Proteomic and biochemical analysis of maize anthers after cold pretreatment and induction of androgenesis reveals an important role of anti-oxidative enzymes

In stress conditions, microspores and young pollen grains can be switched from their normal pollen development toward an embryogenic pathway via a process called androgenesis. Androgenic embryos can produce completely homozygous, haploid or double-haploid plants. This study aimed to investigate changes in the abundance of protein species during cold pretreatment and subsequent cultivation of maize anthers on induction media using gel-based proteomics. Proteins upregulated on the third day of anther induction were identified and discussed here. Simultaneous microscopic observations revealed that the first division occurred in microspores within this period. Using 2-D electrophoresis combined with MALDI TOF/TOF MS/MS analysis 19 unique proteins were identified and classified into 8 functional groups. Proteins closely associated with metabolism, protein synthesis and cell structure were the most abundant ones. Importantly, ascorbate peroxidase, an enzyme decomposing hydrogen peroxide, was also upregulated. Isozyme analysis of peroxidases validated the proteomic data and showed increased peroxidase activities during androgenic induction. Further, the isozyme pattern of SOD revealed increased activity of the MnSOD, which could provide hydrogen peroxide as a substrate for in vivo peroxidase reactions (including ascorbate peroxidase). Together, these data reveal the role of enzymes controlling oxidative stress during induction of maize androgenesis.     

Prevention of wear particle-induced osteolysis by a novel V-ATPase inhibitor saliphenylhalamide through inhibition of osteoclast bone resorption

Wear particle-induced peri-implant loosening (Aseptic prosthetic loosening) is one of the most common causes of total joint arthroplasty. It is well established that extensive bone destruction (osteolysis) by osteoclasts is responsible for wear particle-induced peri-implant loosening. Thus, inhibition of osteoclastic bone resorption should prevent wear particle induced osteolysis and may serve as a potential therapeutic avenue for prosthetic loosening. Here, we demonstrate for the first time that saliphenylhalamide, a new V-ATPase inhibitor attenuates wear particle-induced osteolysis in a mouse calvarial model. In vitro biochemical and morphological assays revealed that the inhibition of osteolysis is partially attributed to a disruption in osteoclast acidification and polarization, both a prerequisite for osteoclast bone resorption. Interestingly, the V-ATPase inhibitor also impaired osteoclast differentiation via the inhibition of RANKL-induced NF-κB and ERK signaling pathways. In conclusion, we showed that saliphenylhalamide affected multiple physiological processes including osteoclast differentiation, acidification and polarization, leading to inhibition of osteoclast bone resorption in vitro and wear particle-induced osteolysis in vivo. The results of the study provide proof that the new generation V-ATPase inhibitors, such as saliphenylhalamide, are potential anti-resorptive agents for treatment of peri-implant osteolysis.     

Lapatinib,a Dual EGFR and HER2 Tyrosine Kinase Inhibitor,Downregulates Thymidylate Synthase by Inhibiting the Nuclear Translocation of EGFR and HER2

Background

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has been shown to exert a synergistic antitumor effect when combined with fluoropyrimidine. This synergy may be attributable to the downregulation of thymidylate synthase (TS), which is frequently overexpressed in fluoropyrimidine-resistant cancer cells. However, the molecular mechanism underlying the downregulation of TS has yet to be clearly elucidated.

Methodology and Principal Findings

In this study, we demonstrate that lapatinib, a dual TKI of EGFR and HER2 downregulates TS via inhibition of the nuclear translocation of EGFR and HER2. From our cDNA microarray experiments, we determined that a variety of nucleotide synthesis-related genes, including TS, were downregulated with lapatinib, and this was apparent in HER2-amplified cells. Targeted and pharmacologic inhibition assays confirmed that the dual inhibition of EGFR and HER2 is required for the more effective reduction of TS as compared to what was observed with gefitinib or trasutuzumab alone. Additionally, we determined that co-transfected EGFR and HER2 activate the TS gene promoter more profoundly than do either EGFR or HER2 alone. The translocation of EGFR and HER2 into the nucleus and the subsequent activation of the TS promoter were inhibited by lapatinib.

Conclusions and Significance

These results demonstrate that lapatinib inhibits the nuclear translocation of EGFR and HER2 and downregulates TS, thus sensitizing cancer cells to fluoropyrimidine.     

The first crystal structures of a family 19 class IV chitinase: the enzyme from Norway spruce

Chitinases help plants defend themselves against fungal attack, and play roles in other processes, including development. The catalytic modules of most plant chitinases belong to glycoside hydrolase family 19. We report here x-ray structures of such a module from a Norway spruce enzyme, the first for any family 19 class IV chitinase. The bi-lobed structure has a wide cleft lined by conserved residues; the most interesting for catalysis are Glu113, the proton donor, and Glu122, believed to be a general base that activate a catalytic water molecule. Comparisons to class I and II enzymes show that loop deletions in the class IV proteins make the catalytic cleft shorter and wider; from modeling studies, it is predicted that only three N-acetylglucosamine-binding subsites exist in class IV. Further, the structural comparisons suggest that the family 19 enzymes become more closed on substrate binding. Attempts to solve the structure of the complete protein including the associated chitin-binding module failed, however, modeling studies based on close relatives indicate that the binding module recognizes at most three N-acetylglucosamine units. The combined results suggest that the class IV enzymes are optimized for shorter substrates than the class I and II enzymes, or alternatively, that they are better suited for action on substrates where only small regions of chitin chain are accessible. Intact spruce chitinase is shown to possess antifungal activity, which requires the binding module; removing this module had no effect on measured chitinase activity.     

The use of ebselen for radioprotection in cultured cells and mice

Ionizing radiation induces the production of reactive oxygen species (ROS), which play an important causative role in cell death. Therefore, compounds that control the level of ROS may confer radioprotective effects. Ebselen, a seleno-organic compound, has been shown to protect against cell injury caused by ROS. The objective of this study was to examine the effects of ebselen on radiation-dependent toxicity. We investigated the protective role of ebselen against ionizing radiation in U937 cells and mice. Upon exposure to 20 Gy of γ-irradiation, there was a distinct difference between untreated cells and the cells pretreated with 5 μM ebselen for 2 h with respect to viability, cellular redox status, and oxidative damage to cells. When cells were exposed to 2 Gy of γ-irradiation, there was a distinct difference between the untreated cells and the cells pretreated with ebselen with respect to apoptotic features and mitochondrial function. Ebselen administration for 14 days at a daily dosage of 10 mg/kg provided substantial protection against killing and oxidative damage to mice exposed to whole-body irradiation. These data indicate that ebselen may have great potential as a new class of in vivo, non-sulfur-containing radiation protector.     

Phylogenomics of Unusual Histone H2A Variants in Bdelloid Rotifers

Rotifers of Class Bdelloidea are remarkable in having evolved for millions of years, apparently without males and meiosis. In addition, they are unusually resistant to desiccation and ionizing radiation and are able to repair hundreds of radiation-induced DNA double-strand breaks per genome with little effect on viability or reproduction. Because specific histone H2A variants are involved in DSB repair and certain meiotic processes in other eukaryotes, we investigated the histone H2A genes and proteins of two bdelloid species. Genomic libraries were built and probed to identify histone H2A genes in Adineta vaga and Philodina roseola, species representing two different bdelloid families. The expressed H2A proteins were visualized on SDS-PAGE gels and identified by tandem mass spectrometry. We find that neither the core histone H2A, present in nearly all other eukaryotes, nor the H2AX variant, a ubiquitous component of the eukaryotic DSB repair machinery, are present in bdelloid rotifers. Instead, they are replaced by unusual histone H2A variants of higher mass. In contrast, a species of rotifer belonging to the facultatively sexual, desiccation- and radiation-intolerant sister class of bdelloid rotifers, the monogononts, contains a canonical core histone H2A and appears to lack the bdelloid H2A variant genes. Applying phylogenetic tools, we demonstrate that the bdelloid-specific H2A variants arose as distinct lineages from canonical H2A separate from those leading to the H2AX and H2AZ variants. The replacement of core H2A and H2AX in bdelloid rotifers by previously uncharacterized H2A variants with extended carboxy-terminal tails is further evidence for evolutionary diversity within this class of histone H2A genes and may represent adaptation to unusual features specific to bdelloid rotifers.     

Absence of Escherichia coli Phylogenetic Group B2 Strains in Humans and Domesticated Animals from Jeonnam Province,Republic of Korea

Multiplex PCR analyses of DNAs from genotypically unique Escherichia coli strains isolated from the feces of 138 humans and 376 domesticated animals from Jeonnam Province, South Korea, performed using primers specific for the chuA and yjaA genes and an unknown DNA fragment, TSPE4.C2, indicated that none of the strains belonged to E. coli phylogenetic group B2. In contrast, phylogenetic group B2 strains were detected in about 17% (8 of 48) of isolates from feces of 24 wild geese and in 3% (3 of 96) of isolates obtained from the Yeongsan River in Jeonnam Province, South Korea. The distribution of E. coli strains in phylogenetic groups A, B1, and D varied depending on the host examined, and there was no apparent seasonal variation in the distribution of strains in phylogenetic groups among the Yeongsan River isolates. The distribution of four virulence genes (eaeA, hlyA, stx₁, and stx₂) in isolates was also examined by using multiplex PCR. Virulence genes were detected in about 5% (38 of 707) of the total group of unique strains examined, with 24, 13, 13, and 9 strains containing hlyA, eaeA, stx₂, and stx₁, respectively. The virulence genes were most frequently present in phylogenetic group B1 strains isolated from beef cattle. Taken together, results of these studies indicate that E. coli strains in phylogenetic group B2 were rarely found in humans and domesticated animals in Jeonnam Province, South Korea, and that the majority of strains containing virulence genes belonged to phylogenetic group B1 and were isolated from beef cattle. Results of this study also suggest that the relationship between the presence and types of virulence genes and phylogenetic groupings may differ among geographically distinct E. coli populations.Escherichia coli is a normal inhabitant of the lower intestinal tract of warm-blooded animals and humans. While the majority of E. coli strains are commensals, some are known to be pathogenic, causing intestinal and extraintestinal diseases, such as diarrhea and urinary tract infections (42). Phylogenetic studies done using multilocus enzyme electrophoresis and 72 E. coli strains in the E. coli reference collection showed that E. coli strains can be divided into four phylogenetic groups (A, B1, B2, and D) (20, 41, 48). Recently, a potential fifth group (E) has also been proposed (11). Since multiplex PCR was developed for analysis of phylogenetic groups (6), a number of studies have analyzed a variety of E. coli strains for their phylogenetic group association (10, 12, 17, 18, 23, 54). Duriez et al. (10) reported the possible influence of geographic conditions, dietary factors, use of antibiotics, and/or host genetic factors on the distribution of phylogenetic groups among 168 commensal E. coli strains isolated from human stools from three geographically distinct populations in France, Croatia, and Mali. Random-amplified polymorphic DNA analysis of the intraspecies distribution of E. coli in pregnant women and neonates indicated that there was a correlation between the distribution of phylogenetic groups, random-amplified polymorphic DNA groups, and virulence factors (54). Moreover, based on comparisons of the distribution of E. coli phylogenetic groups among humans of different sexes and ages, it has been suggested that E. coli genotypes are likely influenced by morphological, physiological, and dietary differences (18). In addition, climate has also been proposed to influence the distribution of strains within E. coli phylogenetic groups (12). There are now several reports indicating that there is a potential relationship between E. coli phylogenetic groups, age, and disease. For example, E. coli isolates belonging to phylogenetic group B2 have been shown to predominate in infants with neonatal bacterial meningitis (27) and among urinary tract and rectal isolates (55). Also, Nowrouzian et al. (39) and Moreno et al. (37) reported that strains belonging to phylogenetic group B2 persisted among the intestinal microflora of infants and were more likely to cause clinical symptoms.Boyd and Hartl (2) reported that among the E. coli strains in the E. coli reference and the diarrheagenic E. coli collections, strains in phylogenetic group B2 carry the greatest number of virulence factors, followed by those in group D. Virulence factors carried by group B2 strains are thought to contribute to their strong colonizing capacity; a greater number of virulence genes have been detected in resident strains than in transient ones (38). Moreover, a mouse model of extraintestinal virulence showed that phylogenetic group B2 strains killed mice at greater frequency and possessed more virulence determinants than strains in other phylogenetic groups, suggesting a link between phylogeny and virulence genes in E. coli extraintestinal infection (45). In contrast, Johnson and Kuskowski (25) suggested that a group B2 ancestral strain might have simply acquired virulence genes by chance and that these genes were vertically inherited by group members during clonal expansion. However, numerous studies published to date suggest that there is a relationship between the genomic background of phylogenetic group B2 and its association with virulence factors (12, 28, 35, 39, 45).Both enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC, respectively) strains are among the most important food-borne pathogens worldwide, often causing severe gastrointestinal disease and fatal infections (13). While EPEC strains cause diarrhea and generally do not produce enterotoxin, they possess an adherence factor which is controlled by the chromosomal gene eaeA, encoding intimin (8). Unlike the EPEC strains, however, the EHEC strains typically contain the hlyA, stx₁, and stx₂ virulence genes, encoding hemolysins and Shiga-like type 1 and 2 toxins, respectively, and eaeA. The ability to detect EHEC has been greatly facilitated by the use of multiplex PCR (13, 44, 53). Several studies have shown that strains producing Shiga-like toxin 2 are more frequently found in cases of hemolytic-uremic syndrome than are those containing Shiga-like toxin 1 (30, 43, 46, 49).In the study reported here, we examined the distribution of phylogenetic groups and the prevalence of virulence genes in 659 genotypically unique E. coli strains isolated from humans and domestic animals in South Korea. In addition, we also tested 48 and 96 nonunique E. coli isolates from wild geese and the Yeongsan River, respectively, for phylogenetic distribution and virulence gene profiles. Here, we report that contrary to what has been previously reported in other parts of the world, no E. coli strains belonging to phylogenetic group B2 were found in domesticated animals and in humans from Jeonnam Province, South Korea. We also report that among the strains we examined, virulence genes were mainly found in phylogenetic group B1 strains isolated from beef cattle. Results of these studies may prove to be useful for the development of risk management strategies to maintain public health.