Kurien, Biji T. | Porter, Andrew | Dorri, Yaser | Iqbal, Saqib | D'Souza, Anil | Singh, Anil | Asfa, Sima | Cartellieri, Marc | Matsumoto, Hiroyuki | Bachmann, Michael | Hensley, Kenneth | Scofield, R. Hal
Our previous work showed that immunization of rabbits with 4-hydroxy 2-nonenal-modified Ro60 (HNE-Ro60) accelerates autoimmunity. We extended this model into mice, hypothesizing that the severity of autoimmunity would be dependent on degree of HNE-modification of Ro60. Five groups of BALB/c mice (ten/group) were used. Group I was immunized with Ro60. Groups II to IV were immunized with Ro60 modified with 0.4 mM (low), 2 mM (medium) and 10 mM (high) HNE respectively. Group V controls received Freund's adjuvant. A rapid abrogation of tolerance to Ro60/La antigens occurred in mice immunized with HNE-modified Ro60, especially in the low and medium HNE-Ro60 groups. Lymphocytic infiltration and significantly high decrement in salivary flow (37%) compared to controls was observed only in high HNE-Ro60 group, suggesting induction of a SS-like condition in this group. Anti-dsDNA occurred only in mice immunized with medium HNE-Ro60. This group did not have significant decrement in salivary flow, suggesting induction of SLE-like manifestation in this group. Significantly high antibodies to Ro60 were found in saliva of mice in low and medium HNE-Ro60, and Ro60 groups as well as anti-HNE Ro60 in low and medium HNE-Ro60 groups. Understanding the mechanism of this differential induction may help delineate between these two autoimmune diseases.
doi:10.1016/j.freeradbiomed.2010.10.687
PMCID: PMC3591494
PMID: 20946951
Sjögren's syndrome; SLE; autoimmunity; epitope spreading; autoantibodies; antigens; 4-hydroxy-2-nonenal; oxidative damage
Here, we report the draft genome sequence of crude oil-degrading Pseudomonas aeruginosa strain N002, isolated from a crude oil-polluted soil sample from Geleky, Assam, India. Multiple genes potentially involved in crude oil degradation were identified.
doi:10.1128/genomeA.00104-12
PMCID: PMC3569314
PMID: 23405324
The present work was conducted to understand the basis of adaptation in Caragana jubata in its niche environment at high altitude cold desert of Himalaya. Molecular data showed predominance of genes encoding chaperones and those involved in growth and development at low temperature (LT), a major cue operative at high altitude. Importantly, these genes expressed in C. jubata in its natural habitat. Their homologues in Arabidopsis thaliana, Oryza sativa, and Glycine max did not exhibit similar trend of gene expression at LT. Constitutive expression and a quick up-regulation of the above genes suggested the ability of C. jubata to adjust its cellular machinery to maintain growth and development in its niche. This was reflected in LT50 (the temperature at which 50% injury occurred) and LT mediated photosynthetic acclimatory response. Such molecular and physiological plasticity enables C. jubata to thrive in the high altitude cold desert of Himalayas.
doi:10.1038/srep01022
PMCID: PMC3535672
PMID: 23289064
Tegla, Cosmin A. | Cudrici, Cornelia | Rozycka, Monika | Soloviova, Katerina | Ito, Takahiro | Singh, Anil K. | Khan, Aamer | Azimzadeh, Philippe | Andrian-Albescu, Maria | Khan, Anver | Niculescu, Florin | Rus, Violeta | Judge, Susan I. V. | Rus, Horea
Voltage-gated potassium (Kv) channels play an important role in the regulation of growth factor-induced cell proliferation. We have previously shown that cell cycle activation is induced in oligodendrocytes (OLGs) by complement C5b-9, but the role of Kv channels in these cells had not been investigated. Differentiated OLGs were found to express Kv1.4 channels, but little Kv1.3. Exposure of OLGs to C5b-9 modulated Kv1.3 functional channels and increased protein expression, whereas C5b6 had no effect. Pretreatment with the recombinant scorpion toxin rOsK-1, a specific Kv1.3 inhibitor, blocked the expression of Kv1.3 induced by C5b-9. rOsK-1 inhibited Akt phosphorylation and activation by C5b-9 but had no effect on ERK1 activation. These data strongly suggest a role for Kv1.3 in controlling the Akt activation induced by C5b-9. Since Akt plays a major role in C5b-9-induced cell cycle activation, we also investigated the effect of inhibiting Kv1.3 channels on DNA synthesis. rOsK-1 significantly inhibited the DNA synthesis induced by C5b-9 in OLG, indicating that Kv1.3 plays an important role in the C5b-9-induced cell cycle. In addition, C5b-9-mediated myelin basic protein and proteolipid protein mRNA decay was completely abrogated by inhibition of Kv1.3 expression. In the brains of multiple sclerosis patients, C5b-9 co-localized with NG2+ OLG progenitor cells that expressed Kv1.3 channels. Taken together, these data suggest that Kv1.3 channels play an important role in controlling C5b-9-induced cell cycle activation and OLG dedifferentiation, both in vitro and in vivo.
doi:10.1016/j.yexmp.2011.04.006
PMCID: PMC3139709
PMID: 21540025
Voltage-gated potassium channels; Oligodendrocyte; Complement activation; C5b-9; Multiple sclerosis
Cone vision mediated by photoreceptor cyclic nucleotide-gated (CNG) channel is essential for central and color vision and visual acuity. Cone CNG channel is composed of two structurally related subunit types, CNGA3 and CNGB3. Naturally occurring mutations in cone CNG channel are associated with a variety of cone diseases including achromatopsia, progressive cone dystrophy, and some maculopathies. Nevertheless, our understanding of the structure of cone CNG channel is quite limited. This is, in part, due to the challenge of studying cones in a rod-dominant mammalian retina. We have demonstrated a robust expression of cone CNG channel and lack of rod CNG channel in the cone-dominant Nrl−/− retina and shown that the Nrl−/− mouse line is a valuable model to study cone CNG channel. This work examined the complex structure of cone CNG channel using infrared fluorescence Western detection combined with chemical cross-linking and blue native-PAGE. Our results suggest that the native cone CNG channel is a heterotetrameric complex likely at a stoichiometry of three CNGA3 and one CNGB3.
doi:10.1007/978-1-4614-0631-0_98
PMCID: PMC3370941
PMID: 22183405
retina; photoreceptor; cone; CNG channel; achromatopsia
US Food and Drug Administration has recently approved transoral robotic surgery for the treatment of some benign tumors and selected malignant tumors of the head and neck. Robotically-assistance in ear, nose and throat surgery is established and will play an increasingly large role in the future of surgical practice. Anesthesiologists need to modify their management and familiarize themselves with the upcoming robotic procedures to ensure better patient outcomes and improve patient safety.
doi:10.4103/0970-9185.94895
PMCID: PMC3339730
PMID: 22557748
Da Vinci surgical system; head and neck cancer; TORS; transoral robotic surgery
Pyogenic granuloma is an overzealously proliferative non-neoplastic lesion of connective tissue origin, found commonly in oral cavity and is secondary to chronic low grade local irritation, poor oral hygiene, and hormonal disturbances. The term is misnomer because a lesion is unrelated to infection. It is characterized by excessive and exuberant tissue repair response with varied inflammatory component. Since it is a benign lesion, choice of treatment is surgical excision with removal of underlying cause if any. This article aims at presenting a case of pyogenic granuloma in an extremely old patient which is unusual as it attained a very large size and also has caused mild resorption of underlying alveolar bone of mandible.
doi:10.4103/0975-5950.102170
PMCID: PMC3513816
PMID: 23251065
Inflammatory hyperplasia; proliferative lesions; pyogenic granuloma
Mycobacterium abscessus is an emerging human pathogen responsible for lung infections, skin and soft-tissue infections and disseminated infections in immunocompromised patients. It may exist either as a smooth (S) or rough (R) morphotype, the latter being associated with increased pathogenicity in various models. Genetic tools for homologous recombination and conditional gene expression are desperately needed to allow the study of M. abscessus virulence. However, descriptions of knock-out (KO) mutants in M. abscessus are rare, with only one KO mutant from an S strain described so far. Moreover, of the three major tools developed for homologous recombination in mycobacteria, only the one based on expression of phage recombinases is working. Several conditional gene expression tools have recently been engineered for Mycobacterium tuberculosis and Mycobacterium smegmatis, but none have been tested yet in M. abscessus. Based on previous experience with genetic tools allowing homologous recombination and their failure in M. abscessus, we evaluated the potential interest of a conditional gene expression approach using a system derived from the two repressors system, TetR/PipOFF. After several steps necessary to adapt TetR/PipOFF for M. abscessus, we have shown the efficiency of this system for conditional expression of an essential mycobacterial gene, fadD32. Inhibition of fadD32 was demonstrated for both the S and R isotypes, with marginally better efficiency for the R isotype. Conditional gene expression using the dedicated TetR/PipOFF system vectors developed here is effective in S and R M. abscessus, and may constitute an interesting approach for future genetic studies in this pathogen.
doi:10.1371/journal.pone.0029306
PMCID: PMC3240655
PMID: 22195042
Athmaram, TN | Saraswat, Shweta | Santhosh, SR | Singh, Anil Kumar | Suryanarayana, VVS | Priya, Raj | Gopalan, N | Parida, Manmohan | Lakshmana Rao, PV | Vijayaraghavan, R
Currently available vaccines for the pandemic Influenza A (H1N1) 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA) based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI) activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat.
doi:10.1186/1743-422X-8-524
PMCID: PMC3251546
PMID: 22126628
Hemagglutinin; H1N1; Pichia pastoris; secreted expression; Influenza recombinant vaccine
Objective:
To investigate standardized ethyl acetate fraction of Rhododendron arboreum (EFRA) flowers for antidiarrheal activity in experimental animals.
Materials and Methods:
A simple sensitive high performance thin layer chromatography (HPTLC) method was used for the determination of hyperin in EFRA. The standardized fraction was investigated for castor oil, magnesium sulfate-induced diarrhea, measurement of gastrointestinal transit using charcoal and castor oil-induced enteropooling.
Results:
The concentration of hyperin in flowers of R. arboreum was found to be 0.148% by HPTLC. Oral administration of EFRA at 100, 200 and 400 mg/kg exhibited dose-dependent and significant (P<0.05-0.001) antidiarrheal potential in castor oil and magnesium sulfate-induced diarrhea. EFRA at doses of 100, 200 and 400 mg/kg also produced significant (P<0.05-0.001) dose-dependent reduction in propulsive movement in castor oil-induced gastrointestinal transit using charcoal meal in rats. EFRA was found to possess an antienteropooling in castor oil-induced experimental animals by reducing both weight and volume of intestinal content significantly.
Conclusion:
These findings demonstrate that standardized ethyl acetate fraction of R. arboreum flowers has potent antidiarrheal activity thus justifying its traditional use in diarrhea and have great potential as a source for natural health products.
doi:10.4103/0253-7613.89827
PMCID: PMC3229786
PMID: 22144775
Antidiarrheal; enteropooling; gastrointestinal motility; high performance thin layer chromatography; Rhododendron arboreum
BACKGROUND:
The protease-antiprotease hypothesis proposes that inflammatory cells and oxidative stress in chronic obstructive pulmonary disease (COPD) produce increased levels of proteolytic enzymes (neutrophil elastase, matrix metalloproteinases [MMP]) which contribute to destruction of parenchyma resulting in progressive decline in forced expiratory volume in one second. Doxycycline, a tetracycline analogue, possesses anti-inflammatory properties and inhibits MMP enzymes.
OBJECTIVES:
To assess the effect of 4 weeks doxycycline in a dose of 100 mg once a day in patients of moderate to severe COPD with stable symptoms.
METHODS:
In an interventional, randomized, observer-masked, parallel study design, the effect of doxycycline (100 mg once a day for 4 weeks) was assessed in patients of COPD having stable symptoms after a run-in period of 4 weeks. The study participants in reference group did not receive doxycycline. The parameters were pulmonary functions, systemic inflammation marker C-reactive protein (CRP), and medical research council (MRC) dyspnea scale. Use of systemic corticosteroids or antimicrobial agents was not allowed during the study period.
RESULTS:
A total of 61 patients completed the study (31 patients in doxycycline group and 30 patients in reference group). At 4 weeks, the pulmonary functions significantly improved in doxycycline group and the mean reduction in baseline serum CRP was significantly greater in doxycycline group as compared with reference group. There was no significant improvement in MRC dyspnea scale in both groups at 4 weeks.
CONCLUSION:
The anti-inflammatory and MMP-inhibiting property of doxycycline might have contributed to the improvement of parameters in this study.
doi:10.4103/1817-1737.84777
PMCID: PMC3183640
PMID: 21977068
Anti-inflammatory; C-reactive protein; doxycycline; dyspnea; matrix metalloproteinase; respiratory function tests
The term unicystic ameloblastoma (UA) refers to those cystic lesions that show clinical, radiographic, or gross features of a jaw cyst, but on histologic examination show a typical ameloblastomatous epithelium lining part of the cyst cavity, with or without luminal and/or mural tumor growth. Although the histology suggests that cystic ameloblastomas follow a biologically low-grade course, recent evidence suggests that they may often behave clinically as aggressive tumors. This is supported by the high incidence of cortical perforation, tooth resorption, increase in lesion size, bony destruction, and a high rate of recurrence after simple enucleation. Here, the authors present a case report on unicystic variant of ameloblastoma in the maxilla. An attempt has been made to emphasize that it can involve the maxillary jaw, which is rarely affected and could be more aggressive than previously thought. A literature review on the topic has been added along with the case report. It is important to remember that a proper and timely diagnosis of the character and extent of a UA (with a thorough histopathologic examination of the entire specimen) can help in the overall long-term well-being of the patient.
doi:10.4103/0975-5950.94472
PMCID: PMC3343406
PMID: 22639505
Ameloblastoma; maxilla; unicystic
Tuberculosis (TB) is a communicable disease caused by Mycobacterium tuberculosis, which is transmitted by aerosolized saliva droplets among individuals in close contact with expelled sputum of a diseased patient. However, TB lesions of the oral cavity are often overlooked in the differential diagnosis. We report here a case of tuberculosis of oral cavity affecting the gingiva of a 24-year-old male.
doi:10.4103/0975-5950.94476
PMCID: PMC3343410
PMID: 22639508
Gingiva; oral mucosa; tuberculous ulcer
Objective:
To evaluate the hepatoprotective potential of ethyl acetate fraction of Rhododendron arboreum (Family: Ericaceae) in Wistar rats against carbon tetrachloride (CCl4)-induced liver damage in preventive and curative models.
Materials and Methods:
Fraction at a dose of 100, 200, and 400 mg/kg was administered orally once daily for 14 days in CCl4-treated groups (II, III, IV, V and VI). The serum levels of glutamic oxaloacetic transaminase (SGOT), glutamate pyruvate transaminase (SGPT), alkaline phosphatase (SALP), γ-glutamyltransferase (γ -GT), and bilirubin were estimated along with activities of glutathione S-transferase (GST), glutathione reductase, hepatic malondialdehyde formation, and glutathione content.
Result and Discussion:
The substantially elevated serum enzymatic activities of SGOT, SGPT, SALP, γ-GT, and bilirubin due to CCl4 treatment were restored toward normal in a dose-dependent manner. Meanwhile, the decreased activities of GST and glutathione reductase were also restored toward normal. In addition, ethyl acetate fraction also significantly prevented the elevation of hepatic malondialdehyde formation and depletion of reduced glutathione content in the liver of CCl4-intoxicated rats in a dose-dependent manner. Silymarin used as standard reference also exhibited significant hepatoprotective activity on post-treatment against CCl4-induced hepatotoxicity in rats. The biochemical observations were supplemented with histopathological examination of rat liver sections. The results of this study strongly indicate that ethyl acetate fraction has a potent hepatoprotective action against CCl4-induced hepatic damage in rats.
doi:10.4103/0253-7613.81518
PMCID: PMC3113381
PMID: 21713093
Hepatotoxicity; phytochemicals; Rhododendron arboreum (Ericaceae) carbon tetrachloride
Aim:
To monitor and evaluate the pattern of ADRs to oseltamivir in pediatric population suffering from H1N1 influenza at a tertiary care hospital.
Materials and Methods:
Children offered oseltamivir for treatment and chemoprophylaxis were monitored for adverse events by direct questioning for symptoms and clinical examination on day 5 and day 10. Assessment of neurological events was done by asking the parents or guardians regarding development of specific symptoms. Adverse events obtained were analyzed for severity, causality and age-group wise.
Results:
Out of 191 children (median age, 3 years), 69 (36.1%) developed ADRs. Most common symptoms were vomiting (16.2%) followed by diarrhea (12.0%), ear disorders (8.9%), and insomnia (6.8%). The incidence of neuropsychiatric symptoms was 12.6% which were mild-to-moderate on severity scale. There was no significant difference in the incidence of adverse events between children less than 1 year and other age groups.
Conclusion:
Oseltamivir is well tolerated in Indian children with suspected or confirmed H1N1 influenza. Our study also indicates safety of oseltamivir in infants.
doi:10.4103/0976-500X.81901
PMCID: PMC3127335
PMID: 21772769
Adverse drug reactions; antiviral agent; child; infant; oseltamivir
Fakhri, Asghar A | Thai, Ngoc L | Oliva, Jose | Dishart, Michael K | Veynovich, Bryan | Yamrozik, June A | Williams, Ronald B | Grant, Saundra B | Poydence, Jacqueline | Rathi, Vikas K | Singh, Anil | Tom, Kusum B | Nathan, Swami | Biederman, Robert WW
doi:10.1186/1532-429X-13-S1-P137
PMCID: PMC3106952
Multiple sclerosis (MS) is chronic inflammatory demyelinating disease of the central nervous system that is mediated by activated lymphocytes, macrophages/microglia, and complement. In MS, the myelin-forming oligodendrocytes (OLGs) are the targets of the immune attack. Experimental evidence indicates that C5b-9 plays a role in demyelination during the acute phase of experimental allergic encephalomyelitis (EAE). Terminal complement C5b-9 complexes are capable of protecting OLGs from apoptosis. During chronic EAE complement C5 promotes axonal preservation, remyelination and provides protection from gliosis. These findings indicate that the activation of complement and C5b-9 assembly can also have protective roles during demyelination.
doi:10.1016/j.jneuroim.2009.06.006
PMCID: PMC2756021
PMID: 19577811
Oligodendrocyte; C5b-9 complement complex; apoptosis; experimental allergic encephalomyelitis; multiple sclerosis; insulin-like growth factor binding proteins
Background
Resistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin.
Methodology/Principal Finding
We show that the minimal promoter of human resistin lies within ∼80 bp sequence upstream of the transcriptional start site (−240) whereas binding sites for cRel, CCAAT enhancer binding protein α (C/EBP-α), activating transcription factor 2 (ATF-2) and activator protein 1 (AP-1) transcription factors, important for induced expression, are present within sequences up to −619. Specificity Protein 1(Sp1) binding site (−276 to −295) is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPARγ) is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPARγ in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA) upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPARγ on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPARγ interaction. Chromatin immunoprecipitation (ChIP) assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPARγ, chromatin modifier histone deacetylase 1 (HDAC1) and the acetylated form of histone H3 but not cRel, C/EBP-α and phospho c-Jun during resistin gene transcription.
Conclusion
Our findings suggest a complex interplay of Sp1 and PPARγ along with other transcription factors that drives the expression of resistin in human monocytic U937 cells.
doi:10.1371/journal.pone.0009912
PMCID: PMC2848011
PMID: 20360975
Background:
Portable ultrasound machines are highly valuable in ICUs, where a patient's condition might not permit shifting the patient to the USG department for imaging. Traditionally central lines are put blindly using anatomical landmarks, which often result in complications such as difficulty in access, misplaced lines, pneumothorax, bleeding from inadvertent arterial punctures, etc. Ultrasonography provides “real time” imaging, i.e., the needle can be visualized entering the vein.
Aims:
We performed a study to compare USG guided central venous cannulation (CVC) and conventional anatomical landmark approach to CVC, in terms of ease of cannulation, time consumed, and associated complications.
Settings and Design:
The study was performed in a 16-bed open ICU. Eighty patients were randomly divided in two groups.
Materials and Methods:
The right internal jugular vein (IJV) was cannulated in all. In Group I, a portable ultrasound machine was used during cannulation. The vessels were visualized in the transverse section with the internal carotid artery (ICA) identified as a circular pulsatile structure, while the IJV as a lateral, oval nonpulsatile structure). The needle was inserted perpendicular to the skin under visualization on the US screen. Central venous line was then inserted by the Seldinger technique. In Group II, CVC was performed by the conventional landmark approach. The parameters studied included time for insertion, attempts required, and complications encountered.
Statistical Analysis:
The database of all parameters was analyzed using SPSS software version 10.5.
Results:
The mean time to successful insertion was 145 and 176.4 sec in groups I and II, respectively (p = 0.00). An average of 1.2 attempts per cannulation was required for group I, while 1.53 for group II (p = 0.03): 10% witnessed arterial puncture and 2.5% pneumothorax in group I and none in group II.
Conclusion:
USG-guided CVC is thus easier, quicker, and safer than landmark approach.
doi:10.4103/0972-5229.60174
PMCID: PMC2856149
PMID: 20436690
central venous cannulation; intensive care unit; ultrasound
Background
In Arabidopsis thaliana (L.) Heynh and Oryza sativa L., a large number of genes encode proteins of unknown functions, whose characterization still remains one of the major challenges. With an aim to characterize these unknown proteins having defined features (PDFs) in plants, we have chosen to work on proteins having a cystathionine β-synthase (CBS) domain. CBS domain as such has no defined function(s) but plays a regulatory role for many enzymes and thus helps in maintaining the intracellular redox balance. Its function as sensor of cellular energy has also been widely suggested.
Results
Our analysis has identified 34 CBS domain containing proteins (CDCPs) in Arabidopsis and 59 in Oryza. In most of these proteins, CBS domain coexists with other functional domain(s), which may indicate towards their probable functions. In order to investigate the role(s) of these CDCPs, we have carried out their detailed analysis in whole genomes of Arabidopsis and Oryza, including their classification, nomenclature, sequence analysis, domain analysis, chromosomal locations, phylogenetic relationships and their expression patterns using public databases (MPSS database and microarray data). We have found that the transcript levels of some members of this family are altered in response to various stresses such as salinity, drought, cold, high temperature, UV, wounding and genotoxic stress, in both root and shoot tissues. This data would be helpful in exploring the so far obscure functions of CBS domain and CBS domain-containing proteins in plant stress responses.
Conclusion
We have identified, classified and suggested the nomenclature of CDCPs in Arabidopsis and Oryza. A comprehensive analysis of expression patterns for CDCPs using the already existing transcriptome profiles and MPSS database reveals that a few CDCPs may have an important role in stress response/tolerance and development in plants, which needs to be validated further through functional genomics.
doi:10.1186/1471-2164-10-200
PMCID: PMC2694836
PMID: 19400948
NorM, a putative efflux pump of Vibrio cholerae, is a member of the multidrug and toxic compound extrusion family of transporters. We demonstrate that NorM confers resistance to norfloxacin, ciprofloxacin, and ethidium bromide. Inactivation of norM rendered V. cholerae hypersensitive towards these fluoroquinolones. Multiple sequence alignment of members of its family identified several regions of high sequence conservation. The topology of NorM was determined using β-lactamase and chloramphenicol acetyltransferase fusions. The amino acid residues G184, K185, G187, P189, E190, G192, and G195 in the periplasmic loops and L381, R382, G383, Y384, K385, and D386 in the cytoplasmic loops, as well as all the acidic and cysteine residues of NorM, were mutated. Mutants G184V, G184W, K185I, P189S, E190K, and E190A lost the norfloxacin resistance-imparting phenotype characteristic of NorM. Mutants E124V, D155V, G187V, G187R, C196S, Y384H, Y384S, and Y384F exhibited partial resistance to norfloxacin. Mutants with replacements of G184 or G187 by A, K185 by R, and E190 by D retained the norfloxacin resistance phenotype of NorM. Analysis of the accumulation of norfloxacin in intact cells of Escherichia coli expressing NorM or its mutants in the presence or absence of carbonyl cyanide m-chlorophenylhydrazone supported the results obtained through susceptibility testing and argued in favor of NorM-mediated efflux as the determining factor in norfloxacin susceptibility in the genetically manipulated strains. Taken together, these results suggested that E124, D155, G184, K185, G187, P189, E190, C196, and Y384 are likely involved in NorM-dependent norfloxacin efflux. Except for D155, C196, and Y384, all of these residues are located in periplasmic loops.
doi:10.1128/AAC.00460-06
PMCID: PMC1635172
PMID: 16954325
Background
The extracellular matrix can have a profound effect upon the phenotype of cancer cells. Previous work has shown that growth of bladder cancer cells on a matrix derived from normal basement membrane suppresses many malignant features that are displayed when the cells are grown on a matrix that has been modified by malignant tumors. This work was undertaken to investigate proteome-level changes as determined by a new commercially available proteome display involving 2-dimensional chromatography for bladder cancer cells grown on different extracellular matrix preparations that modulate the expression of the malignant phenotype.
Results
Depending on the matrix, between 1300 and 2000 distinct peaks were detected by two-dimensional chromatographic fractionation of 2.1 – 4.4 mg of total cellular protein. The fractions eluting from the reversed-phase fractionation were suitable for mass spectrometric identification following only lyophilization and trypsin digestion and achieved approximately 10-fold higher sensitivity than was obtained with gel-based separations. Abundant proteins that were unique to cells grown on one of the matrices were identified by mass spectrometry. Following concentration, peaks of 0.03 AU provided unambiguous identification of protein components when 10% of the sample was analyzed, whereas peaks of 0.05 AU was approximately the lower limit of detection when the entire sample was separated on a gel and in-gel digestion was used. Although some fractions were homogeneous, others were not, and up to 3 proteins per fraction were identified. Strong evidence for post-translational modification of the unique proteins was noted. All 13 of the unique proteins from cells grown on Matrigel were related to MYC pathway.
Conclusion
The system provides a viable alternative to 2-dimensional gel electrophoresis for proteomic display of biological systems. The findings suggest the importance of MYC to the malignant phenotype of bladder cancer cells.
doi:10.1186/1477-5956-4-13
PMCID: PMC1534010
PMID: 16749926
The circadian periodicity of plasma lipid peroxide levels and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were studied in 50 clinically, bacteriologically and radiologically proven fresh cases of pulmonary tuberculosis (age: 21–45 years) and 60 age-matched healthy volunteers with diurnal activity from 06∶00 to about 22∶00 and nocturnal rest. A marked circadian variation in plasma lipid peroxide level was recorded in healthy subjects and pulmonary tuberculosis patients with significant amplitude and acrophase around 16∶21 and 17∶12 respectively. The acrophase tended to be delayed in tuberculosis patients. Furthermore, a statistically significant circadian rhythm was found in SOD, CAT and GPx activities in normal volunteers and pulmonary tuberculopsis patients. SOD and CAT enzyme activity was noted to be maximum at 06∶00 and minimum at 00∶00 in tuberculosis patients. The circadian acrophase for GPx activity was recorded at 16∶15 in normals and around 22∶45 in patients. Moreover, the activity was found to be decreased at all sampling hours during 24-hours sleep-awake period in patients in comparison to healthy counterparts. The MESOR and circadian amplitude also decreased markedly. The decreased activity of measured antioxidant enzymes in pulmonary tuberculosis patients could probably be associated with oxidative stress and/or decreased anti-oxidant defensive mechanism in such patients.
doi:10.1007/BF02872382
PMCID: PMC3453924
PMID: 23105419