Changes in absorbance at 413 nm in plasma from three rodent species exposed to phosgene

Mice, rats and guinea pigs were exposed to phosgene (COCl2), a highly irritating and oxidizing gas. Animals were exposed to 87 mg/m3 phosgene for 20 min in a whole-body exposure chamber. Within 55-65 minutes after the start of exposure, plasma was scanned spectrophotometrically from 200-600 nm. A distinct and significant increase in area under the curve in the Soret band region at 413 nm was observed in plasma from phosgene-exposed animals when compared with air-exposed controls in all three species. These peaks were consistent with hemoglobin, an indication that the integrity of the erythrocyte membrane had been compromised by exposure. An erythrocyte osmotic fragility assay on blood from mice exposed to phosgene indicated that 30% less NaCl was needed to cause 50% hemolysis compared to air-exposed mice. These results suggest a new mechanism of phosgene-induced acute lung injury that may be linked, in part, to a direct attack on the erythrocyte membrane.     

Adjacent guanines as preferred sites for strand breaks in plasmid DNA irradiated with 193 nm and 248 nm UV laser light

Kainate-preferring glutamate receptors may contribute to the glutamatergic responses to seizures. The cloning of their encoding genes overcomes limitations of the receptor ligands available for their investigation. We have examined the expression of the high affinity kainate receptor subunits KA1 and KA2 mRNAs in the rat hippocampus, using electroconvulsive shock (ECS) as a seizure paradigm not confounded by neurotoxicity. A single shock reduced the levels of KA1 mRNA in the CA3c region, while increasing the expression of KA2 mRNA in the dentate gyrus. Following repeated ECS (5 shocks over 10 days), KA1 mRNA was reduced in CA3c and in CA3a-b but was unchanged in dentate gyrus. KA2 mRNA, on the other hand, significantly increased in dentate gyrus, and to a lesser extent in CA3c and CA1. All changes in KA1 and KA2 mRNAs had returned to baseline 3 weeks after the last shock. We also measured the expression of cyclophilin mRNA, and found it to be reduced in all hippocampal subfields, and in the parietal cortex, after a single ECS. It returned to control levels after repeated ECS but was again reduced following 3 weeks recovery from repeated ECS. These results indicate that the expression of KA1 and KA2 not only change in opposite directions in the rat hippocampus after ECS, but that the alterations are anatomically and temporally regulated. In the respect that cyclophilin is regarded as a housekeeping gene, the reduction in its mRNA suggests that ECS may have more persistent and widespread effects on brain gene expression than previously suspected.     

Changes of erythrocyte membrane lipids in ethanol induced hyperlipidemia (Zieve's syndrome) (author's transl)

In 11 patients with alcohol-induced hyperlipemia, of whom 6 showed a Zieve Syndrome increased phospholipids, triglycerides and total cholesterol were found in the red cells stromal. The gasliquid chromatographic analysis of the phospholipid fatty acids showed increased contents of saturated and monounsaturated fatty acids C 16-C 18 smaller contents of longchain highly unsaturated fatty acids. The changes in the fatty acid pattern mainly occurred in patients with Zieve Syndrome and could be important for the mechanism of the hemolytic anemia. Theses findings could be in connexion with vitamin E deficiency.     

An ATP-dependent As(III)-glutathione transport system in membrane vesicles of Leishmania tarentolae

Membrane preparations enriched in plasma membrane vesicles prepared from promastigotes of Leishmania tarentolae were shown to accumulate thiolate derivatives of 73As(III). Free arsenite was transported at a low rate, but rapid accumulation was observed after reaction with reduced glutathione (GSH) conditions that favor the formation of As(GS)3. Accumulation required ATP but not electrochemical energy, indicating that As(GS)3 is transported by an ATP-coupled pump. Pentostam, a Sb(V)-containing drug that is one of the first-line therapeutic agents for treatment of leishmaniasis, inhibited uptake after reaction with GSH. Vesicles prepared from a strain in which both copies of the pgpA genes were disrupted accumulated As(GS)3 at wild-type levels, demonstrating that the PgpA protein is not the As(GS)3 pump. These results have important implications for the mechanism of drug resistance in the trypanosomatidae, suggesting that a plasma membrane As(GS)3 pump catalyzes active extrusion of metal thiolates, including the Pentostam-glutathione conjugate.     

Effect of substituted benzoylglycines (hippurates) and phenylacetylglycines on p-aminohippurate transport in dog renal membrane vesicles

The effect of substituted benzoylglycines (hippurates) and phenylacetylglycines on the transport of p-aminohippurate (PAH) was studied in basolateral (BLMV) and brush border membrane vesicles (BBMV) isolated from dog kidney cortex. The probenecid-sensitive part of 100 microM [3H]PAH uptake into BLMV and BBMV was measured in the presence and absence of 5 mM glycine conjugate. The benzoyl- and phenylacetylglycines studied were substituted in the 2-, 3-, or 4-position with an H, CH3, OCH3 or OH group. Benzoylglycines were stronger inhibitors of PAH transport than phenylacetylglycines and the inhibitory potency of the conjugates was in general lower against the transporter in BBMV than BLMV. The specificities of the transporters in both membranes appear to be very similar. The inhibitory potency of the benzoylglycines, expressed as the apparent inhibition constant (logKi), did not show a linear relationship with their lipophilicity as determined by reversed phase HPLC. Deviation from linearity was caused mainly by the 3-OH and 4-OH analogs, which showed a greater inhibitory potency than expected from their lipophilicity. Phenylacetylglycines only showed a small variation in logKi values, indicating that insertion of a CH2 group between the ring and the carbonyl practically abolishes the influence of the ring and its substituents. In conclusion, both hydrophobic and electronic properties are important determinants of affinity for the PAH transport system. An additional partially negative hydroxyl group in the ring, located preferably at the 3- or 4-position, increases the interaction with the transport system.     

Effect of vitamin D deficiency on lipid composition and calcium transport in basolateral membrane vesicles from chick intestine

Vitamin D deficiency affects the lipid composition and Ca2+ uptake of intestinal basolateral membranes from chick intestine. The increased cholesterol content causes an increase in the molar ratio cholesterol/phospholipid. Phospholipid classes remain unchanged, but the percentages of arachidonic acid from the from the major phospholipid fractions are increased. After 24 hours of oral administration of 2,000 IU of cholecalciferol to vitamin D-deficient chicks, the cholesterol values do not change, but the amount of arachidonic acid returns to normal values. Ca2+ uptake driven by ATP is diminished in vesicles from intestinal basolateral membranes of vitamin D-deficient chicks. Cholecalciferol treatment returns these values to the controls which might be due mainly to the increased number of Ca2+ pump units. In conclusion, changes in lipid composition and in Ca2+ pump caused by vitamin D deficiency seems to play a role in the decrease of vesicular Ca2+ transport. A single dose of cholecalciferol restores only partially the lipid-protein changes produced by vitamin D deficiency.     

Graft decentration in penetrating keratoplasty: nonmechanical trephination with the excimer laser (193 nm) versus the motor trephine

Seven right-handed participants performed bimanual circling movements in either a symmetrical or an asymmetrical coordination mode. Movements were paced with an auditory metronome at predetermined frequencies corresponding to transition frequency, where asymmetrical patterns became unstable, or at two-thirds transition frequency where both symmetrical and asymmetrical patterns were stable. The pacing tones were presented in either a high (1000 Hz) or low (500 Hz) pitch, and the percentage of high-pitched tones during a 20 s trial varied between 0% and 70%. Participants were instructed to count the number of high-pitched pacing tones that occurred during a trial of bimanual circling. Overall, the symmetrical pattern was more stable than the asymmetrical pattern at both frequencies. Errors on the tone-counting task were significantly higher during asymmetrical circling than symmetrical circling but only at the transition movement frequency. The results suggest that cognitive processes play a role in maintaining coordination patterns within regions of instability.     

Dense-cored vesicles, smooth endoplasmic reticulum, and mitochondria are closely associated with non-specialized parts of plasma membrane of nerve terminals: implications for exocytosis and calcium buffering by intraterminal organelles

To determine whether there are anatomical correlates for intraterminal Ca2+ stores to regulate exocytosis of dense-cored vesicles (DCVs) and whether these stores can modulate exocytosis of synaptic vesicles, we studied the spatial distributions of DCVs, smooth endoplasmic reticulum (SER), and mitochondria in 19 serially reconstructed nerve terminals in bullfrog sympathetic ganglia. On average, each bouton had three active zones, 214 DCVs, 26 SER fragments (SERFs), and eight mitochondria. DCVs, SERFs and mitochondria were located, on average, 690, 624, and 526 nm, respectively, away from active zones. Virtually no DCVs were within "docking" (i.e., < or = 50 nm) distances of the active zones. Thus, it is unlikely that DCV exocytosis occurs at active zones via mechanisms similar to those for exocytosis of synaptic vesicles. Because there were virtually no SERFs or mitochondria within 50 nm of any active zone, Ca2+ modulation by these organelles is unlikely to affect ACh release evoked by a single action potential. In contrast, 30% of DCVs and 40% of SERFs were located within 50 nm of the nonspecialized regions of the plasma membrane. Because each bouton had at least one SERF within 50 nm of the plasma membrane and most of these SERFs had DCVs, but not mitochondria, near them, it is possible for Ca2+ release from the SER to provide the Ca2+ necessary for DCV exocytosis. The fact that 60% of the mitochondria had some part within 50 nm of the plasma membrane means that it is possible for mitochondrial Ca2+ buffering to affect DCV exocytosis.     

Kinetic analysis of secretory protein traffic and characterization of golgi to plasma membrane transport intermediates in living cells

Quantitative time-lapse imaging data of single cells expressing the transmembrane protein, vesicular stomatitis virus ts045 G protein fused to green fluorescent protein (VSVG-GFP), were used for kinetic modeling of protein traffic through the various compartments of the secretory pathway. A series of first order rate laws was sufficient to accurately describe VSVG-GFP transport, and provided compartment residence times and rate constants for transport into and out of the Golgi complex and delivery to the plasma membrane. For ER to Golgi transport the mean rate constant (i.e., the fraction of VSVG-GFP moved per unit of time) was 2.8% per min, for Golgi to plasma membrane transport it was 3.0% per min, and for transport from the plasma membrane to a degradative site it was 0.25% per min. Because these rate constants did not change as the concentration of VSVG-GFP in different compartments went from high (early in the experiment) to low (late in the experiment), secretory transport machinery was never saturated during the experiments. The processes of budding, translocation, and fusion of post-Golgi transport intermediates carrying VSVG- GFP to the plasma membrane were also analyzed using quantitative imaging techniques. Large pleiomorphic tubular structures, rather than small vesicles, were found to be the primary vehicles for Golgi to plasma membrane transport of VSVG-GFP. These structures budded as entire domains from the Golgi complex and underwent dynamic shape changes as they moved along microtubule tracks to the cell periphery. They carried up to 10,000 VSVG-GFP molecules and had a mean life time in COS cells of 3.8 min. In addition, they fused with the plasma membrane without intersecting other membrane transport pathways in the cell. These properties suggest that the post-Golgi intermediates represent a unique transport organelle for conveying large quantities of protein cargo from the Golgi complex directly to the plasma membrane.     

Single-laser approach for fluorescence guidance of excimer laser angioplasty at 308 nm: evaluation in vitro and during coronary angioplasty

BACKGROUND AND OBJECTIVE: Spectroscopic guidance of laser angioplasty has been attempted using a diagnostic He-Cd laser in addition to the therapeutic laser system. This study evaluated a single-laser approach for simultaneous ablation and fluorescence excitation. STUDY DESIGN/MATERIALS AND METHODS: A spectroscopy system was coupled to a clinical XeCl excimer laser. Ablation of 162 human aortic samples in saline and blood with 45 mJ/mm2 per pulse yielded 676 fluorescence spectra validated histologically. The same equipment was used in 16 patients for angioplasty of 18 coronary stenoses applying 500 to 1,725 pulses with 45 to 60 mJ/mm2 under saline flushing. A total of 783 spectra were recorded and validated by intracoronary ultrasound (categories: atheroma, fibrous plaque, calcified lesion). RESULTS: In vitro, 5 types of spectra could be differentiated: (1) atheroma, (2) fibrous plaque, (3) calcified lesion in saline, (4) media, and (5) calcified lesion in blood. Discriminant analysis prospectively classified 576 validation spectra with the following sensitivity and specificity for each type: (1) 83.5 and 97.1%, (2) 85.7 and 96.8% (3) 100 and 98.5%, (4) 98.1 and 99.3%, (5) 98.9 and 100%, respectively. In vivo type 1, 2, 3, and 5 spectra were also observed, but not the media spectrum. The predominant sonographic category also prevailed in spectroscopy. Calcified lesions yielded type 3 and 5 as well as mixed spectra. CONCLUSIONS: Using an excimer laser for angioplasty allows combining ablation and fluorescence excitation without a diagnostic laser. Principal types of atherosclerotic lesions and the media can be differentiated spectroscopically with this approach.     

Hydroperoxide-induced increases in intracellular calcium due to annexin VI translocation and inactivation of plasma membrane Ca2+-ATPase

Oxidative stress can cause changes in intracellular free calcium concentration ([Ca2+]i) that resemble those occurring under normal cell signaling. In the alveolar macrophage, hydroperoxide-induced elevation of [Ca2+]i modulates the respiratory burst and other important physiologic functions. The source of Ca2+ released by hydroperoxide is intracellular but separate from the endoplasmic reticulum pool released by receptor-mediated stimuli (Hoyal, C. R., Gozal, E., Zhou, H., Foldenauer, K., and Forman, H. J. (1996) Arch. Biochem. Biophys. 326, 166-171). Previous studies in other cells have suggested that mitochondria are a potential source of oxidant-induced [Ca2+]i elevation. In this study we have identified another potential source of hydroperoxide-releasable intracellular calcium, that bound to annexin VI on the inner surface of the plasma membrane. Translocation of annexin VI from the membrane during exposure to t-butyl hydroperoxide matched elevation of [Ca2+]i as a function of time and t-butyl hydroperoxide concentration. The translocation was possibly due to a combination of ATP depletion and oxidative modification of membrane lipids and proteins. A sustained increase in [Ca2+]i occurring > 50 pmol/10(6) cells (50 microM under these conditions) appeared to be a consequence of membrane Ca2+-ATPase dysfunction. These results suggest that exposure to oxidative stress results in early alterations to the plasma membrane and concomitant release of Ca2+ into the cytosol. In addition it suggests a mechanism for participation of annexin VI translocation that may underlie the alterations in macrophage function by oxidative stress.     

Messenger ribonucleic acid metabolism in mammalian mitochondria. Discrete poly(adenylic acid) lacking messenger ribonucleic acid species associated with mitochondrial polysomes

The mRNA species released from mitochondrial polysomes prepared by the Mg2+ precipitation technique have been further characterized using various analytical techniques. Mitochondrial polysomes were dissociated by treatment with puromycin and chemically labeled with (3H) dimethyl sulfate. About 51% of steady-state mitochondrial mRNA bind to oligo(dT)-cellulose indicating the presence of poly(adenylic acid)(poly(A)) in this fraction. The poly(A)-containing mRNAs resolve into discrete bands of 9-16 Se, while the RNA fraction unable to bind to oligo(dT)-cellulose representing poly(A)-lacking mRNA contains 8-12 Se species. About 90% of poly(A) lacking RNA hybridizes with mitochondrial DNA and less than 7% hybridizes with nuclear DNA. The extent of hybridization of poly(A)-lacking RNA with mitochondrial DNA was not significantly affected by the presence of excess mitochondrial rRNA, cytoplasmic rRNA, or a tenfold concentration of poly(A)-containing RNA isolated from total mitochondrial RNA. Possible differences in sequence properties between poly(A)-containing and -lacking mitochondrial mRNAs were further verified using a solid phase-bound cDNA procedure. Poly(A)-containing mRNA released from mitochondrial polysomes shows over 85% sequance homology with oligo(dT)-cellulose-bound cDNA prepared against total mitochondrial poly(A)-lacking mitochondrial mRNA hybridizes with the cDNA providing direct evidence for the distinct sequence properties of the two mRNA species.     

Adenosine triphosphate-dependent transport of estradiol-17beta(beta-D-glucuronide) in membrane vesicles by MDR1 expressed in insect cells

MDR1, an ABC transporter that confers multidrug resistance in tumor cells, is constitutively expressed in normal liver canalicular membrane. Human MDR1-expressing multidrug-resistant cells display increased resistance to estradiol-17beta(beta-D-glucuronide) (E217G). MDR1 substrates/modulators inhibit adenosine triphosphate (ATP)-dependent transport of E217G in the rat canalicular membrane and protect against E217G-mediated cholestasis in isolated perfused rat liver. The present studies were designed to determine if E217G is a substrate for MDR1 using a baculovirus expression system and if other estrogen glucuronides interact with MDR1. ATP-dependent transport of E217G (10 micromol/L) was linear for up to 2 minutes and yielded a rate of 45.6 pmol/min/mg protein in membrane vesicles from Sf9 cells infected with MDR1-baculovirus. This transport was saturable (Km = 62 micromol/L) and occurred into an osmotically sensitive space. ATP-dependent transport of E217G (10 micromol/L) was inhibited 63% by 10 micromol/L daunomycin, but not by 100 micromol/L S-(2,4-dinitrophenyl)glutathione (GS-DNP) (a substrate for canalicular multispecific organic anion transporter [cMOAT]). Glucuronide conjugates of the estrogen D-ring (100 micromol/L), estriol-17beta(beta-D-glucuronide) (E317G) and estriol-16(beta-D-glucuronide) (E316G), inhibited MDR1-mediated E217G transport by 58% and 35%, respectively. In contrast, noncholestatic glucuronides, estradiol-3-(beta-D-glucuronide) (E23G) or estradiol-3-sulfate-17beta(beta-D-glucuronide) (E23SO417G), had no effect. E217G neither stimulated MDR1 ATPase activity nor inhibited verapamil-stimulated ATPase activity. Infusion of 1.5 micromol/L doxorubicin or 1 micromol/L taxol protected against cholestasis induced by E316G and E317G in isolated perfused rat liver. These studies identify E217G, and probably E316G and E317G, as endogenous substrates for MDR1.     

Green, orange, and red upconversion luminescence in Nd~(3 )/Yb~(3 ): Cs_2NaGdCl_6 powders under 785 nm laser excitation

Nd3 :Cs2NaGdCl6 and Nd3 , Yb3 :Cs2NaGdCl6 polycrystalline powder samples were prepared by Morss method E. Under 785 nm semiconductor laser pumping, the upconversion luminescence of Nd3 ions in Cs2NaGdCl6 was investigated at room temperature, and three upconversion emissions near 538 nm (Green), 603 nm (Orange), and 675 nm (Red) were observed and assigned to 4G7/2→4I9/2, (4G7/2→4I11/2; 4G5/2→4I9/2), and (4G7/2→4I13/2; 4G5/2→4I11/2), respectively. The dependences of these upconverted emissions on laser power and Nd3 ion concentration were investigated, to explore the upconversion mechanism. The effect of doping Yb3 ions on the upconversion luminescence of Nd3 in Cs2NaGdCl6 was also studied under 785 nm laser excitation. The energy transfer processes were discussed as the possible mecha-nism for the above upconversion emissions.     

Irreversible inhibition of plasma membrane (Ca2+ + Mg2+)-ATPase and Ca2+ transport by 4-OH-2,3-trans-nonenal

4-OH-2,3-trans-nonenal (HNE), a major aldehydic lipid peroxidation product, has been shown to cause cellular toxicities and has been linked to a number of pathophysiological processes including atherogenesis. Specifically, in vitro exposure of erythrocyte plasma membrane preparations to HNE resulted in the inhibition of membrane transport function and integrity. To characterize the nature of the inhibitory effects of HNE on plasma membrane regulatory mechanisms, we investigated its effects on substrate and calmodulin (CaM) stimulation on erythrocyte Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities. Concentration-effect relationship analysis in erythrocyte membrane "ghosts" and inside-out vesicles (IOVs) yielded purely noncompetitive kinetics for Ca2+, ATP, and CaM activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport. Reductions of Vmax from direct addition of 0.1 mM HNE to the assay incubation mixtures ranged from 23 to 41%. Similarly, pretreatment with HNE of both membrane ghosts and IOVs resulted in a concentration-dependent inactivation of ATPase and transport activities without changes in affinity for Ca2+, ATP, or CaM. Conversely, pretreatment of CaM itself did not impair its ability to stimulate (Ca2+ + Mg2+)-ATPase activity threefold. Moreover, HNE-pretreated membranes exhibited unaltered acetylcholinesterase activity compared to sham-pretreated membranes. Together, these results suggest that HNE may structurally, and thus irreversibly, modify one or more functionally important sites on the transport protein itself.     

Characterization of the intracellular and the plasma membrane Ca2+-ATPases in fractionated pig brain membranes using calcium pump inhibitors

The Ca2+-ATPase activity of isolated membranes and purified plasma membrane ATPase from pig brain was measured in the presence of specific inhibitors. The inhibition of the enzymatic activity by vanadate presents a lower affinity in microsomes than in the synaptic plasma membrane vesicles, showing K0.5 of 0.4 and 0.2 microM, respectively. The purified enzyme showed a higher sensitivity to vanadate with a K0.5 of 0.10 microM. Thapsigargin (Tg) and 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) were stronger inhibitors of the Ca2+-ATPase activity in microsomes than in the synaptic membrane vesicles. The activity of the purified enzyme was not affected by Tg and only partially by BHQ. Cyclopiazonic acid inhibited the enzymatic activity in all fractions, being more sensitive in microsomes. The microsome preparation incorporated 32P from [gamma-32P]ATP into two main proteins that appear at approx 110,000 and 140,000. According to the inhibition pattern, the lower phosphorylated band was identified as the sarco(endo)plasmic reticulum Ca2+-ATPase, being in a higher percentage than the upper band. Synaptic membrane vesicles also incorporated radioactive 32P into two protein bands. The 140,000 protein (upper band) shows the typical behavior of the purified plasma membrane Ca2+-ATPase, being more abundant in this preparation than the organellar Ca2+-pump (lower band). This study highlights the heterogeneous nature of the Ca2+-ATPase activity measured in brain membrane fractions.