No abstract

High-temperature induction of male sterility during floral organogenesis is a critical problem for barley (Hordeum vulgare L.) crops that the molecular basis is incompletely understood. Gene expression and differentiation of anthers were examined under normal (20°C day/15°C night) and elevated (30°C day/25°C night) temperatures. Serial analysis of gene expression analysis displayed contrasting profiles of gene expression in early panicles between control and high-temperature conditions. Several transcripts dramatically upregulated before development and differentiation of anther wall layers in normal temperatures, including histone H3, H4 and glycine-rich RNA-binding protein genes, were not upregulated at elevated temperatures and typically abundant mRNAs, such as 60S ribosomal protein L27a and glyoxalase I, appeared to be downregulated. Instead, development and differentiation of tapetum cells and pollen mother cells were completely aborted. Failure of transcriptional reactivation with return to normal temperatures increases with duration of elevated temperatures and is strongly correlated with observation of male sterility. Hyper-phosphorylation of the ser-5 residue of the C-terminal domain of the largest subunit of RNA polymerase II (RPB1) was noted to occur under high-temperature conditions. These results indicate that early development and differentiation of barley anthers are very sensitive to high-temperature stress causing major alterations in gene expression.

The yeast Saccharomyces cerevisiae Cdc7p/Dbf4p protein kinase complex was purified to near homogeneity from insect cells. The complex efficiently phosphorylated yeast Mcm2p and less efficiently the remaining Mcm proteins or other replication proteins. Significantly, when pretreated with alkaline phosphatase, Mcm2p became completely inactive as a substrate, suggesting that it must be phosphorylated by other protein kinase(s) to be a substrate for the Cdc7p/Dbf4p complex. Mutant Cdc7p/Dbf4p complexes containing either Cdc7-1p or Dbf4-1ϳ5p were also partially purified from insect cells and characterized in vitro. Furthermore, the autonomously replicating sequence binding activity of various dbf4 mutants was also analyzed. These studies suggest that the autonomously replicating sequencebinding and Cdc7p protein kinase activation domains of Dbf4p collaborate to form an active Cdc7p/Dbf4p complex and function during S phase in S. cerevisiae. It is shown that Rad53p phosphorylates the Cdc7p/Dbf4p complex in vitro and that this phosphorylation greatly inhibits the kinase activity of Cdc7p/Dbf4p. This result suggests that Rad53p controls the initiation of chromosomal DNA replication by regulating the protein kinase activity associated with the Cdc7p/Dbf4p complex.Initiation of chromosomal DNA replication and cell cycle progression are tightly regulated in eukaryotes. In the yeast Saccharomyces cerevisiae, several cdc (cell division cycle) mutants that block initiation of chromosomal DNA replication have been isolated and characterized (1, 2), for example, cdc28, cdc4, cdc6, and cdc7. The stepwise assembly of proteins at origins of DNA replication is a crucial part of regulating entry into S phase. Two key factors that mediate such cell cycle regulation are Cdc6 protein level and availability and the presence of an active cyclin-dependent kinase (Cdk) (see Ref. 3 for review). The origin recognition complex is bound to origins of DNA replication at all stages of the cell cycle in S. cerevisiae (4 -6). However, Cdc6 is not recruited to origins until late in M phase and is required for the association of the Mcm2-7 family proteins at origins to form a prereplicative complex (6 -8).The Cdc6 protein-dependent stage of the assembly reaction is inhibited by active Clb-Cdks (6, 9, 10). Because Cdc6 protein is synthesized only from late M phase until late G1 (11), prereplicative complexes can only be assembled during this period of the cell cycle. S phase cyclin-Cdk (Cdc28p/Clb5p or Cdc28p/ Clb6p) activity is required for the chromatin association of Cdc45p just before the initiation of chromosomal DNA replication (12). The previous results demonstrated that Cdc28 protein-Clb kinase is required throughout S phase to activate origins when they are scheduled to fire (13).The Cdc7/Dbf4 complex is a Cdk-like protein kinase (see Refs. 14 and 15 for review) that is also required for entry into S phase at a very late stage. CDC7 transcript levels are constant throughout the cell cycle, whereas DBF4 transcription is cell cycle regulated a...

This study investigated whether the consumption of a diet in which high-beta-glucan barley replaced rice would reduce the visceral fat area as well as the serum low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) in hypercholesterolemic Japanese men. A randomized, double-blinded, placebo-controlled intervention study was conducted in 44 hypercholesterolemic Japanese men with a body mass index (BMI) >22 kg/m2. The subjects were randomly assigned to groups consuming either rice (placebo group) or a mixture of rice and pearl barley with a high beta-glucan content (test group, 7.0 g beta-glucan per day) for 12 weeks. Blood samples were taken, and CT scan obtained before the trial and every four weeks during the trial. The pearl barley intake significantly reduced serum concentrations of LDL-C (P = 0.041) and TC (P = 0.037) during the trial. Significant differences between the test and placebo groups were found for the visceral fat (P = 0.039), BMI (P = 0.015), and waist circumference (P = 0.011) at the end point. The consumption of pearl barley with a high beta-glucan content reduces not only LDL-C but also visceral fat area.

Pancreatic cancer is among the most lethal malignancies worldwide. We aimed to identify novel prognostic markers by applying mass spectrometry (MS)-based proteomic analysis to formalin-fixed paraffin-embedded (FFPE) tissues. Resectable, node positive pancreatic ductal adenocarcinoma (PDAC) with poor (n 5 4) and better (n 5 4) outcomes, based on survival duration, with essentially the same clinicopathological backgrounds, and noncancerous pancreatic ducts (n 5 5) were analyzed. Cancerous and noncancerous cells collected from FFPE tissue sections by laser microdissection (LMD) were processed for liquid chromatography (LC)-tandem MS (MS/MS). Candidate proteins were identified by semiquantitative comparison and then analyzed quantitatively using selected reaction monitoring (SRM)-based MS. To confirm the associations between candidate proteins and outcomes, we immunohistochemically analyzed a cohort of 87 cases. In result, totally 1,229 proteins were identified and 170 were selected as candidate proteins for SRM-based targeted proteomics. Fourteen proteins overexpressed in cancerous as compared to noncancerous tissue showed different expressions in the poor and better outcome groups. Among these proteins, we found that three novel proteins ECH1, OLFM4 and STML2 were overexpressed in poor group than in better group, and that one known protein GTR1 was expressed reciprocally. Kaplan-Meier analysis showed high expressions of all four proteins to correlate with significantly worse overall survival (p < 0.05). In conclusion, we identified four proteins as candidates of prognostic marker of PDAC. The combination of shotgun proteomics verified by SRM and validated by immunohistochemistry resulted in the prognostic marker discovery that will contribute the understanding of PDAC biology and therapeutic development.