Cave insects with sex‐reversed genitalia had their most recent common ancestor in West Gondwana (Psocodea: Prionoglarididae: Speleketorinae)

The divergence date and ancestral distributional area of the psocid subfamily Speleketorinae, which includes taxa with reversed genitalia (female penis and male vagina of Afrotrogla and Neotrogla, tribe Sensitibillini), were estimated. The most basal divergence of the subfamily (between the North American Speleketor and the tribe Sensitibillini) was estimated to have occurred according to the separation between the North American continent and Gondwana, ca. 175 Ma. The most basal divergence of Sensitibillini (between African Afrotrogla + Sensitibilla and Brazilian Neotrogla) was estimated to have occurred according to the split of West Gondwana (separation between the African and South American continents), ca. 127 Ma. The biome of the ancestral distributional area of Sensitibillini (inland of West Gondwana) is believed to be arid to semi‐arid, which might strengthen the reversed sexual selection and then facilitate the origin of preadaptive features related to the evolution of a female penis. All extant Sensitibillini species inhabit carbonatic caves, but geological evidence suggested independent shifts of these genera to the carbonatic cave habitat in the Tertiary/Quaternary.     

Electrochemical screening of recombinant protein solubility in Escherichia coli using scanning electrochemical microscopy (SECM)

A microbial array chip with collagen gel spots entrapping living Escherichia coli (E. coli) DH5alpha was applied for the screening of recombinant protein solubilities. The alpha-fragment of beta-galactosidase (betaGal) was fused to the target protein, namely, maltose-binding protein (MBP), to monitor the solubility of MBP. Scanning electrochemical microscopy (SECM) was used to detect the release of p-aminophenol from E. coli cells catalyzed by intracellular betaGal. Comparison of the SECM-based method with the Western blotting-based method indicated that the current response obtained using SECM increased with an increase in the betaGal activity and therefore, with the soluble fraction of MBP in the host cells.     

alpha-Tomatine, the major saponin in tomato, induces programmed cell death mediated by reactive oxygen species in the fungal pathogen Fusarium oxysporum

The tomato saponin alpha-tomatine has been proposed to kill sensitive cells by binding to cell membranes followed by leakage of cell components. However, details of the modes of action of the compound on fungal cells are poorly understood. In the present study, mechanisms involved in alpha-tomatine-induced cell death of fungi were examined using a filamentous pathogenic fungus Fusarium oxysporum. alpha-Tomatine-induced cell death of F. oxysporum (TICDF) occurred only under aerobic conditions and was blocked by the mitochondrial F(0)F(1)-ATPase inhibitor oligomycin, the caspase inhibitor D-VAD-fmk, and protein synthesis inhibitor cycloheximide. Fungal cells exposed to alpha-tomatine showed TUNEL-positive nuclei, depolarization of transmembrane potential of mitochondria, and reactive oxygen species (ROS) accumulation. These results suggest that TICDF occurs through a programmed cell death process in which mitochondria play a pivotal role. Pharmacological studies using inhibitors suggest that alpha-tomatine activates phosphotyrosine kinase and monomeric G-protein signaling pathways leading to Ca(2+) elevation and ROS burst in F. oxysporum cells.     

Pathogenic mitochondrial DNA-induced respiration defects in hematopoietic cells result in anemia by suppressing erythroid differentiation

Anemia is a symptom in patients with Pearson syndrome caused by the accumulation of mutated mitochondrial DNA (mtDNA). Such mutated mtDNAs have been detected in patients with anemia. This suggested that respiration defects due to mutated mtDNA are responsible for the anemia. However, there has been no convincing experimental evidence to confirm the pathophysiological relation between respiration defects in hematopoietic cells and expression of anemia. We address this issue by transplanting bone marrow cells carrying pathogenic mtDNA with a large-scale deletion (ΔmtDNA) into normal mice. The bone marrow-transplanted mice carried high proportion of ΔmtDNA only in hematopoietic cells, and resultant the mice suffered from macrocytic anemia. They show abnormalities of erythroid differentiation and weak erythropoietic response to a stressful condition. These observations suggest that hematopoietic cell-specific respiration defects caused by mtDNAs with pathogenic mutations are responsible for anemia by inducing abnormalities in erythropoiesis.     

Time course of vocal modulation during isolation in common marmosets (Callithrix jacchus)

Common marmosets vocalize phee calls as isolation calls, which seem to facilitate their reunion with family groups. To identify multiple acoustic properties with different time courses, we examined acoustic modulations of phees during different social contexts of isolation. Subject marmosets were totally isolated in one condition, were visually isolated and could exchange vocalizations in another condition, and were visually isolated and subsequently totally isolated in a third condition. We recorded 6,035 phees of 10 male–female marmoset pairs and conducted acoustic analysis. The marmosets frequently vocalized phees that were temporally elongated and louder during isolation, with varying time courses of these changes in acoustic parameters. The vocal rates and sound levels of the phees increased as soon as the marmosets saw their pair mates being taken away, and then gradually calmed down. The phee duration was longer in conditions during which there were no vocal responses from their pair mates. Louder vocalizations are conspicuous and seem to be effective for long‐distance transmission, whereas shorter call duration during vocal exchanges might avoid possible vocal overlap between mates. Am. J. Primatol. 72:681–688, 2010. © 2010 Wiley‐Liss, Inc.     

Cell cycle-dependent proteolysis and phosphorylation of human Mcm10

Mcm10 (Dna43) is an essential protein for chromosomal DNA replication in Saccharomyces cerevisiae. Recently, we identified a human Mcm10 homolog that interacts with the mammalian Orc2 and Mcm2-7 complex. We additionally demonstrated that human Mcm10 binds nuclease-resistant nuclear structures during S phase and dissociates from them in G(2) phase. In this study, we have further characterized the subcellular localization, modification, and expression levels of human Mcm10 protein throughout the cell cycle. Human Mcm10 protein decreased in late M phase, remained low during G(1) phase, started to accumulate, and bound chromatin at the onset of S phase. Proteasome inhibitors stabilized Mcm10 levels, suggesting that proteolysis is involved in the down-regulation of the protein in late M/G(1) phase. Dissociation of Mcm10 from chromatin in G(2)/M phase was concomitant with alterations in the electrophoretic mobility of the protein. Treatment with lambda phosphatase revealed that mobility shifts were due to hyperphosphorylation. These results indicate that human Mcm10 is regulated by proteolysis and phosphorylation in a cell cycle-dependent manner. It is further suggested that mammalian Mcm10 is involved in S phase progression, and not the formation of a prereplicative complex, as previously proposed from data on the S. cerevisiae protein.     

Leukotriene B4: metabolism and signal transduction

Leukotriene B4 (LTB4) is known as one of the most potent chemoattractants and activators of leukocytes and is involved in inflammatory diseases. Enzymes involved in the biosynthesis and metabolism of LTB4 have been cloned, and their properties are well understood. Two G-protein-coupled receptors (BLT1 and BLT2) have been cloned and characterized. BLT1 and BLT2 are high- and low-affinity LTB4 receptors, respectively, and form a gene cluster in human and mouse. In this article recent findings on the metabolism of and the receptors for LTB4 are reviewed. We also discuss briefly a coreceptor role of BLT in HIV infection, and ion channel modification by LTB4.     

Increase in the molecular weight and radius of gyration of apocalmodulin induced by binding of target peptide: evidence for complex formation

Small-angle X-ray scattering was used to investigate a complex state of apocalmodulin induced by the binding of a Ca(2+)/calmodulin-dependent protein kinase IV calmodulin target site. Upon binding of the peptide, the molecular weight for apocalmodulin increased by 8.4%, which provides direct evidence for the formation of a calmodulin/target peptide complex. Comparison of the radius of gyration and Kratky plots of the apocalmodulin/peptide complex with those of apocalmodulin indicates that the overall conformation remains unchanged but the flexibility of the central linker decreases. An analysis of residue pairs between calmodulin and the target peptides suggests that the complex formation is induced by electrostatic interactions and subsequent van der Waals interactions.     

Homogeneous tetracycline-regulatable gene expression in mammalian fibroblasts

The expression of transfected genes in mammalian cells is rapidly repressed by epigenetic mechanisms such that, within a matter of weeks, only a fraction of the cells in most clonal populations still exhibit detectable expression. This problem can become prohibitive when one wants to express two ectopically introduced genes, as is necessary to establish cell lines that harbor genes regulated by the tetracycline-controlled transactivators. We describe an approach to establish Chinese hamster ovary (CHO) cell lines that stably induce a tet-responsive reporter gene in all cells of a transfected clonal population. Screening of more than 100 colonies resulting from a standard co-transfection of the tetracycline transactivator (tTA) with a green fluorescent protein (GFP) reporter plasmid failed to identify a single colony that could induce GFP in more than 20% of cells. The presence of chromatin insulator sequences, previously shown to protect some transfected genes from epigenetic silencing, moderately improved stability but was not sufficient to produce homogeneous transformants. However, when cell lines were first established in which selection could be maintained either for the expression of tTA activity (co-transfection with a tTA-responsive selectable marker) or the presence of tTA mRNA (bicistronic message encoding a selectable marker), these cell lines could be subsequently transfected with the GFP reporter construct, and nearly 10% of the resulting colonies exhibited stable homogeneous tet-responsive GFP expression in 100% of the expanded clonal cell population.