Effects of progesterone on gonadotropin-releasing hormone receptor concentration in cultured estrogen-primed female rat pituitary cells

Acute (0.5–4 h) treatment of estradiol (E)-primed female rat pituitary cells with progesterone (P) augments gonadotropin-releasing hormone (GnRH)-induced LH release, whereas chronic (48 h) P-treatment reduces pituitary responsiveness to the hypothalamic decapeptide. Dispersed E-primed (48 h, 1 nM) rat pituitary cells were cultured for 4 or 48 h in the presence of 100 nM P to assess the effects of the progestagen on GnRH receptors and on gonadotrope responsiveness to the decapeptide. P-treatment (4 h) significantly augmented GnRH-receptor concentrations (4.44 ± 0.6 fmol/10⁶ cells) as compared to cells treated only with E (2.6 ± 0.5fmol/10⁶ cells). Parallel significant changes in GnRH-induced LH secretion were observed. The acute increase in GnRH-receptor number was nearly maximal (180% of receptor number in cells treated with E alone) within 30 min of P addition. Chronic P-treatment (48 h) significantly reduced pituitary responsiveness to GnRH as compared to E-treatment. The GnRH-receptor concentrations (3.9 ± 0.6 fmol/10⁶ cells), however, remained elevated above those in E-primed cells. GnRH-receptor affinity was not influenced by any of the different treatments. These results indicate that the acute facilitatory P-effect on GnRH-induced LH release is at least chronologically closely related to an increase in GnRH-receptor concentration. The chronic negative P-effect on pituitary responsiveness to GnRH, however, shows no relation to changes in available GnRH receptors.     

A major difference between the divergence patterns within the lines-1 families in mice and voles

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.      

Stimulation of adenosine 3':5'-monophosphate formation by prostaglandins in human astrocytoma cells. Inhibition by nonsteroidal anti-inflammatory agents.

Prostaglandins (PG) of the E series and catecholamines stimulate adenosine 3':5'-monophosphate (cAMP) formation in human astrocytoma cells (1321N1). These two classes of effectors activated adenylate cyclase upon interaction with different receptor systems. No evidence for a mediatory role for PG in the action of catecholamines was found. PG interacted with 1321N1 cells with an order of potency of PGE1 = PGE2 greater than PGA1 greater than PGF2 alpha. The effect of combinations of the various PG indicated that all efficacious PG interacted with a common receptor. 7-Oxa-13-prostynoic acid and indomethacin were shown to be competitive inhibitors of the effect of PGE1 with Ki values of 4 and 150 micron, respectively. These two compounds did not inhibit the effect of isoproterenol. Polyphloretin phosphate caused a complex pattern of inhibition of the effects of PGE1 and at higher concentrations also inhibited the effects of isoproterenol. The mefenamate class of nonsteroidal anti-inflammatory agents was found to inhibit the effects of PGE1 with a potency order of meclofenamic acid greater than flufenamic acid = mefenamic acid. The inhibitory action of meclofenamic acid was complex involving specific, but partial, insurmountable antagonism of PGE1 as well as competitive inhibition of PGE1 effects. At higher concentrations of meclofenamic acid a nonspecific inhibition of the effects of both PGE1 and isoproterenol was observed. These studies suggest that the inhibition by nonsteroidal anti-inflammatory agents of the physiological effects of PGE1 in animals may occur, at least in part, at the level of adenylate cyclase. The possibility that multiple classes of adenylate cyclase-linked PGE receptors might exist in nature is discussed.     

Predominant T cell receptor gene elements in TNP-specific cytotoxic T cells.

H-2b class I-restricted, TNP-specific CTL clones were obtained by limiting dilution cloning of either short term polyclonal CTL lines or spleen cells of TNP-immunized mice directly ex vivo. Sequence analyses of mRNA coding for TCR alpha- and beta-chains of 11 clones derived from CTL lines from individual C57BL/6 mice revealed that all of them expressed unique but clearly nonrandom receptor structures. Five alpha-chains (45%) employed V alpha 10 gene elements, and four of those (36%) were associated with J beta 2.6-expressing beta-chains. The alpha-chains from these four TCR, moreover, contained an acidic amino acid in position 93 of their N or J region-determined sequences. Clones isolated directly from spleen cells carried these types of receptors at lower frequency, 27% V alpha 10 and 19% J beta 2.6, indicating that bulk in vitro cultivation on Ag leads to selection for these particular receptors. However, even in TNP-specific CTL cloned directly ex vivo, V alpha 10 usage was increased about fivefold over that in Ag-independently activated T cells in H-2b mice (4 to 5%). The selection for V alpha 10/J beta 2.6-expressing cells was obtained repeatedly in other TNP-specific CTL lines from C57BL/6 mice but not in FITC-specific CTL from the same strain or in TNP-specific CTL lines from B10.BR (H-2k) or B10.D2 (H-2d) mice. We conclude from this (a) that the selection for V alpha 10/J beta 2.6+ T cells is driven by the complementarity of these receptors to a combination of TNP and MHC epitopes and (b) that predominant receptor structures reflect the existence of a surprisingly limited number of "T cell-relevant" hapten determinants on the surface of covalently TNP-modified cells.     

Synthetic peptides anchor T cell-specific TNP epitopes to MHC antigens.

Several TNP-specific, H-2Kb-restricted mouse CTL clones were identified which specifically lysed target cells in the presence of tryptic digests of TNP-modified BSA. Glutaraldehyde fixation of cells revealed that the tryptic fragments did not require further cellular processing. Chromatographic fractionation of digested TNP-BSA identified the peptide TNP-BSA222-231, containing a TNP-modified lysine at BSA position 227, as the antigenic entity. The corresponding synthetic peptide was immunologically cross-reactive with the digest. All clones reactive with TNP-BSA222-231 cross-reacted with a similar peptide from mouse serum albumin (TNP-MSA126-135), favoring the assumption that TNP-BSA222-231 represents an artificial determinant, cross-reacting with some as yet unidentified, TNP-modified, Kb-associated self-peptides. Some of our clones also cross-reacted with tryptic digests of TNP-OVA or TNP-keyhole limpet hemocyanin. We interpret these findings to indicate that 1) a significant proportion of hapten (TNP) determinants for T cells are anchored to MHC via peptides; and 2) the amino acid sequence of these peptides may only partly define the specificity of the T cell-relevant hapten epitope, implying a particularly repetitive nature of these determinants. The production of T cell-antigenic hapten-peptide conjugates will hopefully open new roads to study immune responses to environmental allergens.     

Settlement of the diazotrophic,phytoeffective bacterial strain Pantoea agglomerans on and within winter wheat: An investigation using ELISA and transmission electron microscopy

The aim of this study was to investigate the ability of Pantoea agglomerans, a plant growth-promoting bacterium, to colonize various regions and tissues of the wheat plant (Triticum aestivum L.) by using different inoculation methods and inoculum concentrations. In addition, the enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM) were used to determine: (a) the ability of the bacterial cells to grow and survive both on the surface and within internal tissue of the plant and (b) the response of the plant to bacterial infection. After inoculation, cells of the diazotrophic bacterial strain P. agglomerans were found to be located in roots, stems and leaves. Colony development of bacterial cells was only detected within intercellular spaces of the root and on the root surface. However, single bacterial cells were observed in leaves and stems on the surface of the epidermis, in the vicinity to stomatal cells, within intercellular spaces of the mesophyll and within xylem vessels. Inoculated bacterial cells were found to be able to enter host tissues, to multiply in the plant and to maintain a delicate relationship between endophyte and host. The density of bacterial settlement in the plant in all experiments was about 10⁶ to 10⁷ cells per mL root or shoot sap. Establishment was confirmed by a low coefficient of variation of ELISA means at these concentrations.     

Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo

In higher eukaryotes a quality control system monitoring the folding state of glycoproteins is located in the ER and is composed of the proteins calnexin, calreticulin, glucosidase II, and UDP-glucose: glycoprotein glucosyltransferase. It is believed that the innermost glucose residue of the N- linked oligosaccharide of a glycoprotein serves as a tag in this control system and therefore performs an important function in the protein folding pathway. To address this function, we constructed Saccharomyces cerevisiae strains which contain nonglucosylated (G0), monoglucosylated (G1), or diglucosylated (G2) glycoproteins in the ER and used these strains to study the role of glucose residues in the ER processing of glycoproteins. These alterations of the oligosaccharide structure did not result in a growth phenotype, but the induction of the unfolded protein response upon treatment with DTT was much higher in G0 and G2 strains as compared to wild-type and G1 strains. Our results provide in vivo evidence that the G1 oligosaccharide is an active oligosaccharide structure in the ER glycoprotein processing pathway of S.cerevisiae. Furthermore, by analyzing N- linked oligosaccharides of the constructed strains we can directly show that no general glycoprotein glucosyltransferase exists in S. cerevisiae.