Population survey and management strategies of free-roaming dogs (Canis familiaris) on Saipan,Commonwealth of the Northern Mariana Islands

The island of Saipan, Commonwealth of the Northern Mariana Islands, is populated by free-roaming dogs who were introduced during World War II. The local nonhuman animal control agency manages this population; however, the demographic information and public perception of this population remain unknown. To characterize the free-roaming dog population, an island-wide survey on Saipan was conducted. Photographic, mark-recapture data were used to estimate the population size per land type. Age, sex, sociality, and behavior per land type were documented and associations between these variables were tested using G tests. The effects of land type and urbanization on dog abundance were also analyzed. Lastly, in-person surveys provided data on the public perception of free-roaming dogs. Four main findings are reported: (a) The population size of free-roaming dogs on Saipan is estimated at 21,316. (b) Most dogs encountered were sentry adults. (c) Dog abundance increased with urbanization level and was highest in urban areas. (d) The public perceived free-roaming dogs as a health concern and suggested the implementation of leash laws and sheltering initiatives. This article discusses potential and existing population management strategies for free-roaming dogs on Saipan.     

The osmotic regulator OmpR is involved in the response of Yersinia enterocolitica O:9 to environmental stresses and survival within macrophages

Various environmental signals control the expression of the virulence factors in pathogenic Yersinia enterocolitica strains. The role of the osmotic regulator OmpR protein in controlling the production of Yop proteins, virulence determinants in Y. enterocolitica O:9 (European type) has been studied. An ompR deletion mutant was constructed via allelic exchange with an ompR gene of Y. enterocolitica mutagenized in vitro by a reverse genetic polymerase chain reaction (PCR)-based strategy. The ompR mutant showed a reduced ability to survive under conditions of various environmental stresses in vitro. In particular, low pH stress resulted in increased cell mortality levels. Under conditions of high osmolarity, the wild strain's Yop protein production was reduced, whereas protein levels from the mutant strain remained constant regardless of osmolarity variance. In J774A.1 macrophage cell culture survival of the ompR mutant was decidedly lower than that of the wild-type strain, suggesting that the OmpR protein may play a significant role in protecting cells against intracellular conditions associated with macrophage phagocytosis.     

Bioactive aldehydes from diatoms block the fertilization current in ascidian oocytes

The effects of bioactive aldehydes from diatoms, unicellular algae at the base of the marine food web, were studied on fertilization and early development processes of the ascidian Ciona intestinalis. Using whole-cell voltage clamp techniques, we show that 2-trans-4-trans-decadienal (DD) and 2-trans-4-cis-7-cis-decatrienal (DT) inhibited the fertilization current which is generated in oocytes upon interaction with the spermatozoon. This inhibition was dose-dependent and was accompanied by inhibition of the voltage-gated calcium current activity of the plasma membrane. DD and DT did not inhibit the subsequent contraction of the cortex. Moreover, DD specifically acted as a fertilization channel inhibitor since it did not affect the steady state conductance of the plasma membrane or gap junctional (GJ) communication within blastomeres of the embryo. On the other hand, DD did affect actin reorganization even though the mechanism of action on actin filaments differed from that of other actin blockers. Possibly this effect on actin reorganization was responsible for the subsequent teratogenic action on larval development. The effect of DD was reversible if oocytes were washed soon after fertilization indicating that DD may specifically target certain fertilization mechanisms. Thus, diatom reactive aldehydes such as DD may have a dual effect on reproductive processes, influencing primary fertilization events such as gating of fertilization channels and secondary processes such as actin reorganization which is responsible for the segregation of cell lineages. These findings add to a growing body of evidence on the antiproliferative effects of diatom-derived aldehydes. Our results also report, for the first time, on the action of a fertilization channel blocker in marine invertebrates.     

In Vivo Protein Interactions within the Escherichia coli DNA Polymerase III Core

The mechanisms that control the fidelity of DNA replication are being investigated by a number of approaches, including detailed kinetic and structural studies. Important tools in these studies are mutant versions of DNA polymerases that affect the fidelity of DNA replication. It has been suggested that proper interactions within the core of DNA polymerase III (Pol III) of Escherichia coli could be essential for maintaining the optimal fidelity of DNA replication (H. Maki and A. Kornberg, Proc. Natl. Acad. Sci. USA 84:4389–4392, 1987). We have been particularly interested in elucidating the physiological role of the interactions between the DnaE (α subunit [possessing DNA polymerase activity]) and DnaQ ( subunit [possessing 3′→5′ exonucleolytic proofreading activity]) proteins. In an attempt to achieve this goal, we have used the Saccharomyces cerevisiae two-hybrid system to analyze specific in vivo protein interactions. In this report, we demonstrate interactions between the DnaE and DnaQ proteins and between the DnaQ and HolE (θ subunit) proteins. We also tested the interactions of the wild-type DnaE and HolE proteins with three well-known mutant forms of DnaQ (MutD5, DnaQ926, and DnaQ49), each of which leads to a strong mutator phenotype. Our results show that the mutD5 and dnaQ926 mutations do not affect the subunit-α subunit and subunit-θ subunit interactions. However, the dnaQ49 mutation greatly reduces the strength of interaction of the subunit with both the α and the θ subunits. Thus, the mutator phenotype of dnaQ49 may be the result of an altered conformation of the protein, which leads to altered interactions within the Pol III core.     

Diatom-Derived Polyunsaturated Aldehydes Activate Cell Death in Human Cancer Cell Lines but Not Normal Cells

Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs) that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD), 2-trans,4-trans-octadienal (OD) and 2-trans,4-trans-heptadienal (HD) on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1) and Fas Associated Death Domain (FADD) leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP). The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms.     

Dual role of mitofilin in mitochondrial membrane organization and protein biogenesis

The mitochondrial inner membrane consists of two domains, inner boundary membrane and cristae membrane that are connected by crista junctions. Mitofilin/Fcj1 was reported to be involved in formation of crista junctions, however, different views exist on its function and possible partner proteins. We report that mitofilin plays a dual role. Mitofilin is part of a large inner membrane complex, and we identify five partner proteins as constituents of the mitochondrial inner membrane organizing system (MINOS) that is required for keeping cristae membranes connected to the inner boundary membrane. Additionally, mitofilin is coupled to the outer membrane and promotes protein import via the mitochondrial intermembrane space assembly pathway. Our findings indicate that mitofilin is a central component?of MINOS and functions as a multifunctional regulator of mitochondrial architecture and protein biogenesis.     

Dipoid-Specific Genome Stability Genes of S. cerevisiae: Genomic Screen Reveals Haploidization as an Escape from Persisting DNA Rearrangement Stress

Maintaining a stable genome is one of the most important tasks of every living cell and the mechanisms ensuring it are similar in all of them. The events leading to changes in DNA sequence (mutations) in diploid cells occur one to two orders of magnitude more frequently than in haploid cells. The majority of those events lead to loss of heterozygosity at the mutagenesis marker, thus diploid-specific genome stability mechanisms can be anticipated. In a new global screen for spontaneous loss of function at heterozygous forward mutagenesis marker locus, employing three different mutagenesis markers, we selected genes whose deletion causes genetic instability in diploid Saccharomyces cerevisiae cells. We have found numerous genes connected with DNA replication and repair, remodeling of chromatin, cell cycle control, stress response, and in particular the structural maintenance of chromosome complexes. We have also identified 59 uncharacterized or dubious ORFs, which show the genome instability phenotype when deleted. For one of the strongest mutators revealed in our screen, ctf18Δ/ctf18Δ the genome instability manifests as a tendency to lose the whole set of chromosomes. We postulate that this phenomenon might diminish the devastating effects of DNA rearrangements, thereby increasing the cell''s chances of surviving stressful conditions. We believe that numerous new genes implicated in genome maintenance, together with newly discovered phenomenon of ploidy reduction, will help revealing novel molecular processes involved in the genome stability of diploid cells. They also provide the clues in the quest for new therapeutic targets to cure human genome instability-related diseases.     

Novel deletion of RPL15 identified by array-comparative genomic hybridization in Diamond–Blackfan anemia

Diamond–Blackfan anemia (DBA) is an inherited red blood cell aplasia that usually presents during the first year of life. The main features of the disease are normochromic and macrocytic anemia, reticulocytopenia, and nearly absent erythroid progenitors in the bone marrow. The patients also present with growth retardation and craniofacial, upper limb, heart and urinary system congenital malformations in ~30–50 % of cases. The disease has been associated with point mutations and large deletions in ten ribosomal protein (RP) genes RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, RPS26, and RPL26 and GATA1 in about 60–65 % of patients. Here, we report a novel large deletion in RPL15, a gene not previously implicated to be causative in DBA. Like RPL26, RPL15 presents the distinctive feature of being required both for 60S subunit formation and for efficient cleavage of the internal transcribed spacer 1. In addition, we detected five deletions in RP genes in which mutations have been previously shown to cause DBA: one each in RPS19, RPS24, and RPS26, and two in RPS17. Pre-ribosomal RNA processing was affected in cells established from the patients bearing these deletions, suggesting a possible molecular basis for their pathological effect. These data identify RPL15 as a new gene involved in DBA and further support the presence of large deletions in RP genes in DBA patients.     

Pleiotropic effects of a Yersinia enterocolitica ompR mutation on adherent-invasive abilities and biofilm formation

The OmpR regulator positively influences flagella synthesis and negatively regulates invasin expression in Yersinia enterocolitica. To determine the physiological consequences of this inverse regulation, we analyzed the effect of the ompR mutation on the ability of Y. enterocolitica Ye9 (serotype O9, biotype 2) to adhere to and invade human epithelial HEp-2 cells and to form biofilms. Cell culture assays with ompR, flhDC and inv mutant strains, which vary in their motility and invasin expression, confirmed the important contribution of flagella to the adherent-invasive abilities of Y. enterocolitica Ye9. However, the loss of motility in the ompR strain was apparently not responsible for its low adhesion ability. When the nonmotile phenotype of the ompR mutant was artificially eliminated, an elevated level of invasion, exceeding that of the wild-type strain, was observed. Confocal laser microscopy demonstrated a decrease in the biofilm formation ability of the ompR strain that was only partially correlated with its loss of motility. These data provide evidence that OmpR promotes biofilm formation in this particular strain of Y. enterocolitica, although additional OmpR-dependent factors are also required. In addition, our findings suggest that OmpR-dependent regulation of biofilm formation could be an additional aspect of OmpR regulatory function.     

The roles of PCNA SUMOylation, Mms2-Ubc13 and Rad5 in translesion DNA synthesis in Saccharomyces cerevisiae

Mms2, in concert with Ubc13 and Rad5, is responsible for polyubiquitination of replication processivity factor PCNA. This modification activates recombination-like DNA damage-avoidance mechanisms, which function in an error-free manner. Cells deprived of Mms2, Ubc13 or Rad5 exhibit mutator phenotypes as a result of the channelling of premutational DNA lesions to often error-prone translesion DNA synthesis (TLS). Here we show that Siz1-mediated PCNA SUMOylation is required for the stimulation of this TLS, despite the presence of PCNA monoubiquitination. The stimulation of spontaneous mutagenesis by Siz1 in cells carrying rad5 and/or mms2 mutations is connected with the known role of PCNA SUMOylation in the inhibition of Rad52-mediated recombination. However, following UV irradiation, Siz1 is engaged in additional, as yet undefined, mechanisms controlling genetic stability at the replication fork. We also demonstrate that in the absence of PCNA SUMOylation, Mms2-Ubc13 and Rad5 may, independently of each other, function in the stimulation of TLS. Based on this finding and on an analysis of the epistatic relationships between SIZ1, MMS2 and RAD5, with respect to UV sensitivity, we conclude that PCNA SUMOylation is responsible for the functional differences between the Mms2 and Rad5 homologues of Saccharomyces cerevisiae and Schizosaccharomyces pombe.