Biomedical subjects
S K Das
Publications and source records attributed to S K Das.
Selective serotonin reuptake inhibitors-- few pitfalls.
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Induction of pro-renin converting enzyme mk9 by thyroid hormone in the guinea-pig liver.
Kallikreins are a group of specific serine proteases and are an integral part of kallikrein-kinin system. The kallikrein-kinin system is hypotensive in nature and counteracts with the renin-angiotensin system in the maintenance of normal blood pressure. So far, four kallikrein-like enzymes, namely, mK9, mK13, mK22, and mK26, have been known to convert the inactive pro-renin into biologically active renin. Some of these enzymes are induced by the thyroid hormone. In the proposed study, we investigated the effects of thyroid hormone on the expression of genes for mk9, mk13, and mk22 enzymes. We used guinea pigs as models because these animals share many characteristics in common to humans. Male adult guinea pigs were intramuscularly injected with 2 mg/kg body weight of thyronine. Forty-eight hours following the last injection, the liver was processed for Northern blot analysis using labeled mK9, mK13, and mK22 specific RNA probes. Only mK9 was found to be transcriptionally regulated by the hormone.
Long-term efficacy of botulinum toxin A in treatment of various movement disorders over a 10-year period.
Although botulinum toxin A (BTX) has been licensed in Canada for treatment of various movement disorders since 1990, few clinical studies regarding its long-term efficacy and side effects have been reported. We conducted a retrospective analysis of 235 patients who received BTX from our movement disorders clinic over a 10-year period (January 1990 to December 1999). A total of 2,616 treatment cycles (multiple injections) were administered to 235 patients with cervical dystonia (CD), hemifacial spasm (HS), blepharospasm (BP), and other movement disorders. Substantial benefit at 5 years was seen in most patients (90% in BP, 88% in HS, 63% in CD, 100% in jaw closing and lower limb dystonia, and 56% in writer's cramp). Benefit was maintained for up to 10 years in CD, HS, and BP data, with a 75.8% benefit reported. Twenty-eight percent of patients discontinued treatment during the follow-up period due to a variety of reasons. Of these, 9.1% of patients developed primary resistance, and 7.5% of patients secondary resistance. Adverse effects, mostly minor, developed in 27% of patients at any one time, occurring over 4.5% of treatment cycles. These were most frequently reported in blepharospasm (22 of 36 patients in 40 cycles), followed by hemifacial spasm (21 of 70 patients in 46 cycles), and cervical dystonia (17 of 106 in 28 cycles). Only 1.3% of patients discontinued therapy due intolerable adverse effects. The results show that BTX is a safe and effective treatment of various types of movement disorders, and most side effects are well tolerated. Discontinuation for any reason was also low after 5 years. Efficacy was maintained after long periods of treatment with high degree of patient satisfaction.
A randomized controlled trial to evaluate the slow-acting symptom modifying effects of a regimen containing colchicine in a subset of patients with osteoarthritis of the knee.
OBJECTIVE: As crystals may contribute to inflammation in osteoarthritis (OA), it is hypothesized that colchicine may have symptom/disease modifying effects in OA. The objective of this study was to evaluate the symptomatic benefit of addition of colchicine to a regimen of intraarticular steroids and piroxicam in patients with knee OA with inflammation. DESIGN: 39 patients with OA of the knee with persisting inflammation, despite at least 2 weeks of piroxicam, were subjected to intraarticular steroid injection and randomly assigned to receive colchicine 0.5 mg twice daily or placebo in a randomized, double blind, placebo-controlled trial over 5 months. RESULTS: VAS for index knee pain (VAS-pain) and total KGMC score (a modified WOMAC index) at 16 and 20 weeks were significantly better in the colchicine group than the corresponding scores in controls. The benefit persisted on multivariate analysis at 16 weeks (Hotellings T(2)=18.6, F(5,33)=3.3154, P=0.015). The proportion of patients who had 30% or greater response at 16 weeks was significantly higher in the colchicine group in VAS-pain (69% vs 15%) and total KGMC scores (74% vs 45%) and the significance persisted on combined analysis using Mantel-Haenszel test (M-H Risk=5.9, 95% C.I.: 2.08 to 16.73). At 20 weeks, benefit of colchicine therapy was seen on pooled analysis only (M-H risk=3.71, 95% C.I.: 1.07=8.02). CONCLUSION: The addition of colchicine produced significantly greater symptomatic benefit at 16 and 20 weeks than intraarticular steroid and piroxicam alone in patients with knee OA with inflammation.
Formulation approaches for orally administered poorly soluble drugs.
Despite having pharmacodynamic or target activity, many drugs fail in the drug development process due to poor bioavailability, and presently marketed conventional dosage forms of poorly soluble drugs employ high doses leading to potential toxicity. The introduction of the Biopharmaceutic Classification System (BCS) has provided a basis to categorize drugs based on the two major parameters affecting absorption, solubility and permeability. Several techniques can be employed to enhance the absorption and bioavailability of poorly soluble and poorly permeable drugs based on the BCS concept. This article is an attempt to summarize the development of various formulation approaches that are currently employed to enhance bioavailability of orally administered poorly soluble drugs.
Synthesis of conformationally locked L-iduronic acid derivatives: direct evidence for a critical role of the skew-boat 2S0 conformer in the activation of antithrombin by heparin.
We have used organic synthesis to understand the role of L-iduronic acid conformational flexibility in the activation of antithrombin by heparin. Among known synthetic analogues of the genuine pentasaccharidic sequence representing the antithrombin binding site of heparin, we have selected as a reference compound the methylated anti-factor Xa pentasaccharide 1. As in the genuine original fragment, the single L-iduronic acid moiety of this molecule exists in water solution as an equilibrium between three conformers 1C4, 4C1 and 2S0. We have thus synthesized three analogues of 1, in which the L-iduronic acid unit is locked in one of these three fixed conformations. A covalent two atom bridge between carbon atoms two and five of L-iduronic acid was first introduced to lock the pseudorotational itinerary of the pyranoid ring around the 2S0 form. A key compound to achieve this connection was the D-glucose derivative 5 in which the H-5 hydrogen atom has been replaced by a vinyl group, which is a progenitor of the carboxylic acid. Selective manipulations of this molecule resulted in the 2S0-type pentasaccharide 23. Starting from the D-glucose derivative 28, a covalent two atom bridge was now built up between carbon atoms three and five to lock the L-iduronic acid moiety around the 1C4 chair form conformation, and the 1C4-type pentasaccharide 43 was synthesized. Finally the L-iduronic acid containing disaccharide 58 which, due to the presence of the methoxymethyl substituent at position five adopts a 4C1 conformation, was directly used to synthesize the 4C1-type pentasaccharide 61. The locked pentasaccharide 23 showed about the same activity as the reference compound 1 in an antithrombin-mediated anti-Xa assay, whereas the two pentasaccharides 43 and 61 displayed very low activity. These results clearly establish the critical importance of the 2S0 conformation of L-iduronic acid in the activation of antithrombin by heparin.
Radiation-induced pulmonary toxicity: a dose-volume histogram analysis in 201 patients with lung cancer.
PURPOSE: To relate lung dose-volume histogram-based factors to symptomatic radiation pneumonitis (RP) in patients with lung cancer undergoing 3-dimensional (3D) radiotherapy planning. METHODS AND MATERIALS: Between 1991 and 1999, 318 patients with lung cancer received external beam radiotherapy (RT) with 3D planning tools at Duke University Medical Center. One hundred seventeen patients were not evaluated for RP because of <6 months of follow-up, development of progressive intrathoracic disease making scoring of pulmonary symptoms difficult, or unretrievable 3D dosimetry data. Thus, 201 patients were analyzed for RP. Univariate and multivariate analyses were performed to test the association between RP and dosimetric factors (i.e., mean lung dose, volume of lung receiving >or=30 Gy, and normal tissue complication probability derived from the Lyman and Kutcher models) and clinical factors, including tobacco use, age, sex, chemotherapy exposure, tumor site, pre-RT forced expiratory volume in 1 s, weight loss, and performance status. RESULTS: Thirty-nine patients (19%) developed RP. In the univariate analysis, all dosimetric factors (i.e., mean lung dose, volume of lung receiving >or=30 Gy, and normal tissue complication probability) were associated with RP (p range 0.006-0.003). Of the clinical factors, ongoing tobacco use at the time of referral for RT was associated with fewer cases of RP (p = 0.05). These factors were also independently associated with RP according to the multivariate analysis (p = 0.001). Models predictive for RP based on dosimetric factors only, or on a combination with the influence of tobacco use, had a concordance of 64% and 68%, respectively. CONCLUSIONS: Dosimetric factors were the best predictors of symptomatic RP after external beam RT for lung cancer. Multivariate models that also include clinical variables were slightly more predictive.
Nonequilibrium dynamics in the complex Ginzburg-Landau equation.
Results from a comprehensive analytical and numerical study of nonequilibrium dynamics in the two-dimensional complex Ginzburg-Landau equation have been presented. In particular, spiral defects have been used to characterize the domain growth law and the evolution morphology. An asymptotic analysis of the single-spiral correlation function shows a sequence of singularities-analogous to those seen for time-dependent Ginzburg-Landau models with O(n) symmetry, where n is even.
Mimicking oxide surfaces: different types of defects and ligand coordination at well defined positions of a molybdenum oxide based nanocluster.
The mixed valence cluster anion of the compound (NH4)32[MoVI110MoV28O416H6(H2O)58(CH3CO2)6].xH2O 1 (x approximately 250), synthesized under one-pot conditions, contains well-defined different types of defects--missing groups compared to the complete parent (Mo154) type cluster with full D7d symmetry--and acetate ligand coordination; this proves that the giant-wheel type anion can be considered as an object with a variety of nanoscale structural features ("nanostructured landscape") allowing reactions at a variety of well defined centers.
The 1.2 A structure of a novel quorum-sensing protein, Bacillus subtilis LuxS.
In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing. The autoinducer-2 production protein LuxS, is involved in a novel quorum-sensing system and is thought to catalyse the degradation of S-ribosylhomocysteine to homocysteine and the autoinducer molecule 4,5-dihydroxy-2,3-pentadione. The crystal structure of Bacillus subtilis LuxS has been determined at 1.2 A resolution, together with the binary complexes of LuxS with S-ribosylhomocysteine and homocysteine to 2.2 and 2.3 A resolution, respectively. These structures show that LuxS is a homodimer with an apparently novel fold based on an eight-stranded beta-barrel, flanked by six alpha-helices. Each active site contains a zinc ion coordinated by the conserved residues His54, His58 and Cys126, and includes residues from both subunits. S-ribosylhomocysteine binds in a deep pocket with the ribose moiety adjacent to the enzyme-bound zinc ion. Access to the active site appears to be restricted and possibly requires conformational changes in the protein involving the movement of residues 125-129 and those at the N terminus. The structure contains an oxidised cysteine residue in the active site whose role in the biological process of LuxS has not been determined. The autoinducer-2 signalling pathway has been linked to aspects of bacterial virulence and pathogenicity. The structural data on LuxS will provide opportunities for targeting this enzyme for the rational design of new antibiotics.
Nonequilibrium dynamics of the complex Ginzburg-Landau equation: analytical results.
We present a detailed analytical and numerical study of nonequilibrium dynamics for the complex Ginzburg-Landau equation. In particular, we characterize evolution morphologies using spiral defects. This paper is the first in a two-stage exposition. Here, we present analytical results for the correlation function arising from a single-spiral morphology. We also critically examine the utility of the Gaussian auxiliary field ansatz in characterizing a multispiral morphology. In the next paper of this exposition we will present detailed numerical results.
Global gene expression analysis to identify molecular markers of uterine receptivity and embryo implantation.
Infertility and spontaneous pregnancy losses are an enduring problem to women's health. The establishment of pregnancy depends on successful implantation, where a complex series of interactions occurs between the heterogeneous cell types of the uterus and blastocyst. Although a number of genes are implicated in embryo-uterine interactions during implantation, genetic evidence suggests that only a small number of them are critical to this process. To obtain a global view and identify novel pathways of implantation, we used a dual screening strategy to analyze the expression of nearly 10,000 mouse genes by microarray analysis. Comparison of implantation and interimplantation sites by a conservative statistical approach revealed 36 up-regulated genes and 27 down-regulated genes at the implantation site. We also compared the uterine gene expression profile of progesterone-treated, delayed implanting mice to that of mice in which delayed implantation was terminated by estrogen. The results show up-regulation of 128 genes and down-regulation of 101 genes after termination of the delayed implantation. A combined analysis of these experiments showed specific up-regulation of 27 genes both at the implantation site and during uterine activation, representing a broad diversity of molecular functions. In contrast, the majority of genes that were decreased in the combined analysis were related to host immunity or the immune response, suggesting the importance of these genes in regulating the uterine environment for the implanting blastocyst. Collectively, we identified genes with recognized roles in implantation, genes with potential roles in this process, and genes whose functions have yet to be defined in this event. The identification of unique genetic markers for the onset of implantation signifies that genome-wide analysis coupled with functional assays is a promising approach to resolve the molecular pathways required for successful implantation.
Cloning, purification, crystallization and preliminary crystallographic analysis of Bacillus subtilis LuxS.
LuxS of Bacillus subtilis is a member of a novel family of proteins with a potential role in quorum sensing, controlling important aspects of cellular physiology in a range of microbial species. B. subtilis luxS was cloned, expressed in Escherichia coli, purified and crystallized using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant. The crystals belong to one of the enantiomorphic space groups P6(1)22 or P6(5)22, with approximate unit-cell parameters a = b = 63.6, c = 151.5 A and one subunit in the asymmetric unit, corresponding to a packing density of 2.5 A(3) Da(-1). The crystals diffract X-rays to at least 1.55 A resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the key determinants of function of this class of proteins, for which no structures are currently available.
Development and characterization of cholinephosphotransferase antibody.
In the present study, we generated antibodies in rabbits against two synthetic peptides, one based on peptide sequence from yeast CPT cDNA (position 86 to 98 of the amino acid sequence) and the other from our guinea pig CPT cDNA (it corresponds to amino acid positions 119 to 130 according to yeast CPT gene). The antibody titers were measured by both dot blot analysis and ELISA using Keyhole limpets hemocyanin coupled CPT peptides. The CPT antibody recognized a single band by Western blot analysis of proteins from guinea pig liver mitochondria and microsomes. The molecular weight of the protein recognized by Western blot analysis is close to the predicted molecular weight (46 kDa) of yeast CPT. Further analysis revealed that the antibody inhibited CPT activity in both subcellular fractions in a dose dependent manner, thus confirming the specificity of the antibody against both subcellular CPT.
Acute urinary toxicity following transperineal prostate brachytherapy using a modified Quimby loading method.
PURPOSE: To examine the acute urinary toxicity following transperineal prostate implant using a modified Quimby loading method with regard to time course, severity, and factors that may be associated with a higher incidence of morbidity. METHODS AND MATERIALS: One hundred thirty-nine patients with prostate adenocarcinoma treated with brachytherapy from 1997 through 1999 had follow-up records available for review. Patients considered for definitive brachytherapy alone included those with prostate specific antigen (PSA) < or = 6, Gleason score (GS) < or = 6, clinical stage < T2b, and prostate volumes generally less than 40 cc. Patients with larger prostate volumes were given neoadjuvant antiandrogen therapy. Those with GS > 6, PSA > 6, or Stage > T2a were treated with external beam radiation therapy followed by brachytherapy boost. Sources were loaded according to a modified Quimby method. At each follow-up, toxicity was graded based on a modified RTOG urinary toxicity scale. RESULTS: Acute urinary toxicity occurred in 88%. Grade I toxicity was reported in 23%, grade II in 45%, and grade III in 20%, with 14% requiring prolonged (greater than 1 week) intermittent or indwelling catheterization. Overall median duration of symptoms was 12 months. There was no difference in duration of symptoms between patients treated with I-125 or Pd-103 sources (p = 0.71). After adjusting for GS and PSA, multivariate logistic regression analysis showed higher incidence of grade 3 toxicity in patients with larger prostate volumes (p = 0.002), and those with more seeds implanted (p < 0.001). Higher incidence of prolonged catheterization was found in patients receiving brachytherapy alone (p = 0.01), with larger prostate volumes (p = 0.01), and those with more seeds implanted (p < 0.001). CONCLUSION: Interstitial brachytherapy for prostate cancer leads to a high incidence of acute urinary toxicity, most of which is mild to moderate in severity. A prolonged need for catheterization can occur in some patients. Patients receiving brachytherapy alone, those with prostate volumes greater than 30 cc, and those implanted with a greater number of seeds have the highest incidence of significant toxicity.
Regulation of cholinephosphotransferase by thyroid hormone.
We have demonstrated earlier that thyroid hormone (T3) regulates the activity of cholinephosphotransferase (CPT) in guinea pig lung. This effect of T3 is not organ specific because we found T3 also regulates CPT activity in the guinea pig liver. Northern blot analysis using two oligonucleotide probes, one synthesized on the basis of the yeast CPT gene sequence and another on the basis of partial cDNA clone from guinea pig CPT clone, revealed that T3 stimulates the expression of new CPT mRNA. Studies with transcriptional and translational inhibitors indicated that T3 enhanced the translation of the CPT mRNA as well as translocation of preformed CPT enzyme protein from cytosol to mitochondria. Furthermore, it strengthens our previous finding that yeast CPT and guinea pig CPT have high homology in their sequence as both the oligonucleotide probes gave the similar type of Northern blot in the present study.