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Manabu Kurokawa

Publications and source records attributed to Manabu Kurokawa.

At least 19 recordsLinked to original sources
PubMed2006

Calcium oscillations and mammalian egg activation.

Fertilization in all species studied to date induces an increase in the intracellular concentration of free calcium ions ([Ca2+]i) within the egg. In mammals, this [Ca2+]i signal is delivered in the form of long-lasting [Ca2+]i oscillations that begin shortly after fusion of the gametes and persist beyond the time of completion of meiosis. While not fully elucidated, recent evidence supports the notion that the sperm delivers into the ooplasm a trigger of oscillations, the so-called sperm factor (SF). The recent discovery that mammalian sperm harbor a specific phospholipase C (PLC), PLCzeta has consolidated this view. The fertilizing sperm, and presumably PLCzeta promote Ca2+ release in eggs via the production of inositol 1,4,5-trisphosphate (IP3), which binds and gates its receptor, the type-1 IP3 receptor, located on the endoplasmic reticulum, the Ca2+ store of the cell. Repetitive Ca2+ release in this manner results in a positive cumulative effect on downstream signaling molecules that are responsible for the completion of all the events comprising egg activation. This review will discuss recent advances in our understanding of how [Ca2+]i oscillations are initiated and regulated in mammals, highlight areas of discrepancies, and emphasize the need to better characterize the downstream molecular cascades that are dependent on [Ca2+]i oscillations and that may impact embryo development.

Animals↗
PubMed2006

Identification of the ETT2 locus in human diarrheagenic Escherichia coli by multiplex PCR.

An Escherichia coli type III secretion system 2 (ETT2) locus was discovered in enterohemorrhagic E. coli O157:H7. To determine presence or absence of the ETT2 locus in diarrheagenic E. coli, a multiplex polymerase chain reaction (PCR) encoding Shiga toxins (stx1 and stx2), intimin (eaeA), and ETT2 (etrA) was developed for rapid detection. The ETT2 locus was identified not only in Shiga toxin-producing E. coli (STEC) but also in various non-STEC.

Diarrhea↗
PubMed2006

Unusual Corynebacterium mucifaciens isolated from ear and nasal specimens.

OBJECTIVE: Corynebacterium mucifaciens was first proposed as a novel Corynebacterium species in 1997. This report describes our experience with the unexpectedly frequent isolation of C mucifaciens. STUDY DESIGN: By using agar supplemented with several antibiotics, microbiological studies were performed on specimens collected from the ear and nose. SETTING: A primary and secondary referral hospital. RESULTS: The organisms grew slowly. Five strains of C mucifaciens were found in specimens of middle ear effusion (MEE) from patients with otitis media with effusion (OME), and four strains were isolated from the nasal polyps and nasal discharge of patients with chronic sinusitis (CRS). CONCLUSIONS: C mucifaciens was isolated from nine otorhinological specimens. SIGNIFICANCE: This is the first report about the isolation of C mucifaciens from MEE, nasal polyps, and nasal discharge. Although its pathogenetic role is unknown, the characteristics of this microorganism might make it a potential cause of OME and/or CRS, so further studies are necessary. EBM RATING: C-4.

Adolescent↗
PubMed2006

Helicobacter pylori associated gastroduodenal problem among the Nepalese.

Present study was undertaken during a period of eighteen months (April 2001 to October 2002) to see an association of Helicobacter pylori with the gastric problems among the Nepalese. This study comprised of 203 subjects (Males: 154 and Females: 49) aged 21 to 71 years with gastric problems. The biopsy samples taken from the stomach and duodenum using endoscope were tested for the presence of H. pylori by in-house rapid urease test (RUT) and culture. Representative samples were also tested by PCR. Of the total 203, 60 (29.5%) were RUT positive. Marginally high RUT positive rate was observed in males compared with females (P>0.95). Same was true with ethnic group with a marginally higher positive rate among Tibeto-Burmans compared with Indo-Aryans (P>0.05). Age-wise, higher incidence (34.4%) was observed in the age group of 36 to approximately 50 years followed by over 50 years (29.0%), and 21 to approximately 35 years (24.4%) (P>0.05). Both RUT and culture positive rate increased with the severity of the problem. RUT results were confirmed also by PCR. All RUT positive patients were given a full course of new triple therapy (combination of two antibiotics: Clarythromycin and Amoxycillin and a proton-pump inhibitor: Omeprazole) for two weeks followed by extended monotherapy with Omeprazole for up to six weeks. Of the total, 168 (82.7%) patients were available for follow-up and all of them showed an improved condition with negative RUT.

Adult↗
PubMed2005

Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm.

A cytosolic sperm protein(s), referred to as sperm factor (SF), is delivered into eggs by the sperm during mammalian fertilization to induce repetitive increases in the intracellular concentration of free Ca2+ ([Ca2+]i) that are referred to as [Ca2+]i oscillations. [Ca2+]i oscillations are essential for egg activation and early embryonic development. Recent evidence shows that the novel sperm-specific phospholipase C (PLC), PLCzeta, may be the long sought after [Ca2+]i oscillation-inducing SF. Here, we demonstrate the complete extraction of SF from porcine sperm and show that regardless of the method of extraction a single molecule/complex appears to be responsible for the [Ca2+]i oscillation-inducing activity of these extracts. Consistent with this notion, all sperm fractions that induced [Ca2+]i oscillations, including FPLC-purified fractions, exhibited high in vitro PLC activity at basal Ca2+ levels (0.1-5 microM), a hallmark of PLCzeta. Notably, we detected immunoreactive 72-kDa PLCzeta in an inactive fraction, and several fractions capable of inducing oscillations were devoid of 72-kDa PLCzeta. Nonetheless, in the latter fractions, proteolytic fragments, presumably corresponding to cleaved forms of PLCzeta, were detected by immunoblotting. Therefore, our findings corroborate the hypothesis that a sperm-specific PLC is the main component of the [Ca2+]i oscillation-inducing activity of sperm but provide evidence that the presence of 72-kDa PLCzeta does not precisely correspond with the Ca2+ releasing activity of porcine sperm fractions.

Animals↗
PubMed2004

Transgenic RNA interference reveals role for mouse sperm phospholipase Czeta in triggering Ca2+ oscillations during fertilization.

A sperm-specific phospholipase (PL) C, termed PLCzeta, is proposed to be the soluble sperm factor that induces Ca(2+) oscillations in mammalian eggs and, thus, initiates egg activation in vivo. We report that sperm from transgenic mice expressing short hairpin RNAs targeting PLCzeta mRNA have reduced amounts of PLCzeta protein. Sperm derived from these transgenic mice trigger patterns of Ca(2+) oscillations following fertilization in vitro that terminate prematurely. Consistent with the perturbation in patterns of Ca(2+) oscillations is the finding that mating of transgenic founder males to females results in lower rates of egg activation and no transgenic offspring. These data strongly suggest that PLCzeta is the physiological trigger of Ca(2+) oscillations required for activation of development.

Animals↗
PubMed2004

Cell cycle-coupled [Ca(2+)](i) oscillations in mouse zygotes and function of the inositol 1,4,5-trisphosphate receptor-1.

Sperm entry in mammalian eggs initiates oscillations in the concentration of free calcium ([Ca(2+)](i)). In mouse eggs, oscillations start at metaphase II (MII) and conclude as the zygotes progress into interphase and commence pronuclear (PN) formation. The inositol 1,4,5-trisphosphate receptor (IP(3)R-1), which underlies the oscillations, undergoes degradation during this transition, suggesting that one or more of the eggs' Ca(2+)-releasing machinery components may be regulated in a cell cycle-dependent manner, thereby coordinating [Ca(2+)](i) responses with the cell cycle. To ascertain the site(s) of interaction, we initiated oscillations at different stages of the cell cycle in zygotes with different IP(3)R-1 mass. In addition to sperm, we used two other agonists: porcine sperm factor (pSF), which stimulates production of IP(3), and adenophostin A, a non-hydrolyzable analogue of IP(3). None of the agonists tested induced oscillations at interphase, suggesting that neither decreased IP(3)R-1 mass nor lack of production or excessive IP(3) degradation can account for the insensitivity to IP(3) at this stage. Moreover, the releasable Ca(2+) content of the stores did not change by interphase, but it did decrease by first mitosis. More importantly, experiments revealed that IP(3)R-1 sensitivity and possibly IP(3) binding were altered at interphase, and our data demonstrate stage-specific IP(3)R-1 phosphorylation by M-phase kinases. Accordingly, increasing the activity of M-phase kinases restored the oscillatory-permissive state in zygotes. We therefore propose that the restriction of oscillations in mouse zygotes to the metaphase stage may be coordinated at the level of IP(3)R-1 and that this involves cell cycle stage-specific receptor phosphorylation.

Adenosine↗
PubMed2004

Mammalian fertilization: from sperm factor to phospholipase Czeta.

In mammalian eggs, the fertilizing sperm evokes intracellular Ca2+ ([Ca2+]i) oscillations that are essential for initiation of egg activation and embryonic development. Although the exact mechanism leading to initiation of [Ca2+]i oscillations still remains unclear, accumulating studies suggest that a presently unknown substance, termed sperm factor (SF), is delivered from the fertilizing sperm into the ooplasm and triggers [Ca2+]i oscillations. Based on findings showing that production of inositol 1,4,5-trisphosphate (IP3) underlies the generation of [Ca2+]i oscillations, it has been suggested that SF functions either as a phospholipase C (PLC), an enzyme that catalyzes the generation of IP3, or as an activator of a PLC(s) pre-existing in the egg. This review discusses the role of SF as the molecule responsible for the production of IP3 and the initiator of [Ca2+]i oscillations in mammalian fertilization, with particular emphasis on the possible involvement of egg- and sperm-derived PLCs, including PLCzeta, a novel sperm specific PLC.

Animals↗
PubMed2004

Evidence that activation of Src family kinase is not required for fertilization-associated [Ca2+]i oscillations in mouse eggs.

Recent evidence in marine invertebrate, frog, and zebrafish eggs suggests the involvement of a Src family kinase (SFK) in fertilization-induced Ca2+ release. In the present study, we have investigated whether activation of an SFK is required for initiation of intracellular Ca2+ ([Ca2+]i) oscillations in mouse fertilization. We detected a Hck-like protein and tyrosine-phosphorylated proteins in soluble and insoluble sperm fractions, respectively. However, the presence of these proteins did not correspond to the active fractions of porcine sperm extracts (pSE). Moreover, [Ca2+]i oscillations induced by pSE in mouse eggs were unaltered by pre-incubation of pSE with specific SFK inhibitors such as 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]-pyrimidine (PP2) or lavendustin A, despite the fact that the inhibitors were shown to be active both in vivo and in vitro. Another SFK inhibitor, peptide A, blocked oscillations when incubated with pSE prior to injection into eggs, but this inhibition required more than ten times the concentration reportedly required to inhibit SFK activity. In addition, pre-injection or pre-incubation of eggs with these inhibitors did not affect the ability of pSE to trigger [Ca2+]i oscillations in mouse eggs. Microinjection of a recombinant c-Src protein or mRNAs encoding constitutively active Src proteins did not induce [Ca2+]i release. Finally, when sperm and eggs, both of which were pre-treated with PP2, were fertilized, [Ca2+]i oscillations occurred normally. We can therefore conclude that activation of an SFK is neither necessary nor sufficient for triggering fertilization-induced [Ca2+]i oscillations.

Animals↗
PubMed2004

Detection of diarrheagenic viruses from diarrheal fecal samples collected from children in Kathmandu, Nepal.

Diarrhea causing viruses (Rotavirus. Adenovirus and Norovirus) were investigated in diarrheal fecal samples collected from children in Kathmandu, Nepal in Janury 2004 using both real time PCR and immuno-chromatogaphic techniques. Of the total 12 diarrheal samples investigated, 8 (66.7%) were positive for Rotavirus, 1 (8.3%) was positive for Adenovirus and none was positive for Norovirus (Norwalk like virus). The Adenovirus positive sample was also positive for Rotavirus. Similar results were obtained by immuno-chromatographic technique. All of Rotavirus detected belonged to Group-A. Results indicated that immuno-chromatographic technique was equally good in the detection of diarrhea causing viruses in fecal samples. Furthermore, it was simple, cost-effective and less time consuming (15 minutes) compared with the PCR. Immuno-chromatographic technique, therefore, appeared to be useful for rapid diagnosis of viral gastroenteritis in developing countries like Nepal.

Adenovirus Infections, Human↗
PubMed2004

Evaluation of rapid diagnostic methods for pediatric viral diarrhea using samples collected in Nepal and Japan.

We evaluated real time PCR, ELISA and immunochromatography (ICG) test systems for detection of diarrheagenic viruses (Rotavirus, Adenovirus and Norovirus) in pediatric diarrheal fecal samples collected in Kathmandu, Nepal (n = 23) and in Kobe, Japan (n = 25) in summer of 2004. Of the 23 samples collected in Nepal, one (4.3%) was positive for Rotavirus and two (8.7%) for Adenovirus whereas six out of 25 samples (24.0%) collected in Japan were positive for Norovirus. Of the Japanese samples, 11 (44.0%) showed diarrheagenic bacteria (Campylobacter jejuni, Salmolella enteritidis) while six (26.1%) samples from Nepal showed parasites like Ascaris lumbricoides, Giardia intestinalis, Trichuris trchiura and Vampirolepis nana. Viral pathogens were not common during summer season in both Nepal and Japan. Real time RT-PCR was found to be relatively more sensitive compared with ELISA for Norovirus. However, immunological methods are simple and less time consuming (three hours for ELISA, 15 minutes for ICG) and appeared to be useful for rapid diagnosis of viral gastroenteritis especially in developing countries.

Adenoviridae↗
PubMed2003

Patterns of intracellular calcium oscillations in horse oocytes fertilized by intracytoplasmic sperm injection: possible explanations for the low success of this assisted reproduction technique in the horse.

In all species studied, fertilization induces intracellular Ca2+ ([Ca2+]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca2+]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single motile stallion sperm. Few oocytes displayed [Ca2+]i responses regardless of oocyte source and we hypothesized that this may result from insufficient release of the sperm-borne active molecule (sperm factor) into the oocyte. However, permeabilization of sperm membranes with Triton-X or by sonication did not alleviate the deficient [Ca2+]i responses in mare oocytes. Thus, we hypothesized that a step downstream of release, possibly required for sperm factor function, is not appropriately accomplished in horse oocytes. To test this, ICSI-fertilized horse oocytes were fused to unfertilized mouse oocytes, which are known to respond with [Ca2+]i oscillations to injection of stallion sperm, and [Ca2+]i monitoring was performed. Such pairs consistently displayed [Ca2+]i responses demonstrating that the sperm factor is appropriately released into the ooplasm of horse oocytes, but that these are unable to activate and/or provide the appropriate substrate that is required for the sperm factor delivered by ICSI to initiate oscillations. These findings may have implications to improve the success of ICSI in the equine and other livestock species.

Animals↗
PubMed2003

Release of the Ca(2+) oscillation-inducing sperm factor during mouse fertilization.

A cytosolic sperm protein(s), referred to as the sperm factor (SF), is thought to induce intracellular calcium ([Ca(2+)](i)) oscillations during fertilization in mammalian eggs. These oscillations, which are responsible for inducing complete egg activation, persist for several hours. Nevertheless, whether a protracted release of SF is responsible for the duration of the oscillations is unknown. Using a combination of intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), sperm removal, reinjection of the withdrawn sperm, and [Ca(2+)](i) monitoring, we determined that 30 min was necessary for establishing oscillations. Importantly, a significant portion of the Ca(2+) activity became dissociated from the sperm within 15-60 min after entry, and by 120 min post-ICSI or IVF, sperm were unable to induce oscillations. The initiation of oscillations coincided with exposure and solubilization of the perinuclear theca (PT), as evidenced by transmission electron microscopy, although disassembly of the PT was not required for commencement of the [Ca(2+)](i) responses. Remarkably, despite its complete release into the ooplasm, SF associated with nuclear structures at the time of pronuclear formation. Lastly, release of SF was not affected by the cell cycle. We conclude that mouse sperm serves as a carrier for SF, which is rapidly and completely solubilized to establish [Ca(2+)](i) oscillations.

Animals↗
PubMed2003

Reconstitution of Src-dependent phospholipase Cgamma phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs.

We reported previously that egg membrane rafts serve as a subcellular microdomain for sperm-dependent tyrosine kinase signaling in Xenopus fertilization. Moreover, we demonstrated that raft-associated Src tyrosine kinase was activated by sperm in vitro. Here we show that egg rafts incubated with sperm or hydrogen peroxide (H2O2) can promote Src-dependent phosphorylation of phospholipase Cgamma (PLCgamma) and transient calcium release in the extracts of unfertilized Xenopus eggs. In vivo egg activation by sperm or H2O2 also promotes tyrosine phosphorylation and raft-translocalization of PLCgamma. Immunodepletion of PLCgamma from the egg extracts inhibits the raft-dependent calcium release. Rafts prepared from H2O2-activated eggs also promote Src-dependent dephosphorylation of p42 mitogen-activated protein kinase and cell cycle transition from metaphase II to interphase in egg extracts. PLCgamma phosphorylation and calcium release in egg extracts can be promoted by rafts prepared from COS-7 cells expressing the Xenopus Src gene. These results demonstrate that the signaling events elicited by fertilization in Xenopus eggs can be reconstituted in vitro. The development of such experimental platforms will allow us to dissect the molecular mechanism of sperm-dependent activation of raft-associated Src and subsequent up-regulation of PLCgamma and egg activation machinery in Xenopus eggs.

Animals↗
PubMed2003

Phospholipase Cdelta4 is required for Ca2+ mobilization essential for acrosome reaction in sperm.

Zona pellucida (ZP)-induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCdelta4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we report that the [Ca2+]i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCdelta4-/- sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCdelta4-/- sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca2+]i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca2+]i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCdelta4-/- sperm. These results indicate that PLCdelta4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.

Acrosome↗
PubMed2003

ICSI-generated mouse zygotes exhibit altered calcium oscillations, inositol 1,4,5-trisphosphate receptor-1 down-regulation, and embryo development.

ICSI bypasses not only fusion of the gametes but also a series of signalling events that occur in the sperm prior to and during interaction with the oocyte's vestments. The effect of this altered encounter of the gametes on the fertilization-associated intracellular calcium ([Ca2+]i) oscillations has not been thoroughly investigated. Here, ICSI and IVF were performed using gametes from two mouse strains, B6D2F1 and CD1, and in-vitro development, pattern of [Ca2+]i oscillations and down-regulation of inositol 1,4,5-trisphosphate receptor-1 (IP3R-1) in the produced embryos were compared. ICSI and IVF resulted in comparable rates of activation and pre-implantation development. However, ICSI-generated zygotes cleaved at a slower rate, had lower cell numbers and lower hatching rates. The deleterious effects of ICSI could not be exclusively attributed to the injury by the injection since sham-injected IVF zygotes only exhibited delayed progression to the blastocyst stage. ICSI and IVF induced similar initial [Ca2+]i responses, although ICSI zygotes exhibited shorter durations of [Ca2+]i oscillations and showed diminished degradation of IP3R-1. Importantly, sperm manipulation affected the pattern of oscillations, which further decreased pre-implantation developmental rates. Our results demonstrate that ICSI-induced [Ca2+]i responses are not equivalent to those initiated by IVF and that this may have developmental consequences.

Animals↗
PubMed2003

Acute hemorrhagic conjunctivitis epidemic--2003 in Nepal.

This rapid communication present the findings of nationwide outbreak of acute hemorrhagic conjunctivitis (AHC) epidemic occurred in Nepal during summer 2003. Only ten percent of samples showed bacterial and no fungus were grown. On the contrary, viruses were detected in 60.0% cases studied. Of the viruses detected, Adenovirus (ADV), Coxsackie virus (CA24v) and both accounted for 83.3% (30/36), 11.1% (4/36) and 5.5% (2/36), respectively. Interestingly, ADV was detected also from one of the ten a Rupees bill.

Adenovirus Infections, Human↗