Multiple infection with Wolbachia inducing different reproductive manipulations in the butterfly Eurema hecabe

Wolbachia are rickettsial intracellular symbionts of arthropods and nematodes. In arthropods, they act as selfish genetic elements and manipulate host reproduction, including sex-ratio distortion and cytoplasmic incompatibility (CI). Previous studies showed that infection of feminizing Wolbachia and CI Wolbachia sympatrically occurred in the butterfly Eurema hecabe. We demonstrate that feminization-infecting individuals can rescue sperm modified by CI-infecting males. Phylogenetic analysis revealed that feminized individuals are infected with two distinct Wolbachia strains: one is shared with CI-inducing matrilines, and the other is only found in feminized matrilines. Therefore, the simultaneous double manipulation, CI rescue and feminization, is caused by different Wolbachia strains in feminized individuals, not by a single Wolbachia with two functions. This is the first finding of double infection of Wolbachia with different reproductive manipulations.     

Efficient asymmetric syntheses, determination of absolute configurations and biological activities of 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine as a novel potent dopamine uptake inhibitor in the central nervous system

An efficient asymmetric synthesis of the chiral N-(3-chloro-2-hydroxypropyl)anilines (2a and 2b) was achieved through the regioselective ring-opening reaction of chiral epichlorohydrin with aniline. This was applied to an asymmetric synthesis of the enantiomers of 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine 1 as a novel potent dopamine uptake inhibitor. Both enantiomers as trihydrochlorides, 4a.3HCl and 4b.3HCl, could be synthesized in good total yields and optical purities of 100% ee in three steps synthesis, respectively. The absolute configurations of 4a.3HCl and 4b.3HCl were determined using the modified Mosher's method with the related compounds, the intermediates (2a and 2b) and the free bases (4a and 4b). The analytical results indicated that 4a.3HCl and 4b.3HCl have the (S)- and (R)-configuration, respectively, and a series of reactions to provide them proceeded without the apparent influence on the stereochemistry at the chiral centers. In in vitro pharmacological evaluations, 4a.3HCl and 4b.3HCl showed potent dopamine transporter binding affinities, high dopamine, moderate serotonin, and weak norepinephrine uptake inhibitory activities, and 4a.3HCl exhibited a more potent and selective dopamine uptake inhibition over the serotonin or norepinephrine uptake inhibition as compared with 4b.3HCl. An ex vivo evaluation revealed that the oral administrations of both enantiomers at a dose of 30 mg/kg in rats displayed apparent dopamine uptake inhibitory activities and 4a.3HCl had a stronger tendency to inhibit dopamine uptake compared with 4b.3HCl.     

Pegylated interferon-alpha protects type 1 pneumocytes against SARS coronavirus infection in macaques

The primary cause of severe acute respiratory syndrome (SARS) is a newly discovered coronavirus. Replication of this SARS coronavirus (SCV) occurs mainly in the lower respiratory tract, and causes diffuse alveolar damage. Lack of understanding of the pathogenesis of SARS has prevented the rational development of a therapy against this disease. Here we show extensive SCV antigen expression in type 1 pneumocytes of experimentally infected cynomolgus macaques (Macaca fascicularis) at 4 d postinfection (d.p.i.), indicating that this cell type is the primary target for SCV infection early in the disease, and explaining the subsequent pulmonary damage. We also show that prophylactic treatment of SCV-infected macaques with the antiviral agent pegylated interferon-alpha (IFN-alpha) significantly reduces viral replication and excretion, viral antigen expression by type 1 pneumocytes and pulmonary damage, compared with untreated macaques. Postexposure treatment with pegylated IFN-alpha yielded intermediate results. We therefore suggest that pegylated IFN-alpha protects type 1 pneumocytes from SCV infection, and should be considered a candidate drug for SARS therapy.     

Role of MMPs and plasminogen activators in angiogenesis after transmyocardial laser revascularization in dogs

We examined the role of matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs), and plasminogen activator (PA) in transmyocardial laser revascularization (TMLR)-induced angiogenesis. TMLR was accomplished with a carbon dioxide laser in seven dogs whose left anterior descending coronary artery (LAD) was ligated. Seven control dogs underwent only LAD ligation, and four dogs underwent a sham operation, consisting only of a left thoracotomy. Two weeks later, transmural myocardial samples were harvested from the distributions of the LAD and the left circumflex artery for substrate zymography, immunohistochemical staining, and in situ zymography. MMP-1, MMP-2, TIMP-1, TIMP-2, and urokinase-type PA levels in the distribution of the LAD were higher in the laser group than in the control or sham group. Counts of von Willebrand factor-positive microvessels and smooth muscle alpha-actin-positive arterioles demonstrated that the angiogenesis and ateriogenesis was promoted in the laser group and correlated directly with the number of MMP-stained microvessels. We conclude that TMLR induces the expression of MMPs, TIMPs, and urokinase-type PA and that these proteinases play an important role in angiogenesis after TMLR.     

An oxidized purine nucleoside triphosphatase,MTH1, suppresses cell death caused by oxidative stress

MTH1 hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP) and thus protects cells from damage caused by their misincorporation into DNA. In the present study, we established MTH1-null mouse embryo fibroblasts that were highly susceptible to cell dysfunction and death caused by exposure to H2O2, with morphological features of pyknosis and electron-dense deposits accumulated in mitochondria. The cell death observed was independent of both poly(ADP-ribose) polymerase and caspases. A high performance liquid chromatography tandem mass spectrometry analysis and immunofluorescence microscopy revealed a continuous accumulation of 8-oxo-guanine both in nuclear and mitochondrial DNA after exposure to H2O2. All of the H2O2-induced alterations observed in MTH1-null mouse embryo fibroblasts were effectively suppressed by the expression of wild type human MTH1 (hMTH1), whereas they were only partially suppressed by the expression of mutant hMTH1 defective in either 8-oxo-dGTPase or 2-OH-dATPase activity. Human MTH1 thus protects cells from H2O2-induced cell dysfunction and death by hydrolyzing oxidized purine nucleotides including 8-oxo-dGTP and 2-OH-dATP, and these alterations may be partly attributed to a mitochondrial dysfunction.     

Origin of higher affinity to RNA of the N-terminal RNA-binding domain than that of the C-terminal one of a mouse neural protein,musashi1, as revealed by comparison of their structures,modes of interaction,surface electrostatic potentials,and backbone dynamics

Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in maintenance of the character of progenitor cells. Musashi1 contains two ribonucleoprotein-type RNA-binding domains (RBDs), RBD1 and RBD2, the affinity to RNA of RBD1 being much higher than that of RBD2. We previously reported the structure and mode of interaction with RNA of RBD2. Here, we have determined the structure and mode of interaction with RNA of RBD1. We have also analyzed the surface electrostatic potential and backbone dynamics of both RBDs. The two RBDs exhibit the same ribo-nucleoprotein-type fold and commonly make contact with RNA on the beta-sheet side. On the other hand, there is a remarkable difference in surface electrostatic potential, the beta-sheet of RBD1 being positively charged, which is favorable for binding negatively charged RNA, but that of RBD2 being almost neutral. There is also a difference in backbone dynamics, the central portion of the beta-sheet of RBD1 being flexible, but that of RBD2 not being flexible. The flexibility of RBD1 may be utilized in the recognition process to facilitate an induced fit. Thus, comparative studies have revealed the origin of the higher affinity of RBD1 than that of RBD2 and indicated that the affinity of an RBD to RNA is not governed by its fold alone but is also determined by its surface electrostatic potential and/or backbone dynamics. The biological role of RBD2 with lower affinity is also discussed.