Involvement of gamma protein kinase C in estrogen-induced neuroprotection against focal brain ischemia through G protein-coupled estrogen receptor

The neuroprotective effects of estrogen were studied in the ischemic model mice by 90 min transient unilateral middle cerebral artery occlusion (MCAO) followed by 22.5 h reperfusion. The total infarct size in C57BL/6 female mice after MCAO and reperfusion was significantly smaller than that in male mice. Intraperitoneal injection of estrogen after the start of reperfusion significantly reduced the infarct volume in the male mice. However, no significant gender difference was found in total infarct size in gamma protein kinase C (PKC)-knockout mice, suggesting that the neuroprotective effects of estrogen are due to the activation of a specific subtype of PKC, gammaPKC, a neuron-specific PKC subtype, in the brain. We demonstrated that exogenous estrogen-induced neuroprotection was attenuated in gammaPKC-knockout mice. Immunocytochemical study showed that gammaPKC was translocated to nerve fiber-like structures when observed shortly after MCAO and reperfusion. We also visualized the rapid and reversible translocation of gammaPKC-GFP (green fluorescent protein) by estrogen stimulation in living CHO-K1 cells. These results suggest that the activation of gammaPKC through the G-protein-coupled estrogen receptors on the plasma membrane is involved in the estrogen-induced neuroprotection against focal brain ischemia.     

Involvement of N-terminal-extended form of sphingosine kinase 2 in serum-dependent regulation of cell proliferation and apoptosis

Sphingosine kinase (SPHK) 1 is implicated in the regulation of cell proliferation and anti-apoptotic processes by catalyzing the formation of an important bioactive messenger, sphingosine 1-phosphate. Unlike the proliferative action of SPHK1, another isozyme, SPHK2, has been shown to possess anti-proliferative or pro-apoptotic action. Molecular mechanisms of SPHK2 action, however, are largely unknown. The present studies were undertaken to characterize the N-terminal-extended form of SPHK2 (SPHK2-L) by comparing it with the originally reported form, SPHK2-S. Real-time quantitative PCR analysis revealed that SPHK2-L mRNA is the major form in several human cell lines and tissues. From sequence analyses it was concluded that SPHK2-L is a species-specific isoform that is expressed in human but not in mouse. At the protein level it has been demonstrated by immunoprecipitation studies that SPHK2-L is the major isoform in human hepatoma HepG2 cells. SPHK2-L, when expressed in human embryonic kidney (HEK) 293 cells, did not show any inhibition of DNA synthesis in the presence of serum, whereas it showed marked inhibition in the absence of serum. Moreover, serum deprivation resulted in the translocation of SPHK2-L into the nuclei. In addition, serum deprivation induced SPHK2-L expression in HEK293 cells. Furthermore, suppression of SPHK2 by small interfering RNA treatment prevented serum deprivation- or drug-induced apoptosis in HEK293 cells. Taken together, these results indicate that a major form of SPHK2 splice variant, SPHK2-L, in human cells does not inhibit DNA synthesis under normal conditions and that SPHK2-L accumulation in the nucleus induced by serum deprivation may be involved in the cessation of cell proliferation or apoptosis depending on the cell type.     

Role of human heme oxygenase-1 in attenuating TNF-alpha-mediated inflammation injury in endothelial cells

Heme oxygenase (HO) is the rate-limiting enzyme in the formation of bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator and a vasodilator. Cyclooxygenase (COX) is a hemeprotein that catalyzes the conversion of arachidonic acid (AA) to various prostanoids, which play an important role in the regulation of vascular endothelial function in normal and disease states. The influence of suppression or overexpression of HO isoforms on COX expression and synthesis of prostanoids is of considerable physiological importance. Consequently, the goal of the present study was to determine whether the heme-HO system regulates COX enzyme expression and activity in vascular endothelial cells in the absence and presence of TNF-alpha (100 ng/ml). Endothelial cells stably transfected with the retrovirus containing the human HO-1 gene exhibited a several-fold increase in HO-1 protein levels, which was accompanied by an increase in HO activity and a marked decrease in PGE(2) and 6-keto PGF(1alpha) levels. We also assessed the effect of retrovirus-mediated HO-1 gene transfer in the sense and antisense orientation on HO-1 expression and cell cycle progression in human endothelial cells. The levels of CO and HO activity were increased in cells transduced with the HO-1 sense and were greatly suppressed in cells transduced with HO-1 antisense as compared to control sham-transduced cells (P < 0.05). The percentage of the G(1)-phase in cells transduced with HO-1 significantly increased (41.4% +/- 9.1) compared with control endothelial cells (34.8% +/- 4.9). We measured COX activity by determining the levels of PGI(2) and PGE(2). The levels of PGI(2) decreased in cells transduced with HO-1 sense and increased in cells transduced with HO-1 in antisense orientation. The expression of p27 was also studied and showed a marked decrease in cells transduced with HO-1 sense and a marked increase in the HO-1 antisense transduced cells. Cell cycle analysis of endothelial cell DNA distributions indicated that the TNF-alpha-induced decrease in the proportion of G(1)-phase cells and increase in apoptotic cells in control cultures could be abrogated by transfection with HO-1 in the sense orientation. Tin mesoporphyrin (SnMP) reversed the protective effect of HO-1. These results demonstrate that overexpressing HO-1 mitigated the TNF-alpha-mediated changes in cell cycle progression and apoptosis, perhaps by a decrease in the levels of COX activity.     

Differential expression of annexin V during spermatogenesis in the newt Cynops pyrrhogaster

 In order to isolate genes whose expression is up-regulated after the initiation of meiosis, we screened a cDNA expression library of newt testes with antiserum against homogenates of testes derived from the spermatogonial and spermatocyte stages. We report the isolation of spermatocyte-specific cDNA clones encoding a newt homologue of the calcium-dependent phospholipid-binding protein, annexin V. Northern blot analysis showed that newt annexin V mRNA was 1.7 kb in length and was expressed strongly in testes, but weakly in other organs. In situ hybridization revealed that the expression of newt annexin mRNA was barely observed in spermatogonia, but increased significantly in leptotene-zygotene primary spermatocytes and reached a maximum level in pachytene spermatocytes and round spermatids. The newt annexin V cDNA predicted a 323-amino acid protein and had a 68% homology to human annexin V. The predicted amino acid sequence contained a conserved 4-fold internal repeat of approximately 70 residues like other annexin proteins. Immunoblot analysis using the monoclonal antibody against newt annexin V showed that the protein was expressed scarcely in spermatogonia but was abundantly expressed in stages from primary spermatocytes to spermatids; this pattern was consistent to that of the mRNA. Immunohistochemical analysis revealed that newt annexin V was localized in the cytoplasm of the spermatogenic cells, but not in somatic cells such as Sertoli cells or pericystic cells. These results indicate that the expression of newt annexin V is up-regulated in the spermatogenic cells after the initiation of meiosis and suggest that newt annexin V plays an important role in spermatogenesis. Received: 8 December 1995 / Accepted: 12 February 1996 Edited by H. Shimada/D. Tautz     

9-Deoxy-Δ9-prostaglandin D2, a prostaglandin D2 derivative with potent antineoplastic and weak smooth muscle-contracting activities

We have recently described a potential antineoplastic effect of prostaglandin D₂ (PGD₂) (Fukushima, M., Kato, T., Ueda, R., Ota, K., Narumiya, S., and Hayaishi, O. (1982) Biochem. Biophys. Res. Commun. $\text{105}$, 956–964). In the present study three analogues of PGD₂ were examined for their antineoplastic and other biological activities. 9-Deoxy-Δ⁹-PGD₂, a dehydrate of PGD₂, had about 3 times stronger growth inhibitory effect than PGD₂ on L1210 leukemia cultured cells. The IC₅₀ value of this compound was 0.5–1.0 μg/ml (2μ$\text{M}$). In contrast to PGD₂, this compound lacked a contracting activity on colonic smooth muscle and caused less diarrhea. The antiaggregatory action of the compound on blood platelet was about 10 times weaker than PGD₂. The growth inhibitory activity was also observed with cyclopentenone itself; the IC₅₀ value was about 100 μM. On the other hand, BW245C which had the antiaggregatory activity comparable with PGD₂ showed no growth inhibition. These results suggest that cyclopentenone structure is an essential moiety of prostaglandin derivaties for cell growth inhibition and that the growth inhibitory activity could be dissociated from other biological activities of prostaglandins.     

Specific Monoclonal Antibody Overcomes the Salmonella enterica Serovar Typhimurium’s Adaptive Mechanisms of Intramacrophage Survival and Replication

Salmonella-specific antibodies play an important role in host immunity; however, the mechanisms of Salmonella clearance by pathogen-specific antibodies remain to be completely elucidated since previous studies on antibody-mediated protection have yielded inconsistent results. These inconsistencies are at least partially attributable to the use of polyclonal antibodies against Salmonella antigens. Here, we developed a new monoclonal antibody (mAb)-449 and identified its related immunogen that protected BALB/c mice from infection with Salmonella enterica serovar Typhimurium. In addition, these data indicate that the mAb-449 immunogen is likely a major protective antigen. Using in vitro infection studies, we also analyzed the mechanism by which mAb-449 conferred host protection. Notably, macrophages infected with mAb-449-treated S. Typhimurium showed enhanced pathogen uptake compared to counterparts infected with control IgG-treated bacteria. Moreover, these macrophages produced elevated levels of pro-inflammatory cytokine TNFα and nitric oxide, indicating that mAb-449 enhanced macrophage activation. Finally, the number of intracellular bacteria in mAb-449-activated macrophages decreased considerably, while the opposite was found in IgG-treated controls. Based on these findings, we suggest that, although S. Typhimurium has the potential to survive and replicate within macrophages, host production of a specific antibody can effectively mediate macrophage activation for clearance of intracellular bacteria.     

A phage display‐selected peptide inhibitor of Agrobacterium vitis polygalacturonase

Agrobacterium vitis, the causal agent of crown gall of grapevine, is a threat to viticulture worldwide. A major virulence factor of this pathogen is polygalacturonase, an enzyme that degrades pectin components of the xylem cell wall. A single gene encodes for the polygalacturonase gene. Disruption of the polygalacturonase gene results in a mutant that is less pathogenic and produces significantly fewer root lesions on grapevines. Thus, the identification of peptides or proteins that could inhibit the activity of polygalacturonase could be part of a strategy for the protection of plants against this pathogen. A phage‐displayed combinatorial peptide library was used to isolate peptides with a high binding affinity to A. vitis polygalacturonase. These peptides showed sequence similarity to regions of Oryza sativa (EMS66324, Japonica) and Triticum urartu (NP_001054402, wild wheat) polygalacturonase‐inhibiting proteins (PGIPs). Furthermore, these panning experiments identified a peptide, SVTIHHLGGGS, which was able to reduce A. vitis polygalacturonase activity by 35% in vitro. Truncation studies showed that the IHHL motif alone is sufficient to inhibit A. vitis polygalacturonase activity.     

Structure-activity relationships of novel piperazines as antagonists for the melanocortin-4 receptor

During the investigation of antagonists for the MC4 receptor, we found that 10ab having a naphthyl group showed almost the same binding affinity for the MC4 receptor as that of the lead compound 1 with a benzoyl group. We also developed a new type of compounds, namely, bis-piperazines, and found that the bis-piperazines 10 exhibited a high affinity for the MC4 receptor. In particular, (-)-10bg exhibited the highest affinity for the MC4 receptor with an IC50 value of 8.13nM. In this paper, we present the design, synthesis, and structure-activity relationships of the novel bis-piperazines as MC4 receptor antagonists.     

1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity and tyrosinase inhibitory effects of constituents of sugarcane molasses

In the course of our work into the use of cane by-products, we have studied the isolation and structural determination of bioactive compounds in sugarcane molasses. In this study, three stereo isomers of syringyl glycerol 3'-O-beta-D-glucopyranoside, three stereo isomers of guaiacyl glycerol 3'-O-beta-D-glucopyranoside, a syringyl glycerol 2'-O-beta-D-glucopyranoside, tachioside and a 2,3-dihydro-3,5-dihydroxy-6-methyl-4-(H)-pyran-4-one (DDMP) were isolated from the 25% methanol eluate by Amberlite XAD-2 column chromatography of sugarcane molasses. The structures of these compounds were determined on the basis of spectroscopic evidence. These isolated compounds were examined for their scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical species, and for their inhibitory activity against mushroom tyrosinase. All of the isolated compounds showed DPPH radical scavenging activity, while DDMP and tachioside showed mushroom tyrosinase inhibitory activity.     

A significant role for the heme oxygenase-1 gene in endothelial cell cycle progression

Heme oxygenase (HO) catalyzes the conversion of heme to biliverdin with the release of iron and carbon monoxide. HO-1 is inducible by inflammatory conditions, which cause oxidative stress in endothelial cells. Overexpression of human HO-1 in endothelial cells may have the potential to provide protection against a variety of agents that cause oxidative stress. We investigated the physiological significance of human HO-1 overexpression, using a retroviral vector, on cell cycle progression in the presence and absence of pyrrolidine dithiocarbamate (PDTC). The addition of PDTC (25 and 50 microM) to human microvessel endothelial cells over 24 h resulted in significant (P < 0.05) abnormalities in DNA distribution and cell cycle progression compared to cells overexpressing the HO-1 gene. The addition of PDTC resulted in a significantly decreased G(1) phase and an increased G(2)/M phase in the control cells, but not in cells transduced with the human HO-1 gene (P < 0.05). Further, PDTC had a potent effect on DNA distribution abnormalities in exponentially grown cells compared to subconfluent cells. Upregulation of HO activity in endothelial cells, as a result of overexpressing human HO-1, prevented PDTC-mediated abnormalities in DNA distribution. Inhibition of HO activity by tin-mesoporphyrin (SnMP) (30 microM) resulted in enhancement of PDTC-mediated abnormalities in cell cycle progression. Bilirubin or iron did not mediate DNA distribution. We conclude that an increase in endothelial cell HO-1 activity with subsequent generation of carbon monoxide, elicited by gene transfer, reversed the PDTC-mediated abnormalities in cell cycle progression and is thus a potential therapeutic means for attenuating the effects of oxidative stress-causing agents.