Genomic Analysis of Mycobacterium abscessus Strain M139, Which Has an Ambiguous Subspecies Taxonomic Position

Mycobacterium abscessus is a ubiquitous, rapidly growing species of nontuberculous mycobacteria that colonizes organic surfaces and is frequently associated with opportunistic infections in humans. We report here the draft genome sequence of Mycobacterium abscessus strain M139, which shows genomic features reported to be characteristic of both Mycobacterium abscessus subsp. abscessus and Mycobacterium abscessus subsp. massiliense.     

DAAM1 Is a Formin Required for Centrosome Re-Orientation during Cell Migration

Background

Disheveled-associated activator of morphogenesis 1 (DAAM1) is a formin acting downstream of Wnt signaling that is important for planar cell polarity. It has been shown to promote proper cell polarization during embryonic development in both Xenopus and Drosophila. Importantly, DAAM1 binds to Disheveled (Dvl) and thus functions downstream of the Frizzled receptors. Little is known of how DAAM1 is localized and functions in mammalian cells. We investigate here how DAAM1 affects migration and polarization of cultured cells and conclude that it plays a key role in centrosome polarity.

Methodology/Principal Findings

Using a specific antibody to DAAM1, we find that the protein localizes to the acto-myosin system and co-localizes with ventral myosin IIB-containing actin stress fibers. These fibers are particularly evident in the sub-nuclear region. An N-terminal region of DAAM1 is responsible for this targeting and the DAAM1(1-440) protein can interact with myosin IIB fibers independently of either F-actin or RhoA binding. We also demonstrate that DAAM1 depletion inhibits Golgi reorientation in wound healing assays. Wound-edge cells exhibit multiple protrusions characteristic of unpolarized cell migration. Finally, in U2OS cells lines stably expressing DAAM1, we observe an enhanced myosin IIB stress fiber network which opposes cell migration.

Conclusions/Significance

This work highlights the importance of DAAM1 in processes underlying cell polarity and suggests that it acts in part by affecting the function of acto-myosin IIB system. It also emphasizes the importance of the N-terminal half of DAAM1. DAAM1 depletion strongly blocks centrosomal re-polarization, supporting the concept that DAAM1 signaling cooperates with the established Cdc42 associated polarity complex. These findings are also consistent with the observation that ablation of myosin IIB but not myosin IIA results in polarity defects downstream of Wnt signaling. The structure-function analysis of DAAM1 in cultured cells parallels more complex morphological events in the developing embryo.     

Reduction of PrPC in human cerebrospinal fluid after spinal cord injury

It has been estimated that cerebrospinal fluid (CS F) contains approximately 80 proteins that significantly increase or decrease in response to various clinical conditions. Here we have evaluated the CS F protein PrP^C (cellular prion protein) for possible increases or decreases following spinal cord injury. The physiological function of PrP^C is not yet completely understood; however, recent findings suggest that PrP^C may have neuroprotective properties. Our results show that CS F PrP^C is decreased in spinal cord injured patients 12 h following injury and is absent at 7 days. Given that normal PrP^C has been proposed to be neuroprotective, we speculate that the decrease in CS F PrP^C levels may influence neuronal cell survival following spinal cord injury.Key words: CSF, PrP^C, Hsp25, crystallin domain, spinal cord injury     

PP2A1 binding, cell transducing and apoptotic properties of Vpr(77-92): a new functional domain of HIV-1 Vpr proteins

Background

The hallmark of HIV-1 pathogenesis is the progressive CD4⁺ T cell depletion and high propensity of CD4⁺ T cells to apoptosis. HIV-1 viral protein R (Vpr) is a major pro-apoptotic gene product. A first Vpr-mediated apoptotic mechanism that requires a physical interaction of HIV-1 Vpr_71-82 mitochondriotoxic domain containing the conserved sequence _71-HFRIGCRHSRIG_-82 with the Adenine Nucleotide Translocator (ANT) has been characterized. The family of Ser/Thr protein phosphatase PP2A interacts with several viral proteins to regulate cell growth and apoptotic pathways. Previous studies based on yeast two hybrid assays and mutational experiments indicated that PP2A₁ is involved in the induction of G2 arrest by HIV-1 Vpr.

Principal Findings

Experiments combining pull-down, cell penetration and apoptosis analyses in distinct human cells indicate that the PP2A₁ binding sequence from Vpr_77–92 is a new cell penetrating apoptotic sequence. We also found that the I84P mutation or the IIQ/VTR_83–85 and T89A substitutions in the Vpr_77–92 sequence prevent PP2A₁ binding, cell penetration and apoptosis. In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr_71–82 domain, has no effect on the biological properties of the Vpr_77–92 domain.

Conclusion

Together our data provide evidence for the first time that the Vpr_77–92 sequence delineates a biological active domain of Vpr with PP2A₁ binding and pro-apopototic capacities and, it is conceivable that this cell penetrating sequence may account for the Vpr internalization in uninfected cells. Finally, our data also implicate the existence of two partially overlapping pro-apoptotic domains in the Vpr C-terminal part, a redundancy that represents a new approach to address the question of biological relevance of HIV-1 Vpr. In this context, future studies will be required to determine the functional relevance of the Vpr_77–92 domain in full length Vpr protein and also in entire HIV provirus.     

Substance P upregulates cyclooxygenase-2 and prostaglandin E metabolite by activating ERK1/2 and NF-kappaB in a mouse model of burn-induced remote acute lung injury

Acute lung injury (ALI) is a major cause of mortality in burn patients, even without direct inhalational injury. Identification of early mediators that instigate ALI after burn and of the molecular mechanisms by which they work are of high importance but remain poorly understood. We previously reported that an endogenous neuropeptide, substance P (SP), via binding neurokinin-1 receptor (NK1R), heightens remote ALI early after severe local burn. In this study, we examined the downstream signaling pathway following SP-NK1R coupling that leads to remote ALI after burn. A 30% total body surface area full-thickness burn was induced in male BALB/c wild-type (WT) mice, preprotachykinin-A (PPT-A) gene-deficient mice, which encode for SP, and PPT-A(-/-) mice challenged with exogenous SP. Local burn injury induced excessive SP-NK1R signaling, which activated ERK1/2 and NF-κB, leading to significant upregulation of cyclooxygenase (COX)-2, PGE metabolite, and remote ALI. Notably, lung COX-2 levels were abrogated in burn-injured WT mice by L703606, PD98059, and Bay 11-7082, which are specific NK1R, MEK-1, and NF-κB antagonists, respectively. Additionally, burn-injured PPT-A(-/-) mice showed suppressed lung COX-2 levels, whereas PPT-A(-/-) mice injected with SP showed augmented COX-2 levels postburn, and administration of PD98059 and Bay 11-7082 to burn-injured PPT-A(-/-) mice injected with SP abolished the COX-2 levels. Furthermore, treatment with parecoxib, a selective COX-2 inhibitor, attenuated proinflammatory cytokines, chemokines, and ALI in burn-injured WT mice and PPT-A(-/-) mice injected with SP. To our knowledge, we show for the first time that SP-NK1R signaling markedly elevates COX-2 activity via ERK1/2 and NF-κB, leading to remote ALI after burn.     

LOW GENETIC VARIABILITY OF SARGASSUM MUTICUM (PHAEOPHYCEAE) REVEALED BY A GLOBAL ANALYSIS OF NATIVE AND INTRODUCED POPULATIONS1

Sargassum muticum (Yendo) Fensholt is one of the most well‐known invasive species in the world. There have, however, been few genetic investigations on both its introduced and native populations. There are also some questions about the taxonomic status of this species. This study is the first to assess the genetic diversity of S. muticum on a global scale, by utilizing one marker each from the extranuclear genomes, namely, plastidial RUBISCO and mitochondrial TrnW_I spacers, as well as the nuclear internal transcribed spacer 2 (ITS2). Based on the markers investigated, both the invasive as well as the native populations of this species appeared very homogenous, when compared with other invasive and brown macroalgae. No variation in ITS2 and RUBISCO spacer was revealed in S. muticum populations, including those from its native ranges in Asia and the introduced ranges in Europe and North America. Two TrnW_I spacer haplotypes with a fixed two‐nucleotide difference were found between the populations of eastern Japan and the other 15 populations examined. This study confirms that there is no cryptic diversity in the introduced range of this species. All the materials collected globally are indeed S. muticum. Results depicting the distribution range of the two TrnW_I spacer haplotypes also support the earlier suggestion that the source of the introduced S. muticum populations is most likely western and central Japan (Seto Inland Sea), where the germlings of S. muticum were likely to have been transported with the Pacific oysters previously introduced for farming in Canada, UK, and France in earlier years.     

Antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine mastitis between 2003 and 2008 in Korea

A total of 1,444 coagulase-negative staphylococci (CNS) isolates from bovine mastitic milk samples collected during 2003-2008 in Korea were identified to the species level. Of 14 species identified, S. simulans, S. haemolyticus, and S. sciuri accounted for over 60% of the isolates. All the CNS isolates were tested for susceptibility to eight antimicrobials commonly used in dairy cattle. With a few exceptions, similar resistance patterns were observed among the CNS species: penicillin and ampicillin showed the lowest activity while amikacin, cephalothin, and gentamycin were highly effective. About 39% (557/1,444) of the CNS isolates were pan-susceptible, while 12% (175/1,444) showed resistance to four or more antimicrobials tested.     

Behaviors of ATP‐dependent chromatin remodeling factors during maturation of bovine oocytes in vitro

The mammalian oocyte undergoes dynamic changes in chromatin structure to reach complete maturation. However, little known is about behaviors of ATP‐dependent chromatin remodeling factors (ACRFs) during meiosis. Here, we found that respective ACRFs may differently behave in the process of oocyte maturation in the bovine. All ACRFs interacted with oocytic chromatin at the germinal vesicle (GV) stage. Mi‐2 and hSNF2H disappeared from GV‐chromatin within 1 hr of in vitro culture whereas Brg‐1 and BAF‐170 were retained throughout germinal vesicle break down (GVBD). Brg‐1 was localized on the condensed chromatin outside, whereas BAF‐170 was entirely excluded from condensed chromatin. Thereafter, Brg‐1 and BAF‐170 interacted with metaphase I and metaphase II chromosomes. These results imply that Mi‐2 and hSNF2H may initiate the meiotic resumption, and Brg‐1 and BAF‐170 may support chromatin condensation during meiosis. In addition, DNA methylation and methylation of histone H3 at lysine 9 (H3K9) seem to be constantly retained in the oocyte chromatin throughout in vitro maturation. Inhibition of ACRF activity by treatment with the inhibitor apyrase led to retarded chromatin remodeling in bovine oocytes, thereby resulting in poor development of fertilized embryos. Therefore, these results indicate that precise behaviors of ACRFs during meiosis are critical for nuclear maturation and subsequent embryonic development in the bovine. Mol. Reprod. Dev. 77: 126–135, 2010. © 2009 Wiley‐Liss, Inc.