Antimalarial activity of endoperoxide compound 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol

Plasmodium falciparum, the major causative parasite for the disease, has acquired resistance to most of the antimalarial drugs used today, presenting an immediate need for new antimalarial drugs. Here, we report the in vitro and in vivo antimalarial activities of 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) against P. falciparum and Plasmodium berghei parasites. The N-251 showed high antimalarial potencies both in the in vitro and the in vivo tests (EC₅₀ 2.3 × 10⁻⁸ M; ED₅₀ 15 mg/kg (per oral)). The potencies were similar to that of artemisinin in vitro and greater than artemisinin's activity in vivo (p.o.). In addition, N-251 has little toxicity: a single oral administration at 2000 mg/kg to a rat gave no health problems to it. Administration of N-251 to mice bearing 1% of parasitemia (per oral 68 mg/kg, 3 times a day for 3 consecutive days) resulted in a dramatic decrease in the parasitemia: all the 5 mice given N-251 were cured without any recurrence, with no diarrhea or weight loss occurring in the 60 days of experiment. N-251 deserves more extensive clinical evaluation, desirably including future trials in the human.     

Culture and hybridization experiments on an ulva clade including the Qingdao strain blooming in the yellow sea

In the summer of 2008, immediately prior to the Beijing Olympics, a massive green tide of the genus Ulva covered the Qingdao coast of the Yellow Sea in China. Based on molecular analyses using the nuclear encoded rDNA internal transcribed spacer (ITS) region, the Qingdao strains dominating the green tide were reported to be included in a single phylogenetic clade, currently regarded as a single species. On the other hand, our detailed phylogenetic analyses of the clade, using a higher resolution DNA marker, suggested that two genetically separate entities could be included within the clade. However, speciation within the Ulva clade has not yet been examined. We examined the occurrence of an intricate speciation within the clade, including the Qingdao strains, via combined studies of culture, hybridization and phylogenetic analysis. The two entities separated by our phylogenetic analyses of the clade were simply distinguished as U. linza and U. prolifera morphologically by the absence or presence of branches in cultured thalli. The inclusion of sexual strains and several asexual strains were found in each taxon. Hybridizations among the sexual strains also supported the separation by a partial gamete incompatibility. The sexually reproducing Qingdao strains crossed with U. prolifera without any reproductive boundary, but a complete reproductive isolation to U. linza occurred by gamete incompatibility. The results demonstrate that the U. prolifera group includes two types of sexual strains distinguishable by crossing affinity to U. linza. Species identification within the Ulva clade requires high resolution DNA markers and/or hybridization experiments and is not possible by reliance on the ITS markers alone.     

Ceramide 1-phosphate, a mediator of phagocytosis

The agonist-stimulated metabolism of membrane lipids produces potent second messengers that regulate phagocytosis. We studied whether human ceramide kinase (hCERK) activity and ceramide 1-phosphate formation could lead to enhanced phagocytosis through a mechanism involving modulation of the membrane-structural order parameter. hCERK was stably transfected into COS-1 cells that were stably transfected with the FcgammaRIIA receptor. hCERK-transfected cells displayed a significant increase in phagocytic index in association with increased ceramide kinase activation and translocation to lipid rafts after activation with opsonized erythrocytes. When challenged with opsonized erythrocytes, hCERK-transfected cells increased phagocytosis by 1.5-fold compared with vector control and simultaneously increased ceramide 1-phosphate levels 2-fold compared with vector and unstimulated control cells. Control and hCERK-transfected cells were subjected to cellular fractionation. Utilizing an antibody against hCERK, we observed that CERK translocates during activation from the cytosol to a lipid raft fraction. The plasma membrane-structural order parameter of the transfectants was measured by labeling cells with Laurdan. Cells transfected with hCERK showed a higher liquid crystalline order than control cells with stimulation, conditions that are favorable for the promotion of membrane fusion at the sites of phagocytosis. The change in the structural order parameter of the lipid rafts probably contributes to phagocytosis by promoting phagosome formation.     

Modulation of ICa-L by alpha1-adrenergic stimulation in rat ventricular myocytes

We found when L-type calcium current (ICa-L) was recorded with the perforated patch-clamp method in rat ventricular myocytes that bath application of phenylephrine (with propranolol) evoked a biphasic response characterized by an initial transient suppression followed by a sustained potentiation. The transient suppression occurred 30-60 s after phenylephrine perfusion and reached peak inhibition at approximately 2 min. The biphasic modulation of ICa-L was also elicited by methoxamine, and the effects of phenylephrine were blocked by prazosin, indicating that the responses were mediated through alpha1-adrenoceptors. Pretreatment of cells with H7 (100 micromol/L), a broad-spectrum protein kinase inhibitor that inhibits both protein kinase C and A, eliminated potentiation but did not affect transient suppression. The transient suppression occurred concurrently with the acceleration of the fast component of ICa-L inactivation. Depletion of intracellular Ca2+ stores by ryanodine plus caffeine or thapsigargin eliminated the transient suppression. When ICa-L was recorded with whole-cell patch-clamp and with 0.05 mmol/L EGTA in the pipette solution to allow intracellular Ca2+ to fluctuate, phenylephrine evoked a transient suppression as in the perforated patch recordings. Heparin, a specific blocker of IP3 (inositol 1,4,5-trisphosphate) receptors, eliminated the phenylephrine-induced transient suppression of ICa-L when added to the pipette solution. Intensive chelation of intracellular Ca2+ by 5 mmol/L BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) in the pipette solution also eliminated the phenylephrine-induced transient suppression of ICa-L. We conclude that transient increase in the concentration of intracellular calcium ([Ca2+]i) caused by Ca2+ release from intracellular stores underlies the transient suppression of ICa-L, whereas the potentiation of ICa-L is a result of activation of protein kinases.     

PDK1 is required for the hormonal signaling pathway leading to meiotic resumption in starfish oocytes

Meiotic resumption is generally under the control of an extracellular maturation-inducing hormone. It is equivalent to the G2-M phase transition in somatic cell mitosis and is regulated by cyclin B-Cdc2 kinase. However, the complete signaling pathway from the hormone to cyclin B-Cdc2 is yet unclear in any organism. A model system to analyze meiotic resumption is the starfish oocyte, in which Akt/protein kinase B (PKB) plays a key mediator in hormonal signaling that leads to cyclin B-Cdc2 activation. Here we show in starfish oocytes that when PDK1 activity is inhibited by a neutralizing antibody, maturation-inducing hormone fails to induce cyclin B-Cdc2 activation at the meiotic G2-M phase transition, even though PDK2 activity becomes detectable. These observations assign a novel role to PDK1 for a hormonal signaling intermediate toward meiotic resumption. They further support that PDK2 is a molecule distinct from PDK1 and Akt, and that PDK2 activity is not sufficient for the full activation of Akt in the absence of PDK1 activity.     

Conformational transition state is responsible for assembly of microtubule-binding domain of tau protein

In the brains of Alzheimer's disease patients, the tau protein dissociates from the axonal microtubule and abnormally aggregates to form a paired helical filament (PHF). One of the priorities in Alzheimer research is to clarify the mechanism of PHF formation. Although several reports on the regulation of tau assembly have been published, it is not yet clear whether in vivo PHFs are composed of beta-structures or alpha-helices. Since the four-repeat microtubule-binding domain (4RMBD) of the tau protein has been considered to play an essential role in PHF formation, its heparin-induced assembly propensity was investigated by the thioflavin fluorescence method to clarify what conformation is most preferred for the assembly. We analyzed the assembly propensity of 4RMBD in Tris-HCl buffer with different trifluoroethanol (TFE) contents, because TFE reversibly induces the transition of the random structure to the alpha-helical structure in an aqueous solution. Consequently, it was observed that the 4RMBD assembly is most significantly favored to proceed in the 10-30% TFE solution, the concentration of which corresponds to the activated transition state of 4RMBD from a random structure to an alpha-helical structure, as determined from the circular dichroism (CD) spectral changes. Since such an assembly does not occur in a buffer containing TFE of < 10% or > 40%, the intermediate conformation between the random and alpha-helical structures could be most responsible for the PHF formation of 4RMBD. This is the first report to clarify that the non-native alpha-helical intermediate in transition from random coil is directly associated with filament formation at the start of PHF formation.     

Inhibition of c-Jun NH2-terminal kinase activity improves ischemia/reperfusion injury in rat lungs

Although c-Jun NH(2)-terminal kinase (JNK) has been implicated in the pathogenesis of transplantation-induced ischemia/reperfusion (I/R) injury in various organs, its significance in lung transplantation has not been conclusively elucidated. We therefore attempted to measure the transitional changes in JNK and AP-1 activities in I/R-injured lungs. Subsequently, we assessed the effects of JNK inhibition by the three agents including SP600125 on the degree of lung injury assessed by means of various biological markers in bronchoalveolar lavage fluid and histological examination including detection of apoptosis. In addition, we evaluated the changes in p38, extracellular signal-regulated kinase, and NF-kappaB-DNA binding activity. I/R injury was established in the isolated rat lung preserved in modified Euro-Collins solution at 4 degrees C for 4 h followed by reperfusion at 37 degrees C for 3 h. We found that AP-1 was transiently activated during ischemia but showed sustained activation during reperfusion, leading to significant lung injury and apoptosis. The change in AP-1 was generally in parallel with that of JNK, which was activated in epithelial cells (bronchial and alveolar), alveolar macrophages, and smooth muscle cells (bronchial and vascular) on immunohistochemical examination. The change in NF-kappaB qualitatively differed from that of AP-1. Protein leakage, release of lactate dehydrogenase and TNF-alpha into bronchoalveolar lavage fluid, and lung injury were improved, and apoptosis was suppressed by JNK inhibition. In conclusion, JNK plays a pivotal role in mediating lung injury caused by I/R. Therefore, inhibition of JNK activity has potential as an effective therapeutic strategy for preventing I/R injury during lung transplantation.     

Enhanced tumor-specific long-term immunity of hemagglutinating [correction of hemaggluttinating] virus of Japan-mediated dendritic cell-tumor fused cell vaccination by coadministration with CpG oligodeoxynucleotides

Immunization with dendritic cells (DCs) using various Ag-loading approaches has shown promising results in tumor-specific immunotherapy and immunoprevention. Fused cells (FCs) that are generated from DCs and tumor cells are one of effective cancer vaccines because both known and unknown tumor Ags are presented on the FCs and recognized by T cells. In this study, we attempted to augment antitumor immunity by the combination of DC-tumor FC vaccination with immunostimulatory oligodeoxynucleotides containing CpG motif (CpG ODN). Murine DCs were fused with syngeneic tumor cells ex vivo using inactivated hemagglutinating virus of Japan (Sendai virus). Mice were intradermally (i.d.) immunized with FCs and/or CpG ODN. Coadministration of CpG ODN enhanced the phenotypical maturation of FCs and unfused DCs, and the production of Th1 cytokines, such as IFN-gamma and IL-12, leading to the induction of tumor-specific CTLs without falling into T cell anergy. In addition, immunization with FCs + CpG ODN provided significant protection against lethal s.c. tumor challenge and spontaneous lung metastasis compared with that with either FCs or CpG ODN alone. Furthermore, among mice that rejected tumor challenge, the mice immunized with FCs + CpG ODN, but not the mice immunized with FCs or CpG ODN alone, completely rejected tumor rechallenge, indicating that CpG ODN provided long-term maintenance of tumor-specific immunity induced by FCs. Thus, the combination of DC-tumor FCs and CpG ODN is an effective and feasible cancer vaccine to prevent the generation and recurrence of cancers.