Taposh K. Das

Molecular effects of uptake of gold nanoparticles in HeLa cells.

Mellow gold: Toxicogenomics of nanoparticles can expose subtle yet clinically relevant alterations in the intracellular environment that arise from downstream effects of nanoparticle accumulation inside cells. Genome-wide expression profiling by using DNA microarrays revealed that internalization of gold nanoparticles is not associated with gross changes in transcription in HeLa cells.

Inhibition of protein aggregation: supramolecular assemblies of arginine hold the key.

Background Aggregation of unfolded proteins occurs mainly through the exposed hydrophobic surfaces. Any mechanism of inhibition of this aggregation should explain the prevention of these hydrophobic interactions. Though arginine is prevalently used as an aggregation suppressor, its mechanism of action is not clearly understood. We propose a mechanism based on the hydrophobic interactions of arginine. Methodology We have analyzed arginine solution for its hydrotropic effect by pyrene solubility and the presence of hydrophobic environment by 1-anilino-8-naphthalene sulfonic acid fluorescence. Mass spectroscopic analyses show that arginine forms molecular clusters in the gas phase and the cluster composition is dependent on the solution conditions. Light scattering studies indicate that arginine exists as clusters in solution. In the presence of arginine, the reverse phase chromatographic elution profile of Alzheimers amyloid beta 1-42 (Aβ1-42) peptide is modified. Changes in the hydrodynamic volume of Aβ1-42 in the presence of arginine measured by size exclusion chromatography show that arginine binds to Aβ1-42. Arginine increases the solubility of Aβ1-42 peptide in aqueous medium. It decreases the aggregation of Aβ1-42 as observed by atomic force microscopy. Conclusions Based on our experimental results we propose that molecular clusters of arginine in aqueous solutions display a hydrophobic surface by the alignment of its three methylene groups. The hydrophobic surfaces present on the proteins interact with the hydrophobic surface presented by the arginine clusters. The masking of hydrophobic surface inhibits protein-protein aggregation. This mechanism is also responsible for the hydrotropic effect of arginine on various compounds. It is also explained why other amino acids fail to inhibit the protein aggregation.

Disruption of response regulator gene, devR, leads to attenuation in virulence of Mycobacterium tuberculosis

The devR-devS two-component system of Mycobacterium tuberculosis was identified earlier and partially characterized in our laboratory. A devR::kan mutant of M. tuberculosis was constructed by allelic exchange. The devR mutant strain showed reduced cell-to-cell adherence in comparison to the parental strain in laboratory culture media. This phenotype was reversed on complementation with a wild-type copy of devR. The devR mutant and parental strains grew at equivalent rates within human monocytes either in the absence or in the presence of lymphocytic cells. The expression of DevR was not modulated upon entry of M. tuberculosis into human monocytes. However, guinea pigs infected with the mutant strain showed a significant decrease in gross lesions in lung, liver and spleen; only mild pathological changes in liver and lung; and a nearly 3 log lower bacterial burden in spleen compared to guinea pigs infected with the parental strain. Our results suggest that DevR is required for virulence in guinea pigs but is not essential for entry, survival and multiplication of M. tuberculosis within human monocytes in vitro. The attenuation in virulence of the devR mutant in guinea pigs together with DevR-DevS being a bona fide signal transduction system indicates that DevR plays a critical and regulatory role in the adaptation and survival of M. tuberculosis within tissues.

Role of Mycobacterium tuberculosis Ser/Thr Kinase PknF: Implications in Glucose Transport and Cell Division

Protein kinases have a diverse array of functions in bacterial physiology, with a distinct role in the regulation of development, stress responses, and pathogenicity. pknF, one of the 11 kinases of Mycobacterium tuberculosis, encodes an autophosphorylating, transmembrane serine/threonine protein kinase, which is absent in the fast-growing, nonpathogenic Mycobacterium smegmatis. Herein, we investigate the physiological role of PknF using an antisense strategy with M. tuberculosis and expressing PknF and its kinase mutant (K41M) in M. smegmatis. Expression of PknF in M. smegmatis led to reduction in the growth rate and shortening and swelling of cells with constrictions. Interestingly, an antisense strain of M. tuberculosis expressing a low level of PknF displayed fast growth and a deformed cell morphology compared to the wild-type strain. Electron microscopy showed that most of the cells of the antisense strain were of a smaller size with an aberrant septum. Furthermore, nutrient transport analysis of these strains was conducted using 3H-labeled and 14C-labeled substrates. A significant increase in the uptake of D-glucose but not of glycerol, leucine, or oleic acid was observed in the antisense strain compared to the wild-type strain. The results suggest that PknF plays a direct/indirect role in the regulation of glucose transport, cell growth, and septum formation in M. tuberculosis.

mymA operon of Mycobacterium tuberculosis: its regulation and importance in the cell envelope

Mycobacterium tuberculosis faces various stressful conditions inside the host and responds to them through a coordinated regulation of gene expression. We had previously reported identification of the virS gene of M. tuberculosis (Rv3082c) belonging to the AraC family of transcriptional regulators. In the current study, we show that the seven genes (Rv3083-Rv3089) which are present divergently to virS (Rv3082c) constitute an operon designated the mymA operon. Further investigation on the regulation of this operon showed that transcription of the mymA operon is dependent on the presence of VirS protein. A four-fold induction of the mymA operon promoter occurs specifically in wild-type M. tuberculosis and not in the virS mutant of M. tuberculosis (MtbDeltavirS) when exposed to acidic pH. Expression of the mymA operon was also induced in infected macrophages by 10-fold over a 6-day period. To gain an insight into the function of the proteins encoded by this operon, we carried out a bioinformatic analysis, which suggested the involvement of these proteins in the modification of fatty acids required for cell envelope. This was supported by altered colony morphology and cell envelope structure displayed by the virS mutant of M. tuberculosis (MtbDeltavirS).

Possible role of amyloid-beta, adenine nucleotide translocase and cyclophilin-D interaction in mitochondrial dysfunction of Alzheimer's disease.

Alzheimers disease (AD) is a common neurodegenerative disease characterized by both extra- as well as intracellular deposition of amyloid beta peptides (Aβ). The accumulation of Aβ in mitochondria is associated with mitochondrial dysfunction and oxidative stress in AD. Recent evidences suggest the involvement of Aβ interaction with mitochondrial proteins such as cyclophilin-D (CypD) in oxidative stress, mitochondrial permeability transition (MPT) and Alzheimers associated neurodegeneration. The present study is an effort to elucidate the molecular interaction of Aβ with other proteins involved in MPT like adenine nucleotide translocase (ANT). Based on our prediction for sub-cellular localization using WolfPSORT and other experimental evidences, we suggest that Aβ molecules localize in mitochondrial inner membrane in close vicinity with ANT. Our simulation study for protein–protein interaction clearly suggests that the ANT-Aβ interaction is stronger than CypD-Aβ interaction. Further the lipophilic nature and evidences regarding the localization of Aβ in the mitochondrial inner-membrane also support the possibility of strong interaction between ANT and Aβ. Interaction between ANT and Aβ may affect normal physiological function of ANT i.e. transport of ATP and ADP. Since both the CypD-Aβ as well as ANT-Aβ interaction are energetically favorable and both CypD and ANT are associated with the regulation of MPT, the functional impact of both these interactions warrants more in-depth investigations for elucidating the mechanisms involved in Aβ-induced oxidative stress.

Gastroduodenal manifestations in patients with skeletal fluorosis

A prospective case-controlled study was performed to evaluate the gastrointestinal symptoms and mucosal abnormalities occurring in patients with osteofluorosis. Ten patients with documented osteofluorosis and ten age- and sex-matched healthy volunteers were included in the study. Clinical evaluation, real-time ultrasound, and upper gastrointestinal endoscopy and biopsy from the gastric antrum and duodenum were performed in all subjects. The biopsies were subjected to a rapid urease test and light and electron microscopic examinations. Ionic fluoride levels were estimated in the drinking water, serum, and urine using an ION 85 ion analyzer. All patients with osteofluorosis had gastrointestinal symptoms, the most common being abdominal pain. Endoscopic abnormalities were found in seven patients with osteofluorosis. In all 7 of these patients, chronic atrophic gastritis was seen on histology. Electron microscopic abnormalities were observed in all 10 patients with osteofluorosis. These included loss of microvilli, cracked-clay appearance, and the presence of surface abrasions on the mucosal cells. None of the control subjects had any clinical symptoms or mucosal abnormalities. It was concluded that gastrointestinal symptoms as well as mucosal abnormalities are common in patients with osteofluorosis.

Mitochondrial Perturbance and Execution of Apoptosis in Platelet Mitochondria of Patients With Amyotrophic Lateral Sclerosis

ABSTRACT Role of platelets have been evinced as a systemic tool in a variety of neurological disorders. Oxidative phosphorylation contributes approximately 80% of total adenosine–tri-phosphate (ATP) production in resting platelets suggesting potential dependence of platelets on modest mitochondrial functioning. Since mitochondria play a pivotal role in regulating metabolic and apoptotic pathways in various neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), we assessed mitochondrial membrane potential (MMP) associated alterations and apoptotic status of platelet mitochondria in ALS patients using case-control approach. Confocal microscopy reflected heterogeneous distribution of JC-1 aggregates and monomers indicating altered MMP in ALS platelets. Our flow cytometry results confirmed greater percentage of mitochondrial depolarization in ALS platelets. Greater exposure of phosphatidyl serine (PS) residue vindicated by annexin V binding and lesser accumulation of mitotracker red in mitochondrial matrix demonstrated initiation of apoptosis in ALS platelets. Our findings corroborate mitochondrial abnormalities such as perturbance of MMP, mitochondrial depolarization, and apoptosis in ALS platelet mitochondria. In conclusion, our study further evinces the involvement of mitochondrial dysfunction in the pathogenesis of ALS and suggests implication of cell death in peripheral tissues apart from motor neurons in ALS.

Identification of Autoantibodies against Transthyretin for the Screening and Diagnosis of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic, autoimmune, systemic and inflammatory rheumatic disease that leads to inflammation of the joints and surrounding tissues. Identification of novel protein(s) associated with severity of RA is a prerequisite for better understanding of pathogenesis of this disease that may also have potential to serve as novel biomarkers in the diagnosis of RA. Present study was undertaken to compare the amount of autoantigens and autoantibodies in the plasma of RA patients in comparison to healthy controls. Plasma samples were collected from the patients suffering from RA, Osteoarthritis (OA), Systemic lupus erythematosus (SLE) and healthy volunteers. The screening of plasma proteins were carried out using 2-dimensional gel electrophoresis followed by identification of differentially expressed protein by MALDI-TOF MS/MS. Among several differentially expressed proteins, transthyretin (TTR) has been identified as one of the protein that showed significantly up regulated expression in the plasma of RA patients. The results were further validated by Western blot analysis and ELISA. In comparison to OA synovium, an exclusive significantly high expression of TTR in RA has been validated through IHC, Western blotting and IEM studies. Most importantly, the increase in expression of TTR with the progression of severity of RA condition has been observed. The autoantibodies against TTR present in the RA plasma were identified using immunoprecipitation-Western methods. The significant production of autoantibodies was validated by ELISA and Western blot analysis using recombinant pure protein of TTR. Hence, these novel observations on increase in TTR expression with the increase in severity of RA conditions and significant production of autoantibodies against TTR clearly suggest that a systematic studies on the role TTR in the pathogenesis of RA is immediately required and TTR may be used as a serum diagnostic marker together with other biochemical parameters and clinical symptoms for RA screening and diagnosis.

Ultrastructural Variations in Platelets and Platelet Mitochondria: A Novel Feature in Amyotrophic Lateral Sclerosis

Platelets are characterized as a systemic tool to elucidate mitochondria-allied perturbance in neurological diseases. The authors studied ultrastructural changes in platelets and platelet mitochondria using a case-control approach in amyotrophic lateral sclerosis (ALS). Subjects were sporadic ALS cases (n = 22) and age- and sex-matched controls (n = 16). Phlebotomy was performed, platelet concentrates (PCs) were prepared, and mitochondria were extracted. PCs and mitochondria were processed for ultrastructure study using transmission electron microscopy. Image analysis was done using Image-J. Transmission electron microscopy demonstrated both qualitative and quantitative variations in ALS platelets and platelet mitochondria. Heterogeneous distribution of granules, formation of vacuoles, blebs, pseudopodia, loose demarcation of cell membrane with a significant increase in area (20.3%), perimeter (17.82%), integrated density (21.44%), electron-lucent granules (41.79%), and vacuoles (36.58%) were observed in ALS platelets. Conversely, control platelets exhibited an increase of circularity (11.7%) and electron-dense granules (36.89%). In parallel, nonuniformity of matrix, faint cristae, greater lysosomal bodies, and lesser intramitochondrial granules were seen in ALS platelet mitochondria. Significantly greater area (26.88%), perimeter (15%), circularity (3.76%), and integrated density (25.18%) were observed in control platelet mitochondria. Ultastructural divergence in platelets of ALS patients underlines a potential dependence of platelets on modest mitochondrial functioning. These observations also support the view that systemic involvement might be a novel feature in ALS pathophysiology.