Expression of Rho-family GTPases (Rac, cdc42, RhoA) and their association with p-21 activated kinase in adult rat peripheral nerve

To clarify the presence of the Rho family of small GTPases p21-activated kinase (pak) signaling pathway in the PNS, we have examined their expression, the association between the small GTPases and pak and the pak kinase activity in the PNS using immunoblot analysis, immunohistochemistry, co-immunoprecipitation study, and in vitro kinase assay. Immunoblot analysis showed the expression of Rac, cdc42, RhoA and pak in the dorsal root ganglion (DRG) and sciatic nerve. The localization of these proteins in the DRG neurons and axons and Schwann cells of the sciatic nerve was confirmed by immunohistochemistry. Co-immunoprecipitation studies indicated the in vivo associations of pak with Rac and cdc42, but not with RhoA, in both the DRG and sciatic nerve. The autophosphorylation of pak and phosphorylation of histone H4 by pak were also found in the DRG and sciatic nerve as well as in the CNS. These results suggest that the Rac/cdc42-pak signaling pathway exists and functions in the PNS and may mediate some intracellular signals.     

Identification and the role of soluble antigens detected in bile from Eimeria stiedai-infected rabbits

Antibodies against Eimeria stiedai sporozoites and merozoites were detected in the sera of rabbits immunized with bile obtained from infected rabbits on the 15th day post-infection. The trails made by gliding sporozoites were also detected by the sera. After penetration into the host cell, an antibody-binding region was observed on the parasitophorous vacuole membranes of the parasites. Rabbits administered a combination of the bile and cholera toxin shed fewer oocysts in the feces after infection than control rabbits. The immunized rabbits developed a high level of IgA antibody against soluble antigens in the bile. By immunoblotting, antigens with molecular masses of 32, 37, and 49 kDa were detected in the bile obtained from infected rabbits on the 15th day postinfection. Absorption treatment with sporozoites reduced or abolished the antibody reactivity to the 32-kDa antigen of merozoites and the bile antigens. However, antibody reactivity to the 37- and 49-kDa antigens still remained. These results indicate that soluble antigens are present in the bile of rabbits in the acute phase of infection, and these may be produced and released by merozoites during the host cell invasion process.     

A phosphatidylinositol 3-kinase docking site in the cytoplasmic tail of the Jaagsiekte sheep retrovirus transmembrane protein is essential for envelope-induced transformation of NIH 3T3 cells

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer of sheep known as ovine pulmonary carcinoma. Recently, we have found that the expression of the JSRV envelope (Env) is sufficient to transform mouse NIH 3T3 cells in classical transformation assays. To further investigate the mechanisms of JSRV oncogenesis, we generated a series of envelope chimeras between JSRV and the JSRV-related endogenous retroviruses of sheep (enJSRVs) and assessed them in transformation assays. Chimeras containing the exogenous JSRV SU region and the enJSRV TM region were unable to transform NIH 3T3 cells. Additional chimeras containing only the carboxy-terminal portion of TM (a region that we previously identified as VR3) of the endogenous envelope with SU and the remaining portion of TM from the exogenous JSRV were also unable to transform NIH 3T3 cells. The VR3 region includes the putative membrane-spanning region and cytoplasmic tail of the JSRV TM glycoprotein; this suggested that the cytoplasmic tail of the JSRV Env mediates transformation, possibly via a cell signaling mechanism. Mutations Y590 and M593 in the cytoplasmic tail of the JSRV envelope were sufficient to inhibit the transforming abilities of these constructs. Y590 and M593 are part of a Y-X-X-M motif that is recognized by the phosphatidylinositol 3-kinase (PI-3K). PI-3K initiates a cell signaling pathway that inhibits apoptosis and is required for a number of mitogens during the G(1)-to-S-phase transition of the cell cycle. PI-3K activates Akt by phosphorylation of threonine 308 and serine 473. We detected by Western blot analysis phosphorylated Akt in serum-starved MP1 cells (NIH 3T3 cells transformed by JSRV) but not in the parental NIH 3T3 cells. These data indicate that the cytoplasmic tail of the JSRV TM is necessary for cell transformation and suggest a new mechanism of retroviral transformation. In addition, the ability to dissociate the function of the JSRV envelope to mediate viral entry from its transforming capacity has direct relevance for the design of JSRV-based vectors that target the differentiated epithelial cells of the lungs.     

Determination of bufalin-like immunoreactivity in serum of humans and rats by time-resolved fluoroimmunoassay for using a monoclonal antibody

The existence of a mammalian natriuretic substance or endogenous digitalis-like factor, which inhibits Na+,K+-ATPase and thereby regulates body fluid volume, has been speculated for a long time but has yet to be defined. We established in the present study a simple and highly sensitive procedure to measure bufalin, a constituent of toad venom preparation and a specific inhibitor of Na+,K+-ATPase by time-resolved fluoroimmunoassay (TR-FIA) and using a monoclonal antibody. The antibody was specific to bufalin and resembled bufadienolides but showed no cross-reactivity with digitoxin and ouabain. A bufalin-like immunoreactivity was detectable in serum of humans and rats by the proposed TR-FIA. The levels of bufalin-like immunoreactivity in serum of healthy volunteers were significantly correlated with their systolic blood pressure. Moreover, bufalin-like immunoreactivity in serum of Dahl-S rats increased in parallel with a period of high-salt diet. These results suggest that increased bufalin-like immunoreactivity may be associated with certain types of hypertension.     

Cloning and characterization of an inhibitor of apoptosis protein (IAP) from Bombyx mori

We cloned a novel inhibitor of apoptosis protein (IAP) family member, BmIAP, from Bombyx mori BmN cells. BmIAP contains two baculoviral IAP repeat (BIR) domains followed by a RING domain. BmIAP shares striking amino acid sequence similarity with lepidopteran IAPs, SfIAP and TnIAP, and with two baculoviral IAPs, CpIAP and OpIAP, suggesting evolutionary conservation. BmIAP blocks programmed cell death (apoptosis) in Spodoptera frugiperda Sf-21 cells induced by p35 deficient Autographa californica nucleopolyhedrovirus (AcMNPV). This anti-apoptotic function requires both the BIR domains and RING domain of BmIAP. In mammalian cells, BmIAP inhibits Bax induced but not Fas induced apoptosis. Further biochemical data suggest that BmIAP is a specific inhibitor of mammalian caspase-9, an initiator caspase in the mitochondria/cytochrome-c pathway, but not the downstream effector proteases, caspase-3 and caspase-7. These results suggest that suppression of apoptosis by lepidopteran IAPs in insect cells may involve inhibition of an upstream initiator caspase in the conserved mitochondria/cytochrome-c pathway for apoptosis.     

Molecular cloning of PalBH, a mammalian homologue of the Aspergillus atypical calpain PalB

A mammalian homologue of the Aspergillus atypical calpain PalB, PalBH, was identified and its cDNA sequences were determined in human and mouse. The PalBH mRNA was expressed nearly ubiquitously throughout mammalian tissues. When expressed in COS cells, PalBH was enriched in the nucleus, suggesting its role is distinct from that of conventional calpains.     

Functional analyses of Bph-Tod hybrid dioxygenase, which exhibits high degradation activity toward trichloroethylene

Biphenyl dioxygenase (BphDox) in Pseudomonas pseudoalcaligenes KF707 is a multicomponent enzyme consisting of an iron-sulfur protein (ISP) that is composed of alpha (BphA1) and beta (BphA2) subunits, a ferredoxin (FD(BphA3)), and a ferredoxin reductase (FDR(BphA4)). A recombinant Escherichia coli strain expressing hybrid Dox that had replaced BphA1 with TodC1 (alpha subunit of toluene dioxygenase (TolDox) of Pseudomonas putida) exhibited high activity toward trichloroethylene (TCE) (Furukawa, K., Hirose, J., Hayashida, S., and Nakamura, K. (1994) J. Bacteriol. 176, 2121-2123). In this study, ISP, FD, and FDR were purified and characterized. Reconstitution of the dioxygenase components consisting of purified ISP(TodC1BphA2), FD(BphA3), and FDR(BphA4) exhibited oxygenation activities toward biphenyl, toluene, and TCE. Native polyacrylamide gel electrophoresis followed by the Ferguson plot analyses demonstrated that ISP(TodC1BphA2) and ISP(BphA1A2) were present as heterohexamers, whereas ISP(TodC1C2) was present as a heterotetramer. The molecular activity (k(0)) of the hybrid Dox for TCE was 4.1 min(-1), which is comparable to that of TolDox. The K(m) value of the hybrid Dox for TCE was 130 microm, which was lower than 250 microm for TolDox. These results suggest that the alpha subunit of ISP is crucial for the determination of substrate specificity and that the change in the alpha subunit conformation of ISP from alpha(2)beta(2) to alpha(3)beta(3) results in the acquisition of higher affinity to TCE, which may lead to high TCE degradation activity.     

Molecular identification and immunohistochemical localization of atrial natriuretic peptide in the heart of the dromedary camel (Camelus dromedarius)

Atrial and B-type natriuretic peptide (ANP and BNP) are cardiac hormones synthesized and secreted by the myoendocrine cells of the heart. They exert potent actions on body fluid balance. Since various body organs including the heart are under high physiological stress during water and food deprivation in the desert nomads, we intended to perform molecular biological and histological studies of ANP in the heart of the dromedary camel Camelus dromedarius. Initially, we isolated cDNAs encoding ANP from the atrium and BNP from the atrium and ventricle of the dromedary camel. Putative mature ANP, deduced from the cDNA sequence, was identical to that of human and pig ANP, but the putative mature BNP was more diverse and was most similar to pig BNP (94% identity). Thus, we used antisera raised against human ANP that did not cross-react with pig BNP in the subsequent immunohistochemical studies. The ANP-expressing myoendocrine cells are most concentrated in the right atrium, to a lesser extent in the left atrium, and almost absent in the left ventricle. The immuno-positive cells are scattered uniformly in each region and are characterized by the presence of immunoreactive granular deposits around the nucleus. The left atrium comprises some ramifications of conductive cells (Purkinje fibers), some of which also contained ANP-immunoreactive granules. At the electron microscopic level, myoendocrine cells possessed secretory granules primarily in the perinuclear zone and a well-developed Golgi apparatus. The present study is the first comprehensive report dealing with the molecular cloning and immunohistochemical localization of ANP in the heart of a desert dwelling mammal.     

SMC6 is required for MMS-induced interchromosomal and sister chromatid recombinations in Saccharomyces cerevisiae

SMC6 (RHC18) in Saccharomyces cerevisiae, which is a homologue of the Schizosaccharomyces pombe rad18+ gene and essential for cell viability, encodes a structural maintenance of chromosomes (SMC) family protein. In contrast to the rest of the SMC family of proteins, Smc1-Smc4, which are the components of cohesin or condensin, little is known about Smc6. In this study, we generated temperature sensitive (ts) smc6 mutants of budding yeast and characterized their properties. One ts-mutant, smc6-56, ceased growth soon after up-shift to a non-permissive temperature, arrested in the late S and G2/M phase, and gradually lost viability. smc6-56 cells at a permissive temperature showed a higher sensitivity than wild-type cells to various DNA damaging agents including methyl methanesulfonate (MMS). The rad52 smc6-56 double mutant showed a sensitivity to MMS similar to that of the rad52 single mutant, indicating that Smc6 is involved in a pathway that requires Rad52 to function. Moreover, no induction of interchromosomal recombination and sister chromatid recombination was observed in smc6-56 cells, which occurred in wild-type cells upon exposure to MMS.