to use computer-assisted nonlinear regression analysis.

Plasma membrane from Candida tropicalis grown on glucose or hexadecane. II. Biochemical properties and substrate-induced alterations.

Isolated plasma membranes from the yeast Candida tropicalis grown on two different carbon sources (glucose or hexadecane), had similar contents of protein (60% of total dry weight), lipid (21-24%) and carbohydrates (16-21%). Sodium dodecyl sulphate gel electrophoresis of the membrane proteins revealed 17 and 19 protein bands, respectively, for glucose and hexadecane grown cells. There were marked differences in RF values and relative peak heights between the two gels. Sterols and free fatty acids were the major components of the plasma membrane lipids. Phospholipid content was less than 2% of total plasma membrane lipids. Membrane microviscosity, as determined by fluorescence polarization, was very high (16.6 P). Fatty acid determination of membrane lipids by gas chromatography showed a significant increase of C16 fatty acids in plasma membranes of cells grown on hexadecane. Reduced-oxidized difference spectra demonstrated the presence of a b-type cytochrome in both Saccharomyces cerevisiae and C. tropicalis plasma membranes. Its concentration in C. tropicalis plasma membranes was three-fold greater in cells grown on hexadecane than in glucose grown cells.

Vanadium uptake by yeast cells.

During incubation with vanadyl, Saccharomyces cerevisiae yeast cells were able to accumulate millimolar concentrations of this divalent cation within an intracellular compartment. The intracellular vanadyl ions were bound to low molecular weight substances. This was indicated by the isotropic nature of the electron paramagnetic resonance (EPR) spectra of the respective samples. Accumulation of intracellular vanadyl was dependent on presence of glucose during incubation. It could be inhibited by various di- and trivalent metal cations. Of these cations lanthanum displayed the strongest inhibitory action. If yeast cells were exposed to more than 50 microM vanadyl sulfate at a pH higher than 4.0, a potassium loss into the medium was detected. The magnitude of this potassium loss suggests a damage of the plasma membrane caused by vanadyl. Upon addition of vanadate to yeast cells surface-bound vanadyl was detectable after several minutes by EPR. This could be the consequence of extracellular reduction of vanadate to vanadyl. The reduction was followed by a slow accumulation of intracellular vanadium, which could be inhibited by lanthanum or phosphate. Therefore, permeation of vanadyl into the cells can be assumed as one mechanism of vanadium accumulation by yeast during incubation with vanadate.

Phloretin keto-enol tautomerism and inhibition of glucose transport in human erythrocytes (including effects of phloretin on anion transport).

Under various pH conditions phloretin demonstrates keto-enol tautomerism with a pK value of 7.26 +/- 0.06. As Wilbrandt has shown ((1950) Arch. Exp. Pathol. Pharmacol. 212, 9-29) phloretin added to erythrocytes inhibits glucose efflux, but not glucose influx. At pH 6.5 a Ki value of 0.36 and at pH 9 of 22.7 microM was measured; only the ketonic form of phloretin contributes to the inhibition of glucose efflux. This was also the case for inhibition of galactose efflux and anion exchange. The geometry optimization of a large number of conformations of the ketonic and enolic forms of phloretin demonstrates different shapes of the molecules. Only the ketonic form shows several overlapping structures with beta-D-glucopyranose. Considering surplus binding of phloretin under glucose efflux conditions as being equivalent to the number of glucose transporters, a number of about 200,000 molecules was determined. By two independent methods 210,000 and 171,000 molecules per cell were calculated. This result is in close agreement with the number of glucose transporter sites of the erythrocyte.

Sugar transport and potassium permeability in yeast plasma membrane vesicles.

Plasma membrane vesicles were isolated from homogenised yeast cells by filtration, differential centrifugation and aggregation of the mitochondrial vesicles at pH 4. As judged by biochemical, cell electrophoretic and electron microscopic criteria a pure plasma membrane vesicle preparation was obtained. The surface charge density of the plasma membrane vesicles is similar to that of intact yeast cells with an isoelectric point below pH 3. The mitochondrial vesicles have a higher negative surface charge density in the alkaline pH range. Their isoelectric point is near pH 4.5, where aggregation is maximal. The yield of vesicles sealed to K+ was maximal at pH 4 and accounted for about one third of the total vesicle volume. The plasma membrane vesicles demonstrate osmotic behaviour, they shrink in NaCl solutions when loosing K+. As in intact yeast cells the entry and exit of sugars like glucose or galactose in plasma membrane vesicles is inhibited by UO22+. Counter transport in plasma membrane vesicles with glucose and mannose and iso-counter transport with glucose suggests that a mobile carrier for sugar transport exists in the plasma membrane. After galactose pathway induction in the yeast cells and subsequent preparation of plasma membrane vesicles the uptake of galactose into the vesicles increased by almost 100% over the control value without galactose induction. This increase is explained by the formation of a specific galactose carrier in the plasma membrane.

Plasma membranes from Candida tropicalis grown on glucose or hexadecane. I. Isolation, identification and purification.

Plasma membranes from Candida tropicalis grown on glucose or hexadecane were isolated using a method based on the difference in surface charge of mitochondria and plasma membranes. After mechanical disruption of the cells, a fraction consisting of mitochondrial and plasma membrane vesicles was obtained by differential centrifugation. Subsequently the mitochondria were separated from the plasma membrane vesicles by aggregation of the mitochondria at a pH corresponding to their isoelectric point. Additional purification of the isolated plasma membrane vesicles was achieved by osmolysis. Surface charge densities of mitochondria and plasma membranes were determined and showed substrate-dependent differences. The isolated plasma membranes were morphologically characterized by electron microscopy and, as a marker enzyme, the activity of Mg2+-dependent ATPase was determine. By checking for three mitochondrial marker enzymes the plasma membrane fractions were estimated to be 94% pure with regard to mitochondrial contamination.

The mechanism of vanadium action on selective K+-permeability in human erythrocytes.

Low concentrations of chelating agents such as EDTA prevent the air oxidation of vanadyl (VO2+, +4 oxidation state) to vanadate (VO3-, +5 oxidation state). Under these conditions, the ionophore A23187 mediates the rapid entry of vanadyl into human erythrocytes. In the presence of A23187, vanadyl at concentrations in excess of EDTA gives rise to a dramatic increase in K+ permeability, which is very similar to the Gardos Ca2+-induced K+ permeability increase with respect to ion selectivity, response to inhibitors, effects of pH, and stimulation by external K+. In ultrapure media with very low Ca2+, however, vanadyl has no effect on K+ permeability. These experiments suggest that Ca2+ is displaced from EDTA by vanadyl and then enters the cell via A23187 where it triggers the increase in K+ permeability. This hypothesis is confirmed by experiments demonstrating that vanadyl does displace Ca2+ from EDTA. Vanadate, an inhibitor of Ca2+-ATPase, causes a selective increase in K+ permeability in metabolically depleted cells, but the increase is abolished by low concentrations of EDTA, indicating that this effect is also due to entry of extracellular Ca2+. Earlier observations of effects of vanadyl and vanadate on erythrocyte K+ permeability can thus be explained on the basis of inhibition of the Ca2+ pump by vanadium, leading to an increase in intracellular Ca2+ concentration.

The anion-transport inhibitor H2DIDS cross-links hemoglobin interdimerically and enhances oxygen unloading.

Human hemoglobin treated with equal concentrations of the anion-transport inhibitor H2DIDS produces a right shift in the oxygen dissociation curve. concomitantly, the Hill coefficient is reduced from n = 2.7 to 2.1. When higher concentrations of H2DIDS are applied (H2DIDS: hemoglobin = 5:0.5 mM), the Hill coefficient decreases further to 1.5 and the oxygen dissociation curve of hemoglobin is shifted slightly to the left of the control. Similar results were also obtained with DIDS instead of H2DIDS. SDS-PAGE shows that H2DIDS cross-links hemoglobin monomers mainly into dimers. Cross-linking is more effective under anaerobic conditions. With tritiated H2DIDS the larger part of the radioactivity is found in the dimer position of hemoglobin. Separation of the alpha and beta units of hemoglobin reacted with tritiated H2DIDS demonstrated a stoichiometry of 2.2 and 2.4 molecules H2DIDS per molecule alpha and beta unit hemoglobin, leading to about 8-9 H2DIDS molecules per native hemoglobin. The right shift produced in the hemoglobin oxygen dissociation curve and the cross-linking of monomers into dimers, especially under anaerobic condition, suggest that H2DIDS can also react with those amino groups of hemoglobin which are involved in 2,3-DPG binding. A comparison of H2DIDS, DIDS and 2,3-DPG at three different concentrations close to the hemoglobin concentration revealed a concentration dependent right shift in the oxygen dissociation curve with the order of potency 2,3-DPG greater than H2DIDS greater than DIDS. The Hill coefficients (n) at the three concentrations of 2,3-DPG demonstrated no changes, but H2DIDS and DIDS reduced in a concentration-dependent manner the cooperativity of hemoglobin. Again, H2DIDS is more potent than DIDS, especially at the low concentration. These anion-transport inhibitors provide novel approaches to the exploration of hemoglobin function.

Modulation of the Ca2+- or Pb2+-activated K+-selective channels in human red cells. I. Effects of propranolol.

To study the effect of propranolol on the Ca2+- or Pb2+-activated K+ permeability in human erythrocytes, K+ effluxes were compared with single-channel currents. The results demonstrate that propranolol has a twofold effect: (1) it renders the channel protein more sensitive to Ca2+ or Pb2+; and (2) it simultaneously inhibits channel activity and slightly reduces single-channel conductance. The number of active channels is not affected.

The effect of ferricyanide with iodoacetate in calcium-free solution on passive cation permeability in human red blood cells: comparison with the Gardos-effect and with the influence of PCMBS on passive cation permeability.

Freshly prepared human red blood cells incubated with 5 mM ferricyanide, 0.2 mM iodoacetate and 2 mM adenosine in the presence of 5 mM EGTA demonstrate comparable increases in Na+ and K+ permeability (ferricyanide effect). This effect is unrelated to the Ca2+-activated K+ channel (Gardos effect) since influx of Ca2+ from outside the cell is excluded. Also this effect is different from the non-specific Na+ and K+ permeability change elicited by PCMBS. These differences become obvious by using various reagents. For example, A23187 and quinidine exert opposite effects in Gardos and ferricyanide experiments, where A23187 and atebrin react oppositely in the latter and in PCMBS experiments. The ferricyanide effect described here does not involve formation of nonspecific channels. The change in Na+ permeability separately from K+ permeability under certain circumstances suggests a more specific effect.

Modulation of the Ca2+- or Pb2+-activated K+-selective channels in human red cells. II. Parallelisms to modulation of the activity of a membrane-bound oxidoreductase.

Modulation of Ca2+-activable K+ permeability was compared with modulation of a membrane-bound oxidoreductase activity in human erythrocytes. Changes in the K+ permeability were monitored by flux measurements and single-channel recordings. The enzyme activity was detected by measuring reduction of ferricyanide. Pb2+, Atebrin and menadione had parallel effects on the channel protein and the enzyme. In contrast, propranolol stimulates K+ permeability, but is without effect on enzyme activity. The results demonstrate that the K+ channel and the enzyme are distinct membrane proteins but that the enzyme activity may influence channel gating.

Different activation energies in glucose uptake in Saccharomyces cerevisiae DFY1 suggest two transport systems.

The analysis of initial glucose uptake in Saccharomyces cerevisiae at 25 degrees, 20 degrees, 15 degrees and 10 degrees C by computer-assisted nonlinear regression analysis predicts two transport systems. The first demonstrates Michaelis-Menten kinetics and the second shows first order behaviour. The activation energies of these two systems were calculated by the Arrhenius equation at four different growth phases, namely early exponential (EE), middle exponential (ME2), late exponential (LE) and early stationary (ES) with 2% glucose in the batch medium. The activation energies calculated from the V(m) values in EE, ME, LE and ES growth phases were 15.8 +/- 1.7, 13.5 +/- 1.0, 15.1 +/- 0.8 and 13.5 +/- 0.7 kcal/mol. These values are in agreement with activation energies calculated for the first mechanism, facilitated diffusion, which is the mechanism deduced from countertransport experiments. The activation energies derived for the second transport system from the first order rate constants in cells grown to EE, ME2, LE and ES were 8.0 +/- 2.1, 8.1 +/- 1.3, 9.6 +/- 3.0 and 7.5 +/- 2.6 kcal/mol. These values are still significantly higher than for free diffusion of glucose in water and lower as predicted for passage of glucose through the lipid phase. Therefore, we assume in addition to carrier-mediated facilitated diffusion the entrance of glucose into the cell through a pore.

Effects of vanadate, menadione and menadione analogs on the Ca2+-activated K+ channels in human red cells. Possible relations to membrane-bound oxidoreductase activity.

The modulation of the Ca2+- (or Pb2+-)activated K+ permeability in human erythrocytes by vanadate, menadione and chloro-substituted menadione analogs was investigated by measurements of K+ fluxes and single-channel currents. Vanadate and menadione stimulate the K+ permeability by increasing the probability of channel openings; the menadione analogs, on the other hand, inhibit the K+ permeability by increasing the probability of channel closings. The compounds used in these experiments also interact with oxidoreductases; it is demonstrated that menadione analogs in contrast to menadione strongly inhibit the membrane-bound dehydrogenase in the erythrocytes. Concentrations of Pb2+ above 10 mumol/l, but not of Ca2+, inhibit the enzyme activity as well as the K+ permeability. The parallel effects on dehydrogenase activity and the K+ channels suggest a direct relationship between these two systems in the membrane of erythrocytes.

Lead-induced activation and inhibition of potassium-selective channels in the human red blood cell.

The selective increase of net K+ permeability in human red cells brought about by either Ca2+ or lead was studied using a light scattering technique to measure net K+ fluxes in cell suspensions and the patch-clamp technique to study K+ transport in individual K+-selective channels of the red cell membrane. Using ultrapure solutions it was demonstrated that the effect of lead is neither the indirect consequence of a lead-induced increase of the accessibility of the receptor sites of the K+-selective channels to traces of Ca2+ that are present as contamination in analytical grade reagents nor to the release of Ca2+ from intracellular Ca2+ stores. It is further shown that in cell-free membrane patches low concentrations of lead (10 microM) in Suprapur solutions evoke the same single-channel events as added Ca2+ and that this activity can be inhibited by high concentrations of lead (100 microM), similar to the net KCl efflux measured by means of the light scattering technique. It is concluded, therefore, that both Ca2+ and lead independently activate the same K+-selective channels in the red cell membrane.

Purification and properties of a phospholipid acyl hydrolase from plasma membranes of Saccharomyces cerevisiae.

The properties of a phospholipid acyl hydrolase bound to yeast plasma membranes are described in detail. The enzyme is capable of splitting all phospholipids which can be extracted from yeast cells. The specific activity with lysophosphatidylcholine as substrate was much higher than with phosphatidylcholine. With dipalmitoyl phosphatidylcholine as substrate a broad pH optimum was measured between pH 3.0 and 4.5. The membrane-bound enzyme was activated strongly by the anionic detergents SDS, deoxycholate and, to a lesser extent, by cholate. The uncharged detergent Triton X-100 and the zwitterionic detergent SB12 exerted an only slightly activating effect. KCl, NaCl, MgCl2, and CaCl2 were inhibitory in the presence of glycine/acetic acid buffer at pH 4.0. THe enzyme was solubilized by cholate or by SB12 in an active form from the plasma membrane and purified by acetone and (NH4)2SO4 precipitation or gel filtration with Sephadex G-200. THe phospholipid acyl hydrolase was identified as a glycoprotein with an apparent molecular weight of 145,000 by SDS slab gel electrophoresis.