Cytosolic free calcium dependent regulation of osteoclast bone resorbing activity

Osteoclasts are sensitive to KCl-induced depolarization and to increased extracellular calcium concentration, and respond to these treatments with cytosolic calcium increase. In this study we evaluated the possibility that these experimental conditions could affect osteoclast bone resorption. We found that, incubating osteoclasts with 3H-proline previously labeled bone particles the resorbing activity was inhibited by both depolarization and extracellular calcium concentration increase. The released radioactivity was, in fact, 48% and 52% respectively compared to the untreated cultures. These data demonstrated that cytosolic calcium increase is one of the messengers of the pathway that inhibits, in this condition, bone resorption. Furthermore, as in parathyroid cells, extracellular calcium acts with a negative direct feedback mechanism that controls osteoclast activity.     

Influence of the spacer length on the activity of enzymes immobilised on nylon/polyGMA membranes: Part 2: Non-isothermal conditions

β-Galactosidase has been immobilised through spacers of different length on nylon membranes grafted with glycidyl methacrylate. Hexamethylendiamine, ethylendiamine or hydrazine have been separately used as spacers.

The behaviour of the catalytic membranes has been studied in a bioreactor operating under non-isothermal conditions as a function of the applied temperature difference ΔT.

Comparison of the enzyme reaction rates under isothermal and non-isothermal conditions resulted in percentage activity increases (PAI) and reduction of the production time (τ_r) proportional to the size of the applied ΔT. Both these parameters increased with the increase of the spacer length.

Results have been discussed in the frame of reference of the process of thermodialysis which reduces the limitations to the diffusion of substrate and reaction products across the catalytic membrane, limitations introduced by the grafting and immobilisation process.

The advantages of employing non-isothermal bioreactors in biotechnological productive processes have been outlined.     

HGF and M-CSF modulate adhesion of MDA-231 breast cancer cell by increasing osteopontin secretion

Recent studies reported an increased expression of osteopontin (OPN) in metastatic breast cancer cells, but the mechanisms modulating OPN production and the interaction of the cells with the secreted protein are far from clear. In this work, we utilized as an experimental system the cell line MDA-231 and we showed that HGF and M-CSF significantly enhance their adhesion onto OPN. Furthermore, in the presence of HGF and M-CSF, MDA-231 cells can adhere when plated onto BSA via increased OPN secretion. Moreover HGF and M-CSF induce de novo synthesis of OPN. In conclusion, these data suggest that HGF and M-CSF stimulate OPN production by MDA-231 cells, and that OPN is subsequently used as a substrate for cell adhesion.     

Hollow-fiber enzyme reactor operating under nonisothermal conditions

A hollow-fiber enzyme reactor, operating under isothermal and nonisothermal conditions, was built employing a polypropylene hollow fiber onto which beta-galactosidase was immobilized. Hexamethylenediamine and glutaraldehyde were used as spacer and coupling agent, respectively. Glucose production was studied as a function of temperature, substrate concentration, and size of the transmembrane temperature gradient. The actual average temperature differences across the polypropylene fiber, to which reference was done to evaluate the effect of the nonisothermal conditions, were calculated by means of a mathematical approach, which made it possible to know, using computer simulation, the radial and axial temperature profiles inside the bioreactor and across the membrane. Percent activity increases, proportional to the size of the temperature gradients, were found when the enzyme activities under nonisothermal conditions were compared to those measured under comparable isothermal conditions. Percent reductions of the production times, proportional to the applied temperature gradients, were also calculated. The advantage of employing nonisothermal bioreactors in biotechnological industrial process was discussed.     

Evaluation of a butorphanol, detomidine, and midazolam combination for immobilization of captive Nile lechwe antelopes (Kobus magaceros)

Field immobilization of captive antelope may be required for medical examination, blood sample collection, and animal identification. The aim of this study was to evaluate the effects of a combination of butorphanol, detomidine, and midazolam (BDM) and its partial reversibility in Nile lechwe antelope (Kobus megaceros). Nine captive lechwes, weighing 28-64 kg, were immobilized, in February 2011, with butorphanol 0.20 ± 0.05 (mean ± SD) mg/kg, detomidine 0.20 ± 0.05 mg/kg, and midazolam 0.31 ± 0.08 mg/kg administered intramuscularly (IM) with a blowpipe. Physiologic parameters and depth of anesthesia were recorded when the animals became recumbent at 19.55 ± 8.36 min after darting (T0) and after 10 (T10), 20 (T20), and 30 (T30) min. An arterial blood sample was collected at T20. At the end of the procedures, immobilization was partially reversed with atipamezole 0.25 mg/kg IM. Quality of induction, immobilization, and recovery was scored. The BDM combination induced immobilization and lateral recumbency in 13.44 ± 5.61 min. Median induction score (scored 1 [excellent] to 4 [poor]) was 1 (range 1-2). Heart rate varied 40-104 beats/min, respiratory rate 16-108 breaths/min, and rectal temperature 36.5-40.3 C. Hyperthermia was observed and rapidly treated in three animals that demonstrated insufficient immobilization after darting. Arterial blood gas analyses revealed a mean pH of 7.43 ± 0.07, partial arterial pressure of CO(2) of 44.1 ± 6.0 mmHg, partial arterial pressure of O(2) of 74.0 ± 13.5 mmHg, and an arterial O(2) saturation of 94.77 ± 3.96%. Recovery was smooth and animals were walking in 13.44 ± 7.85 min. Median recovery score (1 = excellent to 4 = poor) was 1 (range 1-2). The BDM was effective in immobilizing captive healthy lechwes with minimal cardiorespiratory changes.     

Intracellular acidification induces decrease of cytosolic calcium in isolated osteoclasts

In this study the effects of changes in intracellular pH on cytosolic Ca2+ were examined in single isolated osteoclasts. Alkalinization, performed by incubation in HCO3 containing buffer, induced increases in [Ca2+]. Conversely acidification, obtained by incubation in Na-butyrate-containing buffer induced a rapid and sustained decrease of [Ca2+]. The decrease of [Ca2+], during acidification with Na-butyrate was inhibited by VO4, an inhibitor of the Ca2+-ATPase. All these pieces of information indicate that changes in pH1 can modulate osteoclast activity together with modifications of [Ca2+], probably acting as intracellular signals.     

Regulation of podosomes by intracellular pH in avian osteoclasts

The effects of changes in intracellular pH (pH1) on the organization of the clear zone of isolated avian osteoclasts in culture were studied. The distribution of podosomes, the close contact areas that mediate the adhesion of osteoclasts to the substrate, was investigated by decoration of microfilaments with fluorescent phalloidin. Intracellular acidification by butyric acid induces significant increase of podosome formation at the level of the clear zone compared to controls. Conversely, alkalinization by HCO3- reduces the percentage of osteoclasts with podosomes. A role of pH1 on the adhesion of the osteoclasts to the substrate is hypothesized.     

Cytoplasmic pH is regulated in isolated avian osteoclasts by a Cl-/HCO3- exchanger

Osteoclast resorb bone in an acid compartment formed by the bone-attachment site. The low pH of the resorption compartment provides a lysosome-like milieu suitable for acid proteases to degrade collagen. Solubilization of the hydroxyapatite that makes up bone mineral consumes about 2 moles of protons per moles of calcium dissolved, requiring a massive proton flux to maintain a low pH in the resorption compartment. In order to determine how the osteoclast maintains a physiological cytoplasmic pH while secreting massive amounts of acid, we studied the intracellular pH of osteoclasts using esterified fluorescein derivatives while controlling the electrolyte composition of the medium. The principal finding is that osteoclasts have a high capacity for chloride/bicarbonate exchange which enables them to maintain normal intracellular pH in the face of a large loading of base equivalents. Thus, the overall process of proton secretion during bone resorption is similar to the polarized acid elimination by renal epithelia, involving a proton pump on one surface of the cell, and a Cl-/HCO3- exchange to maintain cytoplasmic pH.     

Isothermal and non-isothermal bioreactors in the detoxification of waste waters polluted by aromatic compounds by means of immobilised laccase from Rhus vernicifera

Laccase from Rhus vernicifera was immobilised on a nylon membrane chemically grafted with glycidyl methacrylate (GMA). Hexamethylenediamine (HMDA) and glutaraldehyde (GLU) were used as spacer and bifunctional coupling agent, respectively. Quinol was used as substrate.

To know how the immobilisation procedures affected the enzyme reaction rate the catalytic behaviour of soluble and insoluble laccase was studied under isothermal conditions as a function of pH, temperature and substrate concentration. From these studies, two main singularities emerged from the experimental data: (i) the narrower pH-activity profile of the insoluble enzyme in comparison to that of the soluble counterpart; (ii) the increase of the affinity of the immobilised enzyme for its substrate.

The behaviour of the catalytic membrane was also studied in a non-isothermal bioreactor as a function of substrate concentration and size of the applied transmembrane temperature difference. It was found that, under non-isothermal conditions and keeping constant the average temperature of the bioreactor, the enzyme reaction rate linearly increases with the increase of the temperature difference. These results have been discussed in the frame of reference of the process of thermodialysis driving thermodiffusive transmembrane substrate fluxes, which add to the diffusive ones.

The advantages of the catalytic process carried out under non-isothermal conditions have been thrown in relief through the evaluation of the reduction of the production times and of the percentage increases of the enzyme activity.