Grazing of nonindigenous bacteria by nano-sized protozoa in a natural coastal system

Mesocosms (4.5 m³) situated in a closed bay area were used to investigate the effect of protozoan predation on nonindigenous bacteria. Pseudomonas fluorescens strain Agl was released into mesocosms as a single inoculum of 1 × 10⁵ cells ml^–1 (final concentration) or as four inocula (same concentration each) at intervals of 3 days. Mesocosms that had received growth media corresponding to the inoculum served as controls. Numbers of P. fluorescens Ag1 decreased rapidly whether released as single or multiple inocula. Direct estimation of protozoan predation using fluorescently labeled P. fluorescens from log phase and starved cultures, respectively, revealed that natural populations of heterotrophic nanoflagellates consumed substantial amounts of the nonindigenous bacterial strain. The volume of fluorescently labeled cells prepared from starved cells was 68% of log phase cell volume, but the individual clearance of the small cells was five to seven times higher than that of the log phase bacteria. The natural populations of nanoflagellates consumed 34–62% of P. fluorescens Ag1 daily if starved bacteria were offered as food, and 3–13% if the cells were in the logarithmic growth phase. This suggests that the effect of protozoan predation on nonindigenous bacterial strains is substantial because cultured bacteria are likely to starve in natural environments. The addition of P. fluorescens Ag1 and the growth medium enhanced the abundance of natural bacteria, chlorophyll a, heterotrophic nanoflagellates, and ciliates, but it did not improve the growth conditions for the released strain. The effects on the indigenous populations were more pronounced after addition of fresh medium than following inoculation with cells, which possibly was due to the lower nutrient content of spent medium. However, these results, based on direct estimation of protozoan predation on log phase and starved nonindigenous bacteria, point to the conclusion that mortality induced by bacterivorous predators is the key factor determining removal of nonindigenous bacteria introduced in natural aquatic systems. Correspondence to: K. Christoffersen.     

Effect of ethylene and carbon dioxide on potato metabolism: stimulation of tuber and mitochondrial respiration, and inducement of the alternative path

The respiration of potato tubers (Solanum tuberosum var. Russet Burbank) which have been kept at room temperature for 10 days is stimulated upon subsequent treatment with C₂H₄ (10 microliters per liter) and O₂. The respiratory rise reaches a peak in 24 to 30 hours and thereafter declines. Coincident with the rise in tuber respiration is an increase in the respiratory rates of fresh slices and isolated mitochondria. Slices and mitochondria from C₂H₄- and O₂-treated tubers also display substantial resistance to CN, and the resistant respiration is inhibited by hydroxamates.

The longer the tubers are stored after harvest, the less effective is C₂H₄ in causing CN resistance in slices and mitochondria from treated tubers. Addition of 10% CO₂ to the C₂H₄-O₂ mixture, however, causes extensive CN resistance to develop, even in slices and mitochondria from old tubers. The results show that C₂H₄, O₂, and CO₂ act synergistically to induce alternative path development in potatoes.

     

Horizontal Transfer of Archaeal Genes into the Deinococcaceae: Detection by Molecular and Computer-Based Approaches

Members of the Deinococcaceae (e.g., Thermus, Meiothermus, Deinococcus) contain A/V-ATPases typically found in Archaea or Eukaryotes which were probably acquired by horizontal gene transfer. Two methods were used to quantify the extent to which archaeal or eukaryotic genes have been acquired by this lineage. Screening of a Meiothermus ruber library with probes made against Thermoplasma acidophilum DNA yielded a number of clones which hybridized more strongly than background. One of these contained the prolyl tRNA synthetase (RS) gene. Phylogenetic analysis shows the M. ruber and D. radiodurans prolyl RS to be more closely related to archaeal and eukaryal forms of this gene than to the typical bacterial type. Using a bioinformatics approach, putative open reading frames (ORFs) from the prerelease version of the D. radiodurans genome were screened for genes more closely related to archaeal or eukaryotic genes. Putative ORFs were searched against representative genomes from each of the three domains using automated BLAST. ORFs showing the highest matches against archaeal and eukaryotic genes were collected and ranked. Among the top-ranked hits were the A/V-ATPase catalytic and noncatalytic subunits and the prolyl RS genes. Using phylogenetic methods, ORFs were analyzed and trees assessed for evidence of horizontal gene transfer. Of the 45 genes examined, 20 showed topologies in which D. radiodurans homologues clearly group with eukaryotic or archaeal homologues, and 17 additional trees were found to show probable evidence of horizontal gene transfer. Compared to the total number of ORFs in the genome, those that can be identified as having been acquired from Archaea or Eukaryotes are relatively few (approximately 1%), suggesting that interdomain transfer is rare.     

Terrigenous sediment-dominated reef platform infilling: an unexpected precursor to reef island formation and a test of the reef platform size–island age model in the Pacific

Low-lying coral reef islands are considered highly vulnerable to climate change, necessitating an improved understanding of when and why they form, and how the timing of formation varies within and among regions. Several testable models have been proposed that explain inter-regional variability as a function of sea-level history and, more recently, a reef platform size model has been proposed from the Maldives (central Indian Ocean) to explain intra-regional (intra-atoll) variability. Here we present chronostratigraphic data from Pipon Island, northern Great Barrier Reef (GBR), enabling us to test the applicability of existing regional island evolution models, and the platform size control hypothesis in a Pacific context. We show that reef platform infilling occurred rapidly (~4–5 mm yr⁻¹) under a “bucket-fill” type scenario. Unusually, this infilling was dominated by terrigenous sedimentation, with platform filling and subsequent reef flat formation complete by ~5000 calibrated years BP (cal BP). Reef flat exposure as sea levels slowly fell post highstand facilitated a shift towards intertidal and subaerial-dominated sedimentation. Our data suggest, however, a lag of ~1500 yr before island initiation (at ~3200 cal BP), i.e. later than that reported from smaller and more evolutionarily mature reef platforms in the region. Our data thus support: (1) the hypothesis that platform size acts to influence the timing of platform filling and subsequent island development at intra-regional scales; and (2) the hypothesis that the low wooded islands of the northern GBR conform to a model of island formation above an elevated reef flat under falling sea levels.

     

Effects of Cell-Bound Microcystins on Survival and Feeding of Daphnia spp.

The influence of cell-bound microcystins on the survival time and feeding rates of six Daphnia clones belonging to five common species was studied. To do this, the effects of the microcystin-producing Microcystis strain PCC7806 and its mutant, which has been genetically engineered to knock out microcystin synthesis, were compared. Additionally, the relationship between microcystin ingestion rate by the Daphnia clones and Daphnia survival time was analyzed. Microcystins ingested with Microcystis cells were poisonous to all Daphnia clones tested. The median survival time of the animals was closely correlated to their microcystin ingestion rate. It was therefore suggested that differences in survival among Daphnia clones were due to variations in microcystin intake rather than due to differences in susceptibility to the toxins. The correlation between median survival time and microcystin ingestion rate could be described by a reciprocal power function. Feeding experiments showed that, independent of the occurrence of microcystins, cells of wild-type PCC7806 and its mutant are able to inhibit the feeding activity of Daphnia. Both variants of PCC7806 were thus ingested at low rates. In summary, our findings strongly suggest that (i) sensitivity to the toxic effect of cell-bound microcystins is typical for Daphnia spp., (ii) Daphnia spp. and clones may have a comparable sensitivity to microcystins ingested with food particles, (iii) Daphnia spp. may be unable to distinguish between microcystin-producing and -lacking cells, and (iv) the strength of the toxic effect can be predicted from the microcystin ingestion rate of the animals.     

Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus

Hepatitis E virus (HEV) causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4). Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp), X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1) and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient model of the virus.     

The involvement of the docking protein Gab1 in mitogenic signalling induced by EGF and HGF in rat hepatocytes

Grb2-assosiated binder (Gab) family proteins are docking molecules that can interact with receptor tyrosine kinases (RTKs) and cytokine receptors and bind several downstream signalling proteins. Studies in several cell types have shown that Gab1 may have a role in signalling mediated by the two RTKs epidermal growth factor (EGF) receptor (EGFR) and Met, the receptor for hepatocyte growth factor (HGF), but the involvement of Gab1 in EGFR and Met signalling has not been directly compared in the same cell. We have studied mechanisms of activation and role in mitogenic signalling of Gab1 in response to EGF and HGF in cultured rat hepatocytes. Gab1, but not Gab2, was expressed in the hepatocytes and was phosphorylated upon stimulation with EGF or HGF. Depletion of Gab1, using siRNA, decreased the ERK and Akt activation, cyclin D1 expression, and DNA synthesis in response to both EGF and HGF. Studies of mechanisms of recruitment to the receptors showed that HGF induced co-precipitation of Gab1 and Met while EGF induced binding of Gab1 to Grb2 but not to EGFR. Gab1 activation in response to both EGF and HGF was dependent on PI3K. While EGF activated Gab1 and Shc equally, within the same concentration range, HGF very potently and almost exclusively activated Gab1, having only a minimal effect on Shc. Collectively, our results strongly suggest that although Gab1 interacts differently with EGFR and Met, it is involved in mitogenic signalling mediated by both these growth factor receptors in hepatocytes.     

Risk factors for <Emphasis Type="Italic">Coxiella burnetii</Emphasis> antibodies in bulk tank milk from Danish dairy herds

The aim was to identify risk factors associated with Coxiella burnetii antibody positivity in bulk tank milk (BTM) samples from 100 randomly selected Danish dairy cattle herds. Antibody levels were measured by an enzyme-linked immuno-sorbent assay. Before testing the herds, the farm managers were interviewed about hired labour, biosecurity, housing and herd health during the 12 months prior to the study. Variables considered important for C. burnetii antibody positivity in multivariable logistic regression analysis included the sharing of machines between farms (OR?=?3.6), human contacts (OR?=?4.2), artificial insemination by other people than artificial insemination technicians (OR?=?7.7), routine herd health contract with the veterinarian (OR?=?4.3) and hygiene precautions taken by veterinarians (OR?=?5). In addition, herd size, hired labour, trading of cattle between farms, quarantine and use of calving and disease pens also showed significant association in univariable analysis. This study demonstrates that strict biosecurity is important for the prevention of infections with C. burnetii.     

BRCA1-directed,enhanced and aberrant homologous recombination: Mechanism and potential treatment strategies

Despite intense studies, questions still remain regarding the molecular mechanisms leading to the development of hereditary breast and ovarian cancers. Research focused on elucidating the role of the breast cancer susceptibility gene 1 (BRCA1) in the DNA damage response may be of the most critical importance to understanding these processes. The BRCA1 protein has an N-terminal RING domain possessing E3 ubiquitin-ligase activity and a C-terminal BRCT domain involved in binding specific phosphoproteins. These domains are involved directly or indirectly in DNA double-strand break (DSB) repair. As the two terminal domains of BRCA1 represent two separate entities, understanding how these domains communicate and are functionally altered in regards to DSB repair is critical for understanding the development of BRCA1-related breast and ovarian cancers and for developing novel therapeutics. Herein, we review recent findings of how altered functions of these domains might lead to cancer through a mechanism of increased aberrant homologous recombination and possible implications for the development of BRCA1 inhibitors.Key words: BRCT, DNA repair, peptide, radiation, RING, ubiquitylation