Identification and characterization of canine growth differentiation factor-9 and its splicing variant

Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor-β (TGF-β) superfamily, is expressed exclusively in the oocyte within the ovary and plays essential roles in the ovarian function in mammals. However, a possible involvement of GDF-9 in canine ovarian physiology that has a unique ovulation process among mammals has not been studied. Interestingly, we have isolated two types of cDNA clones generated by an alternative splicing from a canine ovarian total RNA. The predominant long form cDNA shares a common precursor structure with GDF-9s in other species whereas the minor short form cDNA has a 172 amino acid truncation in the proregion. Using a transient expression system, we found that the long form cDNA has a defect in mature protein production whereas the short form cDNA readily produces mature protein. However, mutations at one or two N-glycosylation sites in the mature domain of the short form GDF-9 caused a loss in mature protein production. These results suggest that the prodomain and N-linked glycosylation of the mature domain regulate proper processing and secretion of canine GDF-9. Based on the biological functions of GDF-9, these characteristics of canine GDF-9 could be causatively linked to the unique ovulation process in the Canidae.     

Highly sensitive and quantitative FRET–FLIM imaging in single dendritic spines using improved non-radiative YFP

Two-photon fluorescence lifetime imaging microscopy (TPFLIM) enables the quantitative measurements of fluorescence resonance energy transfer (FRET) in small subcellular compartments in light scattering tissue. We evaluated and optimized the FRET pair of mEGFP (monomeric EGFP with the A206K mutation) and REACh (non-radiative YFP variants) for TPFLIM. We characterized several mutants of REACh in terms of their "darkness," and their ability to act as a FRET acceptor for mEGFP in HeLa cells and hippocampal neurons. Since the commonly used monomeric mutation A206K increases the brightness of REACh, we introduced a different monomeric mutation (F223R) which does not affect the brightness. Also, we found that the folding efficiency of original REACh, as measured by the fluorescence lifetime of a mEGFP-REACh tandem dimer, was low and variable from cell to cell. Introducing two folding mutations (F46L, Q69M) into REACh increased the folding efficiency by approximately 50%, and reduced the variability of FRET signal. Pairing mEGFP with the new REACh (super-REACh, or sREACh) improved the signal-to-noise ratio compared to the mEGFP-mRFP or mEGFP-original REACh pair by approximately 50%. Using this new pair, we demonstrated that the fraction of actin monomers in filamentous and globular forms in single dendritic spines can be quantitatively measured with high sensitivity. Thus, the mEGFP-sREACh pair is suited for quantitative FRET measurement by TPFLIM, and enables us to measure protein-protein interactions in individual dendritic spines in brain slices with high sensitivity.     

Effects of magnesium deficiency on magnesium uptake activity of rice root,evaluated using <Superscript>28</Superscript> Mg as a tracer

Aims

The mechanisms underlying magnesium (Mg) uptake by plant roots remain to be fully elucidated. In particular, there is little information about the effects of Mg deficiency on Mg uptake activity. A Mg uptake kinetic study is essential for better understanding the Mg uptake system.

Methods

We performed a Mg uptake tracer experiment in rice plants using ²⁸?Mg.

Results

Mg uptake was mediated by high- and low-affinity transport systems. The K _m value of the high-affinity transport system was approximately 70 μM under Mg-deficient conditions. The Mg uptake activity was promoted by Mg deficiency, which in turn fell to the basal level after 5- min of Mg resupply. The induced uptake rate was inhibited by ionophore treatment, suggesting that an energy-dependent uptake system is enhanced by Mg deficiency.

Conclusions

The Mg uptake changes rapidly with Mg conditions in rice, as revealed by a ²⁸?Mg tracer experiment. This technique is expected to be applicable for Mg uptake analyses, particularly in mutants or other lines.

     

Preparation and characterization of polyurethane foams using a palm oil-based polyol

Polyurethane (PU) foams were prepared using a palm oil-based polyol (PO-p). At the first stage, palm oil was converted to monoglycerides as a new type of polyol by glycerolysis. A yield of the product reached 70% at reaction temperature of 90 degrees C by using an alkali catalyst and a solvent. At the second stage, PU foams were prepared from mixtures of the polyol and polyethylene glycol (PEG) or diethylene glycol (DEG) and an isocyanate compound. Characterization of the foams was carried out by thermal and mechanical analyses. The analyses showed that the chain motion of polyurethane becomes more flexible at the higher PO-p content in the whole polymer, which indicates that the monoglyceride molecules work as soft segments. The study here may lead to a development of a new type of polyurethane foams using palm oil as a raw material.     

Potent transglutaminase inhibitors, dithio β-aminoethyl ketones

Potent transglutaminase inhibitors were obtained from disulfide compounds, cystamine, dimethyl cystine, and dimethyl homocystine. The disulfide bond and thiophene ring play an important role in inhibitory activity of synthesized aryl β-amino ketones.     

Roles of TRPM2 in oxidative stress

Reactive oxygen species (ROS) play critical roles in cell death, diseases, and normal cellular processes. TRPM2 is a member of transient receptor potential (TRP) protein superfamily and forms a Ca²⁺-permeable nonselective cation channel activated by ROS, specifically by hydrogen peroxide (H₂O₂), and at least in part via second-messenger mechanisms. Accumulating evidence has indicated that TRPM2 mediates multiple cellular responses, after our finding that Ca²⁺ influx via TRPM2 regulates H₂O₂-induced cell death. Recently, we have demonstrated that Ca²⁺ influx through TRPM2 induces chemokine production in monocytes and macrophages, which aggravates inflammatory neutrophil infiltration in mice. However, understanding is still limited for in vivo physiological or pathophysiological significance of ROS-induced TRPM2 activation. In this review, we summarize mechanisms underlying activation of TRPM2 channels by oxidative stress and downstream biological responses, and discuss the biological importance of oxidative stress-activated TRP channels.     

The E3 ubiquitin ligase activity of Trip12 is essential for mouse embryogenesis

Protein ubiquitination is a post-translational protein modification that regulates many biological conditions. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12(mt/mt)) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12(mt/mt) embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16. In contrast, Trip12(mt/mt) ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12(mt/mt) ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.     

Adoption of lycaenid Niphanda fusca (Lepidoptera: Lycaenidae) caterpillars by the host ant Camponotus japonicus (Hymenoptera: Formicidae)

Caterpillars of the parasitic lycaenid butterfly are often adopted by host ants. It has been proposed that this adoption occurs because the caterpillars mimic the cuticular hydrocarbons of the host ant. This study aimed to examine whether caterpillars of the Japanese lycaenid butterfly Niphanda fusca induce adoption by mimicking their host ant Camponotus japonicus. Behavioral observations conducted in the laboratory showed that most second‐instar caterpillars were not adopted, whereas most third‐instar caterpillars were successfully adopted by host workers. A chemical comparison detected no characteristic differences in the cuticular hydrocarbon profiles between second‐ and third‐instar caterpillars. However, morphological features of the caterpillars differed between the second and third instars; third‐instar caterpillars developed exocrine glands (ant organs) such as tentacle organs and a dorsal nectary organ. These results suggest that multiple chemical signatures, not only cuticular hydrocarbons, may be important for invasion of the host ant nest.     

Multiple translocation of the AVR-Pita effector gene among chromosomes of the rice blast fungus Magnaporthe oryzae and related species

Magnaporthe oryzae is the causal agent of rice blast disease, a devastating problem worldwide. This fungus has caused breakdown of resistance conferred by newly developed commercial cultivars. To address how the rice blast fungus adapts itself to new resistance genes so quickly, we examined chromosomal locations of AVR-Pita, a subtelomeric gene family corresponding to the Pita resistance gene, in various isolates of M. oryzae (including wheat and millet pathogens) and its related species. We found that AVR-Pita (AVR-Pita1 and AVR-Pita2) is highly variable in its genome location, occurring in chromosomes 1, 3, 4, 5, 6, 7, and supernumerary chromosomes, particularly in rice-infecting isolates. When expressed in M. oryzae, most of the AVR-Pita homologs could elicit Pita-mediated resistance, even those from non-rice isolates. AVR-Pita was flanked by a retrotransposon, which presumably contributed to its multiple translocation across the genome. On the other hand, family member AVR-Pita3, which lacks avirulence activity, was stably located on chromosome 7 in a vast majority of isolates. These results suggest that the diversification in genome location of AVR-Pita in the rice isolates is a consequence of recognition by Pita in rice. We propose a model that the multiple translocation of AVR-Pita may be associated with its frequent loss and recovery mediated by its transfer among individuals in asexual populations. This model implies that the high mobility of AVR-Pita is a key mechanism accounting for the rapid adaptation toward Pita. Dynamic adaptation of some fungal plant pathogens may be achieved by deletion and recovery of avirulence genes using a population as a unit of adaptation.     

Availability of Energy in Alcohols,Aldehydes and Ketones by Growing Chicks

Biological availability of 23 alcohols, 16 aldehydes and 2 ketones was compared by the mini-test with chicks. Chicks can utilize methanol and ethanol, but not the alcohols of carbon chain from 3 to 9. Lauryl and myristyl alcohols were well utilized but those of higher carbon chain than 14 were not, mainly due to low digestibility. Glycerol and D-sorbitol were well utilized but not 4 other poly alcohols tested. Aldehydes and ketones with free carbonyl group showed low availability or even toxicity, but their derivatives with masked group showed no detrimental effect. Acetaldehyde, acetal and aldehydes of carbon chain longer than 9 were partially utilized.