Stimulation of rat luteinizing hormone-beta messenger RNA levels by gonadotropin releasing hormone. Apparent role for protein kinase C

The ability of gonadotropin releasing hormone (GnRH) to elevate cellular levels of mRNA for beta-subunit of luteinizing hormone (LH) has been examined in monolayer cultures from rat pituitary. Low concentrations of GnRH (100 pM) induced a 6.8-fold increase in LH-beta mRNA, while higher concentrations of GnRH were less effective. The low concentrations of GnRH (100 pM) did not result in altered GnRH receptor levels (92 +/- 12% compared to controls) after 24 h treatment but did increase protein kinase C activity to 249 +/- 16%. The protein kinase C activator, phorbol 12-myristate 13-acetate, at concentrations (2-20 nM) which did not deplete protein kinase C, stimulated LH-beta mRNA levels 2-5-fold after 24 h. Higher concentrations of phorbol 12-myristate 13-acetate, which depleted protein kinase C activity, substantially reduced the ability of 100 pM GnRH to stimulate increases in LH-beta mRNA levels. As previously observed, protein kinase C-depleted cells exhibited normal LH release in response to GnRH stimulation. These studies demonstrate that low concentrations of GnRH may have an important role in regulation of gonadotropin biosynthesis. Furthermore, the results suggest that activation of protein kinase C is sufficient to stimulate increases in LH-beta mRNA levels and that protein kinase C is necessary for normal GnRH stimulation of LH-beta mRNA levels. Accordingly, we postulate that protein kinase C may mediate the action of GnRH on LH-beta mRNA levels.     

Isolation and characterization of a variant somatostatin-14 and two related somatostatins of 34 and 37 residues from lamprey (Petromyzon marinus)

Three major forms of somatostatin were isolated from pancreas of the lamprey (Petromyzon marinus). One of the two major forms is a 14-residue somatostatin (SS-14) having the sequence AGCKNFFWKTFSSC. The homologous substitution Ser for Thr in position 12 is the first example of SS-14 from vertebrate preprosomatostatin gene I having a divergent sequence. The longest form is 37 residues in length (SS-37) and has the sequence ALRAAAVAGSPQQLLPLGQRERKAGCKNFFWKTFSSC. A 34-residue form (SS-34) identical in sequence but truncated at a single Arg residue at position 3 of SS-37 was also isolated. The yields of the three forms were SS-37 (0.43 nmol/g), SS-34 (134 nmol/g), and SS-14 (51.5 nmol/g). The identification of this nested series of somatostatins suggests that prosomatostatin processing in lamprey more closely resembles that observed for procholecystokinin than that of mammalian or other piscine prosomatostatins. Somatostatin-producing cells in the lamprey pancreas were identified by immunostaining using antiserum against SS-34 and anti-serum against mammalian SS-14.     

Genetic polymorphism of alpha 2HS-glycoprotein.

A genetic polymorphism of the human serum glycoprotein, alpha 2HS-glycoprotein, can be recognized using isoelectric focusing in polyacrylamide, followed by silver-stain immunofixation. In a North American Caucasian population, two common alleles and one rare allele have been recognized, with frequencies as follows: AHSG*1: .6419, AHSG*2: .3535, and AHSG*3: .0046; polymorphism information content (PIC): .36. A black population from various islands of the Caribbean has the two most common alleles, plus a variant (B) not found in the white population. Allele frequencies in the blacks were: AHSG*1: .6901, AHSG*2: .2606, AHSG*B: .0493; PIC: .396. Family studies confirmed the allele designations. Alleles in both populations were in Hardy-Weinberg equilibrium. This polymorphism will be useful as a marker on chromosome 3q and for forensic studies. The serum concentration associated with AHSG*1 may be somewhat greater than that associated with AHSG*2. Differences between the allele products remained after removal of sialic acid from the glycoprotein with neuraminidase. The silver-stain immunofixation technique used for this polymorphism has wide application for the study of polymorphisms where the protein is present in low concentration or where only low titer antiserum is available.     

Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein

After synthesis on membrane-bound ribosomes, the variant surface glycoprotein (VSG) of Trypanosoma brucei is modified by: (a) removal of an N-terminal signal sequence, (b) addition of N-linked oligosaccharides, and (c) replacement of a C-terminal hydrophobic peptide with a complex glycolipid that serves as a membrane anchor. Based on pulse-chase experiments with the variant ILTat-1.3, we now report the kinetics of three subsequent processing reactions. These are: (a) conversion of newly synthesized 56/58-kD polypeptides to mature 59-kD VSG, (b) transport to the cell surface, and (c) transport to a site where VSG is susceptible to endogenous membrane-bound phospholipase C. We found that the t 1/2 of all three of these processes is approximately 15 min. The comparable kinetics of these processes is compatible with the hypotheses that transport of VSG from the site of maturation to the cell surface is rapid and that VSG may not reach a phospholipase C-containing membrane until it arrives on the cell surface. Neither tunicamycin nor monensin blocks transport of VSG, but monensin completely inhibits conversion of 58-kD VSG to the mature 59-kD form. In the presence of tunicamycin, VSG is synthesized as a 54-kD polypeptide that is subsequently processed to a form with a slightly higher Mr. This tunicamycin-resistant processing suggests that modifications unrelated to N-linked oligosaccharides occur. Surprisingly, the rate of VSG transport is reduced, but not abolished, by dropping the chase temperature to as low as 10 degrees C.     

Binding and internalization of heparin by vascular smooth muscle cells

Previous work from our laboratory has demonstrated that heparin specifically inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. In this paper, we examine the binding and mode of internalization of heparin by smooth muscle cells. For these studies, radiolabeled and fluoresceinated (FITC) heparin probes were synthesized that retained their antiproliferative capacity. Binding of 3H-heparin to these cells occurs via specific, high-affinity binding sites (Kd = 10(-9) M, 100,000 binding sites per cell). Approximately 80% of the heparin bound to the cell surface was shed into the culture medium within 2 hr. The heparin that was left on the cell surface was internalized with biphasic kinetics. Approximately 50% of the bound material was internalized within 2 hr. After this initial rapid uptake, the rate slowed substantially, with the remaining heparin requiring 1-2 days to be internalized. Binding and uptake of FITC heparin was monitored using video image intensification fluorescence microscopy. When smooth muscle cells were exposed to FITC heparin at 4 degrees C, a diffuse surface staining pattern was observed. After warming the cells to 37 degrees C, intensely fluorescent vesicles were seen superimposed over the diffuse surface staining within 2 min. After 15 min at 37 degrees C, numerous large punctate vesicles were seen inside the cell. After 2 hr these vesicles had concentrated in the perinuclear region. This pattern of uptake, when considered along with the presence of specific, high-affinity binding sites and the initial rapid uptake of 3H-heparin, suggests that heparin enters smooth muscle cells by both receptor-mediated and other endocytic pathways.     

Genotype dependent variation in mycorrhizal colonization and response to inoculation of pearl millet

Summary Genotypes of pearl millet (Pennisetum americanum L. Leeke) were examined for differences in vesicular-arbuscular mycorrhizal (VAM) colonization and response to inoculation. For thirty genotypes tested across three field locations there was a range of mycorrhizal colonization intensity between 25 and 56%. In another experiment with two male-sterile lines, restorer lines and their derived crosses, grown in pots filled with non-sterilized soil there were significant differences between genotypes for colonization by mycorrhiza. This showed hostgenotype dependence for mycorrhizal colonization.Root growth rates, mycorrhizal root length, percentage root colonization and plant growth and P uptake were studied in ten genotypes. A set of 3 genotypes with similar root lengths varied significantly with regard to mycorrhizal root length and the percentage colonization. This supports the suggestion that VAM colonization and spread is dependent on the host genotype. The growth responses differed significantly between the genotypes and they also differed in their responses to P uptake and VAM inoculation. The utility of host-genotype dependent differences in VAM symbiosis in plant breeding is discussed.Journal Article No. 453     

A Mouse β-Globin Mutant That Is an Exact Model of Hemoglobin Rainier in Man

A mutation induced by ethylnitrosourea in a spermatogonial stem cell of a 101/H mouse has resulted in a structurally altered beta-diffuse major globin in one of his offspring. The mutant hemoglobin is associated with polycythemia, rubor, increased oxygen affinity and decreased hem-hem interaction. The mutant haplotype has been designated Hbbd4, polycythemia. Amino acid analysis of the mutant globin has shown that a single substitution beta 145 Tyr----Cys has occurred, and it is proposed that ethylnitrosourea induced an A----G transition in the tyrosine codon (TAC----TGC). This murine polycythemia is homologous with hemoglobin Rainier in man, in which the amino acid substitution is also beta 145 Tyr----Cys and which is associated with similar physiological consequences.     

Genetic analysis of Escherichia coli oligopeptide transport mutants.

The composition of the outer membrane channels formed by the OmpF and OmpC porins is important in peptide permeation, and elimination of these proteins from the Escherichia coli outer membrane results in a cell in which the primary means for peptide permeation through this cell structure has been lost. E. coli peptide transport mutants which harbor defects in genes other than the ompF/ompC genes have been isolated on the basis of their resistance to toxic tripeptides. The genetic defects carried by these oligopeptide permease-negative (Opp-) strains were found to map in two distinct chromosomal locations. One opp locus was trp linked and mapped to the interval between att phi 80 and galU. Complementation studies with F'123 opp derivatives indicated that this peptide transport locus resembles that characterized in Salmonella typhimurium as a tetracistronic operon (B. G. Hogarth and C. F. Higgins, J. Bacteriol. 153:1548-1551, 1983). The second opp locus, which we have designated oppE, was mapped to the interval between dnaC and hsd at 98.5 min on the E. coli chromosome. The differences in peptide utilization, sensitivity and resistance to toxic peptides, and the L-[U-14C]alanyl-L-alanyl-L-alanine transport properties observed with these Opp-E. coli strains demonstrated that the transport systems encoded by the trp-linked opp genes and by the oppE gene(s) have different substrate preferences. Mutants harboring defects in both peptide transport loci defined in this study would not grow on nutritional peptides except for tri-L-methionine, were totally resistant to toxic peptides, and would not actively transport L-[U-14C]alanyl-L-alanyl-L-alanine.     

Isolation and structures of glucagon and glucagon-like peptide from catfish pancreas

Both glucagon and the structurally similar glucagon-like peptide proteolytically derived from preproglucagon were purified from the endocrine pancreas of the channel catfish (Ictalurus punctata). This study represents the first report of the isolation of glucagon-like peptide from any source. Peptide sequences of glucagon-like peptide from other species have only been deduced from the cDNA sequences for preproglucagon. The sequence of the 34-residue glucagon-like peptide was found to be HADGTYTSDVSSYLQDQAAKDFITWLKSGQPKPE. Catfish glucagon-like peptide shares sequence identity at 26 of 31 residues with the putative glucagon-like peptide from anglerfish preproglucagon II. The mass of catfish glucagon-like peptide was found by fast atom bombardment-mass spectrometry to be 3785, identical with the value predicted by sequence analysis. This suggests that no post-translational modification occurs beyond proteolytic processing. The sequence of catfish glucagon was determined to be HSEGTFSNDYSKYLETRRAQDFVQWLM(N,S). Catfish glucagon exhibits a high degree of immunologic similarity with porcine glucagon by radioimmunoassay, whereas catfish glucagon-like peptide does not.     

The FLP recombinase of the 2 micron circle DNA of yeast: interaction with its target sequences

We have studied the interaction of purified FLP protein with restriction fragments from the substrate 2mu circle DNA of yeast. We find that FLP protects about 50 bp of DNA from nonspecific nuclease digestion. The protected site consists of two 13 bp inverted repeat sequences separated by an 8 bp spacer region. A third 13 bp element is also protected by binding of the FLP protein. We demonstrate that FLP introduces single- and double-strand breaks into the substrate DNA. This site-specific cleavage occurs at the margins of the spacer region, generating 8 bp 5' protruding ends with 5'-OH and 3'-protein-bound termini. Binding to mutant sites and half-sites demonstrates that the third symmetry element is not important for binding and cleavage by the FLP protein. The integrity of the core region is important for the cleavage activity of FLP.