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Ionic actuators have attracted much attention due to their remarkably large strain under low-voltage stimulation. Here, we investigate a highly biocompatible ionic polymer actuator, which consists of multi-walled carbon nanotube (MCNT) film as a double electrode layer and an electrolyte layer equipped with chitosan polymer skeleton and ionic liquid. As a result, with the thickness increase of the electrolyte layer and the electrode layer, the membrane capacitance values are obviously improved, which are 0.01 F (membrane thickness of 1.3 mm) and 0.4 F (0.25 mm). The blocking force and its response speed show peak values of 5.75 mN (1.1 mm) and 5.1 mN (0.25 mm), while reverse increases for the displacement and its response speed are observed, which present maximum values of 10.3 mm (0.3 mm) and 13.3 mm (0.15 mm). Furthermore, for various thicknesses of the electrode layers under applied direct current of 5 V, the generated strain of 0.15 mm thickness (59%) is 4.92 times greater than that of the 0.25 mm thickness. This is against the strain difference on the electrode surface due to the growing stiffness of the electrode layer. Additionally, from the experiments of the electromechanical energy efficiency of various electrode layers and electrolyte layers, our actuator exhibits excellent electromechanical energy efficiency under a high electrical conductivity of the electrode layer, which enhance the specific electromechanical energy up to 9.95%.

Fos family proteins form stable heterodimers with Jun family proteins, and each heterodimer shows distinctive transactivating potential for regulating cellular growth, differentiation, and development via AP-1 binding sites. However, the molecular mechanism underlying dimer specificity and the molecules that facilitate transactivation remain undefined. Here, we show that BAF60a, a subunit of the SWI.SNF chromatin remodeling complex, is a determinant of the transactivation potential of Fos/Jun dimers. BAF60a binds to a specific subset of Fos/Jun heterodimers using two different interfaces for c-Fos and c-Jun, respectively. Only when the functional SWI.SNF complex is present, can c-Fos/c-Jun (high affinity to BAF60a) but not Fra-2/JunD (no affinity to BAF60a) induce the endogenous AP-1-regulated genes such as collagenase and c-met. These results indicate that a specific subset of Fos/Jun dimers recruits SWI.SNF complex via BAF60a to initiate transcription.

High frequency, medium power gyrotrons (Gyrotron FU series) have been developed at the Fukui University as radiation sources convering a broad frequency band from millimeter to submillimeter wave region. We have performed cw operation of the Gyrotron FU IV. However, the output power was not stable (approximately several tens %) due to the fluctuation of the cathode potential (∼40 V). The output power was stabilized within several percent by controlling the anode potential. Such a technique was also used for amplitude modulation of the gyrotron output. Improved stabilization of output power and effective modulation will be achieved by stabilizing the cathode potential.

We examined the transduction efficiency of a VSV-G (vesicular stomatitis virus G protein)-pseudotyped vector encoding β-galactosidase (lacZ) into human solid tumor cell lines and murine fibroblasts, compared with that of an amphotropic vector carrying the same RNA sequence. The ratio of cells transduced with the VSV-G-pseudotyped vector corresponded closely to 1 − e−m.o.i., as predicted from a Poisson distribution of transduction to the entire cellular population, while this was not the case for the amphotropic vector. Here m.o.i. (multiplicity of infection) is defined as the ratio of input infectious units (titrated on the corresponding cell line) to the number of cells used for the transduction. At high m.o.i.s (values greater than 3), the VSV-G-pseudotyped vector transduced approximately 95% of the culture population of all cell lines examined. The transduction efficiency of the amphotropic vector, however, was not dose-dependent and reached a plateau or even decreased, especially at high m.o.i.; this may be attributable at least in part to the presence of envelope protein and noninfectious particles that compete for the receptor of infectious amphotropic virus. The copy number of integrated vector proviral DNA and the expression level of lacZ increased almost linearly with the dose of the VSV-G-pseudotyed vector, which could readily achieve multiple transduction of more than 10 copies per cell and afforded about 100-fold more transgene product than could be achieved with the amphotropic vector. These features of both the VSV-G-pseudotyped vector and the amphotropic vector were essentially unaffected by purification using centrifugation. These properties of the vector should be highly advantageous for gene transfer into entire populations of human tumor cell lines at a designed dosage.

We report here on stable prepackaging cell lines which can be converted into packaging cell lines for high-titer vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors by the introduction of Cre recombinase-expressing adenovirus. The generated prepackaging cell lines constitutively express the gag-pol genes and contain an inducible transcriptional unit for the VSV-G gene. From this unit, the introduced Cre recombinase excised both a neomycin resistance (Neo r ) gene and a poly(A) signal flanked by a tandem pair of loxP sequences and induced transcription of the VSV-G gene from the same promoter as had been used for Neo r expression. By inserting an mRNA-destabilizing signal into the 3′ untranslated region of the Neo r gene to reduce the amount of Neo r transcript, we were able efficiently to select the clones capable of inducing VSV-G at high levels. Without the introduction of Cre recombinase, these cell lines produce neither VSV-G nor any detectable infectious virus at all, even after the transduction of a murine leukemia virus-based retrovirus vector encoding β-galactosidase. They reproducibly produced high-titer virus stocks of VSV-G-pseudotyped retrovirus (1.0 × 10 6 infectious units/ml) from 3 days after the introduction of Cre recombinase. We also present evidence that VSV-G-producing cells are still fully susceptible to transduction by VSV-G pseudotypes. However, in this vector-producing system, which regulates VSV-G pseudotype production in an all-or-none manner, the integration of vector DNA into packaging cell lines would be minimized. We further show that heparin significantly inhibits retransduction of VSV-G pseudotypes in the culture fluids of packaging cell lines, leading to a two- to fourfold increase in the yield of the pseudotypes after induction. This vector-producing system was very stable and should be advantageous in human gene therapy.

Spontaneously activated MDV is rarely included in MDV-transformed cells, while it may influence the results of transcriptional analysis. A population consisting of 10(3) MDV-transformed cells probably did not include spontaneously activated MDV, since the estimated frequency of MDV-transformed cells including activated MDV was below 0.01% according to limiting-dilution polymerase chain reaction (PCR) and the presence of the major early antigen pp38 in 6 transformed cell lines. Reverse transcriptase-PCR (RT-PCR) products corresponding to ICP27, pol, TK, US3, A41, gA, gB and UL50 genes were undetectable in 10(3) cells by Southern hybridization of the RT-PCR products. Transcripts of the VP16 and SORF2 genes were detected in the 10(3) cells of MSB-1, and the pp14 gene transcript was found in 10(3) cells of RPL-1 but not in 10(3) cells of HPRS-1, MOGA-2, MSB-1 or MTB-1. A transcript corresponding to the ICP4 sequence was detected as a 0.7 kbp RT-PCR product in 10(3) cells of these MDV cell lines but not in the retrovirus-transformed 1104B1 cell line. The transcript corresponding to the 0.7 kbp RT-PCR product suggested a splice by its size and sequence. Thus, transcriptional analysis of 10(3) MDV-transformed cells revealed that the transcript corresponding to the ICP4 sequence was a common transcript in latently infected MDV-transformed cells, while most of the genes did not transcribe in these cells.

An exact solution is obtained for the problem of the radiation of sound from a semi-infinite duct, one plate of which is hard and the other soft. The solution of the problem results in a matrix Wiener–Hopf problem, which is solved by carrying out an exact matrix factorization. Some numerical results are given for the reflection coefficient, when the lowest mode propagates. The problem can be considered as a first approximation to an acoustically lined duct. Resume : Nous obtenons une solution exacte pour la radiation sonore d’un conduit semi-infini dont un cote duquel est dur alors que l’autre est mou. La solution du probleme passe par le probleme matriciel de Wiener-Hopf qui est resolu par factorisation matricielle exacte. Nous donnons quelques resultats numeriques pour le coefficient de reflexion lors de la propagation du mode fondamental. La solution de ce probleme est une premiere approximation dans la description d’un conduit avec enduit interne acoustique. [Traduit par la redaction]

It has been shown that inhibition of phosphatidylinositol (PI) 3-kinase blocks neurite outgrowth of PC12 cells stimulated with nerve growth factor. To further assess the role of PI 3-kinase, the active form of PI 3-kinase was expressed in PC12 cells by the adenovirus mediated introduction of a site-specific recombinase, Cre. After expression of the active PI 3-kinase, elevation of the levels of PI 3,4-diphosphate and PI 3,4,5-trisphosphate as well as formation of neurite-like processes was observed. The process formation was inhibited by wortmannin, a selective inhibitor of PI 3-kinase, which suggests that a high activity of PI 3-kinase was responsible for the formation of these processes. The processes lacked accumulation of F-actin and GAP43 at the growth cone, which suggests that the processes were incomplete compared with neurites. Instead, the bundling of microtubules was enhanced, which suggests that organization of the microtubules might be driving the process of elongation in the cells expressing the active PI 3-kinase. Induction of active PI 3-kinase resulted in activation of Jun N-terminal kinase but not of mitogen-activated protein kinase or protein kinase B/Rac protein kinase/Akt. These results suggest that PI 3-kinase is involved in neurite outgrowth in PC12 cells and that activation of Jun N-terminal kinase cascade may be involved in the cell response.

Chicken embryo fibroblasts (CEF) transformed with v-src were previously reported to revert to normal phenotype after the introduction of dominant-negative mutants of Fos or Jun, indicating that endogenous AP-1 activity is essential for the cellular transformation. The major changes in the expression levels of fos and jun family genes induced by v-src were the elevation of fra-2 and c-jun transcripts. We show here that extensive phosphorylation of the AP-1 component Fra-2 is a major qualitative change in v-src transformed CEF and that several Ser and Thr residues in a C-terminal region of Fra-2 (amino acids 266-323) are phosphorylated specifically. The induced kinase activity was detected at the position of 42 kDa by in gel kinase assay using the Fra-2 C-terminal region as a substrate, and it was identified as chicken ERK2. JNK1 and JNK2, other members of the MAP kinase family, were not significantly activated in v-src transformed CEF and Fra-2 was not a good substrate for JNKs. fra-2 promoter analysis indicated that this promoter activity is elevated in v-src transformed CEF via two AP-1 binding sites and CRE-like sequence. We propose that phosphorylation of Fra-2 by ERK2 converts it from an inefficient transcriptional activator to an active one and further that fra-2 expression is autoregulated in response to the phosphorylation status of its gene product.

A temperature-sensitive mutant defective in DNA replication, tsFT848, was isolated from the mouse mammary carcinoma cell line FM3A. In mutant cells, the DNA-dependent ATPase activity of DNA helicase B, which is a major DNA-dependent ATPase in wild-type cells, decreased at the nonpermissive temperature of 39 degrees C. DNA synthesis in tsFT848 cells at the nonpermissive temperature was analyzed in detail. DNA synthesis measured by incorporation of [3H]thymidine decreased to about 50% and less than 10% of the initial level at 8 and 12 h, respectively. The decrease in the level of thymidine incorporation correlated with a decrease in the number of silver grains in individual nuclei but not with the number of cells with labeled nuclei. DNA fiber autoradiography revealed that the DNA chain elongation rate did not decrease even after an incubation for 10 h at 39 degrees C, suggesting that initiation of DNA replication at the origin of replicons is impaired in the mutant cells. The decrease in DNA-synthesizing ability coincided with a decrease in the level of the DNA-dependent ATPase activity of DNA helicase B. Partially purified DNA helicase B from tsFT848 cells was more heat sensitive than that from wild-type cells. Inactivation of DNA-dependent ATPase activity of DNA helicase B from mutant cells was considerably reduced by adding DNA to the medium used for preincubation, indicating that the DNA helicase of mutant cells is stabilized by binding to DNA.

Human cell lines express two genetically distinct isoforms of DNA topoisomerase (topo II) II: topo II alpha (p170) and topo II beta (p180). We detected a higher molecular weight form with an apparent molecular mass of about 190 kDa in M phase-arrested HeLa cells (Kimura, K., Saijo, M., Ui, M., and Enomoto, T. (1994) J. Biol. Chem. 269, 1173-1176). In this study we confirmed, using anti-topo II alpha and topo II beta monoclonal antibodies, that this higher molecular weight form is topo II beta and consists of doublet bands around 190 kDa. We confirmed that the doublet bands constituted an M phase-specific phenomenon and were not an artifact of the procedure used to accumulate mitotic cells. Digesting the immunoprecipitated materials from mitotic cell extracts with alkaline phosphatase resulted in the disappearance of the doublet bands and the appearance of the 180-kDa band with the concomitant disappearance of 32P label in the region of the doublet bands. Neither heat-inactivated alkaline phosphatase nor phosphodiesterase affected the doublet bands and the 32P label. Topo II beta in interphase cells was also phosphorylated, but the shift in apparent molecular weight was very slight after alkaline phosphatase digestion. Analysis of the labeled phosphoamino acids present in topo II beta from M phase and logarithmically growing cells indicated that phosphorylation occurred mainly on serine and fairly on threonine residues in both topo II beta isoforms. These results indicated that topo II beta is phosphorylated at specific sites in M phase, resulting in the formation of the doublet bands.

Significant activity of phosphatidylinositol 3-kinase (PI 3-kinase) was detected in the membrane fractions, or in the immunoprecipitates prepared with anti-phosphotyrosine antibodies, from rat adipocytes that had been incubated with insulin for 20 min. The PI 3-kinase activity in these preparations as well as in the whole cell lysates of adipocytes not treated with insulin was inhibited by the addition of wortmannin, a fungal metabolite, to the enzyme assay mixture. The inhibition was dependent on the inhibitor concentration with IC50 being less than 10 nM and perfect inhibition at 100 nM. The effect of insulin to induce membrane PI 3-kinase activity was mostly abolished, but its effects to tyrosine-phosphorylate the beta-subunit of the insulin receptor or other cellular substrate proteins including insulin-receptor-substrate-1 were not at all antagonized, by wortmannin added to the cell incubation medium. Insulin stimulation of cellular 2-deoxyglucose uptake and inhibition of isoproterenol-induced lipolysis observable in adipocytes under the same conditions were also antagonized by wortmannin added in the same concentration range as used for the inhibition of insulin-susceptible PI 3-kinase. It is concluded, therefore, that activation of wortmannin-sensitive PI 3-kinase plays a pivotal role in the intracellular signaling pathways arising from the insulin receptor autophosphorylation and leading to certain metabolic responses.

The fluctuations in the expression of two forms of DNA topoisomerase II, p170 and p180, were examined by immunoblotting during the transition of the growth state and the cell cycle in Swiss 3T3 cells. The level of p170 expression was higher during the exponentially growing phase than in the stationary phase, and the level of p180 did not appear to change when the cells changed the growth state. When quiescent cells were stimulated with serum, the p170 level began to increase at 12 h and reached a maximal level at 24-28 h, corresponding to the G2 phase. In contrast, the level of p180 was almost constant during the cell cycle, and the p180 band became obscure when the number of mitotic cells increased. Immunoblotting of the samples prepared from metaphase-synchronized HeLa cells did not have a p180 band, but did exhibit a band with a higher molecular weight. This band increased and p180 decreased in parallel with the increase in the number of mitotic cells. When the cells exited from the M phase, p180 reappeared and the higher molecular weight band disappeared.

The DNA polymerase alpha-primase complex purified from mouse FM3A cells is composed of four polypeptides with molecular masses of 180, 68, 54, and 46 kDa. The largest subunit has DNA polymerase activity, the two smallest subunits have DNA primase activity, and the function of the 68-kDa subunit is unknown. We have isolated the cDNAs of the four subunits by low stringency hybridization and reverse transcription polymerase chain reaction and determined their nucleotide sequences. The predicted amino acid sequence of the 180-kDa subunit shows 88, 38, 34, and 32% identity to those of the catalytic subunits of human, Drosophila melanogaster, Schizosaccharomyces pombe, and Saccharomyces cerevisiae DNA polymerase alpha, respectively, and contains seven regions whose orders and sequences are highly conserved among viral and other eukaryotic DNA polymerases. The deduced amino acid sequence of the 68-kDa subunit shows 25% identity to that of the 73-kDa subunit of D. melanogaster DNA polymerase alpha-primase, shows no significant sequence similarity to any other protein in the data bases, but contains a potential phosphorylation site(s) for cdc2 kinase. The amino acid sequence of the 54-kDa subunit shows 32% identity to that of the large subunit of S. cerevisiae DNA primase. During activation of quiescent Swiss mouse 3T3 cells to proliferate, the levels of mRNA of the four subunits of the DNA polymerase alpha-primase complex increased before DNA synthesis. In growing mouse FM3A cells, the transcripts of the four subunits are present throughout the cell cycle and increase slightly prior to the S phase.

In rabbit peritoneal neutrophils prelabeled with [3H] lyso platelet-activating factor, a protein kinase C inhibitor, staurosporine (> 1 microM), increased [3H]phosphatidylethanol ([3H]PEt) level in the presence of ethanol in a concentration- and time-dependent manner, providing evidence for staurosporine activation of phospholipase D (PLD). The staurosporine activation of the enzyme absolutely required both extracellular calcium and cytochalasin B, and was almost completely inhibited by pretreatment of the cells with pertussis toxin (IAP). In a reconstituted system where the purified Gi1 had been incorporated into phospholipid vesicles, staurosporine activated GTPase activity of Gi1 in a concentration-dependent fashion, with a maximal 4-5-fold effect. ADP-ribosylation by IAP of Gi1 in vesicles significantly suppressed the staurosporine activation. As with the GTPase activity of Gi1, GTPase activities of other purified IAP-sensitive G proteins, such as Gi2 and G(o), were significantly stimulated by staurosporine, but the cholera toxin substrate Gs was appreciably less sensitive to the staurosporine stimulation. The staurosporine activation of GTPase was also observed in rabbit neutrophil membranes from control cells, but not in membranes from IAP-treated neutrophils. From these results, we conclude that the staurosporine activation of PLD in rabbit neutrophils is attributed to the direct activation of an IAP-sensitive G protein in a similar manner to receptors occupied by agonists. By contrast, staurosporine failed to activate phosphoinositide-specific phospholipase C (PI-PLC) under the conditions in which it activated PLD, indicating that there exists a PLD activation pathway independent of PI-PLC. Furthermore, it was found that N-acetyl-beta-glucosaminidase release from the granules of intact neutrophils was evoked by staurosporine to almost the same extent as by fMLP (100 nM), but O2- generation was not affected. These results suggest a possibility that PLD pathway plays an important role in enzyme release, but is not sufficient for O2- generation, in rabbit peritoneal neutrophils.

In our previous study, we identified four chromatographically distinct DNA-dependent ATPases, B, C1, C2, and C3, in mouse FM3A cells (Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., and Yamada, M. (1984) Biochemistry 23, 529-533). The DNA-dependent ATPase C1 has been purified and characterized in detail. A divalent cation and a polynucleotide cofactor were required for the ATPase activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Almost no ATPase activity was observed with S1 nuclease-treated native DNA. ATPase C1 hydrolyzed ATP only among the ribo- and deoxyribonucleoside triphosphates tested, and this fact distinguished ATPase C1 from ATPases B, C2, and C3, because the latter enzymes are capable of hydrolyzing both ATP and dATP. The purified DNA-dependent ATPase C1 fraction was shown to have a DNA helicase activity that was dependent on hydrolysis of ATP. The helicase activity and DNA-dependent ATPase activity cosedimented at 5.2 S on glycerol gradient centrifugation. Both activities showed similar preferences for nucleoside 5'-triphosphates and similar requirements for divalent cations. The DNA helicase activity was inhibited by the addition of single-stranded DNAs that served as cofactor for the ATPase activity. The efficiency of a single-stranded DNA to inhibit DNA helicase activity correlated well with the capacity of the DNA to serve as cofactor for DNA-dependent ATPase activity. The helicase was shown to migrate along the DNA strand in the 5' to 3' direction, which is the same direction of migration of the mouse DNA helicase B (Seki, M., Enomoto, T., Yanagisawa, J., Hanaoka, F., and Ui, M. (1988) Biochemistry 27, 1766-1771).

Membrane fractions from the guinea pig lung had high- and low-affinity binding sites for LTD4 with Kd values of 0.016 and 9.1 nM, respectively. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or by prior treatment of the membrane with islet-activating protein (IAP), the high-affinity site shifted to a low-affinity state. Consistently, a 41-kDa protein was ADP-ribosylated by treatment of the lung membranes with IAP, and this event was inhibited by the addition of GTP gamma S. We solubilized the LTD4 receptor from the lung membranes in an active form with 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 10% glycerol. On a gel filtration column, the binding activity was eluted at the volume corresponding to a Mr of 70,000 or over 500,000 in the presence or absence of Mg2+ (5-20 mM), respectively, in solubilizing buffers. The Kd value of [3H]LTD4 binding to the 70-kDa protein was similar to the low-affinity binding constant of the membrane and was insensitive to GTP gamma S. The preparation solubilized in the absence of Mg2+ showed both high- and low-affinity binding sites for LTD4, and the addition of GTP gamma S shifted the high-affinity site to a low-affinity one. Thus, 1) the LTD4 receptor is coupled to an IAP-sensitive GTP-binding protein, 2) this GTP-binding protein is dissociable from the receptor by solubilizing the lung membrane with CHAPS and Mg2+, and 3) the receptor associated to or dissociated from a GTP-binding protein exhibited a high- or low-affinity state, respectively. These data provide an insight into the molecular mechanism of regulation of the LTD4 receptor signaling process by association and dissociation with an IAP-sensitive GTP-binding protein.

A direct interaction of alpha beta gamma trimeric GTP binding proteins (G proteins; G0 and Gs) with nucleoside diphosphate kinase (NDP kinase) was investigated with homogeneously purified proteins. There was a progressive release of 32Pi from [gamma-32P]ATP when GDP-bound G0 was incubated together with NDP kinase. The Pi release induced by the interaction of G0 with NDP kinase was not accompanied by the dissociation of GDP bound to the alpha-subunit of G0. This was a sharp contrast to G protein-catalyzed GTP hydrolysis observed with GTP as the substrate; the dissociation of bound GDP was essentially required for the following binding of the substrate, GTP, to be hydrolyzed. A kinetic analysis displayed different properties for the substrate of NDP kinase between free GDP and G protein-bound GDP. NDP kinase-dependent phosphorylation of GDP on G0 was indeed demonstrated with adenosine 5'-(3-O-thio)triphosphate as the phosphate donor; there was a formation of guanosine 5'-(3-O-thio)triphosphate-bound G0 from the ATP analogue. Moreover, purified Gs was readily ADP-ribosylated by cholera toxin in the presence of NDP kinase, ATP, and an ADP-ribosylation factor, also suggesting that the nucleotide form on Gs was certainly GTP. These results indicate that NDP kinase can transfer the gamma-phosphate of ATP directly to GDP bound to G proteins and that this phosphorylation results in the activation of the signal-coupling proteins. A possible role of the new activation mechanism of G proteins is discussed in comparison with the previously characterized GDP-GTP exchange pathway by the agonist-receptor complex.

A soluble system was developed that could support DNA replication in simian virus 40 (SV40) chromosomes. DNA synthesis in this system required the presence of purified SV40 large tumor antigen, SV40 chromosomes prepared from virus-infected monkey cells, a crude extract from HeLa cells, and several low-molecular-weight components. In comparison to the replication of purified SV40 form I DNA, the rate of DNA synthesis was 15 to 20% in this system. DNA synthesis started near the replication origin of SV40 and proceeded bidirectionally in a semiconservative manner. Micrococcal nuclease digestion experiments revealed that the replicated DNA produced in this system became organized into a regularly spaced array of nucleosome core particles when an appropriate amount of purified HeLa core histones was added to the reaction mixture. SV40 form I DNA replicating under the same conditions was also assembled into nucleosomes, which were arranged in a rather dispersed manner and formed an aberrant chromatin structure.

The rat insulin-like growth factor II (IGF-II) receptor develops transmembrane signaling functions by directly coupling to a guanine nucleotide-binding protein (G protein) having a 40-kDa alpha subunit, Gi-2, whereas recent studies have indicated that the IGF-II receptor is a molecule identical to the cation-independent mannose 6-phosphate receptor (CI-MPR), a receptor implicated in lysosomal enzyme sorting. In this study, by using vesicles reconstituted with the clonal human CI-MPR and G proteins, we indicated that the CI-MPR could stimulate guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding and GTPase activities of Gi proteins in response to IGF-II. The stimulatory effect of IGF-II on Gi-2 depended on the reconstituted amount of the CI-MPR; it could not be found in vesicles reconstituted with Gi-2 alone; and it was also observed on Gi-1 reconstituted with the CI-MPR in phospholipid vesicles. Of interest, such stimulatory effect was not reproduced by Man-6-P in CI-MPR vesicles reconstituted with either G protein. Furthermore, the affinity for Man-6-P-mediated beta-glucuronidase binding to several kinds of native cell membranes was not reduced by 100 microM GTP gamma S. Instead, however, Man-6-P dose-dependently inhibited IGF-II-induced Gi-2 activation with an IC50 of 6 microM in vesicles reconstituted with the CI-MPR and Gi-2. The action of 100 nM IGF-II was completely abolished by 1 mM Man-6-P. Such an inhibitory effect of Man-6-P was reproduced by 4000 times lower concentrations of beta-glucuronidase or similar concentrations of fructose 1-phosphate, but not by mannose or glucose 6-phosphate. These results indicate that the human CI-MPR has two distinct signaling functions that positively or negatively regulate the activity of Gi-2 in response to the binding of IGF-II or Man-6-P.

A large number of mutants that are temperature sensitive (ts) for growth have been isolated from mouse mammary carcinoma FM3A cells by an improved selection method consisting of cell synchronization and short exposures to restrictive temperature. The improved method increased the efficiency of isolating DNA ts mutants, which showed a rapid decrease in DNA-synthesizing ability after temperature shift-up. Sixteen mutants isolated by this and other methods were selected for this study. Flow microfluorometric analysis of these mutants cultured at a nonpermissive temperature (39 degrees C) for 16 h indicated that five clones were arrested in the G1 to S phase of the cell cycle, six clones were in the S to G2 phase, and two clones were arrested in the G2 phase. The remaining three clones exhibited 8C DNA content after incubation at 39 degrees C for 28 h, indicating defects in mitosis or cytokinesis. These mutants were classified into 11 complementation groups. All the mutants except for those arrested in the G2 phase and those exhibiting defects in mitosis or cytokinesis showed a rapid decrease in DNA synthesis after temperature shift-up without a decrease in RNA and protein synthesis. The polyomavirus DNA cell-free replication system, which consists of polyomavirus large tumor antigen and mouse cell extracts, was used for further characterization of these DNA ts mutants. Among these ts mutants, only the tsFT20 strain, which contains heat-labile DNA polymerase alpha, was unable to support the polyomavirus DNA replication. Analysis by DNA fiber autoradiography revealed that DNA chain elongation rates of these DNA ts mutants were not changed and that the initiation of DNA replication at the origin of replicons was impaired in the mutant cells.

1. We have examined the effects of L-glutamate and gamma-aminobutyric acid (GABA) on the presynaptic membrane of spiny lobster by the use of intra-axonal recording near the nerve terminals. 2. Application of glutamate to the synaptic region produced hyperpolarization in the presynaptic membrane but depolarization in the postsynaptic membrane. The presynaptic glutamate potential (PGP) is generated by an activation of K+ channels, as evidenced by its dependence on external K+ concentration. 3. The PGP was not affected by a spider toxin (JSTX), which blocks the postsynaptic glutamate receptor. By contrast, pertussis toxin (IAP) effectively blocked the PGP without affecting the resting conductance channels or action potentials in the presynaptic membrane. 4. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a hydrolysis-resistant analogue of GTP, blocked the PGP, suggesting the involvement of a G protein in the generation of K+ current. 5. Application of GABA induced depolarization or hyperpolarization in the presynaptic axon depending on the resting membrane potential. By reducing external Cl-, GABA-induced hyperpolarizations were converted to depolarizations, indicating that they are mainly mediated by Cl-. 6. In contrast to GABA, baclofen consistently induced hyperpolarization in low Cl- solution as well as in normal solution. Baclofen-induced hyperpolarization was blocked by IAP, indicating the mediation of G protein. 7. These results suggest that the presynaptic membrane of lobster neuromuscular synapse has entirely different types of amino-acid receptors from those in the postsynaptic membrane. Both the excitatory and the inhibitory axonal membrane have glutamate ("glutamateB") and GABAB receptors, which activate K+ channels via G protein.(ABSTRACT TRUNCATED AT 250 WORDS)

The Radial Injection Gas Dynamic Spray (RIGDS) technology of powder wear resistant coatings deposition is considered. A coating is created by injection of powders with variable composition into the supersonic air jet and deposited on the substrate. Functionally graded coatings with advanced mechanical and corrosion properties were produced and tested. It is shown that operational parameters of supersonic powder-gas jet have a significant influence on the coating’s microstructure, thus defining the high performance of the coating. Compaction and bonding of particles were analyzed. Shear localization features of the GDS process are also discussed.

The gyrotrons developed at Fukui University ("Gyrotron FU series") are high frequency, medium power devices, covering a broad frequency band in the millimeter and submillimeter wavelength region. The gyrotrons from the FU series were successfully used in plasma scattering experiments, electron spin resonance (ESR) spectroscopy and in in-depth studies of mode competition and mode cooperation. In the paper, we illustrate some of the achievements of the Gyrotron FU series.

We investigated apoptosis induced by in vitro infection with the chimeric virus of simian immunodeficiency virus and human immunodeficiency virus (SHIV). Macaque and human peripheral blood mononuclear cells (PBMCs) were infected with pathogenic SHIV-89.6p (89.6p) or nonpathogenic SHIV-NM-3rN (NM-3rN). In macaque PBMCs, the extent of virus production and apoptosis induction in CD4(+) cells was much greater in 89.6p infection than in NM-3rN infection. The result was consistent with our previous study of in vivo SHIV infection. In human PBMCs, 89.6p replicated and induced apoptosis more extensively than did NM-3rN, when the cells were infected with the same infectious doses of the viruses. However, in cells infected with a high dose of NM-3rN, the levels of virus production and apoptosis induction were comparable to those in 89.6p infection. There was no significant difference in the extent of apoptosis induction between 89.6p and NM-3rN infection when growth curves of the two viruses matched. Thus, apoptosis induction by SHIV might depend quantitatively on the amount of virus production rather than on the strains of the virus. Moreover, the correlation between the extent of apoptosis induction and virus pathogenicity in macaque PBMCs has also been found in SHIV-infected macaques. This suggests that the profiles of SHIV infection in vitro reflect the in vivo phenomena. Therefore, the in vitro evaluation of apoptosis induction by SHIV could be useful as a safety test for the development of live-attenuated vaccines.

Short-term incubation of adult rat hepatocytes with epidermal growth factor (EGF) caused tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 when the cells had been submitted to primary culture from 1-18 h. Tyrosine-phosphorylated IRS-1 and IRS-2 bound to the regulatory subunit (p85) of phosphatidylinositol (PtdIns) 3-kinase, thereby activating the enzymic activity. Tyrosine phosphorylation of the IRSs and activation of PtdIns 3-kinase in 3 h cultured hepatocytes both proceeded similarly to the same actions of insulin; the activation was rapid and transient, with peak values at 15-30 s and with similar EC(50)s in the nM range in both cases. A possible involvement of insulin receptors in these insulin-like actions of EGF was excluded by the following three lines of evidence. Insulin caused tyrosine phosphorylation of the insulin receptor beta-subunit but EGF did not. In contrast, the EGF receptor was phosphorylated by EGF, but the insulin receptor was not. The actions of EGF, but not those of insulin, were inhibited by AG1478, a selective inhibitor of EGF receptor tyrosine kinase. Cultured hepatocytes exposed to insulin or insulin-like growth factor-I (IGF-I) for a short period responded to the subsequent addition of EGF, whereas EGF-treated cells responded to insulin. The cells, however, displayed receptor desensitization under the same conditions, that is, no response was observed upon repeated addition of the same agonist, EGF, insulin or IGF-I. Thus, the EGF receptor-initiated signalling was mediated by PtdIns 3-kinase associated with tyrosine-phosphorylated IRSs in short-term cultured rat hepatocytes.

We investigated the role of endogenous AP-1 in human tumor cell lines by introducing SupJunD-1, a dominant-negative mutant of AP-1, using vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors. Single inoculation of six human tumor cell lines, originating from osteosarcomas, non-small cell lung carcinomas or cervical carcinomas, with recombinant SupJunD-1 virus at a high multiplicity of infection readily inhibited colony formation in soft agar. We detected no significant changes in expression levels of AP-1 components c-Jun or Fra-1, adhesion molecules CD44 or E-cadherin, or cell cycle regulator p53, which are encoded by genes previously reported to be under the control of AP-1 in some mouse or human cell lines. By varying the dosage of VSV-G-pseudotyped retrovirus, we were able to change the proviral copy number of supjunD-1 from 1 to approximately 10 and monitor suppression of endogenous AP-1 function as assessed by growth characteristics of the tumor cell lines, we found a SupJunD-1 dosage which significantly suppressed anchorage-independent growth without affecting the cellular growth in monolayer cultures at all. We conclude that endogenous AP-1 levels necessary for oncogenic activity are much higher than those sufficient to support normal growth.

Fcgamma receptors (FcgammaR) of guinea pig neutrophils were ligated and anti-Cbl immunoprecipitates prepared therefrom were assayed for the associated protein tyrosine kinase activity, which increased upon ligation of FcgammaR. The increases were overcome upon activation of cellular protein kinase C by simultaneous addition of phorbol 12-myristate 13-acetate (PMA) to the ligated cells. Syk proved to be the most important tyrosine kinase bound to Cbl that served as the major substrate; essentially no tyrosine phosphorylation occurred in the anti-Cbl immunoprecipitates prepared from the cell lysate that had been depleted of Syk by prior immunoprecipitation with anti-Syk antibodies. Exposure of the (32)P-labeled cells to PMA resulted in phosphorylation of cellular Cbl on serine residues. Thus, protein kinase C-induced serine phosphorylation of Cbl suppressed its tyrosine phosphorylation by Syk as a result of tyrosine kinase inhibition by unknown mechanisms, leading to inhibition of Cbl-mediated signaling such as phosphatidylinositol 3-kinase activation.

It is difficult to establish stable packaging cell lines producing retrovirus vectors for the expression of anti-oncogenes with cytotoxic or cytostatic potential, because these genes would also affect the growth of the packaging cell lines. To overcome this problem, we designed a transcriptional unit pBabeLPL for vector RNA production, in which the transcription of the exogenous genes is completely suppressed by the presence of a preceding insertion containing the puromycin resistance gene (puro) and a poly(A) addition signal. This insertion is flanked by a tandem pair of loxP, and is designed to be excised after the introduction of Cre recombinase, when transcription of the exogenous gene will be started from the 5′-LTR. The transcriptional unit car- rying LacZ or p53 as the exogenous gene was introduced into a previously constructed prepackaging cell lines PtG-S2, in which the expression of VSV-G is also designed to be initiated by the introduction of Cre recombinase, while the gag-pol gene is expressed continuously. After the introduction of Cre recombinase by an adenovirus vector, LacZ- or p53-expressing VSV-G-pseudotyped retrovirus vectors with the designed structure were produced at high virus titers. The p53 virus was shown to be able to transduce p53 into the entire population of several human cancer cell lines and to induce their growth arrest at the G1 phase, indicating that this vector-producing system will be advantageous for human gene therapy.

The increase in cellular cAMP concentration during 10-min incubation of rat hepatocytes with glucagon or forskolin was enhanced markedly when the hepatocytes had been cultured for several hours with herbimycin A. This effect of herbimycin was accompanied by inhibition of tyrosine-phosphorylation of cellular proteins including alpha-tubulin, antagonized by coaddition of Na3VO4 plus H2O2, which also antagonized the herbimycin-induced tyrosine phosphorylation, and overcome by the addition to the 10-min incubation medium of a certain inhibitor of cAMP phosphodiesterase (PDE), which caused a huge accumulation of cAMP. The effective PDE inhibitors were 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram) and 4-(3-butyloxy-4-methoxyphenyl)-2-imidazolidinone (Ro-20-1724, a PDE4 inhibitor), in addition to 3-isobutyl-1-methylxanthine (a nonselective inhibitor). Rapid breakdown of the once-accumulated cAMP in cultured hepatocytes during the subsequent incubation without PDE inhibitors was progressively prevented when the concentration of herbimycin was increased from 0.3 to 10 microM during prior culture. This effect of herbimycin to inhibit PDE activity in intact cells was abolished by coaddition of a microtubule-disrupting agent, either colchicine or vinblastine, into the culture, but remained unchanged if the vinblastine-containing medium was further supplemented with taxol, a microtubule-stabilizing agent, which by itself mimicked the effect of herbimycin. None of these agents, which thus affected PDE activity in intact cells, inhibited the PDE activity assayable in the cell lysates. The taxol-like and vinblastine-suppressible action of herbimycin to stimulate microtubular assembly was antagonized by Na3VO4/H2O2, as confirmed by confocal microscopic images of the cells stained with fluorescein-bound anti-(alpha-tubulin). Thus, 4-h culture of hepatocytes with herbimycin inhibits phosphorylation of the C-terminal tyrosine residue of alpha-tubulin, thereby stimulating formation of a microtubular network which is responsible for the inhibition of PDE4 in the intact cells by an unknown mechanism.

The jun D gene encodes one component of the transcription factor, AP-1. Since two forms of JunD protein have been reported, we analyzed here the molecular mechanisms involved in the isoform production. Immunochemical analysis indicated that the longer and shorter forms of mouse JunD (JunD-L and JunD-S, with apparent molecular weights of 44 and 39 kDa, respectively) differ in their content of an N-terminal peptide. Mutational analysis further indicated that JunD-S is the translational product initiated at the third AUG located 144 bp from the first AUG, at which JunD-L translation starts. Such production of two jun D isoforms from a single mRNA using the same reading frame seems to be conserved in human, rat, and chicken. To examine the functional differences between the isoforms, each type of JunD was exclusively expressed by the use of retrovirus vectors harboring the mutated jun D gene. The exogenous expression of either one of these forms did not cause cellular transformation of NIH3T3, but suppressed the anchorage-independent growth of NIH3T3 transforbold by the activated K- ras or v- src gene. These two isoforms were expressed in all the mouse tissues examined and in various cell lines established from human tumors, though the expression ratio between JunD-L and JunD-S varied, suggesting that some factor(s) modulate the alternative usage of the initiation codon of the jun D gene.

Yeast GST1 gene, whose product is a GTP-binding protein structurally related to polypeptide chain elongation factor-1alpha (EF1alpha), was first described to be essential for the G1 to S phase transition (GSPT) of the cell cycle, and the product was recently reported to function as a polypeptide chain release factor 3 (eRF3) in yeast. Although we previously cloned a human homologue (renamed as GSPT1) of the yeast gene, it has remained to be determined whether GSPT1 also functions as eRF3 or if another GSPT may have such a function in mammalian cells. In the present study, we isolated two mouse GSPT genes, the counterpart of human GSPT1 and a novel member of the GSPT gene family, GSPT2. Both the mouse GSPTs had a two-domain structure characterized as an amino-terminal no-homologous region (approximately 200 amino acids) and a carboxyl-terminal conserved eukaryotic elongation factor-1alpha-like domain (428 amino acids). Messenger RNAs of the two GSPTs could be detected in all mouse tissues surveyed, although the level of GSPT2 message appeared to be relatively abundant in the brain. The mouse GSPT1 was expressed in a proliferation-dependent manner in Swiss 3T3 cells, whereas the expression of GSPT2 was constant during the cell-cycle progression. Immunoprecipitation assays in COS-7 cells expressing flag epitope-tagged proteins demonstrated that not only GSPT1 but also GSPT2 was capable of interacting with eRF1. Such interaction between GSPT2 and eRF1 was also confirmed by yeast two-hybrid analysis. Taken together, these data indicated that the novel GSPT2 may interact with eRF1 to function as eRF3 in mammalian cells.

Agonist-induced sequestration, recycling, and resensitization of platelet-activating factor (PAF) receptor were characterized in transfected Chinese hamster ovary cells. Exposure of the cells to PAF led to rapid sequestration of the receptors into the intracellular compartment and desensitization of the response to PAF. The sequestration was inhibited by pretreatments that perturbed the clathrin-mediated pathway. Subsequent removal of PAF by washing with receptor antagonists led to rapid recycling of the sequestered receptors to the cell surface accompanied by resensitization to PAF. To evaluate the potential role of phosphorylation in the receptor cytoplasmic tail during these processes, mutant receptors in which the tails were truncated or substituted, so as to lack serine/threonine residues, were created. PAF phosphorylated the wild-type receptor rapidly and strongly, but the mutants did not. The maximal extent of sequestration of each mutant was lower than that of the wild-type, and one of the substituted mutants showed no sequestration. Furthermore, the sequestration-defective mutant showed evidence of desensitization after agonist stimulation but not resensitization after agonist removal. Thus, agonist-induced phosphorylation of the cytoplasmic tail facilitates but is not essential for receptor sequestration, and sequestration/recycling appears important in receptor resensitization.

COS-7 cells were transiently transfected with human thyrotropin receptor and dog A1 adenosine receptor cDNAs. An A1 agonist, N6-(L-2-phenylisopropyl) adenosine (PIA), which is ineffective alone, enhanced the thyrotropin (TSH)-induced inositol phosphate production, reflecting phospholipase C (PLC) activation, but inhibited the TSH-induced cAMP accumulation, reflecting adenylyl cyclase inhibition. These PIA-induced actions were completely inhibited by pertussis toxin (PTX) treatment. Moreover, in the cells expressing a PTX-insensitive mutant of Gi2alpha or Gi3alpha, in which a glycine residue was substituted for a cysteine residue to be ADP-ribosylated by PTX, at the fourth position of the C terminus, PIA effectively exerted both stimulatory and inhibitory effects on the TSH-induced actions although the cells were treated with the toxin. Overexpression of the betagamma subunits of the G proteins enhanced the TSH-induced inositol phosphate production without any significant effect on the cAMP response; under these conditions, PIA did not further increase the elevated inositol phosphate response to TSH. On the contrary, overexpression of a constitutively active mutant of Gi2alpha, in which the guanosine triphosphatase activity is lost, inhibited the TSH-induced cAMP accumulation but hardly affected the inositol phosphate response; under these conditions, PIA never exerted further inhibitory effects on the cAMP response to TSH. In contrast to the case of the TSH-induced inositol phosphate response, the response to a constitutively active G11alpha mutant was not appreciably affected, and that to NaF was rather inhibited by PIA and overexpression of the betagamma subunits. Taken together, these results suggest that a single type of PTX-sensitive G protein mediates the A1 adenosine receptor-linked modulation of two signaling pathways in collaboration with an activated thyrotropin receptor; alpha subunits of the PTX-sensitive G proteins mediate the inhibitory action on adenylyl cyclase, and the betagamma subunits mediate the stimulatory action on PLC. In the case of the latter stimulatory action on PLC, the betagamma subunits may not directly activate PLC. The possible mechanism by which betagamma subunits enhance the TSH-induced PLC activation is discussed.

It has been shown that inhibition of phosphatidylinositol (PI) 3-kinase blocks neurite outgrowth of PC12 cells stimulated with nerve growth factor. To further assess the role of PI 3-kinase, the active form of PI 3-kinase was expressed in PC12 cells by the adenovirus mediated introduction of a site-specific recombinase, Cre. After expression of the active PI 3-kinase, elevation of the levels of PI 3,4-diphosphate and PI 3,4,5-trisphosphate as well as formation of neurite-like processes was observed. The process formation was inhibited by wortmannin, a selective inhibitor of PI 3-kinase, which suggests that a high activity of PI 3-kinase was responsible for the formation of these processes. The processes lacked accumulation of F-actin and GAP43 at the growth cone, which suggests that the processes were incomplete compared with neurites. Instead, the bundling of microtubules was enhanced, which suggests that organization of the microtubules might be driving the process of elongation in the cells expressing the active PI 3-kinase. Induction of active PI 3-kinase resulted in activation of Jun N-terminal kinase but not of mitogen-activated protein kinase or protein kinase B/Rac protein kinase/Akt. These results suggest that PI 3-kinase is involved in neurite outgrowth in PC12 cells and that activation of Jun N-terminal kinase cascade may be involved in the cell response.

Adrenergic responses of rat hepatocytes were studied by measuring Ins(1,4,5)P3(for the response via alpha 1-subtype receptors) and cAMP (for beta-subtype response) generation during brief incubation of cells with respective agonists. Hepatocytes from young rats with an age of 1 week displayed a very high beta response without a significant alpha 1 response. The beta response decreased and the alpha 1 response increased progressively as the age increased; the response was almost exclusively via alpha 1 receptors in hepatocytes of adult rats 9 weeks or more old. The beta response developed, again at the expense of the alpha 1 response, in hepatocytes from adult rats during the primary culture at low cell densities [(1-2.5) x 10(4) cells/cm2]. Such "alpha 1 to beta subtype switching' of adrenergic responses in vitro was totally inhibited by adding plasma membranes prepared from adult rat liver into the low-cell-density culture, but not inhibited at all by membranes from young rat liver. The inhibitory effect of adult rat liver membranes was lost when the membranes had been exposed to endoglycosidase F or beta-galactosidase but was not affected by prior treatment with sialidase. On the contrary, young rat liver membranes became inhibitory to "alpha 1 to beta subtype switching' after prior treatment with sialidase. Thus glycoproteins with unsialylated galactosyl termini on the surface of adult rat hepatocytes are likely to function as a determinant of the relative development of alpha 1/beta subtypes of adrenergic responses; the beta response is predominant in hepatocytes in the juvenile, presumably as a result of sialylation of the galactosyl termini of the functional glycoproteins.

We expressed the Gly203--Thr (G203T) mutant of Gi2alpha, which was expected to show a dominant-negative phenotype in Gi2-mediated signal transduction, in baculovirus-inefected Sf9 cells and purified the mutant alpha subunit for its characterization. The rate of dissociation of GDP from G203T Gi2alpha was 3- to 4-fold faster than that from wild type Gi2alpha, but their kappacat values for GTP hydrolysis were almost the same. The affinities of the two Gi2alpha proteins for the beta gamma subunits of G proteins to form alpha beta gamma timers, which served as substrates for pertussis toxin-catalyzed ADP-ribosylation, were the same. In marked contrast, G203T Gi2alpha was unable to form a tight complex with a non- hydrolyzable analog (GTP[gammaS) of GTP; bound GTP[gammaS] was readily released from the mutant Gi2alpha even in the presence of a high concentration of Mg2+. Its susceptibility to tryptic digestion also revealed that GTP[gammaS]-bound G203T Gi2alpha formed a conformation apparently different from that of the GTP[gammaS]-bound form of wild-type Gi2alpha. Both the G203T and wild-type Gi2alpha proteins were capable of coupling with membrane-bound alpha2-adrenergic receptors, resulting in the formation of receptor-G protein complexes with high affinity for agonists. However, GTP[gammaS]-dependent uncoupling from high-affinity receptors was markedly attenuated in the case of G203T Gi2alpha. Thus, G203T-mutated Gi2alpha had a unique property in terms of coupling to membrane receptors, in addition to the previously expected defect in the active conformation of the GTP-bound form of Gi2alpha.

We established a modified cell-free system for polyomavirus (PyV) DNA replication, which was supplemented with Escherichia coli single-stranded DNA binding protein (SSB). DNA synthesis in this system was enhanced by 1.4- to over 15-fold depending upon the amount of cell extracts contained in the reaction mixture. By supplementing with E. coli SSB, we were able to reduce the amount of cell extracts in the reaction mixture, and to lower the concentrations of creatine phosphate and Tris, rendering this system more resistant to salts than the conventional PyV DNA replication system. The modified system was characterized using mutant cell extracts which had heat-inactivated DNA polymerase alpha. DNA synthesis in the system was dependent on PyV T antigen, the PyV origin of DNA replication, mutant cell extracts, and DNA polymerase alpha-primase complex purified from wild-type cells. The DNA polymerase alpha-primase complex was not replaced by DNA polymerase alpha, indicating that this system requires a functional DNA polymerase alpha-primase complex. This system exhibited species-specificity in the requirement for DNA polymerase alpha-primase; only mouse DNA polymerase alpha-primase but not human DNA polymerase alpha-primase functioned in this system.

PLC activity was stimulated either by 1-100 microM of GTP or by 100-3,000 microM Ca2+ in lysed synaptosomal membranes of the guinea pig cerebellum. The kappa-opioid receptor agonist selectively inhibited the PLC activity stimulated by 100 microM GTP, but not by 100-3,000 microM Ca2+. Pretreatment of membranes with PTX abolished such a kappa-agonist-induced inhibition of PLC activity. The reconstitution of Gi1, but not of Go purified from porcine brains with PTX-treated membranes showed a complete recovery of the kappa-agonist-inhibition of PLC activity. These findings suggest that a novel subtype kappa-receptor mediates inhibition of PLC through inhibiting the intrinsic activity of PTX-substrate G-proteins.

The GTP-binding protein (G protein) is a heterotrimeric protein composed of an alpha-, a beta- and a gamma-subunit. The G protein is functionally located between membrane receptors whose structures are characterized by seven membrane-spanning domains and effectors that are enzymes responsible for the generation of intracellular second messengers or ionic channels, thereby playing its essential role as a molecular switch for intracellular signal initiation. The switch turns on when GTP binds, in exchange for prebound GDP, to the alpha-subunit (G alpha), whereas it turns off upon the GTP hydrolysis due to the G alpha GTPase activity. The beta gamma-component plays a supporting role for the molecular switching and is also involved in signal transduction to certain effectors. One of the most exciting subjects to be currently studied as to the physiological roles of G proteins will be the mechanism by which the G protein-mediated second messenger system interacts (crosstalks) with the tyrosine kinase-mediated signaling system arising from other types of growth factor receptors.

Culture of mouse resident peritoneal macrophages with retinoic acid resulted in increased expression of the tissue transglutaminase gene as revealed by increases in the maximal velocity of the enzyme reaction in the cytosol and in the enzyme mRNA level. Protein kinase C-activating phorbol esters and okadaic acid, both of which were without effect on the enzyme induction by themselves, enhanced the retinoic acid-induced gene expression, which was in turn inhibited partially by pertussis toxin and totally by inhibitors of protein kinase C in either the presence or absence of phorbol esters. Retinoic acid was more effective in the "conditioned" medium, in which macrophages had been cultured for a time longer than 4 h, than in the "fresh" medium. The retinoic acid induction of transglutaminase was accompanied by increased phosphatidylinositol turnover and phosphatidic acid generation, which were efficiently suppressed by prior exposure of cells to pertussis toxin. It is likely that certain autocrine factor(s) liberated during culture of macrophages may afford conditions favorable for retinoic acid-induced gene expression, presumably via pertussis toxin-sensitive G protein-mediated phosphoinositide metabolism leading to activation of protein kinase C.

Complementation group C of xeroderma pigmentosum (XP) represents one of the most common forms of this cancer-prone DNA repair syndrome. The primary defect is located in the subpathway of the nucleotide excision repair system, dealing with the removal of lesions from the non-transcribing sequences ('genome-overall' repair). Here we report the purification to homogeneity and subsequent cDNA cloning of a repair complex by in vitro complementation of the XP-C defect in a cell-free repair system containing UV-damaged SV40 minichromosomes. The complex has a high affinity for ssDNA and consists of two tightly associated proteins of 125 and 58 kDa. The 125 kDa subunit is an N-terminally extended version of previously reported XPCC gene product which is thought to represent the human homologue of the Saccharomyces cerevisiae repair gene RAD4. The 58 kDa species turned out to be a human homologue of yeast RAD23. Unexpectedly, a second human counterpart of RAD23 was identified. All RAD23 derivatives share a ubiquitin-like N-terminus. The nature of the XP-C defect implies that the complex exerts a unique function in the genome-overall repair pathway which is important for prevention of skin cancer.

Wortmannin, a fungal metabolite, inhibited 32P labeling of phosphatidylinositol trisphosphate, a product of phosphatidylinositol 3-kinase (PI 3-kinase), selectively in formyl peptide-stimulated 32P-loaded guinea pig neutrophils. The inhibition was of the same concentration dependence (with the half-maximal inhibition around 50 nM) as was observed for the simultaneous inhibition of formyl peptide-induced superoxide anion production. Wortmannin inhibited all three of the PI 3-kinase activities found in the cytosol fraction of guinea pig neutrophils, with a similar dose dependence (the half-maximal effects at 5 nM). Wortmannin was also effective on an immunologically purified preparation of the enzyme. The inhibition was of a noncompetitive type with regard to ATP and was observed consistently when PI, PI monophosphate, or PI bisphosphate was used as substrate. PI 4-kinase activity was not affected. It is concluded, therefore, that wortmannin abolished the formyl peptide-induced stimulation of neutrophils as a result of the inhibition of PI 3-kinase. An essential role of PI 3-kinase in receptor-mediated signaling in neutrophils thus evidenced with the use of wortmannin will be expanded to other cellular signaling systems.

The fluctuations in the expression of two forms of DNA topoisomerase II, p170 and p180, were examined by immunoblotting during the transition of the growth state and the cell cycle in Swiss 3T3 cells. The level of p170 expression was higher during the exponentially growing phase than in the stationary phase, and the level of p180 did not appear to change when the cells changed the growth state. When quiescent cells were stimulated with serum, the p170 level began to increase at 12 h and reached a maximal level at 24-28 h, corresponding to the G2 phase. In contrast, the level of p180 was almost constant during the cell cycle, and the p180 band became obscure when the number of mitotic cells increased. Immunoblotting of the samples prepared from metaphase-synchronized HeLa cells did not have a p180 band, but did exhibit a band with a higher molecular weight. This band increased and p180 decreased in parallel with the increase in the number of mitotic cells. When the cells exited from the M phase, p180 reappeared and the higher molecular weight band disappeared.

We examined the role of 185-kDa insulin receptor substrate-1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase) in the signaling pathway of insulin-stimulated GLUT4 translocation. We had already developed a novel cell line to detect GLUT4 on the cell surface, directly and sensitively (Kanai, F., Nishioka, Y., Hayashi, H., Kamohara, S., Todaka, M., and Ebina, Y. (1993) J. Biol. Chem. 268, 14523-14526). We stably expressed a mutant insulin receptor in which Tyr972 was replaced with phenylalanine. Insulin-stimulated tyrosyl phosphorylation of IRS-1 and GLUT4 translocation were decreased in cells expressing the mutant receptor, as compared to findings in cells expressing the normal receptor. Wortmannin, an inhibitor of PI3-kinase, inhibits the insulin-stimulated PI3-kinase activity and GLUT4 translocation at 50 nM, but not the NaF-stimulated GLUT4 translocation. These results suggest that the tyrosine phosphorylation of IRS-1 and activation of PI3-kinase may be involved in the signaling pathway of the insulin-stimulated GLUT4 translocation.

The DNA polymerase alpha-primase complex purified from mouse FM3A cells is composed of four polypeptides with molecular masses of 180, 68, 54, and 46 kDa. The largest subunit has DNA polymerase activity, the two smallest subunits have DNA primase activity, and the function of the 68-kDa subunit is unknown. We have isolated the cDNAs of the four subunits by low stringency hybridization and reverse transcription polymerase chain reaction and determined their nucleotide sequences. The predicted amino acid sequence of the 180-kDa subunit shows 88, 38, 34, and 32% identity to those of the catalytic subunits of human, Drosophila melanogaster, Schizosaccharomyces pombe, and Saccharomyces cerevisiae DNA polymerase alpha, respectively, and contains seven regions whose orders and sequences are highly conserved among viral and other eukaryotic DNA polymerases. The deduced amino acid sequence of the 68-kDa subunit shows 25% identity to that of the 73-kDa subunit of D. melanogaster DNA polymerase alpha-primase, shows no significant sequence similarity to any other protein in the data bases, but contains a potential phosphorylation site(s) for cdc2 kinase. The amino acid sequence of the 54-kDa subunit shows 32% identity to that of the large subunit of S. cerevisiae DNA primase. During activation of quiescent Swiss mouse 3T3 cells to proliferate, the levels of mRNA of the four subunits of the DNA polymerase alpha-primase complex increased before DNA synthesis. In growing mouse FM3A cells, the transcripts of the four subunits are present throughout the cell cycle and increase slightly prior to the S phase.

In rabbit peritoneal neutrophils prelabeled with [3H] lyso platelet-activating factor, a protein kinase C inhibitor, staurosporine (1 microM), increased [3H]phosphatidylethanol ([3H]PEt) level in the presence of ethanol in a concentration- and time-dependent manner, providing evidence for staurosporine activation of phospholipase D (PLD). The staurosporine activation of the enzyme absolutely required both extracellular calcium and cytochalasin B, and was almost completely inhibited by pretreatment of the cells with pertussis toxin (IAP). In a reconstituted system where the purified Gi1 had been incorporated into phospholipid vesicles, staurosporine activated GTPase activity of Gi1 in a concentration-dependent fashion, with a maximal 4-5-fold effect. ADP-ribosylation by IAP of Gi1 in vesicles significantly suppressed the staurosporine activation. As with the GTPase activity of Gi1, GTPase activities of other purified IAP-sensitive G proteins, such as Gi2 and G(o), were significantly stimulated by staurosporine, but the cholera toxin substrate Gs was appreciably less sensitive to the staurosporine stimulation. The staurosporine activation of GTPase was also observed in rabbit neutrophil membranes from control cells, but not in membranes from IAP-treated neutrophils. From these results, we conclude that the staurosporine activation of PLD in rabbit neutrophils is attributed to the direct activation of an IAP-sensitive G protein in a similar manner to receptors occupied by agonists. By contrast, staurosporine failed to activate phosphoinositide-specific phospholipase C (PI-PLC) under the conditions in which it activated PLD, indicating that there exists a PLD activation pathway independent of PI-PLC. Furthermore, it was found that N-acetyl-beta-glucosaminidase release from the granules of intact neutrophils was evoked by staurosporine to almost the same extent as by fMLP (100 nM), but O2- generation was not affected. These results suggest a possibility that PLD pathway plays an important role in enzyme release, but is not sufficient for O2- generation, in rabbit peritoneal neutrophils.

The alpha-subunit of Gi-2, in addition to that of Gs (GTP-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in HL-60 cell membranes when a chemotactic receptor was stimulated by formyl-Met-Leu-Phe (fMLP), and the sites modified by cholera and pertussis toxins on the alpha-subunit of Gi-2 were different (Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 21394-21400). In order to investigate how the functions of Gi-2 were modified by cholera toxin, the ADP-ribosylated and unmodified proteins were purified from HL-60 cell membranes that had been incubated in the presence and absence of cholera toxin, respectively. The modified Gi-2 displayed unique properties as follows. 1) The ADP-ribosylated alpha-subunit had a more acidic pI than the unmodified one, leading to a partial resolution of the modified Gir2 trimer from the unmodified protein by an anion column chromatography. 2) When the purified proteins were incubated with [gamma-32P]GTP, the radioactivity was more greatly retained in the modified Gi-2 than in the unmodified protein. 3) The actual catalytic rate (kcat) of GTP hydrolysis was, indeed, markedly inhibited by cholera toxin-induced modification. 4) There was an increase in the apparent affinity of Gi-2 for GDP by cholera toxin-induced modification. 5) The modified Gi-2 exhibited a low substrate activity for pertussis toxin-catalyzed ADP-ribosylation. 6) A high-affinity fMLP binding to HL-60 cell membranes was more effectively reconstituted with the ADP-ribosylated Gi-2 than with the unmodified protein. These results suggested that the agonist-fMLP receptor complex was effectively coupled with the ADP-ribosylated Gi-2, resulting in the GTP-bound form, and that the hydrolysis of GTP on the modified alpha-subunit was selectively attenuated. Thus, cholera toxin ADP-ribosylated Gi-2 appeared to be not only a less sensitive pertussis toxin substrate but also an efficient signal transducer between receptors and effectors.