11-Hydroxyeicosatetraenoics induces cellular hypertrophy in an enantioselective manner

BackgroundR/S enantiomers of 11-hydroxyeicosatertraenoic acid (11-HETE) are formed from arachidonic acid by enzymatic and non-enzymatic pathways. 11-HETE is predominately formed by the cytochrome P450 1B1 (CYP1B1). The role of CYP1B1 in the development of cardiovascular diseases is well established.ObjectivesThis study aimed to assess the cellular hypertrophic effect of 11-HETE enantiomers in human RL-14 cardiomyocyte cell line and to examine their association with CYP1B1 levels.MethodsHuman fetal ventricular cardiomyocyte, RL-14 cells, were treated with 20 µM (R) or (S) 11-HETE for 24 h. Thereafter, cellular hypertrophic markers and cell size were then determined using real-time polymerase chain reaction (RT-PCR) and phase-contrast imaging, respectively. The mRNA and protein levels of selected CYPs were determined using RT-PCR and Western blot, respectively. In addition, we examined the effect of (R) and (S) 11-HETE on CYP1B1 catalytic activity using human recombinant CYP1B1 and human liver microsomes.ResultsBoth (R) and (S) 11-HETE induced cellular hypertrophic markers and cell surface area in RL-14 cells. Both enantiomers significantly upregulated CYP1B1, CYP1A1, CYP4F2, and CYP4A11 at the mRNA and protein levels, however, the effect of the S-enantiomer was more pronounced. Furthermore, 11(S)-HETE increased the mRNA and protein levels of CYP2J and CYP4F2, whereas 11(R)-HETE increased only CYP4F2. Only 11(S)-HETE significantly increased the catalytic activity of CYP1B1 in recombinant human CYP1B1, suggesting allosteric activation in an enantioselective manner.ConclusionOur study provides the first evidence that 11-HETE can induce cellular hypertrophy in RL-14 cells via the increase in CYP1B1 mRNA, protein, and activity levels

Application of Single-Radial Immuno-diffusion test for evaluation of uropathogenic Escherichia coli sonicated immunogen in chick embryo

This study intended to evaluate the effect of whole cell sonicated uropathogenic Escherichia coli antigen (WCS.Ag) against the challenged of the infectious dose 50 (ID50) in chicken embryos. Twenty chicken embryos / one day age were divided into 2 equal groups. The first group was inoculated with 0.1 ml of WSC, Ag, the second group (control) was injected with 0.1 ml phosphate buffer saline (PBS). Blood samples were collected at 2 and 4weeks after inoculations. Single Radial Immuno-Diffusion assay (SRID) was used to test the significance of the effects of treatments. Fifty groups of eggs (10 eggs per group) were used to determine the infectious dose 50 (ID50). All chicken embryos of the treated and control groups were challenged with 3ID50 (2×106) of virulent uropathogenic Escherichia coli. The SRIT showed significant increase in antibody titers in the experimental group compared with the control group. The post challenge with ID50 of E. coli revealed that all chicken embryos in control group suffered from severe clinical signs of colibacillosis. In conclusion, this study indicated the ability of WSC. Ag to protect chick embryo against the challenged of the infectious dose 50 (ID50)

Alterations in the transcriptome and antibiotic susceptibility of Staphylococcus aureus grown in the presence of diclofenac

<p>Abstract</p> <p>Background</p> <p>Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) which has been shown to increase the susceptibility of various bacteria to antimicrobials and demonstrated to have broad antimicrobial activity. This study describes transcriptome alterations in <it>S. aureus </it>strain COL grown with diclofenac and characterizes the effects of this NSAID on antibiotic susceptibility in laboratory, clinical and diclofenac reduced-susceptibility (Dc^RS) <it>S. aureus </it>strains.</p> <p>Methods</p> <p>Transcriptional alterations in response to growth with diclofenac were measured using <it>S. aureus </it>gene expression microarrays and quantitative real-time PCR. Antimicrobial susceptibility was determined by agar diffusion MICs and gradient plate analysis. Ciprofloxacin accumulation was measured by fluorescence spectrophotometry.</p> <p>Results</p> <p>Growth of <it>S. aureus </it>strain COL with 80 μg/ml (0.2 × MIC) of diclofenac resulted in the significant alteration by ≥2-fold of 458 genes. These represented genes encoding proteins for transport and binding, protein and DNA synthesis, and the cell envelope. Notable alterations included the strong down-regulation of antimicrobial efflux pumps including <it>mepRAB </it>and a putative <it>emrAB/qacA</it>-family pump. Diclofenac up-regulated <it>sigB </it>(σ^B), encoding an alternative sigma factor which has been shown to be important for antimicrobial resistance. <it>Staphylococcus aureus </it>microarray metadatabase (SAMMD) analysis further revealed that 46% of genes differentially-expressed with diclofenac are also σ^B-regulated. Diclofenac altered <it>S. aureus </it>susceptibility to multiple antibiotics in a strain-dependent manner. Susceptibility increased for ciprofloxacin, ofloxacin and norfloxacin, decreased for oxacillin and vancomycin, and did not change for tetracycline or chloramphenicol. Mutation to Dc^RSdid not affect susceptibility to the above antibiotics. Reduced ciprofloxacin MICs with diclofenac in strain BB255, were not associated with increased drug accumulation.</p> <p>Conclusions</p> <p>The results of this study suggest that diclofenac influences antibiotic susceptibility in <it>S. aureus</it>, in part, by altering the expression of regulatory and structural genes associated with cell wall biosynthesis/turnover and transport.</p

Alterations In the Transciptome and Antibiotic Susceptibility of \u3ci\u3eStaphylococcus aureus\u3c/i\u3e Grown In the Presence of Diclofenac

Background Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) which has been shown to increase the susceptibility of various bacteria to antimicrobials and demonstrated to have broad antimicrobial activity. This study describes transcriptome alterations in S. aureus strain COL grown with diclofenac and characterizes the effects of this NSAID on antibiotic susceptibility in laboratory, clinical and diclofenac reduced-susceptibility (DcRS) S. aureus strains. Methods Transcriptional alterations in response to growth with diclofenac were measured using S. aureus gene expression microarrays and quantitative real-time PCR. Antimicrobial susceptibility was determined by agar diffusion MICs and gradient plate analysis. Ciprofloxacin accumulation was measured by fluorescence spectrophotometry. Results Growth of S. aureus strain COL with 80 μg/ml (0.2 × MIC) of diclofenac resulted in the significant alteration by ≥2-fold of 458 genes. These represented genes encoding proteins for transport and binding, protein and DNA synthesis, and the cell envelope. Notable alterations included the strong down-regulation of antimicrobial efflux pumps including mepRAB and a putative emrAB/qacA-family pump. Diclofenac up-regulated sigB (σB), encoding an alternative sigma factor which has been shown to be important for antimicrobial resistance. Staphylococcus aureus microarray metadatabase (SAMMD) analysis further revealed that 46% of genes differentially-expressed with diclofenac are also σB-regulated. Diclofenac altered S. aureus susceptibility to multiple antibiotics in a strain-dependent manner. Susceptibility increased for ciprofloxacin, ofloxacin and norfloxacin, decreased for oxacillin and vancomycin, and did not change for tetracycline or chloramphenicol. Mutation to DcRS did not affect susceptibility to the above antibiotics. Reduced ciprofloxacin MICs with diclofenac in strain BB255, were not associated with increased drug accumulation. Conclusions The results of this study suggest that diclofenac influences antibiotic susceptibility in S. aureus, in part, by altering the expression of regulatory and structural genes associated with cell wall biosynthesis/turnover and transport

Immunohistochemical evaluation of human epidermal growth factor receptor 2 and estrogen and progesterone receptors in breast carcinoma in Jordan

INTRODUCTION: Although breast carcinoma (BC) is the most common malignancy affecting Jordanian females and the affected population in Jordan is younger than that in the West, no information is available on its biological characteristics. Our aims in this study are to evaluate the expression of estrogen receptor (ER) and progesterone receptor (PR) and Her-2/neu overexpression in BC in Jordan, and to compare the expression of these with other prognostic parameters for BC such as histological type, histological grade, tumor size, patients' age, and number of lymph node metastases. METHOD: This is a retrospective study conducted in the Department of Pathology at Jordan University of Science and Technology. A confirmed 91 cases of BC diagnosed in the period 1995 to 1998 were reviewed and graded. We used immunohistochemistry to evaluate the expression of ER, PR, and Her-2. Immunohistochemical findings were correlated with age, tumor size, grade and axillary lymph node status. RESULTS: Her-2 was overexpressed in 24% of the cases. The mean age of Her-2 positive cases was 42 years as opposed to 53 years among Her-2 negative cases (p = 0.0001). Her-2 expression was inversely related to ER and PR expression. Her-2 positive tumors tended to be larger than Her-2 negative tumors with 35% overexpression among T3 tumors as opposed to 22% among T2 tumors (p = 0.13). Her-2 positive cases tended to have higher rates of axillary metastases, but this did not reach statistical significance. ER and PR positive cases were seen in older patients with smaller tumor sizes. CONCLUSION: Her-2 overexpression was seen in 24% of BC affecting Jordanian females. Her-2 overexpression was associated with young age at presentation, larger tumor size, and was inversely related to ER and PR expression. One-fifth of the carcinomas were Her-2 positive and ER negative. This group appears to represent an aggressive form of BC presenting at a young age with large primary tumors and a high rate of four or more axillary lymph node metastases

Rarity of monodominance in hyperdiverse Amazonian forests.

Tropical forests are known for their high diversity. Yet, forest patches do occur in the tropics where a single tree species is dominant. Such "monodominant" forests are known from all of the main tropical regions. For Amazonia, we sampled the occurrence of monodominance in a massive, basin-wide database of forest-inventory plots from the Amazon Tree Diversity Network (ATDN). Utilizing a simple defining metric of at least half of the trees ≥ 10 cm diameter belonging to one species, we found only a few occurrences of monodominance in Amazonia, and the phenomenon was not significantly linked to previously hypothesized life history traits such wood density, seed mass, ectomycorrhizal associations, or Rhizobium nodulation. In our analysis, coppicing (the formation of sprouts at the base of the tree or on roots) was the only trait significantly linked to monodominance. While at specific locales coppicing or ectomycorrhizal associations may confer a considerable advantage to a tree species and lead to its monodominance, very few species have these traits. Mining of the ATDN dataset suggests that monodominance is quite rare in Amazonia, and may be linked primarily to edaphic factors

DIFFERENTIATION BETWEEN Nocardia spp. AND Mycobacterium spp.: CRITICAL ASPECTS FOR BACTERIOLOGICAL DIAGNOSIS

New methodologies were developed for the identification of Nocardia but the initial diagnosis still requires a fast and accurate method, mainly due to the similarity to Mycobacterium, both clinical and bacteriologically. Growth on Löwenstein-Jensen (LJ) medium, presence of acid-fast bacilli through Ziehl-Neelsen staining, and colony morphology can be confusing aspects between Nocardia and Mycobacterium. This study describes the occurrence of Nocardia spp. in a mycobacterial-reference laboratory, observing the main difficulties in differentiating Nocardia spp. from Mycobacterium spp., and correlating isolates with nocardiosis cases. Laboratory records for the period between 2008 and 2012 were analyzed, and the isolates identified as Nocardia sp. or as non-acid-fast filamentous bacilli were selected. Epidemiological and bacteriological data were analyzed as well. Thirty-three isolates identified as Nocardia sp. and 22 as non-acid-fast bacilli were selected for this study, and represented 0.12% of isolates during the study period. The presumptive identification was based on macroscopic and microscopic morphology, resistance to lysozyme and restriction profiles using the PRA-hsp65 method. Nocardia spp. can grow on media for mycobacteria isolation (LJ and BBL MGIT™) and microscopy and colony morphology are very similar to some mycobacteria species. Seventeen patients (54.8%) were reported and treated for tuberculosis, but presented signs and symptoms of nocardiosis. It was concluded that the occurrence of Nocardia sp. during the study period was 0.12%. Isolates with characteristics of filamentous bacilli, forming aerial hyphae, with colonies that may be pigmented, rough and without the BstEII digestion pattern in PRA-hsp65 method are suggestive of Nocardia spp. For a mycobacterial routine laboratory, a flow for the presumptive identification of Nocardia is essential, allowing the use of more accurate techniques for the correct identification, proper treatment and better quality of life for patients

Leptin and leptin receptor polymorphisms are associated with increased risk and poor prognosis of breast carcinoma

BACKGROUND: Leptin (LEP) has been consistently associated with angiogenesis and tumor growth. Leptin exerts its physiological action through its specific receptor (LEPR). We have investigated whether genetic variations in LEP and LEPR have implications for susceptibility to and prognosis in breast carcinoma. METHODS: We used the polymerase chain reaction and restriction enzyme digestion to characterize the variation of the LEP and LEPR genes in 308 unrelated Tunisian patients with breast carcinoma and 222 healthy control subjects. Associations of the clinicopathologic parameters and these genetic markers with the rates of the breast carcinoma-specific overall survival (OVS) and the disease free survival (DFS) were assessed using univariate and multivariate analyses. RESULTS: A significantly increased risk of breast carcinoma was associated with heterozygous LEP (-2548) GA (OR = 1.45; P = 0.04) and homozygous LEP (-2548) AA (OR = 3.17; P = 0.001) variants. A highly significant association was found between the heterozygous LEPR 223QR genotype (OR = 1.68; P = 0.007) or homozygous LEPR 223RR genotype (OR = 2.26; P = 0.001) and breast carcinoma. Moreover, the presence of the LEP (-2548) A allele showed a significant association with decreased disease-free survival in breast carcinoma patients, and the presence of the LEPR 223R allele showed a significant association with decreased overall survival. CONCLUSION: Our results indicated that the polymorphisms in LEP and LEPR genes are associated with increased breast cancer risk as well as disease progress, supporting our hypothesis for leptin involvement in cancer pathogenesis