Expression of a bean storage protein 'phaseolin minigene' in foreign plant tissues

Using the phaseolin gene and its cDNA counterpart we constructed a mutant phaseolin gene lacking the five introns but retaining its natural 5' and 3' plant-regulatory sequences. This mutant phaseolin gene (minigene) was inserted into the Ti-plasmid of Agrobacterium tumefaciens strain 15955 which allowed its transfer and integration into the tobacco genome. Full-length and correctly initiated phaseolin mRNA was found among the poly(A)+RNA isolated from plant callus transformed with the minigene construction by using RNA-DNA hybridization and S1 nuclease mapping techniques. The presence of phaseolin polypeptides in soluble protein extracts from transformed tobacco tissues was confirmed by immunological methods. These results demonstrate that phaseolin gene introns and intron splicing are not a necessary requirement for biogenesis of stable phaseolin mRNA and that no alternative splice site was introduced by the removal of five introns.     

Determination of Pore-Lining Residues in the Hepatitis C Virus p7 Protein

The p7 protein from hepatitis C virus is critical for the assembly and secretion of infectious virus, making it an attractive drug target. It is thought to be a viroporin with a demonstrated ion channel activity when reconstituted into planar lipid bilayers. Electron microscopy experiments suggest that p7 oligomers coexist as hexamers and heptamers. Proposed models of p7 oligomers assume the N-terminal helix to be the pore lining helix. Here, we demonstrate, via electrophysiology, that Cu²⁺ has an inhibitory effect on the p7 ion channel and that the amino acid responsible for this inhibition is one histidine in each monomer. This information coupled with the p7 sequence data suggests that the N-terminal helix of p7 does indeed form the transmembrane pore and that this histidine is pore-lining. The information will aid in the construction of oligomeric pore-models and the interpretation of electron microscopy data.     

Recombinant Human erythropoietin reduces viability of MCF-7 breast cancer cells from 3D culture without caspase activation

Recombinant human erythropoietin (rHuEPO) is the erythropoiesis-stimulating hormone that is being used concurrently with chemotherapeutic drugs in the treatment of anemia of cancer. The effect of rHuEPO on cancer cells in 3-dimensional (3D) cultures is not known. The objective of the study was to determine the effect of rHuEPO on the viability of MCF-7 breast cancer cells from 2-dimensional (2D) and 3D cell cultures. The monolayer MCF-7 cells from 2D culture and MCF-7 cell from 3D culture generated by ultra-low adhesive microplate technique, were treated with 0, 0.1, 10, 100 or 200 IU/mL rHuEPO for 24, 48 or 72 h. The effects of rHuEPO on MCF-7 cell viability and proliferation were determined using the (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT), neutral red retention time (NRRT), trypan blue exclusion assay (TBE), DNA fragmentation, acridine orange/propidium iodide staining (AO/PI) assays. The MCF-7 cells for 3D culture were also subjected to caspase assays and cell cycle analysis using flow cytometry. rHuEPO appeared to have greater effect at lowering the viability of MCF-7 cells from 3D than 2D cultures.rHuEPO significantly (p < 0.05) decreased viability and down-regulated the caspase activities of 3D MCF-7 cells in dose- and time-dependent manner. The cell cycle analysis showed that rHuEPO caused MCF-7 cells to enter the subG0/G1 phase. Thus, the study suggests that rHuEPO has a cytostatic effect on the MCF-7 breast cancer cells from 3D culture.     

Early induction of functional SARS-CoV-2-specific T cells associates with rapid viral clearance and mild disease in COVID-19 patients

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Independent Bottlenecks Characterize Colonization of Systemic Compartments and Gut Lymphoid Tissue by Salmonella

Vaccination represents an important instrument to control typhoid fever in humans and protects mice from lethal infection with mouse pathogenic serovars of Salmonella species. Mixed infections with tagged Salmonella can be used in combination with probabilistic models to describe the dynamics of the infection process. Here we used mixed oral infections with tagged Salmonella strains to identify bottlenecks in the infection process in naïve and vaccinated mice. We established a next generation sequencing based method to characterize the composition of tagged Salmonella strains which offers a fast and reliable method to characterise the composition of genome-tagged Salmonella strains. We show that initial colonization of Salmonella was distinguished by a non-Darwinian selection of few bacteria setting up the infection independently in gut associated lymphoid tissue and systemic compartments. Colonization of Peyer''s patches fuels the sustained spread of bacteria into mesenteric lymph nodes via dendritic cells. In contrast, infection of liver and spleen originated from an independent pool of bacteria. Vaccination only moderately reduced invasion of Peyer''s patches but potently uncoupled bacterial populations present in different systemic compartments. Our data indicate that vaccination differentially skews the capacity of Salmonella to colonize systemic and gut immune compartments and provide a framework for the further dissection of infection dynamics.     

Forty-eight hours hypothermic storage of whole canine pancreas allografts. Improved preservation with a colloid hyperosmolar solution.

This study indicates that crystalloid (Sacks') and colloid (modified silica gel fraction of plasma, MSGF) hyperosmolar solutions are edequate means to preserve whole pancreas allografts under hypothermia (4 to 7 °C) for 24 hr. These groups of preserved canine pancreas allografts survived as long as the fresh transplanted pancreas. However, when the preservation was extended to 48 hr, the crystalloid hyperosmolar solution did not protect the pancreas allografts, but the colloid hyperosmolar solution gave similar results to fresh pancreas allografts.     

Effects of Amyloid Precursor Protein Overexpression on NF-κB,Rho-GTPase and Pro-Apoptosis Bcl-2 Pathways in Neuronal Cells

Background:Alzheimer’s disease (AD) is a neurodegenerative disorder that causes cognitive dysfunction. Previous studies have suggested that amyloid plaques, mainly comprising of amyloid-beta peptides, play a pivotal role in AD pathophysiology. This study focuses on the evaluation of the effects of amyloid precursor protein (APP) overexpression on NF-κB, Rho-GTPase and Bcl-2 mediated pro-apoptotic pathways in neuronal cells. Methods:A lentiviral transduction system was used to generate SH-SY5Y cells overexpressing APP. Immunoblotting was conducted to determine expression levels of NF-κB, Rho-GTPase, and Bcl-2 family proteins in the APP overexpressed cells.Results:In the NF-κB signaling pathway, APP-overexpressing SH-SY5Y cells showed that there was a reduction of p-NF-κB (p< 0.05) and IKKα. Subsequently, there was upregulation of protein expression of NF-Κb, IKKβ and IκBα. On the other hand, protein expression of RhoC (p< 0.05) and Rac1/2/3 was upregulated as compared to the control group. Meanwhile, a decrease in RhoA, Cdc42 (p< 0.05) and p-Rac1/cdc42 protein levels was observed in the APP-overexpressed group. Lastly, in the pro-apoptotic pathway, the expression of Bcl-2, Bid, Bok and Puma (p< 0.05) was up regulated in the APP-overexpressed group. Downregulation of Bad and Bim expression was observed in the APP-overexpressed as compared to the control group, and Bax expression remained unchanged in the APP-overexpressed group.Conclusion:APP overexpression regulated signaling in the NF-κB, Rho-GTPase and Bcl-2 family pathways in neuronal cells, suggesting that these are involved in promoting neuronal survival and modulating synaptic plasticity in AD. However, further studies are essential to elucidate the APP-mediated mechanism of action.Key Words: Alzheimer’s disease, Amyloid precursor protein, Bcl-2 family proteins, NF-κB, Rho-GTPase     

Hybridization properties of immobilized nucleic acids.

The 5'-end attachment of oligonucleotides to dextran supports facilitates the study of the hybridization properties of an immobilized oligonucleotide system. The hybridization properties which were studied include: hybridization capacity and kinetics, hybridization-complex stability, and reagents influencing hybridization efficiency. Results of these experiments reveal that the hybridization efficiencies of support-bound oligonucleotides were 75-80% and 40-50% for single-stranded oligonucleotide targets and long double-stranded targets, respectively. These hybridization efficiencies are dependent upon prehybridizing the support-bound oligonucleotides with dextran sulfate. In addition, comparisons of the relative hybridization efficiencies of the support-bound oligonucleotide and nitrocellulose-based systems have been made which indicate a retention of 13-28% of target sequences on the filters and a detection efficiency of 8-20%.