Urinary protein pattern and quantitative determination of total urinary protein by HPLC

Conclusion GPC has been found valuable to differentiate between physiological, tubular and glomerular types of proteinuria. Though protein discriminating might be further improved, the speed of analysis, the ability for automatisation, the favourable qualitative and quantitative results advocate this method for routine use.

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Urinproteinmuster und quantitative Proteinbestimmung im Urin durch HPLC

     

Proteomic analysis reveals changes in the hippocampus protein pattern of rats exposed to dietary zinc deficiency

An experiment was performed to observe protein changes in the hippocampus of zinc‐deficient (ZD) rats. Twenty‐four male weanling Wistar rats were randomly assigned to ZD (n=12) and control groups (n=12). After 4‐wk treatment, we used 2‐DE and MALDI‐TOF‐MS to analyze the proteomes of hippocampus in the two groups. One of the important differential proteins, ubiquitin C‐terminal hydrolase L1 (Uch‐L1), was confirmed by Western blot assays. The results demonstrated that compared with the controls, ZD rats had significantly reduced plasma zinc concentration and alkaline phosphatase activity. The latency period in passive avoidance performance was also significantly shorter for the ZD rats. Nine proteins were differentially expressed between the two groups. Eight of them were identified. Tubulin β chain and voltage‐dependent anion channel 1 were upregulated, while mitochondrial aldehyde dehydrogenase, α‐enolase, dimethylarginine dimethylaminohydrolase 1, F‐actin capping protein α‐2 subunit, pyruvate dehydrogenase β and Uch‐L1 were downregulated, respectively. Importantly, some of the identified proteins (e.g. Uch‐L1) are known to be involved in cognitive impairment. Western blot analysis of hippocampus Uch‐L1 expression confirmed the proteomic findings. The data indicated that there may be common mechanisms or pathways in cognitive dysfunction between neurodegenerative diseases and zinc deficiency.     

Investigation of protein foams obtained by bubbling

Summary The foaminesses of bovine serum albumin solutions, (BSA) with and without buffer, salt and alcohol additives were measured by bubbling and their surface tensions were obtained as function of the timet. The timet _DG , which is necessary to attain the equilibrium surface tension is long (15 h). The area requirement of a single surface adsorbed moleculeA _j0 was obtained fromd/dc, wherec the surfactant concentration, by the Gibbs relation. By assuming the existence of a hydration complex, which consists of the surfactant andX water molecules, the,coordination numbers were estimated toX=12±1 fromA _j0 and the surface requirement of the hydrophilic group of the surfactant. The dependences of CMC andX on the additives are discussed. A good relation prevails between andt _DG . By applying the phase change model of Avrami for the adsorption and surface denaturation of theBS A, simple relation was found between the dimensionless surface tensionV = (₀ - _s) (_st-_s) and the timet:log (2.3 logV) =n logt + logb The good relations between andn as well as between andnb indicate the applicability of this model.

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Zusammenfassung Das Schaumbildungsvermögen von Rinderserumalbumin-Lösungen (BSA), mit und ohne Zusätze wurde gemessen und ihre Oberflächenspannung als Funktion der Meßzeit ermittelt. Die Einstellzeitt _DG der Gleichgewichtsoberflächenspannung _eq ist lang. Zwischen undt _DG läßt sich eine einfache Beziehung aufstellen. Aus der Änderung von _eq mit der bedarf des adsorbierten Molekel ermitteln. Durch die Anwendung des Modells von Steinbach kann gezeigt werden, daß sich der Flächenbedarf durch die Bildung eines Assoziates erklären läßt, bei dem das Wasser komplexartig um das Protein-Molekül lagert. Für die Koordinationszahl der WassermoleküleX ergab sichX=12±1.Durch das Kristallwachstumsmodell von Avrami, das von überreiter auf Polymeradsorption angewendet wurde, läßt sich die Zeitabhängigkeit der Oberflächenspannung beschreiben. Das Schaumbildungsvermögen kann man als einfache Funktion der Konstanten dieser Avrami-Gleichung darstellen.

     

Investigation of Aluminate Solutions by water activity measurement

Isopiestic measurements were used to determine the change in water activity due to the variation of the aluminium content of alkaline aluminate solutions. This parameter was found suitable to indicate the concentration dependence of the species present in the solution. It was established that the increase of water activity due to the increased aluminium concentration can be attributed to two processes: 1. the coordination of hydroxide ion to aluminium, 2. dimerization connected with the dehydration of the monomeric species. On the basis of the Gibbs-Duhem equation the water activity data gave an indication of the concentration ranges in Which the sodium aluminate solution exists mainly as (I) NaOH + monomeric Al(OH)₄^? and ( H ) NaOH + dimeric Al₂ O(OH)₆ ^2? ions.     

The scaled volume as an image analysis variable for detecting changes in protein expression levels by silver stain.

A new variable for measuring the relative intensities of silver stained protein spots on two-dimensional gels is described. The scaled volume (SV) more accurately measures the intensity of protein spots and accounts for differences frequently encountered when trying to compare two gels than other variables such as relative volume ratio, optical density, or relative optical density. The SV scales the signal of interest by the noise (gel background) with secondary signals removed (spots not of interest, e.g., technical artifacts). The SV of spot intensities offers a better dynamic response to protein amount for the model proteins studied here. Depending on the quantity of protein loaded onto gels, we have observed a coefficient of variation range of 0.2 to 1.3. We also observe that the SV silver stain response follows a characteristic exponential profile for different proteins.     

Inhibition of extracellular signal-regulated kinase 1/2 activity of the breast cancer cell line MDA-MB-231 leads to major alterations in the pattern of protein expression

Biochemical and genetic strategies have implied that aberrant signaling in the extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase pathway contributes significantly to transformed phenotypes. Using PD98059, an inhibitor of the ERK-kinase MEK1, we have here assessed the effects of ERK inhibition on the pattern of protein expression in the metastatic human breast cancer cell line MDA-MB-231. At a concentration of inhibitor which did not significantly affect cell growth, PD98059 induced large changes in the expression of MDA-MB-231 polypeptides. The majority of these changes were due to decreased expression of low-abundance proteins. Decreases of more abundant proteins such as glutathione-S-transferase pi, hsp80 and hsp100 were also recorded. The levels of a few proteins increased, among them cytokeratin 8. We conclude that PD98059 treatment of MDA-MB-231 cells induces large changes in protein expression.     

Assessing protease activity pattern by means of multiple substrate ESI-MS assays

The development of a simultaneous multiple substrate enzymatic assay based on electrospray ionization mass spectrometry (ESI-MS) detection is described. This multiplexing assay scheme was employed in a parallel proteolytic enzyme activity screening. As model systems, the respective activities of trypsin, thrombin, chymotrypsin, bromelain, ficin and elastase towards seven different substrates were assessed. The resulting activity patterns were evaluated semi-quantitatively ranking the enzymatic activities in five classes of activity (very high, high, medium, low and no activity) with respect to the individual substrates. The validity of the MS-based multiplexing assay scheme was proved by comparison with the results obtained from single substrate assays detected by means of UV/vis absorption at 405 nm, showing good agreement of the resulting activity patterns and classifications.     

Investigation of the origin of archaeological glass artefacts by means of pattern recognition

The origin of medieval glass artefacts is studied by using a supervised learning technique, which is shown to be helpful when samples cannot be identified by typical design and appearance. A set of seventy pieces of glass was analyzed for ten trace elements by optical emission spectrography. The data matrix of 33 known objects from five origins was evaluated by multivariate variance and discriminant analysis in a training step. The extracted non-elementary discriminant functions were used to classify the 37 unidentified samples. The classification result is discussed in terms of its cultural/historical information content.     

Investigation of plant hormone level changes in shoot tips of longan (Dimocarpus longan Lour.) treated with potassium chlorate by liquid chromatography-electrospray ionization mass spectrometry

The endogenous levels of indole-3-acetic acid (IAA), gibberellins (GAs), abscisic acid (ABA) and cytokinins (CKs) and their changes were investigated in shoot tips of ten longan (Dimocarpus longan Lour.) trees for off-season flowering until 60 days after potassium chlorate treatment in comparison with those of ten control (untreated) longan trees. These analytes were extracted and interfering matrices removed with a single mixed-mode solid phase extraction under optimum conditions. The recoveries at three levels of concentration were in the range of 72-112%. The endogenous plant hormones were separated and quantified by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). Detection limits based on the signal-to-noise ratio ranged from 10 ng mL⁻¹ for gibberellin A4 (GA4) to 200 ng mL⁻¹ for IAA. Within the first week after potassium chlorate treatment, dry weight (DW) amounts in the treated longan shoot tips of four gibberellins, namely: gibberellin A1(GA1), gibberellic acid (GA3), gibberellin A19 (GA19) and gibberellin A20 (GA20), were found to increase to approximately 25, 50, 20 and 60 ng g⁻¹ respectively, all of which were significantly higher than those of the controls. In contrast, gibberellin A8 (GA8) obtained from the treated longan was found to decrease to approximately 20 ng g⁻¹ DW while that of the control increased to around 80 ng g⁻¹ DW. Certain CKs which play a role in leaf bud induction, particularly isopentenyl adenine (iP), isopentenyl adenosine (iPR) and dihydrozeatin riboside (DHZR), were found to be present in amounts of approximately 20, 50 and 60 ng g⁻¹ DW in the shoot tips of the control longan. The analytical results obtained from the two-month off-season longan flowering period indicate that high GA1, GA3, GA19 and GA20 levels in the longan shoot tips contribute to flower bud induction while high levels of CKs, IAA and ABA in the control longan contribute more to the vegetative development.     

Profiling stage-dependent changes of protein expression in Caenorhabditis elegans by mass spectrometric proteome analysis leads to the identification of stage-specific marker proteins

Proteome maps obtained by synchronization of the wild-type Caenorhabditis elegans development reflected stage-dependent molecular differences and revealed dynamic cytoskeletal processes during ontogenesis. Distinct protein spots that may function as molecular markers for the corresponding developmental stages were mass spectrometrically identified. The amount of the Cu(2+)- Zn(2+) superoxide dismutase (CE23550) and an aspartyl proteinase (CE21681) was highest in the first larval stage (L1) and decreased during the ontogenesis from the first larval stage to the adult. Tropomyosin III (CE29059) was prominently present in the first and second larval stage (L1/L2). Abundances of actin 1 or 4 (CE12358 or CE13148) and tropomyosin I (CE28782) were particularly high in multiple spots in the third larval stage (L3). Interestingly, the amount of DIM-1 protein (CE27706), reflected by two spots, was the lowest in this stage. A particular splicing factor (CE31089) was detected only in the fourth larval stage (L4), whereas a spot with high abundance representing the cuticle collagen (CE02272) was only found highly expressed in adult animals (A). In addition, a Ca(2+)-binding protein (CE12368) and one protein spot which has not yet been identified, both reached their maximal spot intensities in the adult stage (A). Moreover, the ASP-1, CCT-5, GPD-1, GPD-2, HSP-6, HSP-16.2, IFB-2, LEC-2, LIN-53, LMN-1, MDH-1, NUD-1, RPA-0, RSP-12, SOD-1, TBB-1, TBB-2, TMY-1, UNC-60, and VIT-2 proteins for which mutants are available and two still unidentified protein spots which were present in all developmental stages, have been reproducibly localized in proteome maps of distinct ontogenesis states.     

Investigation of structural changes in semi-crystalline polymers during deformation by synchrotron X-ray scattering

The mechanical behavior of polymer materials is strongly dependent on polymer structure and morphology of the material. The latter is determined mainly by processing and thermal history. Temperature-dependent on-line X-ray scattering during deformation enables the investigation of deformation processes, fatigue and failure of polymers. As an example, investigations on polypropylene are presented. By on-line X-ray scattering with synchrotron radiation, a time resolution in the order of seconds and a spatial resolution in the order of microns can be achieved. The characterization of the crystalline and amorphous phases as well as the study of cavitation processes were performed by simultaneous SAXS and WAXS. The results of scattering experiments are complemented by DSC measurements and SEM investigations. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 1574–1586, 2010     

Detection of irradiated food by the changes in protein molecular mass distribution

Protein constituents were extracted from chicken drumstick and chicken white meat, separated according to the molecular mass (Mm), using discontinuous SDS–polyacrylamide electrophoresis (SDS–PAGE) and quantified by scanning densitometry. The obtained profiles were compared with the profiles corresponding to the meat samples which were irradiated at −20, +4, and +20°c. It was observed that the irradiation of chicken white meat led to protein scission which was partially unselective (e.g. the amount of proteins whose Mm>20 kDa decreased, while the amount of those whose Mm<20 kDa increased), and partially selective (e.g. the appearance of Mm16 kDa fragment). In the case of chicken drumstick meat the irradiation caused both protein scission and protein cross-linking (unselective and selective, and appearance of 16 kDa fragment). However, in the case of aerated dehydrated egg white proteins, irradiation led only to unselective protein scission. The obtained results are discussed in view of possible application of discontinuous analytical SDS–PAGE combined with laser scanning densitometry for detection of previously irradiated foodstuff.     

Conformational changes of protein adsorbed on polyurethane studied by ftir-atr spectroscopy

We have studied the adsorption of human serum albumin and human gamma globulins onto a polyurethane (Pellethane 2363-80A) using an attenuated total reflectance (ATR) flow cell. Spectra of the proteins adsorbed onto the polyurethane surface, and of the same proteins in solution were collected for comparison. Significant spectral differences between the solution and surface adsorbed spectra of both proteins were observed.     

Purification of canine prolactin and growth hormone by fast protein liquid chromatography.

Prolactin and growth hormone are two peptide hormone with a very similar structure. A simple method is described for the simultaneous purification of these two peptides from canine pituitary extract by fast protein liquid chromatography. After extraction at pH 5.0 and 9.6 and anion-exchange chromatography on a MonoQ column, prolactin and growth hormone are then separated by gel filtration chromatography on two Superose columns coupled in series. The different fractions of the purification scheme are checked for the presence of the peptide hormones by sodium dodecyl sulphate gel electrophoresis in the Pharmacia PhastSystem. Each hormone is also characterized by its behaviour in a Western Blotting Detection System.     

The influence of lysis buffer composition on the expression and activity of protein tyrosine phosphatase

Lysis conditions are crucial in the extraction and solubilization of phosphotyrosine-containing proteins in a form that is immunoreactive, undegraded and enzymatically active. To establish optimal experimental conditions, we evaluated protein tyrosine phosphatase enzyme activity and the detection of PTP-1 B protein in human acute promyelocytic leukaemic cells, both before and after retinoic acid treatment. We found that the composition of the lysing buffer greatly influenced the efficiency of solubilization, resulting in major alterations in the activity of protein tyrosine phosphatases and on the mobility of PTP-1 B protein.     

Investigation of changes in crystal and electronic structures by hydrogen within LaNi5 from first-principles

Hydrogenation of LaNi₅ P6/mmm, up to saturation brings modifications of the crystal structure with two possible space groups for LaNi₅H₇, i.e. P6₃mc and P31c, determined experimentally. The energetics from computations within DFT allows pointing to the former as the most favorable one within a doubled cell along the c-axis with respect to LaNi₅. Results also show a compensation of the volume expansion effects favorable to the onset of magnetization by the chemical bonding involving Ni–H bond leading to vanishing moments.