Accumulation of infectious mutants in stocks during the propagation of fiber-modified recombinant adenoviruses

In infected cells, replication errors during viral proliferation generate mutations in adenoviruses (Ads), and the mutant Ads proliferate and evolve in the intracellular environment. Genetically fiber-modified recombinant Ads (rAd variants) were generated, by modification of the fiber gene, for therapeutic applications in host cells that lack or express reduced levels of the Coxsackievirus and adenovirus receptor. To assess the genetic modifications of rAd variants that might induce the instability of Ad virions, we examined the frequencies of mutants that accumulated in propagated stocks. Seven of 41 lines of Ad variants generated mutants in the stocks and all mutants were infectious. Moreover, all the mutations occurred in the modified region that had been added at the 3' end of the fiber gene. Our results show that some genetic modifications at the carboxyl terminus of Ad fiber protein lead to the instability of Ad virions.     

Fetal cells in mother rats contribute to the remodeling of liver and kidney after injury

Fetal microchimerism indicates a mixture of cells of maternal and fetal origin seen in maternal tissues during and after pregnancy. Controversy exists about whether persistent fetal microchimerism is related with some autoimmune disorders occurring during and after pregnancy. In the current experiment, an animal model in which EGFP positive cells were taken as fetal-origin cells was designed to detect the fetal microchimerism in various maternal organs. Ethanol drinking and gentamicin injection were adopted to induce liver and kidney injury simultaneously. EGFP positive cells were engrafted not only in the maternal circulation and bone marrow, but also in the liver and kidney as hepatocytes and tubular cells, respectively. These results indicate that fetal cells are engrafted to maternal hematopoietic system without apparent injury and they also contribute to the repairing process of maternal liver and kidney.     

Emerging Antifungal Drug Resistance in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> and Among Other Species of <Emphasis Type="Italic">Aspergillus</Emphasis>

Purpose of Review

The purpose of this review is to give an overview of recent findings on antifungal resistance in Aspergillus fumigatus (the major causative agent of aspergillosis) and sibling Aspergillus species, which can be hidden agents of aspergillosis.

Recent Findings

Azole resistance by Cyp51A mutation in A. fumigatus is a growing problem worldwide. The resistance can occur in patients or in the environment. The former occurs by drug selection in the host, inducing mutations in Cyp51A. The latter is characterized by a tandem repeat in the promoter region of cyp51A gene and mutation(s) in Cyp51A. Environmental resistant strains are prevailing rapidly and globally. Moreover, efflux pump and biofilm formation are closely related with antifungal resistance of A. fumigatus. Finally, sibling species of Aspergillus are described with regard to antifungal resistance.

Summary

Environmental azole-resistant strains have newly emerged and been dispersed globally, and continuous survey and countermeasures are urgently needed against these strains. Although the contributions of Cyp51A and efflux pumps to antifungal resistance are becoming clear, other resistance mechanisms remain unclear. Further investigations including genome comparisons will help to clarify the novel resistant mechanisms and to develop countermeasures or novel antifungal drugs against resistant strains of A. fumigatus and other Aspergillus species that have low susceptibility to antifungal therapeutics.

     

Characterization of morphological conversion of Helicobacter pylori under anaerobic conditions

Helicobacter pylori (H. pylori), a gram‐negative microaerophilic bacterial pathogen that colonizes the stomachs of more than half of all humans, is linked to chronic gastritis, peptic ulcers and gastric cancer. Spiral‐shaped H. pylori undergo morphologic conversion to a viable but not culturable coccoid form when they transit from the microaerobic stomach into the anaerobic intestinal tract. However, little is known about the morphological and pathogenic characteristics of H. pylori under prolonged anaerobic conditions. In this study, scanning electron microscopy was used to document anaerobiosis‐induced morphological changes of H. pylori, from helical to coccoid to a newly defined fragmented form. Western blot analysis indicated that all three forms express certain pathogenic proteins, including the bacterial cytotoxin‐associated gene A (CagA), components of the cag‐Type IV secretion system (TFSS), the blood group antigen‐binding adhesin BabA, and UreA (an apoenzyme of urease), almost equally. Similar urease activities were also detected in all three forms of H. pylori. However, in contrast to the helical form, bacterial motility and TFSS activity were found to have been abrogated in the anaerobiosis‐induced coccoid and fragmented forms of H. pylori. Notably, it was demonstrated that some of the anaerobiosis‐induced fragmented state cells could be converted to proliferation‐competent helical bacteria in vitro. These results indicate that prolonged exposure to the anaerobic intestine may not eliminate the potential for H. pylori to revert to the helical pathogenic state.

     

Iron and fluorescent dissolved organic matter in an estuarine and coastal system in Japan

Terrestrially derived dissolved organic matter (DOM) plays an important role in providing ligands that regulate iron transport to coastal ecosystems. Fluorescent DOM (FDOM) is widely used to evaluate DOM behavior, but the relationship between iron and FDOM distributions in estuarine and coastal systems is poorly understood. In this study, we investigated the distributions of dissolved iron (DFe < 0.7-µm fraction) and FDOM as measured using fluorescence excitation-emission matrices in the Yura River–Tango Bay system. The DFe distribution was not closely correlated with that of the humic-like FDOM, indicating that humic-like FDOM is not the main regulator of the DFe concentration in this system. The relatively low fluorescence intensity of humic-like FDOM demonstrates that colloidal inorganic iron is the major component of riverine DFe. A large proportion of the riverine DFe (> 94%) was removed in the mixing process, but humic-like FDOM likely supports the transport of DFe to the coastal zone.     

Temperature Changes in Brown Adipocytes Detected with a Bimaterial Microcantilever

Mammalian cells must produce heat to maintain body temperature and support other biological activities. Methods to measure a cell’s thermogenic ability by inserting a thermometer into the cell or measuring the rate of oxygen consumption in a closed vessel can disturb its natural state. Here, we developed a noninvasive system for measuring a cell’s heat production with a bimaterial microcantilever. This method is suitable for investigating the heat-generating properties of cells in their native state, because changes in cell temperature can be measured from the bending of the microcantilever, without damaging the cell and restricting its supply of dissolved oxygen. Thus, we were able to measure increases in cell temperature of <1 K in a small number of murine brown adipocytes (n = 4–7 cells) stimulated with norepinephrine, and observed a slow increase in temperature over several hours. This long-term heat production suggests that, in addition to converting fatty acids into heat energy, brown adipocytes may also adjust protein expression to raise their own temperature, to generate more heat. We expect this bimaterial microcantilever system to prove useful for determining a cell’s state by measuring thermal characteristics.     

Mitochondrial inhibitor sodium azide inhibits the reorganization of mitochondria‐rich cytoplasm and the establishment of the anteroposterior axis in ascidian embryo

In ascidian eggs, cytoplasmic and cortical reorganization, previously called ooplasmic segregation, occurs in two phases during the first cell cycle. In the second phase of reorganization, the mitochondria‐rich cytoplasm (myoplasm) moves to the future posterior side, concurrent with sperm aster migration along the egg cortex. Although this reorganization is the critical step for establishing the anteroposterior axis, its molecular mechanism is not fully understood. In this study, we showed that low concentrations of the mitochondrial inhibitor sodium azide (NaN₃), which showed the low toxicity in sperm, inhibited the second phase of reorganization without the microtubule depolymerization. In the NaN₃‐treated embryo, the sperm aster was not attracted to the cortex and altered its migration pathway; therefore, the myoplasm remained at the vegetal pole. Consequently, the anteroposterior axis was not established. Another mitochondrial inhibitor, oligomycin, did not affect these processes. These results suggest that NaN₃ inhibits unknown molecules that are important for the second phase of reorganization. Identifying the target molecule of NaN₃ will lead to a molecular understanding of cytoplasmic and cortical reorganization.     

Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.