In the present study we have used a novel, comprehensive mRNA profiling technique (GeneCalling) for determining differential gene expression profiles of human endothelial cells undergoing ...differentiation into tubelike structures. One hundred fifteen cDNA fragments were identified and shown to represent 90 distinct genes. Although some of the genes identified have previously been implicated in angiogenesis, potential roles for many new genes, including OX-40, white protein homolog, KIAA0188, a homolog of angiopoietin-2, ADAMTS-4 (aggrecanase-1), and stanniocalcin were revealed. Support for the biological significance was confirmed by the abrogation of the changes in the expression of angiogenesis inhibitors and
in situ hybridization studies. This study has significantly extends the molecular fingerprint of the changes in gene expression that occur during endothelial differentiation and provides new insights into the potential role of a number of new molecules in angiogenesis.
Summary
Blocking the cofactor function of human tissue factor may be beneficial in various coagulation-mediated diseases. The murine antibody D3 binds to the membrane proximal substrate interaction ...region of human tissue factor and blocks tissue factor function even in the presence of bound factor VIIa. The cloned murine D3 antibody was humanized and affinity matured by exchanging amino acids in the complementarity determining regions as well as in the antibody framework. The humanized antibody, D3H44, bound to tissue factor with a 100-fold increased affinity (K
D
0.1 nM) as compared to the original murine and chimeric versions. Depending on the particular disease, different pharmacokinetic properties of the antibody may be required and, therefore, several antibody variants – F(ab), F(ab’)
2
, IgG2, IgG4 and IgG4b – were generated.
In vitro
, the humanized D3 antibodies displayed potent inhibition of plasma clotting and tissue factor: factor VIIa-mediated activation of factors IX and X (e. g. D3H44-F(ab’)
2
, IC50 (F.X) 47 pM). In addition, D3H44-F(ab’)
2
completely prevented fibrin deposition in a human
ex vivo
thrombosis model under venous blood flow conditions (IC
50
37 nM). The humanized D3 antibodies may be utilized for treatment of cardiovascular diseases which involve tissue factor activity, e. g. acute coronary syndrome and venous thrombosis.
The onset of cardiac hypertrophy is associated with characteristic changes in myocardial gene expression that are thought to recapitulate a developmental gene program. We report here the first gene ...expression profile of the murine myocardium, using a rapid method of quantitative expression analysis based on real-time analytical RT-PCR. This assay was used to measure expression levels of 29 genes in (1) late stage development as represented by day 1 neonatal ventricles, (2) normal cardiac growth in 3 and 18 month old mice, and (3) cardiac hypertrophy following pressure overload by aortic constriction. For males and females normal growth is not associated with differential expression although there is elevated expression of skeletal and smooth muscle actin mRNA's in males compared to females. Using normal adult ventricles as a reference, there are many qualitative and quantitative differences between the day 1 neonatal myocardium and experimental cardiac hypertrophy. These data suggest that the response to POL involves a subset of re-expressed developmental genes together with altered expression of genes not necessarily associated with cardiac development.
A large-scale effort, termed the Secreted Protein Discovery Initiative (SPDI), was undertaken to identify novel secreted and transmembrane proteins. In the first of several approaches, a biological ...signal sequence trap in yeast cells was utilized to identify cDNA clones encoding putative secreted proteins. A second strategy utilized various algorithms that recognize features such as the hydrophobic properties of signal sequences to identify putative proteins encoded by expressed sequence tags (ESTs) from human cDNA libraries. A third approach surveyed ESTs for protein sequence similarity to a set of known receptors and their ligands with the BLAST algorithm. Finally, both signal-sequence prediction algorithms and BLAST were used to identify single exons of potential genes from within human genomic sequence. The isolation of full-length cDNA clones for each of these candidate genes resulted in the identification of >1000 novel proteins. A total of 256 of these cDNAs are still novel, including variants and novel genes, per the most recent GenBank release version. The success of this large-scale effort was assessed by a bioinformatics analysis of the proteins through predictions of protein domains, subcellular localizations, and possible functional roles. The SPDI collection should facilitate efforts to better understand intercellular communication, may lead to new understandings of human diseases, and provides potential opportunities for the development of therapeutics.
Abstract
Traditional methods for flow cytometry (FCM) data processing have relied on manual gating of cell events to define cell populations for statistical analysis. However, this approach has ...become increasingly problematic with the advances in instrumentation and reagents that allow for evaluation of larger numbers of cell properties. Recently several groups have developed computational methods for automatically identifying cell populations in multidimensional FCM data obviating the need for manual gating. In order to compare the performance of these methods, the Flow Cytometry: Critical Assessment of Population Identification Methods (FlowCAP) competition was established to make available a common set of FCM data together with manual gating results for comparative analysis. The first FlowCAP competition included 5 different data sets with data from 12-30 samples containing 5000-100,000 cell events stained with 3-10 fluorochrome markers. We received 36 analysis result submissions from 14 research groups. Both model fitting and density-based clustering methods were found to perform well in comparison with manual gating by domain experts as the gold standard, using statistical tests to measure and rank algorithm performance. In addition, combining results using a computational “ensemble” method was found to outperform all individual methods. These results suggest that, in the near future, automated computational methods may become an integral part of routine FCM data analysis.
We report the cloning and expressing of rat natriuretic peptide receptor-C (NPR-C), which binds naturally occurring and synthetic
ligands with higher affinity than human NPR-C. Using rat/human ...hybrids and site-directed mutagenesis, we identified residue
188, Ala for rat and Ile for human, which modulates hormone binding. Orthologous mutagenesis at position 188 for either rNPR-C
or hNPR-C results in a complete reversal of the pharmacology. To our knowledge, this is the first example of a single transmembrane
domain receptor for which a single residue dictates the ligand binding properties; previous examples are limited to seven
transmembrane receptors.
We determined the nucleotide sequence of mouse natriuretic peptide receptor-A (NPR-A) cDNA and compared the revised deduced
amino acid sequence with those of rat and human NPR-A. The ligand ...selectivity of these three receptor/guanylyl cyclases was
examined by whole-cell stimulation of cGMP production. The 28-amino acid atrial natriuretic peptide (ANP) has only one difference
among these three species, i.e., human Met-12 versus rat and mouse Ile-12. However, despite the nearly invariant ANP sequence
among these species, ANP analogs have marked differences in ED50 values and maximal cGMP responses among the three receptors.
With the natriuretic peptide analogs we tested, human NPR-A is less sensitive than rat or mouse NPR-A to changes in the 17-amino
acid, disulfide-bonded ring of ANP and to the species differences in brain natriuretic peptide (BNP) but is more sensitive
to deletions in the carboxyl tail of ANP. The ANP determinants of agonist potency have therefore changed for different species
of NPR-A. This is reflected in the amino acid sequence divergence in the receptor extracellular domains and in the divergence
and specificity of BNP among species. Our results suggest that the coevolution of NPR-A and BNP has thus been constrained
within the context of the conserved ANP sequence.
Libraries of monovalent display-phage expressing mutant human B-type natriuretic peptide (hBNP) were used to identify variants that preferentially bind natriuretic peptide receptor-A (NPR-A) compared ...to receptor-C (NPR-C). Position 19 was a significant determinant of receptor specificity for hBNP display phage. The synthetic hBNP variant S19R had a 265-fold improved NPR-A binding over NPR-C, analogous to the atrial natriuretic peptide (ANP) specificity mutation G16R. Mutation of the last three residues of the hBNP disulfide ring, G23F/L24W/G25R, resulted in about 9-fold improved selectivity. The analogous mutations in ANP decreased NPR-A binding, suggesting divergence in the mechanism of NPR-A recognition.
Abstract Background Distal femur fractures continue to be a complex surgical problem for which the incidence is increasing. Presently, there is a need for different constructs to address these ...complex fractures. This study attempts to define the biomechanical properties of several implants. Methods A novel, prototype locking retrograde intramedullary nail and the Russell–Taylor femoral retrograde nail were tested at non-destructive, physiological, axial mode load strength using a young, synthetic bone model for a medial segmental shaft defect in the supracondylar region of the distal femur (medial gap of 10 mm, 65 mm proximal to the distal joint and parallel to the knee axis). Each specimen was compressively loaded and unloaded to the peak load for 80,000 cycles at a 0.5 Hz frequency. These were compared to the results from the same lab of the retrograde Trigen intramedullary nail. Motion and peak displacement were measured across the fracture site as a reflection of construct stability. Findings Previous testing demonstrated that Trigen intramedullary nail had significantly less motion across the gap and increased overall stiffness of the construct ( P < 0.05) compared to both Russell–Taylor and prototype nails. Interpretation Locking technology used in a nail biomechanically appears to lead to more micro-motion across the fracture gap and to less stiffness in this construct. Further research needs to be invested into intramedullary, locking technology before introducing it into clinical practice.
Research examining exercise with blood flow restriction (BFR) has indicated positive findings for the use of this novel training modality in both health and performance-related outcomes. However, ...eccentric resistance training combined with BFR has yet to be fully examined. Therefore, the objective of this pilot study was to investigate the muscular strength and hypertrophic responses before and following a training intervention involving resistance exercise utilizing only eccentric muscle actions with or without BFR. Eighteen resistance-trained males were randomly assigned to one of three training conditions following pre-testing measures: eccentric resistance training only, eccentric resistance training with BFR, and a control training group. Our findings suggest that four weeks of submaximal eccentric resistance training performed to repetition failure, with or without BFR, may represent alternative means of obtaining increases in muscle cross-sectional area (CSA) in resistance-trained males. Although the addition of BFR did reduce training volume, it did not result in superior adaptations.
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