Binding of calpain fragments to calpastatin

Their cDNA-derived amino acid sequences predict that the 80-kDa subunits of the micromolar and millimolar Ca(2+)-requiring forms of the Ca(2+)-dependent proteinase (mu- and m-calpain, respectively) each consist of four domains and that the 28-kDa subunit common to both mu- and m-calpain consists of two domains. The calpains were allowed to autolyze to completion, and the autolysis products were separated and were characterized by using gel permeation chromatography, calpastatin affinity chromatography, and sequence analysis. Three major fragments were obtained after autolysis of either calpain. The largest fragment (34 kDa for mu-calpain, 35 kDa for m-calpain) contains all of domain II, the catalytic domain, plus part of domain I of the 80-kDa subunit of mu- or m-calpain. This fragment does not bind to calpastatin, a competitive inhibitor of the calpains, and has no proteolytic activity in either the absence or presence of Ca2+. The second major fragment (21 kDa for mu-calpain and 22 kDa for m-calpain) contains domain IV, the calmodulin-like domain, plus approximately 50 amino acids from domain III of the 80-kDa subunit of mu- or m-calpain. The third major fragment (18 kDa) contains domain VI, the calmodulin-like domain of the 28-kDa subunit. The second and third major fragments bind to a calpastatin affinity column in the presence of Ca2+ and are eluted with EDTA. The second and third fragments are noncovalently bound, so the 80- and 28-kDa subunits of the intact calpain molecules are noncovalently bound at domains IV and VI. After separation in 1 M NaSCN, the 28-kDa subunit binds completely to calpastatin, approximately 30-40% of the 80-kDa subunit of mu-calpain binds to calpastatin, and the 80-kDa subunit of m-calpain does not bind to calpastatin in the presence of 1 mM Ca2+.     

m-Calpain is required for preimplantation embryonic development in mice

Background  

μ-calpain and m-calpain are ubiquitously expressed proteases implicated in cellular migration, cell cycle progression, degenerative processes and cell death. These heterodimeric enzymes are composed of distinct catalytic subunits, encoded by Capn1 (μ-calpain) or Capn2 (m-calpain), and a common regulatory subunit encoded by Capn4. Disruption of the mouse Capn4 gene abolished both μ-calpain and m-calpain activity, and resulted in embryonic lethality, thereby suggesting essential roles for one or both of these enzymes during mammalian embryogenesis. Disruption of the Capn1 gene produced viable, fertile mice implying that either m-calpain could compensate for the loss of μ-calpain, or that the loss of m-calpain was responsible for death of Capn4 ^-/- mice.     

Identification of μ-, m-calpains and calpastatin and capture of μ-calpain activation in endothelial cells

The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 μM Ca²⁺, comparable to the Ca²⁺ requirement of m-calpain. By Western blot analysis, the large subunit of μ-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of μ- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of μ-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of μ-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of μ-calpain, while the fully autolyzed postautolysis form of μ-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that μ-calpain may be involved in endothelial cell function mediated by Ca²⁺ via the limited proteolysis of various proteins. J. Cell. Biochem. 66:197-209, 1997. © 1997 Wiley-Liss, Inc.     

Localization of m-calpain and calpastatin and studies of their association in pulmonary smooth muscle endoplasmic reticulum

Calpain and calpastatin have been demonstrated to play many physiological roles in a variety of systems. It, therefore, appears important to study their localization and association in different suborganelles. Using immunoblot studies, we have identified 80 kDa m-calpain in both lumen and membrane of ER isolated from bovine pulmonary artery smooth muscle. Treatment of the ER with Na(2)CO(3) and proteinase K demonstrated that 80 kDa catalytic subunit and 28 kDa regulatory subunit (Rs) of m-calpain, and the 110-kDa and 70-kDa calpastatin (Cs) forms are localized in the cytosolic side of the ER membrane. Coimmunoprecipitation studies revealed that m-calpain is associated with calpastatin in the cytosolic face of the ER membrane. We have also identified m-calpain activity both in the ER membrane and lumen by casein-zymography. The casein-zymogram has also been utilized to demonstrate differential pattern of the effects of reversible and irreversible cysteine protease inhibitors on m-calpain activity. Thus, a potential site of Cs regulation of m-calpain activity is created by positioning Cs, 80 kDa and 28 kDa m-calpain in the cytosolic face of ER membrane. However, such is not the case for the 80-kDa m-calpain found within the lumen of the ER because of the conspicuous absence of 28 kDa Rs of m-calpain and Cs in this locale.     

Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns

Apoptosis, which is anti-inflammatory, and necrosis, which is pro-inflammatory, represent the extremes of the cell death spectrum. Cell death is complex and both apoptosis and necrosis can be observed in the same cells or tissues. Here, we introduce a novel combined mode of cellular demise – caspase-dependent regulated necrosis. Most importantly, it is mainly characterized with release of marked amount of oligo- or poly-nucleosomes and their attached damage-associated molecular patterns (DAMPs) and initiated by caspase activation. Caspase-activated DNase has dual roles in nucleosomal release as it can degrade extracellularly released chromatin into poly- or oligo-nucleosomes although it prohibits release of nucleosomes. In addition, osmotically triggered water movement following Cl⁻ influx and subsequent Na⁺ influx appears to be the major driving force for nucleosomal and DAMPs release. Finally, Ca²⁺-activated cysteine protease, calpain, is an another essential factor in nucleosomal and DAMPs release because of complete reversion to apoptotic morphology from necrotic one and blockade of nucleosomal and DAMPs release by its inhibition.Apoptosis is characterized by membrane blebbing, cellular shrinkage, nuclear condensation, nuclear fragmentations, oligo-nucleosomal DNA fragmentation and formation of apoptotic bodies. These characteristics are attributed mainly to the caspase family of cysteine proteases.^1,2 Necrosis is distinguished from apoptosis by cellular swelling, plasma membrane rupture, absence of oligo-nucleosomal degradation and, finally, rapid lysis of cells and cellular constituents including damage-associated molecular patterns (DAMPs) are massively exuded extracellularly to activate inflammatory and immune responses. ^{3, 4, 5}Calpains are a family of Ca²⁺-activated cysteine proteases consisting of 15 genes. Among them, μ-calpain (calpain I) and m-calpain (calpain II) are ubiquitously expressed in most cells as a heterodimer consisting of a large subunit (80 kDa; calpain 1 of μ-calpain and calpain 2 of m-calpain) and a common small subunit (29 kDa; calpain S1), which is processed into a smaller heterodimer (18–78 kDa) upon activation by Ca²⁺. Ubiquitous calpains are regulated by an endogenous inhibitor, calpastatin.⁶It has long been observed that both apoptosis and necrosis can be simultaneously detected in tissues or cell culture. Therefore, apoptosis and necrosis have been assumed to be two extremes of the cell death spectrum capable of inter-conversion by key regulators.^5,7 In this study, we introduce a novel mode of cell death involving the combination of apoptosis and necrosis, being a caspase-dependent process with necrotic morphology, involving the active release of DAMPs bound to nucleosomes.     

Effect of exercise training on calpain systems in lean and obese Zucker rats

Exercise training plays a major role in the improving physiology of diabetes. Herein we aimed to investigate the influence of exercise upon the calcium-dependent calpain-isoform expressions of lean or obese Zucker rats, a model of obesity and type II diabetes (NIDDM). Five-month-old rats were divided: (1) obese sedentary (OS, n=7); (2) obese exercise (OE, n=7); (3) lean sedentary (LS, n=7); (4) lean exercise (LE, n=7). After 2-month exercise (treadmill running), the body weight (BW) and expression of calpain 10, μ-calpain, and m-calpain in skeletal muscles were determined by RT-PCR, using β-actin as internal standard. We found exercise is useful for BW lossing, especially in the obese rats. The BW difference between OS and OE rats (69 g vs. 18.2 g) was more significantly than that between LS and LE rats (41.8 g vs. 28.7g). The calpain 10 expression of LS rats (0.965) was lower than that of LE rats (1.006), whereas those of OS and OE were comparable. The μ- or m-calpain expressions of sedentary groups (OS, LS) was significantly higher than those of exercise groups (OE, LE). The μ-calpain expression (1.13/0.92) and m-calpain expression (1.01/0.99) of OS/LS rats was significantly higher than those of OE/LE rats [1.07/0.9 (μ-calpain); 0.97/0.95 (m-calpain)]. We concluded that the μ- or m-calpains in skeletal muscle are regulated by exercise in both lean and obese Zucker rats. Exercise and BW controlling might improve the physiopathology of obesity and diabetes. Both μ- or m-calpains might become useful markers for prognoses of diabetes.     

Immunological detection of m- and μ-calpains in the skeletal muscle of Marchigiana cattle

Calpains are Ca²⁺-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15^th day post mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.Key words: m-calpain, µ-calpain, skeletal muscle, Marchigiana cattle, immunohistochemistry, Electron Microscopy.     

The control of transmembrane helix transverse position in membranes by hydrophilic residues

The ability of hydrophilic residues to shift the transverse position of transmembrane (TM) helices within bilayers was studied in model membrane vesicles. Transverse shifts were detected by fluorescence measurements of the membrane depth of a Trp residue at the center of a hydrophobic sequence. They were also estimated from the effective length of the TM-spanning sequence, derived from the stability of the TM configuration under conditions of negative hydrophobic mismatch. Hydrophilic residues (at the fifth position in a 21-residue hydrophobic sequence composed of alternating Leu and Ala residues and flanked on both ends by two Lys) induced transverse shifts that moved the hydrophilic residue closer to the membrane surface. At pH 7, the dependence of the extent of shift upon the identity of the hydrophilic residue increased in the order: L < GYT < RH < S < P < K < EQ < N < D. By varying pH, shifts with ionizable residues fully charged or uncharged were measured, and the extent of shift increased in the order: L < GYH^oT < E^oR < S < P < K⁺< QD^oH⁺ < NE^– < D^–. The dependence of transverse shifts upon hydrophilic residue identity was consistent with the hypothesis that shift magnitude is largely controlled by the combination of side chain hydrophilicity, ionization state, and ability to position polar groups near the bilayer surface (snorkeling). Additional experiments showed that shift was also modulated by the position of the hydrophilic residue in the sequence and the hydrophobicity of the sequence moved out of the bilayer core upon shifting. Combined, these studies show that the insertion boundaries of TM helices are very sensitive to sequence, and can be altered even by weakly hydrophilic residues. Thus, many TM helices may have the capacity to exist in more than one transverse position. Knowledge of the magnitudes of transverse shifts induced by different hydrophilic residues should be useful for design of mutagenesis studies measuring the effect of transverse TM helix position upon function.     

Nav1.7 sodium channel-induced Ca influx decreases tau phosphorylation via glycogen synthase kinase-3β in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells expressing Na_v1.7 sodium channel isoform, veratridine increased Ser⁴⁷³-phosphorylation of Akt and Ser⁹-phosphorylation of glycogen synthase kinase-3β by 217 and 195%, while decreasing Ser³⁹⁶-phosphorylation of tau by 36% in a concentration (EC₅₀ = 2.1 μM)- and time (t_1/2 = 2.7 min)-dependent manner. These effects of veratridine were abolished by tetrodotoxin or extracellular Ca²⁺ removal. Veratridine (10 μM for 5 min) increased translocation of Ca²⁺-dependent conventional protein kinase C-α from cytoplasm to membranes by 47%; it was abolished by tetrodotoxin, extracellular Ca²⁺ removal, or Gö6976 (an inhibitor of protein kinase C-α), and partially attenuated by LY294002 (an inhibitor of phosphatidylinositol 3-kinase). LY294002 (but not Gö6976) abrogated veratridine-induced Akt phosphorylation. In contrast, either LY294002 or Gö6976 alone attenuated veratridine-induced glycogen synthase kinase-3β phosphorylation by 65 or 42%; however, LY294002 plus Gö6976 completely blocked it. Veratridine (10 μM for 5 min)-induced decrease of tau phosphorylation was partially attenuated by LY294002 or Gö6976, but completely blocked by LY294002 plus Gö6976; okadaic acid or cyclosporin A (inhibitors of protein phosphatases 1, 2A, and 2B) failed to alter tau phosphorylation. These results suggest that Na⁺ influx via Na_v1.7 sodium channel and the subsequent Ca²⁺ influx via voltage-dependent calcium channel activated (1) Ca²⁺/protein kinase C-α pathway, as well as (2) Ca²⁺/phosphatidylinositol 3-kinase/Akt and (3) Ca²⁺/phosphatidylinositol 3-kinase/protein kinase C-α pathways; these parallel pathways converged on inhibitory phosphorylation of glycogen synthase kinase-3β, decreasing tau phosphorylation.     

Bottom-up controls on a mixed-species HAB assemblage: A comparison of sympatric Chattonella subsalsa and Heterosigma akashiwo (Raphidophyceae) isolates from the Delaware Inland Bays, USA

Recent novel mixed blooms of several species of toxic raphidophytes have caused fish kills and raised health concerns in the highly eutrophic Inland Bays of Delaware, USA. The factors that control their growth and dominance are not clear, including how these multi-species HAB events can persist without competitive exclusion occurring. We compared and contrasted the relative environmental niches of sympatric Chattonella subsalsa and Heterosigma akashiwo isolates from the bays using classic Monod-type experiments. C. subsalsa grew over a temperature range from 10 to 30 °C and a salinity range of 5–30 psu, with optimal growth occurring from 20 to 30 °C and 15 to 25 psu. H. akashiwo had similar upper temperature and salinity tolerances but also lower limits, with growth occurring from 4 to 30 °C and 5 to 30 psu and optimal growth between 16 and 30 °C and 10 and 30 psu. These culture results were confirmed by field observations of bloom occurrences in the Inland Bays. Maximum nutrient-saturated growth rates (μ_max) for C. subsalsa were 0.6 d⁻¹ and half-saturation concentrations for growth (K_s) were 9 μM for nitrate, 1.5 μM for ammonium, and 0.8 μM for phosphate. μ_max of H. akashiwo (0.7 d⁻¹) was slightly higher than C. subsalsa, but K_s values were nearly an order of magnitude lower at 0.3 μM for nitrate, 0.3 μM for ammonium, and 0.2 μM for phosphate. H. akashiwo is able to grow on urea but C. subsalsa cannot, while both can use glutamic acid. Cell yield experiments at environmentally relevant levels suggested an apparent preference by C. subsalsa for ammonium as a nitrogen source, while H. akashiwo produced more biomass on nitrate. Light intensity affected both species similarly, with the same growth responses for each over a range from 100 to 600 μmol photons m⁻² s⁻¹. Factors not examined here may allow C. subsalsa to persist during multi-species blooms in the bays, despite being competitively inferior to H. akashiwo under most conditions of nutrient availability, temperature, and salinity.     

EXTRA-LYSOSOMAL PROTEOLYSIS AND EXPRESSION OF CALPAINS AND CALPASTATIN IN CULTURED THYROID CELLS

Proteolysis at neutral pH in the soluble fraction of cultured pig thyroid epithelial cells was examined using a synthetic calpain substrate, succinyl-Leu-Tyr-7-amino-4-methylcoumarin. The Ca²⁺-independent proteolytic activity was largely inhibited by substances known to affect cysteine- and metalloproteases, whereas no or little effects were obtained with inhibitors affecting serine- and aspartic proteases. Addition of Ca²⁺did not significantly alter the rate of substrate degradation. Biochemical separation via hydrophobic interaction chomatography and Western blotting demonstrated the presence of both m-calpain (40% of total calpain) and μ-calpain (60%) in confluent thyrocytes. Determination of calpastatin activity indicated a 30 times higher level of the inhibitor as compared to total calpain activity. Western blotting showed the presence of a 110kD calpastatin form with additional low mol wt forms possibly representing fragmentation products. In immunofluorescent stainings, m-calpain had a diffuse cytoplasmic distribution whereas μ-calpain was located both in the cytoplasm and at the cell—cell contacts. Calpastatin immunoreactivity was mainly granular and located close to the nucleus, although a fibrillar distribution was also observed. The results show the presence of all components of the calpain/calpastatin system and indicate a strict control of calpain activity in cultured thyrocytes. The different subcellular distributions of calpains and calpastatin suggests that they are compartmentalized and require mobilization to interact.     

Trends in aluminium export from a mountainous area to surface waters, from deglaciation to the recent: Effects of vegetation and soil development, atmospheric acidification, and nitrogen-saturation

We reconstructed the history of terrestrial export of aluminium (Al) to Plešné Lake (Czech Republic) since the lake origin 12,600 year BC, and predicted Al export for 2010–2050 on the basis of previously published and new data on mass budget studies, palaeolimnological data, and MAGIC modelling. We focused on three major Al forms; ionic Al (Al_i), organically-bound Al (Al_o), and particulate Al hydroxide [Al(OH)₃]. In early post-glacial time, Plešné Lake received high terrestrial export of Al, but with a minor proportion of Al(OH)₃ (4–25 μM), and concentrations of Al_i and Al_o were negligible. Since the forest and soil development (9900–9000 year BC), erosion has declined and soil organic acids increased export of Al_o from soils. The terrestrial Al_o leaching (7.5 μM) persisted throughout the Holocene until the industrial period. Then, Al_i concentrations continuously increased (up to 28 μM in the mid-1980s) due to atmospheric acidification; the Al_i leaching was mostly associated with sulphate. The proportion of Al_i associated with nitrate has been increasing since the beginning of lake recovery from acidification after 1990 due to reduction in sulphur deposition and nitrogen-saturation of the catchment, leading to persistent nitrate leaching. Currently, nitrate has become the dominant strong acid anion and the major Al_i carrier. Al_o (5.5 μM) is predicted to dominate Al concentrations around 2050, but the predicted Al_i concentrations (4 μM) are uncertain because of uncertainty associated with the future nitrate leaching and its effect on soils.     

Synthesis and calpain inhibitory activity of peptidomimetic compounds with constrained amino acids at the P2 position

The effect of incorporating α,α′-diethylglycine and α-aminocyclopentane carboxylic acid at the P₂ position of inhibitors on μ-calpain inhibition was studied. Compound 3 with α,α′-diethylglycine was over 20-fold more potent than 2 with α-aminocyclopentane carboxylic acid. Additionally, 3 was over 35-fold selective for μ-calpain compared to cathepsin B, while 2 was 3-fold selective for cathepsin B compared to μ-calpain. Thus, the conformation induced by the P₂ residue influenced the activities of the compounds versus the closely related cysteine proteases, and suggests an approach to the discovery of selective μ-calpain inhibitors.     

Digestion of mu- and m-calpain by trypsin and chymotrypsin

Proteolytic digestion by trypsin and chymotrypsin was used to probe conformation and domain structure of the mu- and m-calpain molecules in the presence and the absence of Ca(2+). Both calpains have a compact structure in the absence of Ca(2+); incubation with either protease for 120 min results in only three or four major fragments. A 24-kDa fragment was produced by removal of the Gly-rich area in domain V of the 28-kDa subunit. The other fragments were from the 80-kDa subunit. Except for trypsin digestion of m-calpain, the region between amino acids 245 and 265 (human sequence) was very susceptible to cleavage by both proteases in the absence of Ca(2+); this region is in domain II (IIb of the crystallographic structure). Although no proteolytically active fragments could be isolated from either tryptic or chymotryptic digests, the calpain molecule can remain assembled in a proteolytically active complex even after the 80-kDa subunit has been completely degraded. The results suggest that interaction among different regions of the entire calpain molecule is required for its full proteolytic activity. In the presence of 1 mM Ca(2+), both calpains are degraded to fragments less than 40-kDa in less than 5 min. The C-terminal ends of both subunits, from amino acids 503 to 506 to the end of the 80-kDa subunit and from amino acids 85 to 88 to the end of the 28-kDa subunit, were resistant to degradation by either protease in the presence or in the absence of Ca(2+). Hence, this part of the calpain molecule is in a compact structure that does not change significantly in the presence of Ca(2+).     

Calpain对细胞骨架蛋白tau降解作用的研究(英文)

Calpain是钙依赖性中性蛋白酶 ,根据其对钙敏感性的不同 ,可分为m 和 μ calpain两型 .分别用不同浓度CaCl2 溶液孵育Wistar大鼠脑皮质匀浆液 ,并用蛋白质印迹和定量图像分析技术检测不同亚型calpain对tau蛋白的降解作用 .研究发现 :在 3 7℃用 1mmol/LCa2 孵育底物 15min ,可见tau蛋白明显降解 ,并在分子质量为 2 9ku处出现tau蛋白降解片段 ;当Ca2 浓度为 5mmol/L时 ,tau蛋白几乎全部被降解 ;这种tau蛋白降解可被calpain特异性抑制剂完全逆转 .进一步的研究发现 ,分别用 μ calpain抑制剂 (0 0 5μmol/Lcalpastatin) ,m calpain抑制剂 (10 0 μmol/LcalpaininhibitorⅣ )或总calpain抑制剂 (552 μmol/Lcalpeptin)与 1mmol/LCa2 共同孵育Wistar大鼠脑皮质匀浆液 ,Ca2 激活的tau蛋白降解分别被抑制8 6% ,92 5%和 97 8% .结果表明一定浓度的Ca2 可同时激活 μ calpain和m calpain ,这两种亚型calpain均参与降解tau蛋白 ,但m calpain的作用比 μ calpain更强     

The role of particles on the biogeochemical cycling of domoic acid and its isomers in natural water matrices

The adsorption of dissolved domoic acid (DA) and its geometrical isomers was assessed in aqueous solutions containing various types of particles. In one series of experiments carried out in coastal seawater, detectable net adsorption of 100 nM DA occurred only onto natural seawater particles (unfiltered seawater) and 0.5 g L⁻¹ chromatographic silica (18%) in 0.2 μm-filtered seawater. Some net adsorption (<5%) also occurred in the 0.5 g L⁻¹ suspension of estuarine sediment and 0.5 g L⁻¹ solution of humic acid in filtered seawater. No losses were seen in 0.5 g L⁻¹ suspensions of illite, kaolinite, montmorillonite, and silica sand. Biological degradation accounted for small losses (8–10%) in filtered seawater without particles. A second series of experiments using organic-free, <5 μm fractions of kaolinite and montmorillonite in deionized water (DIW) demonstrated that 70% of DA adsorbed onto kaolinite, but only 5% onto montmorillonite. Geometrical isomers of DA (iso-DA D, E, and F) showed negligible adsorption (0–8%) onto a variety of particles in filtered seawater, suggesting that major ions in seawater neutralize electrostatic attractions between particles and DA isomers. These results suggest that DA and its isomers are relatively hydrophilic and not particle reactive. Our data suggest that photochemical and biological degradation of dissolved DA and its isomers appears to occur in bulk surface seawater and its transport to bottom sediments must be mainly biologically driven.     

Expression of human, mouse, and rat m-calpains in Escherichia coli and in murine fibroblasts

The two best known calpains, micro- and m-calpain, are Ca(2+)-dependent cysteine proteases found in all mammalian tissues. They are probably involved in many Ca(2+)-linked signal pathways, although the details are not yet clear. The enzymes are heterodimers of a specific large subunit (micro-80k or m-80k) and a common small subunit (28k). Recombinant calpains have been obtained by co-expression of large and small subunits in Escherichia coli and in Sf9 cells, with variable success. Expression with the 28k subunit is very low, but is much higher with a C-terminal 21k fragment of this subunit. Rat m-calpain (m-80k/21k) is well expressed in E. coli but mouse m-calpain (m-80k/21k) is poorly expressed, even though the amino acid sequences of rat-m-80k and mouse-m-80k are 92% identical. It had also been reported that human m-calpain could be expressed in Sf9 cells but not in E. coli. To investigate these differences, hybrid rat/mouse and rat/human m-calpains were cloned and expressed in E. coli. It was shown that Ile-6 and Pro-127, which are specific to the mouse m-80k sequence, caused poor expression. High expression of human m-calpain in E. coli could be achieved by providing the correct Shine-Dalgarno ribosome binding site. The results provide a simple method to obtain approximately 10mg amounts of human m-calpain and a slightly modified mouse m-calpain. Expression of m-80k-EGFP fusions was also studied, both in E. coli and in mammalian cells, varying both the small subunit and the promoters. m-80k-EGFP alone was not active, but with 21k or 28k subunits was active in both cell types. The EGFP domain was partially cleaved during expression, releasing an active m-80k/21k calpain.     

Synthesis of novel ketoconazole derivatives as inhibitors of the human Pregnane X Receptor (PXR; NR1I2; also termed SXR, PAR)

PXR, pregnane X receptor, in its activated state, is a validated target for controlling certain drug–drug interactions in humans. In this context, there is a paucity of inhibitors directed toward activated PXR. Using prior observations with ketoconazole as a PXR inhibitor, the target compound 3 was synthesized from (s)-glycidol with overall 56% yield. (+)-Glycidol was reacted with 4-bromophenol and potassium carbonate in DMF to yield the ring opened compound 6. This was then heated to reflux in benzene along with 2′, 4′-difluoroacetophenone and catalytic amount of para-toluene sulfonic acid to yield 8. The resultant acetal 8 was then functionalized using Palladium chemistry to yield the target compound 3. The activity of the compound was compared with ketoconazole and UCL2158H. However, in contrast with ketoconazole (IC₅₀  0.020 μM; 100% inhibition), 3 has negligible effects on inhibition of microsomal CYP450 (maximum 20% inhibition) at concentrations >40 μM. In vitro, micromolar concentration of ketoconazole is toxic to passaged human cell lines, while 3 does not exhibit cytotoxicity up to concentrations 100 μM (viability >85%). This is the first demonstration of a chemical analog of a PXR inhibitor that retains activity against activated PXR. Furthermore, in contrast with ketoconazole, 3 is less toxic in human cell lines and has negligible CYP450 activity.