Population structure of the Japanese eel, Anguilla japonica

Mitochondrial DNA (mtDNA) sequences that include (a) a part of the cytochrome b gene, (b) two tRNA genes, and (c) a part of the noncoding D-loop region of 31 Anguilla japonica (Japanese eel) and 1 A. marmorata collected from Taiwan, Japan, and mainland China were determined to evaluate the population structure of Japanese eel. Among 30 genotypes identified from the 31 Japanese eel mtDNAs sequenced, there are 58 variable sites, predominantly clustered at the D-loop region. The phylogenetic tree constructed by the unweighted pair-group method with arithmetic mean shows neither significant genealogical branches nor geographic clusters. Furthermore, the sequence-statistics test reveals little, if any, significant genetic differentiation. These results indicate that the 31 Japanese eels might come from a single population. Analysis of sequence variation in mtDNA by using the relationship between the number of segregating sites and the average number of nucleotide differences under the neutral mutation hypothesis reveals that neutral mutation acts as a major factor influencing the evolutionary divergence of the Japanese eel mitochondrial genome sequenced, especially in the noncoding region.      

Visualization of protein interactions in living Caenorhabditis elegans using bimolecular fluorescence complementation analysis

The bimolecular fluorescence complementation (BiFC) assay is a powerful tool for visualizing and identifying protein interactions in living cells. This assay is based on the principle of protein-fragment complementation, using two nonfluorescent fragments derived from fluorescent proteins. When two fragments are brought together in living cells by tethering each to one of a pair of interacting proteins, fluorescence is restored. Here, we provide a protocol for a Venus-based BiFC assay to visualize protein interactions in the living nematode, Caenorhabditis elegans. We discuss how to design appropriate C. elegans BiFC cloning vectors to enable visualization of protein interactions using either inducible heat shock promoters or native promoters; transform the constructs into worms by microinjection; and analyze and interpret the resulting data. When expression of BiFC fusion proteins is induced by heat shock, the fluorescent signals can be visualized as early as 30 min after induction and last for 24 h in transgenic animals. The entire procedure takes 2-3 weeks to complete.     

Probing FinO-FinP RNA interactions by site-directed protein-RNA crosslinking and gelFRET

The conjugative transfer of F-plasmids is repressed by a two-component system, which consists of the antisense RNA FinP and the protein FinO. FinO binds FinP, protecting it from endonucleolytic degradation and facilitating duplex formation between FinP and its complementary RNA. Here we present the results of site-specific protein-RNA cross-linking and gel-based fluorescence resonance energy transfer (gelFRET) experiments used to probe the structure of a complex of FinO bound to an RNA target consisting of a duplex with 5' and 3' single-stranded tails. The crosslinking experiments reveal that an extensive, largely positively charged surface on FinO contacts RNA. The gelFRET measurements indicate that the 5' single-stranded tail of the RNA is in closer contact with much of the protein than the distal, blunt end of the RNA duplex. These data suggest that significant conformational adjustments in the protein and/or the RNA accompany complex formation.     

Bimolecular fluorescence complementation analysis of eukaryotic fusion products

Background information. Cell fusion is known to underlie key developmental processes in humans and is postulated to contribute to tissue maintenance and even carcinogenesis. The mechanistic details of cell fusion, especially between different cell types, have been difficult to characterize because of the dynamic nature of the process and inadequate means to track fusion products over time. Here we introduce an inducible system for detecting and tracking live cell fusion products in vitro and potentially in vivo. This system is based on BiFC (bimolecular fluorescence complementation) analysis. In this approach, two proteins that can interact with each other are joined to fragments of a fluorescent protein and are expressed in separate cells. The interaction of said proteins after cell fusion produces a fluorescent signal, enabling the identification and tracking of fusion products over time. Results. Long‐term tracking of fused p53‐deficient cells revealed that hybrid cells were capable of proliferation. In some cases, proliferation was preceded by nuclear fusion and division was asymmetric (69%±2% of proliferating hybrids), suggesting chromosomal instability. In addition, asymmetric division following proliferation could give rise to progeny indistinguishable from unfused counterparts. Conclusions. These results support the possibility that the chromosomal instability characteristic of tumour cells may be incurred as a consequence of cell fusion and suggest that the role of cell fusion in carcinogenesis may have been masked to this point for lack of an inducible method to track cell fusion. In sum, the BiFC‐based approach described here allows for comprehensive studies of the mechanism and biological impact of cell fusion in nature.     

Genetic analysis of carbamoylphosphate synthesis in Rhizobium meliloti 104A14

We have previously isolated ineffective (Fix-) mutants of Rhizobium meliloti 104A14 requiring both arginine and uracil, and thus probably defective in carbamoylphosphate synthetase. We describe here the molecular and genetic analysis of the R. meliloti genes coding for carbamoylphosphate synthetase. Plasmids that complement the mutations were isolated from a R. meliloti gene bank. Restriction analysis of these plasmids indicated that complementation involved two unlinked regions of the R. meliloti chromosome, carA and carB. Genetic complementation between the plasmids and mutants demonstrated a single complementation group for carA, but two overlapping complementation groups for carB. The cloned R. meliloti genes hybridize to the corresponding E. coli carA and carB genes which encode the two subunits of carbamoylphosphate synthetase. Transposon Tn5 mutagenesis was used to localize the carA and carB genes on the cloned R. meliloti DNA. The cloned R. meliloti carA and carB genes were unable to complement E. coli carA or carB mutants alone or in combination. We speculate on the mechanism of the unusual pattern of genetic complementation at the R. meliloti carB locus.     

Tono Reservoir fishery contribution to poverty reduction among fishers in northern Ghana

Fishery characteristics and livelihood status of fishers at Tono Reservoir, Ghana, were investigated between January 2015 and June 2016. Data on fisher demography, fishing gears, fishing methods, perceptions of the state of fish stocks, management practices, income and consumption of fishers were obtained through structured interviews. Censuses of fishers and fishing gears were conducted through direct observation and counts. The population of fishers was 950 and the majority (74%) of the sampled respondents fell within the ages of 24–41 years. Gillnet, cast net, trap and hook and line were the four main gears utilised. Illegal methods of fishing observed included the use of mosquito nets (nets with mesh <1.0 cm) and the use of brewer’s waste (pito mash) as bait. Brycinus nurse, Synodontis spp., Parailia spiniserrata and Chrysichthys spp. were perceived to have disappeared from the reservoir. The fishers were unaware of the existence of any fisheries regulations, hence there was no adherence to management practices. Their daily income was derived mainly from fishing. The incidence of poverty among fishers was low (8%). The Tono Reservoir has a great potential for supporting livelihood if it is properly managed.     

Granulocyte-CSF induced inflammation-associated cardiac thrombosis in iron loading mouse heart and can be attenuated by statin therapy

Background  

Granulocyte colony-stimulating factor (G-CSF), a hematopoietic cytokine, was recently used to treat patients of acute myocardial infarction with beneficial effect. However, controversy exists as some patients developed re-stenosis and worsened condition post G-CSF delivery. This study presents a new disease model to study G-CSF induced cardiac thrombosis and delineate its possible mechanism. We used iron loading to mimic condition of chronic cardiac dysfunction and apply G-CSF to mice to test our hypothesis.