The 70-kilodalton adenylyl cyclase-associated protein is not essential for interaction of Saccharomyces cerevisiae adenylyl cyclase with RAS proteins.

In the yeast Saccharomyces cerevisiae, adenylyl cyclase is regulated by RAS proteins. We show here that the yeast adenylyl cyclase forms at least two high-molecular-weight complexes, one with the RAS protein-dependent adenylyl cyclase activity and the other with the Mn(2+)-dependent activity, which are separable by their size difference. The 70-kDa adenylyl cyclase-associated protein (CAP) existed in the former complex but not in the latter. Missense mutations in conserved motifs of the leucine-rich repeats of the catalytic subunit of adenylyl cyclase abolished the RAS-dependent activity, which was accompanied by formation of a very high molecular weight complex having the Mn(2+)-dependent activity. Contrary to previous results, disruption of the gene encoding CAP did not alter the extent of RAS protein-dependent activation of adenylyl cyclase, while a concomitant decrease in the size of the RAS-responsive complex was observed. These results indicate that CAP is not essential for interaction of the yeast adenylyl cyclase with RAS proteins even though it is an inherent component of the RAS-responsive adenylyl cyclase complex.     

Synthetic oligonucleotides with particular base sequences from the cDNA encoding proteins of Mycobacterium bovis BCG induce interferons and activate natural killer cells.

Thirteen kinds of 45-mer single-stranded oligonucleotide, having sequence randomly selected from the known cDNA encoding BCG proteins, were tested for their capability to augment natural killer (NK) cell activity of mouse spleen cells in vitro. Six out of the 13 oligonucleotides showed the activity, while the others did not. In order to know the minimal and essential sequence(s) responsible for the biological activity, 2 kinds of 30-mer and 5 kinds of 15-mer oligonucleotide fragments of an active 45-mer nucleotide were tested for their activity. One of the 30-mer oligonucleotides, designated BCG-A4a, was active, but the other 30-mer was inactive. All of the 15-mer oligonucleotide fragments were inactive. The BCG-A4a also stimulated the spleen cells to produce interferon (IFN)-alpha and -gamma. An experiment using anti-IFN antisera showed that the NK cell activation by the oligonucleotide was ascribed to the IFN-alpha produced. It was noticed that all of the biologically active oligonucleotides possessed one or more palindrome sequence(s), and the inactive ones did not, with an exception of a 45-mer inactive oligonucleotide containing overlapping palindrome sequences (GGGCCCGGG). These findings strongly suggest that certain palindrome sequences, like GACGTC, GGCGCC and TGCGCA, are essential for 30-mer oligonucleotides, like BCG-A4a, to induce IFNs.     

The utility of side-chain cyclization in determining the receptor-bound conformation of peptides: cyclic tripeptides and angiotensin II.

The effect of side-chain cyclization on accessible backbone conformations of tripeptides, X-Ala-Y (X and/or Y = Cys, Hcy (Hcy: homocysteine), cis 4-mercaptoproline (MPc), and trans 4-mercaptoproline (MPt)), was elucidated using two variants of systematic conformational search. In addition to cyclization through a disulfide bond, the thioether (-S-CH2-) and amide (-CO-NH-) side-chain analogues of Cys-Ala-Cys and Hcy-Ala-Hcy were evaluated. The number of valid backbone conformations and the allowed phi, psi space were evaluated for each compound, and the ability of the cyclic tripeptides to accommodate beta-turn conformations was examined in order to assess the value of cyclization in limiting conformational freedom. Based on the number of conformations, cyclization was highly effective in reducing the backbone degree of freedom: in order of decreasing number of conformations, Ala-Ala-Ala 1 > Hcy-Ala-Hcy 2 > Cys-Ala-Hcy 3 approximately equal to Hcy-Ala-Cys 4 > MPc-Ala-Hcy 5, 7 > Cys-Ala-Cys 6 > MPc-Ala-Cys 8 > Hcy-Ala-MPt 9 > Cys-Ala-MPt 10 approximately equal to MPc-Ala-MPt 11. Although Hcy-Ala-Hcy 2 had the greatest number of conformations of the cyclic peptides studied, it was still greatly constrained relative to its linear analogue 1. The bicyclic ring system introduced by MP was even more effective in constraining the cycle, having greater impact at position 3 than at position 1. Under the conditions of the study, cyclization of MP-containing analogues could be effected only with the cis isomer (MPc) at position 1 and/or the trans isomer (MPt) at position 3. Sterically allowed conformations of Ala2 for the cyclic tripeptides 2-4 were generally similar to those of the linear tripeptide 1, while those of Cys-Ala-Cys 6 and MPc-Ala-Hcy 7 were restricted to a smaller region of phi 2, psi 2 space: the right- and left-handed alpha-helical conformation and the beta-conformation. This trend was even more pronounced for Hcy-Ala-MPt 9, Cys-Ala-MPt 10, and MPc-Ala-MPt 11, in which Ala2 was severely restricted to a very small region of phi, psi space: the left-handed alpha-helical conformation for 9-11, plus the beta conformation for 9. This suggests that MP at the 3-position is incompatible with a right-handed alpha-helical conformation at position 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Small-angle X-ray scattering studies of calmodulin mutants with deletions in the linker region of the central helix indicate that the linker region retains a predominantly alpha-helical conformation

Two mutant forms of calmodulin were examined by small-angle X-ray scattering in solution and compared with the wild-type protein. Each mutant has deletions in the linker region of the central helix: one lacks residues Glu-83 and Glu-84 (Des2) and the other lacks residues Ser-81 through Glu-84 (Des4). The deletions change both the radii of gyration and the maximum dimensions of the molecules. In the presence of Ca2+, the observed radii of gyration are 22.4 A for wild-type bacterially expressed calmodulin, 19.5 A for Des2 calmodulin, and 20.3 A for Des4 calmodulin. A reduction in the radius of gyration by 1-2 A on removal of calcium, previously observed in the native protein, was also found in the wild type and the Des4 mutant; however, no significant size change was observed in the Des2 mutant. The large calcium-dependent conformational change in calmodulin induced by the binding of melittin [Kataoka, M., Head, J.F., Seaton, B.A., & Engelman, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6944-6948] was observed in all the bacterially expressed proteins. Each protein appears to undergo a transition from a dumbbell shape to a more globular conformation on binding melittin in the presence of calcium, although quantitatively the changes in the wild-type and Des4 proteins greatly exceed those in Des2. Modeling shows the central linker region of the molecule. Thus, the structure of the linker region is stable enough to maintain the average orientation and separation of the lobes yet flexible enough to permit the lobes to approach each other upon binding a peptide.     

Purification and characterization of two classes of neurotoxins from the funnel web spider, Agelenopsis aperta

Two classes of paralytic toxins were isolated from the venom of Agelenopsis aperta and their chemical and larvicidal properties characterized. Five acylpolyamine toxins (alpha-agatoxins) of molecular masses 452, 488, 489, 504, and 505 Da produce immediate but reversible paralysis in Manduca sexta following injection. Six insecticidal peptides (mu-agatoxins) produce a gradual but irreversible paralysis. The complete amino acid sequences (36-38 residues) of the mu-agatoxins are presented. These peptides contain eight half-cystines and are quite similar in sequence. At least four of these toxins are amidated at the carboxyl terminus. The secondary structure of one of these toxins (mu-Aga V) was investigated.     

The methanol-induced transition and the expanded helical conformation in hen lysozyme.

Methanol-induced conformational transitions of hen egg white lysozyme were investigated with a combined use of far- and near-UV CD and NMR spectroscopies, ANS binding and small-angle X-ray scattering. Addition of methanol induced no global change in the native conformation itself, but induced a transition from the native state to the denatured state which was highly cooperative, as shown by the coincidence of transition curves monitored by the far- and near-UV CD spectroscopy, by isodichroic points in the far- and near-UV CD spectra and by the concomitant disappearance of individual 1H NMR signals of the native state. The ANS binding experiments could detect no intermediate conformer similar to the molten globule state in the process of the methanol denaturation. However, at high concentration of methanol, e.g., 60% (v/v) methanol/water, a highly helical state (H) was realized. The H state had a helical content much higher than the native state, monitored by far-UV CD spectroscopy, and had no specific tertiary structure, monitored both by near-UV CD and NMR spectroscopy. The radius of gyration in the H state, 24.9 angstroms, was significantly larger than that in the native state (15.7 angstroms). The Kratky plot for the H state did not show a clear peak and was quite similar to that for the urea-denatured state, indicating a complete lack of globularity. Thus we conclude that the H state has a considerably expanded, flexible broken rod-like conformation which is clearly distinguishable from the "molten globule" state. The stability of both N and H states depends on pH and methanol concentration. Thus a phase diagram involving N and H was constructed.     

Impaired Motor Coordination in Mice Lacking Prion Protein

1. Prion protein (PrP^C) is a host-encoded glycoprotein constitutively expressed on the neuronal cell surface. Accumulation of its protease-resistant isoform is closely related to pathologic changes and prion propagation in the brain tissue of a series of prion diseases. However, the physiological role of PrP^C remains to be elucidated.2. After long-term observation, we noted impaired motor coordination and loss of cerebellar Purkinje cells in the aged mice homozygous for a disrupted PrP gene, a finding which strongly suggests that PrP^C plays a role in the long-term survival of Purkinje cells.3. We also describe the resistance of the PrP null mice to the prion, indicating the requirement of PrP^C for both the development of prion diseases and the prion propagation.     

H-Ras signals to cytoskeletal machinery in induction of integrin-mediated adhesion of T cells

The adhesive function of integrins is regulated through cytoplasmic signaling. The present study was performed to investigate the relevance of cytoplasmic signaling and cytoskeletal assembly to integrin-mediated adhesion induced by chemokines. Adhesion of T cells induced by chemokines macrophage inflammatory protein (MIP)-1alpha and MIP-1beta was inhibited by pertussis toxin, wortmannin, and cytochalasin B, suggesting that both G protein-sensitive phosphatidylinositol (PI) 3-kinase activation and cytoskeletal assemblies are involved. The chemokine-induced T cell adhesion could be mimicked by expression of small G proteins, fully activated H-RasV12, or H-RasV12Y40C mutant, which selectively binds to PI 3-kinase, in T cells, inducing activated form of LFA-1alpha and LFA-1-dependent adhesion to ICAM-1. H-Ras expression also induced F-actin polymerization which colocalized with profilin in T cells. Adult T cell leukemia (ATL) cells spontaneously adhered to ICAM-1, which depended on endogenous MIP-1alpha and MIP-1beta through activation of G protein-sensitive PI 3-kinase. H-Ras signal pathway, leading to PI 3-kinase activation, also induced active configuration of LFA-1 and LFA-1-mediated adhesion of ATL cells, whereas expression of a dominant-negative H-Ras mutant failed to do. Profilin-dependent spontaneous polymerization of F-actin in ATL cells was reduced by PI 3-kinase inhibitors. In this paper we propose that H-Ras-mediated activation of PI 3-kinase can be involved in induction of LFA-1-dependent adhesion of T cells, which is relevant to chemokine-mediated signaling, and that profilin may form an important link between chemokine- and/or H-Ras-mediated signals and F-actin polymerization, which results in triggering of LFA-1 on T cells or leukemic T cells.     

Bombyxin secretion in the adult silkmoth Bombyx mori: sex-specificity and its correlation with metabolism

Changes in the hemolymph bombyxin titer of the adult silkmoth Bombyx mori were investigated by time-resolved fluoroimmunoassay. Immediately after eclosion, hemolymph bombyxin titers were low in both males and females, and then increased steeply in males to a very high level and this high titer was maintained for at least 3 h, whereas the titer increment in females was small and transient. The difference in the change of bombyxin titer between males and females suggests that bombyxin is responsible for the regulation of physiological changes underlying sexually different activities of the adult moths. However, no evidence was obtained that bombyxin controls adult metabolism as far as the effects of bombyxin on the concentrations of carbohydrates and lipids in the hemolymph were investigated. The change in the hemolymph trehalose concentration was almost the same between sexes, and between intact and neck-ligated moths. Furthermore, bombyxin injection did not affect the hemolymph trehalose concentration nor trehalase activity in the muscle. Although the hemolymph lipid concentration rose after eclosion in males, it was not influenced by bombyxin. These results exhibit striking contrast to the results of our previous study, in which bombyxin showed hypotrehalosemic activity in the larval stage, thus indicating that the action of bombyxin changes during metamorphosis.