Identification and Mapping of Two New Genes Conferring Resistance to Powdery Mildew from Aegilops tauschii （Coss,） Schmal

Two powdery mildew resistance genes were Identified from Aegilops tauschll accessions Y201 and Y212 and mapped using two different F2 populations derived from the crosses between susceptible accession Y2272 and Y201, and susceptible accession Y2263 and Y212. Genetic analysis of resistance to powdery mildew Indicated that the resistance of Y201 was controlled by a single dominant gene, whereas the resistance of Y212 was controlled by a single recessive gene. We have temporarily designated these genes as PmY201 and PmY212, respectively. By bulk segregation analysis, six mlcrosatelllte markers Including Xgwm174, cfd26, cfd57, cfdl02, Xgwm583 and Xgwm639 were found to be linked to PraY201 with genetic distances of 5.2, 7.7, 9.6, 12.5, 20.2 and 22.1 cM, respectively. Five SSR markers, including cfd57, Xgwm182, cfd7, cfd102, and cfd12, were found to be linked to PmY212 with distances of 5.6, 7.2, 11.5, 14.7, and 18.5 cM, respectively. According to the locations of the linked markers, the two resistance genes were located In the 5DL region. Based on the chromosomal locations and the resistance patterns of the two genes, we propose that PmY201 and PmY212 are two novel powdery mildew resistance genes, and are suitable for marker-assisted selection.     

Proteomic analysis of effect of hyperthermia on spermatogenesis in adult male mice

We characterized cellular and molecular mechanisms involved in spermatogenesis following short-term heat exposure of murine testis. For these studies, we utilized a proteomic approach with two-dimensional gel electrophoresis (2DE) analyses and mass spectroscopic identification of proteins with altered expression in mouse testes at different times after heat shock. We established a proteome reference map from 7-wk-old mouse testis linked to a federated proteome database. We used these tools to analyze quantitative variations in the tissue over a time course of 0.5, 2, 6, and 12 h following heat exposure. We separated 108 protein spots expressed differentially between the heat shock tissues and the control mouse testes. Of these spots, we identified 36 by comparing with the control reference map. We then focused on the heterogeneous nuclear ribonucleoproteins (hnRNPs) and the chaperonins containing t-complex polypeptide-1 (CCT). Further analysis in this heat-shocked model suggests numerous potential mechanisms for heat shock-induced spermatogenic disorder.     

Effects of shell morphological traits on the weight traits of Manila clam (Ruditapes philippinarum)

The shell length, height, and width, live body weight, and edible tissue weight of Manila clam of 1, 2, and 3 years of age were measured, and their correlation coefficients were calculated. The shell morphological traits were used as independent variables, and live body weight or edible tissue weigh used as a dependent variable for calculating the path coefficients, correlation index and determination coefficients. The results showed that the correlation coefficients between each shell morphological trait and the live body weight or edible tissue weight were all highly significant (P < 0. 01). The shell height at 1-year old clams was highly correlated with the live body weight and edible tissue weight. The shell width of 2- to 3-year-old clams was strongly associated with the live body weight, while the shell length was closely linked to the edible tissue weight. The results of coefficients of determination for the morphological traits against weight traits agreed well with the results of path analysis. The correlation indices for all morphological traits against weight traits were approximately the same as determination coefficients regardless of clam age. The correlation indices (R²) of morphological traits against the live body weight of clams of all ages and edible tissue weight of 1-year-old clams were larger than 0.85, but R² of morphological traits against the edible tissue weight of 2- and 3-year-old clams was smaller than 0.85, indicating that some other factors might be associated with the edible tissue weight of 2- and 3-year-old clams. Multiple regression equations were obtained to estimate shell length X₁ (cm), shell height X₂ (cm), shell width X₃ (cm) against live body weight Y (g), edible tissue weight Z (g): for 1-year-old clams: Y = ?4.317 + 0.18X₁ + 0.147X₂, (X₁ < 0.01, X₂ < 0.01), Z = ?1.011 + 0.095X₂, (X₂ < 0.01); for 2-year-old clams: Y = ?15.119 + 0.249X₁ + 0.176X₂ + 0.688X₃, (X₁ < 0.01, X₃ < 0.01), Z = ?4.248 + 0.198X₁, (X₁ < 0.05, X₃ < 0.01); and for 3-year-old clams: Y = ?25.013 + 0.415X₁ + 1.184X₃, (X₁ < 0.01, X₃ < 0.01), Z = ?7.082 + 0.119X₁ + 0.332X₃, (X₁ < 0.05, X₃ < 0.01).     

The translocon Sec61beta localized in the inner nuclear membrane transports membrane-embedded EGF receptor to the nucleus

Accumulating evidence indicates that endocytosis plays an essential role in the nuclear transport of the ErbB family members, such as epidermal growth factor receptor (EGFR) and ErbB-2. Nevertheless, how full-length receptors embedded in the endosomal membrane pass through the nuclear pore complexes and function as non-membrane-bound receptors in the nucleus remains unclear. Here we show that upon EGF treatment, the biotinylated cell surface EGFR is trafficked to the inner nuclear membrane (INM) through the nuclear pore complexes, remaining in a membrane-bound environment. We further find that importin β regulates EGFR nuclear transport to the INM in addition to the nucleus/nucleoplasm. Unexpectedly, the well known endoplasmic reticulum associated translocon Sec61β is found to reside in the INM and associate with EGFR. Knocking down Sec61β expression reduces EGFR level in the nucleoplasm portion and accumulates it in the INM portion. Thus, the Sec61β translocon plays an unrecognized role in the release of the membrane-anchored EGFR from the lipid bilayer of the INM to the nucleus. The newly identified Sec61β function provides an alternative pathway for nuclear transport that can be utilized by membrane-embedded proteins such as full-length EGFR.     

Involvement of Inducible 6-Phosphofructo-2-kinase in the Anti-diabetic Effect of Peroxisome Proliferator-activated Receptor γ Activation in Mice

PFKFB3 is the gene that codes for the inducible isoform of 6-phosphofructo-2-kinase (iPFK2), a key regulatory enzyme of glycolysis. As one of the targets of peroxisome proliferator-activated receptor γ (PPARγ), PFKFB3/iPFK2 is up-regulated by thiazolidinediones. In the present study, using PFKFB3/iPFK2-disrupted mice, the role of PFKFB3/iPFK2 in the anti-diabetic effect of PPARγ activation was determined. In wild-type littermate mice, PPARγ activation (i.e. treatment with rosiglitazone) restored euglycemia and reversed high fat diet-induced insulin resistance and glucose intolerance. In contrast, PPARγ activation did not reduce high fat diet-induced hyperglycemia and failed to reverse insulin resistance and glucose intolerance in PFKFB3^+/− mice. The lack of anti-diabetic effect in PFKFB3^+/− mice was associated with the inability of PPARγ activation to suppress adipose tissue lipolysis and proinflammatory cytokine production, stimulate visceral fat accumulation, enhance adipose tissue insulin signaling, and appropriately regulate adipokine expression. Similarly, in cultured 3T3-L1 adipocytes, knockdown of PFKFB3/iPFK2 lessened the effect of PPARγ activation on stimulating lipid accumulation. Furthermore, PPARγ activation did not suppress inflammatory signaling in PFKFB3/iPFK2-knockdown adipocytes as it did in control adipocytes. Upon inhibition of excessive fatty acid oxidation in PFKFB3/iPFK2-knockdown adipocytes, PPARγ activation was able to significantly reverse inflammatory signaling and proinflammatory cytokine expression and restore insulin signaling. Together, these data demonstrate that PFKFB3/iPFK2 is critically involved in the anti-diabetic effect of PPARγ activation.     

RNAi Phenotypes and the Localization of a Protein::GUS Fusion Imply a Role for Medicago truncatula PIN Genes in Nodulation

The symbiosis between legumes and rhizobia results in the development of a new plant organ, the nodule. A role for polar auxin transport in nodule development in Medicago truncatula has been demonstrated using molecular genetic tools. The expression of a DR5::GUS auxin-responsive promoter in uninoculated M. truncatula roots mirrored that reported in Arabidopsis, and expression of the construct in nodulating roots confirmed results reported in white clover. The localization of a root-specific PIN protein (MtPIN2) in normal roots, developing lateral roots and nodules provided the first evidence that a PIN protein is expressed in nodules. Reduced levels of MtPIN2, MtPIN3, and MtPIN4 mRNAs via RNA interference demonstrated that plants with reduced expression of various MtPINs display a reduced number of nodules. The reported results show that in M. truncatula, PIN proteins play an important role in nodule development, and that nodules and lateral roots share some early auxin responses in common, but they rapidly differentiate with respect to auxin and MtPIN2 protein distribution.     

Phospholipase Cgamma2 dosage is critical for B cell development in the absence of adaptor protein BLNK

B cell linker (BLNK) protein and phospholipase Cgamma2 (PLCgamma2) are components of the BCR signalosome that activate calcium signaling in B cells. Mice lacking either molecule have a severe but incomplete block in B lymphopoiesis. In this study, we generated BLNK-/- PLCgamma2-/- mice to examine the effect of simultaneous disruption of both molecules on B cell development. We showed that BLNK-/- PLCgamma2-/- mice had compounded defects in B cell maturation compared with either single mutant, suggesting that these two molecules cooperatively or synergistically signaled B lymphopoiesis. However, Ig H chain allelic exclusion was maintained in single and double mutants, indicating that signals propagated by BLNK and PLCgamma2 were not involved in this process. Interestingly, in the absence of BLNK, B cell development was dependent on plcgamma2 gene dosage. This was evidenced by the proportionate decrease in splenic B cell population and increase in bone marrow surface pre-BCR+ cells in PLCgamma2-diploid, -haploid, and -null animals. Intracellular calcium signaling and ERK activation in response to BCR engagement were also proportionately decreased and delayed, respectively, with stepwise reduction of plcgamma2 dosage in a BLNK(null) background. Thus, these data indicate the importance of BLNK not only as a conduit to specifically channel BCR-signaling pathways and as a scaffold for the assembling of macromolecular complex, but also as an efficient aggregator or concentrator of PLCgamma2 molecules to effect optimal signaling for B cell generation and activation.     

Potential influence of Asp in the Ca2+ coordination position 5 of parvalbumin on the calcium-binding affinity: a computational study

Parvalbumins (PV) are calcium-binding proteins, all sharing the common helix-loop-helix (EF-hand) motif. This motif contains a central twelve-residue Ca(2+)-binding loop with the flanking helices positioned roughly perpendicular to each other. The precise role of these coordination residues has been the subject of intense studies. In this work, we focus on the coordination position 5 in the CD Ca(2+)-binding site of silver hake parvalbumin isoform B (SHPV-B). The most common residue at site 5 of calcium-binding loop in canonical EF-hands is Asp [B.J. Marsden, G.S. Shaw, B.D. Sykes, Biochem. Cell Biol. 68 (1990) 587-601], but in the CD site of PV, this position is almost always serine (Ser). The substitution of Ser with Asp will add the 5th carboxylate residue in the CD coordination sphere. However, as predicted by the acid pair hypothesis, the Ca(2+)-binding affinity would be maximized in an EF-hand motif that has four carboxylate ligands paired along the +/-x, and +/-z-axes [R.E. Reid, R.S. Hodges, J. Theor. Biol. 84 (1980) 401-444]. Molecular dynamics simulations and free energy calculations were employed to investigate the influence of Ser to Asp mutation at position 5 on calcium-binding affinity. We found that the Asp variant exhibited remarkable stability during the entire molecular dynamics simulation, with not only the retention of the Ca(2+)-binding site, but also increased compactness in the coordination sphere. The S55D fragment also accommodated Ca(2+) well. We conclude that the reason why Asp which is the most common residue at site 5 of calcium-binding loop in canonical EF-hands has never been identified at this position experimentally for PVs might be related to its physiological functions.