Systematic characterization of nuclear proteome during apoptosis: a quantitative proteomic study by differential extraction and stable isotope labeling

Identification and characterization of the nuclear proteome is important for detailed understanding of multiple signaling events in eukaryotic cells. Toward this goal, we extensively characterized the nuclear proteome of human T leukemia cells by sequential extraction of nuclear proteins with different physicochemical properties using three buffer conditions. This large scale proteomic study also tested the feasibility and technical challenges associated with stable isotope labeling by amino acids in cell culture (SILAC) to uncover quantitative changes during apoptosis. Analyzing proteins from three nuclear fractions extracted from naive and apoptotic cells generated 780,530 MS/MS spectra that were used for database searching using the SEQUEST algorithm. This analysis resulted in the identification and quantification of 1,174 putative nuclear proteins. A number of known nuclear proteins involved in apoptosis as well as novel proteins not known to be part of the nuclear apoptotic machinery were identified and quantified. Consistent with SILAC-based quantifications, immunofluorescence staining of nucleus, mitochondria, and some associated proteins from both organelles revealed a dynamic recruitment of mitochondria into nuclear invaginations during apoptosis.     

Dusk calling in barbary macaques (Macaca sylvanus): Demand for social shelter

Barbary macaques (Macaca sylvanus) show an increase in vocal activity at dusk. This investigation showed that these vocalizations were mainly produced by juveniles from six to 18 months of age, during their attempts to achieve body contact with their mothers, thereby building up a sleeping cluster. The “dusk calling” consisted of protracted sequences of variable vocal patterns which always ceased when the juvenile joined its preferred sleeping cluster. The length of the sequences corresponded to the time it took the juvenile to be accepted into a sleeping cluster. One-year-old juveniles exhibited the highest vocal activity. The similarity of the results obtained in two outdoor enclosures in France and of those in the field (Morocco) indicated that dusk calling is common to this species, and not a behavior unique to captive animals. © Wiley-Liss, Inc.     

Dipeptidyl peptidase IV in the immune system. Cytofluorometric evidence for induction of the enzyme on activated T lymphocytes.

Dipeptidyl peptidase IV (DP IV) is a membrane peptidase with essential functional significance in thymus derived lymphocytes. This conclusion is drawn from 1) the induction of this enzyme after stimulation of T lymphocytes in vitro and 2) the impairment of T cell functions in presence of active site-specific inhibitors of the enzyme. The first item will be addressed in this paper, whereas the second one will be treated in a forthcoming article. Using flow cytofluorometry we investigated the expression of dipeptidyl peptidase IV on activated lymphocytes and the phenotype of lymphocytes expressing this enzyme. After stimulation by mitogenic lectins the number of epitopes on the cell surface binding polyclonal antibodies against DP IV increases 4 to 6 times. By means of double fluorescence staining the enzyme has been shown to be restricted nearly exclusively to T lymphocytes even after mitogenic stimulation. The highest density of DP IV epitopes has been found in cells coexpressing activation markers like receptors for interleukin 2 or transferrin in a high density.     

Historical Invasion Records Can Be Misleading: Genetic Evidence for Multiple Introductions of Invasive Raccoons (Procyon lotor) in Germany

Biological invasions provide excellent study systems to understand evolutionary, genetic and ecological processes during range expansions. There is strong evidence for positive effects of high propagule pressure and the associated higher genetic diversity on invasion success, but some species have become invasive despite small founder numbers. The raccoon (Procyon lotor) is often considered as a typical example for such a successful invasion resulting from a small number of founders. The species’ largest non-native population in Germany is commonly assumed to stem from a small number of founders and two separate founding events in the 1930s and 1940s. In the present study we analyzed 407 raccoons at 20 microsatellite loci sampled from the invasive range in Western Europe to test if these assumptions are correct. Contrary to the expectations, different genetic clustering methods detected evidence for at least four independent introduction events that gave rise to genetically differentiated subpopulations. Further smaller clusters were either artifacts or resulted from founder events at the range margin and recent release of captive individuals. We also found genetic evidence for on-going introductions of individuals. Furthermore a novel randomization process was used to determine the potential range of founder population size that would suffice to capture all the alleles present in a cluster. Our results falsify the assumption that this species has become widespread and abundant despite being genetically depauperate and show that historical records of species introductions may be misleading.     

Cell growth stimulation by EGF: inhibition through antisense-oligodeoxynucleotides demonstrates important role of casein kinase II

Casein kinase II (CKII) is a highly conserved ubiquitous serine/threonine kinase composed of two catalytically active subunits (alpha and/or alpha') and two presumably regulatory subunits (beta). CKII has numerous cellular functions including a possible role in mitogenic signaling. To address this question, growth-arrested primary human fibroblasts (IMR-90) were exposed prior growth stimulation by epidermal growth factor (EGF) to oligodeoxynucleotides complementary to the translation start region of mRNAs coding for CKII alpha and beta subunits. A significant inhibition of growth stimulation (up to 60%) was observed with both antisense-alpha and antisense-beta. The inhibition was reversible, became decreased with mutated antisense-oligodeoxynucleotides, and neutralized by simultaneous presence of respective sense-oligodeoxynucleotides. The expected down-regulation of CKII protein due to hybrid formation of antisense-oligodeoxynucleotides with target mRNAs was investigated by determination of the intracellular protein level of CKII beta-subunit by immunofluorescence and quantitative image analysis. The protein was revealed to be localized predominantly in the nucleus and to become significantly decreased due to antisense-beta treatment of cells. The maximum decrease coincided with the early phase (first several hours) of growth stimulation by EGF when antisense-beta incubation was started 6-2 h before growth stimulation, the period within which application of antisense-alpha and antisense-beta caused the maximum of inhibition of growth stimulation. Thus CKII obviously plays, with both subunit alpha and subunit beta, an important role in the early phase of mitogenic stimulation.