Effects of platelet-activating factor antagonist SRI 63-441 on endotoxemia in sheep

We investigated whether platelet-activating factor (PAF) mediates endotoxin-induced systemic and pulmonary vascular derangements by studying the effects of a selective PAF receptor antagonist, SRI 63-441, during endotoxemia in sheep. Endotoxin infusion (1.3 micrograms/kg over 0.5 h) caused a rapid, transient rise in pulmonary arterial pressure (Ppa) from 16 +/- 3 to 36 +/- 10 mmHg (P less than 0.001) and pulmonary vascular resistance (PVR) from 187 +/- 84 to 682 +/- 340 dyn.s.cm-5 (P less than 0.05) at 0.5 h, followed by a persistent elevation in Ppa to 22 +/- 3 mmHg and in PVR to 522 +/- 285 dyn.s.cm-5 at 5 h in anesthetized sheep. Arterial PO2 (PaO2) decreased from 341 +/- 79 to 198 +/- 97 (P less than 0.01) and 202 +/- 161 Torr at 0.5 and 5 h, respectively (inspired O2 fraction = 1.0). SRI 63-441, 20 mg.kg-1.h-1 infused for 5 h, blocked the early rise in Ppa and PVR and fall in PaO2, but had no effect on the late phase pulmonary hypertension or hypoxemia. Endotoxin caused a gradual decrease in mean aortic pressure, which was unaffected by SRI 63-441. Infusion of SRI 63-441 alone caused no hemodynamic alterations. In follow-up studies, endotoxin caused an increase in lung lymph flow (QL) from 3.8 +/- 1.1 to 14.1 +/- 8.0 (P less than 0.05) and 12.7 +/- 8.6 ml/h at 1 and 4 h, respectively. SRI 63-441 abolished the early and attenuated the late increase in QL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonism by 8-hydroxy-2(di-n-propylamino)tetraline and other serotonin agonists of muscarinic M1-type receptors coupled to inositol phospholipid breakdown in human IMR-32 and SK-N-MC neuroblastoma cells.

IMR-32 and SK-N-MC cells were found to contain [3H]quinuclidinyl benzilate specific binding sites inhibited by pirenzepine in a manner suggesting the presence of both M1-type and M2-type muscarinic receptor recognition sites. Neither cell had detectable [3H]8-OH-DPAT binding sites. Carbachol stimulated the rate of inositol phospholipid breakdown in IMR-32 and SK-N-MC human neuroblastoma cells with an EC50 value of about 50 microM in both cases. Pirenzepine inhibited the carbachol (100 microM)-stimulated inositol phospholipid breakdown in both cells with Hill slopes of unity and IC50 values of 15 nM (IMR-32) and 12 nM (SK-N-MC). The 5-HT1A receptor agonist 8-OH-DPAT competitively inhibited carbachol-stimulated inositol phospholipid breakdown with pA2 values of 5.78 (IMR-32) and 5.61 (SK-N-MC). These values are consistent with the inhibitory potency of 8-OH-DPAT towards [3H]quinuclidinyl benzilate binding in these cells. The 5-HT agonists 5-MeODMT and buspirone at micromolar concentrations inhibited carbachol-stimulated breakdown in IMR-32 cells. The inhibition by 8-OH-DPAT and 5-MeODMT was not affected by preincubation with (-)alprenolol. 5-HT (10-100 microM) was without effect on either basal or carbachol-stimulated breakdown. It is concluded that IMR-32 and SK-N-MC neuroblastoma cells express muscarinic M1-type but not serotoninergic receptors coupled to phosphoinositide-specific phospholipase C. 8-OH-DPAT acts as a weak antagonist at these muscarinic receptors.     

Mutants of Rhodobacter sphaeroides lacking one or more pigment-protein complexes and complementation with reaction-centre, LH1, and LH2 genes

The photosynthetic apparatus of Rhodobacter sphaeroides is comprised of three types of pigment-protein complex: the photochemical reaction centre and its attendant LH1 and LH2 light-harvesting complexes. To augment existing deletion/insertion mutants in the genes coding for these complexes we have constructed two further mutants, one of which is a novel double mutant which is devoid of all three types of complex. We have also constructed vectors for the expression of either LH1, LH2 or reaction-centre genes. The resulting system allows each pigment-protein complex to be studied either as part of an intact photosystem or as the sole complex in the cell. In this way we have demonstrated that reaction centres can assemble independently of either light-harvesting complex in R. sphaeroides. In addition, the isolation of derivatives of the deletion/insertion mutants exhibiting spontaneous mutations in carotenoid biosynthesis provides an avenue for examining the role of carotenoids in the assembly of the photosynthetic apparatus. We show that the LH1 complex is assembled regardless of the carotenoid background, and that the type of carotenoid present modifies the absorbance of the LH1 bacteriochlorophylls.     

The use of limited proteolysis to probe interdomain and active site regions of beta-galactosidase (Escherichia coli)

Limited proteolysis by pancreatic elastase (EC 3.4.21.36) and chymotrypsin (EC 3.4.21.1) was used to study the domain structure and active site of beta-galactosidase (EC 3.2.1.23) (Escherichia coli). Treatment with elastase resulted in a rapid cleavage between residues Ala-732 and Ala-733. No inactivation accompanied this cleavage suggesting that this bond is in a hinge region of the protein. Some slow cleavages beyond the initial one were observed to occur and were accompanied by inactivation. Treatment of beta-galactosidase with chymotrypsin resulted in cleavages first between Trp-585 and Ser-586 and then between Phe-601 and Cys-602. The first of these cleavages resulted in total inactivation of beta-galactosidase. The presence of monovalent ions or isopropyl-beta-D-thiogalactopyranoside protected against the cleavages but when Mg2+ or Mn2+ was present in the reaction mixture, the bond between Trp-585 and Ser-586 was more susceptible to the action of chymotrypsin. These data demonstrate that the conformation of beta-galactosidase around Trp-585 and Ser-586 is dramatically affected by the binding of ions and isopropyl-beta-D-thiogalactopyranoside. The mutant M15 beta-galactosidase, which is missing residues 11 through 41 and is an inactive dimer rather than an active tetramer, was found to be much more labile to proteases than native beta-galactosidase, but the same initial cleavages were found to occur. In addition, trypsin cleaved the M15 protein between Arg-431 and Trp-432 while native beta-galactosidase was stable to trypsin.     

Eukaryotic DNA diverges at a long and complex pyrimidine.purine tract that can adopt altered conformations

A domain exhibiting major sequence divergences among three cloned repeat units of a complex satellite DNA of the Bermuda land crab contains a repetitive polypyrimidine.polypurine segment consisting of a long C.G tract embedded between runs of CCT.AGG and CGCAC.GTGCG and their variations. The domain adopts at least two types of altered conformations that are markedly affected by pH and negative superhelical density; only one is sensitive to ionic strength. Supercoil-dependent distortions in helical structure are most pronounced at points of interruption in compositional bias in this domain and a similar although less extensive, divergent domain nearby. Since the domain is the site of major sequence divergences among individual satellite repeat units, the altered conformations may be involved in site-specific recombination between repeat units, either those arranged in tandem or those scattered throughout the genome.     

Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O

Nile red is a phenoxazone dye that fluoresces intensely, and in varying color, in organic solvents and hydrophobic lipids. However, the fluorescence is fully quenched in water. The dye acts, therefore, as a fluorescent hydrophobic probe. We utilized this novel property of nile red to develop a sensitive fluorescent histochemical stain for tissue lipids. Nile red was prepared by boiling Nile blue A under reflux for 2 hr in 0.5% H2SO4, and extracting the product into xylene. For staining, the purified dye is dissolved in 75% glycerol (1-5 micrograms/ml) and applied to frozen tissue sections. Tissue lipids then fluoresce yellow-gold to red, depending on their relative hydrophobicity. Using sections of liver and aorta from a cholesterol-fed rabbit, we assessed the value of Nile red as a stain for neutral lipids by comparing the staining pattern obtained with that produced by oil red O, a commonly used dye for tissue cholesteryl esters and triacylglycerols. In the cholesterol fatty liver, Nile red staining was comparable to that of oil red O. In contrast, Nile red staining of rabbit aortic atheroma revealed ubiquitous lipid deposits not observed with oil red O staining. These latter results suggest that Nile red can detect neutral lipid deposits, presumably unesterified cholesterol, not usually seen with oil red O or other traditional fat stains.     

Spectrofluorometric studies of the lipid probe, nile red

We found that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, can be applied as a fluorescent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry (J. Cell. Biol. 1985. 100: 965-973). To understand the selectivity of the staining, we examined the fluorescence properties of nile red in the presence of organic solvents and model lipid systems. Nile red was found to be both very soluble and strongly fluorescent in organic solvents. The excitation and emission spectra of nile red shifted to shorter wavelengths with decreasing solvent polarity. However, the fluorescence of nile red was quenched in aqueous medium. Nile red was observed to fluoresce intensely in the presence of aqueous suspensions of phosphatidylcholine vesicles (excitation maximum: 549 nm; emission maximum: 628 nm). When neutral lipids such as triacylglycerols or cholesteryl esters were incorporated with phosphatidylcholine to form microemulsions, nile red fluorescence emission maxima shifted to shorter wavelengths. Serum lipoproteins also induced nile red fluorescence and produced spectral blue shifts. Nile red fluorescence was not observed in the presence of either immunoglobulin G or gelatin. These results demonstrate that nile red fluorescence accompanied by a spectral blue shift reflects the presence of nile red in a hydrophobic lipid environment and account for the selective detection of neutral lipid by the dye. Nile red thus serves as an excellent fluorescent lipid probe.     

Influence of Seismic Stress on Photosynthetic Productivity, Gas Exchange, and Leaf Diffusive Resistance of Glycine max (L.) Merrill cv Wells II

Relative growth rate (RGR), leaf water potential (Ψw), transpiration rate (Tr), photosynthetic rate (Pn), and stomatal and mesophyll resistances to CO₂ exchange were measured or calculated to determine how periodic seismic (shaking) stress decreased dry weight accumulation by soybean (Glycine max [L.] Merrill cv Wells II). Seismic stress was applied with a gyratory shaker at 240 to 280 revolutions per minute for 5 minutes three times daily at 0930, 1430, and 1930 hours. Fifteen days of treatment decreased stem length 21%, leaf area 17%, and plant dry weight 18% relative to undisturbed plants. Seismic stress also decreased RGR 4%, which was due entirely to decreased net assimilation rate. Transpiration decreased 17% and leaf Ψw increased 39% 30 minutes after treatment. A reduction in Pn began within seconds after the onset of treatment and had declined 16% after 20 minutes, at which time gradual recovery began. Assimilation rate recovered fully before the next seismic treatment 5 hours later. Resistance analysis and calculation of leaf internal CO₂ levels indicated that the transitory decrease in Pn caused by periodic seismic stress was due to increased stomatal resistance on the abaxial leaf surface.     

A histochemical study of the changes occurring in the protein-carbohydrate composition of the cumulus-oocyte complex and zona pellucida in immature mice in response to gonadotrophin stimulation

Summary A histochemical account is presented of the changes that occur in the protein—carbohydrate composition of the cumulus—oocyte complex in immature mice after gonadotrophin treatment. The distribution and nature of the glycosaminoglycans (GAG) present was established by enzymic digestion of tissue sections with testicular orStreptomyces hyaluronidase prior to staining with periodic—acid Schiff (PAS) or Alcian Blue. Treatment with exogenous gonadotrophins [pregnant mare's serum and human chorionic gonadotrophin (hCG)] induced gross changes in the appearance of the zona pellucida (and in the histochemical staining of the cumulus—oocyte complex). A reduction was observed in the amount of PAS-positive material present within the zona pellucida of oocytes located in large Graafian follicles examined 40 h after stimulation with pregnant mare's serum. After the injection of hCG, the zona pellucida was further depleted of PAS-positive naterial. Most of the PAS-positive material became confined to the plasma membrane of the oocyte, while the oocyte itself also became increasingly PAS-positive. All the GAGs disappeared from zona pellucida within 4 h of hCG stimulation. The changes observed in the protein—carbohydrate composition of the zona pellucida in preovulatory oocytes immediately prior to ovulation may be a prerequisite for successful sperm-egg interactions.