The Hsp40 J‐domain modulates Hsp70 conformation and ATPase activity with a semi‐elliptical spring

Regulatory protein interactions are commonly attributed to lock‐and‐key associations that bring interacting domains together. However, studies in some systems suggest that regulation is not achieved by binding interactions alone. We report our investigations on specific physical characteristics required of the Hsp40 J‐domain to stimulate ATP hydrolysis in the Hsp40‐Hsp70 molecular chaperone machine. Biophysical analysis using isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy reveals the importance of helix rigidity for the maintenance of Hsp40 function. Our results suggest that the functional J‐domain acts like a semi‐elliptical spring, wherein the resistance to bending upon binding to the Hsp70 ATPase modulates the ATPase domain conformational change and promotes ATP hydrolysis.     

S100A1 enhances the L-type Ca2+ current in embryonic mouse and neonatal rat ventricular cardiomyocytes

S100A1 is an EF-hand type Ca2+-binding protein with a muscle-specific expression pattern. The highest S100A1 protein levels are found in cardiomyocytes, and it is expressed already at day 8 in the heart during embryonic development. Since S100A1 is known to be involved in the regulation of Ca2+ homeostasis, we tested whether extracellular S100A1 plays a role in regulating the L-type Ca2+ current (I(Ca)) in ventricular cardiomyocytes. Murine embryonic (day 16.5 postcoitum) ventricular cardiomyocytes were incubated with S100A1 (0.001-10 microM) for different time periods (20 min to 48 h). I(Ca) density was found to be significantly increased as early as 20 min (from -10.8 +/- 1 pA/pF, n = 18, to -22.9 +/- 1.4 pA/pF; +112.5 +/- 13%, n = 9, p < 0.001) after the addition of S100A1 (1 microM). S100A1 also enhanced I(Ca) current density in neonatal rat cardiomyocytes. Fluorescence and capacitance measurements evidenced a fast translocation of rhodamine-coupled S100A1 from the extracellular space into cardiomyocytes. S100A1 treatment did not affect cAMP levels. However, protein kinase inhibitor, a blocker of cAMP-dependent protein kinase A (PKA), abolished the S100A1-induced enhancement of I(Ca). Accordingly, measurements of PKA activity yielded a significant increase in S100A1-treated cardiomyocytes. In vitro reconstitution assays further demonstrated that S100A1 enhanced PKA activity. We conclude that the Ca2+-binding protein S100A1 augments transsarcolemmal Ca2+ influx via an increase of PKA activity in ventricular cardiomyocytes and hence represents an important regulator of cardiac function.     

Decimal Place Slope,A Fast and Precise Method for Quantifying 13C Incorporation Levels for Detecting the Metabolic Activity of Microbial Species

The metabolic incorporation of stable isotopes such as ¹³C or ¹⁵N into proteins has become a powerful tool for qualitative and quantitative proteome studies. We recently introduced a method that monitors heavy isotope incorporation into proteins and presented data revealing the metabolic activity of various species in a microbial consortium using this technique. To further develop our method using an liquid chromatography (LC)-mass spectrometry (MS)-based approach, we present here a novel approach for calculating the incorporation level of ¹³C into peptides by using the information given in the decimal places of peptide masses obtained by modern high-resolution MS. In the present study, the applicability of this approach is demonstrated using Pseudomonas putida ML2 proteins uniformly labeled via the consumption of [¹³C₆]benzene present in the medium at concentrations of 0, 10, 25, 50, and 100 atom %. The incorporation of ¹³C was calculated on the basis of several labeled peptides derived from one band on an SDS-PAGE gel. The accuracy of the calculated incorporation level depended upon the number of peptide masses included in the analysis, and it was observed that at least 100 peptide masses were required to reduce the deviation below 4 atom %. This accuracy was comparable with calculations of incorporation based on the isotope envelope. Furthermore, this method can be extended to the calculation of the labeling efficiency for a wide range of biomolecules, including RNA and DNA. The technique will therefore allow a highly accurate determination of the carbon flux in microbial consortia with a direct approach based solely on LC-MS.The metabolic incorporation of stable isotopes such as ¹³C or ¹⁵N into proteins has become a powerful component of qualitative and quantitative proteome studies (1). Incorporation of heavy isotopes can be used to analyze microbial processes such as turnover rates and also to help to establish structure-function relationships within microbial communities. Stable isotope probing (SIP¹) techniques based on DNA-SIP (2) and RNA-SIP (3) have been used for this purpose previously. With the introduction of protein-SIP (4), the need for an accurate alternative method for calculating label incorporation into biomolecules arose. Protein-SIP has several advantages compared with DNA/RNA-SIP, the most important being its capacity to detect dynamic levels of incorporation, whereas only labeled or unlabeled states can be categorized by means of DNA/RNA-SIP because of the need to separate ¹³C-DNA/RNA by density gradient centrifugation. Quantitative analysis of ¹³C incorporation levels is of the utmost importance, especially when unraveling carbon fluxes through either microbial communities or food webs with different trophic levels.In contrast to the incorporation of isotopically labeled amino acids, which is often used in quantitative proteomics (5), metabolic labeling by growth substrates and nutrients (e.g. salts) is often imperfect and makes the processing of mass spectrometry (MS) data difficult. For example, when the incorporation of ¹³C exceeds ∼2 atom %, common database search algorithms fail to identify peptides and proteins. The problem can only be managed successfully if a stable, known degree of ¹³C incorporation can be achieved during the experiment (6). Using a low labeling efficiency of roughly 5 atom %, Huttlin et al. (6) chose the altered envelope chain for calculating the incorporation and simultaneously used the signal intensity for a quantitative comparison with the sample that had a natural abundance of ¹³C. Database approaches for peptide identification can cope only with the natural abundance of carbon isotopes; they fail if the incorporation of ¹³C significantly exceeds the natural isotope abundance or if incorporation patterns occur in unpredictable ways (7).The simplest method for determining the incorporation level is to compare the unlabeled average mass of the monoisotopic peptide with the mass of the labeled protein, as estimated by matrix-assisted laser desorption/ionization or electrospray ionization MS (8, 9). A more advanced approach for determining the isotopic mass distribution of peptides is based on the isotopic distribution of the peaks of a peptide envelope (10, 11). Here, for a given isotopomer, the incorporation efficiency is defined as the percentage of incorporated ¹³C atoms with relation to the total number of carbon atoms with the natural isotope abundance (approximately 1.01 atom % ¹³C). As a reference, the theoretical isotopic distribution of a peptide is calculated based upon an algorithm described elsewhere (12). The isotope distribution of both unlabeled and labeled peptides can subsequently be used to calculate the incorporation level. For this method, an Excel spreadsheet (ProSIPQuant.xls) was developed (4). A similar approach, also based on the calculation of isotopic distributions, has been used in other studies (7). In these studies, however, the identification of the peptides is limited to those that have unlabeled counterparts; in addition, an exact calculation can be hampered by overlapping signals coming from additional peaks with similar masses.In the present study, we describe a new way of determining the isotope incorporation level. Our method makes use of characteristic patterns in the digits after the decimal point of the peptide masses generated by high-accuracy instruments such as the linear ion trap LTQ-Orbitrap (Thermo Fisher Scientific, Bremen, Germany). For tryptic peptides, typical regularities in the decimal places of the monoisotopic masses have been observed (13, 14). These observations have been explored in detail for theoretical and experimental data of proteins originating from Helicobacter pylori (15). As a result, a rule called the “half decimal place rule” (HDPR) was defined; it states that the decimal place is nearly half of the first digit for tryptic peptides with masses in the range of 500–1,000 Da. In other words, the exact mass of a peptide is equal to its nominal mass times ∼1.005. Because the difference between ¹²C and ¹³C is slightly greater than 1 Da, exactly 1.0033548378, the decimal places of a tryptic peptide''s mass are shifted in a regular manner by the incorporation level and lead to a significantly increased slope for the digits in the third and fourth place after the decimal point. This shift can be used to estimate the incorporation level of heavy isotopes into the protein. Detecting such shifts requires the highly accurate measurement possible with modern mass spectrometers such as the LTQ-Orbitrap, the Fourier transform ion cyclotron resonance, or the quadrupole time of flight. In this communication, we demonstrate the applicability of this approach using Pseudomonas putida ML2 proteins labeled uniformly via the consumption of [¹³C₆]benzene with five different substrate concentrations (0, 10, 25, 50, and 100 atom % of ¹³C). The ¹³C incorporation was calculated based on several labeled peptides derived from different proteins in one SDS-PAGE band. By these means, we have established a method that allows the determination of ¹³C incorporation into proteins and can be used to assess the metabolic activity of a given species within a mixed community.     

Modified micro suction cup/rhizobox approach for the in-situ detection of organic acids in rhizosphere soil solution

Root–soil interactions can strongly influence the soil solution chemistry in the rhizosphere. In the present study we propose a modification of the classical rhizobox/micro suction cup system to make it suitable for the collection and analysis of organic acids in the rhizosphere. In order to show the potential of the method, we tested the modified system with Lupinus albus L. as a model plant known to exude large amounts of citrate. The suction cups were installed through the transparent front plate of the rhizoboxes just after the emergence of cluster roots in order to allow optimal localized collection of soil solution. A small dead-volume allowed almost immediate stabilisation with formaldehyde of the sampled soil solutions in the collection container to prevent microbial degradation. The concentrations of organic acids were significantly larger in the rhizosphere soil solution of active cluster roots of Lupinus albus L. than in the bulk soil solution (about 400 μM of citrate versus <0.05 μM). We were able to follow the exudation process in-situ, which occurred during 2–3 days. Also the concentrations of other organic acids and inorganic anions differed between the bulk soil and the rhizosphere of cluster roots, normal roots, and nodules.     

Proteome analysis of rat liver mitochondria reveals a possible compensatory response to endotoxic shock

Organ failure induced by endotoxic shock has recently been associated with affected mitochondrial function. In this study, effects of in vivo lipopolysaccharide-challenge on protein patterns of rat liver mitochondria in treated animals versus controls were studied by two-dimensional electrophoresis (differential image gel electrophoresis). Significant upregulation was found for ATP-synthase alpha chain and superoxide dismutase [Mn]. Our data suggest that endotoxic shock mediated changes in the mitochondrial proteome contribute to a compensatory reaction (adaptation to endotoxic shock) rather than to a mechanism of cell damage.     

The extent and significance of petroleum hydrocarbon contamination in Crater Lake,Oregon

In order to evaluate hydrocarbon inputs to Crater Lake from anthropogenic and natural sources, samples of water, aerosol, surface slick and sediment were collected and analyzed by gas chromatography-mass spectrometry (GC-MS) for determination of their aliphatic and aromatic hydrocarbon concentrations and compositions. Results show that hydrocarbons originate from both natural (terrestrial plant waxes and algae) and anthropogenic (petroleum use) sources and are entering the lake through direct input and atmospheric transport. The concentrations of petroleum hydrocarbons range from low to undetectable. The distributions and abundances of n-alkanes, polycyclic aromatic hydrocarbons (PAH) and unresolved complex mixture (UCM) from petroleum are similar for all surface slick sampling sites. The estimated levels of PAH in surface slicks range from 7–9 ng/m² which are low. Transport of petroleum-derived hydrocarbons from the lake surface has resulted in their presence in some sediments, particularly near the boat operations mooring (total petroleum HC = 1440 μg/kg, dry wt. compared to naturally derived n-alkanes, 240 μg/kg, dry wt.). The presence of biomarkers such as the tricyclic terpanes, hopanes and steranes in shallow sediments further confirms petroleum input from boat traffic. In the deep lake sediments, petroleum hydrocarbon concentrations were very low (16 μg/kg, dry wt.). Very low concentrations of PAH were detected in shallow sediments (17–40 μg/kg at 5 m depth near the boat operations) and deep sediments (3–15 μg/kg at 580 m depth). The individual PAH concentrations in sediments (μg/kg or ppb range) are at least three orders of magnitude less than reported threshold effects levels (mg/kg or ppm range, test amphipod Hyalella azteca). Therefore, no adverse effects are expected to occur in benthic biota exposed to these sediments. Boating activities are leaving a detectable level of petroleum in surface waters and lake sediments but these concentrations are very low.     

A long-term porcine model for measurement of gastrointestinal motility

Animal models have become an essential tool in the investigations of gut motility under experimental conditions. To determine the influence of various anaesthetic drugs on the motility pattern of the gastroduodenal tract, a new long-term model has had to be developed for allowing measurements in conscious and unrestrained as well as in sedated and analgosedated pigs. Since mechanical ventilation influences gut motility, it was necessary that this animal model enabled the investigation of the effect of drugs causing sedation and analgosedation during spontaneous breathing. Seven male, castrated pigs, German landrace, 32-40 kg bodyweight (BW) were investigated in this study. After habituation of the pigs to local housing conditions over 5 days, the animals were trained over 4 days to prepare for experimental situations and investigators. Pigs were inserted with a central venous catheter and with percutaneous enterogastrostomy (PEG) under general anaesthesia. Intestinal motility was measured by intraluminal impedancometry. The catheter was introduced over the PEG into the stomach and positioned into the duodenum by duodenoscopy. Measurements were done in conscious, unrestrained pigs and with sedated, and analgosedated animals on subsequent days. The habituation and training of the pigs to the investigators and for the laboratory conditions took between 7 and 9 days. The initial anaesthesia protocol for the instrumentation using remifentanil/propofol led to pyloric spasm and was thus unsuitable for duodenal intubation with an endoscope. In contrast, a combination of ketamine/propofol enabled this procedure. It was practicable to measure gut motility in conscious, unrestrained pigs. Spontaneous breathing was sufficient under propofol sedation and analgosedation using fentanyl-propofol. Systematically local application of polividon iodine in the area of the subcutaneous catheters avoided the necessity of using systemic prophylactic antibiotics. In conclusion, the habituation and training for 9 days enabled the measurement of gut motility by intraluminal impedancometry in conscious pigs. The insertion of the catheter was done during general anaesthesia using a combination of propofol and ketamine. For the future determination of gut motility performed under general anaesthesia, each sedation and analgosedation concept has to be evaluated to see whether it allows spontaneous breathing or whether mechanical ventilation is necessary.     

Renal microvasculature in the adult pipid frog,Xenopus laevis: A scanning electron microscope study of vascular corrosion casts

We studied the opisthonephric (mesonephric) kidneys of adult male and female Xenopus laevis using scanning electron microscopy (SEM) of vascular corrosion casts and light microscopy of paraplast embedded tissue sections. Both techniques displayed glomeruli from ventral to mid-dorsal regions of the kidneys with single glomeruli located dorsally close beneath the renal capsule. Glomeruli in general were fed by a single afferent arteriole and drained via a single thinner efferent arteriole into peritubular vessels. Light microscopy and SEM of vascular corrosion casts revealed sphincters at the origins of afferent arterioles, which arose closely, spaced from their parent renal arteries. The second source of renal blood supply via renal portal veins varied interindividually in branching patterns with vessels showing up to five branching orders before they became peritubular vessels. Main trunks and their first- and second-order branches revealed clear longish endothelial cell nuclei imprint patterns oriented parallel to the vessels longitudinal axis, a pattern characteristic for arteries. Peritubular vessels had irregular contours and were never seen as clear cylindrical structures. They ran rather parallel, anastomosed with neighbors and changed into renal venules and veins, which finally emptied into the ventrally located posterior caval vein. A third source of blood supply of the peritubular vessels by straight terminal portions of renal arteries (vasa recta) was not found.