Molecular mechanism of Enteroaggregative Escherichia coli induced apoptosis in cultured human intestinal epithelial cells.

Background. Enteroaggregative Escherichia coli (EAEC) is an evolving etiological agent of acute and persistent diarrhoea worldwide. The previous study from our laboratory has reported the apoptosis-inducing activity of EAEC in human small intestinal and colonic epithelial cell lines. In the present investigation, we have explored the underlying mechanism of EAEC-induced apoptosis in human intestinal epithelial cell lines.Methods. INT-407 and HCT-15 cells were infected with EAEC-T8 and EAEC-pT8 (plasmid cured strain of EAEC-T8) separately. Cells cultured in the absence of bacteria served as a negative control in all the experiments. For the subsequent experiments, the molecular mechanism(s) of epithelial cell aposptosis was measured in EAEC infecting both the cell lines by flow cytometry, real-time PCR and Western blotting.Results and conclusions. EAEC was found to activate the intrinsic/mitochondrial apoptotic pathway in both the cell lines through upregulation of pro-apoptotic Bax and Bak, un-alteration/reduction in the level of anti-apoptotic Bcl-2 and Bcl-XL, decrease in mitochondrial transmembrane potential, accumulation of cytosolic cytochrome c leading to activation of procaspase-9 and procaspase-3, which ultimately resulted in DNA fragmentation and apoptosis. Further, an increased expression of Fas, activation of procaspase-8 and upregulation of pro-apoptotic Bid in the EAEC-infected cells indicated the involvement of extrinsic apoptotic pathway too in this process. Our finding has undoubtedly led to an increased understanding of EAEC pathogenesis, which may be helpful to develop an improved strategy to combat the infection.

Enteroaggregative Escherichia coli induced activation of epidermal growth factor receptor contributes to IL-8 secretion by cultured human intestinal epithelial cells.

Enteroaggregative Escherichia coli (EAEC) has been identified as a new enteropathogen that causes acute and chronic diarrhea in children and travelers. One defining aspect of EAEC-pathogenesis is the induction of an inflammatory response in intestinal epithelium. In this study, we have found that EAEC-induced EGFR activation in human small intestinal and colonic epithelial was attenuated in the presence of a specific inhibitor of EGFR (Tyrphostin AG1478). Further, the aggregative stacked-brick type of adherence of this organism to both the cell lines and this pathogen-induced cytoskeletal rearrangement of these cells was also reduced in the presence of Tyrphostin AG1478. Moreover, EAEC-induced activation of downstream effectors (ERK-1/2, PI3K and Akt) of EGFR mediated cell signaling pathways were found to be suppressed in the presence of EGFR inhibitor. A decrease in IL-8 response in EAEC infected both the cell types were also noted in the presence of specific inhibitors of these downstream effectors, transcription factors and Tyrphostin AG1478. We propose that EAEC-induced activation of EGFR is quintessential for stacked-brick adherence of EAEC to human intestinal epithelial cells, their cytoskeletal rearrangements and stimulation of ERK-1/2 and PI3K/Akt mediated signal transduction pathways, resulting in the activation of NF-κB, AP-1, STAT-3 and finally IL-8 secretion by these cells.

Role of TLRs in EGFR-mediated IL-8 secretion by enteroaggregative Escherichia coli-infected cultured human intestinal epithelial cells.

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen associated with persistent diarrhea in travelers, immunocompromised patients and children worldwide. However, the pathogenesis of this organism is yet to be established. In this study, the role of Toll-like receptors (TLRs) was evaluated in epidermal growth factor receptor (EGFR)-mediated IL-8 secretion by EAEC-infected human small intestinal and colonic epithelial cells (INT-407 and HCT-15, respectively). We observed that EAEC-induced upregulation of TLR2, TLR4 and TLR5 transcripts in both types of cells, and the maximum level of these transcripts was seen in cells infected with EAEC-T8 (an invasive clinical isolate). All these TLRs made a significant contribution to the EAEC-T8-mediated EGFR activation in these cells. Furthermore, these TLRs were found to be associated with activation of the downstream effectors (ERK-1/2, PI3 kinase and Akt) and transcription factors (NF-κB, c-Jun, c-Fos and STAT-3) of EGFR-mediated signal transduction pathways. Moreover, the involvement of these TLRs was also noted in IL-8 secretion by both EAEC-T8-infected cell types. Our findings suggest that EAEC-induced upregulation of TLR2, TLR4 and TLR5 is important for the IL-8 response via EGFR-mediated signal transduction pathways in these cells.

Role of ST6GAL1 and ST6GAL2 in subversion of cellular signaling during enteroaggregative Escherichia coli infection of human intestinal epithelial cell lines.

Emerging evidence have suggested that aberrant sialylation on cell-surface carbohydrate architecture may influence host-pathogen interactions. The α2,6-sialyltransferase (ST) enzymes were found to alter the glycosylation pattern of the pathogen-infected host cell-surface proteins, which could facilitate its invasion. In this study, we assessed the role of specific α2,6-ST enzymes in the regulation of enteroaggregative E. coli (EAEC)-induced cell signaling pathways in human intestinal epithelial cells. EAEC-induced expression of α2,6-ST family genes in HCT-15 and INT-407 cell lines was assessed at mRNA level by qRT-PCR. Specific esi-RNA was used to silence the target ST-gene in each of the EAEC-infected cell type. Subsequently, the role of these enzymes in regulation of EAEC-induced cell signaling pathways was unraveled by analyzing the expression of MAPkinases (ERK1/2, p38, JNK) and transcription factors (NFκB, cJun, cFos, STAT) at mRNA and protein levels by qRT-PCR and western immunoblotting, respectively, expression of selected sialoglycoproteins by western immunoblotting along with the secretory IL-8 response using sandwich ELISA. ST6GAL-1 and ST6GAL-2 were efficiently silenced in EAEC-infected HCT-15 and INT-407 cells, respectively. Significant reduction in EAEC-induced activation of MAPKs, transcription factors, sialoglycoproteins, and IL-8 secretion was noted in ST-silenced cells in comparison to the respective control cells. We propose that ST6GAL-1 and ST6GAL-2 are quintessential for EAEC-induced stimulation of MAPK-mediated pathways, resulting in activation of transcription factors, leading to an inflammatory response in the human intestinal epithelial cells. Our study may be helpful to design better therapeutic strategies to control EAEC- infection. KEY POINTS: • EAEC induces α2,6-sialyltransferase (ST) upregulation in intestinal epithelial cells • Target STs (ST6GAL-1 & ST6GAL-2) were efficiently silenced using specific esiRNAs • Expression of MAPKs, transcription factors & IL-8 was reduced in ST silenced cells.

Role of a Disease-associated ST3Gal-4 in Non-small Cell Lung Cancer.

Sialylation promotes tumorigenesis by affecting various cancer-related events, including apoptosis inhibition, cell growth, invasion, migration, metastasis, chemo-resistance, and immunomodulation in favor of tumor progression. An altered expression of sialyltransferase enzymes is responsible for synthesizing various tumor-associated sialylated structures. In the present study, our findings have revealed a significant up-regulation of ST3Gal-4 transcript in the two major subtypes of NSCLC cell lines [squamous cell carcinoma cell line (NCI-H520) and adenocarcinoma cell line (A549)]. Thus, the role of the ST3Gal-4 gene was assessed on cancer-associated signal transduction pathways in these cells in view of proliferation, invasion, and migration. ST3Gal-4 was silenced by transfection of both the cell lines with esi-ST3Gal-4-RNA, which RT-PCR and western immunoblotting confirmed. Silencing of ST3Gal-4 resulted in a decreased expression of MAL-I interacting membrane-HSP60, identified earlier as an α2,3-sialylated glycoprotein, thus pointing towards the possible role of ST3Gal-4 in its sialylation. The proliferation, invasion, and migration of both types of NSCLC cells were reduced significantly in the ST3Gal-4 silenced cells. Our findings were substantiated by the down-regulation of ß-catenin and E-cadherin, a reduced expression of activated AKT1, ERK1/2, and NF-ƙB in these cells. We propose that ST3Gal-4 may be the disease-associated sialyltransferase involved in α2,3 sialylation of the membrane proteins, including HSP60 of the NSCLC cells. This may lead to the conformational alteration of these proteins, required for the activation of E-cadherin/ß-catenin, AKT, and ERK/NF-ƙB mediated signal transduction pathways in these cells, resulting in their proliferation, invasion, and migration.

Enteroaggregative Escherichia coli induces altered glycosylation in membrane proteins of cultured human intestinal epithelial cells.

Emerging evidences have suggested that pathogens are capable of manipulating the glycosylation pattern of host-cell glycoconjugates, which may promote their attachment to these cells. Several enteric pathogens are known to induce such altered glycosylation in intestinal epithelium thereby, facilitating the disease process. Enteroaggregative Escherichia coli (EAEC), is one of such pathogens, known to cause acute and persistent diarrhea worldwide. However, glycosylation modulation due to EAEC infection has not been explored so far. In this study, EAEC-induced glycosylation changes in membrane proteins of human small-intestinal and colonic epithelial cell lines were found as revealed by lectin-overlay transblotting using four lectins, among which Sambucus nigra agglutinin (SNA) was selected for subsequent experiments. Several differentially expressed membrane-proteins were detected on SNA-overlay transblots following 2D-PAGE and identified by MALDI-TOF/TOF mass spectrometric analysis. Among these, voltage-dependent anion-selective channel-protein 2 (VDAC2) and prohibitin 2 (PHB2), common to both the cell lines were chosen for further characterization. Reactivity of these proteins to SNA was substantiated by their presence in SNA-agarose affinity chromatography eluted fractions. The plasma membrane localization of VDAC2 and PHB2 in EAEC infected cell lines was validated by confocal microscopy. These proteins were characterized as sialoglycoproteins by SNA-overlay transblots in presence a specific SNA inhibitor i.e., 6'sialyl lactose and deglycosylation using PNGase F, O-glycosidase and neuraminidase. Membrane localization of these sialoglycoproteins was found to facilitate EAEC adherence to human intestinal epithelial cells. SIGNIFICANCE: Our findings regarding EAEC induced altered glycosylation pattern of host cell membrane proteins may help in better understanding of the disease pathogenesis.

Identification and characterization of non-small cell lung cancer associated sialoglycoproteins.

Aberrantly sialylated cellular glycoconjugates were found to be involved in different processes during tumorigenesis. Such alteration was also noted in case of lung cancer, an important cause of cancer-related death throughout the world. Thus, study on lung cancer associated sialoglycoproteins is of paramount relevance to have a deeper insight into the mechanism of the disease pathogenesis. In the present study, sialic acid specific lectin (Maackia amurensis agglutinin and Sambcus nigra agglutinin)-based affinity chromatography followed by 2D-PAGE and MALDI-TOF/TOF mass spectrometric analysis were done to explore the disease-associated serum proteins of squamous cell carcinoma and adenocarcinoma [the major two subtypes of NSCLC (non-small cell lung carcinoma)] patients. Among seven identified proteins, α1-antitrypsin and haptoglobin-ß were preferred for further studies. These two proteins were characterized as the disease associated serum-sialoglycoproteins of NSCLC-patients by western immunoblotting using each lectin specific inhibitor. The presence of these sialoglycoproteins was found on NSCLC cell lines (NCI-H520 & A549) by confocal microscopy. Both these proteins were also present in tissue samples of NSCLC origin and involved in proliferation, invasion and migration of NSCLC cells. Our findings suggest that α1-antitrypsin and haptoglobin-ß may be the disease-associated sialoglycoproteins in NSCLC, which seem to be involved in disease progression. SIGNIFICANCE: Our contribution regarding the identification of the NSCLC associated sialoglycoproteins may provide a new vision towards the development of clinically useful newer strategies for the treatment of this disease.

Intracranial Aneurysm Biomarker Candidates Identified by a Proteome-Wide Study.

The scientific basis of intracranial aneurysm (IA) formation, its rupture and further development of cerebral vasospasm is incompletely understood. Aberrant protein expression may drive structural alterations of vasculature found in IA. Deciphering the molecular mechanisms underlying these events will lead to identification of early detection biomarkers and in turn, improved treatment outcomes. To unravel differential protein expression in three clinical subgroups of IA patients: (1) unruptured aneurysm, (2) ruptured aneurysm without vasospasm, (3) ruptured aneurysm who developed vasospasm, we performed untargeted quantitative proteomic analysis of aneurysm tissue and serum samples from three subgroups of IA patients and control subjects. Candidate molecules were then validated in a larger cohort of patients using enzyme-linked immunosorbent assay. A total of 937 and 294 proteins were identified from aneurysm tissue and serum samples, respectively. Several proteins that are known to maintain structural integrity of vasculature were found to be dysregulated in the context of aneurysm. ORM1, a glycoprotein, was significantly upregulated in both tissue and serum samples of unruptured aneurysm patients. We employed a larger cohort of subjects (n = 26) and validated ORM1 as a potential biomarker for screening of unruptured aneurysms. Samples from ruptured aneurysms with vasospasm showed significant upregulation of MMP9, a protease, compared with ruptured aneurysms without vasospasm. We validated MMP9 as a potential biomarker for vasospasm in a larger cohort (n = 52). This study reports the first global proteomic analysis of the entire clinical spectrum of IA. Furthermore, this study suggests ORM1 and MMP9 as potential biomarkers for unruptured aneurysm and cerebral vasospasm, respectively.

Heat shock protein 60 is a disease-associated sialoglycoprotein in human non-small cell lung cancer.

The diagnostic and therapeutic potential of Maackia amurensis agglutinin (MAA) have been reported in various malignancies. Earlier, we have found that MAA specifically interacted with human non-small cell lung-cancer (NSCLC) cells and induced apoptosis in these cells. The present study was designed to identify M. amurensis leukoagglutinin (MAL-I, one of the components of MAA, having the same carbohydrate specificity as MAA) interacting membrane sialoglycoprotein(s) of two subtypes of human NSCLC cell lines. Nine proteins were identified using two-dimensional (2D)-polyacrylamide gel electrophoresis (PAGE) followed by MAL-I-overlay transblotting and matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). Among these proteins, HSP60 was selected for further characterization. The sialoglycoprotein nature of membrane-HSP60 of NSCLC cell lines was confirmed by its reduced reactivity with MAL-I in Western blots in the presence of GM2 and by dual staining of the cell lines with MAL-I and HSP60-antibody. These findings were further substantiated by enzymatic analysis of membrane-HSP60 as well as in-silico evidence regarding this protein. Our observations were validated by immunohistochemical analysis of both subtypes of NSCLC tissue sections. Membrane-HSP60 was found to be involved in the inhibition of MAL-I-induced morphological alteration of NSCLC cells and also in the proliferation and migration of these cells, indicating the probable role of sialylated membrane-HSP60 in this disease.

Normal and reconstituted high-density lipoprotein protects differentiated monocytes from oxidized low-density lipoprotein-induced apoptosis.

BACKGROUND: The progression of atherosclerosis is an ongoing struggle between cell division and cell death. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a novel receptor for oxidized low-density lipoprotein (ox-LDL), mediates ox-LDL-induced apoptosis of monocytes. The anti-atherogenic function of plasma high-density lipoprotein (HDL) includes the ability to inhibit apoptosis of macrophage, although the exact mechanism and consequences of apoptosis in the development and progression of this disease are still controversial. Thus, in the present study, the effect of normal HDL (nHDL) and reconstituted HDL (rHDL) on ox-LDL-induced cellular responses in differentiated monocytes in view of apoptosis and LOX-1 receptor expression was investigated. METHODS: The expression of B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), caspase-3, and cytochrome c (cyt c) was assessed and substantiated in 30 hyper-LDL and control subjects. To assess the expression of LOX-1 on differentiated THP-1 cells, western blotting was carried out, followed by statistical analysis in 30 patients and control subjects. RESULTS: nHDL/rHDL inhibited the ox-LDL-induced apoptosis in the differentiated human monocytic cells, THP-1 cells, and differentiated monocytes of patient and control subjects. Enhanced expression of scavenger receptor, LOX-1, in the differentiated monocytes was also downregulated in presence of nHDL/rHDL. nHDL/rHDL could inhibit the ox-LDL-induced mitochondrial apoptotic pathway and aberrant expression of LOX-1 in patients. Double immunostaining using fluorescein isothiocyanate (FITC)-conjugated ox-LDL and LOX-1 in apoptotic cells indicates its significant role in the differentiated monocytes. CONCLUSION: It was observed that nHDL/rHDL could promote macrophage survival by preserving mitochondrial integrity from ox-LDL-induced apoptosis.

Maackia amurensis agglutinin induces apoptosis in cultured drug resistant human non-small cell lung cancer cells.

The emergence of multi drug resistance in non-small cell lung cancer (NSCLC) patients is a major challenge towards the efficacy of chemotherapy. Thus, there is an urgent need for the newer, better clinically targeted strategies to treat this disease. Earlier studies from our laboratory revealed the apoptotic activity of Maackia amurensis agglutinin (MAA) in human NSCLC cells. In this study, the effect of MAA on drug resistant NSCLC cells was investigated. Two Paclitaxel-resistant NSCLC sub-lines (A549/PTX100 and NCI-H460/PTX100) were developed from A549 & NCI-H460 cell lines respectively. The generation of drug resistance phenotype was confirmed by the expression of cell surface MDR-1. Both the drug resistant sub-lines showed distinct morphological alterations. MAA interacted with the cell-surface protein(s) of apparent Mr ~66 kDa and induced apoptosis in both the sub-lines through intrinsic/mitochondrial pathway, involving reduction in mitochondrial trans-membrane potential, up-regulation of Bax, unaltered/decreased expression of Bcl-XL, release of mitochondrial cytochrome c into the cytosol and activation of pro-caspases (-9&-3). Our findings highlighted the potential of this plant agglutinin to serve as an apoptosis inducing agent in drug resistant NSCLC cells.

Airway devices in paediatric anaesthesia.

Airway devices were first used in children since 1940 and thereafter an increasingly large number of paediatric airway devices have come into our armamentarium. To control and protect the airway in children during anaesthesia, in intensive care unit or in emergency department either tracheal intubation is performed under direct or indirect visualization of vocal cords with the help of laryngoscopes or video-laryngoscopes respectively or it can be done blindly or by using special instruments such as fiberoptic laryngoscope, lighted stylet or Bullard laryngoscope to name a few. Airway also can be maintained with the help of Laryngeal mask airways, oropharyngeal and nasopharyngeal airways. Updating our information and knowledge regarding these developments is pivotal to our practice of paediatric anaesthesia. With a thorough search of books, MEDLINE, MEDNET, clinical trials.gov.in, this article aims at focusing and understanding a brief basis of paediatric devices and their use.

A cytosol derived factor of Group B streptococcus prevent its invasion into human epithelial cells.

Group B streptococcus (GBS) or Streptococcus agalactiae, is an opportunistic pathogen causing a wide range of infections like pneumonia, sepsis, and meningitis in newborn, pregnant women and adults. While this bacterium has adapted well to asymptomatic colonization of adult humans, it still remains a potentially devastating pathogen to susceptible infants. Advances in molecular techniques and refinement of in vitro and in vivo model systems have elucidated key elements of the pathogenic process, from initial attachment to the maternal vaginal epithelium to penetration of the newborn blood-brain barrier. Still, the formidable array of GBS virulence factors makes this bacterium at the forefront of neonatal pathogens. The involvement of bacterial components in the host-pathogen interaction of GBS pathogenesis and its related diseases is not clearly understood. In this study we demonstrated the role of a 39 kDa factor from GBS which plays an important role in the process of its invasion. We found a homogeneous 39 kDa factor from the cytosol of GBS after following a combination of sequential purification steps involving molecular sieving and ion exchange chromatography using ACTA-FPLC system. Its N-terminal sequence showed a homology with xenobiotic response element type transcriptional regulator protein, a 40 kDa protein of Streptococcus. This factor leads to inhibition of GBS invasion in HeLa and A549 cells. This protein also showed sensitivity and specific cross reactivity with the antibodies raised against it in New Zealand white rabbits by western immunoblotting. This inhibitory factor was further confirmed tolerant for its cytotoxicity. These results add a novel aspect to bacterial pathogenesis where bacteria's own intracellular protein component can act as a potential therapeutic candidate by decreasing the severity of disease thus promoting its invasion inhibition.

Assessment of Cpa, Scl1 and Scl2 in clinical group A streptococcus isolates and patients from north India: an evaluation of the host pathogen interaction.

Group A streptococcus (GAS) infection remains a major concern due to multiple diseases including pharyngitis, impetigo, acute rheumatic fever (ARF) and rheumatic heart disease (RHD). It uses different adhesins and virulence factors like Cpa (collagen binding protein) and Scl (collagen-like protein) in its pathogenicity. Scl having similarities with human collagen may contribute to inducing autoimmunity in the host. Here we assessed gene expression, antibody titer of Cpa, Scl1 and Scl2 in both clinical GAS isolates (n = 45) and blood (n = 45) obtained from pharyngitis, ARF (acute rheumatic fever) and RHD respectively. Skin isolates (n = 30) were obtained from impetigo patients. The study revealed a total of 27 GAS emm types. Frequency of cpa, scl1, scl2 was high in ARF isolates. The antibody titer of these proteins was high in all isolates, and also in patients with pharyngitis and ARF. All isolates showed high binding affinity toward collagen I and IV, which further indicates a potential host pathogen interaction. Our study reflects a strong association of Cpa and Scls in early and post-GAS pathogenicity. However, the increased antibody titer of Scl1 and Scl2 during ARF may be attributed to a cogent immune response in the host.

Amelioration of Pain on Injection of Propofol: A Comparison of Pretreatment with Granisetron Vs Lignocaine.

INTRODUCTION: Pain during propofol injection is a very commonly and frequently encountered event during induction of anaesthesia. A 5HT3 antagonists like granisetron are commonly used just prior to intravenous propofol as pre anaesthetic medication to prevent emesis in patients. AIM: Comparison of pre treatment with granisetron versus lignocaine with respect to amelioration of pain induced by injection of propofol, in patient admitted for elective surgery with general anaesthesia. MATERIALS AND METHODS: A randomized double blinded controlled study was conducted with patients divided into three groups with (n=30) in each group. Group I (the placebo group) received 2 ml of 0.9% normal saline, Group II received 2 ml of 1% lignocaine and Group III received 2 ml of granisetron (1 mg/ml) as pre treatment medication respectively. The patient's complain regarding pain on intravenous propofol administration was recorded using the Verbal Rating Score. Pulse, BP, SpO2 were noted meticulously on three occasions-immediately after pre-treatment, injecting full dose of propofol (not for pain assessment) and after 10 minutes. The results were analysed using the null hypothesis and two sample t-tests. RESULTS: It was observed and obvious that the relief of pain was significant (p<0.05) when granisetron or lignocaine was compared with the placebo group. But there was insignificant difference (p>0.05) when granisetron was compared with lignocaine in terms of relieve of pain induced by propofol. CONCLUSION: It was concluded that parenteral administration of granisetron can be considered to be superior to lignocaine as pre treatment medication for pain relief after propofol injection along with the advantage of its anti-emetic effect.

In vitro study of virulence potential of Acinetobacter baumannii outer membrane vesicles.

Acinetobacter baumannii has emerged as an important opportunistic pathogen mostly causing nosocomial infections. The virulence factors of this important pathogen are largely unknown. Outer membrane vesicles (OMV) are naturally secreted by many gram negative and gram positive bacteria. These vesicles contain outer membrane proteins, lipids, periplasmic proteins, DNA and RNA. Their role in intracellular and intercellular signaling, transfer of virulence factors and eliciting immune response in host cells has been established in many pathogens. In this study, we investigated OMVs from three multi-drug resistant (MDR) clinical isolates and a non-MDR reference strain of A. baumannii for virulence potential. A. baumannii OMVs showed phospholipase C, hemolytic and leukotoxic activities. We found large variations in virulence potential between OMVs of MDR clinical isolates and non-MDR reference strain. These effector molecules were concentrated in OMVs than whole cell bacterial culture and cell-free supernatant. OMV-mediated phospholipase, hemolytic and leucotoxic activities may have a key role in pathogenicity of A. baumannii infection and may be future targets for therapeutic and preventive strategies. This is, to the best of our knowledge, the first report showing virulence potential of A. baumannii OMVs.

Evaluation of antioxidants and argpyrimidine in normal and cataractous lenses in north Indian population.

AIM: To assess the level of glutathione, thioltransferase, and argpyrimidine in nuclear and cortical cataractous lenses as well as in the clear lenses in the north Indian population. METHODS: Human cataractous lenses were collected from the patients who underwent extracapsular cataract extraction surgery; clear lenses were collected from the freshly donated eye bank eyes. Antioxidant molecules such as glutathione and thioltransferase enzyme activity were measured; simultaneously in these lenses a blue fluorophore argpyrimidine, an advanced glycation end (AGE) product level was assessed using high performance liquid chromatography (HPLC). RESULTS: The protein concentration was found to be present at higher levels in the control lenses compared to cataract lenses. A significant decrease in the glutathione level was observed in the nuclear cataractous lenses compared to cortical cataractous (P=0.004) and clear lenses (P≤0.005), but no significant change in the level of antioxidant enzyme thioltransferase was observed. Further, argpyrimidine a blue fluorophore (AGE) was found to be significantly higher in the nuclear cataract (P=0.013) compared to cortical cataract lenses. CONCLUSION: Antioxidants such as glutathione significantly decrease in age-related nuclear and cortical cataract and an AGE, argpyrimidine are present at significantly higher levels in nuclear cataract.

A silent mutation in human alpha-A crystallin gene in patients with age-related nuclear or cortical cataract.

A cataract is a complex multifactorial disease that results from alterations in the cellular architecture, i.e. lens proteins. Genes associated with the development of lens include crystallin genes. Although crystallins are highly conserved proteins among vertebrates, a significant number of polymorphisms exist in human population. In this study, we screened for polymorphisms in crystallin alpha A (CRYAA) and alpha B (CRYAB) genes in 200 patients over 40 years of age, diagnosed with age-related cataract (ARC; nuclear and cortical cataracts). Genomic DNA was extracted from the peripheral blood. The coding regions of the CRYAA and CRYAB gene were amplified using polymerase chain reaction and subjected to restriction digestion. Restriction fragment length polymorphism (RFLP) was performed using known restriction enzymes for CRYAA and CRYAB genes. Denaturing high performance liquid chromatography and direct sequencing were performed to detect sequence variation in CRYAA gene. In silico analysis of secondary CRYAA mRNA structure was performed using CLC RNA Workbench. RFLP analysis did not show any changes in the restriction sites of CRYAA and CRYAB genes. In 6 patients (4 patients with nuclear cataract and 2 with cortical cataract), sequence analysis of the exon 1 in the CRYAA gene showed a silent single nucleotide polymorphism [D2D] (CRYAA: C to T transition). One of the patients with nuclear cataract was homozygous for this allele. The in silico analysis revealed that D2D mutation results in a compact CRYAA mRNA secondary structure, while the wild type CRYAA mRNA has a weak or loose secondary structure. D2D mutation in the CRYAA gene may be an additional risk factor for progression of ARC.

Cell cycle arrest and apoptosis induced by enteroaggregative Escherichia coli in cultured human intestinal epithelial cells.

PURPOSE: Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen causing diarrhoeal diseases in multiple epidemiological and clinical settings. However, understanding of the pathogenesis of the disease caused by this organism is still suboptimal. Studies have indicated that enteric bacteria induced cell cycle arrest and apoptosis in host intestinal epithelial cells might play a vital role in the pathogenesis caused by these organisms. In this study an attempt was made to assess EAEC-induced apoptosis and cell cycle modulation in human intestinal epithelial cell lines. METHODOLOGY: INT-407 and HCT-15 cells were infected with EAEC-T8 (clinical isolate) as well as plasmid cured variant of EAEC-T8 (EAEC-pT8). Propidium iodide staining was done to select the time of infection and the incubation period of the infected culture. Apoptosis was further assessed in EAEC infected both the cell lines by annexin-V-FLUOS & propidium iodide, cell death detection ELISA, DNA strand breaks and microscopic analysis. Further, the DNA content of the EAEC-infected cells at different phases of cell cycle was also monitored. RESULTS: We have found that EAEC could induce apoptosis in human small intestinal as well as colonic epithelial cell lines, which was assessed by the expression of phosphatidylserine on host cell surface, internucleosomal cleavage of host cell DNA and microscopic analysis of the characteristic apoptotic features of these cells. EAEC was also found to arrest cells at S phase and G2-M phase of the cell cycle. CONCLUSIONS: EAEC-T8 could induce maximum apoptosis and cell cycle modulation in both small intestinal and colonic epithelial cells. Further, we have observed that the plasmid of this organism had maximum contribution to these processes. The outcome of this study has undoubtedly led to a better understanding of the basic mechanism of pathogenesis caused by EAEC.

Crosstalk between microRNA-122 and FOX family genes in HepG2 cells.

MicroRNA-122 (miR-122) is liver specific and plays an important role in physiology as well as diseases including hepatocellular carcinoma (HCC). Downregulation of miR-122 in HCC modulates apoptosis. Similarly, the putative targets of miR-122, the forkhead box (FOX) family genes also play an important role in the regulation of apoptosis. Hence, an interplay between miR-122 and FOX family genes has been explored in this study. Initially, an augmentation of apoptosis was noticed in HepG2 cells after transfection with miR-122. Further, the predicted miR-122 targets, the FOX family genes ( FOXM1b, FOXP1, and FOXO4) were selected via in silico analysis based on their role in apoptosis. We checked the expression of all these genes at transcript level after the transfection of miR-122 and found that the relative expression of FOXP1 and FOXM1b was significantly downregulated (p < 0.005) and that of FOXO4 was upregulated (p < 0.005). Thus, the finding indicates deregulation of these FOX genes as a result of miR-122 augmentation might be involved in the modulation of apoptosis.