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Dysregulation of gene expression at RNA and protein level is a hallmark of Huntington's disease (HD). Altered levels of microRNAs and beta catenin in HD were studied earlier; however, any direct involvement of full length, basally-expressing mutant huntingtin (Htt) remained to be elusive. Here we reported that the gain-of-function mutation of full-length basally-expressing Htt in HD cell Q111 (STHdhQ111/HdhQ111) upregulated microRNA-214 and decreased beta catenin & its transcriptional activity in an aggregate-independent manner. The result was quite opposite of the function of aggregate-forming mutant Htt fragment 83Q-DsRed. Here, we also reported an elevated level of beta catenin phosphorylation in Q111 cell compared to Q7 cell (SThdhQ7/HdhQ7). We showed that in Q111 cell (compared to Q7), beta catenin was more localized in the cytosol than that of the plasma membrane. This is significant as Gsk3beta phosphorylates beta catenin in the cytosol. Hence, for the first time, our study identified beta catenin localization and mutant Htt status as two key factors of beta catenin regulation in HD.

Resveratrol, a trans-stilbene polyphenolic compound and its synthetic analogs are widely used bioactive molecules due to their remarkable chemo-preventive potential. Here, we have identified a novel synthetic trans-stilbene compound, Z-DAN-11 ((Z)-3-(3, 4-dimethoxyphenyl)-2-(3, 4, 5-trimethoxyphenyl) acrylonitrile) which shows remarkable efficacy in blocking tumor growth and progression both in vitro and in vivo. Z-DAN-11 inhibits proliferation of cancer cells in vitro through microtubule depolymerization that induced G2/M arrest and consequently leads to apoptotic cell death. More importantly, Z-DAN-11 shows limited cytotoxicity to normal cells as compared to cancer cells. Quite interestingly, we have found that Z-DAN-11-mediated ROS production helps in dramatic alteration in the mitochondrial redox status which critically contributes to the apoptosis. Mechanistic studies reveal that Z-DAN-11 induces the expression of pro-apoptotic proteins and decreases anti-apoptotic protein expression that decisively helps in the activation of caspase 8, caspase 9, and caspase 3, leading to cleavage of PARP1 and cell death via intrinsic and extrinsic pathways of apoptosis. Moreover, Z-DAN-11-mediated apoptosis of cancer cells is through a partial p53-dependent pathway, since both HCT116 p53−/− cells as well as p53-silenced cells (siRNA) were able to block apoptosis partially but significantly. Importantly, Z-DAN-11 also imparts its anti-tumorigenic effect by inhibiting clonogenic property and anchorage-independent growth potential of cancer cells at concentrations at least 10 times lower than that required for inducing apoptosis. Finally, in vivo study with immuno-competent syngeneic mice shows Z-DAN-11 to be able to impede tumor progression without any adverse side-effects. Hence, we identified a novel, synthetic trans-stilbene derivative with anti-tumorigenic potential which might tremendously help in devising potential therapeutic strategy against cancer.

Background Piroxicam (Px) belongs to the oxicam group of the non-steroidal anti-inflammatory drugs (NSAIDs) and have been shown to exert chemopreventive and chemotherapeutic effects in animal models and cultured animal cells. However, little is known about the mode of action of Px and its cellular targets. Methods We explored the role of Px, in triggering apoptosis and examined the involvement of upstream cellular mechanisms in apoptosis induction by Px. Results and discussion Our studies with human breast cancer cells MCF-7 show that Px induces reactive oxygen species (ROS) generation along with apoptotic cell death. ROS release lead to Akt activation. On evaluation it became evident that ROS mediated apoptosis induction was due to Akt activation (hyper phosphorylation). Silencing the expression of Akt using siRNA and a specific Akt inhibitor, triciribine further confirmed the findings. However Px failed to cause ROS generation, cell death or Akt phosphorylation in another human breast cancer cells MDA-MB-231 which is estrogen receptor negative and more aggressive compared to MCF-7 cells. This suggests that Px has cell type specific effects. Thus we revealed for the first time that Px can induce apoptosis by ROS mediated Akt hyperphosphorylation/activation.

Entamoeba histolytica cysteine protease 6 (EhCP6) is a stress responsive cysteine protease that is upregulated in response to heat shock and during pathogen invasion of the host tissue. In the present study an attempt has been made to express and purify recombinant EhCP6 in order to gain insights into its biochemical properties. The recombinant and refolded protein has been shown to undergo autoproteolysis in the presence of DTT and SDS to give rise to ∼25kDa mature form. The mature form of the protein was found to exhibit a protease activity that is sensitive to E-64, a specific cysteine protease inhibitor. In silico homology modelling of EhCP6 revealed that the protein exhibits conservation of almost all the major structural features of cathepsin-L like cysteine proteases. Further in vivo studies are needed to decipher the function of the protein in response to different stressed conditions.

Intracellular protein homeostasis is largely controlled by Heat shock proteins (Hsp). Heat shock proteins (Hsp) impart an age-old defense mechanism for all forms of life on earth. Misfolded proteins are refolded with the aid of Hsp and proteins which are damaged beyond repair are eliminated with assistance from Hsp. Hsp are known as molecular chaperones for their cytoprotective roles. In cancer cells the Hsp are frequently overexpressed and are assumed to be associated with tumor formation. Hsp demonstrate specific affinity to particular classes of oncogenic peptides and client proteins in cancer cells, and are able to stabilize mutated oncogene proteins. They play a key regulatory role in prevention of apoptotic cell death during tumorigenesis and thereby enhance cell growth and proliferation. They may also promote chemoresistance in cancer cells. Here we present the current knowledge on the role of molecular chaperones in particular heat shock protein 70 (Hsp70) in human gastrointestinal cancers along with their therapeutic targeting. This review will focus on the role of Hsp 70 and related chaperones in several gastrointestinal cancers such as pancreatic, gastric, and liver cancers.

Owing to increasing concern of global climate-change, temperature rise is of great interest which can be primarily evaluated from the seasonal variations in some organisms. Aquatic environment can be extremely stressful to its inhabitants because most of them are poikilothermous. In the present study, attempt was made to evaluate the biological effects of oxidative-stress and adaptive/antioxidant capacities during temperature variations (36–40 °C for 24hrs to 72hrs) in Bellamya bengalensis both in environmental and laboratory conditions by testing some biomarkers like HSP70, catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH) and glutathione reductase (GR). The biomarker potency of the molecules and the anti-oxidative metabolic-network was postulated and extrapolated to find its resemblance to the climate-change associated organismal variations. In a natural and eco-restored environment in the Eastern part of India, 10–20 fold increases in CAT, SOD and HSP70 protein expressions (Western blot results) were noticed in Bellamya paralleling to their increased enzymatic activities (gel zymogram studies) due to the seasonal (summer versus winter) temperature variation. It is evident from the consecutive three years’ study that this variation resulted in the unfavorable physico-chemical changes of water quality parameters like dissolved oxygen, biochemical oxygen demand, alkalinity and consequently decreased the animal density in summer. And that was revived due to their higher reproduction-rate in post rainy/winter season when temperature normalizes resulting in a restoration of favorable environment. In laboratory condition, the reduced GR and increased GPx indicated the oxidative damage as evident by higher tissue MDA level following to higher mortality. Changes in SOD and CAT activities suggest activation of physiological mechanism to scavenge the ROS produced during heat stress. However, when mortality increased at different time points (36 °C − 72 h and 38 °C − 72 h), these enzyme activities also decreased as they failed to save the tissues from ROS. The results suggest that temperature variation does alter the active oxygen metabolism by modulating antioxidant enzyme activities, which can be used as biomarker to detect sub-lethal effects of climate change-associated pollution. The parity in environmental and laboratory experimental results may justify this laboratory experiment as model heat-stress experiment and indicate temperature as a universal stressor which alone or in combination with other water parameters initiates a consistent adapting behavior. The Bellamya bengalensis being the highest faunal representative in its habitat may serve as a good bioindicator species.

Expression of the stress biomarkers 70-kDa heat shock proteins (Hsp70) and manganese superoxide dismutase (MnSOD) was measured as the molecular basis of adaptive response against increased experimen- tal temperatures (32-40°C for a span of 24-72h) on the fresh water molluscan species, Bellamya bengalensis (Lamark 1882). The experimental snail specimens were collected during summer and winter seasons from two contrasting wetlands: an ecorestored (free from human interference) site (SI) and other experiencing anthropo- genic stresses (SII). The mortality rate of the B. bengalensis and the immunoblotting of MnSOD and Hsp70 of their digestive glands were performed at regular intervals during the period of heat stress. The SI provided a lower stress environment based on physico- chemical parameters such as pH, dissolved oxygen (DO), biological oxygen demand (BOD), chemical ox- ygen demand (COD), and alkalinity for the survival of test species, although both sites experienced mortality duetothermalstresses.Theparityinproteinexpressions displayed a uniform mode of adaptive impact to tem- perature elevations in both field and laboratory expo- sure. The Hsp70 expression was minimal at lower ther- mal stress, but increased with a rise in temperature. It is very likely that higher Hsp70 levels are not directly related to survival or adaptation. In contrast, MnSOD levels appeared to be an indicator of adaptive responses vis-a-vis survival of the animals. So, the expression levels of a universal free radical scavenger like MnSOD are recognized as a potential biomarker in a bioindicator species like Bellamya.

Seed priming is a method by which seeds are subjected to different stress conditions to impart stress adaptation in seedlings germinating and growing under stressful situations. Drought stress is a major reason behind failure of crops. We studied the effects of hydropriming, dehydration priming (induced by PEG), and osmopriming (induced by NaCl and KH(2)PO(4)) on subsequent germination, growth and anti-oxidant defense mechanisms of 2-week-old rice seedlings under continuing dehydration stress. Unprimed seeds grown in PEG showed significantly lower germination and growth along with significantly higher reactive oxygen species (ROS) and lipid peroxidation levels. Among the priming methods, 5 % PEG priming was found to be the best in terms of germination and growth rate along with the lowest amount of ROS and lipid peroxidation (malondialdehyde [MDA]) values. MDA levels were reduced significantly by all of the priming methods. Hence, reduction of lipid peroxidation may be a key factor underlying the drought tolerance produced by the priming treatments. Glutathione peroxidase (GPX) activity seemed to bear an excellent correlation with oxidative stress resistance through seed priming. The PEG priming produced minimum peroxidative damage and superior germination and growth rate along with efficient GPX activity, overexpressed MnSOD and maintenance of HSP70 expression in normal as well as in drought condition. Therefore, in PEG-primed seeds the existence of robust protective mechanisms is definitely indicated.

One of the major classes of virulence factors acting in different host-pathogen interaction systems is comprised of proteases. Pathogen-secreted or membrane-associated proteases could be found to participate in different stages of establishment of infection. They are explored as candidate drug targets due to their key participation in the disease development process carried out by the pathogen. In this chapter we present an extensive review of the proteases of different protozoan parasites. Throughout the article we have made an effort to provide a comprehensive list of different proteases from various parasitic protozoa that have been demonstrated to execute major functions in the respective infection processes. Attempts have also been made to present their mode of action with respect to host invasion and disease development.

Benzo(a)pyrene (B(a)P) is an environmental toxicant that induces male germ cell apoptosis. Curcumin and resveratrol are phytochemicals with cytoprotective and anti-oxidative properties. At the same time resveratrol is also a natural Aryl hydrocarbon Receptor (AhR) antagonist. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted the synergistic protective effect of curcumin and resveratrol against B(a)P induced p53 mediated germ cell apoptosis. Curcumin-resveratrol significantly prevented B(a)P induced decrease in sperm cell count and motility, as well as increased serum testosterone level. Curcumin-resveratrol co-treatment actively protected B(a)P induced testicular germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3, 8 and 9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, and Apaf1. B(a)P induced testicular reactive oxygen species (ROS) generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 (Cytochrome P4501A1) expression. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ cells from B(a)P induced apoptosis.

The microaerophilic intestinal parasitic protozoan Entamoeba histolytica has been previously shown to be highly susceptible to oxidative stress induced by hydrogen peroxide. However the mechanism of cell death was not investigated. Studies presented in this paper demonstrate several morphological features in the parasite when exposed to H(2)O(2) which are identical to metazoan apoptotic phenotype indicating a possible apoptosis-like cell death exhibited by E. histolytica in response to H(2)O(2) treatment. Trophozoite cell shrinkage, DNA fragmentation, phosphatidyl serine externalization and increased endogenous reactive oxygen species level have been observed in the protozoan parasite when exposed to 2.0mM H(2)O(2) for different time periods. Although the parasite genome is completely devoid of any of the homologues of mammalian caspases it still codes for a huge number of cysteine proteases which may take over the apoptotic function of the caspases. But the present study indicates the existence of a cysteine protease independent programmed cell death in the parasite since E-64 the specific cysteine protease inhibitor could not rescue the cells from H(2)O(2) induced apoptosis-like cell death.

V79 lung fibroblasts were subjected to repetitive oxidative stress in culture through exposures to 30 microM H(2)O(2) for 4 weeks. Repetitively stressed cells were found to be significantly resistant to apoptosis-inducing agent such as ultraviolet radiation (UVR). Concurrent treatment with Resveratrol completely restored the normal apoptotic response after UVR. p38MAPK became dually phosphorylated during the stress period. Akt also became phosphorylated on Ser(473) in cells subjected to repetitive oxidative stress. In these cells, NFkappaB p65 became phosphorylated and appreciable nuclear localization of p65 was observed. NFkappaB transcriptional activity also became augmented during repetitive stress. Treatment of the repetitively stressed cells concurrently with Resveratrol or SB203580, a p38MAPK inhibitor, robustly blocked activation of p38MAPK, NFkappaB transcriptional activity, phosphorylation and nuclear localization of p65, and Akt phosphorylation. Pre-exposure to short interfering RNA (si RNA) to p38MAPK, resulted in a blockage of the Akt and NFkappaB p65 phosphorylation. However, inhibition of Akt activity through PI3 kinase inhibitor LY294002 did not result in obstruction of p38MAPK phosphorylation by H(2)O(2). Also, Resveratrol was effective as an antioxidant in counteracting a rise in reactive oxygen species (ROS) and p38MAPK activation by H(2)O(2) was completely blocked by antioxidant N-acetyl cysteine (NAC). We conclude that Resveratrol acts as an antioxidant and completely reverses the anti-apoptotic effects of repetitive stress by blocking oxidative stress-induced p38MAPK activation which is the key regulatory step for the activation of down-stream survival elements Akt and NFkappaB.

Human prostate cancer is a complex heterogeneous disease that mainly affects elder male population of the western world with a high rate of mortality. Acquisitions of diverse sets of hallmark capabilities along with an aberrant functioning of androgen receptor signaling are the central driving forces behind prostatic tumorigenesis and its transition into metastatic castration resistant disease. These hallmark capabilities arise due to an intense orchestration of several crucial factors, including deregulation of vital cell physiological processes, inactivation of tumor suppressive activity and disruption of prostate gland specific cellular homeostasis. The molecular complexity and redundancy of oncoproteins signaling in prostate cancer demands for concurrent inhibition of multiple hallmark associated pathways. By an extensive manual curation of the published biomedical literature, we have developed Human Prostate Cancer Hallmarks Map (HPCHM), an onco-functional atlas of human prostate cancer associated signaling and events. It explores molecular architecture of prostate cancer signaling at various levels, namely key protein components, molecular connectivity map, oncogenic signaling pathway map, pathway based functional connectivity map etc. Here, we briefly represent the systems level understanding of the molecular mechanisms associated with prostate tumorigenesis by considering each and individual molecular and cell biological events of this disease process.

Heat Shock Protein 70 (Hsp70) is an evolutionarily conserved family of proteins which carry out multiple cellular functions such as protein biogenesis, protection during stress, prevention of formation of protein aggregates, assistance in protein translocation and many others. Hsp70, being the major cytoprotective molecular chaperone, plays a crucial role in protecting against a stunning array of stresses and in the re-establishment of cellular homeostasis. This book chapter gives an overview of the multifaceted Hsp70s in plants, with special emphasis on their association with plant response to various stress conditions and eventually, stress acclimation. The contribution of plant stress-responsive proteomics studies towards putting Hsp in the spotlight has also been brought forth. The road ahead is to decipher the underlying mechanisms of Hsp70-mediated multiple cross tolerance, that is likely to lead to new strategies to enhance crop tolerance to environmental stress.

Modulation of surface properties of biomembranes by any ligand leading to permeabilization, fusion, rupture, etc. is a fundamental requirement for many biological processes. In this work, we present the interaction of piroxicam, a long acting Non-Steroidal Anti-Inflammatory Drug (NSAID) with isolated mitochondria, membrane mimetic systems, intact cells and a mitochondrial protein cytochrome c. Dye permeabilization study on isolated mitochondria indicates that piroxicam can permeabilize mitochondrial membrane. Direct imaging by Scanning Electron Microscope (SEM) shows that piroxicam induces changes in mitochondrial membrane morphology leading to fusion and rupture. Transmission Electron Microscope (TEM) imaging of piroxicam treated DMPC vesicles and mixed micelles formed from CTAB and SDS show that causing membrane fusion is a general property of piroxicam at physiological pH. In intact cells viz., V79 Chinese Hamster lung fibroblast, piroxicam is capable of releasing cytochrome c from mitochondria into the cytosol in a dose dependent manner along with the enhancement of downstream proapoptotic event viz., increase in caspase-3 activity. We have also shown that piroxicam can reduce cytochrome c within a time frame relevant to its lifetime in blood plasma. UV-visible spectroscopy has been used to study the reaction mechanism and kinetics in detail, allowing us to propose and validate a Michaelis–Menten like reaction scheme. CD spectroscopy shows that small but significant changes occur in the structure of cytochrome c when reduced by piroxicam.

Benzo(a)pyrene [B(a)P] is an environmental toxicant that alters the steroidogenic profile of testis and induces testicular dysfunction. In the present study, we have investigated the molecular signaling of B(a)P and the ameliorative potential of the natural aryl hydrocarbon receptor (AhR) antagonist and antioxidant, resveratrol, on B(a)P-induced male reproductive toxicity. Studies showed that B(a)P treatment resulted in p38 MAPK activation and increased inducible nitric oxide synthase (iNOS) production along with testicular apoptosis and steroidogenic dysfunction. Resveratrol cotreatment maintained testicular redox potential, increased serum testosterone level and enhanced expression of major testicular steroidogenic proteins (CYPIIA1, StAR, 3βHSD, 17βHSD) and prevented subsequent onset of apoptosis. Resveratrol cotreatment resulted inhibition of testicular cytochrome P4501A1 (CYP1A1) expression, which is the major B(a)P metabolizing agent for BPDE-DNA adduct formation. Resveratrol also significantly decreased the B(a)P-induced AhR protein level, its nuclear translocation and subsequent promoter activation, thereby decreased the expression of CYP1A1. Resveratrol also down-regulated B(a)P-induced testicular iNOS production through suppressing the activation of p38 MAPK and ATF2, thus improved the oxidative status of the testis and prevented apoptosis. Our findings cumulatively suggest that resveratrol inhibits conversion of B(a)P into BPDE by modulating the transcriptional regulation of CYP1A1 and acting as an antioxidant thus prevents B(a)P-induced oxidative stress and testicular apoptosis.

Increased activity of β-catenin, an important transcriptional activator for survival and proliferation-associated genes has been linked with many cancers. We examined whether β-catenin is a target of resveratrol and whether its degradation contributes to the pro-apoptotic effects of resveratrol. HeLa cells were exposed to 60 μM resveratrol for 48 h. Apoptosis was confirmed by measurement of annexin V externalization, caspase-3 activation and DNA fragmentation. Induction of apoptosis was observed as early as 12 h, when both caspase-3 activation and PARP (poly ADP ribose polymerase) cleavage occurred. Nuclear β-catenin levels remained unchanged for 48 h during resveratrol exposure. However, extranuclear cell lysate β-catenin underwent a decrease at a late stage of apoptosis namely at 36-48 h. Alterations in the phosphorylation status of Akt/GSK3β were not observed during resveratrol-induced apoptosis. Furthermore, inhibition of GSK3β activity which is. largely responsible for β-catenin degradation failed to influence β-catenin stability. However, inhibition of caspase-3 activity prevented the decline in β-catenin levels at 36-48 h of resveratrol exposure. Lactacystin, a proteosomal inhibitor also prevented the degradation of β-catenin by resveratrol. In conclusion, resveratrol induced apoptosis in HeLa cells in an Akt/GSK3β-independent manner and down-regulated β-catenin levels during apoptosis through action of caspase-3 and proteasomal degradation, independent of GSK3β-mediated phosphorylation.

Purpose: Previously, we have synthesized and characterized a novel Cu(II) complex, copper N-(2-hydroxy acetophenone) glycinate (CuNG). Herein, we have determined the efficacy of CuNG in overcoming multidrug-resistant cancer using drug-resistant murine and human cancer cell lines.Experimental Design: Action of CuNG following single i.m. administration (5 mg/kg body weight) was tested in vivo on doxorubicin-resistant Ehrlich ascites carcinoma (EAC/Dox)–bearing mice and doxorubicin-resistant sarcoma 180–bearing mice. Tumor size, ascitic load, and survival rates were monitored at regular intervals. Apoptosis of cancer cells was determined by cell cycle analysis, confocal microscopy, Annexin V binding, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay ex vivo. IFN-γ and tumor necrosis factor-α were assayed in the culture supernatants of in vivo and in vitro CuNG-treated splenic mononuclear cells from EAC/Dox-bearing mice and their apoptogenic effect was determined. Source of IFN-γ and changes in number of T regulatory marker-bearing cells in the tumor site following CuNG treatment were investigated by flow cytometry. Supernatants of in vitro CuNG-treated cultures of peripheral blood mononuclear cells from different drug-insensitive cancer patients were tested for presence of the apoptogenic cytokine IFN-γ and its involvement in induction of apoptosis of doxorubicin-resistant CEM/ADR5000 cells.Results: CuNG treatment could resolve drug-resistant cancers through induction of apoptogenic cytokines, such as IFN-γ and/or tumor necrosis factor-α, from splenic mononuclear cells or patient peripheral blood mononuclear cells and reduce the number of T regulatory marker-bearing cells while increase infiltration of IFN-γ-producing T cells in the ascetic tumor site.Conclusion: Our results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancers irrespective of multidrug resistance phenotype.

V79 Chinese Hamster lung fibroblasts were subjected to repetitive low-grade stress through multiple exposures to 30 microM H2O2 in culture for 4 weeks. Akt/protein kinase B became phosphorylated at serine473 and threonine308 during this period of repetitive stress. Concurrent exposure of the cells to LY294002 (5 microM), a phosphoinositide-3 kinase inhibitor or 4.5 microM epigallocatechin 3-gallate (EGCG), a tea polyphenol almost completely blocked Akt activation by repetitive stress. Phosphorylation of I kappa B kinase (IKK) and transcriptional activity driven by nuclear factor kappa B (NFkappaB) were significantly enhanced by repetitive oxidative stress. These increases were largely abolished by simultaneous exposure to EGCG. The repetitively stressed cells demonstrated a significant resistance to apoptosis by subsequent acute stress in the form of ultraviolet radiation at 5 J/m2 or H2O2 (7.5 mM). The resistance to apoptosis conferred by repetitive stress was drastically reduced (>80%) by constant exposure to EGCG during the stress period while the presence of LY294002 or the NFkappaB inhibitor SN50 brought about a relatively moderate effect (about 50-65%). Our data indicate that activation of Akt and NFkappaB pro-survival pathways by repetitive low-grade stress results in a strong inhibition of the normal apoptotic response after subsequent acute stress. The tea polyphenol EGCG impedes the activation of both Akt and NFkappaB by repetitive stress and as a result preserves the normal apoptotic response during subsequent acute stress.

A gene encoding mitogen-activated protein kinase (MAPK) from the human enteric parasite, Entamoeba histolytica has been identified. Sequence analyses of the polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) products reveal that the EhMAPK gene is intronless and encodes a protein of 352 amino acids. EhMAPK shows significant homology with other MAPKs and contains the 11 subdomains including the invariant residues characteristic of serine/threonine protein kinases. The MAPK signature residues and motifs are also present in EhMAPK. The atomic model of EhMAPK built with rat ERK2 as template exhibits the conservation of all major secondary structural features. However, a deletion in close proximity to the dual phosphorylation/activation site is of particular interest as it may have functional implications. Phylogenetic analysis indicates that EhMAPK is tightly clustered with Giardia intestinalis ERK2 and Dictyostelium discoideum ERK2. Detailed sequence analysis and phylogenetic study aided us to postulate that EhMAPK belongs to the extracellular signal-regulated kinase (ERK) family. Although EhMAPK bears good homology and phylogenetic closeness with human ERK8 and rat ERK7, sequence analysis indicates that they may be functionally different. The significant differences such as the deletions in the vicinity of the phosphorylation lip, variations in the P+1 specificity pocket, presence of additional acidic amino acids in the common docking domain provide a ground for postulations that activators and substrates for EhMAPK may be to some extent divergent from that of the ERKs of the mammalian host. Although functional characterization of EhMAPK remains to be done, this is the first study of any member of the MAPK signaling system in this organism.

Exposure of Chinese hamster V79 fibroblasts to mild and repetitive H 2 O 2 doses in culture for 15 weeks produced no change in lipid peroxidation status, GSH/GSSG ratio and glutathione peroxidase activity of these cells (VST cells). In contrast, in VST cells catalase levels underwent a prominent increase which could be significantly inhibited and brought down to control levels after treatment with the catalase inhibitor 3-aminotriazole (3-AT). When control (VC) cells were exposed to UV radiation (UVC 5 J/m 2 ) or H 2 O 2 (7.5 mM, 15 min), intracellular reactive oxygen species (ROS) levels rose prominently with significant activation of caspase-3. Marked nuclear fragmentation and lower cell viability were also noted in these cells. In contrast, VST cells demonstrated a significantly lower ROS level, an absence of nuclear fragmentation and an unchanged caspase-3 activity after exposure to UVC or H 2 O 2 . Cell viability was also significantly better preserved in VST cells than VC cells after UV or H 2 O 2 exposures. Following 3-AT treatment of VST cells, UVC radiation or H 2 O 2 brought about significantly higher elevations in intracellular ROS, increases in caspase-3 activity, significantly lowered cell viability and marked nuclear fragmentation, indicating the involvement of high catalase levels in the cytoprotective effects of repetitive stress. Therefore, upregulation of the antioxidant defense after repetitive oxidative stress imparted a superior ability to cope with subsequent acute stress and escape apoptotic death and loss of viability.

Akt/PKB is a serine/threonine kinase, which controls vital cellular functions such as cell survival/apoptosis, cell cycle progression and glucose metabolism. Akt/PKB acts down-stream from growth factors and hormones and is a key mediator of their pro-survival, proliferative and metabolic effects. Akt/PKB carries out these diverse tasks through phosphorylation of a number of cellular substrates. The substrates of Akt/PKB, which promote the inhibition of apoptosis after being phosphorylated by Akt, include the Forkhead transcription factors and the Bcl-2 family member Bad. The cyclin dependent kinase inhibitors are substrates of Akt which when phosphorylated relinquish their inhibitory influence on cell cycle progression. Akt mediates many of the stimulatory effects of insulin on glucose metabolism through deactivation of glycogen synthase kinase, activation of phosphofructokinase, and modulation of glucose transporter activity. Consequently, Akt can be implicated in the pathological processes, which are associated with defects in regulation of apoptosis/survival and energy metabolism.

Role of beta catenin in Huntington's disease (HD) is not clear. Previous studies on HD reported varied levels of beta catenin. In the present study we showed that beta catenin is post transcriptionally down-regulated in mutant huntingtin knock-in cell model STHdhQ111/Q111. This in turn leads to decreased level of wnt/beta catenin responsive genes. We observed that Gsk3beta or Gsk3beta (phospho Ser 9) is unaltered in HD and this down-regulation of beta catenin is independent of proteasomal degradation. Finally, we showed that the overexpression of miR-214 leads to the down-regulation of beta catenin at protein level only and reduces its transcriptional activity. We concluded that, miR-214 contributes to the processes that result in proteasome independent post transcriptional down-regulation of beta catenin in STHdhQ111/Q111, probably through inhibition of protein synthesis from beta catenin mRNA.

A series of glycosyl triazol linked 18β-glycyrrhetinic acid (GA) derivatives have been synthesized using 1,3-dipolar cycloaddition reaction of per-O-acetylated glycosyl azide derivatives (4a-h) with propargyl ester of 18β-glycyrrhetinic acid (GA) (2 and 3) following the concept of 'Click chemistry'. The synthesized triazole derivatives were de-O-acetylated to furnish compounds (7a-h and 8a-c) with free hydroxyl groups in the carbohydrate moieties, which were evaluated for their anticancer potential against human cervical cancer cells (HeLa) and normal kidney epithelial (NKE) cells. GA (1), compound 7d, compound 7g and compound 8c showed promising anticancer activities.

In the present study, we examined the involvement of intracellular ceramide in host pathogen interaction of BALB/c mouse peritoneal macrophages infected with the obligate intracellular protozoan, Leishmania donovani. Our findings indicate that the level of intracellular ceramide was enhanced as a result of the in vitro infection. While the elevated ceramide was largely due to de novo synthesis, activation of the sphingomyelinases was also observed. The enhanced ceramide was responsible for the downregulation of classical PKC activity, upregulation of calcium independent atypical PKC-ζ expression and activity of calcium independent PKC. Ceramide also impaired the phosphorylation of MAPK. Evidently, ceramide suppressed the generation of nitric oxide during leishmanial infection and also facilitated the survival of leishmanial parasites in the intramacrophageal milieu. These data present newer insight to the signaling events in leishmania-infected murine macrophages, which might offer ceramide as a new therapeutic target in the future.

Very little protein tyrosine phosphorylation was observed in growing (exponential-phase) Entamoeba histolytica cells by immunoblotting and quantitative immunofluorescence. After 1 h of serum deprivation, two proteins (42 and 38 kDa in SDS-PAGE) were tyrosine phosphorylated and two more proteins (96 and 63 kDa) also showed tyrosine phosphorylation when examined after 4 h of serum deprivation. Intense enhancements of anti-phosphotyrosine immunofluorescence levels were observed during this period of serum withdrawal. Membrane-associated tyrosine kinase activity reached a peak (3.5-fold increase) 1 h after serum deprivation and decreased thereafter reaching a basal level by 2 h of serum deprivation. Interestingly, tyrosine kinase activities remained unaffected by serum stimulation (2-60 min) of serum-deprived cells. Also, during this period of serum stimulation tyrosine phosphorylated proteins of serum-deprived cells were dephosphorylated. Tyrosine phosphatase activities were suppressed during serum deprivation and on serum addition to serum-deprived cells tyrosine phosphatase activities increased significantly. Our data attest that protein tyrosine phosphorylation was associated with growth inhibition of E. histolytica and serum stimulation of E. histolytica produced tyrosine phosphatase activation and protein tyrosine dephosphorylation.

A family of proteases known as caspases is a key element in the proteolytic machinery involved in apoptosis or programmed cell death. Apart from their involvement in cell death, caspases are also associated with the developmental process and other normal functions of adult organisms. Caspases are named such because they constitute a family of cysteine proteases which always cleave an Asp residue in their substrates. Stroke results from a rapid malfunctioning of the brain due to lack of blood supply and is a major health threat producing mortality and morbidity. Majority of strokes are ischemic (80 % of all strokes) and the rest are hemorrhagic. Both forms of divergent cell death mechanisms, necrosis, and apoptosis are observed at different spatial region of ischemic attack. Involvement of multiple caspases in stroke has been documented with caspases 1, 3, 8, 9, and 11 playing major roles. Many neurodegenerative diseases result from loss of functional neurons from the brain through enhanced death of neurons. Neurodegenerative diseases are usually late onset and progressive. Among the most common neurodegenerative disorders in aging populations worldwide, Alzheimer’s disease (AD) and Parkinson’s disease (PD) definitely warrant mentioning. Abnormal protein deposits in specific regions of the brain give rise to both these diseases triggering reactive oxygen species formation and mitochondrial dysfunction. Caspases are activated by these changes resulting in loss of neurons through cell death. In this review we provide a brief overview of the involvement of caspases in diseases associated with the impairment of brain function.

Imprints of stress response by rice seedlings in terms of expression levels of stress response gene HSP70 are characterised . The response to arsenic and/or heat shock are shown to be additive for the same stress or the combined stresses, indicating a commonality of signalling pathways., Background and aims Plants can withstand many abiotic stresses. Stress adaptation through retention of imprints of previous stress exposure has also been described in plants. We have characterized the imprint or memory of adaptive stress responses of rice seedlings to arsenic (As) and heat stress. Methodology Two-week-old rice seedlings (both with and without As) were given a 45 °C heat shock for 3 h. While under heat shock, the leafy portion of the seedlings was harvested at regular intervals. Subsequently, the seedlings were kept at room temperature for recovery and sampling continued over 3 h. Total RNA and protein were extracted from the leafy portion of the seedlings and complementary DNA (cDNA) was prepared from total RNA. The cDNA was used as a template for the polymerase chain reaction to identify the transcription level of HSP70. Protein extracted from the seedlings was western-blotted. HSP70 and actin (loading control) antibodies were used to recognize the proteins on the same blot. Principal results Our studies reveal that HSP70, a cellular chaperone gene, is over-expressed at the mRNA and protein levels when rice seedlings are exposed to As and heat. The effect is cumulative and increases with the duration of stress for 3 h. During 3 h recovery from heat stress at ambient temperatures for 3 h, the chaperone remains expressed at higher levels in plants pre-exposed to As. Conclusions Our findings demonstrate a retention of the imprint of previous stress exposure, perhaps through sustained activation of the signalling pathways upstream of over-expression of HSP70. Furthermore, stress-induced HSP70 expression was additive/cumulative for continued exposure to similar or different kinds of stress, indicating that a commonality of signal transduction networks is adopted when plants experience more than one stress.

Chinese hamster lung fibroblasts V79 cells were treated with heat stress for 4 weeks with short duration (15 min) heat shock every alternate day in culture. It was observed that Hsp 70 and the antioxidant enzyme MnSOD became overexpressed during the chronic heat stress period. Both p38 MAPK and Akt became phosphorylated by chronic heat stress exposure. Simultaneous exposure to SB203580, a potent and specific p38MAPK inhibitor drastically inhibited the phosphorylation of p38MAPK and Akt. Furthermore, exposure to SB203580 also blocked the increase in Hsp70 and MnSOD levels and the elevated SOD activity brought about by chronic heat stress. Heat shock factor 1 (HSF1) transcriptional activity and nuclear translocation of HSF1 were prominently augmented by chronic heat stress, and this amplification is markedly reduced by concomitant exposure to SB203580. Also, activations of p38MAPK and Akt and upregulations of Hsp70 and MnSOD were observed on exposure to heat shock for a single exposure of longer duration (40 min). siRNA against p38MAPK notably reduced Akt phosphorylation by single exposure to heat stress and drastically diminished the rise in Hsp70 and MnSOD levels. Similarly, siRNA against Akt also eliminated the augmentation in Hsp70 and MnSOD levels but p38MAPK levels remained unaffected. Heat stress produced reactive oxygen species (ROS) in V79 fibroblasts. N-acetyl cysteine blocked the increase in phosphorylation of p38MAPK, amplification of Hsp70, and MnSOD levels by heat stress. Therefore, we conclude that heat stress-activated p38MAPK which in turn activated Akt. Akt acted downstream of p38MAPK to increase Hsp70 and MnSOD levels.Concise summary: Thermal injury of the skin over a long period of time has been associated with development of cancerous lesions. Also, in many cancers, the cytoprotective genes Hsp70 and MnSOD have been found to be overexpressed. Therefore, we considered it important to identify the signaling elements upstream of the upregulated survival genes in heat stress. We conclude that heat stress activated p38MAPK which in turn activated Akt. Akt mediated an augmentation in Hsp70 and MnSOD levels working downstream of p38MAPK.

Chronic myelogenous leukemia (CML) is a myeloproliferative disease associated with a characteristic chromosomal translocation called the Philadelphia chromosome. This results in the expression of the Bcr-Abl fusion protein, a constitutively active protein tyrosine kinase. Although there are a few treatment options with Bcr-Abl kinase inhibitors, drug resistance is often encountered. One of the major obstacles in overcoming drug resistance in CML is the high endogenous levels of heat shock protein 70 (Hsp70). Resveratrol is a phytoalexin produced by several plants. We studied the chemotherapeutic effects and mode of action of resveratrol on K562 (CML) cells. Resveratrol induced apoptosis in K562 cells in a time-dependent manner. This was established by increased annexin V binding, corroborated with an enhanced caspase-3 activity and a rise in the sub-G 0 /G 1 population. Resveratrol treatment also caused suppression of Hsp70 both in mRNA and protein levels. The downregulation of Hsp70 by resveratrol exposure was correlated with a diminished presence of heat shock factor 1 (HSF1) in the nucleus, and the downregulation of transcriptional activity of HSF1. High endogenous levels of Hsp70 have been found to be a deterrent for sensitivity to chemotherapy. We show here that resveratrol could considerably enhance the apoptosis induction in K562 cells by 17-allylamino-17-demethoxygeldanamycin, an anticancer agent that inhibits Hsp90 but augments Hsp70 levels. We conclude that resveratrol significantly downregulated Hsp70 levels through inhibition of HSF1 transcriptional activity and appreciably augmented the pro-apoptotic effects of 17-allylamino-17-demethoxygeldanamycin.

p21-activated kinases (PAKs) are a family of serine/threonine kinases whose activity is regulated by the binding of the small Rho family GTPases as well as by RhoGTPase independent mechanisms. PAKs have wide-ranging functions which include cytoskeletal organisation, cell motility, cell proliferation and survival. We have identified a PAK from Entamoeba histolytica - EhPAK3 that is distributed in the cytoplasm of unstimulated cells and localizes to the caps after induction of capping with Concanavalin A. EhPAK3 contains a GTPase interacting (CRIB) domain, an N-terminal pleckstrin homology (PH) domain and a C-terminal kinase domain. Among the PAKs of E. histolytica studied so far, EhPAK3 bears the maximum similarity to Dictyostelium discoideum PAKC (DdPAKC). Phylogenetic analysis showed that EhPAK3 was closely related to DdPAKC and forms a group with DdPAKA, Dd Myosin I heavy chain kinase (DdMIHCK), and a PAK reported earlier from E. histolytica EhPAK2. Recombinant full-length EhPAK3 undergoes auotophosphorylation and phosphorylates histone H1 in vitro in the absence of any small GTPase. This is the first comprehensive characterization of a PAK protein from E. histolytica, which has constitutive activity and has demonstrated a strong involvement in receptor capping.

Pentavalent antimony complexes, such as sodium stibogluconate and sodium antimony gluconate (SAG), are still the first choice for chemotherapy against various forms of leishmaniasis, including visceral leishmaniasis, or kala-azar. Although the requirement of a somewhat functional immune system for the antileishmanial action of antimony was reported previously, the cellular and molecular mechanism of action of SAG was not clear. Herein, we show that SAG induces extracellular signal-regulated kinase 1 (ERK-1) and ERK-2 phosphorylation through phosphoinositide 3-kinase (PI3K), protein kinase C, and Ras activation and p38 mitogen-activated protein kinase (MAPK) phosphorylation through PI3K and Akt activation. ERK-1 and ERK-2 activation results in an increase in the production of reactive oxygen species (ROS) 3 to 6 h after SAG treatment, while p38 MAPK activation and subsequent tumor necrosis factor alpha release result in the production of nitric oxide (NO) 24 h after SAG treatment. Thus, this study has provided the first evidence that SAG treatment induces activation of some important components of the intracellular signaling pathway, which results in an early wave of ROS-dependent parasite killing and a stronger late wave of NO-dependent parasite killing. This opens up the possibility of this metalloid chelate being used in the treatment of various diseases either alone or in combination with other drugs and vaccines.

V79 fibroblasts were repetitively stressed through multiple exposures to a low dose (30 microM) H2O2 in culture for 4 weeks. Catalase activity, protein levels and mRNA levels increased markedly (5-6-fold) during this time and these augmentations were inhibited by the simultaneous presence of SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38MAPK). p38MAPK became dually phosphorylated and ATF-2, a p38MAPK substrate also became increasingly phosphorylated over the repetitive stress period. Short interfering RNA that induced effective silencing of p38MAPK, was used to silence p38MAPK in V79 fibroblasts. Silencing of p38MAPK drastically hindered the elevation in catalase (protein and mRNA) levels observed after a single low dose (50 microM) of H2O. The rise in catalase mRNA levels induced by low concentration (single and multiple dose) H2O2 treatment was established to be unconnected with transcriptional upregulation but was brought forth primarily by an enhancement in catalase mRNA stability through the action of p38MAPK. Therefore, our data strongly indicate that activation of p38MAPK is a key controlling step in the upregulation of catalase levels by low dose H2O2 treatment.

Akt/PKB is a serine/threonine kinase, which controls vital cellular functions such as cell survival/apoptosis, cell cycle progression and glucose metabolism. Akt/PKB acts down-stream from growth factors and hormones and is a key mediator of their pro-survival, proliferative and metabolic effects. Akt/PKB carries out these diverse tasks through phosphorylation of a number of cellular substrates. The substrates of Akt/PKB, which promote the inhibition of apoptosis after being phosphorylated by Akt, include the Forkhead transcription factors and the Bcl-2 family member Bad. The cyclin dependent kinase inhibitors are substrates of Akt which when phosphorylated relinquish their inhibitory influence on cell cycle progression. Akt mediates many of the stimulatory effects of insulin on glucose metabolism through deactivation of glycogen synthase kinase, activation of phosphofructokinase, and modulation of glucose transporter activity. Consequently, Akt can be implicated in the pathological processes, which are associated with defects in regulation of apoptosis/survival and energy metabolism.

Platelet activation is normally induced by primary agonists such as adenosine diphosphate (ADP), thrombin, and collagen, whereas other agonists, such as epinephrine, can play important accessory roles. It is now reported that the macrophage-derived chemokine (MDC), thymus activation–regulated chemokine (TARC), and stromal cell–derived factor one (SDF-1) are highly effective activators of platelet function under a variety of conditions, stimulating platelet shape change, aggregation, and adhesion to collagen or fibrinogen. Chemokine-mediated platelet activation was rapid and maximal (less than 5 seconds) under arterial flow conditions and depended strongly on the presence of low levels of primary agonists such as ADP or thrombin. Concentrations of ADP (0.05-0.25 μM) or thrombin (0.005-0.02 U/mL) that induced minimal aggregation caused major aggregation acting in combination with the chemokines. The ability of apyrase to block chemokine-dependent aggregation or adhesion was consistent with an important role for ADP. Chemokine-stimulated aggregation was also insensitive to indomethacin, suggesting that the activation of cyclo-oxygenase is not involved. TARC, MDC, and SDF-1 increased intracellular calcium concentrations [Ca2+]iwhen combined with low levels of ADP. The MDC and TARC receptor CCR4 was expressed on platelets, and an anti-CCR4 antibody blocked aggregation induced by TARC or MDC. Treatment of platelets with SDF-1 and MDC rapidly exposed P-selectin (CD62P) on the cell surface but did not induce the secretion of serotonin. These findings suggest that the chemokines MDC, TARC, and SDF-1, which may be produced during inflammatory responses, coupled with low levels of ADP or thrombin, can serve as strong stimuli for activating platelet function.

Chaudhuri, S., and Raha, S. 2000. Inhibition and stimulation of growth of Entamoeba histolytica in culture: Association with PKC activity and protein phosphorylation. Experimental Parasitology 95, 28–35. We studied the role of protein kinase C (PKC) and protein threonine phosphorylation in the inhibition and stimulation of growth of the protozoan parasite Entamoeba histolytica. PKC was activated after serum deprivation in E. histolytica and during this period proteins became threonine phosphorylated. Conversely, on serum stimulation of serum-deprived cells, PKC activation was rapidly reversed and the threonine phosphorylation of proteins quickly declined. Growth of E. histolytica was not affected by either PKC inhibitors H-7 and GF109203X or by down-regulation of PKC by Phorbol 12-Myristate 13-Acetate (PMA). Interestingly, very low doses of PMA which caused activation of PKC and were unable to down-regulate PKC after 48 h of culture, negatively influenced the growth of E. histolytica. Serine/threonine phosphatase inhibitors Okadaic acid and Calyculin A drasti cally inhibited growth of E. histolytica. In conclusion, the growth of E. histolytica is not adversely affected by PKC down-regulation. On the contrary, growth inhibition of E. histolytica is associated with activation of Ca 2+ , Diacylglyceol (DAG)-dependent PKC, and threo nine phosphorylation of proteins.

Our aim was to study whether ultraviolet radiation produced any alterations in the subsequent signaling response of V79 fibroblasts to mitogenic stimulus. In ultraviolet C (UVC)-irradiated V79 fibroblasts, increase in cytosolic calcium in response to thrombin was nearly abolished in the presence of 3 mM external Ca 2+ . UVC-treated V79 cells showed a greatly enhanced permeability to Ca 2+ which was reversed by pretreatment with genistein, a tyrosine kinase inhibitor. Genistein also alleviated the inhibition of thrombin response caused by UVC. In UVC-treated cells, significant activation of protein kinase C (PKC) occurred only on exposure to 3 mM external calcium and PKC inhibitors (H-7 or staurosporine) reversed UVC-induced adverse effects on the thrombin response. Therefore, it is likely that protein tyrosine phosphorylation by UVC may play a role in the subsequent inhibition of thrombin response in V79 cells through increased calcium influx and activation of PKC.

V79 fibroblasts were treated with ultraviolet (UV) C radiation alone as well as in conjunction with chronic oxidative stress. The effects of these treatments on calcium signaling were observed at 30 min post-irradiation. In the absence of extracellular calcium, thrombin released calcium from internal stores of UVC-irradiated V79 fibroblasts even after exposure to neomycin. In neomycin-treated control and chronic oxidative stress cells, no calcium release by thrombin was observed after chelation of external calcium. Calcium release by thrombin from internal stores of UV-irradiated and neomycin-treated cells was completely abolished by pretreatment with N-acetyl cysteine and dexamethasone. Cellular total soluble thiol content which is a good indicator of cellular reduced glutathione (GSH) level was significantly elevated 30 min after ultraviolet radiation, indicating an adaptive response after oxidative stress. Chronic oxidative stress alone resulted in a much smaller increase in GSH but chronic oxidative stress in conjunction with UVC produced a very prominent elevation in GSH levels. Our data suggest that thrombin can cause calcium release from internal stores of ultraviolet-irradiated fibroblasts which is independent of phosphatidylinositol bisphosphate hydrolysis and is directly related to the level of oxidative stress. Involvement of phopholipase A2 and a role for its products as possible mediators of calcium release from intracellular stores, is strongly indicated. ((Mol Cell Biochem 196: 23-30, 1999)

[3H]Inositol tetrakisphosphate (Ins(1,3,4,5)P4) binding sites which were poorly displaced by unlabelled inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) were detected in membrane fractions of Entamoeba histolytica. Similarly, unlabelled Ins(1,3,4,5)P4 was 30-fold less efficient in displacing [3H]Ins(1,4,5)P3 binding. pH sensitivities of binding of the two isomers were markedly different. Scatchard analysis of the data revealed single binding sites and similar receptor densities for each of the two isomers. Formation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in E. histolytica was also demonstrated. Calcium release studies showed that after treatment with a saturating dose of either Ins(1,4,5)P3 or Ins(1,3,4,5)P4 the other inositol polyphosphate could partially revive the response to a subsequent addition of the first inducer. Our data clearly demonstrate that Ins(1,4,5)P3 and Ins(1,3,4,5)P4 are two equally important but independent second messengers in E. histolytica.

Calcium mobilisation from internal stores of the parasitic protozoan Entamoeba histolytica was studied by fluorescence measurements of the calcium indicator quin 2 and 45 Ca 2+ incorporation studies in saponin-permeabilised amoebae. Prior energy-dependent calcium sequestration was found to be necessary for subsequent release of calcium by inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ). Both Ins(1,4,5)P 3 and inositol 2,4,5-trisphosphate (Ins(2,4,5)P 3 ) could release calcium equally well from permeabilised E. histolytica with similar EC 50 (concentration which produced half maximal release) values for calcium release. Ins(1,4,5)P 3 -mediated calcium release occurred from a vesicular store, was sensitive to prior treatment by heparin and was attenuated by prior addition of a lower concentration of Ins(1,4,5)P 3 . cAMP failed to influence inositol trisphosphate induced calcium release, indicating the absence of control mechanisms through cAMP-dependent phosphorylation. GTP neither induced calcium release nor could potentiate inositol trisphosphate mediated calcium mobilisation. A saturating concentration of Ins(1,4,5)P 3 could release 50% of radiolabelled calcium sequestered by energy-dependent mechanisms in E. histolytica . The energy-dependent calcium sequestration was inhibited by vanadate and the calcium antagonist Diltiazem but not by dicyclohexylcarbodiimide (DCCD), suggesting the involvement of an endoplasmic reticulum-like structure in calcium storage. Binding studies showed specific association of [ 3 H]Ins(1,4,5)P 3 to crude membrane fractions of E. histolytica , which was significantly inhibited by heparin in a dose-dependent manner. IC 50 (concentration which produced half-maximal inhibition) values for displacement of radiolabelled Ins(1,4,5)P 3 binding by unlabelled Ins(1,4,5)P 3 and Ins(2,4,5)P 3 were estimated to be 0.99 μM for both isomers. Our results suggested that Ins(1,4,5)P 4 -mediated calcium release from internal stores of E. histolytica most probably occurred in an inositol trisphosphate receptor-dependent manner.

Three subfractions of human blood platelets differing in density and function exhibited also differences when EPR (electron paramagnetic resonance) spin label measurements and fluorescence polarization were performed. The membrane anisotropy in the plasma membranes was higher in low density platelets as compared with that of platelets of intermediate and higher densities. This higher plasma membrane anisotropy correlated with a significantly higher cholesterol-phospholipid (C:P) molar ratio in the plasma membranes of low density platelets. As compared with the other platelet subfractions the low density platelets exhibited the smallest cAMP increase after activation with the aggregation inhibitor prostaglandin (PGE1), and the highest percentage of inositol phosphate accumulation after thrombin stimulation. The results suggest a high correlation between functional parameters and the plasma membrane architecture of human blood platelets.

The hypothesis that ADP and thrombin liberate Ins(1,4,5)P3 in blood platelets, with kinetics consistent for releasing Ca2+ within 2s, was tested by quenched-flow techniques. Both agonists stimulated transient and equal synthesis of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 near 200 ms and later short-lived peaks, which were not correlated with the slower steady increase in intracellular [Ca2+] between 0.5 to 2 s detected by Indo-1. Shear forces alone caused transient liberation of these inositol phosphates within 0.5 s and up to 4 s, yet failed to increase intracellular [Ca2+].

Thrombin and adenosine diphosphate (ADP) are perhaps the two most important activators of platelet function (Stormorken, 1986). Both agents elicit a rapid “shape change” where platelet discoid shape is lest and pseudopodia are extended, followed by aggregation. Thrombin also induces massive granule secretion under a variety of conditions, while ADP is much less potent (Mustard et al., 1975). There is therefore considerable functional similarity for both agents and much research has been directed to understanding the biochemical basis for how they cause the initial events in signal transduction, leading to “shape change”, activation of the glycoprotein IIb/Illa receptor with binding of fibrinogen, and resulting platelet aggregation.

We have previously shown that adhesion of human platelets to immobilized collagen is extremely rapid, with initial rates approaching 3% of single particles adhering per 10 ms. Here, we have investigated adhesion efficiency to collagen as a function of platelet density. Platelet subpopulations: low-density (1.040 < d < 1.065 g/ml), intermediate-density (1.065 < d < 1.070 g/ml) and high-density (1.070 < d < 1.080 g/ml) were separated by Percoll density gradient centrifugation. They constituted 24%, 47% and 29% of the total platelet population and had mean volumes of 6.01, 7.37 and 8.21 fl, respectively. Using a continuous-flow, micro-affinity column, we found that the most dense (large) platelets exhibited initial rate of adhesion 4 times greater than the least dense (small) platelets. They were also less sensitive to inhibition by prostacyclin (PGI2). In contrast, there was no significant difference in aggregation induced by high doses of ADP and collagen, indicating that the most dense platelets were not preferentially involved in aggregation induced by high doses of agonists. These results suggest that normal circulating platelets can be distinctly heterogeneous in their ability to adhere to collagen under arterial-flow conditions. The greater efficiency of high-density platelets may be related to increased content of the glycoprotein Ia/IIa (GPIa/IIa) complex.