Characterization of the Prion Protein in Human Urine

The presence of the prion protein (PrP) in normal human urine is controversial and currently inconclusive. This issue has taken a special relevance because prion infectivity has been demonstrated in urine of animals carrying experimental or naturally occurring prion diseases, but the actual presence and tissue origin of the infectious prion have not been determined. We used immunoprecipitation, one- and two-dimensional electrophoresis, and mass spectrometry to prove definitely the presence of PrP in human urine and its post-translational modifications. We show that urinary PrP (uPrP) is truncated mainly at residue 112 but also at other residues up to 122. This truncation makes uPrP undetectable with some commonly used antibodies to PrP. uPrP is glycosylated and carries an anchor which, at variance with that of cellular PrP, lacks the inositol-associated phospholipid moiety, indicating that uPrP is probably shed from the cell surface. The detailed characterization of uPrP reported here definitely proves the presence of PrP in human urine and will help determine the origin of prion infectivity in urine.     

A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: catalysis of hydrolytic reaction by bacterial-type prolyl-tRNA synthetase

Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNA^Pro. The most important functional element of this catalytic mechanism is the 2′-OH group of the terminal adenosine 76 of Ala-tRNA^Pro, which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3′-O atom of A76.     

Urinary Protein Profiles in a Rat Model for Diabetic Complications

Diabetes mellitus is estimated to affect ∼24 million people in the United States and more than 150 million people worldwide. There are numerous end organ complications of diabetes, the onset of which can be delayed by early diagnosis and treatment. Although assays for diabetes are well founded, tests for its complications lack sufficient specificity and sensitivity to adequately guide these treatment options. In our study, we employed a streptozotocin-induced rat model of diabetes to determine changes in urinary protein profiles that occur during the initial response to the attendant hyperglycemia (e.g. the first two months) with the goal of developing a reliable and reproducible method of analyzing multiple urine samples as well as providing clues to early markers of disease progression. After filtration and buffer exchange, urinary proteins were digested with a specific protease, and the relative amounts of several thousand peptides were compared across rat urine samples representing various times after administration of drug or sham control. Extensive data analysis, including imputation of missing values and normalization of all data was followed by ANOVA analysis to discover peptides that were significantly changing as a function of time, treatment and interaction of the two variables. The data demonstrated significant differences in protein abundance in urine before observable pathophysiological changes occur in this animal model and as function of the measured variables. These included decreases in relative abundance of major urinary protein precursor and increases in pro-alpha collagen, the expression of which is known to be regulated by circulating levels of insulin and/or glucose. Peptides from these proteins represent potential biomarkers, which can be used to stage urogenital complications from diabetes. The expression changes of a pro-alpha 1 collagen peptide was also confirmed via selected reaction monitoring.Diabetic nephropathy (DNP)¹ accounts for ∼44% of new cases of end stage renal disease (ESRD) (1). This high morbidity is the result of the impact of a growing population and longer life expectancy. With an increase in the prevalence of DM and a corresponding reduction in the mortality associated with both type 1 and type 2 DM, patients are living longer and are therefore at higher risk to develop complications such as nephropathy (2). Moreover, Type 1 DM patients who progress to ESRD have a substantial risk of mortality with estimated annual health care costs in the United States to be approximately $1.9 billion (3, 4). Two key therapies for the prevention and management of ESRD are aggressive glycemic control and blood pressure regulation (5, 6). Early intervention is essential in reducing the severity and course of this complication (6), and changes in urine biomarkers have historically been used to diagnose and monitor disease progression. In addition, urine represents a desirable matrix in which to detect biomarkers of nephropathy as urinary protein excretion profiles are reflective of functional changes within the kidney, such as glomerular filtration rate. Clinical determinations of urinary total protein and urinary albumin excretion are commonly used measurements to monitor and/or determine the onset of diabetic nephropathy. Unfortunately, these measurements often lead to improper diagnoses for at risk DM patients (7, 8). Therefore, new prognostic indicators are required to accurately target these patients for therapeutic intervention earlier in the course of the disease as well as identify patients who are unlikely to progress, as therapy may be of little or no benefit to them.Utilizing experimental models to study the pathophysiological changes that occur as function of disease progression has provided an approach for biomarker discovery. In diabetes, animal models have been widely used in the investigation of the progression of diabetes complications such as nephropathy. Research conducted on the association between hyperglycemia and microvascular disease in diabetes as well as the study of the effect of extracellular matrix protein expression on changes in morphology in the diabetic kidney are two such examples (9). In addition, these models have assisted in developing appropriate clinical trials for the prevention and treatment of these complications. One such example is the use of anti-hypertensive treatment regimes in genetically hypertensive rats; these have examined whether early intervention may be renoprotective and therefore delay or prevent the onset of diabetic nephropathy (10–12).STZ-induced hyperglycemia in rodents is the most extensively studied model of diabetic nephropathy and associated complications (9, 13). Hyperglycemia occurs in this model because of the toxin''s destruction of pancreatic Beta-islet cells, which are essential to the production of insulin. STZ-induced hyperglycemia is associated with reliable and consistent structural and functional deficits in specific urogenital organ function (i.e. kidney and bladder). Increased glomerular filtration and hypertrophy, as well as increased urgency and morphology changes, are structural and functional abnormalities that have been observed in the kidney and bladder, respectively, in both in humans and the STZ rat model (14–19).Currently, there are two primary methods used to monitor disease progression in diabetes. The measurement of urinary albumin excretion rates and total protein concentration are routinely used to monitor disease progression as they reflect structural and functional changes in the kidney. Measurements of albumin by immunochemical assays and size exclusion high performance liquid chromatography are routinely employed (7). However, urine consists of a multitude of proteins, many of which are also reflective of pathophysiological changes because of DM urogenital complications (20–23). Proteomics provides a powerful approach for the detection of urinary protein changes as a result of disease, and multiple proteomic techniques are available in large scale protein profiling to discover new biomarkers (24–26). To date, proteomic strategies for biomarker discovery in urine have primarily included top-down approaches, for example two-dimensional gel electrophoresis coupled with mass spectrometry and/or surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) analyses (27–30). In addition a number of studies using capillary electrophoresis mass spectrometry, which have a number of advantages for analysis of urine, have been successfully carried out (31–33). While these approaches can easily detect and quantify a variety of proteinaceous species including isoforms, posttranslational modifications, or degradation products, other methods could be used to expand the number of proteins that are both quantified and identified providing an expanded set of biological targets to understand the complications of disease and its progression. Recent advances in both chromatography and mass spectrometry have enabled bottom-up approaches that identify and quantify at the peptide level (34–36). One advantage of bottom-up proteomics is increased overall proteome coverage. Moreover, most bottom-up methods provide both qualitative and quantitative data in a single run, and quantifying at the peptide level leads directly into a bottom-up confirmation/validation analysis thereby avoiding the peptide selection step in this procedure. Approaches to bottom-up proteomics include specific peptide labeling or label-free analysis. Specific labeling approaches such as isobaric tag for relative and absolute quantification (iTRAQ) and ¹⁸O employ differential stable isotope labeling strategies that create specific mass tags for different samples, which are mixed and then identified and quantified using mass spectrometry (37). The utility of these techniques is that they accommodate a wide range of pre-fractionation strategies thereby improving proteome coverage.The label-free approach capitalizes on the highly reproducible chromatography and high mass accuracy available in current LC/MS systems. This method observes all detectable peptides and if interrogated by MS/MS their corresponding fragment ions. This approach quantifies a peptide by its intensity and groups each peptide across individual samples based on its accurate mass and retention time (38, 39). These intensities associated with specific mass and retention time values are organized into peptide array tables that may be further processed using statistical techniques that accommodate high-dimensional data. As with other bottom-up approaches, this method is also amenable to pre-fractionation strategies, but unlike labeled approaches, the removal of chemical or metabolic labeling steps simplifies the overall approach.Here we use a comparative label-free LC/MS/MS approach to identify and rank candidate biomarkers of urogenital complications from an STZ rat model of diabetes. We describe further technical validation of our approach by confirming the changes observed with the putative biomarker, pro-alpha (2) with an alternative method: selected reaction monitoring (SRM).     

Biomass,production and activity of bacteria in the Black Sea,with special reference to chemosynthesis and the sulfur cycle

Abundance, morphological composition, vertical distribution, production and activity of total bacterioplankton and its specific groups in the Black Sea were investigated in August–September 1989. The total bacterioplankton was highest in the upper mixed layer (0.7–1 × 10⁶ cells ml^–1), corresponding to that in mesotrophic basins. In the N-E shallow part of the sea it attained 3 × 10⁶. Below the thermocline (50–100 m) the total number of bacteria decreased to 0.2–0.4 × 10⁶ ml^–1. In the redox gradient zone (zone of O₂-H₂S interface), it increased again. In deep anoxic waters the bacterioplankton, numbering 0.15–0.2 × 10⁶ ml^–1, was functionally inactive. Its biomass was 12–40 mg C m^–3 in the upper mixed layer, 5–10 mg C m^–3 in the intermediate cold layer (40–100 m depth), and 10–20 mg C^–3 in the redox zone. Maximum production rates occurred in the upper mixed layer (8–20 mg C^–3 d^–1) and in the redox-zone, 80–90% of it was due to chemosynthesis of thiobacilli. Below 200 m, microbial production decreased to about zero in the anoxic zone. Maximum activity of heterotrophic bacteria was recorded in the upper mixed layer, while thiobacilli and methaneoxidezing bacteria were most active in the redox-zone. Here, the maximum rates of H₂S and of thiosulfate oxidation, as well as maximum sulfate reduction were recorded. Chemical oxidation of H₂S was dominant. These results are discussed with respect to the present ecological situation of the Black Sea.     

The Effect of an Alternating Magnetic Field on the Chemiluminescence of Human Peripheral Blood Mononuclear Cells and the Production of Pro-Inflammatory Cytokines

Biophysics - Abstract—It has been shown that in vitro exposure to an alternating magnetic field with a strength of 550 ± 30 A/m at a frequency of 8.6 and 9.0 Hz leads to a...     

The Effect of an Alternating Magnetic Field and Silver Nanoparticles on the Spectral Characteristics of an Aqueous Solution of Human Serum Albumin

Biophysics - In this paper, we discuss experimental data obtained during study of the effect of a low-frequency alternating magnetic field on the conformational transitions of human serum albumin...     

Discomfort-related changes in pup ultrasonic calls of fat-tailed gerbils Pachyuromys duprasi

Rodent pups vocalize when placed in social isolation. We apply a method of “joint calls” for examining discomfort in rodent pup ultrasonic (>20 kHz) calls. Previously, this method has been developed for audible calls of fur farm mammals. Using a repeated measures design to exclude effects of individual identity and age on the analysed variables, we compared the ultrasonic call variables produced by 8–40-day pups of fat-tailed gerbils Pachyuromys duprasi during two subsequent experimental stages, the Isolation Stage and the Handling Stage. We considered that discomfort-related negative emotional arousal increased towards the Handling Stage compared to the Isolation Stage because of cumulative effects of handling and time of pup isolation from the nest. At the Isolation Stage, the call rate (calls/s) was higher from 10 to 18 days of age, whereas both the maximum amplitude frequency and power quartiles of joint calls were lower than at the Handling Stage from 20 to 32 days of age. At the same time, in audible (<20 kHz) vocalizations of a wide range of mammalian species, both the higher call rate and the upward shift of the maximum amplitude frequency and power quartiles indicate the discomfort-related increase of negative emotional arousal. We discuss the advantages of the method of joint calls for express-analyses of power variables for large sequences of ultrasonic vocalizations of complex acoustic structure during experimental trials.     

Expression of a Functional Anaphylatoxin C3a Receptor by Astrocytes

Abstract: Human astrocyte cell lines reportedly contain a specific receptor for the complement anaphylatoxin C3a based on ligand-binding studies, functional responses, and RNA analysis by RT-PCR. Uptake of ¹²⁵I-C3a by astrocytes was specific and reversible. Scatchard analysis indicated the presence of two classes of binding sites. High-affinity binding sites were abundantly expressed (20,000–80,000 sites per cell) with an estimated K _D of 1–2 n M . Low-affinity binding sites with a K _D of 209 n M were largely expressed ( n ≥ 4 × 10⁶ sites per cell) and probably did not reflect a receptor-mediated binding, but rather an ionic interaction between C3a and the membrane. Analysis of astrocyte mRNA by RT-PCR with three different sets of primers covering 60% of the C3a receptor (C3aR) mRNA sequence indicated that glial C3aR was identical to the leukocytic one. Western blot analysis using a specific anti-C3aR evidenced a C3aR with a molecular mass of 60,000 Da. C3a and a superagonist peptide, E7, induced a transient increase of intracellular [Ca²⁺] in primary culture of astrocytes. Treatment of the ligands by carboxypeptidase B to eliminate the C-terminus Arg considerably decreased the [Ca²⁺] response. Moreover, flow cytometry experiments demonstrated the expression of C3aR on normal rat astrocyte membrane. This report brings new insight for the role of the complement system in the brain inflammation response.     

Functional distinctions between the mitochondrial ATP-dependent K+ channel (mitoKATP) and its inward rectifier subunit (mitoKIR)

The ATP-sensitive potassium channel from the inner mitochondrial membrane (mitoK(ATP)) is a highly selective conductor of K(+) ions. When isolated in the presence of nonionic detergent and reconstituted in liposomes, mitoK(ATP) is inhibited with high affinity by ATP (K((1/2)) = 20-30 microM). We have suggested that holo-mitoK(ATP) is a heteromultimer consisting of an inwardly rectifying K(+) channel (mitoKIR) and a sulfonylurea receptor (Grover, G. J., and Garlid, K. D. (2000) J. Mol. Cell. Cardiol. 32, 677-695). Here, we show that a 55-kDa protein isolated by ethanol extraction and reconstituted in bilayer lipid membranes and liposomes is the mitoKIR. This protein, which lacks the sulfonylurea receptor subunit, is inhibited with low affinity by ATP, with K(1/2) approximately 550 microM. ATP inhibition of both mitoKIR and holo-mitoK(ATP) is reversed by UDP (K((1/2))1/2 = 10-15 microM). Holo-mitoK(ATP) is and diazoxide, and the opened by cromakalim flux through the open channel is inhibited by glibenclamide and 5-hydroxydecanoate. None of these agents has any effect upon mitoKIR. We have identified two compounds that act specifically on mitoKIR. p-diethylaminoethylbenzoate reverses inhibition of mitoKIR by ATP and ADP at micromolar concentrations and also opens mitoK(ATP) in isolated mitochondria. Tetraphenylphosphonium inhibits K(+) flux through both mitoKIR and mitoK(ATP) with the same apparent affinity. These findings support the hypothesis that the 55-kDa mitoKIR is the channel component of mitoK(ATP).     

Presence of Hydrogenophilus thermoluteolus DNA in accretion ice in the subglacial Lake Vostok, Antarctica, assessed using rrs, cbb and hox

The 3561 m Vostok ice core sample originating from the subglacial Lake Vostok accretion (frozen lake water) ice with sediment inclusions was thoroughly studied by various means to confirm the presence of the thermophile bacterium Hydrogenophilus thermoluteolus reported earlier in the 3607 m accretion ice sample. PCR and molecular-phylogenetic analyses performed in two independent laboratories were made using different 16S rRNA gene (rrs) targeted primers. As a result, rrs-targeted PCR permitted to recover several very closely related clones with a small genetic distance to Hydrogenophilus thermoluteolus (< 1%). In addition, RubisCO (cbbL or rbcL) and NiFe-Hydrogenase (hoxV or hupL) targeted PCR have also allowed to recover sequences highly related to Hydrogenophilus thermoluteolus. All these results point to the presence of thermophilic chemoautotrophic microorganisms in Lake Vostok accretion ice. They presumably originate from deep faults in the bedrock cavity containing the lake in which episodes of seismotectonic activity would release debris along with microbial cells.