Diffusion of myosin V on microtubules: a fine-tuned interaction for which E-hooks are dispensable

Organelle transport in eukaryotes employs both microtubule and actin tracks to deliver cargo effectively to their destinations, but the question of how the two systems cooperate is still largely unanswered. Recently, in vitro studies revealed that the actin-based processive motor myosin V also binds to, and diffuses along microtubules. This biophysical trick enables cells to exploit both tracks for the same transport process without switching motors. The detailed mechanisms underlying this behavior remain to be solved. By means of single molecule Total Internal Reflection Microscopy (TIRFM), we show here that electrostatic tethering between the positively charged loop 2 and the negatively charged C-terminal E-hooks of microtubules is dispensable. Furthermore, our data indicate that in addition to charge-charge interactions, other interaction forces such as non-ionic attraction might account for myosin V diffusion. These findings provide evidence for a novel way of myosin tethering to microtubules that does not interfere with other E-hook-dependent processes.     

Classification of sharks in the Egyptian Mediterranean waters using morphological and DNA barcoding approaches

The identification of species constitutes the first basic step in phylogenetic studies, biodiversity monitoring and conservation. DNA barcoding, i.e. the sequencing of a short standardized region of DNA, has been proposed as a new tool for animal species identification. The present study provides an update on the composition of shark in the Egyptian Mediterranean waters off Alexandria, since the latest study to date was performed 30 years ago, DNA barcoding was used in addition to classical taxonomical methodologies. Thus, 51 specimen were DNA barcoded for a 667 bp region of the mitochondrial COI gene. Although DNA barcoding aims at developing species identification systems, some phylogenetic signals were apparent in the data. In the neighbor-joining tree, 8 major clusters were apparent, each of them containing individuals belonging to the same species, and most with 100% bootstrap value. This study is the first to our knowledge to use DNA barcoding of the mitochondrial COI gene in order to confirm the presence of species Squalus acanthias, Oxynotus centrina, Squatina squatina, Scyliorhinus canicula, Scyliorhinus stellaris, Mustelus mustelus, Mustelus punctulatus and Carcharhinus altimus in the Egyptian Mediterranean waters. Finally, our study is the starting point of a new barcoding database concerning shark composition in the Egyptian Mediterranean waters (Barcoding of Egyptian Mediterranean Sharks [BEMS], http://www.boldsystems.org/views/projectlist.php?&#Barcoding%20Fish%20%28FishBOL%29).     

Phase I randomised clinical trial of an HIV-1(CN54), clade C, trimeric envelope vaccine candidate delivered vaginally

We conducted a phase 1 double-blind randomised controlled trial (RCT) of a HIV-1 envelope protein (CN54 gp140) candidate vaccine delivered vaginally to assess immunogenicity and safety. It was hypothesised that repeated delivery of gp140 may facilitate antigen uptake and presentation at this mucosal surface. Twenty two healthy female volunteers aged 18-45 years were entered into the trial, the first receiving open-label active product. Subsequently, 16 women were randomised to receive 9 doses of 100 μg of gp140 in 3 ml of a Carbopol 974P based gel, 5 were randomised to placebo solution in the same gel, delivered vaginally via an applicator. Participants delivered the vaccine three times a week over three weeks during one menstrual cycle, and were followed up for two further months. There were no serious adverse events, and the vaccine was well tolerated. No sustained systemic or local IgG, IgA, or T cell responses to the gp140 were detected following vaginal immunisations. Repeated vaginal immunisation with a HIV-1 envelope protein alone formulated in Carbopol gel was safe, but did not induce local or systemic immune responses in healthy women. TRIAL REGISTRATION: ClinicalTrials.gov NCT00637962.     

Loss of cytoplasmic CDK1 predicts poor survival in human lung cancer and confers chemotherapeutic resistance

The dismal lethality of lung cancer is due to late stage at diagnosis and inherent therapeutic resistance. The incorporation of targeted therapies has modestly improved clinical outcomes, but the identification of new targets could further improve clinical outcomes by guiding stratification of poor-risk early stage patients and individualizing therapeutic choices. We hypothesized that a sequential, combined microarray approach would be valuable to identify and validate new targets in lung cancer. We profiled gene expression signatures during lung epithelial cell immortalization and transformation, and showed that genes involved in mitosis were progressively enhanced in carcinogenesis. 28 genes were validated by immunoblotting and 4 genes were further evaluated in non-small cell lung cancer tissue microarrays. Although CDK1 was highly expressed in tumor tissues, its loss from the cytoplasm unexpectedly predicted poor survival and conferred resistance to chemotherapy in multiple cell lines, especially microtubule-directed agents. An analysis of expression of CDK1 and CDK1-associated genes in the NCI60 cell line database confirmed the broad association of these genes with chemotherapeutic responsiveness. These results have implications for personalizing lung cancer therapy and highlight the potential of combined approaches for biomarker discovery.     

Isolation and characterization of a metastatic hybrid cell line generated by ER negative and ER positive breast cancer cells in mouse bone marrow

Background

The origin and the contribution of breast tumor heterogeneity to its progression are not clear. We investigated the effect of a growing orthotopic tumor formed by an aggressive estrogen receptor (ER)-negative breast cancer cell line on the metastatic potential of a less aggressive ER-positive breast cancer cell line for the elucidation of how the presence of heterogeneous cancer cells might affect each other''s metastatic behavior.

Methods

ER positive ZR-75-1/GFP/puro cells, resistant to puromycin and non-tumorigenic/non-metastatic without exogenous estrogen supplementation, were injected intracardiacally into mice bearing growing orthotopic tumors, formed by ER negative MDA-MB-231/GFP/Neo cells resistant to G418. A variant cell line B6, containing both estrogen-dependent and -independent cells, were isolated from GFP expressing cells in the bone marrow and re-inoculated in nude mice to generate an estrogen-independent cell line B6TC.

Results

The presence of ER negative orthotopic tumors resulted in bone metastasis of ZR-75-1 without estrogen supplementation. The newly established B6TC cell line was tumorigenic without estrogen supplementation and resistant to both puromycin and G418 suggesting its origin from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. Compared to parental cells, B6TC cells were more metastatic to lung and bone after intracardiac inoculation. More significantly, B6TC mice also developed brain metastasis, which was not observed in the MDA-MB-231/GFP/Neo cell-inoculated mice. Low expression of ERα and CD24, and high expression of EMT-related markers such as Vimentin, CXCR4, and Integrin-β1 along with high CD44 and ALDH expression indicated stem cell-like characteristics of B6TC. Gene microarray analysis demonstrated a significantly different gene expression profile of B6TC in comparison to those of parental cell lines.

Conclusions

Spontaneous generation of the novel hybrid cell line B6TC, in a metastatic site with stem cell-like properties and propensity to metastasize to brain, suggest that cell fusion can contribute to tumor heterogeneity.     

COMMD1-mediated ubiquitination regulates CFTR trafficking

The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.     

Effect of chronic ethanol administration on hepatic eNOS activity and its association with caveolin-1 and calmodulin in female rats

Although chronic and excessive alcohol consumption is associated with liver disease, the mechanism of alcoholic liver injury is still not clear. Whether reduced hepatic production of nitric oxide, which is evident in models of liver injury, is associated with alcohol-induced liver injury has not been investigated. We measured nitric oxide synthase (NOS) activity in the liver of pair-fed rats receiving liquid diet with or without alcohol [3% (vol/vol)] for 12 wk. Compared with control rats, hepatic NOS activity was significantly reduced in alcohol-treated rats along with the evidence of liver injury. Interestingly, there was no difference in the hepatic expression of endothelial NOS (eNOS) between ethanol-fed and pair-fed rats. We then tested the hypothesis that an imbalance between the binding of eNOS with inhibitory and stimulatory proteins may underlie the reduced activity of eNOS because eNOS catalytic activity is regulated partly through dynamic interactions with the inhibitory protein caveolin-1 and the stimulatory protein calmodulin. We found that hepatic caveolin-1 was markedly increased in alcohol-treated rats compared with control rats, whereas calmodulin remained unaltered. The binding of caveolin-1 and calmodulin with eNOS was increased and decreased, respectively, in alcohol-treated rats. Our results suggest that chronic alcohol intake attenuates hepatic eNOS activity by increasing the expression of the inhibitory protein caveolin-1 and enhancing its binding with eNOS.     

Vegetation: A source of air fungal bio-contaminant

Airborne fungal counts and types were examined in three selected regions in Egypt. Two of the sampling sites are rural areas, one cultivated with chamomile and the second with vegetable. The third site is located in an urban area. A sedimentation method was used to isolate airborne fungal spores. Airborne fungal spore counts averaged 71\pm 19, 64\pm 14 and 175\pm 79 cfu/p/h in the urban, vegetable and chamomile growing areas, respectively. A total of 1486 fungal colonies belonging to 32 genera were identified. Alternaria (7.5–59.9%), Aspergillus (11.2–38.9%), Penicillium (9.5–15%) and Cladosporium (7.78–17.5%) were the predominant fungal genera found in all sampling sites. Alternaria (42–59.9%) and Aspergillus (38.9%) were the common fungal genera in the cultivated and urban areas, respectively. Vegetation is considered the main source of Alternaria, whereas Aspergillus, Penicillium and Cladosporium are related to local microenvironments and urbanization. Acremonium, Aureobasidium, Botrytis, Beauveria, Chlamydomyces, Chalara, Curvularia, Fusarium, Geotrichum, Trichothecium, Oidiodendron, Scopulariopsis, Spicaria, Stachybotrys chartarum, Torula and Thamnidium, were only detected in low percentages (0.11–1.8%) in the cultivated areas. Vegetation adds different fungal types into the air and their numbers vary according to vegetation type and weather conditions. Airborne fungal counts increased with temperature and decreased with rainfall and relative humidity. Airborne fungal spores have many implications in the spread of human and plant diseases. The presence of fungal spores in air, in spite of their counts, may raise arguments about their role in health complaints in a particular region, „i.e., the fungal concentration may be low but the predominant aeroallergen may be dangerous”.     

Invertebrate data predict an early emergence of vertebrate fibrillar collagen clades and an anti-incest model

Fibrillar collagens are involved in the formation of striated fibrils and are present from the first multicellular animals, sponges, to humans. Recently, a new evolutionary model for fibrillar collagens has been suggested (Boot-Handford, R. P., Tuckwell, D. S., Plumb, D. A., Farrington Rock, C., and Poulsom, R. (2003) J. Biol. Chem. 278, 31067-31077). In this model, a rare genomic event leads to the formation of the founder vertebrate fibrillar collagen gene prior to the early vertebrate genome duplications and the radiation of the vertebrate fibrillar collagen clades (A, B, and C). Here, we present the modular structure of the fibrillar collagen chains present in different invertebrates from the protostome Anopheles gambiae to the chordate Ciona intestinalis. From their modular structure and the use of a triple helix instead of C-propeptide sequences in phylogenetic analyses, we were able to show that the divergence of A and B clades arose early during evolution because alpha chains related to these clades are present in protostomes. Moreover, the event leading to the divergence of B and C clades from a founder gene arose before the appearance of vertebrates; altogether these data contradict the Boot-Handford model. Moreover, they indicate that all the key steps required for the formation of fibrils of variable structure and functionality arose step by step during invertebrate evolution.