Inactivation of Staphylococcus hyicus lipase by hexadecylsulfonyl fluoride: evidence for an active site serine

The Staphylococcus hyicus lipase is an acyl hydrolase with broadsubstrate specificity including neutral glycerides and phospholipids.To obtain further insight into the mechanism of action of thisenzyme, we tested several sulfonyl fluorides as active site-directedinhibitors. The enzyme is resistant to the well-known serineprotease/esterase inhibitor phenylmethanesulfonyl fluoride (PMSF),but is rapidly inactivated by hexadecylsulfonyl fluoride. Thekinetics of inactivation were studied in Triton X-100 micelles.Inactivation is fast and the rate of inactivation is constantover the pH range where this lipase is active. Metal ions likeCa²⁺ and Sr²⁺ do not appreciably influence the rate of inactivation,although the enzymatic activity is significantly increased,suggesting a structural role for these ions. The S.hyicus lipasecontains a consensus sequence G-H/Y-S-X-G. Substitution by site-directedmutagenesis of this serine (Ser369) by a cysteine resulted ina mutant with only 0.2% residual activity. The activity of thismutant could not be inhibited with water-soluble sulfhydrylreagents either in the presence or absence of Triton X-100 micelles.In the presence of Triton X-100 micelles, inactivation of themutant occurred with 4-nitrophenylhexadecyl disulfide (t_1/2= 125 min) while the wild-type enzyme does not react at all.We conclude that Ser369 is the active site residue and thatin water this residue is inaccessible. Only after interfacialactivation Ser369 (or Cys369) becomes exposed and reacts withirreversible inhibitors.     

Metabolite interactions in prostatic fluid mimics assessed by 1H NMR

Magnetic Resonance Materials in Physics, Biology and Medicine - Molecular interactions in prostatic fluid are of biological interest and may affect MRI and MRS of the prostate. We investigated the...     

Developments in proton MR spectroscopic imaging of prostate cancer

Magnetic Resonance Materials in Physics, Biology and Medicine - In this paper, we review the developments of 1H-MR spectroscopic imaging (MRSI) methods designed to investigate prostate cancer,...     

Comparing Syntactic and Semantic Action Refinement

The semantic definition of action refinement on labelled configuration structures is compared with the notion ofsyntactic substitution, which can be used as another notion of action refinement in a process algebraic setting. The comparison is done by studying a process algebra equipped with sequential composition, parallel composition with an explicit synchronisation set, and an operator for action refinement. On the one hand, the language (including the refinement operator) is given a configuration structure semantics. On the other hand, a reduction procedure transforms a process termPinto aflatterm (i.e., with the refinement operator not occurring in it)red(P) by means of syntactic substitution, defined in a structural inductive way. The main aim of the paper is to investigate general conditions under which the termsPandred(P) have the same semantics. The results we present are essentially dependent on the question whether the refined action can be synchronised or not. In the latter case,Pandred(P) give rise to isomorphic configuration structures under mild assumptions. The former case is considerably more difficult, since then refinement cannot be expected to distribute over parallel composition. We give necessary and sufficientsemanticconditions under which distribution still holds up to semantic equivalence. Subsequently, we also give sufficient (but not necessary)syntacticconditions for reducible terms. Finally, we generalise these results to a language with recursion.     

Enhancement of phenolic compounds content and antioxidant activity of strawberry (Fragaria × ananassa) juice by block freeze concentration technology

The aim of this study was to evaluate the effects of freeze concentration of strawberry juice on content of total phenolic, anthocyanins content and antioxidant activity of the concentrated fluid and the ice obtained. An increase in the values of phenolic content was observed in the concentrate fraction for all the freeze concentration stages (five stages). The process efficiency presented a reduction when compared with the first stage, and however, the average efficiency remained around 65% in all the stages. The concentration of pelargonidin‐3‐O‐glycoside present in samples was increased in all stages. Furthermore, the antioxidant activity of the concentrated fluid of each stage, measured by DPPH and ABTS methods, was significantly higher in comparison with the juice feed. Also, antioxidant activity can be correlated with the content of pelargonidin‐3‐O‐glycoside presented in the strawberry juice. Freeze concentration can be considered a good alternative for improving the antioxidant activity of strawberry juice.     

Effect of Lowering Asymmetric Dimethylarginine (ADMA) on Vascular Pathology in Atherosclerotic ApoE-Deficient Mice with Reduced Renal Mass

The purpose of the work was to study the impact of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) and its degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH1), on atherosclerosis in subtotally nephrectomized (SNX) ApoE-deficient mice. Male DDAH1 transgenic mice (TG, n = 39) and C57Bl/6J wild-type littermates (WT, n = 27) with or without the deletion of the ApoE gene underwent SNX at the age of eight weeks. Animals were sacrificed at 12 months of age, and blood chemistry, as well as the extent of atherosclerosis within the entire aorta were analyzed. Sham treated (no renal mass reduction) ApoE-competent DDAH1 transgenic and wild-type littermates (n = 11) served as a control group. Overexpression of DDAH1 was associated with significantly lower ADMA levels in all treatment groups. Surprisingly, SNX mice did not exhibit higher ADMA levels compared to sham treated control mice. Furthermore, the degree of atherosclerosis in ApoE-deficient mice with SNX was similar in mice with or without overexpression of DDAH1. Overexpression of the ADMA degrading enzyme, DDAH1, did not ameliorate atherosclerosis in ApoE-deficient SNX mice. Furthermore, SNX in mice had no impact on ADMA levels, suggesting a minor role of this molecule in chronic kidney disease (CKD) in this mouse model.     

Transmit/receive headcoil for optimal <Superscript>1</Superscript>H MR spectroscopy of the brain in paediatric patients at 3 T

¹H magnetic resonance (MR) spectroscopy is a useful tool to obtain metabolic information from the brain in paediatric patients. To detect signals of metabolites at low concentrations or from small volumes, the signal-to-noise ratio (SNR) has to be optimized. The SNR can be increased by going to higher field strengths. However, this leads to higher spectral bandwidths, which increases the chemical shift artefact. Here we present a transmit/receive headcoil which is adapted to the dimensions of the paediatric head and enables PRESS localization with high radio-frequency (RF) bandwidths that minimize the chemical shift displacement to only 5%. In addition, since the pulse lengths are shorter with higher RF bandwidths, the echo time can be reduced to 10 ms improving SNR as well.Parts of this work have been presented at the European Society for Magnetic Resonance in Medicine and Biology in Rotterdam in 2003     

Measurements of T1 and T2 relaxation times of colon cancer metastases in rat liver at 7 T

The purpose of this study was to investigate the magnetic resonance imaging (MRI) characteristics of colon cancer metastases in rat liver at 7 T. A dedicated RF microstrip coil of novel design was built in order to increase the signal-to-noise ratio and, in combination with respiratory triggering, to minimize motion artifacts. T₁- and T₂-weighted MR imaging was performed to follow tumor growth. T₁-weighted images provided a good anatomical delineation of the liver structure, while the best contrast between metastases and normal liver tissue was achieved with T₂-weighted images.Measurements of T₁ and T₂ relaxation times were performed with inversion recovery FLASH and Carr–Purcell–Meiboom–Gill and inversion recovery FLASH imaging sequences, respectively, for quantitative MR characterization of metastases. Both the T₁ and T₂ of the metastases were significantly higher than those of normal liver tissue. Further, an increase in the T₁ relaxation time of the metastases was observed with tumor growth. These findings suggest that quantitative in vivo MR characterization provides information on tumor development and possibly response to therapy, though additional studies are needed to elucidate the correlation between the changes in relaxation times and tumor microenvironment.