A mechanism for heterogeneous endothelial responses to flow in vivo and in vitro

Exposure of endothelium to a nominally uniform flow field in vivo and in vitrofrequently results in a heterogeneous distribution of individual cell responses. Extremes in response levels are often noted in neighboring cells. Such variations are important for the spatial interpretation of vascular responses to flow and for an understanding of mechanotransduction mechanisms at the level of single cells. We propose that variations of local forces defined by the cell surface geometry contribute to these differences. Atomic force microscopy measurements of cell surface topography in living endothelium both in vitro and in situ combined with computational fluid dynamics demonstrated large cell-to-cell variations in the distribution of flow-generated shear stresses at the endothelial luminal surface. The distribution of forces throughout the surface of individual cells of the monolayer was also found to vary considerably and to be defined by the surface geometry. We conclude that the endothelial three-dimensional surface geometry defines the detailed distribution of shear stresses and gradients at the single cell level, and that there are large variations in force magnitude and distribution between neighboring cells. The measurements support a topographic basis for differential endothelial responses to flow observed in vivo and in vitro. Included in these studies are the first preliminary measurements of the living endothelial cell surface in an intact artery.     

Development and characterization in vitro of a catalase-based biosensor for hydrogen peroxide monitoring

There is increasing evidence that hydrogen peroxide (H₂O₂) may act as a neuromodulator in the brain, as well as contributing to neurodegeneration in diseased states, such as Parkinson's disease. The ability to monitor changes in endogenous H₂O₂ in vivo with high temporal resolution is essential in order to further elucidate the roles of H₂O₂ in the central nervous system. Here, we describe the in vitro characterization of an implantable catalase-based H₂O₂ biosensor. The biosensor comprises two amperometric electrodes, one with catalase immobilized on the surface and one without enzyme (blank). The analytical signal is then the difference between the two electrodes. The H₂O₂ sensitivity of various designs was compared, and ranged from 0 to 56 ± 4 mA cm⁻² M⁻¹. The most successful design incorporated a Nafion^® layer followed by a poly-o-phenylenediamine (PPD) polymer layer. Catalase was adsorbed onto the PPD layer and then cross-linked with glutaraldehyde. The ability of the biosensors to exclude interference from ascorbic acid, and other interference species found in vivo, was also tested. A variety of the catalase-based biosensor designs described here show promise for in vivo monitoring of endogenous H₂O₂ in the brain.     

A comparison of in vitro anticancerous activity and mechanism of ethanolic extracts from different Ganoderma genus

Five ethanolic extracts from the mycelia of Ganoderma lucidum, G. tsugae, G. oerstedii, G. subamboinense, and G. resinaceum were respectively studied on their anticancerous activities against leukemic HL-60 cell line in vitro. Results showed that all five extracts potently inhibited HL-60 proliferation. The extract from G. lucidum mycelia exerted the highest activity. Annexin V/PI bivariate flow cytometric analysis further revealed that the five extracts significantly induced early apoptosis in HL-60 cells. The results illustrate that not only G. lucidum but also other Ganoderma species can inhibit cancer cells, and their mechanisms are related to induction of apoptosis. __________ Translated from Journal of Shanghai Normal University (Natural Sciences), 2005, 34(2): 77–81 [译自: 上海师范大学学报 (自然科学版), 2005, 34(2): 77–81]     

Skeletal myotube integration with planar microelectrode arrays in vitro for spatially selective recording and stimulation: a comparison of neuronal and myotube extracellular action potentials

Microelectrode array (MEA) technology holds tremendous potential in the fields of biodetection, lab-on-a-chip applications, and tissue engineering by facilitating noninvasive electrical interaction with cells in vitro. To date, significant efforts at integrating the cellular component with this detection technology have worked exclusively with neurons or cardiac myocytes. We investigate the feasibility of using MEAs to record from skeletal myotubes derived from primary myoblasts as a way of introducing a third electrogenic cell type and expanding the potential end applications for MEA-based biosensors. We find that the extracellular action potentials (EAPs) produced by spontaneously contractile myotubes have similar amplitudes to neuronal EAPs. It is possible to classify myotube EAPs by biological signal source using a shape-based spike sorting process similar to that used to analyze neural spike trains. Successful spike-sorting is indicated by a low within-unit variability of myotube EAPs. Additionally, myotube activity can cause simultaneous activation of multiple electrodes, in a similar fashion to the activation of electrodes by networks of neurons. The existence of multiple electrode activation patterns indicates the presence of several large, independent myotubes. The ability to identify these patterns suggests that MEAs may provide an electrophysiological basis for examining the process by which myotube independence is maintained despite rapid myoblast fusion during differentiation. Finally, it is possible to use the underlying electrodes to selectively stimulate individual myotubes without stimulating others nearby. Potential uses of skeletal myotubes grown on MEA substrates include lab-on-a-chip applications, tissue engineering, co-cultures with motor neurons, and neural interfaces.     

Effect of temperature on the electrical activity of the rat epididymis in vitro

1. 1.|Regional differences in the frequency of electrical activity in rat epididymis were maintained at all temperatures below 39°C.

2. 2.|The change in frequency per deg C increased with temperature and was highest in the temperature region of 34–39°C and the Arrhenius plots of the frequency were linear and parallel in all parts of the epididymis.

3. 3.|The Q₁₀ of the frequency varied between 2.2.–4.3.

4. 4.|The conduction velocity at the cauda epididymis was highest (2.8 mm/s) at 37°C. The Q₁₀ of the conduction velocity was 2.3 in the temperature region of 24–37°C.

Novel synthetic RGD analogs incorporating salicylic acid derivatives show antiplatelet activity in vitro

Continuing our investigational efforts for more active GpIIb/IIIa inhibitors we have synthesized four novel RGD (Arg-Gly-Asp) analogs incorporating salicylic acid derivatives at their N-terminal amino group using the solid phase synthesis.The synthesized compounds 5-Cl-2-HO-C₆H₃-CO-Arg-Gly-Asp(OBzl)-NH₂and 5-Br-2-HO-C₆H₃-CO-Arg-Gly-Asp(OBzl)-NH₂ were tested for their inhibitory activity on human platelet aggregationin vitro and found to be potent inhibitors of the platelet aggregation with 75 and 67% inhibitory activity respectively. In order to confirm our results flow cytometry with monoclonal antibodies against GpIb, GpIIb/IIIa, GpIIIa and GMP140receptors was used.     

An in vitro system for the study of tracheal epithelial cells

Enzymatically dissociated hamster tracheal epithelial (HTE) cells were cultured on collagen coated Millicell filters. Within 3-5 days after being placed in culture large numbers of ciliated and mucus cells began to appear. By 1 week the HTE cells closely resembled those seen in vivo, i.e. columnar morphology and organelle polarity. After 4 weeks in vitro the HTE cultures were still able to maintain the polarity and overall columnar morphology found in in vivo tissue. There was, in addition, no apparent degradation of the collagen substrate. Auto-radiographic data indicated that there was an initial period of high DNA synthesis during the first 3 days in vitro. This was followed by a second phase in which, by day 6, the amount of DNA synthesis was greatly reduced. Analysis of the numbers of ciliated cells relative to non-ciliated cells demonstrated that between days 5 and 8 there was an increase in the percentage of ciliated cells, suggesting that cellular differentiation (i.e. ciliogenesis) follows cellular proliferation. The results of this study show that when HTE cells are grown on collagen-coated Millicell filters there is a significant improvement in cell growth and morphology yielding cells that are very similar to those present under in vivo conditions. Moreover, since there is no degradation of the collagen substrate, HTE cultures may be suitable for long-term studies of respiratory tract epithelia.     

Antioxidant activity and antitumor activity (in vitro) of xyloglucan selenious ester and surfated xyloglucan

Two kinds of xyloglucan derivatives (xyloglucan selenious ester and sulfated xyloglucan) were prepared and evaluated on antioxidant activity and antitumor activity. Compared with xyloglucan, xyloglucan derivatives have new bioactivity against oxidative damage and tumor. Furthermore, xyloglucan selenious ester is more potent than sulfated xyloglucan at antioxidant activity and antitumor activity in vitro. The current data suggest for the first time that selenition of xyloglucan significantly increases its bioactivity and the chemical modification of polysaccharide may allow the preparation of derivatives with new properties and a variety of applications.     

Schistosoma mansoni: Antischistosomal activity of the four optical isomers and the two racemates of mefloquine on schistosomula and adult worms in vitro and in vivo

Recent studies have shown that mefloquine (MQ) reveals interesting antischistosomal properties. We examined the antischistosomal activities of the erythro and threo isomers and racemates of MQ on newly transformed schistosomula (NTS) and adult Schistosoma mansoni in vitro and in mice harbouring adult S. mansoni. The in vitro effects in the presence and absence of haemin were monitored by means of microcalorimetry, scanning electron microscopy and phenotypic evaluation. Incubation of NTS with the erythro derivatives at concentrations of 3 μg/ml and above resulted in convulsions, granularity, decrease in heat flow, and death while NTS incubated with the threo derivatives were only affected at high concentrations (100 μg/ml). Extensive tegumental alterations, decrease in metabolic activity, viability, and death were observed when adult schistosomes had been exposed to 10 μg/ml of the erythro compounds. Moderate tegumental and viability changes but reduced heat production rates were observed with the threo derivatives at 10 μg/ml. In the presence of haemin, all MQ derivatives showed pronounced antischistosomal properties against adult S. mansoni in vitro. In vivo, MQ derivatives achieved statistically significant total and female worm burden reductions ranging between 65.4% and 100%. The highest total worm burden reductions of 93.4% and 90.2% were observed following treatment with the erythro and threo racemates, respectively. In conclusion, the optical isomers and racemates of MQ show only moderate stereoselectivity, in particular in vivo. Our results may enhance our understanding of the mechanism of action and therapeutic profile of MQ derivates on schistosomes.     

Assessment of estrogenic activity of flavonoids from Mediterranean plants using an in vitro short-term test

Six isoflavones, daidzein (4',7,-dihydroxyisoflavone), genistein (4',5,7-trihydroxyisoflavone), genistin (genistein 7-O-beta-D-glucopyranoside), isoprunetin (4',7-dihydroxy, 5-metoxyisoflavone), isoprunetin 7-O-beta-D-glucopyranoside, isoprunetin 4',7-di-O-beta-D-glucopyranoside and four flavones, luteolin (3',4',5,7-tetrahydroxyflavone), luteolin 7-O-beta-D-glucopyranoside, luteolin 4'-O-beta-D-glucopyranoside, licoflavone C (4',5,7-trihydroxy,8-isoprenylflavone) were purified from Mediterranean plants (Genista morisii and Genista ephedroides) and their estrogenic activity was assessed by a yeast reporter gene assay (Saccharomyces cerevisiae RMY326 ER-ERE). Licoflavone C showed a powerful estrogenic activity at 10(-7) M (0.0338 microg/ml) and it was 47.45% than 10(-8) M 17beta-estradiol (0.00272 microg/ml). The estrogenicity of this flavone was found to be comparable to the activity showed by genistein at 10(-6) M (0.27 microg/ml). This study points out that a glucose substituent in flavones and isoflavones modulates the hormone-like activity in a different way. Isoflavone aglycones showed a more estrogenic activity than the corresponding glucosides. Conversely, the glucosidation made estrogenic the flavone luteolin and the position of substitution differently influenced the estrogenic activity of compounds.     

A new method for assessing relative dynamic motion of vertebral bodies during cyclic loading in vitro

A new experimental technique for measuring generalized three-dimensional motion of vertebral bodies during cyclic loading in vitro is presented. The system consists of an orthogonal array of three lasers mounted rigidly to one vertebra, and a set of three mutually orthogonal charge-coupled devices mounted rigidly to an adjacent vertebra. Each laser strikes a corresponding charge-coupled device screen. The mathematical model of the system is reduced to a linear set of equations with consequent matrix algebra allowing fast real-time data reduction during cyclic movements of the spine. The range and accuracy of the system is well suited for studying thoracolumbar motion segments. Distinct advantages of the system include miniaturization of the components, the elimination of the need for mechanical linkages between the bodies, and a high degree of accuracy which is not dependent on viewing volume as found in photogrammetric systems. More generally, the spectrum of potential applications of systems of this type to the real-time measurement of the relative motion of two bodies is extremely broad.     

Tannins determined by various methods as predictors of methane production reduction potential of plants by an in vitro rumen fermentation system

Relationships between chemical constituents, including values obtained with tannin assays (i.e., total phenols, total tannins, condensed tannins and tannin activity using a tannin bioassay) for plant materials (n = 17), and methane production parameters at 24 h of incubation in the in vitro Hohenheim gas method were established. The methane production reduction potential (MRP) was calculated by assuming net methane concentration for the control hay as 100%. The MRP of Bergenia crassifolia leaves and roots, and Peltiphyllum peltatum leaves, was >40%. Amongst the chemical constituents, neutral detergent fibre had a high correlation (r = 0.86) with methane concentration. There was negative relationship between total phenol, total tannins or tannin activity and methane concentration. However, a positive relationship existed between these tannin assays and the MRP, with r-values ranging from 0.54 to 0.79 (P<0.05). A very weak relationship (r = 0.09) occurred between condensed tannins and MRP. Similar results to those with MRP were obtained with the percent increase in methane on addition of polyethylene glycol. The highest correlations, 0.79 and 0.92 (P<0.001), were between tannin activity determined using the tannin bioassay and the MRP, or the percent increase in methane on addition of polyethylene glycol, respectively, suggesting that this tannin assay could be used to identify plants possessing antimethanogenic properties. Leaves of Rheum undulatum, Vaccinium vitis-idaea, B. crassifolia, Rhus typhina and P. peltatum, and roots of B. crassifolia have considerable potential (i.e., >25%) to decrease enteric methane production from ruminants.