Biomedical subjects
S Sharma
Publications and source records attributed to S Sharma.
Hospitalised patients' need for information. A study.
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Biology and applications of mycorrhizal fungi.
Mycorrhizae have been shown to increase growth and yield of plants. They have been identified with both nutrient mobilization and nutrient cycling. Arbuscular (or endo-) mycorrhizae play a significant role in agriculture and most natural ecosystems, whereas ectomycorrhizae have a great potential in forestry and wasteland regeneration. The use of mycorrhizal fungi would reduce dependence on chemical fertilizers besides minimizing environmental pollution. The present review addresses the progress that there has been in the area of the ecto- and endomycorrhizae. It also examines the potential of field applications of mycorrhizal biotechnology in agriculture and forestry.
Ophthaproblem. Glaucomatous damage.
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Non-small cell lung cancer-derived soluble mediators and prostaglandin E2 enhance peripheral blood lymphocyte IL-10 transcription and protein production.
Studies suggest that IL-10 may contribute to tumor-associated immunosuppression. In the current study we evaluated the capacity of human non-small cell lung cancer (NSCLC) cell lines to induce PBL IL-10 production. We observed a 10- to 100-fold increase in human PBL IL-10 production following exposure to NSCLC cell supernatants. The tumor-induced increase in PBL IL-10 production was partially blocked by pretreatment of the tumors with the PG inhibitor indomethacin. NSCLC lines were found to constitutively produce PGE2. Exogenous PGE2 also induced PBL IL-10 production in a dose- and time-dependent manner. Both PGE2 and NSCLC supernatant-induced PBL IL-10 production were due to an increase in the IL-10 mRNA transcriptional rate. To evaluate the significance of tumor-induced lymphocyte IL-10 production, the capacity of PBL to produce IFN-gamma during culture in tumor supernatants was assessed in the presence of specific anti-IL-10 mAb. We found enhanced PBL IFN-gamma production following anti-IL-10 treatment. These in vitro studies imply that NSCLC-induced PBL IL-10 production may serve to shift the Th1/Th2 cytokine axis at the tumor site and thus inhibit cell-mediated anti-tumor immune responses. These findings identify a mechanism by which lung cancer cells may escape host immune surveillance. We conclude that NSCLC-derived soluble mediators, including PGs, may play an immunoregulatory role through induction of lymphocyte IL-10 production.
Adenovirus vector infection of chronic lymphocytic leukemia B cells.
Adenovirus vectors have several features that make them attractive for potential use in gene therapy, including a broad tissue tropism and an ability to infect quiescent or postmitotic cells. In light of this, we examined whether recombinant adenovirus vectors could transfer genes into neoplastic cells of patients with chronic lymphocytic leukemia (CLL), a leukemia of "resting" B cells. Using high-titer recombinant adenovirus vectors, we found we could transfer genes encoding beta-galactosidase or murine CD80 (B7-1) into the CLL B cells of all patients tested (n = 10). The efficiency of gene transduction into CLL B cells was approximately 100 to 1,000-fold lower than into HeLa cells at any given multiplicity of infection (MOI). At a MOI of 500, 10% to 70% of the CLL B cells from different patients were made to express the transgene, as assessed by multiparameter flow cytometric analysis. Sustained levels of expression with little loss in the percentage of infected cells were maintained for up to 9 days, at which point the analysis was stopped. We found that CLL B cells have markedly lower expression levels of integrins that facilitate internalization of adenovirus particles into target cells, perhaps accounting, in part, for the reduced efficiency of adenovirus-mediated gene transfer compared with that in HeLa cells. Although HeLa cells express high levels of alpha(v)beta5, and detectable amounts of alpha(v)beta3, we find CLL cells from all patients tested express only low amounts of alpha(v)beta3, and no detectable alpha(v)beta5. Activation of CLL cells via CD40 cross-linking enhances expression of alpha(v)beta3, and induces expression of alpha(v)beta5. This phenotypic change is associated with a fivefold increase in the efficiency of adenovirus-mediated gene transfer into such activated CLL B cells. This study demonstrates that adenovirus vectors can transduce genes into CLL B cells and that the efficiency of gene transduction is enhanced by activation via CD40 cross-linking. This is the first demonstration that high proportions of CLL B cells can be made to express a selected transgene, suggesting that such gene transfer methods may become useful for the study of the pathogenesis and/or treatment of this disease.
Cloning of a ribosomal phosphoprotein P0 gene homologue from Plasmodium falciparum.
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Transcriptional silencing of a tRNA1Gly copy from within a multigene family is modulated by distal cis elements.
Individual copies of tRNA1Gly from within the multigene family in Bombyx mori could be classified based on in vitro transcription in homologous nuclear extracts into three categories of highly, moderately, or weakly transcribed genes. Segregation of the poorly transcribed gene copies 6 and 7, which are clustered in tandem within 425 base pairs, resulted in enhancement of their individual transcription levels, but the linkage itself had little influence on the transcriptional status. For these gene copies, when fused together generating a single coding region, transcription was barely detectable, which suggested the presence of negatively regulating elements located in the far flanking sequences. They exerted the silencing effect on transcription overriding the activity of positive regulatory elements. Systematic analysis of deletion, chimeric, and mutant constructs revealed the presence of a sequence element TATATAA located beyond 800 nucleotides upstream to the coding region acting as negative modulator, which when mutated resulted in high level transcription. Conversely, a TATATAA motif reintroduced at either far upstream or far downstream flanking regions exerted a negative effect on transcription. The location of cis-regulatory sequences at such farther distances from the coding region and the behavior of TATATAA element as negative regulator reported here are novel. These element(s) could play significant roles in activation or silencing of genes from within a multigene family, by recruitment or sequestration of transcription factors.
Efficient infection of a human T-cell line and of human primary peripheral blood leukocytes with a pseudotyped retrovirus vector.
Peripheral blood lymphocytes (PBLs) are an important target for gene transfer studies aimed at human gene therapy. However, no reproducibly efficient methods are currently available to transfer foreign, potentially therapeutic genes into these cells. While vectors derived from murine retroviruses have been the most widely used system, their low infection efficiency in lymphocytes has required prolonged in vitro culturing and selection after infection to obtain useful numbers of genetically modified cells. We previously reported that retroviral vectors pseudotyped with vesicular stomatitis G glycoprotein (VSV-G) envelope can infect a wide variety of cell types and can be concentrated to titers of greater than 10(9) infectious units/ml. In this present study, we examined the ability of amphotropic and pseudotyped vectors expressing a murine cell surface protein, B7-1, to infect the human T-cell line Jurkat or human blood lymphocytes. Limiting dilution analysis of transduced Jurkat cells demonstrated that the pseudotyped vector is significantly more efficient in infecting T cells than an amphotropic vector used at the same multiplicity of infection (moi). To identify the transduction efficiency on PBLs, we examined the levels of cell surface expression of the B7-1 surface marker 48 to 72 hr after infection. The transduction efficiency of PBLs with the pseudotyped vector increased linearly with increasing moi to a maximum of approximately 16-32% at an moi of 40. This relatively high efficiency of infection of a T-cell line and of blood lymphocytes with VSV-G pseudotyped virus demonstrates that such modified pseudotyped retrovirus vectors may be useful reagents for studies of gene therapy for a variety of genetic or neoplastic disorders.
Fatal Falciparum malaria in Canadian travellers.
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Identification of a novel guanine nucleotide exchange factor for the Rho GTPase.
The Rho GTPase promotes proliferation and cytoskeletal rearrangements in mammalian cells. To understand the regulation of Rho, it is important to characterize guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of GDP and subsequent binding of GTP. Using Rho as an affinity ligand, we have isolated a 115-kDa protein (p115-RhoGEF) that binds specifically to the nucleotide-depleted state. A full-length cDNA encoding p115-RhoGEF was isolated, and its protein product, which exhibited sequence homology to Dbl and Lbc, catalyzed the exchange of GDP for GTP specifically on Rho and not on the Rac, Cdc42, or Ras GTPases. p115-RhoGEF is capable of regulating cell proliferation, as determined by its ability to induce the transformation of NIH 3T3 cells. Northern and Western analysis suggests that p115-RhoGEF is ubiquitously expressed. These results indicate that p115-RhoGEF may be a general regulator of Rho and its associated cellular phenotypes.
Role of TATATAA element in the regulation of tRNA1Gly gene expression in Bombyx mori is position dependent.
Transcription of tRNA genes by RNA polymerase III is controlled by the internal conserved sequences within the coding region and the immediate upstream flanking sequences. A highly transcribed copy of glycyl tRNA gene tRNA1Gly-1 from Bombyx mori is down regulated by sequences located much farther upstream in the region -150 to -300 nucleotides (nt), with respect to the +1 nt of tRNA. The negative regulatory effect has been narrowed down to a sequence motif 'TATATAA', a perfect consensus recognised by the TATA binding protein, TBP. This sequence element, when brought closer to the transcription start point, on the other hand, exerts a positive effect by promoting transcription of the gene devoid of other cis regulatory elements. The identity of the nuclear protein interacting with this 'TATATAA' element to TBP has been established by antibody and mutagenesis studies. The 'TATATAA' element thus influences the transcription of tRNA genes positively or negatively in a position-dependent manner either by recruitment or sequestration of TBP from the transcription machinery.
Cocaine down-regulates IL-2-induced peripheral blood lymphocyte IL-8 and IFN-gamma production.
Cocaine has multiple immunomodulatory effects, including the ability to influence cytokine release in immunoeffector cells. Little is known, however, regarding the effects of cocaine on cytokine production by human peripheral blood lymphocytes (PBL). The effect of cocaine on PBL cytokine profiles and the molecular mechanisms responsible for the modulation of cytokine mRNA expression were investigated. To evaluate the effects of cocaine on cytokine production, conditioned supernatant from IL-2-stimulated PBL was evaluated by cytokine-specific ELISA (IL-4, IL-5, IL-8, IL-10, IFN-gamma, and TGF-beta) following in vitro cocaine exposure. Cocaine abrogated the IL-2-induced production of IFN-gamma and IL-8 in a dose-responsive manner. Cocaine also decreased PBL IFN-gamma and IL-8 mRNA expression as determined by Northern blot and slot blot analysis. Cocaine did not affect the stability of the IFN-gamma and IL-8 mRNA. Nuclear run-on assays revealed that cocaine down-regulated the rate of IFN-gamma and IL-8 transcription. These findings suggest that the immunomodulatory effects of cocaine may be mediated, in part, by modification of lymphocyte cytokine production.
Monitoring pulmonary artery wedge pressure in medical patients.
Pulmonary artery wedge pressure is used in the diagnosis and management of critically ill patients. This measurement provides an accurate assessment of hemodynamic status, cardiac function and venous oxygen saturation. Wedge pressure monitoring has been used in the management of patients with complicated myocardial infarction, refractory heart failure, circulatory shock, pulmonary edema and other critical illnesses. Because measuring the pulmonary artery wedge pressure is an invasive procedure, the value of the additional data provided by monitoring should be considered carefully before choosing this method. Clinical assessment or noninvasive tests, including chest radiographs and echocardiography, often provide information adequate for planning management. If therapy fails, or if noninvasive data are uncertain, pulmonary artery catheterization is appropriate. Risks and complications may be secondary to either catheter insertion or the continued presence of the catheter.
Characterization of a new isoform of the NFAT (nuclear factor of activated T cells) gene family member NFATc.
The cyclosporin A (CsA)/FK506-sensitive nuclear factor of activated T cells (NFAT) plays a key role in the inducible expression of cytokine genes in T cells. Although NFAT has been recently shown to be inducible in several non-T immune cells, the NFAT gene family members characterized to date have been isolated only from T cells. To further characterize NFAT function in human B cells and to demonstrate cytokine gene specificity of NFAT proteins, we report here the isolation and characterization of a cDNA clone from the Raji B cell line. The cDNA clone encodes a new isoform, NFATc.beta, of the NFAT gene family member NFATc (designated here NFATc.alpha). The amino acid sequence of NFATc.beta differs from that of NFATc. alpha in the first NH2-terminal 29 residues and contains an additional region of 142 residues at the COOH terminus. Northern analysis using a probe encompassing a common region of both isoforms showed two mRNA species of 2.7 and 4.5 kilobase pairs, while an NFATc.beta-specific probe detected only the 4.5-kilobase pair mRNA which was preferentially expressed in the spleen. Transient expression of NFATc.beta was capable of activating an interleukin-2 NFAT-driven reporter gene in stimulated Jurkat cells in a CsA-sensitive manner. However, NFATc.beta neither bound to the kappa3 element (an NFAT-binding site) in the tumor necrosis factor-alpha promoter nor activated the tumor necrosis factor-alpha promoter in cotransfection assays. These data suggest that different members or isoforms of NFAT gene family may regulate inducible expression of different cytokine genes.
Pancreatic islet expression of the homeobox factor STF-1 relies on an E-box motif that binds USF.
The commitment of cells to specific lineages during development is determined in large part by the relative expression of various homeodomain (HOX) selector proteins, which mediate the activation of distinct genetic programs. But the mechanisms by which individual HOX genes are themselves targeted for expression in different cell types remain largely uncharacterized. Here, we demonstrate that STF-1, a homeodomain protein that functions in pancreatic morphogenesis and in glucose homeostasis is encoded by an "orphan" homeobox gene on mouse chromosome 5. When fused to a beta-galactosidase reporter gene, a 6.5-kilobase genomic fragment of 5'-flanking sequence from the STF-1 gene shows pancreatic islet specific activity in transgenic mice. Two distinct elements within the STF-1 promoter are required for islet-restricted expression: a distal enhancer sequence located between -3 and -6.5 kilobases and a proximal E-box sequence located at -104, which is recognized primarily by the helix loop helix/leucine zipper nuclear factor USF. As point mutation within the -104 E-box that disrupt USF binding correspondingly impair STF-1 promoter activity, our results demonstrate that USF is an important component of the regulatory apparatus which directs STF-1 expression to pancreatic islet cells.
Transthoracic echocardiographic findings in patients with acute retinal arterial obstruction. A retrospective review. Retinal Emboli of Cardiac Origin Group.
OBJECTIVE: To assess the importance of cardio-embolic (emboli of cardiac origin) risk stratification in the cardiac evaluation of patients with acute retinal arterial obstruction. DESIGN: A multicenter retrospective study of patients with acute retinal arterial obstruction who underwent transthoracic echocardiography. SETTINGS: Four North American hospital centers. PATIENTS: One hundred patients with acute retinal artery obstruction in whom transthoracic echocardiography was performed. MAIN OUTCOME MEASURE: Anticoagulation or cardiac surgery based on abnormalities detected on transthoracic echocardiography. Transesophageal echocardiographic results were not evaluated, as they were rarely performed in our centers. Thus, the outcome measure was determined solely by the results of transthoracic echocardiography. RESULTS: Patients were divided into high- and low-risk groups based on their history of cardioembolic risk factors or the presence of a cardiac murmur. Of 67 patients with no risk factors, 41 (61%) had normal echocardiographic study results and 26 (39%) had abnormalities detected, of whom only 1 (1.5%) received anticoagulation or cardiac surgery. The presence of 1 or more cardio-embolic risk factors increased the likelihood for anticoagulation or cardiac surgery 25 times (odds ratio = 25; 95% confidence interval = 3.04-217.02). Although this result is clinically and statistically significant, it is possible that abnormalities missed by transthoracic methods may have been detected by transesophageal technology. CONCLUSIONS: In patients with acute retinal arterial obstruction at low cardioembolic risk, transthoracic echocardiography resulted in anticoagulation or cardiac surgery in 1 (1.5%) of 67 patients. Routine transthoracic echocardiography without follow-up transesophageal echocardiography for patients identified as having abnormalities such as left ventricular hypertrophy or mitral annular calcification and who are at low cardioembolic risk rarely resulted in anticoagulation or cardiac surgery.
Mycobacterium fortuitum infection of the sternum. Review of the literature and case illustration.
Sternal wound infection with atypical mycobacteria following open heart surgery is a rare occurrence. Previous reports have described infection by Mycobacterium fortuitum, an acid-fast bacillus and member of a larger family of rapidly growing mycobacteria. The source and mode of transmission have not been identified. Surgical debridement and the combination of aminoglycosides and quinolones have been shown to be effective methods of treatment. More recently, clarithromycin has been shown to be the drug of choice against rapidly growing mycobacteria. We describe a 49-year-old woman who underwent infundibular stenosis repair and in whom M fortuitum sternal osteomyelitis developed. Total sternectomy, muscle flap reconstruction, and antibiotic treatment successfully eradicated the infection.