Synthesis of polymeric models of elastin: Polyhexapeptides and an insoluble,hybrid cross-linked polypeptide

The polymer of the hexapeptide sequence, Val-Ala-Pro-Gly-Glu-Gly, was synthesized and demostrated of exhibit a reversible, pH-dependent coacervation in low-pH aqueous solution. In addition, the synthesis of an insoluble, hybrid, cross-linked polypeptide matrix is described. The cross-linking was achieved in the coacervate state during flow orientation of the polymers. The chemical means of covalent cross-linking was intermolecular primary amide bond formation between the lysyl side chains in one polypentapeptide unit and the glutamyl side chains in another polyhexapeptide unit. The carboxyl activating reagent was a water-soluble carbodiimide. The key intermediates in the syntheses, the hexamers and their high polymers, were analyzed by carbon-13 magnetic resonance to verify the correctness of synthesis and to obtain information on conformation.     

The size of the corpus luteum during pseudopregnancy and pregnancy in the rat.

The corpus luteum in mature Sprague Dawley rats was weighted at the various stages of pseudopregnancy and pregancy. The average size of these corpora lutea was 1.0 +/- 0.10 mg, 1.61 +/- 0.69 mg, 1.90 +/- 0.25 mg, 3.69 +/- 0.36 mg, and 4.37 +/- 0.50 mg on day 2 of diestrus, on days 10-15 of psuedopregnancy, on days 9-10, 14, and 20 of pregnancy, respectively. The fact that the average size of the corpus luteum on days 10-15 of pseudopregnancy was larger than that on day 2 of diestrus is thought to drive from prolonged exposure of the corpus luteum to prolactin. The average size of the corpus luteum on days 9-10 of pregnancy had a tendency to be larger than that on days 10-15 of pseudopregnancy and this seems to demonstrate that the placenta secreted placental lactogen by this stage of pregnancy. The average size of the corpus luteum on day 14 of pregnancy was larger than that on days 9-10 of pregnancy. This phenomenon might be attributed to the presence of large amounts of placental lactogen secreted from the placenta between days 10 and 14 of pregnancy. Furthermore, it was noted that the size of the corpus luteum on day 20 of pregnancy was larger than that of day 14, which suggests that further secretion of placental lactogen continued after day 14 of pregnancy. As there was a remarkable decrease in the number of fetuses on day 20 of pregnancy when overiectomy was performed on day 14 of pregnancy, the ovary was considered indispensable in maintaining pregnancy in the rat.     

Site-directed mutagenesis of hepatitis B surface antigen sequence at codon 160 from arginine to lysine for conversion of subtypic determinant from r to w

Site-directed mutagenesis from G to A was induced at nucleotide 479 in the S gene of hepatitis B virus DNA, cloned from an individual carrying the surface antigen of subtype ayr. HepG2 cells were transfected with the plasmid DNA containing the mutant. They produced surface antigen of subtype ayw, unlike HepG2 cells harboring the parent viral DNA that produced surface antigen of subtype ayr. These results indicate that a point mutation from G to A at nucleotide 479 in the S gene, changing codon 160 for arginine to that for lysine, can convert the subtypic determinant of hepatitis B surface antigen from r to its allelic determinant w.     

Continuous requirement of cAMP for pre-spore differentiation in Dictyostelium discoideum

Abstract Using a shaking culture system, we have previously shown that both cell contact and cAMP are required for pre-spore differentiation in Dictyostelium discoideum [2]. In the present study, cAMP was removed from the medium by the use of a hydrolysing enzyme after cells had formed agglomerates. This treatment left the agglomerates unchanged, but caused a rapid decrease in the activity of UDP galactose transferase, a pre-spore-specific enzyme. This result indicates that cAMP is required even after agglomerate formation to maintain pre-spore differentiation.     

Direct control of exocytosis by receptor-mediated activation of the heterotrimeric GTPases Gi and G(o) or by the expression of their active G alpha subunits.

The exocytotic release of potent hormones is a tightly controlled process. Its direct regulation without the involvement of second messengers would ensure rapid signal processing. In streptolysin O-permeabilized insulin-secreting cells, a preparation allowing dialysis of cytosolic macromolecules, activation of alpha 2-adrenergic receptors caused pertussis toxin-sensitive inhibition of calcium-induced exocytosis. This inhibition was mimicked very efficiently by the use of specific receptor-mimetic peptides, indicating the involvement of Gi and, to a lesser extent, of G(o). The regulation was exerted beyond the ATP-dependent step of exocytosis. In addition, low nanomolar amounts of pre-activated Gi/G(o) directly inhibited exocytosis. As transient overexpression of constitutively active mutants of G alpha i1, G alpha i2, G alpha i3 and G alpha o2 but not of G alpha o1 reproduced this regulation, the G alpha subunit alone is sufficient to induce inhibition. These results define exocytosis as an effector for heterotrimeric G-proteins and delineate the properties of the transduction pathway.     

Detection of G protein-activator regions in M4 subtype muscarinic, cholinergic, and alpha 2-adrenergic receptors based upon characteristics in primary structure.

The 14-residue region Arg2410-Lys2423 of the human insulin-like growth factor II receptor possesses the ability to stimulate Gi, the activity being dependent on two structural characteristics: (i) at least two basic residues at the N-terminal side and (ii) the C-terminal motif, B-B-X-B or B-B-X-X-B (where B is a basic residue and X is a non-basic residue). The regions satisfying (i) and (ii) with 10 less than or equal to residue length less than or equal to 26 were located in all of the third inner loops and some of the other intracellular domains of the Gi-coupled M4 sub-type muscarinic cholinergic receptor (M4AChR) and the alpha 2-adrenergic receptor (alpha 2AR). Both the second inner loop 130-147 and the C-terminal portion of the third inner loop 382-400 (MIII) of human M4AChR had the ability to stimulate G proteins with the order Gi approximately Go greater than Gs, but only MIII could activate Gi/Go at nanomolar concentrations. In contrast, the N-terminal portion of the third inner loop 218-228 of human alpha 2AR-C10 activated Gi, Go, and Gs at micromolar concentrations with equal potency, whereas the further C-terminal portion of the third inner loop 301-313 of this receptor lacked the ability to activate any G protein. Among these active regions, only MIII indicated Mg(2+)-dependent Gi-stimulating function. Therefore, the search for the regions satisfying (i) and (ii) was useful to localize the G protein-activating activity of Gi-coupled receptors in limited regions, which were not always in the C-terminal portions of the third intracellular loops and activated G proteins in various modes of actions.     

Wortmannin, a PI3-kinase inhibitor: promoting effect on insulin secretion from pancreatic beta cells through a cAMP-dependent pathway

To determine the role of phosphatidylinositol 3-kinase (PI3-kinase) in the regulation of insulin secretion, we examined the effect of wortmannin, a PI3-kinase inhibitor, on insulin secretion using the isolated perfused rat pancreas and freshly isolated islets. In the perfused pancreas, 10(-8) M wortmannin significantly enhanced the insulin secretion induced by the combination of 8.3 mM glucose and 10(-5) M forskolin. In isolated islets, cyclic AMP (cAMP) content was significantly increased by wortmannin in the presence of 3.3 mM, 8.3 mM, and 16.7 mM glucose with or without forskolin. In the presence of 16.7 mM glucose with or without forskolin, wortmannin promoted insulin secretion significantly. On the other hand, in the presence of 8.3 mM glucose with forskolin, wortmannin augmented insulin secretion significantly; although wortmannin tended to promote insulin secretion in the presence of glucose alone, it was not significant. To determine if wortmannin increases cAMP content by promoting cAMP production or by inhibiting cAMP reduction, we examined the effects of wortmannin on 10(-4) M 3-isobutyl-1-methylxantine (IBMX)-induced insulin secretion and cAMP content. In contrast to the effect on forskolin-induced secretion, wortmannin had no effect on IBMX-induced insulin secretion or cAMP content. Moreover, wortmannin had no effect on nonhydrolyzable cAMP analog-induced insulin secretion in the perfusion study. These data indicate that wortmannin induces insulin secretion by inhibiting phosphodiesterase to increase cAMP content, and suggest that PI3-kinase inhibits insulin secretion by activating phosphodiesterase to reduce cAMP content.     

Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase

The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.     

ALDH2 polymorphism for the risk of cervical carcinogenesis

To investigate the clinical significance of ALDH2 genetic polymorphisms in cervical carcinogenesis. ALDH2 polymorphisms together with human papillomavirus (HPV) types were examined in a total of 195 cervical smear in exfoliated cervical cell samples using Real-Time polymerase chain reaction (PCR) System. The frequency for the AG+AA genotype was seven in the normal group (70.0 %), 16 in the LSIL group (57.1 %), and 27 in the HSIL group (90.0 %). A significant difference was found between the LSIL and HSIL groups (P = 0.0064). Patients with HSIL lesions frequently had high-risk HPV infections and concurrently belonged to the AG+AA group. ALDH2 genotype in cervical cell samples may be associated with more severe precancerous lesions of the cervix in a Japanese population.