Thermal stability and folding of type IV procollagen and effect of peptidyl-prolyl cis-trans-isomerase on the folding of the triple helix

Intact, monomeric type IV procollagen was isolated from the medium of PF-HR9 cells. Its stability was measured by optical rotatory dispersion, differential scanning calorimetry, and trypsin susceptibility of the partially unfolded molecules. At neutral pH, a complex transition between 35 and 42 degrees C and a smaller transition at 48 degrees C are observed by optical rotatory dispersion, using a heating rate of 10 degrees C/h. Reduction of the heating rate to 1.6 degrees C/h resulted in a 1 degree C lowering of the apparent melting temperatures. A similar curve is observed in 10 mM acetic acid, with transitions about 2 degrees C lower. Differential scanning calorimetry revealed transitions at 36.0, 42.1, and 48.0 degrees C at neutral pH, with a total transition enthalpy of 17.1 kJ/mol tripeptide units. In 10 mM acetic acid, transitions at 35.6, 38.9, 41.7, and 50.0 degrees C are observed. The transition enthalpy is 16.4 kJ/mol tripeptide units. The transition enthalpy is similar to values found for interstitial collagens. Results from trypsin digestion experiments are consistent with the stability found by optical methods and calorimetry. The rate and completeness of refolding after melting were measured. In neutral buffer, the initial rate was found to be 0.041 min-1, faster than the refolding rates observed with types pN III and III collagen. Peptidyl prolyl cis-trans-isomerase increased the refolding rate to 0.083 min-1, indicating that cis-trans-isomerization is the rate-limiting step, despite the interruptions in the triple helix. Trypsin digestion experiments indicated that the refolding mechanism is similar in the presence and absence of the enzyme. Refolding was nearly complete in neutral buffer. In 10 mM acetic acid, folding was considerably slower and went to about 74% completion. In both solvents, the refolded material was only slightly less stable than the native material. Electron microscopy of partially refolded samples showed that most refolding started at the COOH terminus, but some was initiated at other sites.     

On the Composition, Deposition and Mobilization of Proteins in the Cotyledons of Castor Bean (Ricinus communis L. cv. Hale) Seeds: Their Role as Storage Proteins

Proteins in the soluble and insoluble fractions, extracted frommature castor bean cv. Hale seed cotyledons, differ quantitativelyand qualitatively from their counterparts extracted from theendosperm. The soluble fraction contains no glycoproteins, andthe lectins RCA₁ and ricin D are absent. While the insolubleproteins are electrophoretically and immunologically similarto those in the endosperm, they do not form the 100 kD subunitdimers which characterize some of the endosperm insoluble crystalloidproteins. Rapid rates of deposition of all of the soluble andinsoluble proteins present in the mature seed cotyledons commences30–35 d after pollination (DAP) and continues until 45DAP. These proteins are mobilized rapidly beginning 1–2d after seed imbibition and this coincides with an increasein specific activity, in the cotyledons, of two aminopeptidasesand a carboxypeptidase. The soluble and insoluble proteins inthe cotyledons of the mature seed probably function as storageproteins and support the growth of the germinated seed priorto the mobilization of the major protein storage reserves ofthe endosperm. Key words: Ricinus communis, Castor bean, Hale cultivar, Cotyledon, Storage protein, Seed development, Seed germination     

Micelle‐enhanced spectrofluorimetric method for determination of lacidipine in tablet form; application to content uniformity testing

A highly sensitive, simple and rapid spectrofluorimetric method was developed for the determination of lacidipine (LCP) in tablets. The proposed method is based on the investigation of the fluorescence spectral behavior of LCP in both sodium dodecyl sulphate (SDS) and the tween‐80 micellar system. In aqueous solutions of acetate buffer (pH 4.5), the fluorescence intensities of LCP were greatly enhanced (ca. 2.4 and 4.3 folds) in the presence of either SDS or tween‐80, respectively. The fluorescence intensity was measured at 444 nm after excitation at 277 nm using either SDS or tween‐80 as a surfactant. The fluorescence–concentration plots were rectilinear over the ranges of 50.0–500.0 ng/ml and 5.0–200.0 ng/ml with lower detection limits of 5.11 and 2.06 ng/ml and lower quantification limits of 17 and 6.87 ng/ml using SDS and tween‐80, respectively. The method was successfully applied to the analysis of LCP in commercial tablets and the results were in good agreement with those obtained with the comparison method. Furthermore, content uniformity testing of pharmaceutical tablets was also conducted. Copyright © 2014 John Wiley & Sons, Ltd.     

Abundance-based Classifier for the Prediction of Mass Spectrometric Peptide Detectability Upon Enrichment (PPA)

The function of a large percentage of proteins is modulated by post-translational modifications (PTMs). Currently, mass spectrometry (MS) is the only proteome-wide technology that can identify PTMs. Unfortunately, the inability to detect a PTM by MS is not proof that the modification is not present. The detectability of peptides varies significantly making MS potentially blind to a large fraction of peptides. Learning from published algorithms that generally focus on predicting the most detectable peptides we developed a tool that incorporates protein abundance into the peptide prediction algorithm with the aim to determine the detectability of every peptide within a protein. We tested our tool, “Peptide Prediction with Abundance” (PPA), on in-house acquired as well as published data sets from other groups acquired on different instrument platforms. Incorporation of protein abundance into the prediction allows us to assess not only the detectability of all peptides but also whether a peptide of interest is likely to become detectable upon enrichment. We validated the ability of our tool to predict changes in protein detectability with a dilution series of 31 purified proteins at several different concentrations. PPA predicted the concentration dependent peptide detectability in 78% of the cases correctly, demonstrating its utility for predicting the protein enrichment needed to observe a peptide of interest in targeted experiments. This is especially important in the analysis of PTMs. PPA is available as a web-based or executable package that can work with generally applicable defaults or retrained from a pilot MS data set.Post-translational modification (PTM)¹ of proteins is a key regulatory mechanism in the vast majority of biological processes. Historically, to follow PTMs, site-specific antibodies had to be generated in a time-consuming and laborious process associated with high failure rates. Mass spectrometry (MS) holds enormous promise in PTM analysis as it is currently the only technique that has the ability to both discover, localize, and quantify proteome-wide modifications (1). Recent advances in instrumentation and method optimization makes it possible to detect the complete yeast proteome within one hour (2), an ever increasing proportion of the human proteome (3–6), and more than 10,000 phosphorylation sites in a single MS experiment (7, 8). As a result one of the major publicly available databases (www.phosphosite.org (9)) has curated >200,000 phosphorylation sites.Although the number of proteins and PTMs that can be identified is impressive, many modifications have still not been identified in any MS-based experiment. The identification and quantification of biologically relevant modifications is challenging for three reasons: (1) many proteins of interest are of very low abundance rendering them difficult to detect and quantify; (2) many modifications sites are present at substoichiometric quantities, further reducing their detectability; and (3) as large scale proteomics is based on the detection of peptides after a proteolytic digest, and the detectability of a peptide is determined by its physiochemical properties (10), many peptides from highly abundant proteins are never detected. This is particularly important, as there is a shift in the use of MS-based proteomics from large scale, unbiased, discovery-focused experiments toward directed experiments for accurate and precise quantification of biologically relevant PTMs. Protein and peptide enrichment strategies and/or targeted MS experiments like single reaction monitoring (SRM) (11) have increased the number of detectable peptides; however, both of these methods are laborious, and often not successful, that is, the peptide carrying the modification of interest is still not observed as it is fundamentally very difficult to detect.Protein enrichment is the method choice for most experimentalists, but there is no current way to determine whether this is likely to succeed prior to engaging in lengthy biochemical and/or analytical experiments. In an effort to gauge the chances of success for detecting a particular peptide we sought to develop an algorithm that can predict both the chances of detecting a particular peptide and, more importantly, what enrichment it would take to detect a particular peptide that is not easily detected. Here we present such a tool that predicts the detectability and estimates an enrichment factor, i.e. an increase in signal over the background that is necessary to actually detect a particular peptide. Our algorithm development was motivated by two premises: (1) In silico methods have been developed that focus on the prediction of easily detectable “proteotypic” peptides (peptides that are likely to provide the best detection sensitivity) with good accuracy (12–15). (2) Comprehensive proteome studies have shown that the number of detected peptides per protein, and thus the sequence coverage, varies with protein abundance (which is the basis for spectral counting-based protein quantification (16, 17)). We find that incorporation of protein abundance in a peptide classification tool improves the accuracy of the prediction of peptide detectability allowing us to predict the detectability of all peptides within a protein as well as the amount of enrichment needed to detect a peptide of interest.We used a set of 120 purified in vitro expressed proteins as a training set to develop a prediction tool. We deliver this in the form of a web-based interface that provides information about: (1) the probability of detecting the different tryptic peptides of a protein, and (2) the fold enrichment that would be required to bring a peptide of interest into the detectable range. This tool will help guide researchers in their efforts to monitor particular peptides and their modified cognates by MS, specifically, in prioritizing their efforts toward enriching proteins where they would be likely to be able to detect a peptide or modification of interest.     

Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.     

The trace fossil Nummipera eocenica from the Tatra Mountains,Poland: morphology and palaeoenvironmental implications

Jach, R., Machaniec, E. & Uchman, A. 2011: The trace fossil Nummipera eocenica from the Tatra Mountains, Poland: morphology and palaeoenvironmental implications. Lethaia, Vol. 45, pp. 342–355. The tubular trace fossil Nummipera eocenica Hölder 1989 occurs in a single stratigraphical horizon in Eocene nummulitic limestones of the Tatra Mountains, Poland. The wall of N. eocenica is built of Discocyclina and Nummulities (larger foraminifera) tests, very rarely of the Ditrupa (Polychaeta) tube fragments, bivalve shell fragments, echinoid spines and coralline algae. Morphotype are distinguished on the basis of wall composition and structure. Morphotype A is dominated by fusiform Discocyclina tests, which were preferentially selected by the trace makers for construction of a well‐constructed and resistant wall. Morphotype B contains more robust tests of Nummulites, while morphotype C is dominated by saddle‐shaped tests of Discocyclina. Nummipera eocenica was produced during a period of seafloor stabilization caused by a deepening. The succession of the morphotypes B, A reflects diminishing energy and increasing water depth. Probably morphotype C represents even lower energy environment than morphotype A. The trace fossil is interpreted as a domichnion, which wall was constructed for protection. The trace maker can be considered between polychaetes and crustaceans; however, comparisons to the closest recent analogues, the polychaete Diopatra cuprea or alpheid shrimps, are not satisfactory. □Bartonian, burrow, Carpathians, large foraminifera, trace fossils.     

A new large philisid (Mammalia,Chiroptera, Vespertilionoidea) from the late Early Eocene of Chambi,Tunisia

Abstract: Among the new dental remains from the late Early Eocene of Chambi (Kasserine area, Tunisia) is a large‐sized upper molar of a new bat species, Witwatia sigei nov. sp. (Chiroptera, Vespertilionoidea, Philisidae), described herein. The locality of Chambi has revealed evidence for an early appearance of two modern microchiropteran superfamilies in Africa: Dizzya exsultans, a Philisidae, which is considered to be an archaic Vespertilionoidea, and an indeterminate Rhinolophoidea. In addition to D. exsultans, the new species, W. sigei, is the second representative of the Philisidae in this locality. W. sigei extends back to the late Early Eocene the occurrence of the genus Witwatia, which was previously only reported from the early Late Eocene of the Fayum (BQ‐2, Egypt). By analogy with the largest extant microbats, the large size of Witwatia suggests a tendency to the opportunistic diet of this taxon, thereby contrasting with the strict insectivory characterizing primitive bats found in other continents in the same epoch.     

Elucidating cell-penetrating peptide mechanisms of action for membrane interaction,cellular uptake,and translocation utilizing the hydrophobic counter-anion pyrenebutyrate

Cell-penetrating peptides (CPPs) are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. To elucidate CPP uptake mechanisms, the effect of pyrenebutyrate on well-recognized CPPs with varying hydrophobicity and arginine content is investigated. The cellular CPP uptake and CPP-mediated oligonucleotide delivery is analyzed by fluorescence activated cell sorting, confocal microscopy, and a cell-based splice-switching assay. The splice-switching oligonucleotide is a mixmer of 2′-O-methyl RNA and locked nucleic acids delivered as a non-covalent complex with 10-fold molar CPP excess. CPP-induced membrane perturbation on large unilamellar vesicles is investigated in calcein release experiments. We observed that pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotide mediated by oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. By combining the different experimental results we conclude that the pathway for cellular uptake of oligoarginine is dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate and we suggest that pyrenebutyrate has different sites of action for the two uptake and translocation mechanisms.