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Pankaj Gupta

Publications and source records attributed to Pankaj Gupta.

34 records · Page 2Linked to original sources
PubMed2004

Mechanistic aspects of mda-7/IL-24 cancer cell selectivity analysed via a bacterial fusion protein.

The human mda-7/IL-24 gene product is normally expressed in melanocytes and certain lymphocyte populations. Loss of expression, a distinctive feature of many tumor suppressor genes, has been documented at RNA and protein levels in association with melanoma progression both in vitro as well as in human tumor-derived material. The MDA-7/IL-24 protein undergoes post-translational processing, including removal of an amino-terminal 48-residue signal peptide and extensive glycosylation prior to its secretion by producing cells. Its inherent cytokine properties have been documented in multiple reports, which have identified and characterized its cognate receptors and activation of the JAK/STAT signaling pathway following ligand/receptor docking. A notable and incompletely understood property of MDA-7/IL-24 is its ability to induce apoptosis in transformed cells, while having marginal growth suppressive effects on normal primary or immortalized cell lines. MDA-7/IL-24 has been delivered to cells, tumor xenografts and patients in clinical trials via a nonreplicating adenovirus (Ad.mda-7). Studies utilizing eukaryotically expressed and purified MDA-7/IL-24 protein from several sources have recapitulated some of the molecule's reported properties including receptor binding and JAK/STAT activation. Here, we report the properties and characteristics of a bacterially expressed and purified GST-MDA-7 fusion protein. These studies reveal that GST-MDA-7 retains its cancer-selective apoptosis-inducing properties, thereby providing a new reagent that will assist in defining the mechanism of action of this novel cytokine. In addition, retention of tumor-specific activity of GST-MDA-7 suggests that this protein may also have therapeutic applications.

Animals↗
PubMed2004

MDA-7 regulates cell growth and radiosensitivity in vitro of primary (non-established) human glioma cells.

We examined the impact of purified bacterially synthesized GST-MDA-7 (IL-24) and ionizing radiation on the proliferation and survival of nonestablished human glioblastoma multiforme (GBM) cells. Glioma cell types expressing mutated PTEN and p53 molecules, activated ERBB1VIII, overexpressing wild type ERBB1 or without receptor overexpression were selected. In MTT assays, GST-MDA-7 caused a dose-dependent reduction in the proliferation of nonestablished glioma cells; however only at higher concentrations did GST-MDA-7 reduce cell viability. The anti-proliferative and cytotoxic effects of GST-MDA-7 were enhanced by radiation in a greater than additive fashion that correlated with JNK1/2/3 activation. The reduction in cell growth and enhancement in cell killing by the combination of GST-MDA-7 and radiation were blocked by an ROS scavenger, N-acetyl cysteine (NAC), a JNK1/2/3 inhibitor SP600125, a pan-caspase inhibitor (zVAD) and by an inhibitor of caspase 9 (LEHD), but not by an inhibitor of caspase 8 (IETD). Low concentrations of either GST-MDA-7 or radiation reduced clonogenic survival, however colony formation ability was significantly further decreased when the two treatments were combined, which was also blocked by inhibition of caspase 9 function. In general agreement with activation of the intrinsic caspase pathway, cell death correlated with reduced BCL-XL expression and with increased levels of the pro-apoptotic proteins BAD and BAX. Inhibition of caspase 9 after combination treatment blunted neither JNK1/2/3 activation nor the enhanced expression of BAD and BAX, but did block caspase 3 cleavage, reduced expression of BCL-XL and inhibition of ERK1/2 activity. In contrast, incubation with NAC blocked JNK1/2/3 activation and cell killing, but not the increases in BAD and BAX expression. These findings argue that after combination treatment JNK1/2/3 activation is a primary pro-apoptotic event and loss of BCL-XL expression and ERK1/2 activity are secondary caspase-dependent processes. This data also argues that GST- MDA-7 induces two parallel pro-apoptotic pathways via ROS-dependent and -independent mechanisms. Infection of primary human astrocytes with a recombinant adenovirus to express MDA-7, Ad.mda-7, but not infection with either Ad.cmv or Ad.mda-7SP- lacking MDA-7 secretion, resulted in the suppression of GBM cell colony formation in soft agar overlay assays, an effect that was enhanced in a greater than additive fashion by radiation. Collectively, our findings demonstrate that MDA-7 reduces proliferation and enhances the radiosensitivity of nonestablished human GBM cells in vitro, and when grown in 3 dimensions, and that sensitization occurs independently of basal EGFR/ERK1/2/AKT activity or the functions of PTEN and p53.

Acetylcysteine↗
PubMed2004

Mechanisms of acute myocardial infarction study (MAMIS).

Acute myocardial infarction (AMI) is a highly dynamic event, which is associated with marked neuroendocrinological dysfunction in addition to cardiac damage. The immediate trigger for AMI is not precisely known. Studies conducted by Lown, Braunwald, Halberg, Otsuka and our group have demonstrated a marked increase in sympathetic activity, oxidative stress, and magnesium and potassium deficiency during AMI. Clinical studies have reported an increased incidence of AMI, sudden death and ischemia during first quarter of the day when there is a rapid withdrawal of vagal activity and increase in sympathetic tone. In one case-control study of 202 patients with AMI, there was a significant (P < 0.02) increase in cardiac events in the second quarter of the day compared to other quarters, respectively (16.8%, 41.0%, 13.8%, 28.2% per quarter). This characteristic remained prevalent in both men and women and among patients with and without known AMI (n = 52), diabetes (n = 53) or hypertension (n = 75). Triggers of AMI were noted among 162 (82.2%) of the patients. Neuropsychological mechanisms were observed as follows: emotional stress (45.5%), sleep deprivation (27.7%), cold climate (29.2%), hot climate (24.7%), large meals (47.5%) and physical exertion (31.2%). These triggering factors are known to enhance sympathetic activity and decrease vagal tone, resulting in an increased secretion of plasma cortisol, noradrenaline, aldosterone, angiotension-converting enzyme (ACE), interleukin (IL)-1, -2, -6, -18, and tumor necrosis factor-alpha (TNF-alpha), all of which are are proinflammatory agents. There is also a deficiency in the serum levels of vitamin A, E, and C and magnesium, potassium, melatonin, and IL-10 (an anti-inflammatory agent). In our study, we found a decrease in magnesium, potassium, vitamin A, E, C and beta carotene combined with an increase in thiobarbituric acid-reactive substances (TBARS), MDA and diene conjugates, TNF-alpha and IL-6, all of which are indicators of oxidative damage and proinflammatory activity, respectively.

Activity Cycles↗
PubMed2004

Leukocoria.

Explore the source record for details and available documents.

Child, Preschool↗
PubMed2003

Mactinin: a modulator of the monocyte response to inflammation.

During inflammatory processes, monocytes leave the blood stream at increased rates and enter inflammation tissue, where they undergo phenotypic transformation to mature macrophages with enhanced phagocytic activity. alpha-Actinin, a cytoskeletal protein, is present in focal adhesion complexes and left in the microenvironment as a result of cell movement. Mactinin, a 31 kDa amino-terminal fragment of alpha-actinin, is generated by the degradation of extracellular alpha-actinin by monocyte-secreted urokinase. We have previously demonstrated that mactinin promotes monocyte/macrophage maturation. We now report that 0.5-10 nM mactinin has significant chemotactic activity for monocytes. Mactinin seems to be present in inflammatory arthritis synovial fluid, because affinity-purified antisera reacted with a protein of the expected molecular mass in various types of arthritis fluids that were immunoaffinity-purified and subjected to Western analysis. Thus, six of seven samples from patients with psoriatic arthritis, reactive arthritis, gout, or ankylosing spondylitis contained mactinin at levels that are active in vitro. Initially, mactinin was not found in affinity-purified rheumatoid arthritis samples. However, it was detectable after the dissociation of immune complexes, suggesting that it was complexed to anti-microfilament auto-antibodies. In addition, mactinin was found in the lavage fluid from the arthritic knee joints of rabbits with antigen-induced arthritis and was absent from the contralateral control knee fluids. We conclude that mactinin is present in several types of inflammatory arthritis and might modulate mononuclear phagocyte response to inflammation.

Actinin↗
PubMed2003

Congenital cystic eye with multiple dermal appendages: a case report.

BACKGROUND: A partial or complete failure in the involution of the primary optic vesicle resulting in the formation of a cyst is an extremely rare anomaly known as congenital cystic eye. The primary optic vesicle is formed but instead of the anterior part of the vesicle involuting to lie in apposition with the posterior part, a cyst persists at birth and replaces the eye. CASE PRESENTATION: We report a case of congenital cystic eye associated with multiple dermal appendages in a 1-day-old female child. This condition presented at birth as a large orbital mass in the left orbit that bulged forwards and stretched the eyelids. No globe or any other ocular structures were identified in the orbit. Multiple dermal appendages were present in the adjacent part of the face below the left orbit and on the upper part of the neck. CONCLUSIONS: Congenital cystic eye is an extremely rare condition and with only 28 previous cases reported in the literature. We present the second case of congenital cystic eye with multiple dermal appendages of the face and neck.

Cysts↗
PubMed2003

Fibrin glue versus N-butyl-2-cyanoacrylate in corneal perforations.

OBJECTIVE: To compare the efficacy of fibrin glue and N-butyl-2-cyanoacrylate in corneal perforations. DESIGN: Randomized, controlled clinical trial. PARTICIPANTS: Forty-one patients (41 eyes) with corneal perforations up to 3 mm in diameter with a positive Seidel's test were randomly assigned to two groups (1 and 2). INTERVENTION: Group 1 comprised 19 eyes treated with fibrin glue, and group 2 comprised 22 eyes treated with N-butyl-2-cyanoacrylate. MAIN OUTCOME MEASURES: Number of eyes with successful healing, time required for healing, status of corneal vascularization, and complications were compared in the two groups. Power calculation was performed at alpha = 0.05. RESULTS: Fifteen (79%) eyes had successful healing of corneal perforation in group 1, compared with 19 (86%) eyes in group 2 (P > 0.05) at 3 months' follow-up. The power to detect a difference between the two groups was 10%. Corneal perforation healed within 6 weeks in 12 (63%) eyes in group 1 and 7 (31.8%) eyes in group 2 (P < 0.05). Reapplication of glue was required in six (31.5%) eyes in group 1 and seven (31.4%) eyes in group 2 during the first 3 months of follow-up. The mean number of applications per eye was 1.37 in group 1 and 1.36 in group 2. An increase in deep corneal vascularization was observed in 2 (10.5%) eyes in group 1 and 10 (45.5%) eyes in group 2 (P < 0.05). Giant papillary conjunctivitis occurred in one (5%) eye in group 1 and eight (36.4%) eyes in group 2 (P < 0.05). CONCLUSIONS: Fibrin glue and cyanoacrylate tissue adhesive are both effective in the closure of corneal perforations up to 3 mm in diameter. Fibrin glue provides faster healing and induces significantly less corneal vascularization, but it requires a significantly longer time for adhesive plug formation.

Adolescent↗
PubMed2003

Synchronous cystic teratomas of the craniofacial region.

A case of synchronous cystic teratomas presented with the complaints of a slow-growing facial mass and forward protrusion of the right eye since birth. The patient was examined thoroughly and subjected to magnetic resonance imaging (MRI) scan. The MRI scan revealed the presence of two separate cysts in the right orbit and right temporal fossa. The patient was operated by a combined effort of ophthalmologists and otorhinolaryngologists. The cysts were excised and subjected to pathological examination. The histopathological survey revealed both the cysts to be mature cystic teratomas similar to each other. This coexistence of a primary temporal fossa teratoma with an orbital teratoma is the first of its kind and to the best of our knowledge has not been reported before.

Adolescent↗
PubMed2002

ATF1 phosphorylation by the ERK MAPK pathway is required for epidermal growth factor-induced c-jun expression.

Epidermal growth factor induction of c-jun expression requires ATF1 and MEF2 sites in the c-jun promoter. We find that activation of the c-jun promoter through the ATF1 site requires phosphorylation of ATF1 at serine 63. A serine 63 to alanine mutation of ATF1 acts to block epidermal growth factor (EGF) induction of a transfected c-jun gene. ATF1 can be phosphorylated by mitogen- and stress-activated protein kinase 1 (MSK1), which is activated by EGF and ERK1/2. Kinase-dead MSK1 mutants blocked EGF induction of a transfected c-jun gene suggesting that MSK1 or a similar family member is required for induced c-jun expression. Use of the MEK1 inhibitor U0126 and dominant negative MEK1 further showed that MSK1 activation and c-jun induction require the ERK pathway. In contrast, a JNK inhibitor blocked EGF induction of c-jun expression but not ATF1 phosphorylation. These results show that the two MAPK pathways, ERK and JNK, are required for EGF-induced c-jun expression and that the ERK pathway acts through downstream phosphorylation of ATF1.

Activating Transcription Factor 1↗
PubMed2002

Identification of an MIP-1alpha -binding heparan sulfate oligosaccharide that supports long-term in vitro maintenance of human LTC-ICs.

We previously showed that heparan sulfate (HS) is required for in vitro cytokine + chemokine-mediated maintenance of primitive human hematopoietic progenitors. However, HS preparations are mixtures of polysaccharide chains of varying size, structure, and protein-binding abilities. Therefore, we examined whether the long-term culture-initiating cells (LTC-IC) supportive capability of HS is attributable to an oligosaccharide of defined length and protein-binding ability. Oligosaccharides of a wide range of sizes were prepared, and their capability to support human marrow LTC-IC maintenance in the presence of low-dose cytokines and a single chemokine, macrophage inflammatory protein-1alpha (MIP-1alpha), was examined. LTC-IC supportive capability of HS oligosaccharides correlated directly with size and MIP-1alpha binding ability. A specific MIP-1alpha-binding HS oligosaccharide preparation of M(r) 10 kDa that optimally supported LTC-IC maintenance was identified. This oligosaccharide had the structure required for MIP-1alpha binding, which we have recently described. The present study defines the minimum size and structural features of LTC-IC supportive HS.

Antigens, CD34↗
PubMed2002

Urokinase is required for the formation of mactinin, an alpha-actinin fragment that promotes monocyte/macrophage maturation.

We have previously shown that lysates from HL-60 myeloid leukemia cells or from peripheral blood monocytes are able to degrade alpha-actinin to form a 31-kDa amino-terminal fragment with monocyte/macrophage maturation promoting activity. In contrast, intact alpha-actinin, which is a 100-kDa actin-binding protein, has no differentiating activity. The aim of this study was to investigate the enzyme responsible for the degradation of alpha-actinin to form this fragment, named mactinin. The ability of cell lysates to degrade [125I]alpha-actinin in the presence of various enzyme inhibitors, including inhibitors of metalloproteinases, cysteine proteinases, and serine proteases, was measured. Phenylmethylsulfonyl fluoride (PMSF) was the only inhibitor able to prevent formation of mactinin by cell lysate degradation of alpha-actinin, suggesting that a serine protease is responsible for the digestion. Of the various serine proteases tested (thrombin, plasmin, and urokinase), only urokinase was able to produce a 31-kDa band. The urokinase-generated 31-kDa band promoted maturation in HL-60 cells. Amiloride, a specific inhibitor of urokinase, inhibited production of the 31-kDa alpha-actinin fragment by HL-60 cell lysates. For in vivo tests, inflammatory fluid (from bronchoalvelolar lavage) was collected from uPA (urokinase) knockout mice and their wild-type counterparts after intratracheal challenge with Pneumocystis carinii. Although most (6 of 8) wild-type mice had mactinin in their inflammatory fluid samples, none (0 of 8) of the uPA knockout mice had mactinin present (P<0.01). These results demonstrate that urokinase is necessary and sufficient for the formation of the monocyte/macrophage maturation promoting fragment, mactinin, in vitro and in vivo. These findings support the role of urokinase in the regulation of monocyte/macrophage functions, such as that occurring in inflammatory reactions.

Actinin↗
PubMedDate not supplied

Constitutive expression of the Fas receptor and its ligand in adult human bone marrow: a regulatory feedback loop for the homeostatic control of hematopoiesis.

Fas ligand (FasL) mediated apoptosis in the bone marrow may contribute to suppression of hematopoiesis in myelodysplastic syndromes (MDS) and in aplastic anemia, and also to the regulation of normal erythropoiesis. To identify potential effector and target cells in this regulatory pathway, we examined the constitutive expression of Fas receptor (Fas) and FasL (total and cell-surface) in myeloid and lymphoid cells and subsets of CD34+ cells in normal healthy adult human bone marrow using multiparameter flow cytometry. A high proportion of CD34+ cells constitutively expressed cell-surface FasL. However, none of the CD34+ cells expressed Fas alone. A reciprocal gradient of expression of FasL and Fas was observed in subsets of CD34+ cells: as compared to primitive CD34+/HLA-DR(-) (DR(-)) cells, a higher proportion of committed CD34+/HLA-DR(++) (DR(++)) cells expressed FasL but fewer expressed Fas; the expression of both molecules was intermediate in CD34+/HLA-DR(dim) cells. Also, the intensity of FasL expression was higher in DR(++) than in DR(-) cells. These results suggest that the homeostatic regulation of myelopoiesis in normal bone marrow is mediated via an autoregulatory feedback loop by myeloid cells and progenitors themselves, at least partly via the Fas-FasL pathway. This notion is also consistent with our recent observation that overexpression of FasL by myeloid cells in MDS correlates directly with anemia, transfusion requirements, and shorter survival, an example of dysregulation of this pathway.

Adult↗
PubMedDate not supplied

The microenvironment of AFT024 cells maintains primitive human hematopoiesis by counteracting contact mediated inhibition of proliferation.

We have previously shown that maintenance of primitive human hematopoietic stem cells is poor when cultured in contact with marrow stromal feeders. However, when separated from stromal contact, human progenitors can be maintained because adhesion mediated proliferation inhibition does not occur. In this study we demonstrate how the murine fetal liver cell line, AFT024, supports primitive human hematopoiesis better in contact cultures compared to primary feeders. We evaluated if better progenitor maintenance in contact with AFT024 cells can be explained by decreased adhesion itself or decreased adhesion mediated inhibition of proliferation. We show that primitive human hematopoietic cells adhered equally well to AFT024 and primary feeders, such as M2-10B4. Further, contact with metabolically inactive AFT024 cells prevented cell cycle progression and decreased maintenance of primitive progenitors to the same extent as contact with M2-10B4 feeders. However, contact with viable AFT024 feeders did not inhibit proliferation, suggesting that AFT024-factors counteract contact mediated inhibition of proliferation. Cytokine production by M2-10B4 and AFT024 cells was similar. Large-size O-sulfated heparan sulfate glycosaminoglycans, known to be important for hematopoietic support, were found only in AFT024-matrix. We hypothesize that these factors may explain, in part, our observations. Finally, we show that more than 100% of primitive myeloid progenitors could be maintained for at least five weeks when cultured in contact with AFT024 feeders in the presence of Interleukin-3 and Macrophage Inflammatory Protein-1alpha. In conclusion, AFT024 cells produce factor(s), that counteract contact induced growth inhibition of primitive human hematopoietic progenitors, leading to expansion of these cells in contact with the microenvironment.

Animals↗
PubMedDate not supplied

Real-time three-dimensional transthoracic echocardiography.

BACKGROUND: Complex anatomy of intra-cardiac structures requires spatial orientation of image in three dimensions for better understanding and enhanced image interpretation. We evaluated the feasibility and efficacy of the emerging 'real-time three-dimensional transthoracic echocardiography' technique for comprehensive assessment of cardiac anatomy, physiology, pathomorphology and pathophysiology in patients with structural heart disease. METHODS AND RESULTS: Patients with structural heart disease (n=152) were evaluated by conventional two-dimensional transthoracic echocardiography and real-time three-dimensional transthoracic echocardiography using standard protocol. Fifty-six cases were of rheumatic etiology with multi-valvular involvement (mitral stenosis: 32; mitral regurgitation: 29; tricuspid regurgitation: 8; aortic valve disease: 11) and 21 cases of non-rheumatic valvular heart disease. A total of 38 congenital heart disease patients were examined including 23 patients with atrial septal defect. Left ventricular function (n=20) and right ventricular function (n=10) were also assessed using dedicated software. CONCLUSIONS: Results of real-time three-dimensional transthoracic echocardiography mitral valve area assessment by planimetery are comparable to two-dimensional transthoracic echocardiography with additional information about surface anatomy of leaflets and the subvalvular apparatus in real time with clear demarcation of commissural fusion and scallops of leaflets. Enface view of atrial septal defect with direct visualization of shape, size and number of defects, tricuspid valve area by planimetery, right ventricular shape, objective assessment of ventricular volumes and regurgitation vena contracta area are the fields where three-dimensional transthoracic echocardiography was of additive value to conventional two-dimensional transthoracic echcardiography. This study proves clinical feasibility of real-time three-dimensional transthoracic echocardiography but requires further validation of quantitative observations.

Adolescent↗