Computational Design of TrkB Peptide Inhibitors and Their Biological Effects on Ovarian Cancer Cell Lines

There are large numbers of different intracellular signaling pathways regulated by Tyrosine kinases (Trk) receptors. Trk receptors, especially TrkB, are also frequently overexpressed in a variety of human malignant tumors. In this study, we have computationally designed small peptide-based inhibitors of TrkB and investigated their effects on the proliferation and apoptosis of two ovarian cancer cell lines. Molecular docking of TrkB with its ligand and antagonist, BDNF and Cyclotraxin B respectively, was carried out using HADDOCK program. A peptide library was constructed based on the critical residues involved in the TrkB binding site. After docking and optimization, two selected peptides were purchased and their effects on the viability and apoptosis of the cells were evaluated by performing MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test and flow cytometry assay. Subsequently, the levels of expression and phosphorylation statues of TrkB and its two downstream genes including MAPK3 and eIF4E were assessed with western blot. We found that designed peptides effectively reduced TrkB, MAPK3 and eIF4E phosphorylation, reduced cell viability and induced apoptosis in the treated cells when compared to untreated cells. In conclusion, the BDNF/TrkB signaling is shown to be attenuated substantially in the presence of peptide inhibitors suggesting a strong inhibitory potential of the designed peptides for Trk family.     

Effects of alternate RNA splicing on glucokinase isoform activities in the pancreatic islet, liver, and pituitary

Different glucokinase isoforms are produced by tissue-specific alternative RNA splicing in the liver and pancreatic islet, the only tissues in which glucokinase activity has been detected. To determine whether differences in protein structure brought about by alternative RNA splicing have an effect on glucose phosphorylating activity, we expressed cDNAs encoding four different hepatic and islet glucokinase isoforms and determined the Km and Vmax of each. When the glucokinase B1 and L1 isoforms were expressed in eukaryotic cells, both high Km glucose phosphorylating activity and immunoreactive protein were detected. However, when the glucokinase B2 and L2 isoforms were expressed, both of which differ by deletion of 17 amino acids in a region between the putative glucose and ATP-binding domains, no high Km glucose phosphorylating activity and much less immunoreactive protein were detected. When the glucokinase B1 and B2 isoforms were expressed in Escherichia coli as fusion proteins with glutathione S-transferase, affinity-purified B1 fusion protein was able to phosphorylate glucose whereas the B2 fusion protein was not, thus indicating that the lack of glucose phosphorylating activity from both the B2 and L2 isoforms is due to lack of intrinsic activity in addition to accumulation of less protein. The Km values of the B1 and L1 isoforms, which differ from each other by 15 amino acids at the NH2 terminus, were similar, but the Vmax of the B1 isoform was 2.8-fold higher than that of the L1 isoform. Mutagenesis of the first two potential initiation codons in the glucokinase B1 cDNA from ATG to GTC (methionine to valine) indicated that the first ATG was crucial for activity and is, therefore, the likely translation initiation codon. Messenger RNAs encoding both the B2 and L2 isoforms of glucokinase were detected in islet and liver by polymerase chain reaction amplification of total cDNA, indicating that mRNAs utilizing this weak alternate splice acceptor site in the fourth exon are normally present in both the liver and islet but as minor components. A regulatory role for weak alternate splice acceptor and donor sites in the glucokinase gene was suggested by examining the expression of the gene in the pituitary and in AtT-20 cells. Interestingly, although glucokinase mRNAs of appropriate sizes were detected in both the AtT-20 cells and rat pituitaries, neither exhibited any detectable high Km glucose phosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Desert hedgehog is a mammal-specific gene expressed during testicular and ovarian development in a marsupial

Background

Sphingosine-1-phosophate (S1P) is a biologically active sphingolipid metabolite that influences cellular events including differentiation, proliferation, and migration. S1P acts through five distinct cell surface receptors designated S1P_1-5R, with S1P₁R having the highest expression level in the developing heart. S1P₁R is critical for vascular maturation, with its loss leading to embryonic death by E14.5; however, its function during early cardiac development is not well known. Our previous studies demonstrated that altered S1P levels adversely affects atrioventricular (AV) canal development in vitro, with reduced levels leading to cell death and elevated levels inhibiting cell migration and endothelial to mesenchymal cell transformation (EMT).

Results

We determined, by real-time PCR analysis, that S1P₁R was expressed at least 10-fold higher than other S1P receptors in the developing heart. Immunohistochemical analysis revealed S1P₁R protein expression in both endothelial and myocardial cells in the developing atrium and ventricle. Using AV canal cultures, we observed that treatment with either FTY720 (an S1P_1,3,4,5R agonist) or KRP203 (an S1P₁R-specific agonist) caused similar effects on AV canal cultures as S1P treatment, including induction of cell rounding, inhibition of cell migration, and inhibition of EMT. In vivo, morphological analysis of embryonic hearts at E10.5 revealed that S1P₁R-/- hearts were malformed with reduced myocardial tissue. In addition to reduced myocardial tissue, E12.5 S1P₁R-/- hearts had disrupted morphology of the heart wall and trabeculae, with thickened and disorganized outer compact layer and reduced fibronectin (FN) deposition compared to S1P₁R+/+ littermates. The reduced myocardium was accompanied by a decrease in cell proliferation but not an increase in apoptosis.

Conclusions

These data indicate that S1P₁R is the primary mediator of S1P action in AV canal cultures and that loss of S1P₁R expression in vivo leads to malformed embryonic hearts, in part due to reduced fibronectin expression and reduced cell proliferation.     

Effect of glucose oxidase on decolorization efficiency of crystal violet by Phanerochaete chrysosporium cultures

An enzymatic reaction using glucose oxidase (GOx) was applied for continues production of hydrogen peroxide and organic acid in Phanerochaete chrysosporium cultures for use simultaneously in catalytic cycle of peroxidases. Decolorization efficiency of crystal violet (CV) as a model pollutant was investigated in 16 d old cultures which overproduced manganese peroxidase (MnP) in response to daily GOx addition and control cultures (i.e. no GOx was added). However, the ability of overproduced cultures in decolorization of CV was not increased significantly, through addition of GOx (300?U/L)?+?glucose (10?Mm) to the culture medium at the start of decolorization, the time needed to obtain 87?±?0.5% removal of CV was reduced 10.7-fold in compared with the control culture. The best GOx concentration in culture medium for more efficient decolorization was obtained to be 300?U/L. These findings indicated that GOx in the presence of glucose could increase the degradation of CV not only by inducing ligninolytic activity in cultures but also as a subsidiary source for in situ H₂O₂ and organic acid production for catalytic activity of peroxidases in P. chrysosporium cultures.     

Doped‐MoSe2 Nanoflakes/3d Metal Oxide–Hydr(Oxy)Oxides Hybrid Catalysts for pH‐Universal Electrochemical Hydrogen Evolution Reaction

Clean hydrogen production is highly promising to meet future global energy demands. The design of earth‐abundant materials with both high activity for hydrogen evolution reaction (HER) and electrochemical stability in both acidic and alkaline environments is needed, in order to enable practical applications. Here, the authors report a non‐noble 3d metal Cl‐chemical doping of liquid phase exfoliated single‐/few‐layer flakes of MoSe₂ for creating MoSe₂/3d metal oxide–hydr(oxy)oxide hybrid HER‐catalysts. It is proposed that the electron‐transfer from MoSe₂ nanoflakes to metal cations and the chlorine complexation‐induced neutralization, as well as the in situ formation of metal oxide–hydr(oxy)oxides on the MoSe₂ nanoflakes' surface, tailor the proton affinity of the catalysts, increasing the number and HER‐kinetics of their active sites in both acidic and alkaline electrolytes. The electrochemical coupling between doped‐MoSe₂/metal oxide–hydr(oxy)oxide hybrids and single‐walled carbon nanotubes heterostructures further accelerates the HER process. Lastly, monolithic stacking of multiple heterostructures is reported as a facile electrode assembly strategy to achieve overpotential for a cathodic current density of 10 mA cm^?2 of 0.081 and 0.064 V in 0.5 m H₂SO₄ and 1 m KOH, respectively. This opens up new opportunities to address the current density versus overpotential requirements targeted in pH‐universal hydrogen production.     

Stem cells or their exosomes: which is preferred in COVID-19 treatment?

Biotechnology Letters - It only took 8 months for the pneumonia caused by a previously unknown coronavirus to turn into a global pandemic of unprecedentedly far-reaching implications. Failure of...