Flavonoid glucuronides from Helicteres isora

Five flavonoid glucuronides were obtained from the fruit of Helicteres isora, three of which were previously unknown compounds: isoscutellarein 4'-methyl ether 8-O-beta-D-glucuronide 6"-n-butyl ester. isoscutellarein 4'-methyl ether 8-O-beta-D-glucuronide 2", 4"-disulfate and isoscutellarein 8-O-beta-D-glucuronide 2",4"-disulfate. The structures were determined on the basis of spectroscopy and hydrolysis experiments.     

Murine colonic mucosa hyperproliferation. I. Elevated CFTR expression and enhanced cAMP-dependent Cl(-) secretion

Fluid transport in the large intestine is mediated by the cystic fibrosis gene product and cAMP-dependent anion channel cystic fibrosis transmembrane conductance regulator (CFTR). cAMP-mediated Cl(-) secretion by gastrointestinal cell lines in vitro has been positively correlated with the insertion of CFTR into the apical membrane of differentiated senescent colonocytes and negatively correlated with the failure of CFTR to insert into the plasma membrane of their undifferentiated proliferating counterparts. In native tissues, this relationship remains unresolved. We demonstrate, in a transmissible murine colonic hyperplasia (TMCH) model, that (8-fold) colonocyte proliferation was accompanied by increased cellular CFTR mRNA and protein expression (8.3- and 2.4-fold, respectively) and enhanced mucosal cAMP-dependent Cl(-) secretion (2. 3-fold). By immunofluorescence microscopy, cellular CFTR expression was restricted to the apical pole of cells at the base of the epithelial crypt. In contrast, increased cellular proliferation in vivo led to increases in both the cellular level and the total number of cells expressing this anion channel, with cellular CFTR staining extending into the crypt neck region. Hyperproliferating colonocytes accumulated large amounts of CFTR in apically oriented subcellular perinuclear compartments. This novel mode of CFTR regulation may explain why high endogenous levels of cellular CFTR mRNA and protein within the TMCH epithelium were not matched with larger increases in transmucosal CFTR Cl(-) current.     

Method optimisation for peptide profiling of microdissected breast carcinoma tissue by matrix-assisted laser desorption/ionisation-time of flight and matrix-assisted laser desorption/ionisation-time of flight/time of flight-mass spectrometry

Appropriate methods for the analysis of microdissected solid tumour tissues by matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF MS) are not yet well established. Optimisation of sample preparation was performed first on undissected tissue slices, representing approximately 200 000 cells, which were solubilised either in urea containing buffer, trifluoroethanol/NH4HCO3, 0.1% sodium dodecyl sulphate (SDS) or in 0.1% RapiGest solution, then trypsin digested and analysed by MALDI-TOF MS. Solubilisation in 0.1% SDS resulted in detection of the highest number of sample specific peak signals. Interestingly, there was little overlap in detectable peaks using the different buffers, implying that they can be used complementarily to each other. Additionally, we fractionated tryptic digests on a monolithic high-performance liquid chromatography column. Fractionation of tryptic digest from whole tissue sections resulted in a four-fold increase in the total number of peaks detected. To prove this principle, we used 0.1% SDS to generate peptide patterns from 2000 microdissected tumour and stromal cells from five different breast carcinoma tumours. The tumour and stroma specific peaks could be detected upon comparison of the peptide profiles. Identification of differentially expressed peaks by MALDI-TOF/TOF MS was performed on fractionated tryptic digests derived from a whole tissue slice. In conclusion, we describe a method that is suitable for direct peptide profiling on small amounts of microdissected cells obtained from breast cancer tissues.     

Arthritis imaging using a near-infrared fluorescence folate-targeted probe

A recently developed near-infrared fluorescence-labeled folate probe (NIR2-folate) was tested for in vivo imaging of arthritis using a lipopolysaccharide intra-articular injection model and a KRN transgenic mice serum induction mouse model. In the lipopolysaccharide injection model, the fluorescence signal intensity of NIR2-folate (n = 12) and of free NIR2 (n = 5) was compared between lipopolysaccharide-treated and control joints. The fluorescence signal intensity of the NIR2-folate probe at the inflammatory joints was found to be significantly higher than the control normal joints (up to 2.3-fold, P < 0.001). The NIR2-free dye injection group showed a persistent lower enhancement ratio than the NIR2-folate probe injection group. Excessive folic acid was also given to demonstrate a competitive effect with the NIR2-folate. In the KRN serum transfer model (n = 4), NIR2-folate was applied at different time points after serum transfer, and the inflamed joints could be detected as early as 30 hours after arthritogenic antibody transfer (1.8-fold increase in signal intensity). Fluorescence microscopy, histology, and immunohistochemistry validated the optical imaging results. We conclude that in vivo arthritis detection was feasible using a folate-targeted near-infrared fluorescence probe. This receptor-targeted imaging method may facilitate improved arthritis diagnosis and early assessment of the disease progress by providing an in vivo characterization of active macrophage status in inflammatory joint diseases.     

Near-infrared fluorescent nanoparticles as combined MR/optical imaging probes

A number of quantitative three-dimensional tomographic near-infrared fluorescence imaging techniques have recently been developed and combined with MR imaging to yield highly detailed anatomic and molecular information in living organisms (1, 2). Here we describe magnetic nanoparticle based MR contrast agents that have a near-infrared fluorescence (NIRF) that is activated by certain enzymes. The probes are prepared by conjugation of arginyl peptides to cross-linked iron oxide amine (amino-CLIO), either by a disulfide linkage or a thioether linker, followed by the attachment of the indocyanine dye Cy5.5. The NIRF of disulfide-linked conjugate was activated by DTT, while the NIRF of thioether-linked conjugate was activated by trypsin. Fluorescent quenching of the attached fluorochrome occurs in part due to the interaction with iron oxide, as evident by the activation of fluorescence with DTT when nanoparticles that have less than one dye attached per particle. With a SC injection of the probe, axillary and brachial lymph nodes were darkened on MR images and easily delineated by NIRF imaging. The probes may provide the basis for a new class of so-called smart nanoparticles, capable of pinpointing their position through their magnetic properties, while providing information on their environment by optical imaging techniques.     

Thymoma: a clinico pathological study of 21 cases and assessment of prognostic features

This is a retrospective and comprehensive study of 21 cases of thymoma treated during a period of 30 years (1954-1984). The tumors were staged into 3 categories: stage 1 for encapsulated completely resectable tumor, stage 2 for nonresectable intrathoracic tumor and stage 3 for tumor with extrathoracic spread. According to their lymphocytic content tumors were separated into 3 groups: 1) predominantly epithelioid (PE); 2) mixed cellular (MC) and predominantly lymphocytic (PL). Incidence of recurrence and survival were correlated with various treatment modalities. The tumor occurred in all age groups with highest incidence in the fourth decade. Six cases were asymptomatic. Myasthenia gravis was present only in one case. The most important prognostic factor was the stage of the tumor. Five-year survival was 69% for stage 2 and 0% for stage 3. All 12 patients who died with evidence of residual disease had PE tumors. Lymphocytic participation might be indicative of a residual functional competence and appears to confer a more favourable prognosis. This is a tumor of uncertain malignant potential which should be excised or debulked, and staged. Post-operative radiotherapy appears to prevent recurrence and improve the prognosis in stage 2. No therapeutic benefits were seen in the stage 3 cases. The value of chemotherapy is uncertain.     

Proteomics Analyses and Morphological Structure of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Inactivated by Pulsed Magnetic Field

Pulsed magnetic field (PMF) technology has emerged as a non-thermal method for inhibition of spoilage microorganism in food. In this study, we evaluate the effect of PMF treatment on the inactivation of Bacillus subtilis. The mechanisms responsible for cell death were also studied using transmission electron microscopy (TEM) and proteome approaches. Results showed that the survival rate of B. subtilis generally decreased with an increase of pulse numbers at the intensity of 3.30 T. The observation of TEM showed damage in cell cytoplasm and cytoplasmic membrane after PMF treatment. Additionally, 18 differentially expressed protein spots were identified by two dimensional gel electrophoresis (2D-GE) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) analysis. The down-regulated outer membrane protein A (OmpA) illustrated that PMF destroyed the cell membrane. Furthermore, Gene ontology (GO) analysis and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were used to characterize the functions of those proteins. That PMF treatment damaged the membrane component, depressed cellular molecular functions and biological process, and decreased the carbohydrate metabolism and energy metabolism, which explain the death of cells. The presented results give the better view into the proteome of food microorganism and provide insight into the nature of PMF inactivation mechanisms.     

Treatment efficiency of a hybrid constructed wetland system for municipal wastewater and its suitability for crop irrigation

Design and implementation of wastewater treatment is inevitable due to toxic effects of wastewater irrigation on crops, soil and human health. Current investigation is the pioneer attempt on full-scale hybrid constructed wetland system (HCWS) built for municipal wastewater treatment from Pakistan. HCWS was comprised of vertical sub-surface flow constructed wetland (VSSF-CW) and five phyto-treatment ponds connected in series. Higher environmental risk was associated with untreated municipal wastewater usage in irrigation as estimated through discharge of metals to recipient soils. Treatment efficiency percentages recorded for HCWS reclaimed water quality parameters were, i.e., EC (56.68), TDS (56.86), alkalinity (39.67), chloride (39.68), sulfate (46.73), Na (28.80), Mn (65.24), Cr (78.07), Ni (81.02), BOD (68.74), total hardness (19.56), Fe (70.09), phosphate (55.40), Pb (80.48), COD (63.64), Mg (17.24), K (60.05), Co (100), Cu (67.73), Zn (59.97), Cd (100), and Ca (21.47) respectively. Wastewater treatment in HCWS was due to aquatic plants [Phragmites australis Cav. Trin. ex Steud., Canna indica L. Typha latifolia L., and Hydrocotyle umbellata L.], microbial activities and substrate based wetland processes. The HCWS treated water was well under irrigation standards and recommended for safer crop production in water scarce regions.     

Genetic characterization of phenicol-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> and role of wild-type repressor/regulator gene (<Emphasis Type="Italic">acrR</Emphasis>) on phenicol resistance

The genetic basis for phenicol resistance was examined in 38 phenicol-resistant clinical Escherichia coli isolates from poultry. Out of 62 isolates, 38 showed resistance for chloramphenicol and nine for florfenicol, respectively. Each strain also demonstrated resistance to a variety of other antibiotics. Molecular detection revealed that the incidence rates of the cat1, cat2, flo, flo-R, cmlA, and cmlB were 32, 29, 18, 13, 0, and 0%, respectively. Nineteen strains were tolerant to organic solvents. PCR amplification of the complete acrR (regulator/repressor) gene of five isolates revealed the amino acid changes in four isolates. DNA sequencing showed the non-synonymous mutations which change the amino acid, silent mutation, and nucleotide deletion in four isolates. MY09C10 showed neither deletion nor mutation in nucleotide. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in these strains. Complementation with a plasmid-borne wild-type acrR gene reduced the expression level of AcrA protein in the mutants and partially restored antibiotic susceptibility one- to fourfold. This study shows that mutations in acrR are an additional genetic basis for phenicol resistance.