Biomedical subjects
Publications and source records attributed to T Das.
We have isolated a novel gene (GLELO) from Mortierella alpina and its homologue (CEELO1) from Caenorhabditis elegans and demonstrate the involvement of their encoded proteins in the elongation of C(18) polyunsaturated fatty acids.
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Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly population. The prevalence is reported to be 1.2-1.4% in several population-based epidemiological studies. Currently 25-30 million people worldwide are blind due to AMD. With the aging world population it is bound to increase significantly, and could become a significant public health problem in next two decades, with serious socio-economic implications. Several strategies are today available to treat the wet form of AMD, which is responsible for significant visual loss. These were until recently confined to laser photocoagulation, and subretinal surgery, but today two other modalities, namely, radiation and photodynamic therapy, are available. These treatment modalities however, are aimed at preservation of vision only, and not at reversing the process of the disease. Further research on antiangiogenic drugs and gene therapy could significantly help AMD patients.
Zidovudine, the anti-AIDS drug, caused inhibition of mitogen-induced proliferation and perturbation of cell-cycle progression of cultured bone marrow cells of mice. There was significant hypoploidy observed in flow cytometric analysis of AZT-treated bone marrow cells. In apo-direct analysis, cells showed apoptosis in G0/G1 phase. In DNA gel analysis, characteristic laddering of apoptosis was observed in AZT-treated bone marrow cells. We demonstrated that, when the animals were pretreated with protein A (PA) of Staphylococcus aureus, the apoptotic changes could be prevented in bone marrow cells of AZT-treated animals. There is a significant (p < 0.05) increase in proliferation of bone marrow cells subjected to mitogen treatment in PA+AZT-treated animals, compared to only AZT-treated animals. However, cell-cycle phase distribution was not hampered and no laddering in DNA gel analysis was also observed in this group. In apo-direct analysis, PA treatment showed significant (p < 0.001) inhibition of AZT-induced apoptosis. These observations indicate that by using a suitable agent such as protein A the toxic side effects of AZT could be minimized.
Apart from many of the biological properties of protein A (PA) of Staphylococcus aureus, it has been recognized recently as a B-cell superantigen. Therefore, we investigated the molecular mechanisms of PA superantigen-induced mice splenic B-cell proliferation. Treatment of resting B cells with PA-evoked cell proliferation. Binding of PA to B cells led to a cascade of signal transduction mechanisms involving tyrosine kinase that activated phospholipase C, which in turn activated protein kinase C (PKC), and translocated it from cytosol to membrane. Mitogen-activated protein (MAP) kinase has been found to be activated down-stream of PKC in this signal pathway, which ultimately caused an activation of serum-responsive factor (SRF). Inhibition at any step of this signaling cascade could block B-cell proliferation. PA could also stimulate the Bcl-2 gene expression at protein level thereby supporting the pro-proliferative effect of PA. Thus, the molecular mechanisms related to PA-induced B cell proliferation has been delineated in this report as tyrosine kinase > PLC > PKC > MAP kinase > SRF > Bcl-2. Knowledge gathered from these observations might be of immense help to study the immune cell proliferation as a part of immunoactivation process. Also, the development of suitable inhibitors of the signaling pathway outlined here might provide clues as to how to abrogate pathologic antibody production in many disease processes.
We examined optokinetic and optomotor responses of 450 zebrafish mutants, which were isolated previously based on defects in organ formation, tissue patterning, pigmentation, axon guidance, or other visible phenotypes. These strains carry single point mutations in >400 essential loci. We asked which fraction of the mutants develop blindness or other types of impairments specific to the visual system. Twelve mutants failed to respond in either one or both of our assays. Subsequent histological and electroretinographic analysis revealed unique deficits at various stages of the visual pathway, including lens degeneration (bumper), melanin deficiency (sandy), lack of ganglion cells (lakritz), ipsilateral misrouting of axons (belladonna), optic-nerve disorganization (grumpy and sleepy), inner nuclear layer or outer plexiform layer malfunction (noir, dropje, and possibly steifftier), and disruption of retinotectal impulse activity (macho and blumenkohl). Surprisingly, mutants with abnormally large or small eyes or severe wiring defects frequently exhibit no discernible behavioral deficits. In addition, we identified 13 blind mutants that display outer-retina dystrophy, making this syndrome the single-most common cause of inherited blindness in zebrafish. Our screen showed that a significant fraction (approximately 5%) of the essential loci also participate in visual functions but did not reveal any systematic genetic linkage to particular morphological traits. The mutations uncovered by our behavioral assays provide distinct entry points for the study of visual pathways and set the stage for a genetic dissection of vertebrate vision.
Protein A (PA) of Staphylococcus aureus is known as an immunomodulator. In a search of the molecular mechanism(s) of PA-induced immunocyte potentiation, we found dose-dependent binding of PA (0.01 to 100 microg/ml PA) to the mice splenic lymphocytes. Interestingly, treatment of 1 microg PA/20 g mice increased the splenic lymphocyte number approximately 5-fold over control but at a 10-microg dose the cell number was decreased compared with a 1-microg dose. Flow cytometric analysis of cell-cycle phase distribution of nuclear DNA in splenic lymphocytes showed that at a 1-microg dose, PA shifted the cell-cycle phases from G0/G1 to S and G2/M supporting the pro-proliferative role of PA. In contrast, the same inducer increased the sub-G1 cell population at a 10-microg dose indicating the breakdown of cellular DNA. These findings were supported by DNA ladder formation and nuclear breakdown at this higher dose. Further studies revealed that at a 1-microg dose, the level of the pro-proliferative/anti-apoptotic protein bcl-2 was increased in splenic lymphocytes whereas at a 10-microg dose it showed a decreasing trend. In contrast, concentrations of proapoptotic proteins, p53 and bax, were increased at a 10-microg dose. A search of the mechanism(s) of such differential action of PA at these two doses revealed that the lower dose of PA upregulated the production of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) to the extent which has already been reported by our laboratory to be beneficial to the host. However, at a larger dose, much higher release of TNF-alpha and interleukin-2 (IL-2) may account for the apoptosis of splenic cells. All these findings indicated that the cross-talk between all these pro- and anti-apoptotic factors may contribute to maintain a balance between growth and death of cells and may be one of the important factors deciding whether a cell would follow a proliferative pathway or an apoptotic pathway.
The encapsidation of vesicular stomatitis virus (VSV) genome RNA, a prerequisite step to the replication process by the nucleocapsid protein (N) was studied by its ability to package VSV leader RNA in vitro in a RNase-resistant form. The VSV leader RNA was derived from the SP6 transcription vector while the N protein was made in rabbit reticulocyte lysate. The in vitro encapsidation was carried out by translating N mRNA in the presence of 32P-labeled presynthesized leader RNA. The RNA encapsidation property of the N protein was completely abrogated when the C-terminal five amino acids (VEFDK-COOH) were deleted. Systematic mutational analyses within the C-terminal five amino acid regions reveal that the RNA encapsidation activity was lost in all mutants except K --> A and K --> R, indicating that C-terminal five amino acids, in particular the lysine residue play critical role in genome RNA encapsidation. To correlate the in vitro encapsidation abilities of these mutant N proteins with genome RNA replication, we have used a full-length cDNA clone of VSV genome RNA to rescue infectious virions from cells expressing L, P, and wt or mutant N proteins and measured the recovery of plaque forming units. The results indicate that the N mutants that are defective in in vitro encapsidation of leader RNA do not support replication, establishing the requirement of C-terminal five amino acids of the N protein in viral replication.
Protein A (PA) of Staphylococcus aureus is known to elicit several cytokines such as IFN gamma, TNF alpha and IL1. However, it has not been delineated yet as to which differentiation pathway lymphocytes follow after stimulation by PA. In this report, we attempted to collect such evidences. Cytokines, such as IFN gamma, IL2, IL4, IL6, IL10, TNF alpha, IL1alpha and IL1beta were measured in serum by ELISA. Our results show that 1 microg dose of PA stimulates the production of IFN gamma (115 +/- 5 pg/ml), TNF alpha (250 +/- 8 pg/ml) and IL1alpha (100 +/- 5 pg/ml) as compared to control levels of, 22 +/- 2, 20 +/- 2 and 35 +/- 3 pg/ml respectively whereas IL2 and IL1beta secretion were less (beyond the lower detection limit of the kit and 25 +/- 1 pg/ml, respectively) as compared to control (28 +/- 2 and 52 +/- 4 pg/ml, respectively). Larger dose of PA (10 microg) increases the expression of IL2 (75 +/- 3 pg/ml), TNF alpha (1380 +/- 120 pg/ml), IL1alpha (495 +/- 10 pg/ml) and IL1beta (110 +/- 7 pg/ml) as compared to controls described above. We also observed that 1 microg dose of PA decreases IL4, IL6 and IL10 secretion to 9 +/- 1, 10 +/- 1 and 10 +/- 2 pg/ml, respectively, whereas 10 microg dose also decreased them to 11 +/- 1, 12 +/- 2 and 30 +/- 4 pg/ml, respectively as compared to the background controls, i.e. 50 +/- 5, 50 +/- 2 and 215 +/- 9 pg/ml respectively. The ratio of IFN gamma to IL4 increased and the peak value at 4 h, came to 13 +/- 1 and 9.6 +/- 0.5 with 1 microg and 10 microg PA, respectively, which is an established parameter indicating a Th1 type response. Flow cytometry analysis of CD4+/CD8+ cells, and c-myc protein expression by splenocytes indicate that 1 microg dose of PA causes 2-fold increase of CD4+ cells with no change in CD8+ cells, and 10-fold increase in c-myc protein, whereas 10 microg dose increases CD4+ cells 4-fold, CD8+ cells 3-fold and c-myc protein 100-fold. The cell cycle data shows an induction of apoptosis in thymocytes and splenocytes with the large dose (10 microg), whereas the 1 microg dose does not show any apoptosis. This report indicates that a Th1 response is induced in mice, after PA inoculation at a dose of 1 microg animal. Thus, cytokine mediated therapeutic strategies should consider the fact that an induction of large concentration of some cytokines might become detrimental to the host.
Protein A (PA) of Staphylococcus aureus is known for its immunostimulatory, anti-cancer, and anti-toxic properties. The present study revealed that PA stimulates specific immunocytes to act as a potential immunorestorer. It has also been shown that the percentage of various cell types bearing different clusters of differentiation markers, e.g., CD4(+), CD8(+), CD19(+), increases considerably after inoculation with PA. It has also been observed that CD34(+) progenitor cells of bone marrow also increased significantly (P < 0.05) upon PA treatment. PA significantly elevated Th-1 cytokines, e.g., IFN-gamma, TNF-alpha, and IL-1alpha. The increased percentages of CD4(+), CD8(+), CD19(+), CD34(+) cells and elevated cytokine levels in PA treated animals may contribute to the reported anti-tumor, anti-fungal, anti-parasitic, and anti-toxic properties of PA. Since in various diseased conditions and during toxic drug therapy lymphocytes bearing such differentiation markers get suppressed, this type of approach could help in immunorestoration of the host. These findings might help in designing therapeutic approaches toward various diseases which cause immunosuppression.
The purpose of this study was to determine the long-term safety of transplanting human fetal neuroretinal cells (14 to 18 week gestational age) into a series of patients with advanced retinitis pigmentosa (RP). After obtaining informed consent, both hosts and mothers of donors were screened for transmissible diseases. Pre- and postoperative clinical exams, visual acuity, electroretinograms, and fluorescein angiograms were performed and visual field testing was attempted in each case. Surgically, an anterior approach through pars plana ciliaris was used. A retinotomy was performed in the paramacular area and a two-function cannula was introduced into the subretinal space to deliver a suspension of donor cells. The cell suspension carried approximately 4000 cells/microl; the volume injected did not exceed 150 microl. The patients were examined for periods ranging from 12 to 40 months posttransplantation. To date, no evidence of inflammation, infection, or overt rejection of the graft was noted in the host eye, neither was any change observed in the contralateral, unoperated eye. In conclusion, neuroretinal cells were injected into the subretinal space of 14 patients with advanced RP with no clinical appearance of detrimental effects at the time of surgery or up to 40 months postinjection except in 1 patient who developed retinal detachment. This sets the stage for a phase II clinical trial to determine the possible beneficial effects of this procedure in patients blinded by degenerative retinal disease.
PURPOSE: To present the microbial spectrum and susceptibilities of isolates in postoperative endophthalmitis. METHOD: Isolates from 206 eyes of 206 patients who underwent vitrectomy for postoperative endophthalmitis were examined. RESULTS: One-hundred twelve (54.4%) of 206 vitreous samples were culture positive and 14 (12.5%) of 112 culture-positive cases were polymicrobial, yielding a total of 126 isolates. Isolates included 59 (46.8%) gram-positive cocci, eight (6.3%) gram-positive bacilli, 33 (26.2%) gram-negative organisms, five (4.0%) Actino-mycetes-related organisms, and 21 (16.7%) fungi. Susceptibilities to amikacin, ceftazidime, chloramphenicol, cefazolin, ciprofloxacin, gentamicin, and vancomycin are reported. CONCLUSIONS: This is the largest, single-center, prospective series on microbial susceptibilities in postoperative endophthalmitis. We report a high prevalence of gram-negative species and fungi, suggesting that empiric therapy should include coverage for gram-negative pathogens and for fungal pathogens in appropriate settings.
PURPOSE: To present the microbial spectrum and susceptibilities of isolates in posttraumatic endophthalmitis. METHOD: Isolates from 182 eyes of 182 patients who underwent vitrectomy for posttraumatic endophthalmitis were examined. RESULTS: One hundred thirteen (62.1%) of 182 vitreous samples were culture-positive, and 23 (20.4%) of 113 culture-positive cases were polymicrobial, including three (2.7%) trimicrobial cases, yielding a total of 139 isolates. Isolates included 63 (45.3%) gram-positive cocci, 24 (17.3%) gram-positive bacilli, 25 (18.0%) gram-negative organisms, seven (5.0%) Actinomycetes-related organisms, and 20 (14.4%) fungi. Susceptibilities to amikacin, ceftazidime, chloramphenicol, cefazolin, ciprofloxacin, gentamicin, and vancomycin are reported. CONCLUSIONS: This study represents a large series on microbial spectrum and susceptibilities in posttraumatic endophthalmitis. We report a high prevalence of gram-positive bacilli species and polymicrobial infections containing gram-negative species, underscoring the importance of broad-spectrum, combination antibiotics in the empiric treatment of posttraumatic endophthalmitis.
Fibroblast growth factors (FGFs) stimulate proliferation, differentiation and motility of different cell types. The cellular effects of FGF are transduced by its interaction with any one of four members of a family of high affinity, cell surface FGF receptors (FGFRs) that have autophosphorylating tyrosine kinase activity. Activation of FGFR causes release of various low molecular weight signaling molecules which are required for the pleotropic effects of FGFs. We report here that basic FGF plays critical role in membrane phospholipid hydrolysis in NIH 3T3 cells that are stably transfected with FGFR1. Upon binding to FGFR1, basic FGF stimulates cytosolic form of phospholipase A2 (cPLA2), phospholipase C-gamma1 (PLC-gamma1) and phospholipase D (PLD), the key enzymes for the production of various lipid second messengers, in a tyrosine kinase-dependent manner. In addition to tyrosine phosphorylation, cPLA2 catalytic activation requires serine phosphorylation by p42 mitogen-activated protein (MAP) kinase and possibly pertussis toxin-sensitive G-protein coupling. On the other hand, phosphatidyl inositol 4,5 bisphosphate (PIP2) hydrolysis requires direct phosphorylation at tyrosine residue of the PLC-gamma1 isozyme. The activation of PLD needs direct or indirect receptor tyrosine kinase and protein kinase C (PKC) activities. Additionally, it also requires botulinum toxin C-sensitive Rho-like G-protein activation. All these results suggest that the pleotropic effects of FGF are exerted through its tyrosine kinase receptors and individual effectors are activated via distinguishable signaling mechanisms according to the cell's need.
PURPOSE: To determine the effects of intravitreal dexamethasone in patients with exogenous fungal endophthalmitis. METHODS: Twenty cases of culture-proven exogenous fungal endophthalmitis following cataract surgery (11/20) and trauma (9/20) were retrospectively analysed for pre- and postoperative visual acuity, anterior chamber and vitreous inflammation and media clarity. All patients were managed with pars plana vitrectomy with intravitreal amphotericin B and oral ketoconazole with (steroid plus group) or without (steroid minus group) intravitreal dexamethasone. Results were analysed by Fisher's exact test. RESULTS: Following vitrectomy 9 of 20 patients (45%) achieved a visual acuity better than counting fingers at 3 m. No statistically significant difference was observed in anatomical and visual outcome between the steroid plus and steroid minus groups, though the number of patients with favourable visual outcome was greater in the steroid plus group. Rate of clearance of inflammation was better in the steroid plus group (40 +/- 15.5 vs 55 +/- 8.6 days). All patients (6/20) with pre-operative vision better than counting fingers showed good anatomical and visual outcome in both groups. CONCLUSIONS: The results of our retrospective study suggest that steroids may be beneficial in promoting faster clearance of inflammation in fungal endophthalmitis. Sensitivity of the fungi to antifungals, dose and timing of steroid and institution of effective antifungal medication prior to the use of steroids are the essential factors which need to be examined further in a prospective manner.
BACKGROUND: Prevention of variceal bleeding, a major cause of morbidity and mortality, is an important goal in the management of patients with portal hypertension (PHT). Although propranolol has been found useful in preventing the first episode of variceal bleeding (primary prophylaxis) in cirrhotic PHT, it has limitations which include side effects, contraindications, non-compliance and failure in some patients. Endoscopic variceal ligation (EVL) has not been used for primary prophylaxis. METHODS: Thirty cirrhotic patients with PHT, grade III to IV oesophageal varices, hepatic venous pressure gradient > or = 12 mmHg and no prior history of upper gastrointestinal bleeding were randomized to receive propranolol (to reduce their pulse rate by 25% from baseline, n = 15) and EVL (weekly to fortnightly until variceal eradication, n = 15). The two groups were comparable. All the patients in EVL group had variceal eradication during 3.8 +/- 2.2 sessions. RESULTS: There was no major complication or interval bleeding. During a follow-up period of 17.6 +/- 4.7 months, varices recurred in three, two of which bled (successfully treated by EVL). In contrast, during this period of follow up one patient in the propranolol group had variceal bleeding (P=NS). Side effects of propranolol included symptomatic bradycardia requiring reduction of dose in one of 15 patients. CONCLUSIONS: Although sample size in this study is small, it seems that EVL may be a good option for primary prophylaxis for variceal bleeding in patients with cirrhotic PHT; further studies on a larger number of patients and longer follow up are required.
The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a key subunit of the viral RNA-dependent RNA polymerase complex. The protein is phosphorylated at multiple sites in two different domains. We recently showed that specific serine and threonine residues within the amino-terminal acidic domain I of P protein must be phosphorylated for in vivo transcription activity, but not for replication activity, of the polymerase complex. To examine the role of phosphorylation of the carboxy-terminal domain II residues of the P protein in transcription and replication, we have used a panel of mutant P proteins in which the phosphate acceptor sites (Ser-226, Ser-227, and Ser-233) were altered to alanines either individually or in various combinations. Analyses of the mutant proteins for their ability to support replication of a VSV minigenomic RNA suggest that phosphorylation of either Ser-226 or Ser-227 is necessary for optimal replication activity of the protein. The mutant protein (P226/227) in which both of these residues were altered to alanines was only about 8% active in replication compared to the wild-type (wt) protein. Substitution of alanine for Ser-233 did not have any adverse effect on replication activity of the protein. In contrast, all the mutant proteins showed activities similar to that of the wt protein in transcription. These results indicate that phosphorylation of the carboxy-terminal domain II residues of P protein are required for optimal replication activity but not for transcription activity. Furthermore, substitution of glutamic acid residues for Ser-226 and Ser-227 resulted in a protein that was only 14% active in replication but almost fully active in transcription. Taken together, these results, along with our earlier studies, suggest that phosphorylation of residues at two different domains in the P protein regulates its activity in transcription and replication of the VSV genome.
AIM: To evaluate the efficacy of intravitreal dexamethasone co-administered with intravitreal antibiotics along with vitrectomy in the management of exogenous bacterial endophthalmitis. METHODS: In a prospective randomised clinical trial, 63 patients (63 eyes) with suspected bacterial endophthalmitis (postoperative and post-traumatic) were treated with vitrectomy and intravitreal antibiotics and randomised to intravitreal dexamethasone (IOAB with = 29 eyes) and no dexamethasone (IOAB without = 34 eyes). Inflammation score (IS) and visual acuity were measured by two masked observers before surgery, and at 1, 4, and 12 weeks after surgery in both the groups. RESULTS: There was significant reduction (p <0.0001) in IS at 1, 4, and 12 weeks after the surgery in the "IOAB with" group; there was temporary but significant increase (p <0.01) in IS at 1 week in the "IOAB without" group, before decline (p <0.001) of IS at 4 and 12 weeks. The magnitude and relative percentage change in IS between the two groups were found to be significant at 1 (p <0.0001), and 4 (p <0.01) weeks, and not at 12 weeks. The visual acuity at 12 weeks was comparable in both the IOAB with and IOAB without groups. CONCLUSION: Intravitreal dexamethasone helps in early reduction of inflammation in exogenous bacterial endophthalmitis, but has no independent influence on the visual outcome. In selected patients with endophthalmitis where oral corticosteroids cannot be given for medical reasons intravitreal corticosteroids could be beneficial; in other situations they could be complementary to oral corticosteroid therapy.