Actin filaments are involved in the maintenance of Golgi cisternae morphology and intra-Golgi pH

Here we examine the contribution of actin dynamics to the architecture and pH of the Golgi complex. To this end, we have used toxins that depolymerize (cytochalasin D, latrunculin B, mycalolide B, and Clostridium botulinum C2 toxin) or stabilize (jasplakinolide) filamentous actin. When various clonal cell lines were examined by epifluorescence microscopy, all of these actin toxins induced compaction of the Golgi complex. However, ultrastructural analysis by transmission electron microscopy and electron tomography/three-dimensional modelling of the Golgi complex showed that F-actin depolymerization first induces perforation/fragmentation and severe swelling of Golgi cisternae, which leads to a completely disorganized structure. In contrast, F-actin stabilization results only in cisternae perforation/fragmentation. Concomitantly to actin depolymerization-induced cisternae swelling and disorganization, the intra-Golgi pH significantly increased. Similar ultrastructural and Golgi pH alkalinization were observed in cells treated with the vacuolar H+ -ATPases inhibitors bafilomycin A1 and concanamycin A. Overall, these results suggest that actin filaments are implicated in the preservation of the flattened shape of Golgi cisternae. This maintenance seems to be mediated by the regulation of the state of F-actin assembly on the Golgi pH homeostasis.     

Temporal variation in the infection dynamics and maturation cycle of Oligogonotylus manteri (Digenea) in the cichlid fish, 'Cichlasoma' urophthalmus, from Yucatán, México

We studied the infection dynamics and maturation cycle of Oligogonotylus manteri in wild and caged 'Cichlasoma' urophthalmus, and determined the potential role of different sources of infection in its transmission in a quarry (MITZA). Metacercariae, and nongravid and gravid stages of O. manteri were present throughout 1 annual cycle. Prevalence, mean intensity, and/or aggregation values peaked around April and June in both wild and caged fish. This period of time includes the start of the rainy season, in which the water temperature reaches its maximum annual values. Because temperature is a major factor triggering 'C.' urophthalmus activity (food intake, growth, and reproduction), and O. manteri metacercariae and adults are trophically transmitted, temperature may be playing an important role in the recruitment of worms to the fish. We also determined that cercariae infect caged fish through a mechanism other than trophic transmission whereby fish consume infected snails, which has been described as the most common mode of transmission to 'C.' urophthalmus.     

Tissue MicroRNAs as Predictors of Outcome in Patients with Metastatic Colorectal Cancer Treated with First Line Capecitabine and Oxaliplatin with or without Bevacizumab

Purpose

We tested the hypothesis that expression of microRNAs (miRNAs) in cancer tissue can predict effectiveness of bevacizumab added to capecitabine and oxaliplatin (CAPEOX) in patients with metastatic colorectal cancer (mCRC).

Experimental Design

Patients with mCRC treated with first line CAPEOX and bevacizumab (CAPEOXBEV): screening (n = 212) and validation (n = 121) cohorts, or CAPEOX alone: control cohort (n = 127), were identified retrospectively and archival primary tumor samples were collected. Expression of 754 miRNAs was analyzed in the screening cohort using polymerase chain reaction (PCR) arrays and expression levels were related to time to disease progression (TTP) and overall survival (OS). Significant miRNAs from the screening study were analyzed in all three cohorts using custom PCR arrays. In situ hybridization (ISH) was done for selected miRNAs.

Results

In the screening study, 26 miRNAs were significantly correlated with outcome in multivariate analyses. Twenty-two miRNAs were selected for further study. Higher miR-664-3p expression and lower miR-455-5p expression were predictive of improved outcome in the CAPEOXBEV cohorts and showed a significant interaction with bevacizumab effectiveness. The effects were strongest for OS. Both miRNAs showed high expression in stromal cells. Higher expression of miR-196b-5p and miR-592 predicted improved outcome regardless of bevacizumab treatment, with similar effect estimates in all three cohorts.

Conclusions

We have identified potentially predictive miRNAs for bevacizumab effectiveness and additional miRNAs that could be related to chemotherapy effectiveness or prognosis in patients with mCRC. Our findings need further validation in large cohorts, preferably from completed randomized trials.     

Photosynthetic acclimation to photon irradiance and its relation to chlorophyll fluorescence and carbon assimilation in the halotolerant green alga Dunaliella viridis

This work describes the long-term acclimation of the halotolerant microalga Dunaliella viridis to different photon irradiance, ranging from darkness to 1500 μmol m⁻² s⁻¹. In order to assess the effects of long-term photoinhibition, changes in oxygen production rate, pigment composition, xanthophyll cycle and in vivo chlorophyll fluorescence using the saturating pulse method were measured. Growth rate was maximal at intermediate irradiance (250 and 700 μmol m⁻² s⁻¹). The increase in growth irradiance from 700 to 1500 μmol m⁻² s⁻¹ did not lead to further significant changes in pigment composition or EPS, indicating saturation in the pigment response to high light. Changes in Photosystem II optimum quantum yield (F_v/F_m) evidenced photoinhibition at 700 and especially at 1500 μmol m⁻² s⁻¹. The relation between photosynthetic electron flow rate and photosyntetic O₂ evolution was linear for cultures in darkness shifting to curvilinear as growth irradiance increased, suggesting the interference of the energy dissipation processes in oxygen evolution. Carbon assimilation efficiencies were studied in relation to changes in growth rate, internal carbon and nitrogen composition, and organic carbon released to the external medium. All illuminated cultures showed a high capability to maintain a C:N ratio between 6 and 7. The percentage of organic carbon released to the external medium increased to its maximum under high irradiance (1500 μmol m⁻² s⁻¹). These results suggest that the release of organic carbon could act as a secondary dissipation process when the xanthophyll cycle is saturated. This revised version was published online in June 2006 with corrections to the Cover Date.     

Electronic characterization of the oxidized state of the blue copper protein rusticyanin by 1H NMR: is the axial methionine the dominant influence for the high redox potential?

The oxidized state of rusticyanin, the blue copper protein with the highest redox potential in its class, has been investigated through (1)H nuclear magnetic resonance applied to its cobalt(II) derivative. The assignment of the protons belonging to the coordinated residues has been performed. Many other amino acids situated in the vicinity of the metal ion, including six hydrophobic residues (isoleucine140 and five phenylalanines) have also been identified. The orientation of the main axes of the magnetic susceptibility tensor for the cobalt(II)-rusticyanin as well as its axial, Deltachi(ax), and rhombic, Deltachi(rh), magnetic susceptibility anisotropy components have been determined. A comparison of the present results with those previously obtained for cobalt(II)azurin [Donaire, A., Salgado, J., Moratal, J. M. (1998) Biochemistry 37, 8659-8673] allows us to provide further insights into the reasons for the high redox potential of this protein. According to our results, the interaction between the metal ion and the thioether Sdelta of the axial methionine is not as influential as the strong destabilizing effect that the hydrophobic residues close to the metal ion undergo in the oxidized state.     

Vertical stratification of subsurface microbial community composition across geological formations at the Hanford Site

Microbial diversity in subsurface sediments at the Hanford Site 300 Area near Richland, Washington state (USA) was investigated by analysing 21 samples recovered from depths of 9-52?m. Approximately 8000 near full-length 16S rRNA gene sequences were analysed across geological strata that include a natural redox transition zone. These strata included the oxic coarse-grained Hanford formation, fine-grained oxic and anoxic Ringold Formation sediments, and the weathered basalt group. We detected 1233 and 120 unique bacterial and archaeal OTUs (operational taxonomic units at the 97% identity level) respectively. Microbial community structure and richness varied substantially across the different geological strata. Bacterial OTU richness (Chao1 estimator) was highest (>?700) in the upper Hanford formation, and declined to about 120 at the bottom of the Hanford formation. Just above the Ringold oxic-anoxic interface, richness was about 325 and declined to less than 50 in the deeper reduced zones. The deeper Ringold strata were characterized by a preponderance (c. 90%) of Proteobacteria. The bacterial community in the oxic sediments contained not only members of nine well-recognized phyla but also an unusually high proportion of three candidate divisions (GAL15, NC10 and SPAM). Additionally, 13 novel phylogenetic orders were identified within the Deltaproteobacteria, a clade rich in microbes that carry out redox transformations of metals that are important contaminants on the Hanford Site.