Developmental capacity of vitrified immature porcine oocytes following ICSI: effects of cytochalasin B and cryoprotectants

In the present study, effects of concentration and pretreatment time of cytochalasin B (CB), and of two types of cryoprotectant solutions on the nuclear maturation of vitrified-warmed porcine oocytes were examined. Also, the developmental capacity of vitrified immature porcine oocytes following intracytoplasmic sperm injection (ICSI) was investigated. The nuclear maturation rate (46.8%) of the vitrified-warmed oocytes treated with 7.5 microg/mL CB for 30 min was significantly higher (P < 0.05) than those (13.9-39.2%) of the vitrified-warmed oocytes treated with 0, 2.5, or 5.0 microg/mL CB for 10 or 30 min. Additionally, the nuclear maturation rate of oocytes treated with CB and vitrified in ethylene glycol (EG) (37.1%) was significantly higher (P < 0.05) than that of EG + dimethyl sulfoxide (Me(2)SO) (23.9%). However, no significant differences were observed in the cleavage and blastocyst development rates among the control (45.2 and 20.0%, respectively), the EG group (37.8 and 13.5%, respectively) and the EG + Me(2)SO group (39.3 and 14.3%, respectively). These results demonstrated that: (1) pretreatment with 7.5 microg/mL CB was beneficial for the vitrification of immature porcine oocytes; (2) the combination of EG and Me(2)SO as a cryoprotectant was not advantageous for in vitro maturation (IVM) of vitrified immature porcine oocytes; and (3) vitrified-warmed porcine oocytes matured after IVM, developed to the blastocyst stage without distinct differences compared to fresh oocytes following ICSI.     

RNA interfering approach for clarifying the PPARgamma pathway using lentiviral vector expressing short hairpin RNA

Peroxisome proliferator-activated receptor γ (PPARγ) plays a central role in adipocyte differentiation and insulin sensitivity. Although PPARγ also appears to regulate diverse cellular processes in other cell types such as lymphocytes, the detailed mechanisms remain unclear. In this study, we established a lentivirus-mediated short hairpin RNA expression system and identified a potent short hairpin RNA which suppresses PPARγ expression, resulting in marked inhibition of preadipocyte-to-adipocyte differentiation in 3T3-L1 cells. Our PPARγ-knockdown method will serve to clarify the PPARγ pathway in various cell types in vivo and in vitro, and will facilitate the development of therapeutic applications for a variety of diseases.     

Electronic spectral studies of dinuclear Cobalt(II) complexes with a phenol-based dinucleating ligand containing four methoxyethyl chelating arms

The electronic spectra of dinuclear cobalt(II) complexes [Co₂(bomp)(MeCO₂)₂]BPh₄ (1) and [Co₂(bomp)(PhCO₂)₂]BPh₄ (2) were studied [H(bomp): 2,6-bis[bis(2-methoxyethyl)aminomethyl]-4-methylphenol]; the spectral components obtained by Gaussian curve analysis were well simulated based on the angular overlap model using the aomx program. The first transition band ⁴T₁ → ⁴T₂(⁴F) of an octahedral high-spin cobalt(II) complex was found to be sensitive to the distortion around the cobalt(II) ion.     

Misexpression screen for genes altering the olfactory map in Drosophila

Despite the identification of a number of guidance molecules, a comprehensive picture has yet to emerge to explain the precise anatomy of the olfactory map. From a misexpression screen of 1,515 P{GS} lines, we identified 23 genes that, when forcibly expressed in the olfactory receptor neurons, disrupted the stereotyped anatomy of the Drosophila antennal lobes. These genes, which have not been shown previously to control olfactory map development, encode novel proteins as well as proteins with known roles in axonal outgrowth and cytoskeletal remodeling. We analyzed Akap200, which encodes a Protein Kinase A-binding protein. Overexpression of Akap200 resulted in fusion of the glomeruli, while its loss resulted in misshapen and ectopic glomeruli. The requirement of Akap200 validates our screen as an effective approach for recovering genes controlling glomerular map patterning. Our finding of diverse classes of genes reveals the complexity of the mechanisms that underlie olfactory map development.     

SDS-dependent proteases induced by ABA and its relation to Rubisco and Rubisco activase contents in rice leaves

Protease activities and its relation to the contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase were investigated in detached leaves of rice (Oryza sativa L.) floated on the solutions containing abscisic acid (ABA) or benzyladenine (BA). Rubisco and Rubisco activase contents were decreased during the time course and the decreases were enhanced by ABA and suppressed by BA. The decrease in Rubisco activase was faster than that in Rubisco. SDS-dependent protease activities at 50–70 kDa (rice SDS-dependent protease: RSP) analyzed by the gelatin containing PAGE were significantly enhanced by ABA. RSPs were also increased in attached leaves during senescence. RSPs had the pH optimum of 5.5, suggesting that RSPs are vacuolar protease. Both decrease in Rubisco and Rubisco activase contents and increase in RSPs activities were suppressed by cycloheximide. These findings indicate that the activities of RSPs are well correlated with the decrease in these protein contents. Immunoblotting analysis showed that Rubisco in the leaf extracts was completely degraded by 5 h at pH 5.5 with SDS where it was optimal condition for RSPs. However, the degradation of Rubisco did not proceed at pH 7.5 without SDS where it is near physiological condition for stromal proteins. Rubisco activase was degraded at similar rate under both conditions. These results suggest that RSPs can functions in a senescence related degradation system of chloroplast protein in rice leaves. Rubisco activase would be more susceptible to proteolysis than Rubisco under physiological condition and this could affect the contents of these proteins in leaves.     

Thermococcus kodakarensis Mutants Deficient in Di-myo-Inositol Phosphate Use Aspartate To Cope with Heat Stress

Many of the marine microorganisms which are adapted to grow at temperatures above 80°C accumulate di-myo-inositol phosphate (DIP) in response to heat stress. This led to the hypothesis that the solute plays a role in thermoprotection, but there is a lack of definitive experimental evidence. Mutant strains of Thermococcus kodakarensis (formerly Thermococcus kodakaraensis), manipulated in their ability to synthesize DIP, were constructed and used to investigate the involvement of DIP in thermoadaptation of this archaeon. The solute pool of the parental strain comprised DIP, aspartate, and α-glutamate. Under heat stress the level of DIP increased 20-fold compared to optimal conditions, whereas the pool of aspartate increased 4.3-fold in response to osmotic stress. Deleting the gene encoding the key enzyme in DIP synthesis, CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, abolished DIP synthesis. Conversely, overexpression of the same gene resulted in a mutant with restored ability to synthesize DIP. Despite the absence of DIP in the deletion mutant, this strain exhibited growth parameters similar to those of the parental strain, both at optimal (85°C) and supraoptimal (93.7°C) temperatures for growth. Analysis of the respective solute pools showed that DIP was replaced by aspartate. We conclude that DIP is part of the strategy used by T. kodakarensis to cope with heat stress, and aspartate can be used as an alternative solute of similar efficacy. This is the first study using mutants to demonstrate the involvement of compatible solutes in the thermoadaptation of (hyper)thermophilic organisms.Hyperthermophilic bacteria and archaea isolated from saline environments accumulate unusual organic solutes in response to osmotic as well as heat stress. Mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, mannosyl-di-myo-inositol phosphate (DIP), diglycerol phosphate, and glycero-phospho-myo-inositol are examples of compatible solutes highly restricted to thermophiles and hyperthermophiles (27, 31). Our team has, over several years, examined the compatible solute composition in a large number of hyperthermophiles and their accumulation under stressful conditions. The data reveal a trend toward specialization of roles in thermoadaptation and osmoadaptation. Indeed, mannosylglycerate and diglycerol phosphate typically accumulate in response to increased NaCl concentration in the growth medium, whereas the levels of DIP and derivatives consistently increase at supraoptimal growth temperatures (11, 16, 17, 27, 31).DIP is widespread among extreme archaeal hyperthermophiles, such as Methanotorris igneus, Aeropyrum pernix, Stetteria hydrogenophila, Pyrodictium occultum, Pyrolobus fumarii, Archaeoglobus spp., and all the members of the Thermococcales examined thus far, except Palaeococcus ferrophilus (5, 7, 11, 13, 16, 18, 31). This organic solute has also been found in representatives of the two hyperthermophilic bacterial genera, Aquifex and Thermotoga (14, 17, 22).The specific chemical nature of solutes encountered in hyperthermophiles, together with their accumulation in response to elevated temperatures, led to the hypothesis that they play a role in thermoprotection of cellular components in vivo. However, there is a lack of convincing experimental evidence, such as that obtained with suitable mutants. Progress toward understanding the physiological functions of these solutes critically depends on two conditions: the availability of genetic tools to manipulate hyperthermophilic organisms and knowledge about the genes and enzymes implicated in the synthesis of these unusual solutes.Thermococcus kodakarensis (formerly Thermococcus kodakaraensis) is a member of the order Thermococcales with an optimal growth temperature of 85°C and is able to grow at temperatures up to 94°C in batch cultures. The NaCl concentration for optimal growth matches that of seawater (1). T. kodakarensis is the only marine hyperthermophile for which a number of genetic tools have been developed, including Escherichia coli-T. kodakarensis shuttle vectors and a reliable gene disruption system (19, 29, 32, 34). The genome of T. kodakarensis possesses a gene encoding CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase (IPCT/DIPPS), a key enzyme in DIP synthesis (2, 25, 26). This enzyme catalyzes the synthesis of CDP-inositol from CTP and inositol-1-phosphate as well as the transfer of the inositol group from CDP-inositol to a second molecule of inositol-1-phosphate to yield a phosphorylated form of DIP (2). Therefore, we set out to investigate whether DIP was involved in thermoadaptation of T. kodakarensis. A DIP-deficient mutant was constructed by deleting the IPCT/DIPPS gene; subsequently, this strain was complemented in this activity by inserting the gene under the control of a constitutive promoter, resulting in a construct with restored ability to synthesize DIP. The effects of heat and osmotic stress on the pattern of solute accumulation and on the growth profiles of the two mutants provided evidence for the involvement of DIP in thermoprotection.     

Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate‐binding module (CBM)–cyan fluorescent protein (CFP)

In enzymatic saccharification of lignocellulosics, the access of the enzymes to exposed cellulose surfaces is a key initial step in triggering hydrolysis. However, knowledge of the structure–hydrolyzability relationship of the pretreated biomass is still limited. Here we used fluorescent‐labeled recombinant carbohydrate‐binding modules (CBMs) from Clostridium josui as specific markers for crystalline cellulose (CjCBM3) and non‐crystalline cellulose (CjCBM28) to analyze the complex surfaces of wood tissues pretreated with NaOH, NaOH–Na₂S (kraft pulping), hydrothermolysis, ball‐milling, and organosolvolysis. Japanese cedar wood, one of the most recalcitrant softwood species was selected for the analysis. The binding analysis clarified the linear dependency of the exposure of crystalline and non‐crystalline cellulose surfaces for enzymatic saccharification yield by the organosolv and kraft delignification processes. Ball‐milling for 5–30 min increased saccharification yield up to 77%, but adsorption by the CjCBM–cyan fluorescent proteins (CFPs) was below 5%. Adsorption of CjCBM–CFPs on the hydrothermolysis pulp were less than half of those for organosolvolysis pulp, in coincidence with low saccharification yields. For all the pretreated wood, crystallinity index was not directly correlated with the overall saccharification yield. Fluorescent microscopy revealed that CjCBM3–CFP and CjCBM28–CFP were site‐specifically adsorbed on external fibrous structures and ruptured or distorted fiber surfaces. The assay system with CBM–CFPs is a powerful measure to estimate the initiation sites of hydrolysis and saccharification yields from chemically delignified wood pulps. Biotechnol. Bioeng. 2010; 105: 499–508. © 2009 Wiley Periodicals, Inc.