Biochemical characteristics and partial amino acid sequence of the receptor-like human B cell and carcinoma antigen CDw40

The Ag CDw40 (p50, Bp50) is a phosphoprotein expressed on the surface of both B lymphocytes and on certain malignant cell types of nonhemopoietic origin. Antibodies to this Ag have been shown to act as a potent co-mitogen for B cells. In order to elucidate the function of this Ag, we have now investigated some of its biochemical characteristics as well as the relationship of B cell derived CDw40 to that derived from urinary bladder carcinoma (transitional cell carcinoma, TCC) cells. CDw40 from normal B cells or from the Burkitt lymphoma line Raji showed a characteristic pattern of three bands when analyzed by SDS-PAGE and Western blotting: a main band of 47 kDa, a degradation product of 43 kDa, and a dimer of 85 kDa. The dimer was disrupted by reduction with 2-ME but was reformed spontaneously from the purified monomers under nonreducing conditions. CDw40 from two bladder cancer cell lines gave a similar pattern but formed little or no dimer. Thirty amino acids of the amino terminal end of CDw40 from Raji and 22 amino acids of that from TCC cells (HU549) were sequenced. The sequences were unusually rich in cysteines and differed only in that the cysteine in position 6 in Raji CDw40 had been replaced by glutamine in HU549. In addition there were two conservative changes in positions 15 and 19. Taken together these results show that CDw40 derived from B cells or from TCC cells are the same or closely related molecules. Comparisons of the amino acid sequence and biochemical characteristics of CDw40 with proteins having receptor functions indicated a close structural resemblance of CDw40 to the nerve growth factor-receptor.     

N-Terminal Protein Characterization by Mass Spectrometry Using Combined Microscale Liquid and Solid-phase Derivatization

A sample-preparation method for N-terminal peptide isolation from protein proteolytic digests has been developed. Protein thiols and primary amines were protected by carboxyamidomethylation and acetylation, respectively, followed by trypsinization. The digest was bound to ZipTip_C18 pipette tips for reaction of the newly generated N-termini with sulfosuccinimidyl-6-[3′-(2-pyridyldithio)-propionamido] hexanoate. The digest was subsequently exposed to hydroxylamine for reversal of hydroxyl group acylation, followed by reductive release of the pyridine-2-thione moiety from the derivatives. The thiol group-functionalized internal and C-terminal peptides were reversibly captured by covalent chromatography on activated thiol sepharose leaving the N-terminal fragment free in solution. The use of the reversed-phase supports as a reaction bed enabled optimization of the serial modification steps for throughput and completeness of derivatization. The use of the sample-preparation method was demonstrated with low picomole amounts of in-solution- and in-gel-digested protein. The N-terminal peptide was selectively retrieved from the affinity support. The sample-preparation method provides for throughput, robustness, and simplicity of operation using standard equipment available in most biological laboratories and is anticipated to be readily expanded to proteome-wide applications.     

Lipid raft targeting of hematopoietic protein tyrosine phosphatase by protein kinase C theta-mediated phosphorylation

Protein kinase C theta (PKC theta) is unique among PKC isozymes in its translocation to the center of the immune synapse in T cells and its unique downstream signaling. Here we show that the hematopoietic protein tyrosine phosphatase (HePTP) also accumulates in the immune synapse in a PKC theta-dependent manner upon antigen recognition by T cells and is phosphorylated by PKC theta at Ser-225, which is required for lipid raft translocation. Immune synapse translocation was completely absent in antigen-specific T cells from PKC theta-/- mice. In intact T cells, HePTP-S225A enhanced T-cell receptor (TCR)-induced NFAT/AP-1 transactivation, while the acidic substitution mutant was as efficient as wild-type HePTP. We conclude that HePTP is phosphorylated in the immune synapse by PKC theta and thereby targeted to lipid rafts to temper TCR signaling. This represents a novel mechanism for the active immune synapse recruitment and activation of a phosphatase in TCR signaling.     

When things don't add up: quantifying impacts of multiple stressors from individual metabolism to ecosystem processing

Ecosystems are exposed to multiple stressors which can compromise functioning and service delivery. These stressors often co‐occur and interact in different ways which are not yet fully understood. Here, we applied a population model representing a freshwater amphipod feeding on leaf litter in forested streams. We simulated impacts of hypothetical stressors, individually and in pairwise combinations that target the individuals' feeding, maintenance, growth and reproduction. Impacts were quantified by examining responses at three levels of biological organisation: individual‐level body sizes and cumulative reproduction, population‐level abundance and biomass and ecosystem‐level leaf litter decomposition. Interactive effects of multiple stressors at the individual level were mostly antagonistic, that is, less negative than expected. Most population‐ and ecosystem‐level responses to multiple stressors were stronger than expected from an additive model, that is, synergistic. Our results suggest that across levels of biological organisation responses to multiple stressors are rarely only additive. We suggest methods for efficiently quantifying impacts of multiple stressors at different levels of biological organisation.     

Time-dependent lectin binding to isolated receptors in model membranes

Binding of lectins to two integral membrane glycoproteins has been measured in lipid bilayer model membranes and in their cell of origin with an eye to clarifying the basis of time-dependence in such processes. Specific binding was monitored as a function of time using an assay that involved membrane exposure to radiolabelled wheat-germ agglutinin or concanavalin A, and subsequent differential centrifugation to remove unbound material. Qualitatively, the time dependence of lectin binding to the isolated receptors in lipid bilayers was found to be similar to that for the same receptors in the intact cell - hence the phenomenon does not depend for its existence upon receptor interaction with other specific native membrane components. Quantitatively, the time-course was sensitive to structural features of the model membrane involved. The results may be understood by viewing the glycopeptide headgroups as deformable structures which rearrange as a direct result of lectin attachment, and it would appear that rearrangement is essential for high-affinity binding. Model membrane structure was examined by light microscopy and freeze-etch electron microscopy in an attempt to assess the applicability of this type of study to a more detailed analysis of the processes involved in lectin binding. Although the freeze-etch technique is a promising one, it was concluded that heterogeneity in receptor arrangement within the lipid bilayer is the most important limitation to correlation of binding curves with membrane structure.     

Location of the sulfhydryl groups involved in disulfide interaction between the neighboring proteins L6 and L29 in mammalian ribosomes. S-cleavage of the cyanylated proteins in polyacrylamide gels after separation by dodecylsulfate gel electrophoresis

Proteins L6 and L29 occupy closely adjacent sites in mammalian 60-S ribosomal subparticles and are easily cross-linked by intermolecular disulfide bond formation. For locating the interacting thiols within the polypeptide chains the dissociated proteins L6 and L29 obtained from the isolated disulfide complex were subjected to S-cleavage following [14C]cyanylation of the two cysteine residues. Four split products of the [14C]cyanylated proteins were isolated by dodecylsulfate gel electrophoresis. Two of these could be identified by autoradiography as the selectively labeled C-terminal fragments. For unequivocal assignment of the fragments to the parent proteins, a simple and generally applicable method of cleaving cyanylated proteins in polyacrylamide gel for subsequent diagonal analysis was developed. The experiments indicated that the sulfhydryl group of L6 interacting with L29 is located at a distance of approximately 80 amino acid residues from the N-terminus. In the intact ribosome this sequence contains a clostripain-sensitive and trypsin-sensitive portion of the protein more or less exposed at the ribosomal surface. In the case of protein L29, the interacting sulfhydryl group was located at a distance of approximately 40 amino acid residues from the C-terminal.     

Reversine ameliorates hallmarks of cellular senescence in human skeletal myoblasts via reactivation of autophagy

Cellular senescence leads to the depletion of myogenic progenitors and decreased regenerative capacity. We show that the small molecule 2,6-disubstituted purine, reversine, can improve some well-known hallmarks of cellular aging in senescent myoblast cells. Reversine reactivated autophagy and insulin signaling pathway via upregulation of Adenosine Monophosphate-activated protein kinase (AMPK) and Akt2, restoring insulin sensitivity and glucose uptake in senescent cells. Reversine also restored the loss of connectivity of glycolysis to the TCA cycle, thus restoring dysfunctional mitochondria and the impaired myogenic differentiation potential of senescent myoblasts. Altogether, our data suggest that cellular senescence can be reversed by treatment with a single small molecule without employing genetic reprogramming technologies.