Relative fitness of near isogenic lines for melanic and typical forms of the oriental armyworm, Mythimna separata (Walker)

Melanism in the oriental armyworm Mythimna separata (Walker) is characterized by a black color in adults instead of the normal body color of off-white. Because inheritance of melanism in this moth follows Mendel's law, with the melanic allele (t) recessive to the normal allele (T) at a single autosomal locus, a backcrossing procedure was used to develop near isogenic lines (NILs) for melanic (NILs-tt) and typical forms (NILs-TT) of this moth. Melanic males (tt) and typical females (TT) were crossed in the laboratory, their progeny (F(1)) were interbred, and F(2) melanic males were backcrossed to typical female offspring of the parallel line. This cycle was repeated five times to create NILs for melanic and typical forms of this moth. The effects of the melanic allele on relative fitness were evaluated in terms of developmental and reproductive characteristics. Life tables of the NILs were constructed to determine net reproductive rate (R(0)). Results indicated that NILs-tt possessed apparent developmental and reproductive advantages, including shorter immature development time, higher larval and pupal survival, a shorter adult preoviposition period, and more eggs laid per female compared with the NILs-TT.     

Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics

Rad51 and Rad54 are key proteins that collaborate during homologous recombination. Rad51 forms a presynaptic filament with ATP and ssDNA active in homology search and DNA strand exchange, but the precise role of its ATPase activity is poorly understood. Rad54 is an ATP-dependent dsDNA motor protein that can dissociate Rad51 from dsDNA, the product complex of DNA strand exchange. Kinetic analysis of the budding yeast proteins revealed that the catalytic efficiency of the Rad54 ATPase was stimulated by partial filaments of wild-type and Rad51-K191R mutant protein on dsDNA, unambiguously demonstrating that the Rad54 ATPase activity is stimulated under these conditions. Experiments with Rad51-K191R as well as with wild-type Rad51-dsDNA filaments formed in the presence of ATP, ADP or ATP-γ-S showed that efficient Rad51 turnover from dsDNA requires both the Rad51 ATPase and the Rad54 ATPase activities. The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA. Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability. These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo.     

In vitro and in silico analysis reveals an efficient algorithm to predict the splicing consequences of mutations at the 5' splice sites

We have found that two previously reported exonic mutations in the PINK1 and PARK7 genes affect pre-mRNA splicing. To develop an algorithm to predict underestimated splicing consequences of exonic mutations at the 5′ splice site, we constructed and analyzed 31 minigenes carrying exonic splicing mutations and their derivatives. We also examined 189 249 U2-dependent 5′ splice sites of the entire human genome and found that a new variable, the SD-Score, which represents a common logarithm of the frequency of a specific 5′ splice site, efficiently predicts the splicing consequences of these minigenes. We also employed the information contents (R_i) to improve the prediction accuracy. We validated our algorithm by analyzing 32 additional minigenes as well as 179 previously reported splicing mutations. The SD-Score algorithm predicted aberrant splicings in 198 of 204 sites (sensitivity = 97.1%) and normal splicings in 36 of 38 sites (specificity = 94.7%). Simulation of all possible exonic mutations at positions −3, −2 and −1 of the 189 249 sites predicts that 37.8, 88.8 and 96.8% of these mutations would affect pre-mRNA splicing, respectively. We propose that the SD-Score algorithm is a practical tool to predict splicing consequences of mutations affecting the 5′ splice site.     

Quantitative detection of BCR–ABL fusion gene and its application in monitoring chronic myeloid leukemia treatment

The BCR–ABL fusion gene in chromosome translocation, t (9; 22), and its product, p210BCR/ABL oncogenic tyrosine kinase, is the underlying molecular mechanism that leads to the development of CML. Quantitative detection of BCR–ABL fusion gene has become a reliable approach to diagnose and monitor CML. The aim of this study was to evaluate a Roche t (9; 22) kit in CML diagnosis, monitoring treatment responses, and identification of relapse. Using BCR–ABL fusion gene-expressing K562 cells, a series of standard samples were prepared and used to establish a curve for the calculation of BCR–ABL fusion gene expression in patient samples. Our results indicate that PCR detection system with aforementioned kit has good reproducibility. In addition, the relative concentration of BCR–ABL measured by PCR was in agreement with the patient’s response to the Imatinib treatment and bone marrow morphology remission. Furthermore, we found that the relative concentration of BCR–ABL fusion gene increased 1–3 months before CML relapse was clinically and cytogenetically diagnosed, suggesting that the PCR-based BCR–ABL fusion gene detection with t (9; 22) kit is able to diagnose the recurrence of CML at least 1 month earlier than the classic cytogenetic analysis. In conclusion, detection of BCR–ABL fusion gene expression in CML using Roche t (9; 22) kit has great clinical value in the primary diagnosis, monitoring treatment responses, and identification of relapse in CML patients.     

KPC1 expression and essential role after acute spinal cord injury in adult rat

KPC1 (Kip1 ubiquitylation-promoting complex 1) is the catalytic subunit of the ubiquitin ligase KPC, which regulates the degradation of the cyclin-dependent kinase inhibitor p27^kip1 at the G1 phase of the cell cycle. To elucidate the expression and role of KPC1 in nervous system lesion and repair, we performed an acute spinal cord contusion injury (SCI) model in adult rats. Western blot analysis showed a significant up-regulation of KPC1 and a concomitant down-regulation of p27^kip1 following spinal injury. Immunohistochemistry and immunofluorescence revealed wide expression of KPC1 in the spinal cord, including expression in neurons and astrocytes. After injury, KPC1 expression was increased predominantly in astrocytes, which highly expressed PCNA, a marker for proliferating cells. Co-immunoprecipitation demonstrated increased interactions between p27^kip1 and KPC1 4 days after injury. To understand whether KPC1 plays a role in astrocyte proliferation, we applied LPS to induce astrocyte proliferation in vitro. Western blot analysis demonstrated that p27^kip1 expression was negatively correlated with KPC1 expression following LPS stimulation. Immunofluorescence analysis showed subcellular localizations of p27^kip1 and KPC1 were also changed following the stimulation of astrocytes with LPS. These results suggest that KPC1 is related to the down-regulation of p27^kip1; this event may be involved in the proliferation of astrocytes after SCI.     

Rho kinase inhibitors stimulate the migration of human cultured osteoblastic cells by regulating actomyosin activity

We investigated the effects of Rho-associated kinase (ROCK) on migration and cytoskeletal organization in primary human osteoblasts and Saos-2 human osteosarcoma cells. Both cell types were exposed to two different ROCK inhibitors, Y-27632 and HA-1077. In the improved motility assay used in the present study, Y-27632 and HA-1077 significantly increased the migration of both osteoblasts and osteosarcoma cells on plastic in a dose-dependent and reversible manner. Fluorescent images showed that cells of both types cultured with Y-27632 or HA-1077 exhibited a stellate appearance, with poor assembly of stress fibers and focal contacts. Western blotting showed that ROCK inhibitors reduced myosin light chain (MLC) phosphorylation within 5 min without affecting overall myosin light-chain protein levels. Inhibition of ROCK activity is thought to enhance the migration of human osteoblasts through reorganization of the actin cytoskeleton and regulation of myosin activity. ROCK inhibitors may be potentially useful as anabolic agents to enhance the biocompatibility of bone and joint prostheses.     

Plk1 Self-Organization and Priming Phosphorylation of HsCYK-4 at the Spindle Midzone Regulate the Onset of Division in Human Cells

Animal cells initiate cytokinesis in parallel with anaphase onset, when an actomyosin ring assembles and constricts through localized activation of the small GTPase RhoA, giving rise to a cleavage furrow. Furrow formation relies on positional cues provided by anaphase spindle microtubules (MTs), but how such cues are generated remains unclear. Using chemical genetics to achieve both temporal and spatial control, we show that the self-organized delivery of Polo-like kinase 1 (Plk1) to the midzone and its local phosphorylation of a MT-bound substrate are critical for generating this furrow-inducing signal. When Plk1 was active but unable to target itself to this equatorial landmark, both cortical RhoA recruitment and furrow induction failed to occur, thus recapitulating the effects of anaphase-specific Plk1 inhibition. Using tandem mass spectrometry and phosphospecific antibodies, we found that Plk1 binds and directly phosphorylates the HsCYK-4 subunit of centralspindlin (also known as MgcRacGAP) at the midzone. At serine 157, this modification creates a major docking site for the tandem BRCT repeats of the Rho GTP exchange factor Ect2. Cells expressing only a nonphosphorylatable form of HsCYK-4 failed to localize Ect2 at the midzone and were severely impaired in cleavage furrow formation, implying that HsCYK-4 is Plk1's rate-limiting target upstream of RhoA. Conversely, tethering an inhibitor-resistant allele of Plk1 to HsCYK-4 allowed furrows to form despite global inhibition of all other Plk1 molecules in the cell. Our findings illuminate two key mechanisms governing the initiation of cytokinesis in human cells and illustrate the power of chemical genetics to probe such regulation both in time and space.     

Enhanced chilling tolerance in Zoysia matrella by pre-treatment with salicylic acid, calcium chloride, hydrogen peroxide or 6-benzylaminopurine

Following leaf application of salicylic acid (SA), calcium chloride, hydrogen peroxide and 6-benzylaminopurine (BA), Manila grass (Zoysia matrella) plants were exposed to day/night temperature of 7/2 °C for 120 h in a growth chamber. The lower content of malondialdehyde (MDA) and H₂O₂ and higher activities of ascorbate peroxidase (APX), guaiacol peroxidase (POD) and catalase (CAT) during exposure to low temperature in pre-treated plants in comparison with control plants demonstrated that these compounds improved the chilling tolerance of Manila grass.     

葛根素对糖尿病心肌细胞的保护及其机制研究

观察葛根素（Puerarin）对链脲佐菌素（streptozotocin,STZ）诱导的糖尿病大鼠心肌细胞的保护作用,并探讨血小板反应素1（Thrombospondin-1,TSP-1）的表达改变及其作用。雄性SD大鼠45只随机分为三组（n=15）：糖尿病组和葛根素治疗组采用一次腹腔注射链脲佐菌素（STZ）65mg／kg制备糖尿病模型,其中葛根素治疗组于造模后葛根素腹腔注射4周（100mg/kg/day）,正常对照组仅腹腔注射等量生理盐水（6ml／kg）,同样喂养4周。四周后各组大鼠处死。H—E染色及透射电子显微镜观察三组大鼠心肌细胞纤维显微结构和超微结构的病理改变．免疫组化和实时荧光定量PCR法观察大鼠心肌细胞中TSP-1蛋白和mRNA表达的变化．同时利用Langendorff离体心脏灌流法测定各组大鼠心室肌细胞功能。结果发现葛根素治疗组较糖尿病组大鼠的体重增加明显,同时血糖下降,有显著性差异（P〈0．01）。H—E染色显示糖尿病大鼠多处心肌肌丝紊乱伴少量炎症细胞浸润,电镜下发现有线粒体嵴消失溶解,肌丝排列紊乱等病理改变,而葛根素治疗组大鼠偶见上述病理变化。免疫组化显示葛根素治疗组心肌内TSP-1阳性细胞密度小于糖尿病大鼠,TSP-1 mRNA表达也比糖尿病大鼠要低。此外葛根素治疗组大鼠的左室收缩末压（LVSEP）、左心室舒张末期压（LVEDP）等心功能指标均明显低于正常组（P〈0．01）,但较糖尿病组有显著改善（P〈0．01）。上述结果显示葛根素能保护糖尿病大鼠心肌细胞的高糖损伤和维持心室肌细胞的功能,而该机制可能与抑制心肌细胞TSP-1表达的水平有关。     

Higher-level salamander relationships and divergence dates inferred from complete mitochondrial genomes

Phylogenetic relationships among the salamander families have been difficult to resolve, largely because the window of time in which major lineages diverged was very short relative to the subsequently long evolutionary history of each family. We present seven new complete mitochondrial genomes representing five salamander families that have no or few mitogenome records in GenBank in order to assess the phylogenetic relationships of all salamander families from a mitogenomic perspective. Phylogenetic analyses of two data sets—one combining the entire mitogenome sequence except for the D-loop, and the other combining the deduced amino acid sequences of all 13 mitochondrial protein-coding genes—produce nearly identical well-resolved topologies. The monophyly of each family is supported, including the controversial Proteidae. The internally fertilizing salamanders are demonstrated to be a clade, concordant with recent results using nuclear genes. The internally fertilizing salamanders include two well-supported clades: one is composed of Ambystomatidae, Dicamptodontidae, and Salamandridae, the other Proteidae, Rhyacotritonidae, Amphiumidae, and Plethodontidae. In contrast to results from nuclear loci, our results support the conventional morphological hypothesis that Sirenidae is the sister-group to all other salamanders and they statistically reject the hypothesis from nuclear genes that the suborder Cryptobranchoidea (Cryptobranchidae + Hynobiidae) branched earlier than the Sirenidae. Using recently recommended fossil calibration points and a “soft bound” calibration strategy, we recalculated evolutionary timescales for tetrapods with an emphasis on living salamanders, under a Bayesian framework with and without a rate-autocorrelation assumption. Our dating results indicate: (i) the widely used rate-autocorrelation assumption in relaxed clock analyses is problematic and the accuracy of molecular dating for early lissamphibian evolution is questionable; (ii) the initial diversification of living amphibians occurred later than recent estimates would suggest, from the Late Carboniferous to the Early Permian (294 MYA); (iii) living salamanders originated during the Early Jurassic (183 MYA), and (iv) most salamander families had diverged from each other by Late Cretaceous. A likelihood-based ancestral area reconstruction analysis favors a distribution throughout Laurasia in the Early Jurassic for the common ancestor of all living salamanders.