Is functional manganese involved in hydrogen-peroxide-stimulated anomalous oxygen evolution in CACl2-washed photosystem II membranes?

When detergent-derived photosystem II (PSII) membranes are treated with CaCl₂ to remove the three extrinsic proteins associated with the O₂-evolving complex, the resulting membranes (CaPSII) can still catalyze water oxidation if sufficient Ca²⁺ and Cl^- are present. When CaPSII membranes are exposed to single turnover flashes on an O₂ rate electrode, anomalous O₂ is produced by the first two flashes. The addition of catalase to the membrane suspension completely inhibits O₂ produced by the first two flashes, but not by subsequent flashes. Exogenous H₂O₂ stimulates anomalous O₂ production by the first few flashes in CaPSII membranes, but not in control PSII membranes. Diuron (DCMU) does not inhibit H₂O₂-stimulated O₂ production by the first flash. However, it does inhibit the O₂ yield of all subsequent flashes, indicating that all flash-induced O₂ signals in CaPSII membranes are dependent on photosystem II electron transport. H₂O₂ stimulation of O₂ yields is inhibited in Tris-, heat-, and EDTA-(ethylenediaminetetraacetic acid)-treated CaPSII. In the presence of high salt, H₂O₂ (but not EDTA) treatment of CaPSII, extracts Mn functional in normal photosynthetic O₂ evolution. The addition of exogenous Mn²⁺ reconstitutes anomalous O₂ production in Tris-and H₂O₂/EDTA-treated CaPSII preparations but only in the presence of H₂O₂. Anomalous H₂O₂-stimulated O₂ production can be observed both with a Clark electrode (steady state) and an O₂ rate electrode (flash sequence). The mechanism involves electron donation from H₂O₂, mediated by free Mn²⁺, to PSII, and the 33-kDa extrinsic protein under some conditions can block this process. Since H₂O₂ can remove functional Mn from CaPSII membranes, its presence can convert functional Mn to the Mn²⁺ mediator state required for anomalous O₂ production. EDTA binds Mn in CaPSII disrupted by H₂O₂ and prevents anomalous O₂ evolution.Abbreviations CaPSII a PSII preparation washed with approximately 1M CaCl₂ - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EDTA ethylenediaminetetraacetic acid - MES 2-[N-morpholino]-ethanesulfonic acid - PSII a detergent-derived photosystem II membrane preparation - RC reaction center - Tris tris(hydroxymethyl)-aminomethane - Y_n oxygen rate electrode flash yield resulting from the nth flash of a sequence of single turnover flashes of light Operated by the Midwest Research Institute for the U.S. Department of Energy under contract DE-AC02-83CH10093.     

Active and passive immunization of mice against larval Dirofilaria immitis

The objective of this study was to determine if Dirofilaria immitis larvae would survive in diffusion chambers implanted in dogs and mice and secondly to determine if mice could be immunized against infection with D. immitis. Dirofilaria immitis third-stage larvae (L3) survived and grew in diffusion chambers implanted in dogs and mice for at least 3 wk. BALB/c mice, which were repeatedly infected with live L3, showed resistance to challenge infections. Dead L3, with or without adjuvants elicited no protective immunity. A correlation was found between the degree of immune protection seen in mice and antibody levels to soluble larval antigen but not to antibody levels to surface antigens. A monoclonal antibody was prepared that reacted with the surface of D. immitis and Onchocerca lienalis L3, but not to the surfaces of other stages and species of various filarial worms. When this antibody was administered to mice prior to challenge no significant reduction in larval survival was observed.     

Filtration rate capacities in 6 species of European freshwater bivalves

Summary Filtration rate capacities in undisturbed freshwater bivalves were determined by means of two different methods (indirect clearance and suction methods) in Anodonta anatina (L.), Unio tumidus Philipsson, Unio pictorum (L.), Unio crassus Philipsson, Dreissena polymorpha (Pallas) and Sphaerium corneum (L.). In A. anatina, D. polymorpha, and S. corneum the filtration rate (FR, 1 h^-1) at 19–20°C as a function of dry tissue weight (DW, g) or ash-free dry weight (AFDW, g) could be expressed by the equations: 1.10 DW^0.78, 6.82 DW^0.88, and 2.14 AFDW^0.92, respectively. In U. tumidus, U. pictorum, and U. crassus filtration rates were comparable with those of A. anatina. In D. polymorpha the b value of the corresponding regression of gill area on dry weight was 0.87. The rates of water transport in freshwater bivalves are 2–8 times lower than in marine bivalves of comparable size. A corresponding difference in the filtration rate per gill area unit is found. The measured filtration rates in undisturbed bivalves are substantially higher (at least 4 times) than previously reported. This indicates that the impact of bivalve water processing on freshwater ecosystems is greater than hitherto suggested.     

Surface-enhanced resonance Raman scattering spectroscopy of bacterial photosynthetic membranes. The carotenoid of Rhodospirillum rubrum

Resonance Raman scattering by the carotenoid, spirilloxanthin (Spx), in a suspension of chromatophores (cytoplasmic side out) isolated from the photosynthetic bacterium, Rhodospirillum rubrum, is greatly enhanced when the membranes are adsorbed onto the surface of an anodized Ag electrode. The phenomenon is the basis for surface-enhanced resonance Raman scattering (SERRS) spectroscopy. The Spx SERRS peaks observed were at 1505-1510, 1150-1155, and 1000-1005 cm-1 with laser excitation wavelengths ranging between 457.9 and 568.2 nm. Similar peaks were not observed with spheroplasts (periplasmic side out) isolated from the same species. The difference in signal detected in chromatophores and spheroplasts is not due to differences in membrane surface charge, presence of residual cell wall on the spheroplast surface, lack of adhesion of spheroplasts to metals, or large differences in pigment content per unit membrane area. Instead, the results indicate an asymmetric distribution of Spx in vivo across the membrane (i.e., it is located on the cytoplasmic side of the membrane). The results also demonstrate that the SERRS effect is extremely distance sensitive, and the thickness of a single bacterial membrane (separating the Ag electrode from the carotenoid) is sufficient to prevent detection of Spx spectra. Studies of chromatophores from the F24 strain (a reaction centerless mutant) have pin-pointed B880 antenna complex as the source of the Spx SERRS spectra, and a schematic model of the minimal structural unit of B880 is presented. This work demonstrates the potential of the SERRS technique as a probe for surface topology of pigmented membranes.     

The hepatic response to Ca2+ is inhibited by Mg2+ and enhanced by phenylephrine or ouabain

Net hepatic Ca2+ efflux, K+ uptake and glycogen breakdown in response to the alpha 1-adrenergic agonist phenylephrine were studied. Rat livers were perfused with CO2/bicarbonate-buffered solutions containing 10 microM Ca2+ and different amounts of Mg2+. K+-free medium and/or ouabain were used to block (Na+ + K+)-ATPase-dependent K+ uptake. In some experiments a sharp increase in extracellular Ca2+ concentrations was produced by infusing CaCl2 into the medium entering the liver. Perfusion with K+-free medium and ouabain enhanced the phenylephrine-induced Ca2+ efflux and diminished the glycogenolytic response, indicating a dissociation of Ca2+ release and glycogenolysis. Exogenous Ca2+ had practically no effect if livers were perfused with regular medium containing 1.2 mM Mg2+. In the presence of phenylephrine and if extracellular Mg2+ concentrations were lowered by omitting Mg2+ from the medium or by preperfusion with EGTA, exogenous Ca2+ was glycogenolytically effective and also produced a transient K+ uptake. Increased extracellular concentrations of Mg2+ inhibited the effects of exogenous Ca2+. In the presence of phenylephrine, higher concentrations of Mg2+ were needed than in the absence of alpha 1-adrenergic agonist to achieve a similar degree of inhibition. In one respect ouabain effects were comparable to those of phenylephrine: the glycoside also increased the metabolic response to exogenous Ca2+ and diminished the sensitivity towards Mg2+. Phenylephrine and ouabain may both enhance the permeability of plasma membranes for Ca2+.     

Changes in left ventricular geometry during spontaneous breathing

The purpose of these experiments was to determine the effects of a spontaneously generated inspiration on the size and shape of the left ventricle (LV) in anesthetized supine dogs. We implanted markers in the LV to establish three perpendicular axes and recorded the motion of these markers using biplane cinefluoroscopy at 60 Hz. The primary changes in LV size that accompanied inspiration occurred at end diastole (ED). The largest change in LVED dimension was a 2.46-mm narrowing of the septal-lateral wall dimension, but the apex-base dimension decreased also, by 0.74 mm. The anteroposterior dimension actually widened by 1.07 mm. The septal-lateral narrowing was caused by both a 1.0-mm narrowing of the distance between the septal marker and the apex-base axis, as well as by a 1.4-mm narrowing between the apex-base axis and the lateral wall marker. Narrowing of the septal portion seemed expected because of presumed enhanced right ventricular filling during inspiration. Narrowing of the lateral portion of the LV, while the anteroposterior dimension widened, was surprising because a change in LVEDV shape is implied. Assuming ventricular homogeneity, this change in LVED shape implies that the forces applied to the epicardial surface were not uniform. There must have been a retraction on the anterior and posterior surface that was not experienced by the lateral LV wall. The net effect of these dimensional changes of the LV at end diastole (estimated from the product of the three ED axes) was a 3.5-cm3 reduction in LVED volume.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diadenosine 5′,5‴-P1,P3-triphosphate in eukaryotic cells: Identification and quantitation

A highly sensitive enzymatic assay for diadenosine 5′,5?-P¹,P³-triphosphate (Ap₃A) has been established on the basis of the coupled luminescence assay for diadenosine 5′,5?-P¹,P³-tetraphosphate (A. Ogilvie (1981)Anal. Biochem.115, 302–307). Snake venom phosphodiesterase splits Ap₃A into AMP plus ADP which can be measured in a luminescence reaction containing pyruvate kinase, phosphoenolpyruvate and luciferin-luciferase. The procedure is linear with Ap₃A levels ranging from 0.1 to 2 pmol. The assay has been used to measure Ap₃A in various eukaryotic cells after ion-exchange chromatography and high-performance liquid chromatography of acidic extracts of the cells. The level of diadenosine triphosphate was higher in all instances than the level of diadenosine tetraphosphate. When growing in the abdominal cavity of mice, Ehrlich ascites tumor cells contained high amounts of Ap₃A ($\text{0.1}\text{nmol}\text{10}{}^6\text{cells}$), allowing direct optical determination in the HPLC chromatography. The quantitative measurement of Ap₃A with the luminescence assay gave identical results. Ap₃A extracted from Ehrlich cells was also chromatographed with authentic nucleotide in two thin-layer systems providing additional proof for the existence of Ap₃A in biological material.     

Desmethylimipramine Enhances the Release of Endogenous GABA and Other Neuro trans mitter Amino Acids from the Rat Thalamus

The influence of desmethylimipramine (DMI) on the release of endogenous gamma-aminobutyric acid (GABA) and some other amino acids from the rat thalamus was studied with a push-pull perfusion technique. Following HPLC the amino acids were fluorimetrically estimated. Added to the perfusion medium at a concentration of 10 mumol L-1, DMI caused a 5- to 10-fold increase in the release of GABA. Similar effects were found with imipramine, trimeprimine, haloperidol, and propranolol. The elevation of GABA release induced by DMI was Ca dependent. The release of aspartate and glutamate was also increased by DMI, but in contrast to K ions, DMI did not reduce the thalamic output of glutamine.     

The complete heart-lead relationship in the einthoven triangle

The relationship between the source strength and the “manifest vector” in the Einthoven Triangle is derived for a line and a point dipole source and confirmed experimentally. The result permits the interpretation of the standard ECG leads in absolute terms and corrected for body size. The manifest vector is shown to be approximately times what it would be in an otherwise similar circular slab which circumscribes the triangle.