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Background. Reduced TCRζ chain surface has been reported in T cells from patients with different inflammatory conditions and cancer. However, the causes of this diminished expression in cancer remain elusive. Methods. T cell-enriched populations of blood or tissue (tumoral and nontumoral) origin from 44 patients with gastric adenocarcinoma and 33 healthy subjects were obtained. Samples were subjected to cytofluorimetry, Western blot analysis, TCRζ cDNA sequencing experiments, measurement of TCRζ mRNA levels, and caspase-3 activity assays. Results. Cytofluorimetry revealed a decreased TCRζ expression in T cells of patients, assessed either as percentage of cells expressing this chain (blood: control subjects 99.8 ± 0.1 % , patients 98.8 ± 1.1 % P < 0.001 ; tissue: control subjects 96.7 ± 0.9 % , patients tumoral tissue 67.9 ± 27.0 % , patients nontumoral tissue 82.8 ± 12.6 % , P = 0.019 ) or mean fluorescence intensity (MFI) value (blood: control subjects 102.2 ± 26.0 ; patients 58.0 ± 12.3 , P = 0.001 ; tissue: control subjects 99.4 ± 21.4 ; patients tumoral tissue 41.6 ± 21.4 ; patients nontumoral tissue 62.3 ± 16.6 , P = 0.001 ). Other chains pertaining to the TCR-CD3 complex (CD3ε) showed no significant differences (MFI values). Subsequent TCRζ cDNA sequencing experiments or measurements of TCRζ mRNA levels disclosed no differences between patients and control subjects. Evaluation of caspase-3 activity showed higher levels in T cell extracts of patients, and this activity could be decreased by 70% with the use of the inhibitor Ac-DEVD-FMK, although CD3ζ expression levels did not recover. Conclusions. These results further place the defect responsible for the low TCRζ expression in cancer at the posttranscriptional level and suggests contrary to what has been proposed in other pathologies that elevated caspase-3 activity is not the causative agent.

The number of death due to cancer-related diseases each year is at the alarming level and is constantly growing. Tools that can effectively and conveniently detect cancer cell apoptosis can play a significant role in cancer research, cancer therapy, and other related industries. Herein, we fabricated, for the first time, an ultrasensitive, disposable, self-enhanced off-on electrochemiluminescence (ECL) biosensor based on ternary Ru-PEI@PCN-333(Al) system to determine caspase-3 activity, the biomarker of apoptosis. The biosensor had a low detection limit of 0.017 pg/mL and was able to enhance the ECL emission and stability. A solid-state (SS) ECL strategy was adopted to overcome the relatively weak ECL emission due to the long distance between electrochemiluminophore and electrode surface. The analysis requires only one incubation step, which can significantly reduce the operational complexity and time. The biosensor had higher sensitivity, and the off-on ECL mode was achieved using caspase-3 as a switch. The on-site and rapid detection capability of the biosensor was achieved by the introduction of disposable screen-printed electrodes (SPEs). This study lays a foundation for the development of more advanced, ingenious, portable and reliable ECL devices for biosensing not only caspase-3, but also other bioanalytes.

Intracellular detection of caspase-3 activity is crucial for the study of cell apoptosis and caspase-3 related diseases. Although various nanomaterials-based biosensors have been constructed for this purpose, they often suffer from poor stability or complicated construction due to the lack of a facile and efficient biofunctionalization method, which decreases their sensing performance and limits their use in the complex physiological environments. As novel two-dimentional (2D) nanomaterials, MoS2 nanosheets (NSs) have shown great potential for biosensing due to their unique properties. Herein, we develop a versatile yet facile covalent biofucntionalization strategy of MoS2 NSs by utilizing polydopamine (PDA) as nano-bio interface, and construct an intracellular fluorescent biosensor (MoS2@PDA-PEG-Peptide, MPPP) for the determination of caspase-3 activity. This covalent biofunctionalization of MoS2 NSs can significantly improve the conjugation efficiency of biomolecules and enhance their stability in complicated environments, which is much better than conventional biofunctionalization by using thiol-metal coordination. Furthermore, this novel caspase-3 biosensor based on peptides biofunctionalized MoS2 NSs shows high sensitivity and selectivity for the detection of caspase-3 with a limit of detection (LOD) of 0.33 ng/mL, and can be used for high-contrast fluorescent imaging of cell apoptosis.

We report here the first observations of time-delayed degradation of the ability to learn and remember in rats after changes in the level of caspase-3 activity in early postnatal ontogeny. Administration of intracerebroventricular caspase inhibitor Ac-DEVD-CHO to 20-day-old rat pups was followed by impairments to memory and learning, along with anomalous increases in caspase-3 activity on post-treatment day 7; impairments were also present 30–45 days after treatment, when the activity level of this enzyme was normal for age. An analogous picture was seen in young and adult rats after prenatal hypoxia (7% O2, 3 h, on E14). In addition, the stronger and longer-lasting increases in caspase-3 activity seen after prenatal hypoxia were found to be accompanied by more significant impairments to memory and learning on testing in a two-level radial maze. The positive correlation found between the magnitude of changes in caspase activity in early ontogeny and impairments to cognitive functions in adults points to the need to maintain optimum caspase-3 activity at the early stage of development to support the normal formation of the brain and behavior during maturation. The fact that these cognitive impairments occurring in conditions of altered caspase-3 in the first month of postnatal development were seen regardless whether or not intense cell death occurred supports the hypothesis that this enzyme has an important nonapoptotic function in early ontogeny.

Background: Vincosamide(Vinco) was first identified in the methanolic extract of the leaves of Psychotria leiocarpa, and Vinco has important anti-inflammatory effects and activity against cholinesterase, Vinco also has a trait to ant-tumor. However, whether Vinco is able to inhibit the malignant behaviors of hepatocellular carcinoma(HCC) cells is still unclear. In the present study, we explored the role of Vinco in suppressing the malignant behaviors of HCC cells.Methods: MTT, trypan blue exclusion assay and the Cell Counting Kit(CCK)-8 analysis were applied to detect the proliferation and death of HCC cells; electron microscopy was performed to observe the change of cellular mitochondrial morphology; scratch repair and Transwell assays were used to analyze the migration and invasion of HCC cells; the expression and localization of proteins were detected by laser confocal microscopy and Western blotting; and the growth of the cancer cells in in vivo was assessed in a mouse tumorous model.Results: At a dose of 10-80 mg/ml, Vinco inhibited the proliferation and promoted death of HCC cells in a dose-independent manner, but had low cytotoxcity effect on normal liver cells. Additionally, 80 mg/ml of Vinco could significantly disrupt the morphology of mitochondria, suppress the migration and invasion of HCC cells. The growth of HCC cells in the animal tumorous model was significantly inhibited after treatment with Vinco (10 mg/kg/day) for 3 days. The results of the present study indicated that Vinco (10-80 mg/ml) played a role in activating caspase-3, promoting the expression of PTEN, and inhibiting the phosphorylation of AKT(Ser473) and mTOR(Thr2448), Vinco also has a trait for suppressing the expression of CXCR4, Src, MMP9, EpCAM, Ras and Oct4 in HCC cells.Conclusions: Vinco has a role in inhibiting the malignant behaviors of HCC cells; the role molecular mechanism of Vinco maybe involve in restraining expression of the growth-, metastasis-related factors Src, Ras, MMP9, EpCAM and CXCR4, and activating the activity of caspase-3. Vinco also could block PI3K/AKT signaling pathway. Thus, Vinco is an available chemotherapy for HCC patients.

Background:The alpha-1 antitrypsin (AAT) protein has an important role in the anti-inflammatory and apoptotic response. AAT inhibits not only serine proteases but also cysteine and aspartic proteases. Apoptosis results from the sequential activation of cysteine proteases of the caspase family. This study aimed to evaluate the effect of AAT on formaldehyde-induced apoptosis of human peritoneal mesothelial cells (HPMCs).Methods:HPMCs were cultured and treated with formaldehyde (250 µM) to induce apoptosis. In the AAT group, the cultured HPMCs were pretreated with AAT (2 mg/mL) for 1 h before formaldehyde treatment. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays to determine cell viability, and flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays to detect apoptosis. The MTT assays were used to find optimal concentrations of formaldehyde and AAT. We measured caspase-3 activity and used Western blotting to estimate Bcl-2 and Bad expression.Results:Flow cytometry and TUNEL assays revealed that formaldehyde exposure significantly increased apoptosis compared with the control treatment, but pretreatment with AAT significantly inhibited this effect. Compared with the control, caspase-3 activity was significantly increased and the ratio of Bcl-2 to Bad expression significantly decreased following treatment with formaldehyde. However, caspase-3 activity was significantly lower and the Bcl-2 to Bad expression ratio higher in the AAT group than in the formaldehyde-only group.Conclusion:AAT inhibits formaldehyde-induced apoptosis of HPMCs via a caspase-mediated pathway. These data support a potential use for AAT as a therapeutic agent for the inhibition of peritoneal cell apoptosis during peritoneal dialysis.

Cocaine is one of the most widely used illicit drugs in the world, and as a result of incomplete removal by sewage treatment plants it is found in surface waters, where it represents a new potential risk for aquatic organisms. In this study we evaluated the influence of environmental concentrations of cocaine on the liver and the kidney of the European eel (Anguilla anguilla). The eels were exposed to 20 ng L−1 of cocaine for fifty days, after which, three and ten days after the interruption of cocaine exposure their livers and kidneys were compared to controls. The general morphology of the two organs was evaluated, as well as the following parameters: cytochrome oxidase (COX) and caspase-3 activities, as markers of oxidative metabolism and apoptosis activation, respectively; glucose-regulated protein (GRP)78 levels, as a marker of endoplasmic reticulum (ER)-stress; blood glucose level, as stress marker; serum levels of alanine aminotransferase (ALT), as a marker of liver injury and serum levels of C-reactive protein (CRP), as a marker of the inflammatory process. The liver showed morphologic alterations such as necrotic areas, karyolysis and pyknotic nuclei, while the kidneys had dilated glomeruli and the renal tubules showed pyknotic nuclei and karyolysis. In the kidney, the alterations persisted after the interruption of cocaine exposure. In the liver, COX and caspase-3 activities increased (COX: P = 0.01; caspase-3: P = 0.032); ten days after the interruption of cocaine exposure, COX activity returned to control levels (P = 0.06) whereas caspase-3 activity decreased further (P = 0.012); GRP78 expression increased only in post-exposure recovery specimens (three days: P = 0.007 and ten days: P = 0.008 after the interruption of cocaine exposure, respectively). In the kidney, COX and caspase-3 activities increased (COX: P = 0.02; caspase-3: P = 0.019); after the interruption of cocaine exposure, COX activity remained high (three days: P = 0.02 and ten days: P = 0.029 after the interruption of cocaine exposure, respectively) whereas caspase-3 activity returned to control values (three days: P = 0.69 and ten days: P = 0.67 after the interruption of cocaine exposure, respectively). Blood glucose and serum ALT and CRP levels increased (blood glucose: P = 0.01; ALT: P = 0.001; CRP: 0.015) and remained high also ten days after the interruption of cocaine exposure (blood glucose: P = 0.009; ALT: P = 0.0031; CRP: 0.036). These results suggest that environmental cocaine concentrations adversely affected liver and kidney of this species.

Objective Investigation of the anticarcinogenic effects of natural products with low toxicity is very important in the development of new therapeutic strategies against cancer. Ginnalin A (GA) is one of the most important phenolic compounds of Acer genus and its anticancer effect has been shown that in various cancer cell lines. SB203580, a p38 MAPK inhibitor, can inhibit cell proliferation independently of p38 MAPK. The objective of this study was to investigate combination effect of GA and SB203580 on Hep-3B cell line. Material and methods Cell viability was determined by using XTT method after the treatment with GA, SB203580 and combination of both. Anticarcinogenic effects of GA and SB203580 both in single and in combination have been analyzed with Caspase-3 activity assay and expression levels of important genes involved in cell cycle and apoptosis were evaluated by qPCR. Results GA and SB203580 have shown additive effect on Hep-3B cells in the combination inhibited 50% of cell viability. And, SB203580 increased the effect of GA on activation of Caspase-3 and expressions of genes important in apoptosis and cell cycle. Conclusion This study indicates that GA and SB203580 can be an effective for development of new therapeutic strategies in hepatocellular carcinoma.

The activation of the apoptosis pathway in tilapia muscle during postmortem storage was studied. Changes in caspase-3 activity, ATP content, cytochrome c levels, and ratio of Bcl-2/Bax levels of tilapia muscle were observed during postmortem storage at 20°C. Caspase-3 activity was found to be significantly increased at first, followed by a decrease (P

The study was conducted in order to compare the in vitro effect of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153) on the number of apoptotic cells and the activity of caspase-3 in chicken ovarian follicles. The ovarian stroma, white (WF) and yellowish (YF) prehierarchical follicles and fragments of the theca and granulosa layers of the 3 largest preovulatory follicles (F3-F1) were in vitro exposed to TCDD (10 nM), PCB 126 (10 nM) and PCB 153 (10 μM) for 24 h. After incubation the number of apoptotic cells and caspase-3 activity were determined by TUNEL method and fluorometric assay, respectively. PCB 126 and PCB 153 increased the number of apoptotic cells in the ovarian stroma while TCDD and PCB 126 elevated it in the WF follicles. Under the control conditions, caspase-3 activity steadily increased along with maturation of the follicles, reaching the highest level in the theca layer of the F1 follicle. The activity of this enzyme in the granulosa layer of F3-F1 follicles was on average 60% lower in comparison to the stroma. Exposure to TCDD elevated caspase-3 activity in prehierarchical follicles and in the granulosa layer of F2 and F1 preovulatory follicles. On the contrary, PCB 126 exerted a suppressive effect on caspase-3 activity in the WF follicles and the granulosa layer of the F2 follicle, and PCB 153 in the theca layer of F2 and F1 and the granulosa layer of the F3 follicle. In conclusion, the results indicate that TCDD and PCBs affect apoptosis in chicken ovarian follicles and in consequence may disrupt follicle development.

Aim: The study evaluated the effects of the combined extracts of Vernonia amygdalina (VA) and Gongronema latifolium (GL) on pancreatic GLUT 2 expression and caspase 3 activity in streptozotocin (STZ, 45 mg/Kg)-induced diabetic rats. Study Design: Fifteen Albino rats were used for the study and were placed in 3 groups of 5 rats each: A - normal control, B – Diabetic control and C – experimental group. Place and Duration of Study: The study was carried out in the department of Anatomy, University of Calabar. Duration: 6 months. Methodology: Half of the diabetic rats were treated with VA+GL (400mg/kg, ratio 1:1, DE group) for 28 days, while the other half was untreated and served as diabetic control (DC). Normal control (NC) rats were untreated. After 28 days, the rats were sacrificed and their blood glucose, serum GLUT 2 and caspase 3 activity were measured. Histochemical evaluation of the pancreas was also carried out. Results: Blood glucose concentrations for the 3 groups were 60.31±7.28, 257.00±4.43, and 116.60±10.11 mg/dl for NC, DC and DE respectively. This represented a 4-fold increase in the DC compared with NC and a significant amelioration in the extract-treated DE group compared with DC group. Serum GLUT 2 concentrations were 70 ng/ml in NC, dropped to 8 ng/ml (p

This paper aimed to investigate the postmortem ageing process of beef M. semitendinosus (ST, just slaughtered muscles) using ultrasound-assisted l-histidine treatment. The treatments with different concentrations of l-histidine solutions (0.1%, 0.15%, and 0.2%, w/v) at 4 °C for 60 min were labeled "LH", "MH" and "HH", respectively. Furthermore, the corresponding treatments with the above l-histidine solutions for 55 min after ultrasound pretreatment for 5 min were labeled "ULH", "UMH" and "UHH", respectively. The results showed that the UMH group had the lowest Warner-Bratzler shear stress. The pH value of the HH and UHH groups was higher than that of the other groups (HH: 6.39 ± 0.02, UHH: 6.52 ± 0.03, P 0.05). The MH and UMH groups showed large fiber spacing, cavities and fractures as well as obviously damaged myofibrils. In the UMH group, the soluble protein concentration (SPC) and caspase-3 activity were the highest, and the turbidity of actomyosin was the lowest. Surprisingly, the Ca

Caspase-3 plays a vital role in intrinsic and extrinsic pathways of programed cell death and in cell proliferation. Its detection is an important tool for early detection of some cancers and apoptosis-related diseases, and for monitoring the efficacy of pharmaceuticals and of chemo- and radiotherapy of cancers. This review (with 72 references) summarizes nanomaterial based methods for signal amplification in optical methods for the determination of caspase-3 activity. Following an introduction into the field, a first large section covers optical assays, with subsections on luminescent and chemiluminescence, fluorometric (including FRET based), and colorimetric assays. Further section summarize methods for bioimaging of caspase-3. A concluding section covers current challenges and future perspectives. Graphical Abstract ᅟ.

This paper reports a novel approach for the simple detection of cell apoptosis using an electrochemical technique. This method uses caspase-3 activity as an indicator of apoptosis. Caspase-3 activity was detected with differential plus voltammetry (DPV) as an alternative to conventional spectrometry. In this method, p-nitroaniline (pNA) released from Asp-Glu-Val-Asp-pNA by caspase-3 enzyme reaction was measured with DPV by using a glassy carbon electrode. Using this method, we successfully detected cell apoptosis occurring inside living HepG2 cells without the need for a cell lysis step. This method provides an easy assay procedure and, more importantly, allows a live cell apoptosis detection format. This novel electrochemical apoptosis assay using living cells instead of typically used cell lysates will expand the applicable range of the apoptosis assay to include cell activity assays for drug discovery and cell transplantation medicine.

The objective of this study is to compare whether caspase-3 is activated in the M.longissimus lumborum of cows, the M.longissimusdorsi of sheep, and the breast and leg muscles of geese during the initial postmortem stage. Samples from cows were obtained at 0, 4, 8, and 24 h after slaughter. Samples from sheep and geese were taken at 0, 3, 6, 9, and 24 h. Pro-caspase-3 was cleaved into active caspase-3 during the initial postmortem stage in cow. Caspase-3 activity was found to increase gradually at 0, 4, and 24 h postmortem in cows. Pro-caspase-3 was activated at 6 and 9 h postmortem in sheep. Caspase-3 activity is not activated postmortem in the breast and leg muscles of geese. The results of this study show that caspase-3 activity differed in different species and that the changes in caspase-3 activity over time also differed in the different species.

Stallion-to-stallion variability in the quality of cryopreserved ejaculates postthaw affects the commercial acceptability of frozen semen and thus is a major constraint for the equine industry. In recent years, the molecular mechanisms associated with sperm damage during cryopreservation have become better understood. Identification of the freezability of the ejaculates before the freezing process is initiated will have a major impact on the equine industry. We studied three markers of oxidative stress in sperm, including 8-iso-PGF2alpha, 8-OH guanosine, and 4-hydroxynonenal (4-HNE); the presence of active caspase 3; and their changes after sperm cryopreservation. Although 4-HNE levels increased after cryopreservation (from 7% to 33%, P < 0.001), 8OH-guanosine and 8-ISO-PGF2alpha levels decreased after cryopreservation (from 130 to 35 arbitrary fluorescence units, P < 0.01, and from 1280 to 1233, P < 0.01, respectively). Postthaw sperm quality was classified as poor, average, or good using the 25th and 75th percentiles of all assays of sperm quality studied (motility, velocity, membrane functionality, and thiol content) as thresholds. Using these values, a sperm postthaw quality index was proposed. Receiver operating characteristic curves and the Youden J statistic were used to investigate the value of the measured parameters in fresh sperm as predictors of potential freezability. Using these techniques, we identified markers of bad freezers (percentages of caspase 3-positive dead sperm [area under the curve (AUC) = 0.820, P < 0.05] and percentages of caspase 3- and 4-HNE-positive sperm [AUC = 0.872, P < 0.05]) and good freezers (percentages of caspase 3-negative live sperm [AUC = 0.815, P < 0.05], percentages of live sperm with high thiol content [AUC = 0.907, P < 0.01], and percentages of 8-ISO-PGF2alpha-positive sperm [AUC = 0.900, P < 0.01]. Moreover, we described for the first time the presence of 8-ISO-PGF2alpha in stallion spermatozoa and revealed the importance of considering different markers of oxidative stress.

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still unclear. Our laboratory has developed a rat model of post-MI syndrome that displays impaired cardiac functions, neuronal loss in the limbic system, cognitive deficits and behavioral signs of depression. At the neuronal level, caspase-3 activation mediates post-MI apoptosis in different limbic regions, such as the amygdala – peaking at 3 days post-MI. Cognitive and behavioral impairments appear 2-3 weeks post-MI and these correlate statistically with measures of caspase-3 activity. The protocol described here is used to induce MI, collect amygdala tissue and measure caspase-3 activity using spectrofluorometry. To induce MI, the descending coronary artery is occluded for 40 min. The protocol for evaluation of caspase-3 activation starts 3 days after MI: the rats are sacrificed and the amygdala isolated rapidly from the brain. Samples are quickly frozen in liquid nitrogen and kept at -80 °C until actual analysis. The technique performed to assess caspase-3 activation is based on cleavage of a substrate (DEVD-AMC) by caspase-3, which releases a fluorogenic compound that can be measured by spectrofluorometry. The methodology is quantitative and reproducible but the equipment required is expensive and the procedure for quantifying the samples is time-consuming. This technique can be applied to other tissues, such as the heart and kidneys. DEVD-AMC can be replaced by other substrates to measure the activity of other caspases.

Background Cigarette smoking is the leading cause of preventable death in the world. It has been implicated in the pathogenesis of pulmonary, oral and systemic diseases. Smoking during pregnancy is clearly a risk factor for the developing fetus and may be a major cause of infant mortality. Moreover, the oral cavity is the first site of exposure to cigarette smoke and may be a possible source for the spread of toxins to other organs of the body. Fibroblasts in general are morphologically heterogeneous connective tissue cells with diverse functions. Apoptosis or programmed cell death is a crucial process during embryogenesis and for the maintenance of homeostasis throughout life. Deregulation of apoptosis has been implicated in abnormal lung development in the fetus and disease progression in adults. Caspases, are proteases which belong to the family of cysteine aspartic acid proteases and are the key components for the downstream amplification of intra-cellular apoptotic signals. Of the 14 caspases known, caspase-3 is the key executioner of apoptosis. Fetal rat lung fibroblasts but not PDL viability is reduced by exposure to CSE. In addition Caspase 3 activity is elevated after CSE exposure in fetal lung fibroblasts but not in PDLs. Expression of caspase 3 is induced in CSE exposed lung fibroblasts but not in PDLs. Caspase 3 was localized to the cytoplasm in both cell types.

This study was designed to determine the possible protective effect of polysaccharides extract of rhizoma atractylodis macrocephalae on heart function in aged rats. Polysaccharides extract of rhizoma atractylodis macrocephalae was administered to aged rats. Results showed that thymus, spleen and cardiac indexs were significantly increased, whereas caspase-3 activity ratio, Smac/DIABLO and HtrA2/Omi protein expression, Smac/DIABLO and HtrA2/Omi mRNA expression levels were markedly reduced. It can be concluded that polysaccharides extract of rhizoma atractylodis macrocephalae may enhance immunity and improve heart function in aged rats.

We studied the effects of the tripeptide pinealon (Lys-Glu-Arg) on behavior and caspase-3 activity in the brain of 21-day-old rats that were subjected to prenatal hypoxia. We found that administration of pinealon to females during the entire course of pregnancy increased the survival of newborn rat rats that were subjected to prenatal hypoxia. In these rats, at the age of 21 days the activity of caspase-3 in brain structures was significantly lower as compared to rats that were not treated with pinealon during the pregnancy period. This was probably a cause of the less expressed cognitive deficit in these rats. Hypoxic hypoxia during the prefetal or fetal periods had worse effects on cognitive functions and rat survival.

As one of the principal cytoplasmic second messengers, the calcium ion (Ca(2+)) is central to a variety of intracellular signal transduction pathways. Accordingly, there is a sustained interest in methods for spatially- and temporally resolved imaging of the concentration of Ca(2+) in live cells using noninvasive methods such as genetically encoded biosensors based on Förster resonance energy transfer (FRET) between fluorescent proteins (FPs). In recent years, protein-engineering efforts have provided the research community with FRET-based Ca(2+) biosensors that are dramatically improved in terms of enhanced emission ratio change and optimized Ca(2+) affinity for various applications. We now report the development and systematic optimization of a pair of spectrally distinct FRET-based biosensors that enable the simultaneous imaging of Ca(2+) in two compartments of a single cell without substantial spectral crosstalk between emission channels. Furthermore, we demonstrate that these new biosensors can be used in conjunction with previously reported caspase-3 substrates based on the same set of FRET pairs.

Increased Lymphocyte Caspase-3 Activity in Patients with SchizophreniaA growing body of evidence indicates that cortical brain cells of schizophrenic patients are vulnerable to apoptosis. As apoptosis is an important mechanism in organism modeling during development, active since the early phase of intrauterine life, it could be involved in the pathogenesis of schizophrenia. To test this hypothesis, caspase-3 activity was determined in peripheral blood mono nuclear cells from 30 patients with schizophrenia and from 30 age and gender matched healthy subjects by a colorimetric commercially available kit. Consistent with increased susceptibility to apoptosis, caspase-3 activity in lymphocytes of patients with schizophrenia was significantly increased (0.111±0.055 μmol/mg protein, p

Could Lymphocyte Caspase-3 Activity Predict Atherosclerotic Plaque Vulnerability?Apoptotic cell death may play a critical role in a variety of cardiovascular diseases, especially in those developing on the basis of atherosclerosis. The goal of this study was to compare the activity of caspase-3 in different forms of ischemic heart disease and to correlate caspase-3 activity with inflammatory and lipid markers as well as risk factors. This enzyme activity was determined in peripheral blood mononuclear cells (PBMC) of 30 patients with stable angina pectoris (SAP), 27 with unstable angina (USAP), 39 with acute ST-elevation myocardial infarction (STEMI) and 27 healthy volunteers by a colorimetric commercially available ELISA method. In the SAP group caspase-3 activity was 0.093±0.036 μmol/mg protein, in patients with STEMI it was 0.110±0.062 μmol/mg protein, and both values were insignificantly higher in comparison with controls (0.092±0.022 μmol/mg protein). In PBMC of USAP patients caspase-3 activity (0.122±0.062 μmol/mg protein) was significantly higher (p

A method to detect an enzymatic reaction in a single living cell using an atomic force microscope equipped with an ultra-thin needle (a nanoneedle) and a fluorescent probe molecule was developed. The nanoneedle enables the low-invasive delivery of molecules attached onto its surface directly into a single cell. We hypothesized that an enzymatic reaction in a cell could be profiled by monitoring a probe immobilized on a nanoneedle introduced into the cell. In this study, a new probe substrate (NHGcas546) for caspase-3 activity based on fluorescent resonance energy transfer (FRET) was constructed and fixed on a nanoneedle. The NHGcas546-modified nanoneedle was inserted into apoptotic cells, in which caspase-3 is activated after apoptosis induction, and a change in the emission spectrum of the immobilized probe could be observed on the surface of the nanoneedle. Thus, we have developed a successful practical method for detecting a biological phenomenon in a single cell. We call the method MOlecular MEter with Nanoneedle Technology (MOMENT).

Chronic reductions in muscle activation and loading are associated with decreased heat shock protein 25 (Hsp25) expression and phosphorylation (pHsp25) which, in turn, may contribute to elevated caspase-3-mediated muscle protein breakdown. Thus, the purpose of the present study was to determine whether there are any changes in Hsp25, pHsp25 and caspase-3 activity among rat muscles having different fibre type compositions and functions [soleus, adductor longus (AL), plantaris and tibialis anterior (TA)] at 0 (control), 1, 8 or 28 days after a complete spinal cord transection (ST). The Hsp25 levels were unaffected on days 1 and 8 in all muscles, except for a significant reduction on day 8 in plantaris. The Hsp25 levels were lower than control values in all muscles except TA on day 28. The pHsp25 levels were lower than control values after 8 and 28 days in plantaris and AL and after 28 days in soleus, but higher than control in TA after 8 and 28 days. Caspase-3 activity was higher in ST than control rats on day 8 in all muscles except TA. Caspase-3 activity was negatively correlated with muscle mass for all muscles. In plantaris, Hsp25 and pHsp25 were negatively correlated with caspase-3 activity and Hsp25 was correlated with muscle mass. These relationships were not observed in other muscles. Thus, the effects of ST on Hsp25 and caspase-3 are muscle specific and time dependent, factors that should be considered in developing any intervention to maintain muscle mass after a spinal cord injury.

Purpose: Radiotherapy followed by total mesorectal excision surgery has been shown to significantly reduce local recurrence rates in rectal cancer patients. Radiotherapy, however, is associated with considerable morbidity. The present study evaluated the use of biochemical detection of enzymatic caspase-3 activity as preoperative marker for apoptosis to preselect patients that are unlikely to develop a local recurrence to spare these patients from overtreatment and the negative side effects of radiotherapy. Experimental Design: Nonirradiated freshly frozen tissue samples from 117 stage III rectal cancer patients were collected from a randomized clinical trial that evaluated preoperative radiotherapy in total mesorectal excision surgery. Additional frozen archival tissues from 47 preoperative biopsies and corresponding resected colorectal tumors were collected. Level of apoptosis was determined by measuring the enzymatic activity of caspase-3 in a biochemical assay. Results: In tumor tissue, caspase-3 activity lower than the median was predictive of 5-year local recurrence (hazard ratio, 7.4; 95% confidence interval, 1.7-32.8; P = 0.008), which was unaffected by adjustment for type of resection, tumor location, and T status (adjusted hazard ratio, 7.5; 95% confidence interval, 1.7-34.1; P = 0.009). Caspase-3 activity in preoperative biopsies was significantly correlated with caspase-3 activity in corresponding resected tumors (r = 0.56; P < 0.0001). Conclusion: Detection of tumor apoptosis levels by measuring caspase-3 activity, for which a preoperative biopsy can be used, accurately predicted local recurrence in rectal cancer patients. These findings indicate that caspase-3 activity is an important denominator of local recurrence and should be evaluated prospectively to be added to the criteria to select rectal cancer patients in which radiotherapy is redundant.

Plant proteases with caspase-like enzymatic activity have been widely studied during the last decade. Previously, we have reported the presence and induction of caspase-3 like activity in the apoplast of potato leaves during Solanum tuberosum- Phytophthora infestans interaction. In this work we have purified and identified a potato extracellular protease with caspase-3 like enzymatic activity from potato leaves infected with P. infestans. Results obtained from the size exclusion chromatography show that the isolated protease is a monomeric enzyme with an estimated molecular weight of 70 kDa approximately. Purified protease was analyzed by MALDI-TOF MS, showing a 100% of sequence identity with the deduced amino acid sequence of a putative subtilisin-like protease from S. tuberosum (Solgenomics protein ID: PGSC0003DMP400018521). For this reason the isolated protease was named as StSBTc-3. This report constitutes the first evidence of isolation and identification of a plant subtilisin-like protease with caspase-3 like enzymatic activity. In order to elucidate the possible function of StSBTc-3 during plant pathogen interaction, we demonstrate that like animal caspase-3, StSBTc-3 is able to produce in vitro cytoplasm shrinkage in plant cells and to induce plant cell death. This result suggest that, StSBTc-3 could exert a caspase executer function during potato- P. infestans interaction, resulting in the restriction of the pathogen spread during plant–pathogen interaction. Fil: Fernández, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Daleo, Gustavo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Guevara, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales; Argentina

Background The role of apoptosis in the development of graft coronary artery disease (GCAD) is poorly understood. We have previously shown that early overexpression of the anti-apoptotic protein Bcl-2 lessens the development of GCAD. We hypothesized that early inhibition of apoptosis with a caspase-3 inhibitor would also lessen the development of GCAD. Methods Heterotopic heart transplantation was performed in 4 groups of rats. Donor hearts were pretreated with 50 μg DEVD-CHO, a cell-permeable caspase-3 inhibitor, or vehicle. Recipient animals were pretreated with 1.7 mg/kg intraperitoneal DEVD-CHO or vehicle. Animals were treated with 7.5 mg/kg/d cyclosporine for 10 days to prevent acute rejection. On post-operative day 90, the animals were sacrificed and the transplanted hearts were assessed morphometrically for evidence of GCAD. Results At 90 days, intimal proliferation was significantly higher in vehicle treated animals than in inhibitor treated animals. Moreover, the percentage of vessels with high-grade occlusion (>50%) was also lower in inhibitor treated animals. Conclusions Early inhibition of caspase-3 activity with cell-permeable DEVD-CHO lessens the development of GCAD. Caspase-3 inhibition may be a useful strategy for prevention of GCAD in clinical transplantation.

A new label-free method for the detection of apoptosis was proposed based on colorimetric assay of caspase-3 activity using an unlabeled Asp-Glu-Val-Asp (DEVD)-containing peptide substrate and unmodified gold nanoparticles (AuNPs).

A novel electrochemical sensing platform for sensitive determination of caspase 3 activity and inhibition was developed by combining the site-specific recognition and cleavage of the DEVD-peptide with quantum dots as signal amplification.

There is substantial evidence to link early graft loss after islet transplantation to isolation-induced islet cell apoptosis. Measurement of caspase 3 activity and detection of the lost cell membrane asymmetry, revealed by annexin V binding, are newly available assays that allow the analysis of early events of apoptosis.

Previous study has demonstrated that squamous cell carcinoma antigen (SCCA) 1 attenuates apoptosis induced by TNFα, NK cell or anticancer drug. In this study, we have examined the effect of SCCA2, which is highly homologous to SCCA1, but has different target specificity, against radiation-induced apoptosis, together with that of SCCA1. We demonstrated that cell death induced by radiation treatment was remarkably suppressed not only in SCCA1 cDNA-transfected cells, but also in SCCA2 cDNA-transfected cells. In these transfectants, caspase 3 activity and the expression of activated caspase 9 after radiation treatment were suppressed. Furthermore, the expression level of phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) was suppressed compared to that of the control cells. The expression level of upstream stimulator of p38 MAPK, phosphorylated MKK3/MKK6, was also suppressed in the radiation-treated cells. Thus, both SCCA1 and SCCA2 may contribute to survival of the squamous cells from radiation-induced apoptosis by regulating p38 MAPK pathway. © 2001 Cancer Research Campaign http://www.bjcancer.com

The hepatitis B virus-encoded HBx protein coactivates transcription of viral and cellular genes, and it is believed to play an important role in hepatitis B virus-related liver cancer. HBx has been shown to alter the coordinated balance between proliferation and programmed cell death, being able to either induce or block apoptosis. Here, we demonstrate for the first time that the HBx is a potent caspase 3 inhibitor. Rat fibroblasts (REV2) and hepatoma cells (Hep) synthesizing the HBx protein were resistant to various apoptotic stimuli such as growth factor depletion, tumor necrosis factor alpha, or anti-Fas antibodies administration. In these cells, HBx prevented DNA fragmentation and cell death in the absence of de novo protein synthesis, with a similar efficiency as the competitive caspase 3 substrates inhibitors VAD-FMK and DEVD-FMK. Protein extracts obtained from the HBx positive cells contained a very low caspase activity, and addition of anti-HBx antibody restored the endogenous caspase activity. To obtain a functional map of the anti-caspase activity of HBx, various cell lines were established that synthesized either N-terminally or C-terminally truncated HBx molecules. These gene dissection experiments revealed that the regions required for the anti-caspase activity overlap with the two known transactivation domains of HBx.

Increased caspase-3 activity in refractory anemias: lack of evidence for Fas pathway implication