Application of DIGE and mass spectrometry in the study of type 2 Diabetes Mellitus (T2DM) mouse models

Knowledge of the differences between the amounts and types of protein that are expressed in diseased compared to healthy subjects may give an understanding of the biological pathways that cause disease. This is the reasoning behind the presented protocol, which uses difference gel electrophoresis to discover up‐ or down‐regulated proteins between mice of different genotypes, or of those fed on different diets, that may thus be prone to develop diabetes‐like phenotypes. Subsequent analysis of these proteins by tandem mass spectrometry typically facilitates their identification with a high degree of confidence

S-adenosyl-L-methionine: (S)-scoulerine 9-O-methyltransferase, a highly stereo- and regio-specific enzyme in tetrahydroprotoberberine biosynthesis

Suspension cultures of Berberis species are useful sources for the detection and isolation of a new enzyme which transfers the methyl group from S-adenosyl-L-methionine specifically to the 9-position of the (S)-enantiomer of scoulerine, producing (S)-tetrahydrocolumbamine. The enzyme was enriched 27-fold; it is not particle bound, has a pH optimum of 8.9, a molecular weight of 63 000 and shows a high degree of substrate specificity

Evaluation of mTOR-regulated mRNA translation.

mTOR, the mammalian target of rapamycin, regulates protein synthesis (mRNA translation) by affecting the phosphorylation or activity of several translation factors. Here, we describe methods for studying the impact of mTOR signalling on protein synthesis, using inhibitors of mTOR such as rapamycin (which impairs some of its functions) or mTOR kinase inhibitors (which probably block all functions).To assess effects of mTOR inhibition on general protein synthesis in cells, the incorporation of radiolabelled amino acids into protein is measured. This does not yield information on the effects of mTOR on the synthesis of specific proteins. To do this, two methods are described. In one, stable-isotope labelled amino acids are used, and their incorporation into new proteins is determined using mass spectrometric methods. The proportions of labelled vs. unlabeled versions of each peptide from a given protein provide quantitative information about the rate of that protein's synthesis under different conditions. Actively translated mRNAs are associated with ribosomes in polyribosomes (polysomes); thus, examining which mRNAs are found in polysomes under different conditions provides information on the translation of specific mRNAs under different conditions. A method for the separation of polysomes from non-polysomal mRNAs is describe

A simple method to assess the oxidative susceptibility of low density lipoproteins

BACKGROUND: Oxidative modification of low density lipoproteins (LDL) is recognized as one of the major processes involved in atherogenesis. The in vitro standardized measurement of LDL oxidative susceptibility could thus be of clinical significance. The aim of the present study was to establish a method which would allow the evaluation of oxidative susceptibility of LDL in the general clinical laboratory. RESULTS: LDL was isolated from human plasma by selective precipitation with amphipathic polymers. The ability of LDL to form peroxides was assessed by measuring thiobarbituric acid reactive substances (TBARS) after incubation with Cu(2+) and H(2)O(2). Reaction kinetics showed a three-phase pattern (latency, propagation and decomposition phases) which allowed us to select 150 min as the time point to stop the incubation by cooling and EDTA addition. The mixture Cu(2+)/H(2)O(2) yielded more lipoperoxides than each one on its own at the same time end-point. Induced peroxidation was measured in normal subjects and in type 2 diabetic patients. In the control group, results were 21.7 ± 1.5 nmol MDA/mg LDL protein, while in the diabetic group results were significantly increased (39.0 ± 3.0 nmol MDA/mg LDL protein; p < 0.001). CONCLUSION: a simple and useful method is presented for the routine determination of LDL susceptibility to peroxidation in a clinical laboratory

Partial purification of alpha-amylase from culture supernatant of Bacillus subtilis in aqueous two-phase systems

The original publication can be found at www.springerlink.comA study was made of the partition and purification of -amylase from a culture supernatant of Bacillus subtilis in the polyethylene glycol (PEG)—citrate aqueous two-phase system (ATPS). Factors that influenced the partition of the protein in this system, including the molecular weight of the PEG, the tie line length of ATPS, the pH value and the sodium chloride concentration, were investigated. Purification of -amylase was attained with a purification factor (PF) of 1.8 and 90% yield at pH 6.0 in a PEG1000-citrate ATPS with short tie line length. By utilizing the salt-out effect of neutral salt, the purification of -amylase was further improved to 2.0 of PF and 80% yield in a PEG3350-citrate ATPS with 4% sodium chloride.Wenbo Zhi, Jiangnan Song, Jingxiu Bi and Fan Ouyan

A cysteine proteinase in the penetration glands of the cercariae of Cotylurus cornutus (Trematoda, Strigeidae)

A cysteine proteinase from the penetration glands of Cotylurus cornutus cercariae was examined with histochemical and biochemical methods. The enzyme hydrolyzed gelatin, azocoll, azocasein, azoalbumin, N-blocked-l-arginine-4-methoxy-2-naphthylamide, and N-blocked-p-nitroanilide, but did not degrade elastin. The metal ion complexane ethylenediamine tetraacetate and the thiol-reducing compound dithioerythritol enhanced the proteinase activity, whereas the thiol-blocking compounds p-hydroxymercuribenzoate and N-ethylmaleimide (NEM) inhibited it. The enzyme was also sensitive to leupeptin but insensitive to soybean trypsin inhibitor. An electrophoretic separation of extract proteins from the cercariae under acidic, non-denaturing conditions and in the presence of 0.1% gelatin in a polyacrylamide gel revealed the presence of two distinct and three weak transparent bands in the gel resulting from a gelatinolytic activity at pH 6.8. The distinct bands apparently resulted from the activity of the glandular enzyme and lysosomal cathepsin B, whereas the weak ones presumably indicated these enzymes partially degraded in the course of the preparative procedure. No gelatinolysis occurred following treatment of an extract sample with 0.1 mM NEM

Inhibition of Fungi and Gram-Negative Bacteria by Bacteriocin BacTN635 Produced by Lactobacillus plantarum sp. TN635

The aim of this study was to evaluate 54 lactic acid bacteria (LAB) strains isolated from meat, fermented vegetables and dairy products for their capacity to produce antimicrobial activities against several bacteria and fungi. The strain designed TN635 has been selected for advanced studies. The supernatant culture of this strain inhibits the growth of all tested pathogenic including the four Gram-negative bacteria (Salmonella enterica ATCC43972, Pseudomonas aeruginosa ATCC 49189, Hafnia sp. and Serratia sp.) and the pathogenic fungus Candida tropicalis R2 CIP203. Based on the nucleotide sequence of the 16S rRNA gene of the strain TN635 (1,540 pb accession no FN252881) and the phylogenetic analysis, we propose the assignment of our new isolate bacterium as Lactobacillus plantarum sp. TN635 strain. Its antimicrobial compound was determined as a proteinaceous substance, stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 3 and 11 with an optimum at pH = 7. The BacTN635 was purified to homogeneity by a four-step protocol involving ammonium sulfate precipitation, centrifugal microconcentrators with a 10-kDa membrane cutoff, gel filtration Sephadex G-25, and C18 reverse-phase HPLC. SDS-PAGE analysis of the purified BacTN635, revealed a single band with an estimated molecular mass of approximately 4 kDa. The maximum bacteriocin production (5,000 AU/ml) was recorded after a 16-h incubation in Man, Rogosa, and Sharpe (MRS) medium at 30 °C. The mode of action of the partial purified BacTN635 was identified as bactericidal against Listeria ivanovii BUG 496 and as fungistatic against C. tropicalis R2 CIP203

Monitoring HSVtk suicide gene therapy: the role of [18F]FHPG membrane transport

Favourable pharmacokinetics of the prodrug are essential for successful HSVtk/ganciclovir (GCV) suicide gene therapy. [F-18] FHPG PET might be a suitable technique to assess the pharmacokinetics of the prodrug GCV noninvasively, provided that [F-18] FHPG mimics the behaviour of GCV. Since membrane transport is an important aspect of the pharmacokinetics of the prodrug, we investigated the cellular uptake mechanism of [F-18] FHPG in an HSVtk expressing C6 rat glioma cell line and in tumour- bearing rats. The nucleoside transport inhibitors dipyridamol, NBMPR and 2- chloroadenosine did not significantly affect the [F-18] FHPG uptake in vitro. Thymidine and uridine significantly decreased [F-18] FHPG uptake by 84 and 58%, respectively, but an enzyme assay revealed that this decline was due to inhibition of the HSVtk enzyme rather than membrane transport. Nucleobase transport inhibitors, thymine and adenine, caused a 58 and 55% decline in tracer uptake, respectively. In vivo, the ratio of [F-18] FHPG uptake in C6tk and C6 tumours decreased from 3.070.5 to 1.070.2 after infusion of adenine. Thus, in our tumour model, [F-18] FHPG transport exclusively occurred via purine nucleobase transport. In this respect, FHPG does not resemble GCV, which is predominantly taken up via the nucleoside transporter, but rather acyclovir, which is also taken up via the purine nucleobase carrier

Knockout studies reveal an important role of <i>plasmodium</i> lipoic acid protein ligase a1 for asexual blood stage parasite survival

Lipoic acid (LA) is a dithiol-containing cofactor that is essential for the function of a-keto acid dehydrogenase complexes. LA acts as a reversible acyl group acceptor and 'swinging arm' during acyl-coenzyme A formation. The cofactor is post-translationally attached to the acyl-transferase subunits of the multienzyme complexes through the action of octanoyl (lipoyl): &lt;i&gt;N&lt;/i&gt;-octanoyl (lipoyl) transferase (LipB) or lipoic acid protein ligases (LplA). Remarkably, apicomplexan parasites possess LA biosynthesis as well as scavenging pathways and the two pathways are distributed between mitochondrion and a vestigial organelle, the apicoplast. The apicoplast-specific LipB is dispensable for parasite growth due to functional redundancy of the parasite's lipoic acid/octanoic acid ligases/transferases. In this study, we show that &lt;i&gt;LplA1&lt;/i&gt; plays a pivotal role during the development of the erythrocytic stages of the malaria parasite. Gene disruptions in the human malaria parasite &lt;i&gt;P.falciparum&lt;/i&gt; consistently were unsuccessful while in the rodent malaria model parasite &lt;i&gt;P. berghei&lt;/i&gt; the &lt;i&gt;LplA1&lt;/i&gt; gene locus was targeted by knock-in and knockout constructs. However, the &lt;i&gt;LplA1&lt;/i&gt; &lt;sup&gt;(-)&lt;/sup&gt; mutant could not be cloned suggesting a critical role of LplA1 for asexual parasite growth &lt;i&gt;in vitro&lt;/i&gt; and &lt;i&gt;in vivo&lt;/i&gt;. These experimental genetics data suggest that lipoylation during expansion in red blood cells largely occurs through salvage from the host erythrocytes and subsequent ligation of LA to the target proteins of the malaria parasite

Protein versus DNA as a marker for peripheral blood mononuclear cell counting

Quantitative analysis of intracellular analytes requires an accurate and precise assay not only for the quantitation of the analytes, but also for the quantitation of the number of cells in which they were determined. In this technical note we compare protein and DNA as markers for the number of peripheral blood mononuclear cells (PBMCs) isolated from whole blood. The protein content of samples was highly influenced by red blood cell contamination and was, therefore, a less suitable marker. The DNA-based method was unaffected by red blood cell contamination and was finally validated over a range from 10 × 106 to 300 × 106 PBMCs/mL