The Fission Yeast Minichromosome Maintenance (MCM)-binding Protein (MCM-BP), Mcb1, Regulates MCM Function during Prereplicative Complex Formation in DNA Replication

The minichromosome maintenance (MCM) complex is a replicative helicase, which is essential for chromosome DNA replication. In recent years, the identification of a novel MCM-binding protein (MCM-BP) in most eukaryotes has led to numerous studies investigating its function and its relationship to the MCM complex. However, the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood; in addition, the functional role of MCM-BP remains controversial and may vary between model organisms. The present study aims to elucidate the nature and biological function of the MCM-BP ortholog, Mcb1, in fission yeast. The Mcb1 protein continuously interacts with MCM proteins during the cell cycle in vivo and can interact with any individual MCM subunit in vitro. To understand the detailed characteristics of mcb1⁺, two temperature-sensitive mcb1 gene mutants (mcb1^ts) were isolated. Extensive genetic analysis showed that the mcb1^ts mutants were suppressed by a mcm5⁺ multicopy plasmid and displayed synthetic defects with many S-phase-related gene mutants. Moreover, cyclin-dependent kinase modulation by Cig2 repression or Rum1 overproduction suppressed the mcb1^ts mutants, suggesting the involvement of Mcb1 in pre-RC formation during DNA replication. These data are consistent with the observation that Mcm7 loading onto replication origins is reduced and S-phase progression is delayed in mcb1^ts mutants. Furthermore, the mcb1^ts mutation led to the redistribution of MCM subunits to the cytoplasm, and this redistribution was dependent on an active nuclear export system. These results strongly suggest that Mcb1 promotes efficient pre-RC formation during DNA replication by regulating the MCM complex.     

Development of the competence of bovine oocytes to release cortical granules and block polyspermy after meiotic maturation

Bovine immature oocytes do not have the ability to block polyspermic penetration. The present study was conducted to determine whether this is correlated to cortical granule (CG) distribution and the competence of oocytes to release CG upon sperm penetration, and whether the ability of bovine oocytes to release CG develops during in vitro maturation. Fluorescein isothiocyanate-conjugated Lens culinaris agglutinin was used for detecting CG in immature and mature oocytes before and after sperm penetration and electric stimulation. The labeled oocytes were examined with laser confocal and fluorescent microscopes. The results show that CG exist as clusters in all immature oocytes. The CG were not released from immature oocytes exposed to electric pulse or penetrated by spermatozoa, resulting in 94% of oocytes being polyspermic. When immature oocytes were cultured for 22h in vitro , 81% extruded the first polar body and reached metaphase II. In mature oocytes, 25% of oocytes showed CG clusters, 42% and 33% of oocytes showed partial and complete CG dispersion, respectively. When mature oocytes were inseminated in vitro , only 15% of oocytes were polyspermic. Cortical granule exocytosis occurred in 97% of oocytes after sperm penetration and 84% of oocytes released all of the CG 18 h after insemination. Electric pulse induced all of the mature oocytes to release CG but only 55% released all of their CG 18 h post stimulation. These results indicate that polyspermy in immature bovine oocytes is the result of the complete failure of the oocyte to release CG after sperm penetration. Bovine oocytes became competent to release CG by sperm penetration and electric stimulation after meiotic maturation. These results provide evidence that CG exocytosis plays an important role(s) in the establishment of the block to polyspermy in bovine oocytes.     

Activation of protein kinase C induces cortical granule exocytosis in a Ca2+-independent manner, but not the resumption of cell cycle in porcine eggs

The effects of protein kinase C (PKC) activation on meiotic resumption and cortical granule (CG) exocytosis as well as its dependence on Ca²⁺ in porcine eggs matured in vitro were studied. Cortical granule release was judged by both confocal laser microscopy after the eggs were labeled with fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and electron microscopy. Meiotic resumption and pronuclear formation were observed after eggs were stained with acetic orcein. When eggs were treated with PKC activators, 1-oleyl-2-acetyl-glycerol (OAG) or phorbol 12-myristate 13-acetate (PMA), the pronuclear formation percentage was significantly lower than that of Ca²⁺ ionophore A23187-treated group, but not statistically different from that in negative control group (P > 0.05), and most of the eggs were still arrested at metaphase II stage, suggesting that PKC activation does not induce the resumption of meiosis and pronuclear formation. In contrast, PKC activation induced 89.1% to 100% of the eggs completely or partially released their CG in different groups, not statistically different from A23187-treated group, and this effect could be overcome by PKC inhibition. When the intracellular free Ca²⁺ was chelated with acetoxymethal ester form of 1,2-bis(0-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), and then treated with PMA or OAG in Ca²⁺-free medium, the proportions of eggs with CG release were 90.9% and 78.1%, respectively, not statistically different from the above-treated groups, suggesting that CG exocytosis induced by PKC activation is independent of Ca²⁺ rise. The results indicate that different events of porcine egg activation may be uncoupled from one another.     

Cloning and Functional Expression of a Novel Geranylgeranyl Pyrophosphate Synthase Gene from Arabidopsis thaliana in Escherichia coli

A gene encoding a novel geranylgeranyl pyrophosphate (GGPP)synthase from Arabidopsis thaliana has been identified and termedGGPS5. The gene has been sequenced and expressed in Escherichiacoli. The deduced amino acid sequence showed 64.5% and 57.5%identity with a putative GGPP synthase from Arabidopsis andCapsicum annuum, respectively. GGPP enzymatic activity was detectedin E. coli cells expressing the GGPS5 gene in two differentways. One was the direct measurement of GGPP synthase activityin cell extracts and the other was the yellow color productionof cells when the GGPS5 gene was co-expressed with crtB, crtI,crtX, crtY and crtZ genes derived from Erwinia uredovora. (Received May 20, 1996; Accepted December 14, 1996)     

Introduction of SLG (S locus glycoprotein) alters the phenotype of endogenous S haplotype,but confers no new S haplotype specificity in Brassica rapa L.

Self-incompatibility (SI) in Brassicaceae is genetically controlled by the S locus complex in which S locus glycoprotein (SLG) and S receptor kinase (SRK) genes have been identified, and these two genes encoding stigma proteins are believed to play important roles in SI recognition reaction. Here we introduced the SLG⁴³ gene of Brassica rapa into a self-incompatible cultivar, Osome, of B. rapa, and examined the effect of this transgene on the SI behavior of the transgenic plants. Preliminary pollination experiments demonstrated that Osome carried S⁵² and S⁶⁰, and both were codominant in stigma, but S⁵² was dominant to S⁶⁰ in pollen. S⁴³ was found to be recessive to S⁵² and codominant with S⁶⁰ in stigma. The nucleotide sequence of SLG⁴³ was more similar to that of SLG⁵² (87.8% identity) than to that of SLG⁶⁰ (74.8% identity). Three of the ten primary transformants (designated No. 1 to No. 10) were either completely (No. 9) or partially (No. 6 and No. 7) self-compatible; the SI phenotype of the stigma was changed from S⁵²S⁶⁰ to S⁶⁰, but the SI phenotype of the pollen was not altered. In these three plants, the mRNA and protein levels of both SLG⁴³ and SLG⁵² were reduced, whereas those of SLG⁶⁰ were not. All the plants in the selfed progeny of No. 9 and No. 6 regained SI and they produced a normal level of SLG⁵². These results suggest that the alteration of the SI phenotype of the stigma in the transformants Nos. 6, 7, and 9 was the result of specific co-suppression between the SLG⁴³ transgene and the endogenous SLG⁵² gene. Three of the transformants (Nos. 5, 8 and 10) produced SLG⁴³ protein, but their SI phenotype was not altered. The S⁶⁰ homozygotes in the selfed progeny of No. 10 which produced the highest level of SLG⁴³ were studied because S⁴³ was codominant with S⁶⁰ in the stigma. They produced SLG⁴³ at approximately the same level as did S⁴³S⁶⁰ heterozygotes, but did not show S⁴³ haplotype specificity at the stigma side. We conclude that SLG is necessary for the expression of the S haplotype specificity in the stigma but the introduction of SLG alone is not sufficient for conferring a novel S haplotype specificity to the stigma.     

Astrocytes with previous chronic exposure to amyloid β‐peptide fragment 1–40 suppress excitatory synaptic transmission

Synaptic dysfunction and neuronal death are responsible for cognitive and behavioral deficits in Alzheimer's disease (AD). It is well known that such neurological abnormalities are preceded by long‐term exposure of amyloid β‐peptide (Aβ) and/or hyperphosphorylated tau prior. In addition to the neurological deficit, astrocytes as a major glial cell type in the brain, significantly participate in the neuropathogenic mechanisms underlying synaptic modulation. Although astrocytes play a significant key role in modulating synaptic transmission, little is known on whether astrocyte dysfunction caused by such long‐term Aβ exposure affects synapse formation and function. Here, we show that synapse formation and synaptic transmission are attenuated in hippocampal‐naïve neurons co‐cultured with astrocytes that have previously experienced chronic Aβ_1‐40 exposure. In this abnormal astrocytic condition, hippocampal neurons exhibit decrements of evoked excitatory post‐synaptic currents (EPSCs) and miniature EPSC frequency. Furthermore, size of readily releasable synaptic pools and number of excitatory synapses were also significantly decreased. Contrary to these negative effects, release probability at individual synapses was significantly increased in the same astrocytic condition. Taken together, our data indicate that lower synaptic transmission caused by astrocytes previously, and chronically, exposed to Aβ1–40 is attributable to a small number of synapses with higher release probability.

     

Metabolic engineering of Synechocystis sp. PCC 6803 for enhanced ethanol production based on flux balance analysis

Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production due to its ability to directly convert atmospheric carbon dioxide into ethanol using photosystems. To enhance ethanol production in Synechocystis sp. PCC 6803, metabolic engineering was performed based on in silico simulations, using the genome-scale metabolic model. Comprehensive reaction knockout simulations by flux balance analysis predicted that the knockout of NAD(P)H dehydrogenase enhanced ethanol production under photoautotrophic conditions, where ammonium is the nitrogen source. This deletion inhibits the re-oxidation of NAD(P)H, which is generated by ferredoxin-NADP⁺ reductase and imposes re-oxidation in the ethanol synthesis pathway. The effect of deleting the ndhF1 gene, which encodes NADH dehydrogenase subunit 5, on ethanol production was experimentally evaluated using ethanol-producing strains of Synechocystis sp. PCC 6803. The ethanol titer of the ethanol-producing ∆ndhF1 strain increased by 145%, compared with that of the control strain.

     

Structure of spontaneous UP and DOWN transitions self-organizing in a cortical network model

Synaptic plasticity is considered to play a crucial role in the experience-dependent self-organization of local cortical networks. In the absence of sensory stimuli, cerebral cortex exhibits spontaneous membrane potential transitions between an UP and a DOWN state. To reveal how cortical networks develop spontaneous activity, or conversely, how spontaneous activity structures cortical networks, we analyze the self-organization of a recurrent network model of excitatory and inhibitory neurons, which is realistic enough to replicate UP–DOWN states, with spike-timing-dependent plasticity (STDP). The individual neurons in the self-organized network exhibit a variety of temporal patterns in the two-state transitions. In addition, the model develops a feed-forward network-like structure that produces a diverse repertoire of precise sequences of the UP state. Our model shows that the self-organized activity well resembles the spontaneous activity of cortical networks if STDP is accompanied by the pruning of weak synapses. These results suggest that the two-state membrane potential transitions play an active role in structuring local cortical circuits.     

Migratory history of the spotted flagtail, <Emphasis Type="Italic">Kuhlia marginata</Emphasis>

The spotted flagtail, Kuhlia marginata (Kuhliidae) is commonly found in streams throughout the Ryukyu Archipelago, including Okinawa Island, Japan. Although it has been suggested that they spawn at sea, little is known about their migratory history. The aim of the study was to clarify their migration history based on otolith microchemistry analysis, size composition and the gonadal development of fish collected from the Genka River on Okinawa Island. All strontium/calcium (Sr/Ca) ratios of otoliths remained high around their cores, and then dropped rapidly at some distance from the core. The estimated standard length corresponding to decreases correlated with the size of new recruits collected in the river (ca. 20 mm in standard length: SL). Multiple increases, the sign for migrations into the high saline area, were recognized intermittently around the otolith margin. As the estimated SL at these increases in the Sr/Ca ratio occurred above the minimum maturation size (female: 95.5 mm SL; male: 83.5 mm SL), such multiple increases in Sr/Ca ratios were caused by spawning migration. Therefore, we conclude that the catadromous pattern of K. marginata is as follows; this species grows in the sea during the early life stage until ca. 20 mm SL and then grow in the freshwater area until maturation, before they migrate again to the sea for spawning. Sr/Ca ratio profiles also suggested that a large number of males did not return to the freshwater area after spawning, whereas females might spawn several times during their lives around the inshore area.