Targeting a newly established spontaneous feline fibrosarcoma cell line by gene transfer

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas.     

Relationships between the flexed-arm hang and select measures of muscular fitness

The flexed-arm hang (FAH) has been used to assess arm and shoulder girdle strength for 35 years despite little evidence to support its use. The purpose of this study was to determine what muscular fitness component, if any, was related to the FAH. The traditional overgrip chin-above-bar test and 5 different variations were compared with absolute strength (1 repetition maximum [1RM] lat pull down), relative strength (1RM.mass(-1)), and muscle endurance (repetitions to failure at 70% of the 1RM). Sixty college-age women volunteered for the study. Relationships were examined using Pearson Product Moment Correlation. No significant relationship was found between any of the FAH variations and absolute strength or muscle endurance; however, all FAH variations correlated significantly with relative strength (1RM.mass(-1)). The strongest relationship was with the undergrip FAH timed to 90 degrees of elbow extension (r = 0.72). Investigators concluded that the FAH is a test of weight-relative muscular strength and appears unrelated to absolute strength or muscle endurance.     

Conserved signature proposed for folding in the lipocalin superfamily

We systematically identify a group of evolutionarily conserved residues proposed for folding in a model beta-barrel superfamily, the lipocalins. The nature of conservation at the structural level is defined and we show that the conserved residues are involved in a network of interactions that form the core of the fold. Exploratory kinetic studies are conducted with a model superfamily member, human serum retinol-binding protein, to examine their role. The present results, coupled with key experimental studies conducted with another lipocalin beta-lactoglobulin, suggest that the evolutionarily conserved regions fold on a faster folding time-scale than the non-conserved regions.     

In Situ Biosurfactant Production by Bacillus Strains Injected into a Limestone Petroleum Reservoir

Biosurfactant-mediated oil recovery may be an economic approach for recovery of significant amounts of oil entrapped in reservoirs, but evidence that biosurfactants can be produced in situ at concentrations needed to mobilize oil is lacking. We tested whether two Bacillus strains that produce lipopeptide biosurfactants can metabolize and produce their biosurfactants in an oil reservoir. Five wells that produce from the same Viola limestone formation were used. Two wells received an inoculum (a mixture of Bacillus strain RS-1 and Bacillus subtilis subsp. spizizenii NRRL B-23049) and nutrients (glucose, sodium nitrate, and trace metals), two wells received just nutrients, and one well received only formation water. Results showed in situ metabolism and biosurfactant production. The average concentration of lipopeptide biosurfactant in the produced fluids of the inoculated wells was about 90 mg/liter. This concentration is approximately nine times the minimum concentration required to mobilize entrapped oil from sandstone cores. Carbon dioxide, acetate, lactate, ethanol, and 2,3-butanediol were detected in the produced fluids of the inoculated wells. Only CO₂ and ethanol were detected in the produced fluids of the nutrient-only-treated wells. Microbiological and molecular data showed that the microorganisms injected into the formation were retrieved in the produced fluids of the inoculated wells. We provide essential data for modeling microbial oil recovery processes in situ, including growth rates (0.06 ± 0.01 h⁻¹), carbon balances (107% ± 34%), biosurfactant production rates (0.02 ± 0.001 h⁻¹), and biosurfactant yields (0.015 ± 0.001 mol biosurfactant/mol glucose). The data demonstrate the technical feasibility of microbial processes for oil recovery.     

P-selectin and CD63 use different mechanisms for delivery to Weibel-Palade bodies

The biogenesis of endothelial-specific Weibel-Palade bodies (WPB) is poorly understood, despite their key role in both haemostasis and inflammation. Biogenesis of specialized organelles of haemopoietic cells is often adaptor protein complex 3-dependent (AP-3-dependent), and AP-3 has previously been shown to play a role in the trafficking of both WPB membrane proteins, P-selectin and CD63. However, WPB are thought to form at the trans Golgi network (TGN), which is inconsistent with a role for AP-3, which operates in post-Golgi trafficking. We have therefore investigated in detail the mechanisms of delivery of these two membrane proteins to WPB. We find that P-selectin is recruited to forming WPB in the trans-Golgi by AP-3-independent mechanisms that use sorting information within both the cytoplasmic tail and the lumenal domain of the receptor. In contrast, CD63 is recruited to already-budded WPB by an AP-3-dependent route. These different mechanisms of recruitment lead to the presence of distinct immature and mature populations of WPB in human umbilical vein endothelial cells (HUVEC).     

Construction of a Genetic Linkage Map and Genetic Analysis of Domestication Related Traits in Mungbean (Vigna radiata)

The genetic differences between mungbean and its presumed wild ancestor were analyzed for domestication related traits by QTL mapping. A genetic linkage map of mungbean was constructed using 430 SSR and EST-SSR markers from mungbean and its related species, and all these markers were mapped onto 11 linkage groups spanning a total of 727.6 cM. The present mungbean map is the first map where the number of linkage groups coincided with the haploid chromosome number of mungbean. In total 105 QTLs and genes for 38 domestication related traits were identified. Compared with the situation in other Vigna crops, many linkage groups have played an important role in the domestication of mungbean. In particular the QTLs with high contribution were distributed on seven out of 11 linkage groups. In addition, a large number of QTLs with small contribution were found. The accumulation of many mutations with large and/or small contribution has contributed to the differentiation between wild and cultivated mungbean. The useful QTLs for seed size, pod dehiscence and pod maturity that have not been found in other Asian Vigna species were identified in mungbean, and these QTLs may play the important role as new gene resources for other Asian Vigna species. The results provide the foundation that will be useful for improvement of mungbean and related legumes.     

Orientation to solar radiation in black wildebeest (<Emphasis Type="Italic">Connochaetes gnou</Emphasis>)

We recorded the body axis orientation of free-living black wildebeest relative to incident solar radiation and wind. Observations were made on three consecutive days, on six occasions over the course of 1 year, in a treeless, predominantly cloudless habitat. Frequency of orientation parallel to incident solar radiation increased, and perpendicular to incident solar radiation decreased, as ambient dry-bulb temperature or solar radiation intensity increased, or wind speed decreased. We believe these changes were mediated via their effect on skin temperature. Parallel orientation behavior was more prominent when the wildebeest were standing without feeding than it was when they were feeding. We calculate that a black wildebeest adopting parallel orientation throughout the diurnal period would absorb 30% less radiant heat than the same animal adopting perpendicular orientation. Parallel orientation was reduced at times when water was freely available, possibly reflecting a shift from behavioral to autonomic thermoregulatory mechanisms. The use of orientation behavior by black wildebeest is well developed and forms part of the suite of adaptations that help them to maintain heat balance while living in a shadeless, often hot, environment.     

N-terminal region of gelsolin induces apoptosis of activated hepatic stellate cells by a caspase-dependent mechanism

Activated hepatic stellate cells (HSCs) are the major source for alteration of extracellular matrix in fibrosis and cirrhosis. Conditioned medium (CM) collected from immortalized human hepatocytes (IHH) have earlier been shown to be responsible for apoptosis of HSCs. In this study, we have shown that antibodies raised against a peptide derived from a linear B-cell epitope in the N-terminal region of gelsolin identified a gelsolin fragment in IHH CM. Analysis of activated stellate cell death by CM collected from Huh7 cells transfected with plasmids encoding gelsolin deletion mutants suggested that the N-terminal half of gelsolin contained sequences which were responsible for stellate cell death. Further analysis determined that this activity was restricted to a region encompassing amino acids 1-70 in the gelsolin sequence; antibody directed to an epitope within this region was able to neutralize stellate cell death. Gelsolin modulation of cell death using this fragment involved upregulation of TRAIL-R1 and TRAIL-R2, and involved caspase 3 activation by extrinsic pathway. The apoptotic activity of N-terminal gelsolin fragments was restricted to activated but not quiescent stellate cells indicating its potential application in therapeutic use as an anti-fibrotic agent. Gelsolin fragments encompassing N-terminal regions in polypeptides of different molecular sizes were detected by N-terminal peptide specific antiserum in IHH CM immunoprecipitated with chronically HCV infected patient sera, suggesting the presence of autoantibodies generated against N-terminal gelsolin fragments in patients with chronic liver disease.