Variability in the proliferative responsiveness of cultured human vascular smooth muscle cells to alpha-thrombin

alpha-Thrombin, a key enzyme of the coagulation cascade, is also a potent mitogen for many cell types. In the present study, the responsiveness to alpha-thrombin of cultured human vascular smooth muscle cells (HVSMC) derived from either vein or normal and atherosclerotic arteries was investigated. All HVSMC populations examined responded mitogenically to alpha-thrombin. However, the extent of this response varied between different cell populations. No significant differences were observed between HVSMC derived from vein versus artery or atherosclerotic versus normal tissues. The responsiveness of a specific HVSMC culture to alpha-thrombin was not affected by cell density and remained constant over several passages. Unlike platelet-derived growth factor BB (PDGF-BB), alpha-thrombin did not exhibit any significant chemotactic effects on HVSMC or induce their anchorage independent growth in semi-solid medium. The hypothesis that the observed variability in HVSMC responsiveness to alpha-thrombin is due to the heterogeneity of cultured HVSMC is raised and discussed.     

Tissue factor pathway inhibitor in endothelial cells colocalizes with glycolipid microdomains/caveolae. Regulatory mechanism(s) of the anticoagulant properties of the endothelium

Tissue factor pathway inhibitor (TFPI), the main downregulator of the procoagulant activity of tissue factor.factor VIIa complex, locates in human endothelial cells (EC) in culture as well-defined clusters uniformly distributed both on the cell surface and intracellularly. We here demonstrate by immunofluorescence that TFPI colocalizes in EC with caveolin, urokinase-type plasminogen activator receptor, and glycosphingolipids. The localization of TFPI in caveolae in resting endothelium is proved by double immunogold electron microscopy for TFPI and caveolin. After ultracentrifugation of rat lung or EC homogenates through density gradients of Nycodenz, TFPI was highly enriched at densities of 1.05 to 1.08 g/mL, together with caveolin and alkaline phosphatase. By ELISA, more than half of the cellular TFPI was detected in Triton X-100-insoluble extracts of EC. TFPI incorporates [1-3H]ethanolamine and is cleaved from the cell surface by phosphatidylinositol-phospholipase C, indicating a specific glycosylphosphatidylinositol-anchorage mechanism for TFPI in the plasma membrane. Clustering of TFPI and its localization in caveolae are dependent on the presence of cholesterol in the membrane. Agonist-induced stimulation of EC caused marked changes of distribution for both TFPI and caveolin at subcellular level, with subsequent increase of the cell surface-associated inhibitory activity toward tissue factor.factor VIIa. Our findings suggest that, beside their function in transcytosis, potocytosis, cell surface proteolysis, and regulation of signal transduction, caveolae also play a direct role in the regulation of EC anticoagulant properties.     

The SwitchWare active network architecture

Active networks must balance the flexibility of a programmable network infrastructure against the safety and security requirements inherent in sharing that infrastructure. Furthermore, this balance must be achieved while maintaining the usability of the network. The SwitchWare active network architecture is a novel approach to achieving this balance using three layers: active packets, which contain mobile programs that replace traditional packets; active extensions, which provide services on the network elements and can be dynamically loaded; and a secure active router infrastructure, which forms a high-integrity base on which the security of the other layers depends. In addition to integrity checking and cryptography-based authentication, security in our architecture depends heavily on verification techniques from programming languages, such as strong type checking     

Diazepam induced early oxidative changes at the subcellular level in rat brain

Binding of calcium to calmodulin (CAM) induces specific structural rearrangements in the whole protein molecule. Ca2+ organizes and stabilizes the four-domains structure of calmodulin in a helical, active conformation that can bind to its target proteins; the central helix remaining flexible is an essential condition for their bio-recognition. The conformation of calmodulin, and its efficacy to interact with target proteins, is profoundly altered when bound to metal ions other than calcium. As recently reported, the local structural changes of CaM, which occur upon aluminium binding, lead to the impairment of protein flexibility and to the loss of its ability to interact with several other proteins, which may decrease or inhibit the regulatory character of calmodulin. In this study we followed conformational changes occurring in the calmodulin molecule after aluminium binding using highly specific monoclonal antibodies (mAbs) able to differentiate between the conformational states of calmodulin, as well as mAbs which recognize aluminium free or bound to proteins. Under the same experimental conditions, mAb CAM-1, a Ca2+ conformation sensitive antibody raised against calmodulin, fails to recognize the calmodulin-aluminium complex, despite the presence of Ca2+, while the anti-Al antibodies show a maximal binding pattern towards their antigen. These data suggest that Al3+ ions bind to calmodulin in the presence of Ca2+ ions, leading to an inactive, reversible conformation, instead of its physiological active form. Alteration of the conformation of calmodulin imposed by Al binding may have possible implications in the neurotoxicity mechanism related to Alzheimer's disease.     

Phosphate diabetes in patients with chronic fatigue syndrome

Phosphate depletion is associated with neuromuscular dysfunction due to changes in mitochondrial respiration that result in a defect of intracellular oxidative metabolism. Phosphate diabetes causes phosphate depletion due to abnormal renal re-absorption of phosphate be the proximal renal tubule. Most of the symptoms presented by patients with phosphate diabetes such as myalgia, fatigue and mild depression, are also common in patients with chronic fatigue syndrome, but this differential diagnosis has not been considered. We investigated the possible association between chronic fatigue syndrome and phosphate diabetes in 87 patients who fulfilled the criteria for chronic fatigue syndrome. Control subjects were 37 volunteers, who explicitly denied fatigue and chronic illness on a screening questionnaire. Re-absorption of phosphate by the proximal renal tubule, phosphate clearance and renal threshold phosphate concentration were the main outcome measures in both groups. Of the 87 patients with chronic fatigue syndrome, nine also fulfilled the diagnostic criteria for phosphate diabetes. In conclusion, we report a previously undefined relationship between chronic fatigue syndrome and phosphate diabetes. Phosphate diabetes should be considered in differential diagnosis with chronic fatigue syndrome; further studies are needed to investigate the incidence of phosphate diabetes in patients with chronic fatigue syndrome and the possible beneficial effect of vitamin D and oral phosphate supplements.     

Topical delivery of tetrahydrocurcumin lipid nanoparticles effectively inhibits skin inflammation: in vitro and in vivo study

Tetrahydrocurcumin (THC) also referred to as ‘white curcumin’, is a stable colorless hydrogenated product of curcumin with superior antioxidant and anti-inflammatory properties. The present study is an attempt to elevate the topical bioavailability of THC, post-incorporation into a nano-carrier system with its final dosage as a hydrogel. Lipid nanoparticles of THC (THC-SLNs) prepared by microemulsification technique were ellipsoidal in shape (revealed in transmission electron microscopy) with a mean particle size of 96.6?nm and zeta potential of ?22?mV. Total drug content and entrapment efficiency of THC-SLNs was 94.51%?±?2.15% and 69.56%?±?1.35%, respectively. Differential scanning calorimetry and X-ray diffraction studies confirmed the formation of THC-SLNs. In vitro drug release studies showed the drug release from THC-SLNs gel to follow Higuchi’s equation revealing a Fickian diffusion. Ex vivo permeation studies indicated a 17 times (approximately) higher skin permeation of THC-SLNs gel as compared with the free THC gel. Skin irritation, occlusion, and stability studies indicated the formulation to be nonirritating, and stable with a desired occlusivity. Pharmacodynamic evaluation in an excision wound mice model clearly revealed the enhanced anti-inflammatory activity of THC-SLNs gel and was further confirmed using biochemical and histopathological studies. It is noteworthy to report here that THC-SLNs gel showed significantly better (p?≤?0.001) activity than free THC in gel. As inflammation is innate to all the skin disorders, the developed product opens up new therapeutic avenues for several skin diseases. To the best of our knowledge, this is the first paper elaborating the therapeutic usefulness of white curcumin-loaded lipidic nanoparticles for skin inflammation.     

Cardiac high molecular weight calmodulin binding protein contains calpastatin activity

A high molecular weight calmodulin binding protein (HMWCaMBP) was previously identified and purified from bovine heart cytosolic fraction [Sharma, R.K. (1990) J. Biol. Chem. 265, 1152-1157]. In this study, we report the biological function of this protein. HMWCaMBP was subjected to peptide mapping and three peptides were sequenced. Two of the three peptide sequences were shown to be highly homologous to the calpain inhibitor, calpastatin. However, the third peptide did not show homology to any known proteins. The Western blot analysis of HMWCaMBP and purified calpastatin from bovine cardiac muscle showed immunoreactivity with polyclonal antibody raised against HMWCaMBP. Furthermore, HMWCaMBP inhibited calpain II and calpain I activities in a dose dependent fashion. Our data based on sequence homology, amino acid analysis, antibody reactivity and calpain inhibition suggests that HMWCaMBP is homologous to calpastatin and may be a CaM-binding form of calpastatin.