Fluorescence spectroscopy of H‐ras transfected murine fibroblasts: A comparison with Monte Carlo simulations

Autofluorescence properties of tissues have been widely used to diagnose various types of malignancies. In this study, we measured the autofluorescence properties of H‐ras transfected murine fibroblasts and the counterpart control cells. The pair of cells is genetically identical except for the transfected H‐ras gene. We applied Monte Carlo simulations to evaluate the relative contributions of Rayleigh and Mie scattering effects towards fluorescence in an in vitro model system of normal and H‐ras transfected fibroblasts. The experimental results showed that fluorescence emission intensity was higher for normal cells than the malignant counterpart cells by about 30%. In normal cells, linearity in emission intensity was observed for cell densities of up to 1.0 × 10⁶ cells/ml whereas for transformed cells it was up to 1.4 × 10⁶ cells/ml. Nuclear volume changes give good account for the differences in the intrinsic fluorescence between normal and malignant cells. The Monte Carlo (MC) code, newly developed for this study, explains both predominant experimental features: the large fluorescence intensity differences between the transfected and the corresponding control cells as well as the phenomena of the red shift in the excitation spectra as a function of cell density. The contribution of Rayleigh scattering was found to be predominant compared to Mie scattering. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 132–140, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

LDL and cAMP cooperate to regulate the functional expression of the LRP in rat ovarian granulosa cells

Rat ovarian granulosa rely heavily on lipoprotein-derived cholesterol for steroidogenesis, which is principally supplied by the LDL receptor- and scavenger receptor class B type I (SR-BI)-mediated pathways. In this study, we characterized the hormonal and cholesterol regulation of another member of the LDL receptor superfamily, low density lipoprotein receptor-related protein (LRP), and its role in granulosa cell steroidogenesis. Coincubation of cultured granulosa cells with LDL and N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (Bt2cAMP) greatly increased the mRNA/protein levels of LRP. Bt2cAMP and Bt2cAMP plus human hLDL also enhanced SR-BI mRNA levels. However, there was no change in the expression of receptor-associated protein, a chaperone for LRP, or another lipoprotein receptor, LRP8/apoER2, in response to Bt2cAMP plus hLDL, whereas the mRNA expression of LDL receptor was reduced significantly. The induced LRP was fully functional, mediating increased uptake of its ligand, alpha2-macroglobulin. The level of binding of another LRP ligand, chylomicron remnants, did not increase, although the extent of remnant degradation that could be attributed to the LRP doubled in cells with increased levels of LRP. The addition of lipoprotein-type LRP ligands such as chylomicron remnants and VLDL to the incubation medium significantly increased the progestin production under both basal and stimulated conditions. In summary, our studies demonstrate a role for LRP in lipoprotein-supported ovarian granulosa cell steroidogenesis.     

Synthesis and characterization of a new cutinase substrate, 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate

Phytopathogenic fungi penetrate plants by breaking down the cuticular barrier with cutinase. Cutinases are extracellular hydrolytic enzymes that degrade cutin, a polyester composed of hydroxy and epoxy fatty acids. Until now, cutinase has been recognized by its ability to release labeled cutin monomers or by a non-specific esterase assay based on the hydrolysis of p-nitrophenyl esters of short fatty acids. In this work, an insoluble p-nitrophenyl derivative was synthesized and purified, and its structure was determined to be 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate (pNMSEH) by nuclear magnetic resonance (H+ NMR) analysis. pNMSEH was tested as a new cutinase substrate with Pseudomonas mandocino cutinase and porcine liver esterase. While a linear release over time of p-nitrophenol (pNP) was recorded in the presence of cutinase, no response was obtained with the esterase. The calculated kinetic parameters of pNMSEH hydrolysis by cutinase revealed a high specificity (Km=1.8mM), albeit a low catalytic rate (Vmax=10.5 micromol min(-l)l(-1)). This new synthetic substrate may be helpful for detecting and assaying cutinase activity in mixed solutions, such as crude fungal extracellular extracts.     

Respiratory‐induced vasoconstriction measured by light transmission and by laser Doppler signal

Changes in finger tissue blood volume (TBV) measured by light transmission and in laser Doppler flow (LDF) were obtained during long breathing (of 12 s period) and associated with the respiratory phases, inspiration and expiration. For fifteen out of sixteen subjects TBV and LDF started to decrease 0–2 s after the start of expiration and increased during inspiration but the start of increase occurred before the start of inspiration, showing that the respiratory‐induced changes in TBV and LDF are mainly associated with the expiration. Decrease of TBV and LDF after expiration was also found during the inspiratory gasps (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Prevalence of NDM‐1 carbapenemase in patients with diarrhoea in Pakistan and evaluation of two chromogenic culture media

Aims

To evaluate two chromogenic media, Brilliance CRE and chromID CARBA, with stool samples referred to the Public Health Laboratories Division of the National Institute of Health in Islamabad, and assess the prevalence of carbapenemase‐producing Enterobacteriaceae (CPE) in this population.

Methods and Results

One hundred and fifty‐two stool samples from patients with diarrhoea were referred to the Microbiology Department and were investigated for the presence of CPE using two chromogenic culture media, Brilliance CRE and chromID CARBA. Thirteen patients (8·6%) were found to be colonized with CPE and all produced NDM‐1 carbapenemase. Twelve of these patients (92%) were found to be colonized by culture on chromID CARBA compared with seven (54%) using Brilliance CRE.

Conclusions

If only coloured colonies were considered as presumptive CPE, the sensitivity, specificity and positive predictive value were 54, 23 and 6% for Brilliance CRE and 85, 85 and 36% for chromID CARBA, respectively.

Significance and Impact of the Study

We conclude that Enterobacteriaceae that produce NDM‐1 carbapenemase can be found in patients from all major provinces of Pakistan and that chromID CARBA was the most effective of the two chromogenic media in this setting.     

Site-directed mutagenesis of human cytosolic sulfotransferase (SULT) 2B1b to phospho-mimetic Ser348Asp results in an isoform with increased catalytic activity

Human SULT2B1b is distinct from other SULT isoforms due to the presence of unique amino (N)- and carboxy (C)-terminal peptides. Using site-directed mutagenesis, it was determined that phosphorylation of Ser348 was associated with nuclear localization. To investigate the effects of this phosphorylation of Ser348 on activity and cellular localization, an in silico molecular mimic was generated by mutating Ser348 to an Asp. The Asp residue mimics the shape and charge of a phospho-Ser and homology models of SULT2B1b-phospho-S348 and SULT2B1b-S348D suggest a similar significant structural rearrangement in the C-terminal peptide. To evaluate the functional consequences of this post-translational modification and predicted rearrangement, 6His-SULT2B1b-S348D was synthesized, expressed, purified and characterized. The 6His-SULT2B1b-S348D has a specific activity for DHEA sulfation ten-fold higher than recombinant 6His-SULT2B1b (209.6 and 21.8pmolmin(-1)mg(-1), respectively). Similar to native SULT2B1b, gel filtration chromatography showed SULT2B1b-S348D was enzymatically active as a homodimer. Stability assays comparing SULT2B1b and SUL2B1b-S348 demonstrated that SULT2B1b is 60% less thermostable than SULT2B1b-348D. The increased stability and sulfation activity allowed for better characterization of the sulfation kinetics for putative substrates as well as the determination of dissociation constants that were difficult to obtain with wild-type (WT) 6His-SULT2B1b. The K(D)s for DHEA and PAPS binding to 6His-SULT2B1b-S348D were 650±7nM and 265±4nM, respectively, whereas K(D)s for binding of substrates to the WT enzyme could not be determined. Characterization of the molecular mimic SULT2B1b-S348D provides a better understanding for the role of the unique structure of SULT2B1b and its effect on sulfation activity, and has allowed for improved kinetic characterization of the SULT2B1b enzyme.