Photochemical Electron Transport in Photosynthetic Reaction Centers from Rhodopseudomonas spheroides: I. Kinetics of the Oxidation and Reduction of P-870 As Affected by External Factors

Photosynthetic reaction centers from Rhodopseudomonas spheroides were prepared with the detergent lauryl dimethylamine oxide (LDAO). In contrast to reaction centers made with Triton X-100, these contained no cytochromes and little or no ubiquinone (UQ). The reduction of P-870, after its photochemical oxidation, was studied in these materials with the following results. In reaction centers made with Triton X-100, slow kinetic components (seconds to minutes) could be attributed to secondary electron acceptors or traps. In reaction centers made with LDAO the kinetics were predominantly fast (half-times, 100 msec or less); slower components could be introduced by adding UQ. Added UQ appeared to become bound to reaction centers made with LDAO, but the binding might have meant only that both components were trapped within detergent micelles. Ferricyanide could retard the reduction of oxidized P-870, apparently by capturing electrons from the reducing side of the photochemical system. Under conditions in which the participation of secondary electron acceptors seemed to have been eliminated, the recovery of P-870 was mainly by a first-order process with a half-time of about 60 msec at room temperature and 20-30 msec at about -80°C and below. The transition with decreasing temperature suggested the presence of a mixed population, exhibiting both the 60 and 20 msec components, but variations in the absorption spectra with temperature did not suggest the presence of a mixed population. Absorption difference spectra in the ultraviolet were compatible with the idea that UQ added to reaction centers became reduced in the light.     

Interet diagnostique d’une injection intracaverneuse de 20 μg de Prostaglandine E1 dans l’impuissance

Intracavernous injection of 20 μg of prostaglandin E1 (PGE1) was carried out in 130 impotent patients. The erectile response was compared to the results of arteriological investigations including nocturnal penile tumescence and rigidity monitoring (NPTR) in 59 patients. The response of 60 patients positively categorized as exclusively psychogenic or vasculogenic was also compared to the pattern of the response to 80 mg of papaverine observed in a previous study by the same authors. The PGE1 test may not discriminate psychogenic from wholly organic patients since its results are not correlated to those of NPTR. It helps for the screening of vasculogenic impotence. Lack of response or a partly rigid response is consistent with this actiology but is not specific for it. A fully response makes it unlikely. Compared to papaverine, PGE1 induces less non rigid responses in psychogenic patients (15% versus 35% with papaverine) and more fully rigid responses in vasculogenic patients (respectively 12% and 5 %). Consequently the specificity of the PGE1 test is higher but its sensitivity lower than that of papaverine so that there is no clear difference in the effectiveness of the tests. Nevertheless the PGE1 test should be preferred, because it is safer. Prolonged erections occured in only 5 patients, and all ceased spontaneously. However 4 presented severely painful erections.     

Non-canonical functions of RGS proteins

Regulators of G protein signalling (RGS) proteins are united into a family by the presence of the RGS domain which serves as a GTPase-activating protein (GAP) for various Galpha subunits of heterotrimeric G proteins. Through this mechanism, RGS proteins regulate signalling of numerous G protein-coupled receptors. In addition to the RGS domains, RGS proteins contain diverse regions of various lengths that regulate intracellular localization, GAP activity or receptor selectivity of RGS proteins, often through interaction with other partners. However, it is becoming increasingly appreciated that through these non-RGS regions, RGS proteins can serve non-canonical functions distinct from inactivation of Galpha subunits. This review summarizes the data implicating RGS proteins in the (i) regulation of G protein signalling by non-canonical mechanisms, (ii) regulation of non-G protein signalling, (iii) signal transduction from receptors not coupled to G proteins, (iv) activation of mitogen-activated protein kinases, and (v) non-canonical functions in the nucleus.     

Effect of neurotransmitters on phospholipid metabolism in rat cerebral-cortex slices: cellular and subcellular distribution

Abstract— Young rat cerebral-cortex slices were incubated with ³²Pi in the absence and presence of ACh plus eserine, norepinephrine, dopamine or serotonin for 1 h. their cellular and subcellular fractions were isolated, and the specific radioactivities of the various phospholipids determined. In the neuronal- and astroglial-enriched fractions ACh plus eserine increased the ³²P-labelling of phosphatidyl inositol (PhI) phosphatidic acid (PhA) and phosphatidylcholine (PhC) by increments which ranged from 108 per cent for PhI to 30 per cent for PhC and in the presence of norepinephrine or dopamine these increments ranged from 180 per cent for PhI to 29 per cent for PhC. In the subcellular fractions ACh plus eserine exerted maximal stimulatory effect on the labelling of the synaptosomal phospholipids, which was 88 per cent for PhI and 79 per cent for PhA, followed by those of microsomes, mitochondria and nuclei. ACh plus eserine exerted no effect on [^l4C]glucose incorporation, but inhibited the incorporation of [¹⁴C]glycerol into phospholipids by amounts which ranged from 30 per cent for PhI to 3 per cent for PhE. Although the rate of incorporation of ³²Pi into phospholipids of 0.2 mm slices was higher than that of the 0.5 mm slices the stimulatory effect of ACh plus eserine on the ³²Pi incorporation into the lipids of the latter was higher. When neuronal- and astroglial enriched fractions were first isolated from the cerebra then incubated with ³²Pi or [¹⁴C]choline, labelling of phospholipids in the neuronal fraction was higher than that of the astroglial fraction; however, ACh plus eserine had no effect on the incorporation of ³²Pi into the lipids of either fraction. ACh plus eserine stimulated the activity of phosphatidic acid phosphatase in the various subcellular fractions by increments which ranged from 13 per cent in nuclei to 37 per cent in microsomes. It was concluded that the nonspecific localization of the neurotransmitter effect could be due to the widespread distribution of the enzymes which appear to be responsive to cholinergic and adrenergic neurotransmitters.     

ATF6alpha optimizes long-term endoplasmic reticulum function to protect cells from chronic stress

In vertebrates, three proteins--PERK, IRE1alpha, and ATF6alpha--sense protein-misfolding stress in the ER and initiate ER-to-nucleus signaling cascades to improve cellular function. The mechanism by which this unfolded protein response (UPR) protects ER function during stress is not clear. To address this issue, we have deleted Atf6alpha in the mouse. ATF6alpha is neither essential for basal expression of ER protein chaperones nor for embryonic or postnatal development. However, ATF6alpha is required in both cells and tissues to optimize protein folding, secretion, and degradation during ER stress and thus to facilitate recovery from acute stress and tolerance to chronic stress. Challenge of Atf6alpha null animals in vivo compromises organ function and survival despite functional overlap between UPR sensors. These results suggest that the vertebrate ATF6alpha pathway evolved to maintain ER function when cells are challenged with chronic stress and provide a rationale for the overlap among the three UPR pathways.     

Reverse-Polynomial Dilution Calibration Methodology Extends Lower Limit of Quantification and Reduces Relative Residual Error in Targeted Peptide Measurements in Blood Plasma

Matrix effect is the alteration of an analyte''s concentration-signal response caused by co-existing ion components. With electrospray ionization (ESI), matrix effects are believed to be a function of the relative concentrations, ionization efficiency, and solvation energies of the analytes within the electrospray ionization droplet. For biological matrices such as plasma, the interactions between droplet components is immensely complex and the effect on analyte signal response not well elucidated. This study comprised of three sequential quantitative analyses: we investigated whether there is a generalizable correlation between the range of unique ions in a sample matrix (complexity); the amount of matrix components (concentration); and matrix effect, by comparing an E. coli digest matrix (∼2600 protein proteome) with phospholipid depleted human blood plasma, and unfractionated, nondepleted human plasma matrices (∼10⁷ proteome) for six human plasma peptide multiple reaction monitoring assays. Our data set demonstrated analyte-specific interactions with matrix complexity and concentration properties resulting in significant ion suppression for all peptides (p < 0.01), with nonuniform effects on the ion signals of the analytes and their stable-isotope analogs. These matrix effects were then assessed for translation into relative residual error and precision effects in a low concentration (∼0–250 ng/ml) range across no-matrix, complex matrix, and highly complex matrix, when a standard addition stable isotope dilution calibration method was used. Relative residual error (%) and precision (CV%) by stable isotope dilution were within <20%; however, error in phospholipid-depleted and nondepleted plasma matrices were significantly higher compared with no-matrix (p = 0.006). Finally a novel reverse-polynomial dilution calibration method with and without phospholipid-depletion was compared with stable isotope dilution for relative residual error and precision. Reverse-polynomial dilution techniques extend the Lower Limit of Quantification and reduce error (p = 0.005) in low-concentration plasma peptide assays and is broadly applicable for verification phase Tier 2 multiplexed multiple reaction monitoring assay development within the FDA-National Cancer Institute (NCI) biomarker development pipeline.Plasma is the overriding human medium sampled for established and novel protein biomarkers (1, 2). As of 2011, 1929 high-confidence proteins have been cataloged by the Human Plasma Proteome Project, with estimates that there are up to 10⁷ unique protein sequences in plasma that span a concentration range across 10 orders of magnitude (1, 3). 99% of the protein mass in plasma is made up of 22 proteins including Albumin, Fibrinogen, and a range of immunoglobulins, leaving more than 1900 known small proteins and essentially the entirety of the projected plasma proteome in the remaining 1% (4). It is these low-mass, low abundance proteins such as the Interleukins, C-Reactive Protein, and Carcinoma Antigen 125 (CA125), that are indicative of many important physiological and pathological processes, and proteomic scientists and clinicians have thus focused their efforts in qualitatively and quantitatively defining this fraction for novel biomarkers (4–6).The development of plasma biomarkers is a large-scale undertaking that spans discovery, verification, and validation phases in a multistage pipeline: Thousands of “discovered” differentiated proteins are evaluated for probability of effect, from which 10–100s of proteins are then selected for targeted quantification in verification phase to evaluate sensitivity and specificity for its intended indication (2, 7). Finally a panel of the strongest marker candidates is progressed to validation phase, and FDA-level validated quantitative assays are used to test the clinical utility of the biomarker panel. Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS)¹ is the most robust analytical method available for proteomic scientists in this pipeline, able to separate complex mixtures and specifically and sensitively identify and quantify its components (2, 7–10), The ability to ionize and evaporate the contents of a liquid sample (coupling LC to MS/MS) is the basis that allows this to happen (9). Electrospray Ionization (ESI) is the most widely used ionization apparatus in LC-MS/MS bioanalysis because of its ionization efficiency and stability and low chemical specificity (9, 10). Although these properties make ESI very robust, the complexity of biological matrices poses a significant challenge for LC-ESI-MS/MS-based quantitation; despite chromatography and nanospray technology, the ESI droplet of a plasma peptide-digest sample (given its immense range of unique protein/peptide sequences and concentrations) can contain an unknown multitude of co-eluting components that “compete” to dissolve from the droplet and reach gas phase, suppressing and varying the signal intensity responses for a given analyte concentration (9–13). These ionization competing elements can also go on to produce isobaric signals in the third quadrupole that interfere with an analyte''s transition signals (14). Termed “matrix effects,” these phenomena of complex sample matrices can significantly impede quantitative accuracy (15). For high-throughput clinical assays, matrix effects are controlled for by preparing calibration standards in the same biological matrix to mimic the conditions of the samples intended for study as per FDA bioanalytical method validation guidelines (16). The catch to this technique is that the signal from the endogenous analyte in the background matrix hinders accuracy when the nominal concentration is close to or below the endogenous signal (14, 17). There is a need for broadly applicable methods of controlling matrix effects and increasing accuracy in low concentration MRM peptide assays for nondepleted, unfractionated plasma that can be adopted for the highly multiplexed, high throughput, “Tier 2” MS assays required in verification phase of the biomarker development pipeline (2, 8). Several simple methods have independently demonstrated the ability to increase accuracy in various hyphenated-MS assays in complex matrices: “Reverse” curves utilize the stable-isotope analog not as an internal standard but as a surrogate calibration analyte to circumvent interference from the endogenous analyte signal and extend assay Lower Limit(s) of Quantification (LLOQ), and nonlinear calibration techniques have proven to more accurately reflect the concentration-MS detector response at the low and high end of concentration gradients (8, 14, 18–21). Specifically in the case of biological matrices, phospholipids are particularly deleterious ion suppressing elements because of their easily ionizable, polar, and hydrophobic moieties that can have complex interactions with co-eluting analytes as well as the chromatography stationary and mobile phases required for most other analytes (22–25). Combination solid-phase extraction (SPE) and phospholipid removal techniques have proved to effectively minimize ion suppression effects in ESI-MS assays (22–25).In this study, we investigated whether there is a generalizable linear correlation between the number of unique ions (complexity) in a biological sample matrix, the amount of ionizable matrix content (concentration), and matrix effects, for six human plasma peptides comparing serial dilutions of an Escherichia Coli (E. coli) peptide-digest against phospholipid-depleted and nondepleted unfractionated human plasma peptide-digest (highly complex) matrices. We examined the influence of matrix effects on relative residual error in a low-concentration (∼0–250 ng/ml) plasma peptide range, and compared the utility of a reverse-polynomial dilution (RPD) calibration method versus standard addition stable-isotope dilution (SID) in phospholipid-depleted and nondepleted unfractionated human plasma. A peptide-centric matrix effect is reported and the effect of the endogenous analyte signal on relative residual error in low-concentration (∼0–250 ng/ml) plasma peptide assays is established. A RPD calibration technique that extends LLOQ and reduces relative residual error in low-concentration plasma peptide MRM assays is presented.