Modulation of calcification of vascular smooth muscle cells in culture by calcium antagonists, statins, and their combination

Background Vascular calcification is an organized process in which vascular smooth muscle cells (VSMCs) are implicated primarily. The purpose of the present study was to assess the effects of calcium antagonists and statins on VSMC calcification in vitro. Methods VSMC calcification was stimulated by incubation in growth medium supplemented with 10 mmol/l β-glycerophosphate, 8 mmol/l CaCl₂, 10 mmol/l sodium pyruvate, 1 μmol/l insulin, 50 μg/ml ascorbic acid, and 100 nmol/l dexamethasone (calcification medium). Calcification, proliferation, and apoptosis of VSMCs were quantified. Results Calcium deposition was stimulated dose-dependently by β-glycerophosphate, CaCl₂, and ascorbic acid (all P < 0.01). Addition of amlodipine (0.01–1 μmol/l) to the calcification medium did not affect VSMC calcification. However, atorvastatin (2–50 μmol/l) stimulated calcium deposition dose-dependently. Combining treatments stimulated calcification to a degree similar to that observed with atorvastatin alone. Both atorvastatin and amlodipine inhibited VSMC proliferation at the highest concentration used. Only atorvastatin (50 μmol/l) induced considerable apoptosis of VSMCs. Conclusion In vitro calcification of VSMCs is not affected by amlodipine, but is stimulated by atorvastatin at concentrations ≥10 μmol/l, which could contribute to the plaque-stabilizing effect reported for statins. J. W. Jukema is an Established Clinical Investigator of The Netherlands Heart Foundation 2001D032.     

Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division.

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.     

Development of a high-performance liquid chromatographic method for the determination of 11-keto-β-boswellic acid in human plasma

A validated HPLC method for the determination of 11-keto-β-boswellic acid (KBA) in human plasma was developed. The method involves the solid-phase extraction of KBA from plasma followed by a separation with reversed-phase HPLC. Calibration was based on external standardisation and ranged between 0.1 and 2.0 μg KBA per ml plasma. Linearity was established over the entire calibration range and in each case the coefficient of correlation (r²) was above 0.99. The recovery of KBA from plasma was 85.7%. It was further demonstrated that the method can be applied successfully to monitor the level of KBA in plasma.     

Serum withdrawal induces a redistribution of intracellular gelsolin towards F-actin in NIH 3T3 fibroblasts preceding apoptotic cell death

The intracellular distribution of gelsolin in NIH 3T3 cells was examined by immunostaining using affinity-purified polyclonal gelsolin antibodies before and after induction of apoptosis by serum withdrawal. Serum deprivation induced detachment of an increasing number of NIH 3T3 cells, but also apoptosis in attached cells as verified morphologically by chromatin condensation, nuclear fragmentation and labelling of their periphery by FITC-annexin V. Ongoing apoptosis was also demonstrated by activation of caspase-3 activity and chromatin cleavage into high-molecular-mass fragments, although no internucleosomal chromatin degradation (DNA-ladder formation) was detected. When cells were maintained in the presence of 10% foetal calf serum, gelsolin immunoreactivity was evenly distributed in the cytoplasm. No obvious co-localisation of gelsolin and the actin-containing stress fibres was detected under these conditions. At day one after serum withdrawal, a redistribution of gelsolin to actin filaments was detected within a few attached cells by double fluorescence staining. The number of cells exhibiting this redistribution increased at days two to four. In addition, the stress fibres increased in thickness and their length was continuously reduced. At day four, many cells contained shortened stress fibres, which had lost their longitudinal orientation. Additionally, the cytoplasm of a number of attached cells was highly condensed around their nuclei and a homogenous distribution of both gelsolin and actin was detected in the remaining cytoplasmic rim. Up to day two, these effects were reversible after re-addition of serum to attached cells. A similar redistribution of gelsolin immunore-activity was observed after induction of apoptosis by cycloheximide, but not after initiation of necrosis by hydrogen peroxide. In NIH 3T3 cells no alteration in the expression of gelsolin at the level of protein (Western blot) or specific mRNA (Northern blot) was observed after serum withdrawal. Using Western blotting, no proteolysis of gelsolin was detected up to day 4, although caspase-3 activity was found to have increased fivefold after serum withdrawal. These results suggested that in these cells F-actin severing might occur in the absence or advance of gelsolin cleavage by caspases. Intact gelsolin on its own may be sufficient for the dissolution of the microfilaments, since micro-injection of gelsolin into primary bovine lens cells led to a transient disappearance of the stress fibres and to a reduction of their attachment area to the substratum. In NIH 3T3 cells similar effects of micro-injected gelsolin were only observed at day one after serum withdrawal.     

Age structure and selected pathological aspects of a series of skeletons of late medieval Bernau (Brandenburg,Germany)

The age distribution of an excavation site from the late Middle Ages (beginning of the 13th century until 1598) in Bernau (Brandenburg, Germany) that contained 252 skeletons shows a mortality maximum at the age classes infants I and mature. The lowest mortality was calculated for the adult age class. 51.6% of the individuals died before the age of 20 years. The mortality rate of young women was higher than that of young men. The life expectancy of the total population was 25 years. Altogether, 87 adult skeletons were examined for degenerative joint diseases. Many of the examined joints showed indications of beginning osteoarthritis; the intensity of the disease was low or medium severe. The highest values were found for the hip joint, followed by the elbow and the knee. For all joints examined, women were less often and to a lesser extent affected than men, which was noticeable best in the upper extremity. Harris lines were found in 75% of the children, 4.7 per average individual. No such lines were found by the age of one year, the peak value of the frequency being recorded at the age between 2 and 3 years. This can be correlated to the weaning period. The comparison to a rural population yielded a higher load with Harris lines in the small village, which, however, was not correlated to a higher mortality. Altogether, the population of Bernau is characterised by good living conditions and a considerable chance of survival for the children, as well as by a labour system, dominated by crafts and farming.     

Integrating phenotypic and expression profiles to map arsenic-response networks

Background  

Arsenic is a nonmutagenic carcinogen affecting millions of people. The cellular impact of this metalloid in Saccharomyces cerevisiae was determined by profiling global gene expression and sensitivity phenotypes. These data were then mapped to a metabolic network composed of all known biochemical reactions in yeast, as well as the yeast network of 20,985 protein-protein/protein-DNA interactions.     

Neural networks: models and neurons show hybrid vigor in real time

Hybrid networks in which living neurons interact with digital or analog model neurons are providing insights into the role of neural and synaptic properties in shaping neural network activity.     

The influence of intercalator binding on DNA triplex stability: correlation with effects on A-tract duplex structure

The triplex form of DNA is of interest because of a possible biological role as well as the potential therapeutic use of this structure. In this paper the stabilizing effects of two intercalating drugs, ethidium and the quinoxaline derivative 9-OH-B220, on DNA triplexes have been studied by thermal denaturation measurements. The corresponding duplex structures of the DNA triplex systems investigated are either A-tract or normal B-DNA. The largest increases in the triplex melting temperatures caused by the intercalators were found for sequences having A-tract duplex structures. Inserting a single base pair with an N2-amino group in the minor groove, e.g. a G-C pair, breaks up the A-tract duplex structure and also reduces the stabilizing effect of the drugs on the triplex melting temperatures. The large drug-induced increase in triplex melting temperature for complexes having an original duplex A-tract structure is correlated with a low initial melting point of the triplex, not with the triplex being unusually stable in the presence of the drug. Hence, we conclude that the large thermal stabilizing effect exhibited by ethidium and 9-OH-B220 on dTn.dAn-dTn triplexes is partly caused by the intercalators breaking up the intrinsic A-tract structure of the underlying duplex.