Protein diffusion in crowded electrolyte solutions

We report on a combined cold neutron backscattering and spin-echo study of the short-range and long-range nanosecond diffusion of the model globular protein bovine serum albumin (BSA) in aqueous solution as a function of protein concentration and NaCl salt concentration. Complementary small angle X-ray scattering data are used to obtain information on the correlations of the proteins in solution. Particular emphasis is put on the effect of crowding, i.e. conditions under which the proteins cannot be considered as objects independent of each other. We thus address the question at which concentration this crowding starts to influence the static and in particular also the dynamical behaviour. We also briefly discuss qualitatively which charge effects, i.e. effects due to the interplay of charged molecules in an electrolyte solution, may be anticipated. Both the issue of crowding as well as that of charge effects are particularly relevant for proteins and their function under physiological conditions, where the protein volume fraction can be up to approximately 40% and salt ions are ubiquitous. The interpretation of the data is put in the context of existing studies on related systems and of existing theoretical models.     

Characterisation of human and murine snRNP proteins by two-dimensional gel electrophoresis and phosphopeptide analysis of U1-specific 70K protein variants.

The proteins of the major human snRNPs U1, U2, U4/U6 and U5 were characterised by two-dimensional electrophoresis, with isoelectric focussing in the first dimension and SDS-polyacrylamide gel electrophoresis in the second. With the exception of protein F, which exhibits an acidic pl value (pl = 3.3), the snRNP proteins are basic. Post-translational modification was found among the proteins associated specifically with the U1 and U2 particles. The most complex modification pattern was observed for the U1-specific 70K protein. This was found in at least 13 isoelectric variants, with pl values ranging from 6.7 to 8.7; these variants differed also in molecular weight. All of the 70K variants are phosphorylated in the cell. Thin-layer analysis of their tryptic phosphopeptides revealed that the 70K variants have four major phosphopeptides in common, in addition to which at least four additional serine residues are phosphorylated to different extents. The comparative phosphopeptide analysis shows that differential phosphorylation alone is not sufficient to explain the occurrence of the many isoelectric variants of 70K, so that the final charge of the 70K variants is determined both by phosphorylation and by other, as yet unidentified posttranslational modifications. By two-dimensional separation of snRNP proteins obtained from mouse Ehrlich ascites tumour cells, it was shown that the pattern of pl values of the mouse proteins was almost identical with the corresponding pattern for human proteins. Even the complex modification patterns of the 70K protein are identical in mouse and man, indicating that the presence in the cell of so many variants of this protein may have functional importance. The major difference between murine and human snRNP proteins is the absence of protein B' from mouse snRNPs. This suggests that the homologous protein B may be able to carry out the task of protein B'.     

Protein product of proto-oncogene c-mil.

Using antipeptide antibodies with specificity for the carboxyl termini of v-raf and v-mil protein products, two proteins with apparent molecular weights of approximately 71,000/73,000 and 215,000 were detected in immunoprecipitates from normal uninfected chicken cells. The 71,000/73,000-molecular-weight protein was identified as the product of the c-mil proto-oncogene by the close structural relationship of its 42,000-molecular-weight carboxyl-terminal domain to the v-mil-encoded domain of the hybrid protein p100gag-mil specified by the avian retrovirus MH2. The amino-terminal domain of the cellular protein is encoded by 5' c-mil sequences that have not been transduced into the genome of MH2. The c-mil protein (p71/73c-mil) was found to be phosphorylated in vivo, and homologous proteins were detected at variable levels in a variety of vertebrate cells, including human cells.     

Characterization of the protein apparently responsible for the elevated tyrosine protein kinase activity in LSTRA cells.

The LSTRA murine thymoma cell line contains an elevated level of tyrosine protein kinase activity. When a microsomal preparation from these cells is incubated in vitro with ATP, the principal tyrosine protein kinase substrate is a 56,000-dalton protein, p56. We have found that an activity phosphorylating p56 on tyrosine can also be detected at low levels in microsomes from most, but not all, T lymphoma cell lines and from normal thymic tissue. Only 1 of 30 other lymphoma cell lines was found to contain an elevated level of such a tyrosine protein kinase. An activity that phosphorylated p56 in vitro was not detectable in the cells of other hematopoietic lineages. Anti-peptide antibodies reactive with the site of in vitro tyrosine phosphorylation of p56 allowed us to determine that the apparent abundance of the p56 polypeptide parallels closely the level of the tyrosine protein kinase activity in the cell lines examined. This suggests that p56 is the protein kinase responsible for the elevated tyrosine protein kinase activity in LSTRA cells and that the phosphorylation of p56 observed in vitro results from autophosphorylation. Two-dimensional tryptic peptide mapping revealed that p56 is distinct from the proteins encoded by the cellular genes which are the progenitors of retroviral tyrosine protein kinases, src, yes, fgr, abl, fes, and ros. Additionally, none of these proto-oncogenes was found to be transcribed at elevated levels in LSTRA or Thy19 cells. Like the catalytic subunit of the cyclic AMP-dependent protein kinase, the cellular and viral forms of p60src, and the protein phosphatase calcineurin B, p56 contains covalently bound fatty acid.     

Positive Facial Affect – An fMRI Study on the Involvement of Insula and Amygdala

Imitation of facial expressions engages the putative human mirror neuron system as well as the insula and the amygdala as part of the limbic system. The specific function of the latter two regions during emotional actions is still under debate. The current study investigated brain responses during imitation of positive in comparison to non-emotional facial expressions. Differences in brain activation of the amygdala and insula were additionally examined during observation and execution of facial expressions. Participants imitated, executed and observed happy and non-emotional facial expressions, as well as neutral faces. During imitation, higher right hemispheric activation emerged in the happy compared to the non-emotional condition in the right anterior insula and the right amygdala, in addition to the pre-supplementary motor area, middle temporal gyrus and the inferior frontal gyrus. Region-of-interest analyses revealed that the right insula was more strongly recruited by (i) imitation and execution than by observation of facial expressions, that (ii) the insula was significantly stronger activated by happy than by non-emotional facial expressions during observation and imitation and that (iii) the activation differences in the right amygdala between happy and non-emotional facial expressions were increased during imitation and execution, in comparison to sole observation. We suggest that the insula and the amygdala contribute specifically to the happy emotional connotation of the facial expressions depending on the task. The pattern of the insula activity might reflect increased bodily awareness during active execution compared to passive observation and during visual processing of the happy compared to non-emotional facial expressions. The activation specific for the happy facial expression of the amygdala during motor tasks, but not in the observation condition, might reflect increased autonomic activity or feedback from facial muscles to the amygdala.     

Beautiful,complicated—and intelligent? Novel aspects of the thigmonastic stamen movement in Loasaceae

In a recent study we investigated the complex mechanisms regulating the pollen release via thigmonastic stamen movement found exclusively in Loasaceae subfamily Loasoideae. We demonstrated that stamen movement is modulated by abiotic (light and temperature) as well as biotic stimuli (pollinator availability and visitation frequency). This is explained as a mechanism to adjust the rate of stamen movement and thus pollen dispensation to different environmental conditions in order to optimize pollen transfer. Stamen movement is rapid and thus a near-immediate response to pollinator visits. However, Loasaceae flowers also show a response to biotic stimuli on a longer time scale, by adjusting the duration of both the staminate and the carpellate phase of the anthesis. We here present two additional data sets on species not previously studied, underscoring the shortening of the staminate phase in the presence of pollinator visits vs. their absence and the shortening of the carpellate phase after pollination. Overall, the plant shows not only a rapid but an “intelligent” reaction to its environment in adjusting anthesis and pollen presentation to a range of factors. The physiological and morphological bases of the stamen movement are poorly understood. Our previous study showed that there is no direct spatial relationship between the place of stimulation in the flower and the stamen bundle activated. We here further show the morphological basis for stamen movement from a reflexed into an erect position: Only the basal part of the filament curves around the receptacle, while the upper part of the filament retains its shape. We hypothesize that the stimulus is transmitted over the entire receptacle and the place of reaction is determined by stamen maturity, not the location of the stimulus.     

Relatively Recent Evolution of Pelage Coloration in Colobinae: Phylogeny and Phylogeography of Three Closely Related Langur Species

To understand the evolutionary processes leading to the diversity of Asian colobines, we report here on a phylogenetic, phylogeographical and population genetic analysis of three closely related langurs, Trachypithecus francoisi, T. poliocephalus and T. leucocephalus, which are all characterized by different pelage coloration predominantly on the head and shoulders. Therefore, we sequenced a 395 bp long fragment of the mitochondrial control region from 178 T. francoisi, 54 T. leucocephalus and 19 T. poliocephalus individuals, representing all extant populations of these three species. We found 29 haplotypes in T. francoisi, 12 haplotypes in T. leucocephalus and three haplotypes in T. poliocephalus. T. leucocephalus and T. poliocephalus form monophyletic clades, which are both nested within T. francoisi, and diverged from T. francoisi recently, 0.46-0.27 (T. leucocephalus) and 0.50-0.25 million years ago (T. poliocephalus). Thus, T. francoisi appears as a polyphyletic group, while T. leucocephalus and T. poliocephalus are most likely independent descendents of T. francoisi that are both physically separated from T. francoisi populations by rivers, open sea or larger habitat gaps. Since T. francoisi populations show no variability in pelage coloration, pelage coloration in T. leucocephalus and T. poliocephalus is most likely the result of new genetic mutations after the split from T. francoisi and not of the fixation of different characters derived from an ancestral polymorphism. This case study highlights that morphological changes for example in pelage coloration can occur in isolated populations in relatively short time periods and it provides a solid basis for studies in related species. Nevertheless, to fully understand the evolutionary history of these three langur species, nuclear loci should be investigated as well.     

A serial lectin approach to the mucin-type O-glycoproteome of Drosophila melanogaster S2 cells

Identification of mucin-type O-glycosylated proteins with known functions in model organisms like Drosophila could provide keys to elucidate functions of the O-glycan moiety and proteomic analyses of O-glycoproteins in higher eukaryotes remain a challenge due to structural heterogeneity and a lack of efficient tools for their specific isolation. Here we report a strategy to evaluate the O-glycosylation potential of the embryonal hemocyte-like Drosophila Schneider 2 (S2) cell line by expression of recombinant glycosylation probes derived from tandem repeats of the human mucin MUC1 or of the Drosophila salivary gland protein Sgs1. We obtained evidence that mucin-type O-glycosylation in S2 cells grown under serum-free conditions is restricted to the Tn-antigen (GalNAcalpha-Ser/Thr) and the T-antigen (Galbeta1-3GalNAcalpha-Ser/Thr) and this structural homogeneity enables unique glycoproteomic strategies. We present a label-free strategy for the isolation, profiling and analysis of O-glycosylated proteins consisting of serial lectin affinity capture, 2-DE-based glycoprotein analysis by O-glycan specific mAbs and protein identification by MALDI-MS. Protein identity and O-glycosylation was confirmed by ESI-MS/MS with detection of diagnostic sugar oxonium-ion fragments. Using this strategy, we established 2-D reference maps and identified 21 secreted and intracellular mucin-type O-glycoproteins. Our results show that Drosophila S2 cells express O-glycoproteins involved in a wide range of biological functions including proteins of the extracellular matrix (Laminin gamma-chain, Peroxidasin and Glutactin), pathogen recognition proteins (Gnbp1), stress response proteins (Glycoprotein 93), secreted proteases (Matrix-metalloprotease 1 and various trypsin-like serine proteases), protease inhibitors (Serpin 27 A) and proteins of unknown function.     

Hygrophorones A-G: fungicidal cyclopentenones from Hygrophorus species (Basidiomycetes)

Twenty new 5-(hydroxyalkyl)-2-cyclopentenone derivatives (hygrophorones) could be isolated from Hygrophorus latitabundus, H. olivaceoalbus, H. persoonii, and H. pustulatus. Their fungicidal activity was exemplarily tested. The hygrophorones have structural similarities to the antibiotic pentenomycin. Chemically, hygrophorones are 2-cyclopentenones with hydroxy or acetoxy substituents at C-4 and/or C-5. An odd-numbered 1' oxidized alkyl chain (C(11), C(13), C(15), or C(17)) is attached at C-5. In addition, from H. persoonii the new gamma-butyrolactone derivative [5-(E)-2-hydroxytetradexylidene-5H-furan-2-one] could be isolated. Some hygrophorones are responsible for the color reaction of the stipes of these fungi upon treatment with potassium hydroxide solution. Structural elucidations are based on 1D ((1)H, (13)C) and 2D (COSY, NOESY, HSQC, HMBC) NMR spectroscopic analyses as well as HR-FT-ICR-MS investigations.     

Removal of Ca2+ channel beta3 subunit enhances Ca2+ oscillation frequency and insulin exocytosis

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.