Identification of a component of rat mononuclear cell SRS as leukotriene D

Slow reacting substance (SRS), produced by rat peritoneal mononuclear cells after stimulation with ionophore A23187, consists of two main components (Bach, M.K. et al. (1979) J. Immunol. 122, 160–165). One of these components was recently identified as leukotriene C-1. The other component has now been identified as leukotriene D.     

Evidence for FUS6 as a component of the nuclear-localized COP9 complex in Arabidopsis.

The pleiotropic CONSTITUTIVE PHOTOMORPHOGENIC (COP), DEETIOLATED (DET), and FUSCA (FUS) loci are essential regulatory genes involved in the light control of seedling developmental patterns in Arabidopsis. Although COP1, DET1, COP9, and FUS6 (also called COP11) have been cloned, their biochemical activities and interactions remain elusive. We have recently suggested that multiple pleiotropic COP, DET, and FUS genes may encode subunits of a large regulatory complex. In this study, we generated specific antibodies against Arabidopsis FUS6 and show that accumulation of both COP9 and FUS6 is coordinated in the pleiotropic cop, det, and fus mutant backgrounds and in wild-type plants throughout development. Both COP9 and FUS6 cofractionated into identical high molecular mass fractions in an analytical gel filtration assay, and neither was found in its monomeric form. Moreover, antibodies raised against either COP9 or FUS6 selectively coimmunoprecipitated both proteins. We have also developed an Arabidopsis protoplast immunolocalization assay and demonstrated that the COP9 complex is localized in the nucleus and that its nuclear localization is not affected by light conditions or tissue types. The integrated genetic and biochemical results strongly support the conclusion that both COP9 and FUS6 are components of the nuclear-localized COP9 complex. Therefore, we have provided the strongest evidence for the conclusion that at least some of the pleiotropic COP, DET, and FUS loci act in the same signaling pathway.     

Evidence for a spectrin-like protein as a major component of the synaptosomal membrane cytoskeleton

After treatment of synaptosomes with Nonidet-containing buffers, a proportion of the proteins remained insoluble. The major component (50%) of the residue was identified as a spectrin-like protein by immunodetection after mono- and bi-dimensional gel electrophoresis and transfer to nitrocellulose paper. Actin was also present.     

Evidence for a cycloheximide-sensitive component in the biological clock of Acetabularia.

An 8-h exposure to cycloheximide (0.1 μg/ml) delays the phase of the photosynthesis rhythm 6–14 h in individual Acetabularia cells monitored at 25 °C, providing the drug is present during the first half of a cell's circadian cycle. Puromycin pulses (50 μg/ml) are like cycloheximide in their effect on phase, but chloramphenicol (100 μg/ml) is ineffective. These results indicate that protein synthesis on 80S ribosomes provides a necessary component for the biochemical mechanism of circadian regulation in Acetabularia.     

Evidence for the participation of aspartate aminotransferase in hepatic glucose synthesis in the suckling newborn rat.

Inhibition of liver aspartate aminotransferase by L-2-amino-4-methoxy-trans-3-butenoic acid in the suckling newborn rat causes a decrease in all gluconeogenic precursors from phosphoenolpyruvate to glucose and an accumulation of lactate but not of pyruvate. This suggests that the aspartate shuttle is operative and confirms the quantitative importance of lactate as a gluconeogenic precursor at this time during development.     

Evidence for a PGE-receptor in the rat kidney.

A membrane preparation derived from homogenates of the rat kidney has been shown to possess a high affinity for prostaglandins of the E-series. Other prostaglandins including PGI2 had characteristic but significantly weaker binding properties. A 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to be associated with the membrane fraction studied. However it was possible to distinguish between this and the "receptor" binding by kinetic studies and by the use of a new inhibitor highly specific for PGDH.     

Experimental disintegration of the nuclear envelope. Evidence for pore- connecting fibrils

The disintegration of the nuclear envelope has been examined in nuclei and nuclear envelopes isolated from amphibian oocytes from amphibian oocytes and rat liver tissue, using different electron microscope techniques (ultrathin sections and negatively or positively stained spread preparations). Various treatments were studied, including disruption by surface tension forces, very low salt concentrations, and nonionic detergents such as Triton C-100 and Nonidet P-40. The highest local stability of the cylinders of nonmembranous pore complex material is emphasized. As progressive disintegration occurred in the membrane regions, a network of fibrils became apparent which interconnects the pore complexes and is distinguished from the pore complex-associated about 15-20 nm thick, located at the level of the inner nuclear membrane, which is recognized in thin sections to bridge the interpore distances. With all disintegraiton treatments a somewhat higher susceptibility of the outer nuclear membrane is notable, but a selective removal does not take place. Final stages of disintegration are generally characterized by the absence of identifiable, membrane- like structures. Analysis of detergent-treated nuclei and nuclear membrane fractions shows almost complete absence of lipid components but retention bo significant amount of glycoproteins with a typical endomembrane-type carbohydrate pattern. Various alternative interpretations of these observations are discussed. From the present observations and those of Aaronson and Blobel (1,2), we favor the notion that threadlike intrinsic membrane components are stabilized by their attachment to the pore complexes, and perhaps also to peripheral nuclear structures,and constitute a detergent-resistant, interpore skeleton meshwork.     

Evidence for common binding sites for ferrichrome compounds and bacteriophage phi 80 in the cell envelope of Escherichia coli.

Mutants ton A and ton B of Escherichia coli K12, known to be resistant to bacteriophage phi80, were found to be insensitive as well to albomycin, an analogue of the specific siderochrome ferrichrome. Ferrichrome at micromolar concentrations strongly inhibited plaque production by phi80. Preincubation with ferrichrome did not inactivate the phage. At a concentration at which ferrichrome allowed 90% inhibition of plaque formation, the chromium analogue of ferrichrome showed no detectable activity. Similarly, ethylenediaminetetraacetic acid, ferrichrome A, and certain siderochromes structurally distinct from ferrichrome, such as ferrioxamine B, schizokinen, citrate, and enterobactin, did not show detectable inhibitory activity. However, rhodotorulic acid showed moderate activity. A host range mutant of phi80, phi80h, was also inhibited by ferrichrome, as was a hybrid of phage lambda possessing the host range of phi80. However, phage lambdacI- and a hybrid of phi80 possessing the host range of lambda were not affected by ferrichrome. Finally, ferrichrome and chromic deferriferrichrome were shown to inhibit adsorption of phi80 to sensitive cells, ferrichrome giving 50% inhibition of adsorption at a minimal concentration of 8 nM. It is suggested that a component of the ferrichrome uptake system may reside in the outer membrane of E. coli K12 and may also function as a component of the receptor site for bacteriophage phi80, and that ferrichrome inhibition of the phage represents a competition for this common site.     

Evidence for circadian rhythmicity in guanyl cyclase of the rat intestinal epithelial cell.

The activity of the enzyme, guanyl cyclase, associated with the rat intestinal brush border membrane, has an endogenous circadian rhythm which is observed in the absence of oral intermittent feeding and of a dark period. This rhythm is cued by the feeding schedule but is essentially unaffected by a light-dark cycle.     

Evidence for a granulosa cell site of dihydrostestosterone metabolism in the adult rat ovary

The ovarian enzyme 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) converts dihydrotestosterone (DHT) to 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol), a reduced androgen that does not bind to the granulosa cell androgen receptor. To determine the relative contribution of the granulosa cells to total ovarian 3 alpha-HSD activity, adult rats treated with either medroxyprogesterone acetate (MPA) or vehicle underwent ovarian microdissection. 3 alpha-Hydroxysteroid dehydrogenase is primarily located in excised follicles and corpora lutea, and is inhibited in the follicles but not corpora lutea by MPA (P less than 0.05). Elimination of healthy granulosa cells while maintaining healthy theca cells by irradiation of the exteriorized ovaries with 6000 rads resulted in a marked reduction in 3 alpha-HSD to 19% of control levels on a per-organ basis (P less than 0.01). The granulosa cell is the major ovarian site for 3 alpha-hydroxylation of ring A-reduced C19 steroids in the adult rat.     

Effects of secA mutations on the synthesis and secretion of proteins in Escherichia coli. Evidence for a major export system for cell envelope proteins

We have followed the synthesis and secretion of a number of periplasmic and outer membrane proteins in three strains of Escherichia coli, a secA amber mutant, a secA temperature-sensitive mutant, and a strain that blocks protein secretion due to a high level of expression of an export-defective hybrid protein between maltose-binding protein and beta-galactosidase (MalE-LacZ). Our results show that after several hours under nonpermissive conditions the specificity and extent of the export blocks in the secA temperature-sensitive mutant and the strain producing the MalE-LacZ hybrid protein are identical, affecting at least four major outer membrane proteins and most but not all periplasmic proteins. The secA gene product, therefore, appears to be an essential component of the major export pathway in E. coli which is used by many envelope proteins independent of whether they are cotranslationally or post-translationally secreted. In contrast, the synthesis of only a subset of these envelope proteins is reduced in the secA amber mutant after shift to the nonpermissive condition. These results indicate that the SecA protein serves roles both in the synthesis and the secretion of certain cell envelope proteins.     

Halococcus morrhuae: a sulfated heteropolysaccharide as the structural component of the bacterial cell wall.

The qualitative and quantitative composition of purifed cell wall of Halococcus morrhuae CCM 859 was determined. Glucose, mannose, galactose; glucuronic and galacturonic acids; glucosamine, galactosamine, gulosaminuronic acid; acetate, glycine and sulfate are found as major constituents. The amino sugars are N-acetylated. It was not possible to fractionate the cell wall in chemically different polymers. Evidence is presented that the major cell wall polymer of this strain is a complex heterolgycan which seems, like the peptidoglycan of most bacteria, to be responsible for the rigidity and stability of the cell wall. In addition it could be proved that this heteroglycan is sulfated and therefore differs considerably from previously described bacterial cell wall polymers.     

Evidence for a separate signal sequence for the carboxy-terminal envelope glycoprotein E1 of Semliki forest virus.

When Semliki Forest virus temperature-sensitive mutant ts-3 was grown at the restrictive temperature an aberrant nascent cleavage of the 130,000-dalton structural polyprotein took place relatively frequently. This cleavage yielded an abnormal 86,000-dalton fusion protein (p86) consisting of the amino-terminal capsid protein linked to the amino acid sequences of envelope protein p62 (a precursor of E3 and E2). The other cleavage product was the carboxy-terminal envelope protein E1. p86 was not glycosylated and was sensitive to the action of protease in the microsomal fraction, whereas E1 was glycosylated and protected from proteases, indicating that it had been segregated into the cysternal side of the microsomal vesicles. All attempts to show the E1 protein at the cell surface have failed so far, suggesting that it remains associated with intracellular membranes. When ts-3-infected cells labeled at the restrictive temperature were shifted to the permissive temperature the only labeled protein released with the virus particles was E1, indicating that E1, synthesized at the restrictive temperature, was competent to participate in the virus assembly. These results suggest strongly that there are two separate signal sequences for the envelope proteins of Semliki Forest virus. One follows the capsid protein as shown previously, and the other is for the carboxy-terminal E1. Even if the insertion of the amino-terminal envelope protein (p62) fails due to a cleavage defect, the other signal sequence can operate independently to guide the E1 through the endoplasmic reticulum membrane.