Purification and characterization of a pp60c-src-related tyrosine kinase that effectively phosphorylates a synthetic peptide derived from p34cdc2.

A protein tyrosine kinase has been purified from the particulate fraction of bovine spleen to a specific activity of 0.217 mumol/min/mg at 100 microM ATP and 3 mM [Val5] angiotensin II. Both the angiotensin phosphorylation activity and immunoreactivity towards an antibody preparation raised against a synthetic peptide containing the autophosphorylation site of pp60c-src, Cys-src(403-421), were monitored during the purification. The purified sample displayed three closely spaced protein bands with molecular weights of 50-55 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All bands could be phosphorylated exclusively on tyrosine residues under autophosphorylation conditions. All reacted on immunoblots with an antibody raised against a synthetic peptide corresponding to the consensus autophosphorylation site of members of the pp60c-src family of tyrosine kinases. Tryptic phosphopeptide maps of the three proteins were essentially indistinguishable. The results suggest that the purified enzyme preparation contained mainly three closely related pp60c-src-family protein tyrosine kinases or a pp60src-family protein tyrosine kinase modified posttranslationally to give three closely spaced protein bands on sodium dodecyl sulfate gel. Neither of these proteins appears to be pp60c-src or p56lck. The spleen protein tyrosine kinase was found to phosphorylate a p34cdc2 kinase peptide, Cys-cdc2(8-20), which contained the regulatory tyrosine residue Tyr-15 about 20 times better than [Val5]angiotensin II or Cys-src(403-421) peptide at a peptide substrate concentration of 1 mM. In contrast, epidermal growth factor receptor kinase partially purified from A431 cells did not show preference for Cys-cdc2(8-20) as its substrate. Although Cys-cdc2(8-20) contained two tyrosine residues, only the tyrosine corresponding to Tyr-15 in p34cdc2 was phosphorylated by the spleen tyrosine kinase. The observation suggests that the primary structure surrounding Tyr-15 of p34cdc2 contains substrate structural determinants specific for the spleen tyrosine kinase.     

Serum-free medium for fermentor cultures of hybridomas

Hybridomas lend themselves particularly well to large scale cultivation techniques since they grow as single cells in suspension without requiring attachment to a substrate. Furthermore, many cell strains have been adapted to grow in serum-free (SF) media to a similar cell density and antibody production as in serum containing media. This review will concern itself mainly with the cultivation of hybridomas in SF-media in bioreactors of various types with the ultimate goal of producing large quantities of monoclonal antibodies (mAb).     

Use of activated clotting time for monitoring anticoagulation during cardiopulmonary bypass in infants and children with congenital heart disease

The use of a fixed dosage schedule was compared with the use of activated clotting time (ACT) for determining heparin and protamine dosages during and after cardiopulmonary bypass disease. Use of the ACT resulted in a statistically significant increase in heparin dosage and a statistically significant reduction of postoperative blood loss. With ACT use, chest tubes were retained for a shorter period of time, and the incidence of serious postoperative hemorrhage was reduced from 44% to 18%. These results confirm the superiority of the ACT method for monitoring intraoperative anticoagulation in pediatric patients with congenital heart disease.     

Mutational analysis in podocin-associated hereditary nephrotic syndrome in Polish patients: founder effect in the Kashubian population

Hereditary nephrotic syndrome is caused by mutations in a number of different genes, the most common being NPHS2. The aim of the study was to identify the spectrum of NPHS2 mutations in Polish patients with the disease. A total of 141 children with steroid-resistant nephrotic syndrome (SRNS) were enrolled in the study. Mutational analysis included the entire coding sequence and intron boundaries of the NPHS2 gene. Restriction fragment length polymorphism (RFLP) and TaqMan genotyping assay were applied to detect selected NPHS2 sequence variants in 575 population-matched controls. Twenty patients (14 %) had homozygous or compound heterozygous NPHS2 mutations, the most frequent being c.1032delT found in 11 children and p.R138Q found in four patients. Carriers of the c.1032delT allele were exclusively found in the Pomeranian (Kashubian) region, suggesting a founder effect origin. The 14 % NPHS2 gene mutation detection rate is similar to that observed in other populations. The heterogeneity of mutations detected in the studied group confirms the requirement of genetic testing the entire NPHS2 coding sequence in Polish patients, with the exception of Kashubs, who should be initially screened for the c.1032delT deletion.     

A Novel Attempt to Standardize Results of CFD Simulations Basing on Spatial Configuration of Aortic Stent-Grafts

Currently, studies connected with Computational Fluid Dynamic (CFD) techniques focus on assessing hemodynamic of blood flow in vessels in different conditions e.g. after stent-graft’s placement. The paper propose a novel method of standardization of results obtained from calculations of stent-grafts'' “pushing forces” (cumulative WSS—Wall Shear Stress), and describes its usefulness in diagnostic process. AngioCT data from 27 patients were used to reconstruct 3D geometries of stent-grafts which next were used to create respective reference cylinders. We made an assumption that both the side surface and the height of a stent-graft and a reference cylinder were equal. The proposed algorithm in conjunction with a stent-graft “pushing forces” on an implant wall, allowed us to determine which spatial configuration of a stent-graft predispose to the higher risk of its migration. For stent-grafts close to cylindrical shape (shape factor φ close to 1) WSS value was about 267Pa, while for stent-grafts different from cylindrical shape (φ close to 2) WSS value was about 635Pa. It was also noticed that deformation in the stent-graft’s bifurcation part impaired blood flow hemodynamic. Concluding the proposed algorithm of standardization proved its usefulness in estimating the WSS values that may be useful in diagnostic process. Angular bends or tortuosity in bifurcations of an aortic implant should be considered in further studies of estimation of the risk of implantation failure.     

Comprehensive review of antimicrobial activities of plant flavonoids

Flavonoids are one of the largest classes of small molecular secondary metabolites produced in different parts of the plant. They display a wide range of pharmacological and beneficial health effects for humans, which include, among others, antioxidative activity, free radical scavenging capacity, coronary heart disease prevention and antiatherosclerotic, hepatoprotective, anti-inflammatory, and anticancer activities. Hence, flavonoids are gaining high attention from the pharmaceutical and healthcare industries. Notably, plants synthesize flavonoids in response to microbial infection, and these compounds have been found to be a potent antimicrobial agent against a wide range of pathogenic microorganisms in vitro. Antimicrobial action of flavonoids results from their various biological activities, which may not seem very specific at first. There are, however, promising antibacterial flavonoids that are able not only to selectively target bacterial cells, but also to inhibit virulence factors, as well as other forms of microbial threats, e.g. biofilm formation. Moreover, some plant flavonoids manifest ability to reverse the antibiotic resistance and enhance action of the current antibiotic drugs. Hence, the development and application of flavonoid-based drugs could be a promising approach for antibiotic-resistant infections. This review aims to improve our understanding of the biological and molecular roles of plant flavonoids, focusing mostly on their antimicrobial activities.