Abstract: Protein kinases participate in a myriad of cellular
processes of major biomedical interest. The in vivo substrate
specificity of these enzymes is a process determined by several
factors, and despite several years of research on the topic, is still
far from being totally understood. In the present work, we have
quantified the contributions to the kinase substrate specificity of
i) the phosphorylation sites and their surrounding residues in the
sequence and of ii) the association of kinases to adaptor or scaffold
proteins. We have used position-specific scoring matrices (PSSMs),
to represent the stretches of sequences phosphorylated by 93 families
of kinases. We have found negative correlations between the number
of sequences from which a PSSM is generated and the statistical
significance and the performance of that PSSM. Using a subset
of 22 statistically significant PSSMs, we have identified specificity
determinant residues (SDRs) for 86% of the corresponding kinase
families. Our results suggest that different SDRs can function as
positive or negative elements of substrate recognition by the different
families of kinases. Additionally, we have found that human proteins
with known function as adaptors or scaffolds (kAS) tend to interact
with a significantly large fraction of the substrates of the kinases to
which they associate. Based on this characteristic we have identified
a set of 279 potential adaptors/scaffolds (pAS) for human kinases,
which is enriched in Pfam domains and functional terms tightly
related to the proposed function. Moreover, our results show that
for 74.6% of the kinase–pAS association found, the pAS colocalize
with the substrates of the kinases they are associated to. Finally, we
have found evidence suggesting that the association of kinases to
adaptors and scaffolds, may contribute significantly to diminish the
in vivo substrate crossed-specificity of protein kinases. In general, our
results indicate the relevance of several SDRs for both the positive
and negative selection of phosphorylation sites by kinase families and
also suggest that the association of kinases to pAS proteins may be
an important factor for the localization of the enzymes with their set
of substrates.
Abstract: Heme oxygenase-1 (HO-1), an enzyme degrading heme to carbon monoxide, iron, and biliverdin, has been recognized as playing a crucial role in cellular defense against stressful conditions, not only related to heme release. In the present study, the effects of TNF-a on the expression of heme oxygenase-1 (HO-1) in human aortic endothelial cells (HAECs) as well as the related mechanisms were investigated. 10 ng/mL TNF-α treatment significantly increased HO-1 expression after 6h, then a further increase at 12h and declined at 24h. Treatment with 2 ng/mL of TNF-a after 12 h resulted in a significant increase in HO-1 expression, which peaked at 10 ng/mL, then declined at 20 ng/mL. TNF-α induced HO-1 expression and then HO-1 expression reduced vascular cell adhesion molecule-1 (VCAM-1) expression. Phosphorylation studies of ERK1/2, JNK, and p38, three subgroups of mitogen-activated protein kinases (MAPKs) demonstrated TNF-α-induced ERK1/2, JNK, and p38 phosphorylation. The increase in HO-1 expression in response to TNF-α treatment was affected by pretreatment with SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor), not with PD98059 (an ERK1/2 inhibitor). The expression of HO-1 was stronger in aortas of TNF-α-treated apo-E deficient mice when compared with control mice. These results suggest that low dose of TNF-α treatment notably induced HO-1 expression was mediated through JNK/p38 phosphorylation and may have a protective potential in cardiovascular diseases and inflammatory response through the regulation of HO-1 expression.
Abstract: This study was conducted in order to determine the physical properties and stability of mayonnaise-like emulsions as affected by modified yam starches. Native yam starch was modified via pre-gelatinization and cross-linking phosphorylation procedures. The emulsions (50% oil dispersed phase) were prepared with 0.3% native potato, native yam, pre-gelatinized yam and cross-linking phosphorylation yam starches. The droplet size of surface weighted mean diameter was found to be significantly (p < 0.05) lower in the sample with cross-linking phosphorylation yam starch as compared to other samples. Moreover, the viscosity of the sample with pregelatinized yam starch was observed to be higher than that of other samples. The phase separation stability was low in the freshly prepared and stored (45 days, 5°C) emulsions containing native yam starch. This study thus generally suggested that modified yam starches were more suitable (i.e. better physical properties and stability) to be used as stabilizers in a similar system i.e. light mayonnaises, rather than a native yam starch.
Abstract: Bones are dynamic and responsive organs, they
regulate their strength and mass according to the loads which they are subjected. Because, the Wnt/β-catenin pathway has profound
effects on the regulation of bone mass, we hypothesized that mechanical loading of bone cells stimulates Wnt/β-catenin signaling, which results in the generation of new bone mass.
Mechanical loading triggers the secretion of the Wnt molecule, which after binding to transmembrane proteins, causes GSK-3β (Glycogen synthase kinase 3 beta) to cease the phosphorylation of β-catenin. β-catenin accumulation in the cytoplasm, followed by its
transport into the nucleus, binding to transcription factors (TCF/LEF)
that initiate transcription of genes related to bone formation. To test this hypothesis, we used TOPGAL (Tcf Optimal Promoter
β-galactosidase) mice in an experiment in which cyclic loads were
applied to the forearm. TOPGAL mice are reporters for cells effected
by the Wnt/β-catenin signaling pathway. TOPGAL mice are genetically engineered mice in which transcriptional activation of β-
catenin, results in the production of an enzyme, β-galactosidase. The
presence of this enzyme allows us to localize transcriptional
activation of β-catenin to individual cells, thereby, allowing us to quantify the effects that mechanical loading has on the Wnt/β-catenin pathway and new bone formation. The ulnae of loaded TOPGAL
mice were excised and transverse slices along different parts of the
ulnar shaft were assayed for the presence of β-galactosidase.
Our results indicate that loading increases β-catenin transcriptional
activity in regions where this pathway is already primed (i.e. where basal activity is already higher) in a load magnitude dependent
manner. Further experiments are needed to determine the temporal and spatial activation of this signaling in relation to bone formation.