Protective treatments for copper alloy artworks: preliminary studies of sodium oxalate and limewater effectiveness against bronze disease.

Nantokite (CuCl) locked inside subsurface micro-pits has been recognised as the driving force to the form of corrosion called bronze disease. The use of the traditional corrosion inhibitor benzotriazole is questioned because of toxicity. So there is a need for alternative conservation treatments. This work is focused on the experimental design to test the effectiveness of sodium oxalate followed by treatment with limewater to face bronze disease on outdoor bronzes. A number of foundry bronze coupons were exposed to weathering at Genoa Experimental Marine Station (GEMS) exposure site and sprayed twice a week with a 5% NaCl solution for the first 124 days. After 562 days of natural weathering, the patinas on coupons were characterised with non-destructive techniques (NDT) and the presence of nantokite was verified. We designed a workflow, as similar as possible to conservation treatments on real artworks, to test a 3% w/v sodium oxalate treatment with two different application times, with or without limewater, on the coupons. The effectiveness of the treatments was analysed by comparison of surface properties by several NDT measurements. A statistical approach and XRD measurements directly on the corroded bronze surfaces are suggested as an effective way to characterise and compare the overall behaviour of bronze disease treatments for conservation.

Microplastics in the Arctic: A case study with sub-surface water and fish samples off Northeast Greenland.

The Arctic is a unique and fragile ecosystem that needs to be preserved and protected. Despite its remoteness, plastic pollution has been documented in this region. In the coming years, it is likely to worsen since, with climate changes and the opening of new shipping routes, the human presence is going to increase in the whole area. Here, we investigated the presence of microplastics (MPs) in sub-surface water and in two mid-trophic level Arctic fishes collected off Northeast Greenland: the demersal bigeye sculpin, Triglops nybelini, and the pelagic polar cod, Boreogadus saida. Plastics debris were found in the water samples at a concentration of 2.4 items/m3 ±0.8 SD which is higher than in most seas at lower latitudes. Both fish species had eaten MPs with different proportion among the species, 34% for T. nybelini (n = 71) and 18% for B. saida (n = 85). The significant difference in the occurrence of MPs between the two species is likely a consequence of their feeding behavior and habitat. Polyethylene was the main plastic polymer for water samples (41%, n = 17) and polyester (34%, n = 156) for fish samples as analyzed by Fourier Transformed Infrared (FT-IR) spectroscopy. Our data underscore that the Arctic regions are turning into a hotspot for plastic pollution, and this calls urgently for precautionary measures.

Calpain-1 resident in lipid raft/caveolin-1 membrane microdomains plays a protective role in endothelial cells.

We are here reporting that calpain-1 is a constitutive component of a distinct lipid raft/caveolin-1 microdomain isolated from bEnd5 cells in association with endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (HSP90). Perturbations in intracellular calcium concentration by Ca2+-ionophore A23187 or prolonged cell exposure to high glucose induce a significant decrease in the level of eNOS accompanied by a recruitment of additional HSP90 molecules at this site. In these conditions the cells are more resistant to cell death by Ca2+ overload. The decrease of eNOS has been due not only to its Ca2+-mediated release from the caveolin-1 aggregates but also to its digestion by calpain-1. The specific involvement of calpain-1 in digestion of eNOS is supported by the preventive effect of a synthetic calpain inhibitor (CI-2) and by the absence of calpain-2 and calpastatin in the caveolin-1 microdomain. These results suggest that the protein adjustments observed in lipid raft/caveolin-1 microdomains could be visualized as a process required to protect the cells against NO overproduction and aberrant calpain activation. Alterations in eNOS, calpain-1 and HSP90 levels have been observed in aorta of Zucker Diabetic Rats (ZDR). The loss of HSP90 occurring in these animals indicates an aberrant activation of calpain and thereby the transition from a physiological to a pathological cell condition.

Necrosis of the nipple-areola complex in breast reduction. Our personal way to solve problem.

Necrosis of the NAC is a condition that penalizes patients who underwent breast reduction surgery or mastopexy. Breast reduction is a widely used technique for over-sized breasts. Breast hypertrophy, in fact, can cause the onset of many issues--both aesthetical and pathological--because of the excessive weight that the breasts exert on the patient's spine. Aim and objective of our study is to suggest a systematic use of diagnostic imaging composed of pre-operative and intraoperative ultrasound with color-Doppler and pre-operative MRI. Trying to solve this problem definitively, we relied on our notions of anatomy on ten fresh cadavers, on whose twenty breasts we could make very detailed dissections. The dissections led us to conclude that, albeit with their anatomic differences, each breast was characterized by a vascular-nervous pedicle coming out from the inter-costal spaces and aimed to the blood supply to the NAC. To overcome the anatomic variations between one subject and another--but also between one breast and the other from the same patient, we relied on diagnostic imaging, both in the pre-operative and in the intra-operative staging. This way we were able to intervene successfully with 15 patients, none of which has complained damages to the vascularity or innervation of the NAC so far. In conclusion we believe that pre and intra operative diagnostic imaging is the only way to completely eliminate any potential risk of NAC necrosis. Only by means of the systematic use of conventional imaging--especially during surgery--it is possible to constantly monitor the position of the NAC's pedicle in a breast that is being reduced in volume.

Clinical severity of ß-thalassaemia/Hb E disease is associated with differential activities of the calpain-calpastatin proteolytic system.

Earlier observations in the literature suggest that proteolytic degradation of excess unmatched α-globin chains reduces their accumulation and precipitation in ß-thalassaemia erythroid precursor cells and have linked this proteolytic degradation to the activity of calpain protease. The aim of this study was to correlate the activity of calpain and its inhibitor, calpastatin, with different degrees of disease severity in ß-thalassaemia. CD34(+) cells were enriched from peripheral blood of healthy individuals (control group) and patients with mild and severe clinical presentations of ß(0)-thalassaemia/Hb E disease. By ex vivo cultivation promoting erythroid cell differentiation for 7 days, proerythroblasts, were employed for the functional characterization of the calpain-calpastatin proteolytic system. In comparison to the control group, enzymatic activity and protein amounts of µ-calpain were found to be more than 3-fold increased in proerythroblasts from patients with mild clinical symptoms, whereas no significant difference was observed in patients with severe clinical symptoms. Furthermore, a 1.6-fold decrease of calpastatin activity and 3.2-fold accumulation of a 34 kDa calpain-mediated degradation product of calpastatin were observed in patients with mild clinical symptoms. The increased activity of calpain may be involved in the removal of excess α-globin chains contributing to a lower degree of disease severity in patients with mild clinical symptoms.

Evidence for alteration of calpain/calpastatin system in PBMC of cystic fibrosis patients.

We are here reporting that in peripheral blood mononuclear cells (PBMC) of patients homozygous for F508del-CFTR the calpain-calpastatin system undergoes a profound alteration. In fact, calpain basal activity, almost undetectable in control PBMC, becomes measurable at a significant extent in cells from cystic fibrosis (CF) patients, also due to a 40-60% decrease in both calpastatin protein and inhibitory activity. Constitutive protease activation in CF patients' cells induces a large accumulation of the mutated cystic fibrosis transmembrane conductance regulator (CFTR) in the 100kD+70kD split forms as well as a degradation of proteins associated to the CFTR complex. Specifically, the scaffolding protein Na(+)/H(+) exchanger 3 regulatory factor-1 (NHERF-1) is converted in two distinct fragments showing masses of 35kD and 20kD, being however the latter form the most represented one, thereby indicating that in CF-PBMC the CFTR complex undergoes a large disorganization. In conclusion, our observations are providing new information on the role of calpain in the regulation of plasma membrane ion conductance and provide additional evidence on the transition of this protease activity from a physiological to a pathological function.

Prosthetic breast implant rupture: imaging--pictorial essay.

In recent years, requests for breast implant surgery have occurred for several reasons. First, the number of diagnosed breast cancer cases has increased, and the number of reconstructive surgeries consequently has multiplied. Second, the number of patients who constantly try to achieve a better physical shape, corresponding in Western countries to the common image of prosperous and tonic breasts, has proliferated. These circumstances have led to an increasingly frequent need for more accurate and sophisticated imaging methods to study prosthetic breast implants and their integrity. Diagnostic imaging for the study of patients with suspected breast implant ruptures uses different techniques of radiologic investigation such as mammography and ultrasonography, even if the current gold standard is magnetic resonance imaging (MRI). This study aimed to draw attention to the main MRI signs capable of highlighting contractures or ruptures of the implants that are not always clinically detectable and thus to provide plastic surgeons with an adequate instrument for discerning any possible alterations in prosthetic implants. Furthermore, it was necessary to stress the importance of teamwork. In fact, proper cooperation and coordination between radiologists and dedicated plastic surgeons are fundamental for the proper management of patients and the complications they may experience.

Calpain digestion and HSP90-based chaperone protection modulate the level of plasma membrane F508del-CFTR.

We are here showing that peripheral mononuclear blood cells (PBMC) from cystic fibrosis (CF) patients contain almost undetectable amounts of mature 170 kDa CF-transmembrane conductance regulator (CFTR) and a highly represented 100 kDa form. This CFTR protein, resembling the form produced by calpain digestion and present, although in lower amounts, also in normal PBMC, is localized in cytoplasmic internal vesicles. These observations are thus revealing that the calpain-mediated proteolysis is largely increased in cells from CF patients. To characterize the process leading to the accumulation of such split CFTR, FRT cells expressing the F508del-CFTR mutated channel protein and human leukaemic T cell line (JA3), expressing wild type CFTR were used. In in vitro experiments, the sensitivity of the mutated channel to the protease is identical to that of the wild type, whereas in Ca(2+)-loaded cells F508del-CFTR is more susceptible to digestion. Inhibition of intracellular calpain activity prevents CFTR degradation and leads to a 10-fold increase in the level of F508del-CFTR at the plasma membrane, further indicating the involvement of calpain activity in the maintenance of very low levels of mature channel form. The higher sensitivity to calpain of the mutated 170 kDa CFTR results from a reduced affinity for HSP90 causing a lower degree of protection from calpain digestion. The recovery of HSP90 binding capacity in F508del-CFTR, following digestion, explains the large accumulation of the 100 kDa CFTR form in circulating PBMC from CF patients.

Role of calpain in the regulation of CFTR (cystic fibrosis transmembrane conductance regulator) turnover.

The level of the mature native 170 kDa form of CFTR (cystic fibrosis transmembrane conductance regulator) at the plasma membrane is under the control of a selective proteolysis catalysed by calpain. The product of this limited digestion, consisting of discrete fragments still associated by strong interactions, is removed from the plasma membrane and internalized in vesicles and subject to an additional degradation. This process can be monitored by visualizing the accumulation of a 100 kDa fragment in a proliferating human leukaemic T-cell line and in human circulating lymphocytes. In reconstructed systems, and in intact cells, the conversion of native CFTR into the 100 kDa fragment linearly correlated with calpain activation and was prevented by addition of synthetic calpain inhibitors. A reduction in Ca2+ influx, by blocking the NMDA (N-methyl-D-aspartate) receptor Ca2+ channel, inhibited the conversion of the native 170 kDa fragment into the 100 kDa fragment, whereas an endosome acidification blocker promoted accumulation of the digested 100 kDa CFTR form. An important role in calpain-mediated turnover of CFTR is exerted by HSP90 (heat-shock protein 90), which, via association with the protein channel, modulates the degradative effect of calpain through a selective protection. Taken together these results indicate that CFTR turnover is initiated by calpain activation, which is induced by an increased Ca2+ influx and, following internalization of the cleaved channel protein, and completed by the lysosomal proteases. These findings provide new insights into the molecular mechanisms responsible for the defective functions of ion channels in human pathologies.

Calpain3 is expressed in a proteolitically active form in papillomavirus-associated urothelial tumors of the urinary bladder in cattle.

BACKGROUND: Calpain 3 (Capn3), also named p94, is a skeletal muscle tissue-specific protein known to be responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Recent experimental studies have hypothesized a pro-apoptotic role of Capn3 in some melanoma cell lines. So far the link between calpain3 and tumors comes from in vitro studies. The objective of this study was to describe Capn3 activation in naturally occurring urothelial tumors of the urinary bladder in cattle. METHODS AND FINDINGS: Here we describe, for the first time in veterinary and comparative oncology, the activation of Capn3 in twelve urothelial tumor cells of the urinary bladder of cattle. Capn3 protein was initially identified with nanoscale liquid chromatography coupled with tandem mass spectrometry (nano LC-MS/MS) in a co-immunoprecipitation experiment on E2F3, known to be a transcription factor playing a crucial role in bladder carcinogenesis in humans. Capn3 expression was then confirmed by reverse transcription polymerase chain reaction (RT-PCR). Finally, the Ca(2+)-dependent proteolytic activity of Capn3 was assayed following ion exchange chromatography. Morphologically, Capn3 expression was documented by immunohistochemical methods. In fact numerous tumor cells showed an intracytoplasmic immunoreactivity, which was more rarely evident also at nuclear level. In urothelial tumors, bovine papillomavirus type 2 (BPV-2) DNA was amplified by PCR and the expression of E5 protein, the major oncogenic protein of BVP-2, was detected by western blotting, immunohistochemistry, and immunofluorescence. E2F3 overexpression and pRb protein downregulation were shown by western blotting. CONCLUSION: The role of capn3 protein in urothelial cancer of the urinary bladder remains to be elucidated: further studies would be required to determine the precise function of this protease in tumor development and progression. However, we suggest that activated Capn3 may be involved in molecular pathways leading to the overexpression of E2F3, which in turn could be responsible for urothelial tumor cell proliferation also in cattle, though other mechanisms are likely to exist. If further studies corroborate the important role of Capn3 in urothelial tumors of the urinary bladder, cattle with urinary tumors may prove useful as animal model for bladder carcinogenesis.

Adaptive modifications in the calpain/calpastatin system in brain cells after persistent alteration in Ca2+ homeostasis.

Persistent dysregulation in Ca(2+) homeostasis is a pervasive pathogenic mechanism in most neurodegenerative diseases, and accordingly, calpain activation has been implicated in neuronal cells dysfunction and death. In this study we examined the intracellular functional state of the calpain-calpastatin system in -G93A(+) SOD1 transgenic mice to establish if and how uncontrolled activation of calpain can be prevented in vivo during the course of prolonged [Ca(2+)](i) elevation. The presented data indicate that 1) calpain activation is more extensive in motor cortex, in lumbar, and sacral spinal cord segments compared with the lower or almost undetectable activation of the protease in other brain areas, 2) direct measurements of the variations of Ca(2+) levels established that the degree of the protease activation is correlated to the extent of elevation of [Ca(2+)](i), 3) intracellular activation of calpain is always associated with diffusion of calpastatin from perinuclear aggregated forms into the cytosol and the formation of a calpain-calpastatin complex, and 4) a conservative fragmentation of calpastatin is accompanied by its increased expression and inhibitory capacity in conditions of prolonged increase in [Ca(2+)](i). Thus, calpastatin diffusion and formation of the calpain-calpastatin complex together with an increased synthesis of the inhibitor protein represent a cellular defense response to conditions of prolonged dysregulation in intracellular Ca(2+) homeostasis. Altogether these findings provide a new understanding of the in vivo molecular mechanisms governing calpain activation that can be extended to many neurodegenerative diseases, potentially useful for the development of new therapeutic approaches.

Calpain-mediated activation of NO synthase in human neuroblastoma SK-N-BE cells.

In resting human neuronal cells, nitric oxide synthase (nNOS) is present in its native 160 kDa form in a quiescent state predominantly co-localized on the plasma membrane, via its PDZ (Psd-95/Discs-large/Zona Occludens) domain, with NMDA receptor (NMDA-R) and in tight association with heat shock protein 90 (HSP90). Following exposure of the cells to Ca(2+)-ionophore or to NMDA, nNOS undergoes proteolytic removal of the PDZ domain being converted into a fully active 130 kDa form. The newly generated nNO synthase form dissociates from NMDA-R and extensively diffuses into the cytosol in direct correlation with NO production. Intracellular redistribution and activation of nNOS are completely prevented in cells preloaded with calpain inhibitor-1, indicating that these processes are triggered by a concomitant activation of calpain. The role of calpain has been confirmed by immunoprecipitation experiments revealing that also mu-calpain is specifically recruited into the NMDA-R-nNOS-HSP90 complex following calcium loading. Thus, the formation of clusters containing HSP90, mu-calpain, nNOS and NMDA-R represents the limiting step for the operation of the mechanism that links an efficient synthesis of NO to a local increase in Ca(2+) influx.

Involvement of exon 6-mediated calpastatin intracellular movements in the modulation of calpain activation.

BACKGROUND: To establish the physiological role of calpain, it is necessary to define how the protease can escape from the effect of its natural inhibitor calpastatin, since both proteins co-localize into the cell cytosol. METHODS: To answer this question, we have overexpressed four fluorescent calpastatin constructs, differing in the composition of their XL- and L-domains, and the intracellular trafficking of this protein inhibitor has been followed by single cell fluorescence imaging. RESULTS AND CONCLUSIONS: By the use of these calpastatin forms differing in the type of exon-derived sequences contained in the XL- and L-domains, we have demonstrated that the sequence coded by exon 6, containing multiple phosphorylation sites, is directly involved in determining the cell localization of calpastatin. In fact, exposure to cAMP promotes the recruitment into aggregates of those calpastatin forms containing the exon 6 sequence. These protein movements are directly related to the level of cytosolic inhibitory capacity and thereby to the extent of intracellular calpain activation. GENERAL SIGNIFICANCE: The recruitment of calpastatin into aggregates allows the translocation and activation of the protease to the membranes; on the contrary, the presence of large amounts of calpastatin in the cytosol prevents both processes, protecting the cell from undesired proteolysis.

Role of the calpain-calpastatin system in the density-dependent growth arrest.

In dividing cells calpastatin diffuses from aggregates into cytosol, indicating the requirement for a tight regulation of calpain. Accordingly, the involvement of the calpain-calpastatin system in cell proliferation and in the density-dependent growth arrest was studied in JA3 cells stably transfected with a calpastatin form permanently localized in cytosol. In calpastatin overexpressing cells, cell cycle rate is 50% reduced, and cells enter the ungrowing, still fully reversible, stage at a 3-fold higher cell density. Furthermore, in cell density growth arrest phase, down regulation of alpha- and theta-PKC isoforms, as well as FAK and talin occurs. In calpastatin overexpressing cells, degradation of these calpain substrate proteins is prevented and delayed. Thus, calpain activity plays a crucial role in inducing the cell entry into a functional quiescent phase.

Functional role of HSP90 complexes with endothelial nitric-oxide synthase (eNOS) and calpain on nitric oxide generation in endothelial cells.

Although several reports have indicated that eNOS is a highly sensitive calpain substrate, the occurrence of a concomitant Ca(2+)-dependent activation of the synthase and of the protease has never been analyzed in specific direct experiments. In this study, we have explored in vivo how eNOS can undergo Ca(2+)-dependent translocation and activation, protected against degradation by activated calpain. Here we demonstrate that following a brief exposure to Ca(2+)-loading, the cytosolic eNOS-HSP90 complex recruits calpain in a form in which the chaperone and the synthase are almost completely resistant to digestion by the protease. Furthermore, in the presence of the HSP90 inhibitor geldanamycin, a significant decrease in NO production and an extensive degradation of eNOS protein occurs, indicating that dissociation from membranes and association with the chaperone is correlated to the protection of the synthase. Experiments with isolated membrane preparations confirm the primary role of HSP90 in dissociation of eNOS from caveolae. Prolonged exposure of cells to Ca(2+)-loading resulted in an extensive degradation of both eNOS and HSP90, accompanied by a large suppression of NO production. We propose that the protective effect exerted by HSP90 on eNOS degradation mediated by calpain represents a novel and critical mechanism that assures the reversibility of the intracellular trafficking and activation of the synthase.

In vivo degradation of nitric oxide synthase (NOS) and heat shock protein 90 (HSP90) by calpain is modulated by the formation of a NOS-HSP90 heterocomplex.

We have shown previously that isolated heat shock protein 90 (HSP90) and nitric oxide synthase (NOS), once associated in a heterocomplex, become completely resistant to calpain digestion. In this study, it is shown that, in vivo, under conditions of calpain activation, the protection of NOS degradation occurs. In addition, the extent of NOS degradation is a function of the level of HSP90 expression. Thus, in rat brain, which contains a large excess of HSP90, almost all neuronal NOS is associated with the chaperone protein. In this condition, neuronal NOS retains its full catalytic activity, although limited proteolytic conversion to still active low-molecular-mass (130 kDa) products takes place. In contrast, in aorta, which contains much smaller amounts of HSP90, endothelial NOS is not completely associated with the chaperone, and undergoes extensive degradation with a loss of protein and catalytic activity. On the basis of these findings, we propose a novel role of the HSP90-NOS heterocomplex in protecting in vivo NOS from proteolytic degradation by calpain. The efficiency of this effect is directly related to the level of intracellular HSP90 expression, generating a high HSP90 to NOS ratio, which favours both the formation and stabilization of the HSP90-NOS heterocomplex. This condition seems to occur in rat brain, but not in aorta, thus explaining the higher vulnerability to proteolytic degradation of endothelial NOS relative to neuronal NOS.

Proteolytic degradation of nitric oxide synthase isoforms by calpain is modulated by the expression levels of HSP90.

Ca2+ loading of Jurkat and bovine aorta endothelium cells induces the degradation of the neuronal and endothelial nitric oxide synthases that are selectively expressed in these cell lines. For neuronal nitric oxide synthase, this process involves a conservative limited proteolysis without appreciable loss of catalytic activity. By contrast, endothelial nitic oxide synthase digestion proceeds through a parallel loss of protein and catalytic activity. The chaperone heat shock protein 90 (HSP90) is present in a large amount in Jurkat cells and at significantly lower levels in bovine aorta endothelium cells. The differing ratios of HSP90/nitric oxide synthase (NOS) occurring in the two cell types are responsible for the conservative or nonconservative digestion of NOS isozymes. Consistently, we demonstrate that, in the absence of Ca2+, HSP90 forms binary complexes with NOS isozymes or with calpain. When Ca2+ is present, a ternary complex containing the three proteins is produced. In this associated state, HSP90 and NOS forms are almost completely resistant to calpain digestion, probably due to a structural hindrance and a reduction in the catalytic efficiency of the protease. Thus, the recruitment of calpain in the HSP90-NOS complexes reduces the extent of the proteolysis of these two proteins. We have also observed that calpastatin competes with HSP90 for the binding of calpain in reconstructed systems. Digestion of the proteins present in the complexes can occur only when free active calpain is present in the system. This process can be visualized as a novel mechanism involving the association of NOS with HSP90 and the concomitant recruitment of active calpain in ternary complexes in which the proteolysis of both NOS isozymes and HSP90 is significantly reduced.

Multiple rat brain calpastatin forms are produced by distinct starting points and alternative splicing of the N-terminal exons.

5'-RACE was performed on rat brain calpastatin mRNA and two new translation initiation ATG's were found. The first one is upstream of the previously designed initiation translation site localized in the rat calpastatin L-domain. The deduced protein sequence of this region is highly homologous to the XL-domain of calpastatin type I in other species. The other ATG has not previously been reported and is localized in exon 8, thus originating a calpastatin isoform constituted only by four repetitive inhibitory units without the XL-L-domains. Transcripts from the rat brain calpastatin gene are also subjected to multiple splicing events involving exons 4, 6, 8 in different combinations. A series of recombinant calpastatin forms was produced that differed in the exons present in the L-domain, and all the variants showed comparable inhibitory efficiency against calpain. It was concluded that the presence of the XL-domain in these isoforms is not relevant for the formation of the calpain/calpastatin complex in the absence of calcium, that is the interaction of calpastatin with inactive calpain. Using exon-specific antisera, specific calpastatin protein isoforms containing the XL-domain have been detected in rat brain homogenates.

Regulation of calpain activity in rat brain with altered Ca2+ homeostasis.

Activation of calpain occurs as an early event in correlation with an increase in [Ca2+]i induced in rat brain upon treatment with a high salt diet for a prolonged period of time. The resulting sequential events have been monitored in the brain of normal and hypertensive rats of the Milan strain, diverging for a constitutive alteration in the level of [Ca2+]i found to be present in nerve cells of hypertensive animals. After 2 weeks of treatment, the levels of the plasma membrane Ca2+-ATPase and of native calpastatin are profoundly decreased. These degradative processes, more pronounced in the brain of hypertensive rats, are progressively and efficiently compensated in the brain of both rat strains by different incoming mechanisms. Along with calpastatin degradation, 15-kDa still-active inhibitory fragments are accumulated, capable of efficiently replacing the loss of native inhibitor molecules. A partial return to a more efficient control of Ca2+ homeostasis occurs in parallel, assured by an early increase in the expression of Ca2+-ATPase and of calpastatin, both producing, after 12 weeks of a high salt (sodium) diet, the restoration of almost original levels of the Ca2+ pump and of significant amounts of native inhibitor molecules. Thus, conservative calpastatin fragmentation, associated with an increased expression of Ca2+-ATPase and of the calpain natural inhibitor, has been demonstrated to occur in vivo in rat brain. This represents a sequential adaptive response capable of overcoming the effects of calpain activation induced by a moderate long term elevation of [Ca2+]i.

Characterization of the calpain/calpastatin system in human hemopoietic cell lines.

As previously suggested by PCR analysis [R. DeTullio, R. Stifanese, F. Salamino, S. Pontremoli, E. Melloni, Characterization of a new p94-like calpain form in human lymphocytes, Biochem. J. 375 (2003) 689-696], a p94-like calpain was now established to be present in six different human cells resembling the various peripheral blood cell types. This protease resulted to be the predominant calpain isoforms whereas the conventional mu- and m-calpains are also expressed although at lower or almost undetectable amounts. The p94-like calpain has been identified by a specific mAb and displays unique features such as: Ca2+ requirement for half maximum activity around 30 microM; no autolytic conversion to a low Ca2+ requiring form and lower sensitivity to calpastatin inhibition. Following cell stimulation, the p94-like calpain undergoes inactivation, a process indicating that the protease is activated and participates in the cell responses to stimuli. The involvement of this protease isoform in immunocompetent cell activation is further supported by its partial recruitment on plasma membranes, the site of action of the conventional calpain forms. The amount of calpain translocated to the membranes correlates to the level of calpastatin which has been shown to control this process through the formation of a complex with calpain, which maintains the protease in the cytosol. These results provide new information on the calpain/calpastatin system expressed in immunocompetent cells and on the functional relationship between the p94-like calpain and the biological function of these cells.