Removal of both Ycf48 and Psb27 in Synechocystis sp. PCC 6803 disrupts Photosystem II assembly and alters QA oxidation in the mature complex

The Photosystem II (PS II) assembly factors Psb27 and Ycf48 are transiently associated with PS II during its biogenesis and repair pathways. We investigated the function of these proteins by constructing knockout mutants in Synechocystis sp. PCC 6803. In ΔYcf48 cells, PS II electron transfer and stable oxygen evolution were perturbed. Additionally, Psb27 was required for photoautotrophic growth of cells lacking Ycf48 and assembly beyond the RC47 assembly complex in ΔYcf48:ΔPsb27 cells was impeded. Our results suggest the RC47 complex formed in ΔYcf48 cells is defective and that this deficiency is exacerbated if CP43 binds in the absence of Psb27.     

Hydrogen sulfide is a reversible inhibitor of the NADH oxidase activity of synaptic plasma membranes

Hydrogen sulfide is now accepted as a neuromodulator, which can be involved in neuronal defence against oxidative stress insults in the brain. In this work we show that concentrations of H₂S within the physiological range reported in the brain produce a reversible inhibition of the NADH oxidase activity and coupled superoxide anion production by synaptic plasma membranes from rat brain. At physiological pH 7 the concentration of H₂S needed for 50% inhibition of the NADH oxidase activity is 5 ± 1 μM, which is within the low range of the reported physiological H₂S concentrations. Thus, the NADH oxidase activity of the neuronal plasma membrane can act as a sensor of local H₂S depletion in neurones. H₂S inhibition of the NADH oxidase activity of the neuronal plasma membrane can be accounted for direct reduction by H₂S of cytochrome b₅. However, H₂S fails to afford a significant protection against the inhibition of this activity by peroxynitrite. In conclusion, our results point out that H₂S is more potent as inhibitor of reactive oxygen species formation than as a sacrificial antioxidant.     

Kinetics and ion specificity of Na/Ca exchange mediated by the reconstituted beef heart mitochondrial Na/Ca antiporter

The Na⁺/Ca²⁺ antiporter was purified from beef heart mitochondria and reconstituted into liposomes containing fluorescent probes selective for Na⁺ or Ca²⁺. Na⁺/Ca²⁺ exchange was strongly inhibited at alkaline pH, a property that is relevant to rapid Ca²⁺ oscillations in mitochondria. The effect of pH was mediated entirely via an effect on the K_m for Ca²⁺. When present on the same side as Ca²⁺, K⁺ activated exchange by lowering the K_m for Ca²⁺ from 2  to 0.9 μM. The K_m for Na⁺ was 8 mM. In the absence of Ca²⁺, the exchanger catalyzed high rates of Na⁺/Li⁺ and Na⁺/K⁺ exchange. Diltiazem and tetraphenylphosphonium cation inhibited both Na⁺/Ca²⁺ and Na⁺/K⁺ exchange with IC₅₀ values of 10 and 0.6 μM, respectively. The V_max for Na⁺/Ca²⁺ exchange was increased about fourfold by bovine serum albumin, an effect that may reflect unmasking of an autoregulatory domain in the carrier protein.     

A new assay for endocytosis in erythrocyte ghosts based on loss of acetylcholinesterase activity

A new method for assaying endocytosis in erythrocyte ghosts is presented. The method involves measuring the percentage loss of acetylcholinesterase activity which occurs when vacuoles form, making the acetylcholinesterase on the vacuole surface inaccessible. This method is compared to other methods of measuring endocytosis in this system, including phase contrast microscope estimation of vesiculation, stereological analysis of electron micrographs to determine vesiculation and loss of sialic acid accessible to neuraminidase due to endocytosis. Comparison of the percentage loss of acetylcholinesterase activity with the electron micrographic and sialic acid methods showed that all three methods gave a quantitative measure of the percentage of total membrane area taken in as vesicles. Since the acetylcholinesterase method was fast, easy, inexpensive, and quantitative, it was the preferred method for assay of endocytosis. The inhibition of endocytosis by Ca²⁺ was observed with this method; the success of this experiment demonstrated the applicability of the method to the study of inhibitors of endocytosis.     

Effect of carbonylcyanide m-chlorophenylhydrazone on the calcium-stimulated ATPase activity of erythrocyte ghosts

Incubation of erythrocyte ghosts with carbonylcyanide m-chlorophenylhydrazone (CCCP) plus Ca²⁺ resulted in inactivation of the Ca²⁺-stimulated ATPase activity. Omission of Ca²⁺ or lowering of the temperature below 25 °C eliminated the inhibitory effect, as also did the presence of ATP during the incubation. On the other hand, the addition of β-mercaptoethanol did not influence the Ca²⁺-dependent inhibition by CCCP. Compared with the level of CCCP which uncouples oxidative phosphorylation, a rather high level (0.5 mM) of CCCP was required to inhibit the ATPase activity in ghosts. However, once the inhibition had been accomplished, almost all of the CCCP could be removed from the ghost membrane by washing with a Ca²⁺-containing solution, without affecting the inhibition of ATPase. If ethylene-glycol-bis(β-aminoethyl acid was included in the washing medium, the inhibition of ATPase was nearly completely reversed by washing. The results indicate that only the Ca²⁺-stimulated, Mg²⁺-ATPase was inhibited by 0.5 mM CCCP, while the remaining components of the ATPase activity were slightly inhibited by higher levels of the uncoupler. Low levels of CCCP (0.1 mM) stimulated the Mg²⁺-ATPase slightly. CCCP was much more specific for the Ca²⁺-stimulated ATPases than N-(1-naphthyl)maleimide, an unusually effective sulfhydryl reagent, and the requirement of Ca²⁺ for inactivation was also quite specific.     

Proline hydroxylation by cell free extract of a streptomycete

Free L-proline was hydroxylated to free L-hydroxyproline by cell free extract of Streptomyces griseoviridus P8648. The hydroxylation reaction required ferrous ion, 2-ketoglutarate and ascorbate. Zinc ion, ethylenediaminetetraacetic acid and alpha,alpha'-dipyridyl inhibited the reaction. Optimum temperature and pH were 25.0 degrees C and 7.5, respectively.     

An intracellular calcium store is present in Leishmania donovani glycosomes

A fourth intracellular Ca2+ pool in Leishmania donovani was identified by permeabilizing plasma membrane with digitonin. In Fura 2 loaded cells Ca2+ was released synergistically when mitochondrial function was blocked by antimycin and oligomycin. Vanadate did not have any effect if applied before incorporation of these mitochondrial poisons. However, the same inhibitor which inhibits Ca2+-ATPase activity of endoplasmic reticulum was able to release Ca2+ at a slow rate when added after antimycin and oligomycin. Alkalization of cytoplasmic pH allowed further release of Ca2+ essentially from the acidocalcisome. Purified glycosomes could mediate Ca2+ uptake mechanism in presence of vanadate whereas bafilomycin, a specific and potent inhibitor of vacuolar proton pump did not have any effect. Glycosomal Ca2+-ATPase activity was optimum at pH 7.5. The apparent Km for calciumin presence of vanadate was 12 nM. Taken together, it may be suggested that a vanadate-insensitive Ca2+-ATPase is present in the membrane of this microbody. Presence of glycosomal Ca2+ was further confirmed by imaging of Ca2+ activity in the Fura 2 loaded purified organelle using confocal laser. Results reveal that newly localized glycosomal calcium may essentially be an effective candidate to play a significant role in cellular function.     

Toxocara canis: Larval migration dynamics, detection of antibody reactivity to larval excretory-secretory antigens and clinical findings during experimental infection of gerbils (Meriones unguiculatus)

In this study, Mongolian gerbils were used to analyse features of Toxocara infection that included larval migration, humoral immune responses to Toxocara canis excretory-secretory antigens (TES) and aspects of host physiology. At day 10 post-infection (p.i.) most larvae were in the intestine and the lungs while later the total number of larvae was higher in the carcass tissue; the number of larvae per gram of tissue was lower elsewhere other than in the brain. Infected animals showed several neurological abnormalities, an early increase in leukocyte and neutrophil levels, two peaks of peripheral eosinophilia (5 and 40 d.p.i.) and high antibody levels against TES in the circulation and in the vitreous humor. A sequential recognition of eight T.canis larval antigens with MW from 24 to 200 kDa was detected by Western blot. The results obtained in this study further support the use of gerbils as an experimental model for systemic, ocular and cerebral toxocariasis.     

NADP and NADPH in glucose-6-phosphate dehydrogenase-deficient erythrocytes under oxidative stimulation

A mild oxidative stimulation of the hexose monophosphate pathway of human glucose-6-phosphate dehydrogenase (EC 1.1.1.49)-deficient erythrocytes (Mediterranean variant) causes a significant drop in NADPH.

These results, other than to confirm that glucose-6-phosphate dehydrogenase deficiency is a product deficiency disorder, demonstrate that under oxidative stimulation glutathione reductase may become functionally impaired and GSSG cannot be reduced at a sufficient rate.