Clinicopathologic Dissociation: Robust Lafora Body Accumulation in Malin KO Mice Without Observable Changes in Home-cage Behavior.

Lafora Disease (LD) is a syndrome of progressive myoclonic epilepsy and cumulative neurocognitive deterioration caused by recessively inherited genetic lesions of EPM2A (laforin) or NHLRC1 (malin). Neuropsychiatric symptomatology in LD is thought to be directly downstream of neuronal and astrocytic polyglucosan aggregates, termed Lafora bodies (LBs), which faithfully accumulate in an age-dependent manner in all mouse models of LD. In this study, we applied home-cage monitoring to examine the extent of neurobehavioral deterioration in a model of malin-deficient LD, as a means to identify robust preclinical endpoints that may guide the selection of novel genetic treatments. At 6 weeks, ~6-7 months and ~12 months of age, malin deficient mice ("KO") and wild type (WT) littermates underwent a standardized home-cage behavioral assessment designed to non-obtrusively appraise features of rest/arousal, consumptive behaviors, risk aversion and voluntary wheel-running. At all timepoints, and over a range of metrics that we report transparently, WT and KO mice were essentially indistinguishable. In contrast, within WT mice compared across timepoints, we identified age-related nocturnal hypoactivity, diminished sucrose preference and reduced wheel-running. Neuropathological examinations in subsets of the same mice revealed expected age dependent LB accumulation, gliosis and microglial activation in cortical and subcortical brain regions. At 12 months of age, despite the burden of neocortical LBs, we did not identify spontaneous seizures during an electroencephalographic (EEG) survey, and KO and WT mice exhibited similar spectral EEG features. Using an in vitro assay of neocortical function, paroxysmal increases in network activity (UP states) in KO slices were more prolonged at 3 and 6 months of age, but were similar to WT at 12 months. KO mice displayed a distinct response to pentylenetetrazole, with a greater incidence of clonic seizures and a more pronounced post-ictal suppression of movement, feeding and drinking behavior. Together, these results highlight a stark clinicopathologic dissociation in a mouse model of LD, where LBs accrue substantially without clinically meaningful changes in overall wellbeing. Our findings allude to a delay between LB accumulation and neurobehavioral decline: one that may provide a window for treatment, and whose precise duration may be difficult to ascertain within the typical lifespan of a laboratory mouse.

Nuclear Inhibitor of Protein Phosphatase 1 (NIPP1) Regulates CNS Tau Phosphorylation and Myelination During Development.

Nuclear inhibitor of protein phosphatase 1 (NIPP1) is a known regulator of gene expression and plays roles in many physiological or pathological processes such as stem cell proliferation and skin inflammation. While NIPP1 has many regulatory roles in proliferating cells, its function in the central nervous system (CNS) has not been directly investigated. In the present study, we examined NIPP1 CNS function using a conditional knockout (cKO) mouse model in which the Nipp1 gene is excised from neural precursor cells. These mice exhibited severe developmental impairments that led to premature lethality. To delineate the neurological changes occurring in these animals, we first assessed microtubule-associated protein tau, a known target of NIPP1 activity. We found that phosphorylation of tau is significantly enhanced in NIPP1 cKO mice. Consistent with this, we found altered AKT and PP1 activity in NIPP1 cKO mice, suggesting that increased tau phosphorylation likely results from a shift in kinase/phosphatase activity. Secondly, we observed tremors in the NIPP1 cKO mice which prompted us to explore the integrity of the myelin sheath, an integral structure for CNS function. We demonstrated that in NIPP1 cKO mice, there is a significant decrease in MBP protein expression in the cortex, along with deficits in both the conduction of compound action potentials (CAP) and the percentage of myelinated axons in the optic nerve. Our study suggests that NIPP1 in neural precursor cells regulates phosphorylation of tau and CNS myelination and may represent a novel therapeutic target for neurodegenerative diseases.

Home-cage behavior in the Stargazer mutant mouse.

In many childhood-onset genetic epilepsies, seizures are accompanied by neurobehavioral impairments and motor disability. In the Stargazer mutant mouse, genetic disruptions of Cacng2 result in absence-like spike-wave seizures, cerebellar gait ataxia and vestibular dysfunction, which limit traditional approaches to behavioral phenotyping. Here, we combine videotracking and instrumented home-cage monitoring to resolve the neurobehavioral facets of the murine Stargazer syndrome. We find that despite their gait ataxia, stargazer mutants display horizontal hyperactivity and variable rates of repetitive circling behavior. While feeding rhythms, circadian or ultradian oscillations in activity are unchanged, mutants exhibit fragmented bouts of behaviorally defined "sleep", atypical licking dynamics and lowered sucrose preference. Mutants also display an attenuated response to visual and auditory home-cage perturbations, together with profound reductions in voluntary wheel-running. Our results reveal that the seizures and ataxia of Stargazer mutants occur in the context of a more pervasive behavioral syndrome with elements of encephalopathy, repetitive behavior and anhedonia. These findings expand our understanding of the function of Cacng2.

Development and validation of chemical kindling in adult zebrafish: A simple and improved chronic model for screening of antiepileptic agents.

BACKGROUND: Zebrafish has emerged as a potential animal model of acute convulsion for early screening of antiepileptic agents. There is a need for alternative chronic zebrafish models of epilepsy with more correlation to the clinical condition. NEW METHOD: Adult zebrafish were repeatedly exposed to subeffective concentrations of pentylenetetrazole (PTZ), until appearance to tonic-clonic seizures, considered as kindled. Valproic acid (VPA) exposure was given during kindling and in kindled fish in 2 different groups. The neurotransmitters level and expression of the genes associated with kindling were studied in the fish brain. RESULTS: There was an increase in seizure severity score at 1.25 mM concentration of PTZ, and 66.66 % of fish achieved kindling after 22 days' exposure. A marked increase in c-fos, crebbpa and crebbpbexpression, and glutamate/GABA level was observed in the brain of kindled fish. VPA inhibited the induction of PTZ-mediated kindling and reduced seizure severity in kindled fish. COMPARISON WITH EXISTING METHOD: In contrast to an existing adult zebrafish kindling method, the present protocol is of longer duration, with more similarity to clinical epilepsy. Moreover, the induction of kindling involves a simple non-invasive technique without the use of anesthesia. The protocol can be used for evaluation of both antiepileptic and antiepileptogenic agents. CONCLUSION: Repeated exposure of 1.25 mM PTZ induced kindling in zebrafish, altering the brain neurotransmitter levels and gene expression. Inhibition of kindling induction and decrease in seizures in normal and kindled fish, respectively by VPA validated application of the model for preclinical testing of agents against epilepsy.

Anticonvulsant action of a selective phosphatidylinositol-3-kinase inhibitor LY294002 in pentylenetetrazole-mediated convulsions in zebrafish.

The role of PI3K/AKT/mTOR signalling pathway has been widely deciphered in pathogenesis of epilepsy. LY294002 is a selective inhibitor of phosphatidylinositol-3-kinase (PI3K). The present study was designed to explore the anticonvulsant potential of LY294002 in a zebrafish model of pentylenetetrazole (PTZ)-mediated convulsions. Zebrafish larvae at 7dpf (days post fertilization) were pre-incubated with varying concentrations of LY294002, prior to PTZ exposure. The adult zebrafish were also exposed to PTZ after intraperitoneal injection with different concentrations of LY294002, followed by gene expression studies in the brain. The hyperactive responses indicated by total distance travelled and the mean speed of larva was drastically decreased, whereas latency to first clonic-like seizure was increased following LY294002 pre-treatment. Additionally, a marked decrease in c-fos expression was also observed in the larvae group exposed to LY294002 in comparison to control. Furthermore, PTZ evoked seizure severity was considerably decreased, while latency to clonic-like seizure was increased in adult zebrafish group treated with 100 nM of LY294002. Furthermore, the occurrence of tonic-like seizures was also reduced in the adult zebrafish treated with LY294002. The mRNA levels of PIK3CA, PIK3R1, AKT1, mTOR, Rps6 and Rps6kb1 in adult zebrafish brain was significantly reduced as compared to vehicle control group. Our results provided conclusive support for the anticonvulsant potential of LY294002.

Spontaneous Recurrent Seizures Mediated Cardiac Dysfunction via mTOR Pathway Upregulation: A Putative Target for SUDEP Management.

BACKGROUND: Alteration in electrophysiology, leading to cardiac dysfunction and subsequently a nontraumatic death is a complication of epilepsy known as "SUDEP" (Sudden Unexpected Death in Epilepsy). AIMS: The present study was designed to understand the molecular changes and cardiac parameters during different phases of epileptogenesis in lithium-pilocarpine (Li-pilo) rat model of epilepsy. METHODS: The animals were exposed to Li-pilo to induce Spontaneous Recurrent Seizures (SRS). Noninvasive blood pressure and electrocardiography was recorded at 7th, 28th and 75th day following pilocarpine administration, considered as latent, initial and late SRS phases, respectively. The serum biochemistry, cardiac histopathology, protein and mRNA expressions were studied, following electrocardiography on day 75. RESULTS: The mean arterial pressure decreased during the latent phase, thereafter it progressively increased during the initial and the late SRS phases, as compared to the basal and the latent phase. Histopathological analysis of the heart sections indicated hypertrophy, degenerative changes and fibrous tissue deposition in epileptic animals, along with increased levels of lactate dehydrogenase and creatine kinase-MB in the serum. The expression of HIF-1α, phospho-S6, phospho-mTOR, TGF-ß, collagen I and Na+/K+-ATPase α1 proteins, and mRNA levels of HIF-1α, mTOR, Rps6, Scn1b, Scn3b, Nav1.5 and TGF-ß were increased in the cardiac tissue of epileptic animals, as compared to control. CONCLUSION: Our results conclusively showed that Li-pilo-induced SRS leads to cardiac dysfunction via mTOR pathway upregulation, thus suggested the regulatory control of mTOR pathway as a potential target for SUDEP management.

Target identification, screening and in vivo evaluation of pyrrolone-fused benzosuberene compounds against human epilepsy using Zebrafish model of pentylenetetrazol-induced seizures.

Pyrrolone-fused benzosuberene (PBS) compounds were semi-synthesized from α,ß,γ-Himachalenes extracted from the essential oil of Cedrus deodara following amino-vinyl-bromide substituted benzosuberenes as intermediates. These PBSs compounds classified as an attractive source of therapeutics. The α-isoform of PI3K which is a pivotal modulator of PI3K/AKT/mTOR signaling pathway, responsible for neurological disorders like epilepsy, found as a potential target molecule against these 17 semi-synthesized PBS compounds using in silico ligand-based pharmacophore mapping and target screening. The compounds screened using binding affinities, ADMET properties, and toxicity that were accessed by in silico docking simulations and pharmacokinetics profiling. Ultimately two compounds viz., PBS-8 and PBS-9 were selected for further in vivo evaluation using a zebrafish (Danio rerio) model of pentylenetetrazol (PTZ)-induced clonic convulsions. Additionally, gene expression studies performed for the genes of the PI3K/AKT/mTOR pathway which further validated our results. In conclusion, these findings suggested that PBS-8 is a promising candidate that could bedeveloped as a potential antiepileptic.

Early α-linolenic acid exposure to embryo reduces pentylenetetrazol-induced seizures in zebrafish larva.

Over the past few years, there has been a tremendous increase in interest of general population toward food-based therapies for management of chronic clinical conditions due to their lesser adverse effects with prolonged use over pharmacotherapies. Foods enriched with omega-3 fatty acids have shown some promising results in case of epilepsy. The present study was envisioned to investigate the effect of early exposure of α-linolenic acid (ALA), an essential omega-3 fatty acid in developing zebrafish (Danio rerio) embryos toward pentylenetetrazol (PTZ)-induced seizure susceptibility. The healthy wild-type zebrafish embryos were incubated in system water or system water containing different ALA concentrations (1-20 µM) till 7 dpf (days post fertilization). Each larva at 7 dpf was placed in 8 mM PTZ solution and seizure event was recorded. ALA incubation at 10 µM and 20 µM concentrations showed a dose-dependent reduction in PTZ-mediated hyperactive responses in larvae indicated by a marked decrease in total distance travelled and speed, as compared to vehicle control. Furthermore, both the treated groups showed increase in the latency to PTZ-induced clonus-like seizures in larvae, as compared to vehicle control. ALA incubated larvae at 10 µM and 20 µM concentrations also showed a significant reduction in c-fos mRNA level. A marked increase in the level of ALA and docosahexaenoic acid was also observed in the larvae incubated at highest effective concentration of ALA. The present study concluded that embryonic exposure of ALA reduced PTZ-induced seizures in zebrafish larva.

Mycophenolate mofetil contributes to downregulation of the hippocampal interleukin type 2 and 1ß mediated PI3K/AKT/mTOR pathway hyperactivation and attenuates neurobehavioral comorbidities in a rat model of temporal lobe epilepsy.

The role of neuroinflammatory mediators has been well established in the pathogenesis of temporal lobe epilepsy (TLE) and associated neurobehavioral comorbidities. Mycophenolate mofetil (MMF) is commonly used as an immunosuppressant in organ transplantations. Its neuroprotective effect is well explored in different preclinical and clinical studies. The present study was designed to investigate the effect of MMF in rat model of lithium pilocarpine (LiPc)-induced spontaneous recurrent seizures and its associated neurobehavioral comorbidities. MMF treatment showed a dose-dependent decrease in seizure severity and reduced aggression in epileptic rats. There was marked improvement in spatial and recognition memory functions, along with substantial decrease in depression-like behavior in MMF treated epileptic rats. There was considerable decrease in mossy fiber sprouting in the dentate gyrus and the cornu ammonis 3 regions of the hippocampus, along with reduction in neuronal death in the treated groups. Furthermore, the hippocampal mRNA level of IL-1ß, IL-2, PI3K, AKT, HIF-1α, RAPTOR, mTOR, Rps6kb1 and Rps6 was found to be decreased in MMF treated animals. mTOR, S6, pS6 and GFAP protein expression was decreased, whereas NeuN was increased in the rat hippocampus of the treated animals. The results concluded that MMF suppress recurrent seizures, and improves its associated behavioral impairments and cognitive deficit in rat model of TLE. The observed effects of MMF be correlated with the inhibition of IL-2 and IL-1ß linked PI3K/AKT/mTOR signaling pathway hyperactivation.

Ginkgo biloba L. attenuates spontaneous recurrent seizures and associated neurological conditions in lithium-pilocarpine rat model of temporal lobe epilepsy through inhibition of mammalian target of rapamycin pathway hyperactivation.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba L. (Ginkgoaceae) has been widely used in traditional medicine for variety of neurological conditions particularly behavioral and memory impairments. AIM OF THE STUDY: The present study was envisaged to explore the effect of a standardized fraction of Ginkgo biloba leaves (GBbf) in rat model of lithium-pilocarpine induced spontaneous recurrent seizures, and associated behavioral impairments and cognitive deficit. MATERIALS AND METHODS: Rats showing appearance of spontaneous recurrent seizures following lithium pilocarpine (LiPc)-induced status epilepticus (SE) were treated with different doses of GBbf or vehicle for subsequent 4 weeks. The severity of seizures and aggression in rats were scored following treatment with GBbf. Further, open field, forced swim, novel object recognition and Morris water maze tests were conducted. Histopathological, protein levels and gene expression studies were performed in the isolated brains. RESULTS: Treatment with GBbf reduced seizure severity score and aggression in epileptic animals. Improved spatial cognitive functions and recognition memory, along with reduction in anxiety-like behavior were also observed in the treated animals. Histopathological examination by Nissl staining showed reduction in neuronal damage in the hippocampal pyramidal layer. The dentate gyrus and Cornu Ammonis 3 regions of the hippocampus showed reduction in mossy fiber sprouting. GBbf treatment attenuated ribosomal S6 and pS6 proteins, and hippocampal mTOR, Rps6 and Rps6kb1 mRNA levels. CONCLUSIONS: The results of present study concluded that GBbf treatment suppressed lithium-pilocarpine induced spontaneous recurrent seizures severity and incidence with improved cognitive functions, reduced anxiety-like behavior and aggression. The effect was found to be due to inhibition of mTOR pathway hyperactivation linked with recurrent seizures.

Crocin Attenuates Kindling Development and Associated Cognitive Impairments in Mice via Inhibiting Reactive Oxygen Species-Mediated NF-κB Activation.

Crocin is a pharmacologically active carotenoid pigment mainly present in the stigmas of Crocus sativus L. (Iridaceae). It has been well explored in experimental animal models of cognitive impairments, depression, anxiety and epilepsy. This study was designed to understand the effect of crocin on pentylenetetrazol (PTZ)-induced kindling development and its associated cognitive deficit in mouse. Crocin treatment at 5, 10 and 20 mg/kg p.o. doses showed a marked reduction in severity of PTZ-induced seizures. There was an increase in novel object preference index and discrimination ratio in the crocin-treated groups in the novel object recognition test. Its treatment also increased percentage spontaneous alternations in T-maze test at all the tested doses. Histopathological examination by Nissl staining showed a reduction in dark neurons in the hippocampal pyramidal layer of crocin-treated animals in contrast to vehicle control, indicating a decrease in neuronal damage. Biochemical estimations showed a significant increase in superoxide dismutase activity and reduced reactive oxygen species (ROS) in the hippocampus of crocin-treated animals. Immunohistochemistry results revealed attenuation in the levels of nuclear factor-κB (NF-κB) and phosphorylated NF-κB in the hippocampal sections of crocin-treated animals. The results of this study concluded that crocin treatment increased seizure threshold, thus inhibiting PTZ-induced kindling development and improving cognitive functions. The effect was found to be due to suppression of seizure-induced ROS generation and its linked NF-κB pathway-associated neuronal damage.

Anticancer Mammalian Target of Rapamycin (mTOR) Signaling Pathway Inhibitors: Current Status, Challenges and Future Prospects in Management of Epilepsy.

The role of phosphatidylinositol 3-kinase linked mammalian target of rapamycin (mTOR) pathway hyperactivation is well established in cancer pathogenesis. Several molecules inhibiting mTOR pathway, leading to inhibition of protein synthesis responsible for angiogenesis of tumor cells have emerged out to be potential anticancers. Similar hyperactivation of mTOR pathway has also reported in epilepsy during latent phase, following precipitating injury causing reorganization of neuronal networks and ultimately leading to induction of seizures. The mTOR inhibitors have also found to attenuate pathological changes in the brain associated with epilepsy, primarily suppression of mossy fiber sprouting. At the same time, a few antiepileptic molecules which have been studied against cancer showed anticancer activity, apart from their principal mechanism of action. These studies suggest mTOR signaling pathway to be a common pathogenic link between cancer and epilepsy. It has been found that, anticancer molecules acting on different molecular targets, that ultimately down regulate the expression of mTOR, can also be used in case of epilepsy to reduce its hyperactivation. There are several unexplored anticancer molecules that act by inhibiting mTOR directly or indirectly available which can be explored as antiepileptic in future. Majority of the molecules which are tested as anticancer do not reach the final phases of clinical trials due to less potency and efficacy, and ultimately a few of them reach the market. Since a lot of experimental/safety studies have already been conducted on such molecules, hence it is worthwhile to test these molecules for other disorders that share common pathogenic pathway like epilepsy, provided their pitfalls have been addressed, as proposed in the present review.

Dwindling of cardio damaging effect of isoproterenol by Punica granatum L. peel extract involve activation of nitric oxide-mediated Nrf2/ARE signaling pathway and apoptosis inhibition.

Punica granatum L. (Punicaceae) peel is often considered as a food waste in-spite of its high bioactive metabolite composition. Primarily it is rich in therapeutically active phenolics that act on multiple cellular sites, through diverse mechanisms. Hence, the present study was envisaged to investigate the effect of standardised peel extract of P. granatum against isoproterenol (ISO)-induced myocardial infarction (MI). ISO administration at a dose of 150 mg/kg; s.c., twice at 24 h interval resulted in electrocardiographic abnormalities with increased heart weight and myocardial tissue damage signifying MI. Pretreatment with the extract at 50, 100 and 200 mg/kg; p.o., for 21 days prior to ISO intoxication (30 min prior to intoxication on day 22 and 23) attenuated the observed changes, along with increased myocardial tissue superoxide dismutase activity, reduced glutathione and nitrite levels, and decreased lipid peroxidation. The extract treated groups also showed reduced serum marker enzymes of MI, showing maximum effect at highest tested dose. Immunohistochemical studies revealed increased myocardial expression of nuclear factor erythroid 2-related factor 2 (Nrf2), endothelial nitric oxide synthase (eNOS) and Bcl-2 proteins in the extract treated groups with decreased Bax expression. From the results it can be concluded that the extract pretreatment prevents ISO-induced MI through increased myocardial expression of eNOS, leading to nitric oxide-mediated Nrf2 activation, thus upregulating antioxidant mechanisms, along with inhibition of apoptosis.

Localization and expression of a 70 kDa protein in goat spermatozoa having Na(+),K(+)-ATPase inhibitory and arylsulphatase A activities.

We have previously isolated and purified a goat sperm protein of 70 kDa molecular weight designated as P70 and characterized it as an inhibitor of Na(+),K(+)-ATPase. Our study reveals that the first 10 amino acid residues from the N-terminal end of P70 has high degree of homology with arylsulphatase A from mice, pig and human. Indirect immunofluorescence study shows the presence of the protein on goat sperm surface. Furthermore, live goat sperm and the extract of peripheral sperm plasma membrane proteins exhibit arylsulphatase A's desulphation activity. The P70 remains on the head surface of in vitro capacitated cauda epididymal sperm as shown by positive immunofluorescence staining of cauda sperm. Immunoblot and flow cytometric studies corroborate the above findings. The presence of P70 on capacitated cauda sperm surface suggest a possible role of this protein in sperm zona pellucida binding. In the present report we demonstrate arylsulphatase A like activity in P70 and describe its localization and expression in goat sperm.

Induction of apoptosis in cells expressing exogenous Hippi, a molecular partner of huntingtin-interacting protein Hip1.

To decipher the pathway of apoptosis induction downstream to caspase-8 activation by exogenous expression of Hippi, an interactor of huntingtin-interacting protein Hip1, we studied apoptosis in HeLa and Neuro2A cells expressing GFP-tagged Hippi. Nuclear fragmentation, caspase-1, caspase-8, caspase-9/caspase-6 and caspase-3 activation were increased significantly in Hippi expressing cells. Cleavage of Bid, release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria were also increased in GFP-Hippi expressing cells. It was observed that caspase-1 and caspase-8 activation was earlier than caspase-3 activation and nuclear fragmentation. Expression of caspase-1, caspase-3 and caspase-7 was increased while anti-apoptotic gene Bcl-2 and mitochondrial genes ND1 and ND4 were reduced in Hippi expressing cells. Besides, the expression SDHA and SDHB, nuclear genes, subunits of mitochondrial complex II were decreased in GFP-Hippi expressing cells. Taken together, we concluded that Hippi expression induced apoptosis by releasing AIF and cytochrome c from mitochondria, activation of caspase-1 and caspase-3, and altering the expression of apoptotic genes and genes involved in mitochondrial complex I and II.

Fas-, caspase 8-, and caspase 3-dependent signaling regulates the activity of the aminophospholipid translocase and phosphatidylserine externalization in human erythrocytes.

Apoptosis and erythrocyte senescence share the common feature of exposure of phosphatidylserine (PS) in the outer leaflet of the cells. Western analysis showed that mature red cells contain Fas, FasL, Fas-associated death domain (FADD), caspase 8, and caspase 3. Circulating, aged cells showed colocalization of Fas with the raft marker proteins Galpha(s) and CD59; the existence of Fas-associated FasL, FADD and caspase 8; and caspase 8 and caspase 3 activity. Aged red cells had significantly lower aminophospholipid translocase activity and higher levels of PS externalization in comparison with young cells. In support of our contention that caspases play a functional role in the mature red cell, the oxidatively stressed red cell recapitulated apoptotic events, including translocation of Fas into rafts, formation of a Fas-associated complex, and activation of caspases 8 and 3. These events were independent of calpain but dependent on reactive oxygen species (ROS) as evident from the effects of the ROS scavenger N-acetylcysteine. Caspase activation was associated with loss of aminophospholipid translocase activity and with PS externalization. ROS was not generated by treatment of cells with t-butyl hydroperoxide at 10 degrees C, and Fas did not translocate into rafts. Concomitantly, neither formation of a Fas-associated signaling complex nor caspase activation could be observed, supporting the view that translocation of Fas into rafts was the trigger for the chain of events leading to caspase 3 activation. Our data demonstrate for the first time the novel involvement of Fas/caspase 8/caspase 3-dependent signaling in an enucleated cell leading to PS externalization, a central feature of erythrophagocytosis and erythrocyte biology.

Entamoeba histolytica: Characterization of human collagen type I and Ca2+ activated differentially expressed genes.

Earlier it was demonstrated that the Entamoeba histolytica trophozoites, when incubated with human collagen and Ca2+, expressed and released the collagenolytic activity [Munoz, M.L., Calderon, J., Rojkind, M., 1982. The collagenase of Entamoeba histolytica. Journal of Experimental Medicine 155, 42-51], a virulence factor involved in the pathogenesis of amoebiasis. In this study, attempts have been made to identify and characterize the gene(s) that are upregulated by the human collagen type I and Ca2+ interaction. A comparative evaluation of gene expression pattern of the parasite before and after treatment with human collagen type I was done using the differential display reverse transcription-PCR technique. The cDNA fragments that were overexpressed in collagen treated trophozoites compared to collagen untreated trophozoites were characterized. Northern blot hybridization and RT-PCR amplification using gene-specific primers validated the differential expression. Sequence analyses and database searches revealed homology with known virulence factor genes of E. histolytica such as amoebapore C and cysteine proteinase 5, along with stress-induced protein HSP70, and ribosomal protein L27a (known to be involved in protein synthesis). The study provides the experimental evidence that interaction of E. histolytica with human collagen type I and Ca2+ triggers the transcriptional activation of at least two important genes responsible for pathogenesis of amoebiasis.