Cross-striated collagen fibers formation with low amplitude, high frequency vibrations

Human Adipose derived Stem Cells (hASCs) are observed to express genes and proteins associated with the osteoblast phenotype under osteogenic conditions and with mechanical stimulation. New investigations have been carried out to understand the effects of Low Amplitude, High frequency vibrations (HFVs) in vitro. In this work hASCs were cultured in proliferative or osteogenic media and daily unstimulated/stimulated at 30 Hz for 45 minutes for 28 days to evaluate the effects of HFVs on the differentiation of hASCs toward bone tissue. We treated four groups of cells (control in proliferative medium-CP, control in osteogenic medium-CO, treated in proliferative medium-TP and treated in osteogenic medium-TO) for 21 and 28 days. The tissue was incubated in digestion buffer and after suspended in DMEM supplemented with 10% FBS. The hASCs were cultured in proliferative medium (PM) at 37°C with 5% CO2. In order to stimulate the cells, we used a custom made “bioreactor”. At the end of the culture period the DNA content was evaluated, Alizarin red test was used to determine the presence of calcium deposition. Quantitative real-time RT-PCR was performed to check the expression of the investigated osteogenic genes. The extracellular matrix (ECM) extraction to evaluate the amount of the ECM constituents. We quantified the level of the most important osteogenic protein: Col-I, Col-III, osteocalcin (OC), osteopontin (OP), alkaline phopsphatase (ALP), osteonectin (ON) and fibronectin (FN). Ultrastructural investigation with TEM were performed after 21 and 28 days. Statistical analysis: a two-way analysis of variance (ANOVA) was performed. Alizarin red test after 21days culture showed strong differences between treated and control samples. The results of osteogenic gene expression reveal the strong increase of their content (Col-I, OP) for the mechanically treated samples, in both media at 21 days. The results obtained from the protein content analysis at 21 days seem to confirm previous observations: both the osteogenic medium and the mechanical treatment induce the early translation of osteogenic genes. TEM observations showed that in the sample treated with HFVs for 21 days, long collagen fibers of indeterminate length are present; at 28 days fibers appear shorter with the classical arrangement cross-striated in the extracellular space. From our results, the HFV treatment seems to have effect only after 21 days treatment and it seems to be ineffective in the long term trial, only collagen fibers appear more structured after 28 days

Effects of cytochalasin B on the morphology of secretory cells of rana esculenta fungiform papilla.

The effects of cytochalasin B on the three types of secreting cells (apical, paracoronal and parietal cells) present in the fungiform papilla of the frog's tongue were studied. Cytochalasin B induced morphological modifications only in the apical secreting cells, localized in the sensorial area, without affecting the paracoronal cells or the parietal cells. The morphological features of treated apical cells showed that the drug induces two different effects: a disappearance of microfilaments and an increase in the exocytotic process. The results are related to previous experimental findings which showed that cytochalasin B produces actin depolymerization and interferes with calcium transport

Can a Bacterial Endotoxin be a Key Factor in the Kinetics of Amyloid Fibril Formation?

Data found in literature have reported that bacterial endotoxins may be involved in the inflammatory and pathological processes associated with amyloidosis and Alzheimer's disease (AD). In fact, it has been observed that the chronic infusion of the bacterial lipopolysaccharide, the outer cell wall component of Gram negative bacteria, into the fourth ventricle of rats reproduces many of the inflammatory and pathological features seen in the brain of AD patients. In this context, a key player in the pathogenesis of AD is the amyloid-β peptide (Aβ) that is capable of aggregating in fibrils that represent the main component of amyloid plaques. These deposits that accumulate among brain cells are indeed one of the hallmarks of AD. This aggregation in fibrils seems to correlate with Aβ toxic effects. However, recent data have shown that amyloid fibril formation not only results in toxic aggregates but also provides biologically functional molecules; such amyloids have been identified on the surface of fungi and bacteria. The aim of this work was to gain insight into the influence of bacterial endotoxins on Aβ fibrillogenesis; factors that influence fibril formation may be important for Aβ toxic potential. Following three days of incubation at 37°C, Aβ was organized in compact fibrils and the in vitro Aβ fibrillogenesis was potentiated by the Escherichia coli endotoxin. This suggests the importance of infectious events in the pathogenesis of AD and proposes a new aspect related to the putative pathological factors that can be implicated in the mechanisms involved in Aβ25-35 fibrillogenesis

Expression of proteins involved in calcium homeostasis during crista ampullaris regeneration

Most of the events related to the mechano-transduction process in hair cell are regulated by cytoplasmic Ca++ concentrations. Ca++ influx trough the mechano-transduction channels regulates the adaptation process in the stereocilia, while Ca++ entering into the cell through the voltage gated channels regulates the neurotransmitter release. Moreover Ca++ activates the K+ channels that dominate the inhibitory postsynaptic potential at efferent synapses. Cytoplasmic Ca++ concentration is regulated by a complex system of proteins, including the Na+/Ca++ exchanger, Ca++ pumps (PMCAs, SERCAs) and a copious supply of diffusible proteins that buffers free Ca++ in specific cell compartments. These proteins intercept Ca++ near its sites of entry and restrict the spread of the free ion. Moreover this buffering limits the period during which Ca++ local concentration are high enough to trigger its physiological effects. In frog crista ampullaris PMCAs seems to be the most relevant mechanism of Ca++ extrusion since the lack of a functional Na+/Ca++ exchanger. We investigated the distribution of different subtypes of PMCAs finding that each isoform has its specific expression domain along the crista. We also demonstrate a similar compartmentalization for the IP3R, which is involved in Ca++ release from the IP3-sensitive intracellular stores, and for some buffering proteins. These data and the morphological identification of diverse hair cell types differentially distributed along the crista ampullaris, suggest the presence in the sensory epithelium of distinct functional domains where the mechanisms of Ca++ homeostasis regulation are not completely overlapping. This hypothesis is confirmed by the observation that the different regions of the crista ampullaris show a different sensibility to the ototoxic damage induced by gentamicin treatment: although the mechanism of actions of this drug has not jet completely understood, aminoglycosides are known to interfere with Ca++ homeostasis. Our studies on frog crista ampullaris regeneration showed that the complete crista functional activity is restored well afterwards its morphological recovery. Indeed, while the appearance of a stereociliary apparatus expressing a correct pattern of Ca++ pumps seems to be sufficient to restore hair cell basal activity, the recovery of the evoked sensory discharge seems to need also the expression of Ca++ buffering proteins. This study try to correlate the appearance of the different proteins involved in Ca++ homeostasis with the complete functional recovery of the sensory epithelium

Nature and expression of dihydropyridine-sensitive and -insensitive calcium currents in hair cells of frog semicircular canals

Ca(2+) currents in hair cells of the frog crista ampullaris were studied using the whole-cell patch-clamp technique. Currents were recorded in situ from hair cells in peripheral, intermediate and central regions of the sensory epithelium. Two types of Ca(2+) currents were found: a partially inactivating current that was expressed by nearly all central cells and by about 65% of intermediate and peripheral cells, and a sustained current expressed by the remaining cell population. The mean Ca(2+) current amplitude was larger in intermediate cells than in central or peripheral cells. The two types of Ca(2+) currents were composed of two components: a large, nifedipine-sensitive (NS) current and a small, nifedipine-insensitive (NI) current. The latter was resistant to SNX-482, omega-conotoxin MVIIC and omega-agatoxin IVA and to omega-conotoxin GVIA, antagonists of R, P/Q and N-type Ca(2+) channels. The amplitude of NS and NI currents varied among peripheral cells, where the current density gradually increased from the beginning of the region toward its end. No significant variation of Ca(2+) current density was detected in hair cells of either intermediate or central regions. These results demonstrate the presence of regional and intraregional variations in the expression of L and non-L Ca(2+) channels in the frog crista ampullaris. Finally, immunocytochemical investigations revealed the presence of Ca(2+) channel subunits of the alpha(1D) type and the unexpected expression of alpha(1B)-subunits

Ecto-ATPase activity sites in vestibular tissue: an ultracytochemical study in frog semicircular canals

The present study describes the localization and distribution of putative ecto-nucleoside-triphosphate-diphosphohydrolases in the frog semicircular canals. These enzymes provide the terminating mechanism of adenosine-5'-triphosphate (ATP) signalling. The localization of the ATP hydrolysis was mapped ultracytochemically using a one-step cerium citrate reaction. Electron-dense precipitates, indicating ecto-adenosine-triphosphatase (ecto-ATPase) activity, were found at the outer surface of plasma membranes of crista hair cells and supporting cells of the sensory epithelium, transitional cells and undifferentiated cells of the ampullar wall and dark cells constituting the secretory epithelium. Non-sensory cells of the ampulla usually exhibited reaction deposits at the level of both apical and basolateral membranes coming into contact with the endolymph and the perilymph respectively, while cells constituting the sensory epithelium showed evident differences in relation to their position. Hair cells and supporting cells of the peripheral regions exhibited clear reaction products both at the level of apical and basolateral membranes, while those of the isthmus region showed abundant reactivity only at the level of their apical membranes. Of particular interest was the observation that hair cell stereocilia exhibited an abundant ecto-ATPase activity, thus suggesting a possible colocalization of enzymatic sites with purinergic receptors and mechanotransduction channels. This strategic expression of ecto-ATPase sites could provide a rapid mechanism of ATP removal able to rapidly restore the sensitivity of transduction channels. In conclusion, the widespread distribution of ecto-ATPase sites at the level of sensory and non-sensory cells of the frog semicircular canals suggests that ATP may have a key role in controlling vestibular function