Calcium-activated Neutral Protease Research Articles

Regionally Distinct Patterns of Calpain Activation and Traumatic Axonal Injury following Contusive Brain Injury in Immature Rats

Impact-induced head injury in infants results in acute focal contusions and traumatic axonal injury (TAI) that are associated with chronic holohemispheric cortical and white matter atrophy and may contribute to poor outcome in brain-injured children less than 4 years of age. Contusive brain trauma in postnatal day (PND) 11 or PND 17 rat pups, ages neurologically equivalent to a human infant and toddler, respectively, leads to cortical tissue loss and white matter atrophy which are associated with cognitive deficits. In adult models of brain trauma and in brain-injured humans, acute and sustained activation of the calpain family of calcium-activated neutral proteases has been implicated in neuronal death and TAI. PND 11 or PND 17 rat pups were subjected to closed head injury over the left hemisphere using the controlled cortical impact device and sacrificed at 6 h, 24 h or 3 days. Hemorrhagic contusions and tissue tears in the cortex and white matter were visible at 6 h, and neuronal loss was evident by 3 days. Calpain activation was observed in cell soma and dendrites of injured neurons at 6 h, and in degenerating dendrites and atrophic neurons at 24 h after injury at both ages. Axonal accumulation of amyloid precursor protein, indicative of TAI, was observed in the corpus callosum and lateral aspects of the white matter below the site of impact, and in the thalamus in PND 11 rats only. Intra-axonal calpain activation was observed to a limited extent in the corpus callosum and subcortical white matter tracts in both brain-injured PND 11 and PND 17 rats. Collectively, these results provide evidence that calpain activation may participate in neuronal loss in the injured cortex, but may not contribute to the pathogenesis of TAI following contusive brain trauma in the immature rat.

Correlation between bovine calpastatin mRNA transcripts and protein isoforms

Calpastatin is a specific calpain protease inhibitor: calpains are a family of calcium-activated neutral proteases, which have been implicated in various processes. Despite all the available data concerning calpastatin, little is known about how this gene is regulated, particularly in bovine. The existence of four types of transcripts differing at their 5′ ends (Type I, II, III, and IV) has been demonstrated. Here, we show that the Type I, II, and III transcripts are ubiquitous while Type IV is testis-specific. In addition, a Northern blot analysis revealed that the Type III transcript may have three different 3′ termini. Using specific anti-peptide anti-sera, a correspondence between a 145 and a 125 kDa isoforms, and Type I and/or II and III transcripts, respectively, has been established. Finally, we discuss the origin of a 70 kDa isoform, recognized by anti-sera directed against the N-terminal region.

Effects of ACE inhibitor and AT blocker on dystrophin-related proteins and calpain in failing heart

Genetic depletion of dystrophin-related protein (DRP) complex causes cardiomyopathy in animals and humans. We found in a previous study that some types of DRP were degraded and that calpain content was increased in rats with non-genetically induced heart failure. The present study was aimed at examining the effects of an angiotensin-I-converting enzyme inhibitor (ACEI) trandolapril (Tra) or an angiotensin II type 1 receptor blocker (ARB) candesartan (Can), both of which are known to improve the pathophysiology of chronic heart failure (CHF) on degradation of DRP in failing hearts. Coronary artery-ligated (CAL) and sham-operated rats (Sham rats) were treated orally with 3 mg/kg/day trandolapril (Tra) or 1 mg/kg/day candesartan (Can) from the 2nd to 8th week after surgery. Hemodynamic parameters of CAL rats at the 8th week after CAL (8w-CAL) indicated heart failure. alpha-Sarcoglycan (SG) and dystrophin in the surviving left ventricle (surviving LV) of 8w-CAL rats decreased, whereas beta-, gamma-, and delta-SGs remained unchanged. Calcium-activated neutral proteases mu-calpain and m-calpain increased in the surviving LV at the 8th week of postmyocardial infarction. Proteolytic activity in the presence of 5 mM Ca2+ markedly increased at the 2nd and 8th weeks, whereas 50 microM Ca2+ slightly but significantly increased proteolysis of casein. Tra or Can treatment improved the hemodynamic parameters, attenuated changes in alpha-SG and dystrophin, and reversed both calpain contents and activities of the failing heart back to sham levels. These results suggest that attenuation in calpain-induced degradation of DRP complex is a possible mechanism for the Tra- or Can-mediated improvement of the pathogenesis of CHF following myocardial infarction.

Assessment of single nucleotide polymorphisms in genes residing on chromosomes 14 and 29 for association with carcass composition traits in Bos indicus cattle1,2

Objective of this study was to assess the association of SNP in the diacylglycerol O-acyltransferase 1 (DGAT1), thyroglobulin (TG), and micromolar calcium-activated neutral protease (CAPN1) genes with carcass composition and meat quality traits in Bos indicus cattle. A population of Brahman calves (n = 479) was developed in central Florida from 1996 to 2000. Traits analyzed were ADG, hip height, slaughter weight, fat thickness, HCW, marbling score, LM area, estimated KPH fat, yield grade, retail yield, sensory panel tenderness score, carcass hump height, and cooked meat tenderness measured as Warner-Bratzler shear force at 7, 14, and 21 d postmortem. Single nucleotide polymorphisms previously reported in the TG and DGAT1 genes were used as markers on chromosome 14. Two previously reported and two new SNP in the CAPN1 gene were used as markers on chromosome 29. One SNP in CAPN1 was uninformative, and another one was associated with tenderness score (P < 0.05), suggesting the presence of variation affecting meat tenderness. All three informative SNP at the CAPN1 gene were associated with hump height (P < 0.02). The TG marker was associated with fat thickness and LMA (P < 0.05), but not with marbling score. No significant associations of the SNP in the DGAT1 gene were observed for any trait. Allele frequencies of the SNP in TG and CAPN1 were different in this Brahman population than in reported allele frequencies in Bos taurus populations. The results suggest that the use of molecular marker information developed in Bos taurus populations to Bos indicus populations may require development of appropriate additional markers.

Association of markers in the bovine CAPN1 gene with meat tenderness in large crossbred populations that sample influential industry sires1,2

Two previously identified single-nucleotide polymorphism markers located within the micromolar calcium-activated neutral protease gene (CAPN1) were evaluated for their association with variation in meat tenderness using one commercial sample of Simmental x Angus crossbred calves and one multibreed, crossbred research herd. The commercial sample included 362 animals sired by 23 registered Simmental bulls bred to unregistered Angus cows and represented current industry animals in which to test the predictive merit of the markers. The second sample was a research herd including 564 steers from the Germplasm Evaluation Cycle VII population at the U.S. Meat Animal Research Center, produced with semen from popular sires of the seven Bos taurus beef breeds with the most registrations in the United States (Angus, Charolais, Gelbvieh, Hereford, Limousin, Red Angus, and Simmental) on Angus, Hereford, and MARC III cows. These animals form a relatively outbred population that constituted a stringent test of the predictive merit of the genetic markers, although small groups were half-sibs. Warner-Bratzler shear force measurements were used to determine tenderness phenotypes for all animals. The populations were genotyped for two markers that predict variation at amino acid positions 316 and 530 of the mu-calpain polypeptide, produced by the CAPN1 gene. Minor allele frequencies for markers 316 and 530 in the commercial sample were 0.17 and 0.37, respectively, and in the Cycle VII animals, were 0.20 and 0.28, respectively. Both markers showed association with shear force in the commercial sample (P = 0.04) and the Cycle VII population (P = 0.02), supporting the hypothesis that they represent potential markers to aid selection for improved meat tenderness in commercial populations of beef cattle in the United States.

Tat-calpastatin fusion proteins transduce primary rat cortical neurons but do not inhibit cellular calpain activity

Excessive activation of calpains (calcium-activated neutral proteases) is observed following spinal cord contusion injury, traumatic brain injury, stroke, and in neurodegenerative disorders including Alzheimer's disease. Calpain inhibition represents an attractive therapeutic target, but current calpain inhibitors possess relatively weak potency, poor specificity, and in many cases, limited cellular and blood–brain barrier permeability. We developed novel calpain inhibitors consisting of the endogenous inhibitor, calpastatin or its inhibitory domain I, fused to the protein transduction domain of the HIV trans-activator (Tat) protein (Tat 47–57). The Tat-calpastatin fusion proteins were potent calpain inhibitors in a cell-free activity assay, but did not inhibit cellular calpain activity in primary rat cortical neurons when applied exogenously at concentrations up to 5 μM. The fusion proteins were able to transduce neurons, but were localized within endosome-like structures. A similar endosomal uptake was observed for Tat-GFP. Together, the results suggest that endosomal uptake of the Tat-calpastatin prevents its interaction with calpain in other cellular compartments. Endosomal uptake of proteins fused to the Tat protein transduction domain severely limits the applications of this methodology.

Selective Activation Induced Cleavage of the NR2B Subunit by Calpain

Although activation of calcium-activated neutral protease (calpain) by the NMDA receptor has been suggested to play critical roles in synaptic modulation and neurologic disease, the nature of its substrates has not been completely defined. In this study, we examined the ability of calpain to cleave the NMDA receptor in cultured hippocampal neurons. Activation of the NMDA receptor by agonist application led to rapid calpain-specific proteolysis of spectrin and decreased levels of NR2A/2B subunits. Cleavage of the NR2A/2B subunit created a 115 kDa product that retained the ability to bind 125I-MK-801 and is predicted to be active. Increases in levels of this product appeared within 5 min of NMDA receptor activation and were stable for periods of >30 min. Subtype-specific antibodies demonstrated that the NR2B subunit was cleaved in these primary cultures, but the NR2A subunit was not. An inhibitor of calpain blocked both the decrease of intact NR2B and the increase of the low molecular weight form, whereas neither caspase nor cathepsin inhibitors had an effect on these events. Cell surface biotinylation experiments demonstrated that the 115 kDa fragment remained on the cell surface. This NR2B fragment was also found in the rat hippocampus after transient forebrain ischemia, showing that this process also occurs in vivo. This suggests that calpain-mediated cleavage of the NR2B subunit occurs in neurons and gives rise to active NMDA receptor forms present on the cell surface after excitotoxic glutamatergic stimulation. Such forms could contribute to excitotoxicity and synaptic remodeling.

174 Cytotoxic effects of 100 toxic compounds on HEP G2 and hela cells

Organophosphorus (OP) used as pesticides and hydraulic fluids can produce acute poisoning known as OP-induced delayed neuropathy (OPIDN), whose effects take long time to recover. Thus a secure therapeutic strategy to prevent the most serious effects of this poisoning would be welcome. In this study, tri-o-cresyl phosphate (TOCP, 500 mg/kg p.o.) was given to hens, followed or not by nimodipine (1 mg/kg i.m.) and calcium gluconate (Ca-glu 5 mg/kg i.v.). Six hours after TOCP intoxication, neuropathy target esterase (NTE) activity inhibition was observed, peaking after 24 h exceeding 80% inhibition. A fall in the plasmatic calcium levels was noted 12 h after TOCP was given and, in the sciatic nerve, Ca2+ fell 56.4% 24 h later; at the same time calcium activated neutral protease (CANP) activity increased 308.7%, an effect that lasted 14 days. Any bird that received therapeutic treatment after TOCP intoxication presented significant signs of OPIDN. These results suggest that NTE may be implicated in the regulation of calcium entrance into cells being responsible for the maintenance of normal function of calcium channels, and that increasing CANP activity is responsible to triggering OPIDN. Thus, with one suitably adjusted dose of nimodipine as well as Ca-glu, we believe that this treatment strategy may be used in humans with acute poisoning by neuropathic OP.

Different expression patterns of calpain isozymes 1 and 2 (CAPN1 and 2) in squamous cell carcinomas (SCC) and basal cell carcinomas (BCC) of human skin.

Calpain, also named CAPN (for calcium-activated neutral protease), is a ubiquitous intracellular cytoplasmic non-lysosomal cysteine endopeptidase that requires calcium ions to exert its activity. Two major isoenzymes are known- micro -calpain (CAPN1) and m-calpain (CAPN2)-requiring micromolar and millimolar calcium concentrations for activation, respectively. Many known substrates of the different calpain isoenzymes, such as the transcription factors c-Fos and c-Jun, the tumour suppressor protein p53, protein kinase C, pp60src, or the adhesion molecule integrin, have been implicated in the pathogenesis of various malignancies including squamous (SCC) and basal (BCC) cell carcinomas of human skin, suggesting an important role of the calpain isoenzymes in malignant diseases. We have analysed the expression of CAP1 and CAPN2 protein and mRNA expression in BCCs and SCCs of human skin. Interestingly, CAPN1 immunoreactivity (streptavidin-peroxidase technique) was markedly reduced in BCCs compared to normal human skin or SCCs, while in contrast CAPN1 mRNA levels (determined by real-time PCR) were markedly elevated in BCCs and SCCs compared to normal human skin. No differences were found analysing CAPN2 protein and mRNA expression in normal human skin, BCCs and SCCs. In conclusion, we have demonstrated for the first time alterations in calpain mRNA expression and protein content in malignant skin tumours that may be of importance for the tumorigenesis and growth characteristics of BCCs and SCCs. However, our results do not allow conclusions on the function of CAPN1 and CAPN2 in BCCs and SCCs. It is not known if the CAPN genes in BCCs or SCCs exhibit functionally inactivating mutations or whether decreased CAPN1 protein expression in BCCs and elevated CAPN1 mRNA in BCCs and SCCs reflect a feedback loop coupled with increased degradation or proteolysis of CAPN1 protein.

Dinitrophenol derivatization of proteolytic products and its application in the assay of protease(s) activity

A spectrophotometric method based on dinitrophenol (DNP) derivatization of proteolytic products was developed for monitoring the increase in NH 2-groups as a function of protease activity. DNP derivatization of amino acids and proteolytic products was carried out at an alkaline pH of 8.8, in presence of 2,4-dinitrofluorobenzene (DNFB), followed by the stabilization of products by adjusting the pH to ∼2.5. Using casein as substrate, under the defined assay conditions for proteases, trichloroacetic acid soluble proteolytic products were derivatized with DNFB reagent. Though alkaline pH favored the DNP derivatization of primary amino compounds, the products formed were found to be unstable. However, upon adjusting the pH to 2.5±0.1, DNP derivatives of amino acids and proteolytic products were found to be stable with identical λ max of 395 nm. The utility of the method was evaluated by assaying the proteolytic activities of trypsin and calcium activated neutral protease (CANP). Proteolytic activity was quantified by employing the molar extinction coefficient of DNP derivatives of an equimolar concentration of glutamate and glycine. By employing this method, CANP activity in different regions of rat brain was determined. The proposed method to monitor the increase in NH 2-end groups as a function of proteolytic activity could be employed to assay the activity of proteases.

Modulation of the activity of calcium-activated neutral proteases (calpains) in chronic lymphocytic leukemia (B-CLL) cells

Decreased susceptibility to apoptosis and impaired proliferative control are thought to be responsible for prolonged life span and accumulation of chronic lymphocytic leukemia (B-CLL) cells. The activity of calpains (calcium-dependent, neutral proteases, active in the cells responding to signals inducing a rise of cytoplasmic Ca(++)) is involved in the regulation of apoptosis of some cell types by interaction with caspase-3. This work verifies the hypothesis of the abnormal activity of calpains and its role in reduced apoptosis of the B-CLL cells. Casein zymography, reverse transcriptase-polymerase chain reaction, and Western blotting were used for identification and quantification of the activity and expression of calpains in B-CLL cells and purified normal B lymphocytes. The activity and expression of mu-calpain (requiring micromolar Ca(++) for activation) are significantly higher in the leukemic than in nonmalignant cells. Contrarily, the activity and expression of m-calpain (requiring millimolar Ca(++)) as well as the expression of calpastatin (an endogenous inhibitor of calpains) are unchanged or reduced in the B-CLL lymphocytes. Correspondingly, the activity of caspase-3 is many times lower in the B-CLL cells than in normal B lymphocytes. Inhibition of overexpressed mu-calpain in living B-CLL cells in vitro results in doubling of the proportion of the cells undergoing spontaneous apoptosis. This observation suggests a possible role for calpains in longer survival of the B-CLL cells and may open new therapeutic possibilities.

Development of an inducible system to assess p94 (CAPN3) function in cultured muscle cells

p94 belongs to the calpain family of enzymes, also called calcium-activated neutral proteases and is mainly expressed in the skeletal muscle. Mutations affecting the gene coding for p94 are responsible for a myopathy syndrome called Limb Girdle Muscular Dystrophy type 2A (LGMD2A). Although the activity of p94 seems necessary for muscle function, the biological role of the enzyme is still unknown. The goal of this study was to develop a muscle cell line in which the expression level of p94 can be regulated, by an inducible way. In this study, a biological system was developed which allowed mimicking, in vitro, of part of the events occurring in patients (i.e. a decrease of p94 activity). The first results indicate that the decrease in p94 activity results in a significant increase of myogenin level, a high specific transcription factor involved in myoblast fusion. This muscle specific inducible system is an interesting biological tool to assess specifically p94 function(s) in cultured muscle cells. According to the present results, p94 seems at least to be involved in a myogenesis regulation pathway via its action on certain proteins belonging to the myogenic regulator factor family.

Ritonavir inhibition of calcium-activated neutral proteases

Calpains (EC 3.4.22.17) are intracellular calcium-activated cysteine proteases that mediate tissue injury following post-ischemic and post-traumatic stress. Both human HIV protease and calpains share a similar secondary structure, where the active site is flanked by hydrophobic regions. The present study demonstrates that ritonavir, a hydrophobic HIV protease inhibitor, also inhibits calpain activity. In PC12 cell extracts assayed for calpain at maximal activity (2 mM calcium), ritonavir exhibited competitive inhibition with a K i of 11±7.0 μM. Experiments with purified enzymes showed inhibition for both m- and μ-calpain isoforms (m-calpain, K i =9.2±1.2 μM; μ-calpain, K i =5.9±1.4 μM). Ritonavir also inhibited calcium-stimulated calpain activity in PC12 cells in situ. These results suggest that ritonavir or analogues of the drug should be investigated as cytoprotective agents in conditions where cell death or injury is mediated via calpain activation.

The purification and characterization of μ-calpain and calpastatin from ostrich brain

Calcium-activated neutral proteinases (CANPs) and their endogenous specific inhibitor calpastatin are found in a wide variety of vertebrate and invertebrate tissues. The CANPs are cysteine proteinases that have an absolute requirement for Ca 2+ for activity. μ-Calpain and calpastatin were purified by successive chromatographic steps on Toyopearl-Super Q 650S and Pharmacia Mono Q HR 5/5 columns. The enzyme has a M r of 84 KDa using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), a M min of 79 KDa from amino acid analysis and an pI of 5.2. Calpastatin has a M r of 323 KDa using denaturing gradient PAGE and a pI of 4.7. The amino acid composition of μ-calpain revealed 689 residues and the pH and temperature optima were found to be 7.5 and 37 °C, respectively. μ-Calpain underwent a Ca 2+-dependent autoproteolysis producing a fragment of 82 KDa. The N-terminal sequence of μ-calpain showed 24 and 18% sequence identity with human and bovine μ-calpain.

Screening the proteins that interact with calpain in a human heart cDNA library using a yeast two-hybrid system.

Calpain, a cytosolic cysteine protease, requires calcium ions for activity. It has been reported that calpain is involved in the degradation of myofibrillar and neurofilament proteins, and the activation of phosphorylase b kinase and protein kinase C. More recently, calpain was shown to participate in apoptosis. In order to understand the calpain-related signal transduction pathway and its changes during hypertrophy, and especially in hypertension, we screened a human heart cDNA library to find proteins that interact with calpain. 1) Using PCR we amplified the full-length, domain II, domain III and domain IV cDNA of calpain (calcium-activated neutral protease, CANP) I large subunit respectively. 2) Then the fragments were cloned into pGBKT7 vector, resulting in 4 bait expression constructs (pGBKT7-CANP, pGBKT7-CANP II, pGBKT7-CANP III, and pG BKT7-CANP IV). 3) After 4 bait vectors were transformed into AH109 by the lithium acetate-mediated method, AH109/pGBKT7-CANP, AH109/pGBKT7-CANP II, AH109/pGBKT7-CANP III, and AH109/pGBKT7-CANP IV were obtained, respectively. 4) After the human heart cDNA library was sequentially transformed into AH109/ pGBKT7-CANP, 1000-1200 positive clones were grown on SD/Trp-Leu-Ade-His-. Only 150 positive clones were obtained through a colony-lift filter assay to detect beta-galactosidase activity. 5) Total 105 clones among above 150 positive clones were eliminated through that the duplicate, pseudopositive and autoactive detection, respectively. 6) Finally, sequencing eliminated clones with a wrong open reading frame (ORF). Eight clones were cancelled with wrong ORF. The remaining 37 positive clones were analyzed using BLAST software available on the Internet and classified as follows: 1. enzymes or proteins related to signal transduction in the cell; 2. contraction proteins 3. matrix proteins 4. unknown proteins. 7) In order to determine which domain of the calpain I large subunit was involved in the interaction with these real clones, the 37 clones were transformed into AH109/pGBKT7-CANP II, AH109/pGBKT7-CANP III or AH109/pGBKT7-CANP IV. Among these 37 clones, 29 clones could interact with domain II, 5 clones could interact with domain III and 6 clones could interact with domain IV. Thus, we successfully constructed 4 bait expression vectors, pGBKT7-CANP, pGBKT7-CANP II, pGBKT7-CANP III and pGBKT7-CANP IV, and obtained 37 real positive clones that interacted with the calpain I large subunit by screening a human heart cDNA library using pGBKT7-CANP as bait. Among them, 29 clones could interact with domain II of the calpain I large subunit, where the active site of calpain is located. Additional studies will be needed to clarify the calpain-related signal transduction pathway in greater detail.

Milli-calpain from sea bass (Dicentrarchus labrax) white muscle: purification, characterization of its activity and activation in vitro.

A calcium-activated neutral cysteine protease was purified to homogeneity from Dicentrarchus labrax white muscle using three steps: hydrophobic interaction, anion exchange, and gel filtration chromatographies. The purified enzyme showed a native molecular weight of 124 kDa with an oligomeric structure (large subunit of 80 kDa and small subunit of 24 kDa). It has been classified as a milli-calpain from its calcium sensitivity. Activity was maximal at pH 7.0, 24 degrees C in Tris buffer without NaCl as determined by means of a two-level experimental design and response surface methodology. Sea bass calpain is neither glycosylated nor phosphorylated and shared some common cleavage specificities and activation and autolysis mechanisms with other typical mammalian or invertebrates calpains. Calcium-induced activation and autolysis of calpain has been characterized together with the effect of the strontium cation acting as a calcium analog. On the basis of its in vitro properties, the contribution of the sea bass milli-calpain to the process of postmortem deterioration of fish muscle is discussed, even though further information such as in vivo regulation or in vitro effects on myofibrils is required.

SNP43 of CAPN10 and the risk of type 2 Diabetes in African-Americans: the Atherosclerosis Risk in Communities Study.

Recently, an A-to-G variant in intron 3 (SNP43) of the calcium-activated neutral protease 10 gene (CAPN10) was identified as a possible type 2 diabetes susceptibility gene through positional cloning in Mexican-Americans. We conducted cross-sectional and prospective studies to evaluate the relation between SNP43 and type 2 diabetes and related traits in middle-aged African-American participants of the Atherosclerosis Risk in Communities Study, a population-based longitudinal study. At baseline, 269 prevalent diabetes cases and 1,159 nondiabetic control subjects were studied. Those with the G/G genotype were more likely to have diabetes than those with the A/G or A/A genotype (odds ratio [OR] 1.41, 95% CI 1.00-1.99, P = 0.05). In the prospective study, 166 of the control subjects developed incident diabetes over 9 years of follow-up. The incidence of diabetes for individuals with the G/G genotype did not differ significantly from those with at least one copy of the A allele (23.3 vs. 19.5 per 1,000 person years, P = 0.29). Pooling prevalent and incident diabetic cases together, individuals with the G/G genotype were approximately 40% more likely to have diabetes than those without (OR 1.38, 95% CI 1.04-1.83, P = 0.03). Because of the high frequency of the G allele (0.88), approximately 25% of the susceptibility to type 2 diabetes in African-Americans may be attributed to the G/G genotype at SNP43 of CAPN10, although most of the subjects with the G/G genotype did not develop diabetes over the 9 years of follow-up. We conclude from this large prospective study that the G allele of SNP43 of CAPN10 or another allele or gene that is in linkage disequilibrium with it increases susceptibility to type 2 diabetes in African-Americans.

The effects of calpain inhibition upon IL-2 and CD25 expression in human peripheral blood mononuclear cells

Calcium is an important contributor to T cell activation; it is also the major factor in the activation of the calcium-activated neutral proteinase, calpain. For this reason, we wanted to investigate if calpain has a role in T cell activation and what aspects of this activation calpain affects. As measured by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), calpain inhibition decreased interleukin-2 (IL-2) and CD25 mRNA expression in a dose-dependent manner, at early time points following the initial activation, and over extended periods of time in activated human peripheral blood mononuclear cells (PBMCs). Using an enzyme-linked immuno-sorbent assay (ELISA) specific for human IL-2, we found that calpain inhibition decreased IL-2 secretion in a dose-dependent manner, shortly after activation, and continuously over time. Inhibiting calpain caused a dose-dependent inhibition of CD25 cell surface expression and also inhibited expression shortly after activation and for at least 48 h. This study showed that calpain has an integral role in the synthesis of the two important T cell activation factors, IL-2 and CD25.

Liver Alcohol Dehydrogenase Is Degraded by the Ubiquitin–Proteasome Pathway

Dihydrotestosterone (DHT) decreases rat liver alcohol dehydrogenase (ADH) due principally to an increased rate of degradation of the enzyme. The pathway of degradation of ADH was investigated. Exposure of hepatocytes in culture to lactacystin or to MG132, which are inhibitors of the ubiquitin–proteasome pathway of protein degradation, resulted in higher ADH. Furthermore, both lactacystin and MG132 prevented the decrease in ADH caused by DHT. By contrast, the lysosomal proteolytic inhibitors 3-methyladenine and leupeptin as well as inhibitors of the calcium-activated neutral protease calpain system had no effect on ADH in the absence or presence of DHT. ADH isolated by immunoprecipitation from hepatocytes exposed to DHT reacted specifically with anti-ubiquitin antibody. Ubiquitinated ADH was also demonstrated in hepatocytes exposed to MG132. The combination of DHT and MG132 resulted in more ubiquitinated ADH than exposure to either compound alone. These results suggest that the ubiquitin–proteasome pathway plays a role in the degradation of ADH and in the enhanced degradation of this enzyme by DHT.

Vaccination with calpain induces a Th1-biased protective immune response against Schistosoma japonicum.

A large subunit of calpain, a calcium-activated neutral proteinase, from Schistosoma japonicum was cloned and expressed in Escherichia coli. When BALB/c mice were immunized with purified recombinant calpain (r-calpain) emulsified in complete Freund's adjuvant, a significant reduction in the number of recovered worms and also in egg production per female worm was observed (P<0.01). Spleen cells of the immunized mice showed enhanced production of gamma interferon (IFN-gamma) by activated CD4(+) T cells. Considering our observation of elevated expression of inducible nitric oxide synthase mRNA in immunized mice, r-calpain-induced IFN-gamma seemed to upregulate the production of nitric oxide by macrophages and subsequently mediated the killing of schistosomulae in the lung. On the other hand, spleen cells of immunized mice showed only faint interleukin-4 production in response to r-calpain in vitro, suggesting that immunization with r-calpain alters the Th1-Th2 balance in murine hosts even during a Th2-promoting S. japonicum infection. Furthermore, histopathological study of the livers of immunized mice showed that granulomas formed around eggs were diminished in both size and number. Egg production by female worms was clearly decreased in immunized mice, suggesting that r-calpain also has antifecundity effects. Taken together, these results point to S. japonicum calpain as a potential vaccine candidate for both worm killing and disease prevention, possibly through the induction of a strong Th1-dominant environment in immunized mice.