RNA binding fragments from nucleolin contain the ribonucleoprotein consensus sequence

Nucleolin (C23 or 100 kDa) is a major nucleolar phosphoprotein whose primary structure has recently been determined (Lapeyre, B., Bourbon, H., and Amalric, F. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1472-1476) and found to be associated with preribosomal RNA (Herrera, A. H., and Olson, M. O. J. (1986) Biochemistry 25, 6258-6263). To identify the RNA binding region of the molecule, cyanogen bromide fragments were tested for binding of 18 S and 28 S ribosomal RNA by a "Western blotting" technique. Fragments with apparent molecular masses of 13, 33, and 47 kDa bound RNA with no preference for either 18 S or 28 S RNA. By protein sequencing, these fragments were localized in the carboxyl-terminal two-thirds of the molecule. The nucleolin sequence was searched for the ribonucleoprotein consensus sequence found in other RNA binding proteins. Four copies of a closely related 11-residue sequence were found within 80-90 residue repeats in the RNA binding region between residues 285 and 629. These results suggest that a highly conserved structure for the binding of different classes of RNA is utilized by several proteins.     

Novel aspects of gonadotropin-releasing hormone action on inositol polyphosphate metabolism in cultured pituitary gonadotrophs

The hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) stimulates luteinizing hormone secretion via receptor-mediated activation of phosphoinositide hydrolysis to yield inositol phosphates and diacylglycerol. Application of anion-exchange high-performance liquid chromatography together with absorbance and radiochemical flow detection has enabled both the characterization and quantitative estimation of pituitary cell inositol phosphates and phosphoinositides. In cultured pituitary cells, GnRH caused a rapid and progressive rise in the formation of inositol 1,4,5-trisphosphate and of higher polyphosphoinositols corresponding to inositol tetrakisphosphate, pentakisphosphate, and hexakisphosphate. The inositol 1,4,5-trisphosphate formed during GnRH action was dephosphorylated predominantly via inositol 4-monophosphate rather than the expected metabolite, inositol 1-monophosphate. The catabolism of inositol 4-monophosphate, like that of inositol 1-monophosphate, was inhibited by lithium. For these reasons and because it was the major metabolite of [3H] inositol 1,4,5-trisphosphate in permeabilized gonadotrophs, inositol 4-monophosphate appears to represent a specific marker for ligand-stimulated inositol polyphosphate formation and metabolism. The marked and sustained elevations of inositol 4-monophosphate and inositol 1,4-bisphosphate in GnRH-stimulated gonadotrophs indicate that polyphosphoinositides rather than phosphatidylinositol are the preferred substrates of phospholipase C during GnRH action.     

Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells

Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing have offered an explanation for fibronectin polymorphism. Here we report on monoclonal antibodies that show specificity for a fibronectin segment (ED) that can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Using these monoclonals, we have quantitatively analyzed the expression of the ED sequence in human fibronectin from different sources. The results demonstrated that, at the protein level, the ED segment is not expressed in plasma fibronectin and that, in fibronectin from the tissue culture medium of tumor-derived or simian virus-40-transformed human cells, the percentage of fibronectin molecules containing the ED segment is about 10 times higher than in fibronectin from normal human fibroblasts. These results suggest that in malignant cells the mechanisms that regulate the splicing of mRNA precursors are altered.     

NOTES ON CHINESE ORDOVICIAN AGNOSTIDS

Before 1985 some 54 Ordovician agnostidspecies and subspecies had been described fromChina.They were assigned to the following ele-ven genera and subgenera:Geragnostus,Trinodus,Pseudagnostus,Corrugatagnostus,Pseudoperonop-sis,Girvanagnostus,Geragnostella,Peziziopsis,Sphaeragnostus,Leiagnostus and Micragnostus.Most of them were based on inadequate materialand the range of morphological variation in theseforms largely unknown.After comparison,thepresent author tentatively retain 43 of the 54 spe-cies pendingfu rther material becoming available.They are now reassigned to the following ninegenera:Geragnostus,Arthrorhachis,Corrugatag-     

Ultrastructure of the pineal gland of the tropical bat Rousettus leschenaulti

The ultrastructure of the pineal organ was studied in the tropical megachiropteran Rousettus leschenaulti. The pineal lies deep beneath the hemispheres adjacent to the third ventricle and is traversed by the habenular commissure anteriorly. Its parenchyma consists of a uniform population of light and occasional dark pinealocytes which appear to differ only in the degree of cytoplasmic staining. Pinealocytes are characterized by well developed Golgi bodies associated with numerous small vesicles, many mitochondria and polyribosomes, and frequent subsurface cisternae. Lipid droplets and elements of smooth endoplasmic reticulum are scant. Cisternae of granular endoplasmic reticulum are occasionally dilated. A distinct feature is the abundance of clear vesicles in the pinealocyte pericapillary terminals, which also frequently contain granular vesicles and a very large vacuole. The pineal is further characterized by the presence of a small number of glial cells and myelinated nerve fibers. A broad perivascular space investing numerous capillaries contains glial-cell and pinealocyte processes, collagen fibrils and abundant unmyelinated nerve fibers. Tortuous extensions of the perivascular space enter the pineal parenchyma where they come in close proximity to branched intercellular channels or canaliculi characterized by specialized junctions and microvilli. Differences between the pineal of the non-hibernating megachiropteran Rousettus and that of the hibernating microchiropteran bats, and structural similarities to the pineal of tropical rodents are discussed.     

Antibody for detection and quantitation of membrane-associated folate-binding protein from Lactobacillus casei

Lactobacillus casei cells contain a 25 kDa, membrane-associated, folate-binding protein (fbp), which is a component of the folate transport system. Polyclonal antibody to fbp (anti-fbp) has been prepared, and conditions have been established for detection and quantitation of the protein. Anti-fbp did not block [3H]folate transport or binding in L. casei cells. As judged by Western blots, the antibody reacted only with fbp on sodium dodecyl sulfate electrophoretograms of Triton X-100 extracts of L. casei membranes. Anti-fbp showed no cross-reactivity with L. casei dihydrofolate reductase, L. casei 5,10-methenyltetrahydrofolate synthetase, L1210 dihydrofolate reductase, rat liver dihydrofolate reductase, or L1210 folate-binding protein. Enzyme-linked immunosorbent assay measurements indicated the presence of an fbp in membranes of Lactobacillus salivarius and two transport-defective sublines of L. casei. Anti-fbp was used to demonstrate selective extraction, with n-butanol, of fbp from a mixture of Triton-solubilized L. casei membrane proteins; repression of fbp in membranes of L. casei cells grown on high levels of folate; and localization of fbp by electron microscopy, using anti-fbp in conjunction with goat anti-rabbit IgG gold conjugate, in L. casei membranes.     

Enzymatic transformation of PGH2 to PGF2 alpha catalyzed by glutathione S-transferases

Glutathione S-transferases (GSTs) purified from both rat liver cytosol and microsomes catalyzed the direct reduction of PGH2 to PGF2 alpha. As much as 40% of the substrate was transformed into a prostanoid whose Rf value corresponded to that of PGF2 alpha. The identification of the reaction product as PGF2 alpha was confirmed by TLC and reverse-phase HPLC as well as by mass spectral analysis. In the absence of GSTs, PGH2 was found to be primarily converted to PGE2 and PGD2. Also, PGF2 alpha formation was completely abolished by decylglutathione, a potent inhibitor of both peroxidase and transferase activity associated with GSTs. These results indicate that the direct reduction of endoperoxide moiety of PGH2 to form PGF2 alpha is an enzymatic process. Interestingly, selenium-dependent glutathione peroxidase (Se-GSH-Px) showed very little PGF2 alpha formation from PGH2. However, this enzyme was very active in the reduction of PGG2 to PGH2. In contrast, GSTs were very poor in the conversion of PGG2 to PGH2. Therefore, it is possible that the relative tissue distribution of Se-GSH-Px and GSTs might play an important role in the tissue specific synthesis of PGF2 alpha.     

Magnesium ion effects on microtubule nucleation in vitro

Much interest has currently been attached to the length distribution of microtubules polymerized in vitro and the related question of their possible 'dynamic instability'. Fundamental to this question is the mechanism of microtubule nucleation, which controls the rates of assembly and disassembly of microtubule protein in vitro. These kinetics are affected by a number of factors, including both the guanine nucleotides, GTP and GDP, and magnesium ion. Mg2+ exerts complex effects, as indicated by the existence of an optimal Mg2+ concentration for the maximum assembly rate of microtubule protein, and we investigate these effects in this report. At [Mg2+] greater than 0.5 mM, the characteristic lag-phase is substantially increased and the rate of assembly is greatly reduced without affecting the critical concentration significantly. We show that increasing [Mg2+] has two effects on the assembly process: nucleation is less efficient and the intrinsic rate constant for the elongation reaction is reduced. Lowering [Mg2+] (less than 0.5 mM) also inhibits nucleation. These effects of varying [Mg2+] can be explained predominantly in terms of enhanced stability of the microtubule-associated protein-containing oligomeric species present in the microtubule protein preparation. [Mg2+] is thus found to be a further important factor in microtubule nucleation, and hence, in determining length distributions in assembling microtubules.     

Investigations on the role of Golgi-mediated, ligand-receptor processing in the activation of granulocytes by chemoattractants: differential effects of monensin

Human granulocytes were exposed to different concentrations of the ionophore monensin for 20 min at 37 degrees C. Subsequent exposure to 50 nM of the chemoattractant fMet-Leu-[3H]Phe for up to 30 min at 37 degrees C resulted in a receptor-mediated uptake that was inhibited 80% at a monensin concentration of 30 microM. 50% inhibition was observed at 1-10 microM monensin with no significant change in fMet-Leu-Phe dose dependency. Subcellular fractionation of cells treated with monensin, indicated that the low density UDP-galactosyltransferase activity associated with internalized receptor-fMet-Leu-Phe complexes in untreated cells was absent. The high density galactosyltransferase activity cosedimenting with specific granule markers, however, was unaffected. Monensin also inhibited chemotaxis toward fMet-Leu-Phe as measured by migration of granulocytes through millipore filters and fMet-Leu-Phe induction of polarized morphology. Incubation of cell suspensions with up to 30 microM monensin, both before and during measurement of fMet-Leu-Phe stimulated superoxide production, did not affect the magnitude, kinetics, or transiency of the radical generation. Monensin did, however, shift the dose dependency of superoxide production of fMet-Leu-Phe to higher concentrations. These differential effects of monensin suggest that endocytosis of complexes of the chemoattractant and receptor is not involved in the activation or termination of the fMet-Leu-Phe stimulated superoxide production. They also are consistent with a role for receptor modulation and processing in the chemotactic response.