Wild type and calmodulin mutants (cam) of Paramecium tetraurelia were examined for cold-sensitive responses. Among mutants tested, cam12 and cam13 mutants, which have substitutions in N-terminal lobe of calmodulin molecule, reduced both responses in the swimming and the membrane potential. Under voltage clamp conditions, the cooling stimulus to the wild type cell induced a transient inward current whose amplitude increased with the rate of temperature drop. The cam12 cell did not induce inward currents in response to cooling with a rate slower than - 0.4°C/s. The reduced current response of cam12 mutant was restored by an external application of a phosphodiesterase inhibitor, theophylline. Also, an intracellular injection of hydrolysis-resistant cyclic nucleotides, either 8-bromoadenosine 3', 5'-cyclic monophosphate (8-Br-cAMP) or 8-bromoguanosine 3, 5'-cyclic monophosphate (8-Br-cGMP), restored the current response. Such restoration was accompanied by shifts of the resting potential to hyperpolarized levels and by an increase in the membrane conductance. The results suggest the possibility that calmodulin and cyclic nucleotide regulate K+ channels responsive to the cooling stimulus.
In the yeast Saccharomyces cerevisiae, autophagy, a bulk protein degradation in the vacuole, is induced in response to nutrient starvation. In a screen for mutations that result in induction of autophagy even in the presence of nutrients, we have isolated four mutants representing two esc complementation groups. These mutants induce autophagy of which activity is represented by activation of truncated alkaline phosphatase that is designed to be expressed in the cytosol. CSC1 was cloned by complementation of loss of viability phenotype of csc1-1 mutant and shown to be identical to END13/VPS4/GRD13. Though csc1-1 mutation is recessive, cells of Δcsc1 do not induce autophagy in rich media, suggesting that csc1-1 allele is not a complete loss-of-function. Csc1p is a member of novel ATPase family named AAA protein including Secl8p/NSF, Cdc48p/p97, and PasSp. Mutation site in cscl-1 is found in the SRH region that is highly conserved among AAA proteins. Cells of csc1-1 show sorting defect of CPY and the appearance of the class E compartment. These mutant phenotypes suggest the role of the protein that is involved in the traffic among the Golgi, endosome, and the vacuole in autophagy.
To investigate neuron-specific antigens, hybridoma cells were produced between mouse spleen cells immunized with human neuroblastoma cells (IMR-32) and mouse myeloma cells. 247 hybridoma clones were harvested and one of them was further cultured for recloning. Eventually, one hybridoma clone was obtained and its antibody was designated N-A8. The characteristics of this antibody were determined by immunostaining and flow cytometry. First, the antibody recognized the surface antigens of IMR-32 cells. Second, unexpectedly, N-A8 was reactive not only with human neuroblastoma cell lines but also with human lung cancer cell lines. As analyzed by immunoprecipitation method and SDS-PAGE, the molecular size of the antigen recognized by N-A8 was 210 kDa. The antigen was then purified by affinity chromatography and identified as neural adhesion molecule LI by amino acid sequence analysis. By the present investigation, it was clearly demonstrated that LI is expressed in human lung cancer cells.
Expression of 105 k-Da heat shock protein (HSP105) during mouse embryo development was investigated. Although HSP105 was detected scarcely in mouse embryos at gestational day (GD) 8, its level of HSP105 increased markedly in the embryos from GD 9 to 11, and then decreased gradually by GD 14. In contrast, 70 k-Da heat shock cognate protein (HSC70) was detected at constantly high levels in embryo from GD 8 to 17 (before birth). The transient increase in level of HSP105 was observed in most embryonic tissues. Since the levels of HSP105 mRNA was detected at constantly high levels in the embryos between GD 8 and 12, the increased expression of HSP105 at GD 9 to 11 seemed to be regulated at posttranscriptional level. Immunohistochemically, HSP105 as well as HSC70 were localized in the cytoplasm and the nuclei of cells in various mouse embryonic tissues. After treatment of tissue sections with proteinase K, HSP105 but not HSC70 was also found to be localized in condensed cells and condensed bodies which showed the typical characteristics of apoptotic cells and apoptotic bodies at the interdigital regions of limbs. These findings suggest that HSP105 plays important roles during mouse embryo development.
Polyploidization of Meth-A and B16-F10 cells by demecolcine was examined using flow cytometry (FCM). In the presence of demecolcine, both cell lines were polyploidized to more than 16c DNA content. A marked difference was observed in the durations of S phase of polyploidy. The S-phase duration of Meth-A cells was doubly increased with ploidy, but that of B16F10 cells remained constant. When the rate of DNA synthesis in the polyploidizing cells was examined through the BrdU-uptake experiments, it was confirmed that the level of DNA-synthesis rate was constant in Meth-A cells but increased in B16F10 cells. The cellular content of c-Myc protein in polyploidized cells was also examined using anti-c-Myc monoclonal antibody. The c-Myc level of Meth-A cells was constant regardless of the ploidy but that of B16F10 cells increased with ploidy. Thus, the c-Myc content seems to be related to the duration of S phase in polyploidy.
When M cells derived from rat liver and then transformed by treatment with 4-dimethylamino-azobenzene were cultured in vitro, the culture became covered with a collagen fiber network. When the M cells were cultured in the presence of more than 10 μg/ml heparin, no collagen fiber formation was observed. The inhibitory effect was evident at 5 μg/ml but was not significant at 1 μg/ml. High performance liquid chromatography (HPLC) showed that about 50% decrease in hydroxyproline content occurred in the presence of 10 μg/ml heparin. The inhibition by heparin reached a plateau at 10 μg/ml. Other glycosaminoglycans such as heparan sulfate, keratan sulfate, chondroitin sulfate B and chondroitin sulfate C did not show a significant effect on hydroxyproline content. Modified heparins slightly decreased hydroxyproline content, but the collagen fibers were still observed. These results indicate that the native structure of heparin is important to attain the complete inhibition of collagen fiber formation; the basic structure, (-GlcUA or IdUA β 1-4 GlcNSO3-)n, is important. HPLC, Northern blot analysis and Western blot analysis for rat type I collagen revealed that collagen synthesis is independent of heparin, but that collagen fiber formation is prevented by heparin.
αB-Crystallin, one of the small heat shock proteins, is constitutively expressed in lens as well as in nonlenticular tissues. It can function as a molecular chaperone for other lens crystallins and some other proteins. Its nonocular function is unknown although some reported one of them is related to cytoskeletal networks and/or components. In the present study, we demonstrate the association of αB-crystallin with tubulin. Imnninoprecipitation experiments using L6 myoblast cell lysate with anti-αB-crystallin antibody resulted in the coprecipitation of α-tubulin, which was apparently temperature-dependent. Further, purified αB-crystallin prevented the turbidity development of purified tubulin molecule at 37°C in vitro. Sucrose gradient centrifugation revealed that this chaperone activity was accompanied by the formation of large complex of αB-crystallin and tubulin dimer. These results indicate that one of the nonlenticular functions of αB-crystallin may be the protection of tubulin subunits of microtubules.
We have found that 1, 9-DideoxyforskoIin (DDF) strongly inhibited the cell death induced by ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in MDCK cells, suggesting that these protein toxins have a DDF-sensitive common pathway leading to cell death. However, no significant effect of forskolin on these toxins was observed, implying that cAMP-independent DDF specific mechanism is responsible for the inhibitory effect. The protective effect of DDF against ricin-induced cell death was significantly reversed by the increase in extracellular Ca2+ concentrations. The addition of brefeldin A (BFA) also reversed the protective effect of DDF, while BFA alone slightly increased the cytotoxicity of ricin. The protein synthesis inhibitory activity of modeccin was strongly inhibited by DDF, while only partial inhibition of the activities of ricin and diphtheria toxin was observed. However, the activity of Pseudomonas toxin was enhanced by DDF rather than inhibited. Thus, the process leading to cell death and protein synthesis inhibition by these toxins may be separately affected by DDF, and the protective effect of DDF against toxin-induced cell death is distinct from its effect on protein synthesis inhibition by toxins. Forskolin and DDF slightly increased rather than inhibited the binding and the internalization of ricin to MDCK cells. Despite the strong inhibitory effect of DDF on toxin-induced cell death, DDF did not block toxin-induced DNA fragmentation. These results suggest that DNA fragmentation and cell death may be triggered through separate pathways during apoptosis caused by these toxins, and that a DDF-sensitive specific step may be present in the pathway leading to cell death.
Valinomycin is a potassium ionophore, and is well known to cause the collapse of the mitochondrial membrane potential. It has been reported that loss of mitochondrial membrane potential is observed in the early stages of apoptosis induced by various agents. Thus, the effects of valinomycin on tumor cells were examined. Valinomycin induced uncoupling of respiration and depolarization of isolated mitochondria. Depolarization of intact mitochondria in AH-130 rat ascites hepatoma cells was also induced by valinomycin. Valinomycin induced apoptosis revealing the typical apoptotic characteristics such as fragmentation and ladder formation of DNA, shrinkage of cells, and formation of pycnotic nucleus. There was a correlation between the depolarization of mitochondria and DNA fragmentation. After depolarization of mitochondria, the activity of caspase-3-like protease but not caspase-1-like protease increased markedly. In contrast, this apoptosis did not involve the release of reactive oxygen species from mitochondria, increase in intracellular calcium concentration, or protein synthesis. In addition, anti-apoptotic members of the Bcl-2 family (Bcl-xL and Bcl-2) were not correlated with apoptosis. These results indicate that valinomycin might induce apoptosis through degradation of the mitochondrial membrane potential. Taken together, these observations suggest that there may be a mechanism that transmits the signal from mitochondrial depolarization to subsequent apoptosis execution steps.
Autoantibodies from a patient with rheumatoid arthritis recognized an antigen localized in the Golgi complex of various cells tested. The autoantibodies were used as a probe for screening rat NRK cDNA library, resulting in identification of an 11 kbp cDNA. The cDNA contained an open reading frame which encodes a 3, 187-residue protein with a calculated mass of 364 kDa. The predicted protein, GCP364 (for a Golgi complex-associated protein of 364 kDa), was found to have no NH2-terminal signal sequence but a single hydrophobic domain at the COOH terminus and characteristically contain many coiled-coil domains with various sizes throughout the entire sequence. The identity of GCP364 with the autoantigen was confirmed by immunofluorescence and immunoblot analysis with the autoantibodies and anti-recombinant GCP364 produced in rabbits and by transfection/expression experiments. Search for the protein sequence data base revealed that GCP364 has 75% identity in amino acid sequence with human GCP372/giantin, indicating that it is a rat homolog of the latter. Immunogold electron microscopy showed that GCP364 was not detected on coated vesicles derived from the Golgi membrane, suggesting no involvement in the formation of transport vesicles. When cells were perforated and incubated with anti-GCP364 serum, the Golgi complex localized at perinuclear regions was dispersed into fragment-like structures as observed in nocodazole-treated cells. Taken together, these results suggest that GCP364 is anchored to the membrane by the COOH-terminal hydrophobic domain and has an extremely long cytopJasmic domain with coiled-coil structures, which may be involved in the formation and/or maintenance of the characteristic Golgi structure.