Aspartic proteinases: Fourier transform infrared spectroscopic studies of a model of the active side.

We synthesized and studied by Fourier transform infrared spectroscopy nine monosalts of diamides as models for the active side of aspartic proteinases. One compound, the monosalt of meta-aminobenzoic acid diamide of fumaric acid (m-FUM), shows the same biological activity as pepsin with regard to the splitting of peptide bonds of the Pro-Thi-Glu-Phe-Phe(4-NO2)-Arg-Leu heptapeptide. The monosalt of m-FUM forms with oxindole a complex in which the carboxylic acid group of the monosalt of m-FUM is strongly hydrogen bonded with the O atom of the peptide bond of oxindole. When one water molecule is added to this complex, the strong field of the carboxylate group destabilizes an O-H bond of the water molecule. The distorted water molecule attacks the carbon atom of the peptide group, and the water proton transfers to the peptide N atom. Simultaneously, the C-N bond of the amide group is broken. Hence it is demonstrated that the catalytic mechanism of aspartic acid proteinases is a base catalysis. The results show that for this catalytic mechanism there are sufficient carboxylic and carboxylate groups, as well as a water molecule in the correct arrangement. It was also demonstrated with other monosalts of dicarboxylic acids that well-defined steric conditions of the carboxylic acid and the carboxylate group must be fulfilled to show hydrolytic activity with regard to oxindole molecules.     

Inhibition of intestinal ischemia/repurfusion induced apoptosis and necrosis via down-regulation of the NF-kB,c-Jun and caspace-3 expression by epigallocatechin-3-gallate administration

Intestinal ischemia/reperfusion (I/R) produces reactive oxygen species (ROS) activating signal transduction and apoptosis. The aim of this study was to evaluate the effect of (?)-epigallocatechin-3-gallate (EGCG) administration in inhibition of apoptosis by attenuating the expression of NF-kB, c-Jun and caspace-3 in intestinal I/R. Thirty male wistar rats were used. Group A sham operation, B I/R, C I/R-EGCG 50 mg/kg ip. Intestinal ischemia was induced for 60 min by clamping the superior mesenteric artery. Malondialdehyde (MDA), myeloperoxidase (MPO), light histology, Fragment End Labelling of DNA (TUNEL), immunocytochemistry for NF-kB, c-Jun and caspace-3 analysis in intestinal specimens were performed 120 min after reperfusion. Apoptosis as indicated by TUNEL and Caspace-3, NF-kB and c-Jun was widely expressed in I/R group but only slightly expressed in EGCG treated groups. MDA and MPO showed a marked increase in the I/R group and a significant decrease in the EGCG treated group. Light histology showed preservation of architecture in the EGCG treated group. In conclusion, EGCG pre-treatment is likely to inhibit intestinal I/R-induced apoptosis by down-regulating the expression of NF-kB, c-Jun and caspase-3.     

Antiproliferative activity of L-asparaginase of Tetrahymena pyriformis on human breast cancer cell lines

Purified L-asparaginase of Tetrahymena pyriformis is a multi-subunit enzyme exhibiting protein kinase activity as well. The enzyme's L-asparaginase activity is affected by its phosphorylation state. Both native and dephosphorylated L-asparaginase show antiproliferative activity on three breast cancer cell lines (T47D, BT20 and MCF-7) and on Walker 256 cells. These cells do not possess measurable L-asparaginase or L-asparagine synthetase activity. When T47D cells are treated for different times with L-asparaginase and then placed in fresh medium, the growth of cells treated for 1, 3, or 6 hours is initiated and parallels control curve, while the growth of cells treated for 24 or 48 hours with L-asparaginase stays at the same inhibitory level (24 h treatment) or continues to drop (48 h treatment). Addition of D-asparagine, a competitive inhibitor of T. pyriformis L-asparaginase, counteracts the antiproliferative activity of L-asparaginase, indicating that L-asparaginase and not the kinase activity is responsible for that effect.     

Resection of Tarsal Coalition in 27 Children with 2 Years Follow-Up – Patient-Reported Outcomes Using the Validated Oxford Ankle Foot Questionnaire

BackgroundPatient Reported Outcome Measures (PROM) after resection of tarsal coalitions are sparse. This cross-sectional study evaluates the outcome after resection of tarsal coalitions in children using the validated Oxford Foot and Ankle Questionnaire (OxAFQ).MethodsTarsal coalition patients between 5-16 years of age from Aarhus University Hospital (Denmark) and The Royal London Hospital (United Kingdom) were included. The patients were identified using patient and theatre register. All patients and proxies filled in the PROM: OxAFQ-C and OxAFQ-proxy respectively. The scores were calculated within each domain and reported as means (95% confidence intervals). Talocalcaneal coalitions were compared to calcaneonavicular coalition with regard to OxAFQ score and re-operation rate.Results27 patients and their proxies returned 54 questionnaires in total regarding 36 feet. Mean time from surgery to filling of the questionnaire was 25 (21-30) months. The relative mean OxAFQ score was higher in the School and Play and Emotional domain than the Physical domain, p = 0.007. The OxAFQ scores and re-operation rates were similar for both coalitions, p=0.63.ConclusionThe OxAFQ PROM showed more encouraging results in playing or emotional health status than the physical health status. The outcome for both types of coalitions is similar.Level of Evidence: IV     

A sequential Monte Carlo framework for haplotype inference in CNV/SNP genotype data

Copy number variations (CNVs) are abundant in the human genome. They have been associated with complex traits in genome-wide association studies (GWAS) and expected to continue playing an important role in identifying the etiology of disease phenotypes. As a result of current high throughput whole-genome single-nucleotide polymorphism (SNP) arrays, we currently have datasets that simultaneously have integer copy numbers in CNV regions as well as SNP genotypes. At the same time, haplotypes that have been shown to offer advantages over genotypes in identifying disease traits even though available for SNP genotypes are largely not available for CNV/SNP data due to insufficient computational tools. We introduce a new framework for inferring haplotypes in CNV/SNP data using a sequential Monte Carlo sampling scheme ‘Tree-Based Deterministic Sampling CNV’ (TDSCNV). We compare our method with polyHap(v2.0), the only currently available software able to perform inference in CNV/SNP genotypes, on datasets of varying number of markers. We have found that both algorithms show similar accuracy but TDSCNV is an order of magnitude faster while scaling linearly with the number of markers and number of individuals and thus could be the method of choice for haplotype inference in such datasets. Our method is implemented in the TDSCNV package which is available for download at http://www.ee.columbia.edu/~anastas/tdscnv.     

An interleukin-6 ZnO/SiO(2)/Si surface acoustic wave biosensor

A novel high sensitivity ZnO/SiO(2)/Si Love mode surface acoustic wave (SAW) biosensor for the detection of interleukin-6 (IL-6), is reported. The biosensors operating at 747.7MHz and 1.586GHz were functionalized by immobilizing the monoclonal IL-6 antibody onto the ZnO biosensor surface both through direct surface adsorption and through covalent binding on gluteraldehyde. The morphology of the IL-6 antibody-protein complex was studied using scanning electron microscopy (SEM), and the mass of the IL-6 protein immobilized on the surface was measured from the frequency shift of the SAW resonator biosensor. The biosensor was shown to have extended linearity, which was observed to improve with higher sensor frequency and for IL-6 immobilization through the monoclonal antibody. Preliminary results of biosensor measurements of low levels of IL-6 in normal human serum are reported. The biosensor can be fully integrated with CMOS Si chips and developed as a portable real time detection system for the interleukin family of proteins in human serum.     

Attenuation of intestinal ischemia/reperfusion induced liver and lung injury by intraperitoneal administration of (-)-epigallocatechin-3-gallate

The aim of this study was to evaluate the effect of ( - )-epigallocatechin-3-gallate (EGCG), a natural antioxidant, on liver and lungs after warm intestinal ischemia/reperfusion (I/R). Thirty male Wistar rats were equally divided into a sham-operation group, an intestinal I/R group and an intestinal I/R group pretreated with EGCG intraperitoneally. Intestinal ischemia was induced by occlusion of the superior mesenteric artery for 60 min followed by reperfusion for 120 min. Immediately after reperfusion, liver, lung and blood samples were collected and analyzed. Results showed that intestinal I/R increased the levels of aspartate (AST) and alanine (ALT) transaminase in serum to 987 and 752 IU/l, respectively. Malondialdehyde (MDA) increased in liver to 1.524 nmol/g in the group subjected to intestinal I/R compared to 0.995 nmol/g in the sham operation group. MDA was also increased in lungs to 1.581 nmol/g compared to 0.896 nmol/g in the sham operation group. Myeloperoxidase (MPO) increased in liver, after intestinal I/R, to 5.16 U/g compared to 1.59 U/g in the sham operation group. MPO was also increased in lungs to 3.89 U/g compared to 1.65 U/g in the sham operation group. Pretreatment with EGCG decreased serum levels of AST and ALT to 236 and 178 IU/l, respectively. It also decreased mean MDA levels in liver and lungs to 1.061 and 1.008 nmol/g, respectively, and mean MPO levels in liver and lungs to 1.88 and 1.71 U/g, respectively. Light microscopy and transmission electron microscopy examinations showed significant alteration in liver and lungs and protection of liver and lung parenchyma in the animals treated with EGCG.