Mammalian phospholipase Czeta induces oocyte activation from the sperm perinuclear matrix

Mammalian sperm-borne oocyte activating factor (SOAF) induces oocyte activation from a compartment that engages the oocyte cytoplasm, but it is not known how. A SOAF-containing extract (SE) was solubilized from the submembrane perinuclear matrix, a domain that enters the egg. SE initiated activation sufficient for full development. Microinjection coupled to tandem mass spectrometry enabled functional correlation profiling of fractionated SE without a priori assumptions about its chemical nature. Phospholipase C-zeta (PLCzeta) correlated absolutely with activating ability. Immunoblotting confirmed this and showed that the perinuclear matrix is the major site of 72-kDa PLCzeta. Oocyte activation was efficiently induced by 1.25 fg of sperm PLCzeta, corresponding to a fraction of one sperm equivalent (approximately 0.03). Immunofluorescence microscopy localized sperm head PLCzeta to a post-acrosomal region that becomes rapidly exposed to the ooplasm following gamete fusion. This multifaceted approach suggests a mechanism by which PLCzeta originates from an oocyte-penetrating assembly--the sperm perinuclear matrix--to induce mammalian oocyte activation at fertilization.     

Novel cytoprotective mechanism of anti-parkinsonian drug deprenyl: PI3K and Nrf2-derived induction of antioxidative proteins

Neuroprotection has received considerable attention as a strategy for the treatment of Parkinson's disease (PD). Deprenyl (Selegiline) is a promising candidate for neuroprotection; however, its cytoprotective mechanism has not been fully clarified. Here, we report a novel cytoprotective mechanism of deprenyl involving PI3K and Nrf2-mediated induction of oxidative stress-related proteins. Deprenyl increased the expression of HO-1, PrxI, TrxI, TrxRxI, gammaGCS, and p62/A170 in SH-SY5Y cells. Deprenyl also induced the nuclear accumulation of Nrf2 and increased the binding activity of Nrf2 to the enhancer region of human genomic HO-1. The Nrf2-mediated induction of antioxidative molecules was controlled by PI3K. Indeed, furthermore, neurotrophin receptor TrkB was identified as an upstream signal for PI3K-Nrf2 activation by deprenyl. These results suggest that the cytoprotective effect of deprenyl is, in part, dependent on Nrf2-mediated induction of antioxidative proteins, suggesting that activation of the PI3K-Nrf2 system may be a useful therapeutic strategy for PD.     

Antioxidant activity of flavonoids evaluated with myoglobin method

Antioxidant activities of four flavonoids (rutin, quercetin, luteolin, and kaempferol) and two non-flavonoids (chlorogenic acid and pyrocatechol) against four reactive oxygen species (ROS) have been measured with a myoglobin method developed by our group. The myoglobin method uses the absorbance changes of myoglobin (a probe molecule) due to the reaction with the ROS as an indicator for the antioxidant activity measurement. Myoglobin protective ratio (MPR) was defined to express the antioxidant activities of the specimens. Antioxidant activities against hypochlorite ion, hydroxyl radical, peroxyl radical, and peroxynitrite were measured with the myoglobin method. The antioxidant activities were comprehensively evaluated by plotting MPR against four ROS and vitamin C equivalent concentration evaluated by DPPH quenching method in 5-axe cobweb charts. The four flavonoids show a very similar pattern in the 5-axe cobweb charts, while the patterns of two non-flavonoids are quite different from that of the flavonoids. This procedure combining the myoglobin method with the cobweb charts is useful in the evaluation of antioxidant activities of plant-derived food, and also can be extended to monitor antioxidant condition of media for plant cell cultures.     

Social memory,amnesia, and autism: Brain oxytocin secretion is regulated by NAD+ metabolites and single nucleotide polymorphisms of CD38

Previously, we demonstrated that CD38, a transmembrane protein with ADP-ribosyl cyclase activity, plays a critical role in mouse social behavior by regulating the release of oxytocin (OXT), which is essential for mutual recognition. When CD38 was disrupted, social amnesia was observed in Cd38 knockout mice. The autism spectrum disorders (ASDs), characterized by defects in reciprocal social interaction and communication, occur either sporadically or in a familial pattern. However, the etiology of ASDs remains largely unknown. Therefore, the theoretical basis for pharmacological treatments has not been established. Hence, there is a rationale for investigating single nucleotide polymorphisms (SNPs) in the human CD38 gene in ASD subjects. We found several SNPs in this gene. The SNP rs3796863 (C > A) was associated with high-functioning autism (HFA) in American samples from the Autism Gene Resource Exchange. Although this finding was partially confirmed in low-functioning autism subjects in Israel, it has not been replicated in Japanese HFA subjects. The second SNP of interest, rs1800561 (4693C > T), leads to the substitution of an arginine (R) at codon 140 by tryptophan (W; R140W) in CD38. This mutation was found in four probands of ASD and in family members of three pedigrees with variable levels of ASD or ASD traits. The plasma levels of OXT in ASD subjects with the R140W allele were lower than those in ASD subjects lacking this allele. The OXT levels were unchanged in healthy subjects with or without this mutation. One proband with the R140W allele receiving intranasal OXT for approximately 3 years showed improvement in areas of social approach, eye contact and communication behaviors, emotion, irritability, and aggression. Five other ASD subjects with mental deficits received nasal OXT for various periods; three subjects showed improved symptoms, while two showed little or no effect. These results suggest that SNPs in CD38 may be possible risk factors for ASD by abrogating OXT function and that some ASD subjects can be treated with OXT in preliminary clinical trials.     

Use of a recycle-type SEC method as a powerful tool for purification of thiosialoside derivatives

An efficient separation between fully acetylated thiosialoside methyl esters and fully acetylated Neu5Ac2en methyl esters was accomplished by means of a size-exclusion chromatography (SEC) method. Purity determinations and structural elucidation of the isolated compounds were performed by a combination of elemental analyses and spectroscopic analyses, including IR, ¹H, and ¹³C NMR, and mass spectroscopic analyses.     

Protective effects of mangafodipir against chemotherapy-induced ovarian damage in mice

Background

Given the seriousness of chemotherapy-induced ovarian injury in female cancer patients, the preservation of fertility, including through the use of cryopreservation technology and pharmaceuticals, requires investigation. Previous studies have shown that damage to the ovaries is related to oxidative stress caused by anticancer drugs. Therefore, superoxide dismutase (SOD) may represent a key factor in the pharmacological protection of the ovaries. The aim of our study was to identify the effects of mangafodipir, a manganese chelate and SOD-mimetic, on suppression of apoptosis in granulosa cells and primordial follicle activation induced by anticancer drugs.

Methods

Cell viability assays using methyltrichlorosilane solutions and immunoblotting for cleaved caspase-3 were performed in in vitro experiments with the simultaneous addition of mangafodipir to human non-luteinized granulosa cell line (HGrC) cultures treated with hydrogen peroxide (H₂O₂), cisplatin, or paclitaxel. Count and morphological analyses of follicles at each developing stage in the ovaries and immunohistochemistry for cleaved caspase-3, Ki67 and 4-hydroxynonenal, a marker for oxidative stress, were also performed using mangafodipir-injected 6-week-old female ICR mice treated with cisplatin or paclitaxel. Further, mangafodipir was injected into 6-week-old female BALB/c mice inoculated with ES-2 to analyze whether mangafodipir inhibits the anti-tumor effects of cisplatin or paclitaxel treatment.

Results

Mangafodipir attenuated apoptosis induced by H₂O₂ and anticancer drugs in vitro. Mangafodipir also decreased the expression of 4-hydroxynonenal and reduced cisplatin- and paclitaxel-induced apoptosis in granulosa cells in vivo. In addition, mangafodipir inhibited the loss of primordial follicles. Tumor xenograft studies in mice showed that mangafodipir did not affect anticancer drug antitumor effects.

Conclusions

Oxidative stress might be one of the mechanisms of cisplatin- and paclitaxel-induced the loss of primordial follicles. Mangafodipir can reduce cisplatin- and paclitaxel-induced apoptosis in granulosa cells and primordial follicle activation partially via its SOD activity. At the same time, mangafodipir might have other potential mechanisms to inhibit the activation of primordial follicles. Further, mangafodipir attenuated the ovarian damage caused by cisplatin and paclitaxel without affecting their antitumor activities. Mangafodipir, therefore, though its efficacy might be limited, may be a new option for the preservation of fertility during anticancer treatment.

     

Addition and reexamination of Japanese species belonging to the genusCercospora and allied general II Species described by Japanese mycologists (1)

In this second report of the present series, six species ofCercospora described in Japan were transferred to the genusPseudocercospora after detailed reexamination. They arePseudocercospora abeliae, P. chionanthi-retusi, P. corylopsidis, P. ehretiae, P. naitoi andP. paulowniae.