Overexpression of the barley aquaporin HvPIP2;1 increases internal CO(2) conductance and CO(2) assimilation in the leaves of transgenic rice plants

The internal conductance for CO(2) diffusion (g(i)) and CO(2) assimilation rate were measured and the related anatomical characteristics were investigated in transgenic rice leaves that overexpressed barley aquaporin HvPIP2;1. This study was performed to test the hypothesis that aquaporin facilitates CO(2) diffusion within leaves. The g(i) value was estimated for intact leaves by concurrent measurements of gas exchange and carbon isotope ratio. The leaves of the transgenic rice plants that expressed the highest levels of Aq-anti-HvPIP2;1 showed a 40% increase in g(i) as compared to g(i) in the leaves of wild-type rice plants. The increase in g(i) was accompanied by a 14% increase in CO(2) assimilation rate and a 27% increase in stomatal conductance (g(s)). The transgenic plants that had low levels of Aq-anti-HvPIP2;1 showed decreases in g(i) and CO(2) assimilation rate. In the plants with high levels of Aq-anti-HvPIP2;1, mesophyll cell size decreased and the cell walls of the epidermis and mesophyll cells thickened, indicating that the leaves had become xeromorphic. Although such anatomical changes could partially offset the increase in g(i) by the aquaporin, the increase in aquaporin content overcame such adverse effects.     

The Pro to glycine mutation of staphylococcal nuclease simplifies the unfolding—folding kinetics

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro¹¹⁷ is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed.     

The effect of platelet activating factor on ovulation.

The mechanism of ovulation has been compared to an inflammatory reaction. Platelet activating factor (PAF) is an important mediator of inflammation as it may induce the production of prostaglandins and lysosomal enzyme. We evaluated the potential role of PAF in PMSG-HCG induced ovulation using CV3988, a specific PAF receptor antagonist in a superovulated ICR mice (9-12 weeks old). CV3988 blocked the ovulation in a dose dependent manner, and the significant reduced ovulatory efficiency was observed at more than 500 micrograms dose (p less than 0.001). The ovulatory efficiency reduced by CV3988 was reversed by PAF in a dose dependent manner. In vitro fertilization (IVF) rate of follicular oocytes with treatment of CV3988 was not different from that of ovulated ova without treatment. These results suggest that PAF may be involved in the ovulation process but the presence of PAF may not be essential for the fertilization of the ova as IVF.     

Oligomeric Hsp33 with enhanced chaperone activity: gel filtration, cross-linking, and small angle x-ray scattering (SAXS) analysis

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress.     

Fibril formation of hsp10 homologue proteins and determination of fibril core regions: differences in fibril core regions dependent on subtle differences in amino acid sequence

Heat shock protein 10 (hsp10) is a member of the molecular chaperones and works with hsp60 in mediating various protein folding reactions. GroES is a representative protein of hsp10 from Escherichia coli. Recently, we found that GroES formed a typical amyloid fibril from a guanidine hydrochloride (Gdn-HCl) unfolded state at neutral pH. Here, we report that other hsp10 homologues, such as human hsp10 (Hhsp10), rat mitochondrial hsp10 (Rhsp10), Gp31 from T4 phage, and hsp10 from the hyperthermophilic bacteria Thermotoga maritima, also form amyloid fibrils from an unfolded state. Interestingly, whereas GroES formed fibrils from either the Gdn-HCl unfolded state (at neutral pH) or the acidic unfolded state (at pH 2.0-3.0), Hhsp10, Rhsp10, and Gp31 formed fibrils from only the acidic unfolded state. Core peptide regions of these protein fibrils were determined by proteolysis treatment followed by a combination of Edman degradation and mass spectroscopy analyses of the protease-resistant peptides. The core peptides of GroES fibrils were identical for fibrils formed from the Gdn-HCl unfolded state and those formed from the acidic unfolded state. However, a peptide with a different sequence was isolated from fibrils of Hhsp10 and Rhsp10. With the use of synthesized peptides of the determined core regions, it was also confirmed that the identified regions were capable of fibril formation. These findings suggested that GroES homologues formed typical amyloid fibrils under acidic unfolding conditions but that the fibril core structures were different, perhaps owing to differences in local amino acid sequences.     

Delayed sleep/wake rhythm and excessive daytime sleepiness correlate with decreased daytime brain activity during cognitive task in university students

Insufficient sleep and irregular sleep/wake rhythm are common problems among university students. We investigated the effect of sleep/wake rhythm and excessive daytime sleepiness (EDS) on the cortical oxygenation as measured by near-infrared spectroscopy (NIRS) and cognitive performance in university students. Peak- and integral values by a word fluency task were measured with NIRS. EDS was evaluated by the Epworth sleepiness scale (ESS), and performance function was evaluated using N-back task. Peak cerebral oxygenation was significantly correlated with ESS, bedtime, wake-up time, and median time of sleep. Accuracy on 2-back task was significantly correlated with integral value. Peak- and integral values were significantly lower, and bedtime and median time of sleep were significantly delayed in the EDS group than in the non-EDS group. EDS accompanied by delayed sleep/wake rhythm and short sleep duration may play an important role in decreasing daytime brain activity and cognitive performance.     

Intracellular uptake and inhibition of gene expression by PNAs and PNA-peptide conjugates

Peptide nucleic acids (PNAs) offer a distinct option for silencing gene expression in mammalian cells. However, the full value of PNAs has not been realized, and the rules governing the recognition of cellular targets by PNAs remain obscure. Here we examine the uptake of PNAs and PNA-peptide conjugates by immortal and primary human cells and compare peptide-mediated and DNA/lipid-mediated delivery strategies. We find that both peptide-mediated and lipid-mediated delivery strategies promote entry of PNA and PNA-peptide conjugates into cells. Confocal microscopy reveals a punctate distribution of PNA and PNA-peptide conjugates regardless of the delivery strategy used. Peptide D(AAKK)(4) and a peptide containing a nuclear localization sequence (NLS) promote the spontaneous delivery of antisense PNAs into cultured cells. The PNA-D(AAKK)(4) conjugate inhibits expression of human caveolin 1 (hCav-1) in both HeLa and primary endothelial cells. DNA/lipid-mediated delivery requires less PNA, while peptide-mediated delivery is simpler and is less toxic to primary cells. The ability of PNA-peptide conjugates to enter primary and immortal human cells and inhibit gene expression supports the use of PNAs as antisense agents for investigating the roles of proteins in cells. Both DNA/lipid-mediated and peptide-mediated delivery strategies are efficient, but the compartmentalized localization of PNAs suggests that improving the cellular distribution may lead to increased efficacy.     

TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation

TNF activates three distinct intracellular signaling cascades leading to cell survival, caspase-8–mediated apoptosis, or receptor interacting protein kinase 3 (RIPK3)–dependent necrosis, also called necroptosis. Depending on the cellular context, one of these pathways is activated upon TNF challenge. When caspase-8 is activated, it drives the apoptosis cascade and blocks RIPK3-dependent necrosis. Here we report the biological event switching to activate necrosis over apoptosis. TAK1 kinase is normally transiently activated upon TNF stimulation. We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation. In addition, we also demonstrated that activation of RIPK1 and RIPK3 promoted TAK1 activation, suggesting a positive feedforward loop of RIPK1, RIPK3, and TAK1. Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro. Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis. TAK1 acts as a switch between apoptosis and necrosis.     

Acetylated Histone H3K9 is associated with meiotic recombination hotspots,and plays a role in recombination redundantly with other factors including the H3K4 methylase Set1 in fission yeast

Histone modifications are associated with meiotic recombination hotspots, discrete sites with augmented recombination frequency. For example, trimethylation of histone H3 lysine4 (H3K4me3) marks most hotspots in budding yeast and mouse. Modified histones are known to regulate meiotic recombination partly by promoting DNA double-strand break (DSB) formation at hotspots, but the role and precise landscape of involved modifications remain unclear. Here, we studied hotspot-associated modifications in fission yeast and found general features: acetylation of H3 lysine9 (H3K9ac) is elevated, and H3K4me3 is not significantly enriched. Mutating H3K9 to non-acetylatable alanine mildly reduced levels of the DSB-inducing protein Rec12 (the fission yeast homologue of Spo11) and DSB at hotspots, indicating that H3K9ac may be involved in DSB formation by enhancing the interaction between Rec12 and hotspots. In addition, we found that the lack of the H3K4 methyltransferase Set1 generally increased Rec12 binding to chromatin but partially reduced DSB formation at some loci, suggesting that Set1 is also involved in DSB formation. These results suggest that meiotic DSB formation is redundantly regulated by multiple chromatin-related factors including H3K9ac and Set1 in fission yeast.