Chronological protein synthesis in regenerating rat liver

Liver regeneration has been studied for decades; however, its regulation remains unclear. In this study, we report a dynamic tracing of protein synthesis in rat regenerating liver with a new proteomic technique, ³⁵S in vivo labeling analysis for dynamic proteomics (SiLAD). Conventional proteomic techniques typically measure protein alteration in accumulated amounts. The SiLAD technique specifically detects protein synthesis velocity instead of accumulated amounts of protein through ³⁵S pulse labeling of newly synthesized proteins, providing a direct way for analyzing protein synthesis variations. Consequently, protein synthesis within short as 30 min was visualized and protein regulations in the first 8 h of regenerating liver were dynamically traced. Further, the 3.5–5 h post partial hepatectomy (PHx) was shown to be an important regulatory turning point by acute regulation of many proteins in the initiation of liver regeneration.     

Isolation, synthesis and structures of cytotoxic ginsenoside derivatives

Four known ginsenosides: ginsenoside-Rb1 (1), Rb3 (2), Rd (3) and Re (4) were isolated from the methanolic extract of the traditional Chinese medicine Panax ginseng C. A. Meyer. Further enzyme reactions and chemical modifications led us to obtain ginsenoside-M1 (5) and synthesize three novel mono-esters of ginsenoside-M1, ginsenoside-DM1 (6), PM1 (7) and SM1 (8) 30 - 50% of yield via a facile and green synthetic strategy. The structures were elucidated on the basis of extensive 1D- and 2DNMR, as well as high resolution ESI-TOF mass spectroscopic analyses. The isolated and synthetic compounds were tested in an anti-tumor bioassay, and compounds 5-8 showed considerable cytotoxicity (SRB) against several human cancer cell lines (breast cancer MCF-7, skin melanoma SK-MEL-2 and human ovarian carcinoma B16), but moderate effects on lung carcinoma COR-L23. The other ginsenosides showed no effects.     

Optimizing protein recovery yield from serum samples treated with beads technology

Proteomics studies are often complicated by the wide dynamic range of the biological fluids, in which few highly abundant proteins obscure the signal of low abundant ones. To overcome this problem, several techniques have been developed on the basis of "depletion principles," namely immuno-subtraction with specific antibodies against the most-abundant proteins. Unfortunately, the probability of codepletion is a noteworthy drawback associated with these strategies. The ProteoMiner (PM) technology is a novel approach, consisting of a combinatorial library of hexapeptide ligands coupled to beads, that allows the capture of all species present in a proteome, but at much reduced protein concentration differences, simultaneously enhancing the concentration of the most dilute species. In this study, we evaluated the compatibility of the PM kit's elution reagent with 2-DE analysis, comparing five different purification methods on serum samples eluted from the beads: the "ReadyPrep 2-D Clean-up kit" and precipitation with organic solvents, as acetone/methanol, TCA/acetone, ACN, and chloroform/methanol. Considering protein recovery yield (quantity) and 2-DE spot pattern (quality), precipitation with ACN offered the most promising approach, showing the best spot resolution in all regions of the pH gradient and the greatest number of protein spots visualized on 2-D gels.     

Metabolite profiling of ginsenoside Rg1 after oral administration in rat

The goal of this study is to investigate the biotransformation of ginsenoside Rg₁ in vivo. A highly sensitive and specific LC‐MS/MS method was developed and used for metabolite identification in rat feces and urine after oral administration of ginsenoside Rg₁. Four metabolites of Rg₁ were detected in rat feces and three metabolites of Rg₁ were detected in rat urine. Deglycosylation and oxygenation were found to be the major metabolic pathways of ginsenoside Rg₁ after oral administration in rat. Except for the reported metabolites Rh₁ and protopanaxatriol, mono‐oxygenated Rg₁ and mono‐oxygenated protopanaxatriol were detected for the first time after oral administration of Rg₁. The in vivo metabolite profiling of ginsenoside Rg₁ in rat was proposed. Viewed collectively, Rg₁ was metabolized to mono‐oxygenated Rg₁, Rh₁, protopanaxatriol and the secondary metabolite mono‐oxygenated protopanaxatriol in rat. Copyright © 2014 John Wiley & Sons, Ltd.     

Altered protein synthesis in rat kidney cells exposed to semiconductor materials

It is thought that the extensive industrial use of arsenic, gallium and indium, which have applications as the materials for III–V semiconductors, will increase human exposure to these compounds in the near future. We have undertaken the development of new biological indicators for assessing exposure to these elements. Element-specific alterations in protein synthesis patterns were expected to occur following exposure to arsenic compounds. We examined alterations in protein synthesis in primary cultures of rat kidney proximal tubule epithelial cells by sodium arsenite, gallium chloride and indium chloride, utilizing two-dimensional gel electrophoresis. After incubation with the chemicals for 20 h, newly synthesized proteins were labeled with [³⁵S]methionine. A protein with a molecular weight (M_r) of 30 000 was markedly induced on exposure to 10 μM arsenite or 300 μM gallium chloride, and synthesis of proteins with M_r values of 85 000, 71 000, 65 000, 51 000, 38 000 and 28 000 were also increased by exposure to arsenite and gallium chloride. No significant changes were observed upon exposure to indium. Some of these increased proteins could be heat-shock proteins.     

A web site for the rat serum protein study group

We describe a site http://users.unimi.it/-ratserum/homeframed.ht ml with clickable maps of serum proteins of control and inflamed rats as well as quantitative data on the expression of such serum proteins under varying physiological and experimental conditions. This information enhances the value of minimally invasive techniques, thus reducing the number of animals to be treated, and eventually sacrificed, in pharmacological/toxicological research projects.     

Stimulation of Erwinia sp. Fumarase and aspartase synthesis by changing medium components

The optimal concentrations of nutrient medium components, aeration conditions, and pH providing for maximum biomass yields, as well as fumarase and l-aspartase activities, during submerged cultivation of Erwinia sp. were determined. The data showed that different concentrations of carbon source (molasses) and pH of the nutrient medium were required to reach the maximum fumarase and l-aspartase activities. Calculations performed by application of the additive lattice model suggested that the combination of these optimized factors would result in 3.2-, 3.4-, and 3.8-fold increases as compared to the experimental means in Erwinia sp. biomass, and l-aspartase and fumarase activities, respectively. The conditions of the fumaric acid biotransformations into l-malic and l-aspartic acids were optimized on the basis of intact Erwinia sp. cells, a fumarase and l-aspartase producer. In the cases of fumarate transformation into l-malic acid and of fumarate transformation into l-aspartic acids, fumarase and l-aspartase activities increased 1.5- and 1.7-fold, respectively. The experimental data were consistent with these estimates to 80% accuracy. In comparison with the additive lattice model, the application of polynomial nonlinear model allowed the between-factor relations to be considered and analyzed, which resulted in 1.1-, 1.27-, and 1.1-fold increases in Erwinia sp. biomass and fumarase and l-aspartase activities for the case of cultivation. In the case of fumarate transformation into l-malic acid, this model demonstrated a 1.7-fold increase in fumarase activity, whereas during fumarate transformation into l-aspartic acid no significant change in aspartase activity was observed.     

Inhibition of polyphosphoinositide breakdown and prostaglandin synthesis in rat mesangial cells by islet-activating protein

Conclusions Our results show that pertussis toxin abolished the angiotensin II-induced breakdown of PIP₂ and also the increase in prostaglandin E₂ synthesis in mesangial cells. It is suggested that a guanine nucleotide binding regulatory protein is somehow involved in receptor-mediated activation of phospholipase C in mesangial cells.

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Islet-activating-Protein hemmt Polyphosphoinositid-Abbau und Prostaglandinsynthese in Rattenmesangialzellen

     

An application of fast protein liquid chromatography (FPLC) in the purification of retinol binding protein from rat serum

Summary Retinol Binding Protein (RBP) is the specific plasma protein for the transport of retinol from liver to peripheral tissues. It is a single polypeptide chain of approximately 21 KDa, and circulates as a 11 molar complex with transthyretin (TTR). The relative low concentration in plasma (40–50 g/ml and its chromatographic behaviour on ionic exchangers render the purification of rat RBP particularly laborious. In this paper we report a simple and semi-automatic method for the preparative purification to homogeneity of rat serum RBP. The method includes: (1) Selective removal of albumin by affinity chromatography on a Blue Sepharose column; (2) Chromatography on a Mono Q strong anion exchange column; (3) Dissociation of the RBP-TTR complex by 3 M urea; (4) Concentration, desalting and freeze drying. The purified RBP has been used for the production in rabbit of antirat RBP specific antibodies for studies on nutritional control of RBP synthesis and metabolism.     

Characteristics of a protein error in determination of serum protein in the presence of inorganic salt.

There is a possibility that the color development of the dye-binding method based on a protein error of a pH indicator is affected by the coexisting inorganic salt. Thus, the author theoretically and experimentally investigated the effect of the inorganic salt on the protein error. In a theoretical analysis, the anion of an inorganic salt, like the dissociated dye and buffer anions, was assumed to react with the protein, forming a colorless anion-protein complex. The calculated results were compared with those obtained by experiments using three pH indicators and various kinds of inorganic salts. The calculated results obtained are as follows: (1) The color development decreases with increasing the concentration of the inorganic salt and the equilibrium constant of the reaction between the inorganic salt and protein; (2) The rate of the absorbance decrease is larger for a lower concentration of the inorganic salt than for a higher one; (3) The larger is the equilibrium constant, the larger is the absorbance decrease. The absorbance decrease was caused by the anion, and was increased by increasing the anion concentration. The magnitude of the effect of the anion was iodide > bromide > chloride, which was associated with their ionic radius. The difference in the effect of the anion was thought to indicate that the equilibrium constant, in other words, the bonding strength of the anion to protein is iodide > bromide > chloride.     

Proteins of rat serum, urine, and cerebrospinal fluid: VI. Further protein identifications and interstrain comparison

We have investigated the biological fluids--serum, cerebrospinal fluid, and urine--of three strains of rats; the present data extend our database (also available on-line) and may be of interest for pharmacological and toxicological investigation. Specifically, we have defined reference maps of the major protein components in cerebrospinal fluid and urine. Compartment-specific isoforms were recognized for transferrin and transthyretin. Mass spectrometric data established the cleavage site of the signal peptide and identified the N-terminal blocking group of prostaglandin D synthase from rat cerebrospinal fluid. A previously undescribed member of the family of low molecular mass rat urinary proteins was characterized as containing a sequence similar, but not identical, to the N-terminal region of rat urinary protein-2 (RUP-2), and divergent from RUP-1.     

Quantitative determination of ginsenoside Rh2 in rat biosamples by liquid chromatography electrospray ionization mass spectrometry

Ginsenoside Rh₂ is a “hot” natural compound with great potential as a new anti-cancer drug based on abundant pharmacological experiments. However, no systemic pharmacokinetic study of Rh₂ was reported because current analysis methods could not fully meet the requirements. Thus, we developed a simple LC/MS method with highly improved sensitivities for the determination of Rh₂ in rat plasma, bile, urine, feces and most tissues. The tissues and feces were firstly homogenized mechanically using buffer and methanol as the media, respectively. Plasma, bile, urine and tissue homogenates were extracted with diethyl ether for sample preparation. Feces homogenates were directly deproteinized with acetonitrile. The subsequent analysis procedures were performed on a Shimadzu LCMS2010A system (electrospray ionization single quadrupole mass analyzer), with an ODS column (150 mm × 2.0-mm i.d., 5 μm) plus a C18 guard column for separation and ammonium chloride (500 μmol) as mobile phase additive. The proportions of mobile phase were changed timely according to gradient programs. Chlorinated adducts of molecular ions [M + Cl]⁻ of Rh₂ at m/z 657.35 and internal standard digitoxin at m/z 799.55 were monitored in selective ion monitoring mode of negative ions. The method was validated to be accurate, precise and rugged with good linearity in all matrices, according to the FDA guidelines. The lower limits of quantitation in rat plasma, urine and feces were 0.2, 0.2 and 20 ng/mL respectively. Stability studies were also performed, indicating that there were no stability-related problems in the analytical procedure of Rh₂. The proposed method was successfully applied to the preclinical pharmacokinetic research of Rh₂ in rats, including plasma kinetics, tissue distribution and excretion studies.