Insulin-like growth factor-1 lowers spreading depression susceptibility and reduces oxidative stress

Spreading depression (SD), the likely cause of migraine aura and perhaps migraine, is triggered by widespread and unfettered neuronal hyperexcitability. Migraine and the initiating hyperexcitability of seizure, which involve oxidative stress (OS), are likely interrelated. Environmental enrichment (EE) decreases seizure and can reduce migraine. EE's well-characterized neuroprotective effect involves insulin-like growth factor-1 (IGF-1). Accordingly, we asked if IGF-1 could mitigate the hyperexcitability that initiates SD using rat hippocampal slice cultures. We demonstrate that IGF-1 significantly decreased SD susceptibility and related OS. We mimicked OS of SD and observed that IGF-1 abolished hyperexcitability from OS. Application of an antioxidant significantly decreased SD susceptibility and co-administration of an antioxidant with IGF-1 produced no additive effect, whereas an oxidizer significantly increased SD, and this effect was abrogated by IGF-1. Moreover, IGF-1 significantly decreased baseline OS, despite seemingly paradoxically increasing CA3 bursting. These results suggest that IGF-1 increased endogenous antioxidants to levels sufficient to buffer against the OS of SD. Insulin similarly mitigated SD susceptibility, but required a far greater dose. Since brain IGF-1 increases with EE, and, like insulin, independently functions as an EE mimetic, we suggest that EE mimetics are a novel source of therapeutics for SD, and by extension, migraine.     

Bacterial multidrug resistance unrelated to multidrug exporters: cell biology insight

Multidrug resistance (MDR) revealed in malignant cell lines was firstly attributed to the activity of multidrug exporters pumping drugs out of the cell. However, mutagenised Escherichia coli develop extraordinary numerous mutants resistant to target inhibitor and we have shown that with mutations mapped around the entire genome most of the mutants were multiple-resistant. In case of one such mutant studied MDR was shown as a sum of individual resistances due to mutations resulted in target and ligand sequestration and induced simultaneously in tightly linked, cassette-like genes. An explanation of local mutagenesis efficiency and the nature of sequestration process is proposed. A cassette-like organization of genes responsible for chemoresistance emergence could promote the local intensity of mutagenesis by a cassette facing the intracellular space and flux and contacting unlike other genes mutagen the first. Target and ligand sequestration could result from clogging the intracellular flux due to cytoplasm geometry alteration attributable to disorder-order transition in natively unfolded proteins affected with mutation.     

CaJOINTLESS is a MADS-box gene involved in suppression of vegetative growth in all shoot meristems in pepper

In aiming to decipher the genetic control of shoot architecture in pepper (Capsicum spp.), the allelic late-flowering mutants E-252 and E-2537 were identified. These mutants exhibit multiple pleiotropic effects on the organization of the sympodial shoot. Genetic mapping and sequence analysis indicated that the mutants are disrupted at CaJOINTLESS, the orthologue of the MADS-box genes JOINTLESS and SVP in tomato and Arabidopsis, respectively. Late flowering of the primary and sympodial shoots of Cajointless indicates that the gene functions as a suppressor of vegetative growth in all shoot meristems. While CaJOINTLESS and JOINTLESS have partially conserved functions, the effect on flowering time and on sympodial development in pepper, as well as the epistasis over FASCICULATE, the homologue of the major determinant of sympodial development SELF-PRUNING, is stronger than in tomato. Furthermore, the solitary terminal flower of pepper is converted into a structure composed of flowers and leaves in the mutant lines. This conversion supports the hypothesis that the solitary flowers of pepper have a cryptic inflorescence identity that is suppressed by CaJOINTLESS. Formation of solitary flowers in wild-type pepper is suggested to result from precocious maturation of the inflorescence meristem.     

The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome: IDENTIFICATION, GLYCOSITE OCCUPANCY, AND MEMBRANE ORIENTATION*

Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and β-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes.Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function. One of the best examples of endogenous repair mechanisms involves skeletal muscle, which has innate regenerative capacity (for reviews, see Refs. 1–4). Skeletal muscle repair begins with satellite cells, a heterogeneous population of mitotically quiescent cells located in the basal lamina that surrounds adult skeletal myofibers (5, 6), that, when activated, rapidly proliferate (7). The progeny of activated satellite cells, known as myogenic precursor cells or myoblasts, undergo several rounds of division prior to withdrawal from the cell cycle. This is followed by fusion to form terminally differentiated multinucleated myotubes and skeletal myofibers (7, 8). These cells effectively repair or replace damaged cells or contribute to an increase in skeletal muscle mass.The molecular mechanisms that regulate differentiation of satellite cells and myoblasts toward myofibers are not fully understood, although it is known that the cell surface proteome plays an important biological role in skeletal muscle differentiation. Examples include how cell surface proteins modulate myoblast elongation, orientation, and fusion (for a review, see Ref. 8). The organization and fusion of myoblasts is mediated, in part, by cadherins (for reviews, see Refs. 9 and 10), which enhance skeletal muscle differentiation and are implicated in myoblast fusion (11). Neogenin, another cell surface protein, is also a likely regulator of myotube formation via the netrin ligand signal transduction pathway (12, 13), and the family of sphingosine 1-phosphate receptors (Edg receptors) are known key signal transduction molecules involved in regulating myogenic differentiation (14–17). Given the important role of these proteins, identifying and characterizing the cell surface proteins present on myoblasts in a more comprehensive approach could provide insights into the molecular mechanisms involved in skeletal muscle development and repair. The identification of naturally occurring cell surface proteins (i.e. markers) could also foster the enrichment and/or characterization of cell intermediates during differentiation that could be useful therapeutically.Although it is possible to use techniques such as flow cytometry, antibody arrays, and microscopy to probe for known proteins on the cell surface in discrete populations, these methods rely on a priori knowledge of the proteins present on the cell surface and the availability/specificity of an antibody. Proteomics approaches coupled with mass spectrometry offer an alternative approach that is antibody-independent and allows for the de novo discovery of proteins on the surface. One approach, which was used in the current study, exploits the fact that a majority of the cell surface proteins are glycosylated (18). The method uses hydrazide chemistry (19) to immobilize and enrich for glycoproteins/glycopeptides, and previous studies using this chemistry have successfully identified soluble glycoproteins (20–24) as well as cell surface glycoproteins (25–28). A recently optimized hydrazide chemistry strategy by Wollscheid et al. (29) termed cell surface capturing (CSC)¹ technology, reports the ability to identify cell surface (plasma membrane) proteins specifically with little (<15%) contamination from non-cell surface proteins. The specificity stems from the fact that the oligosaccharide structure is labeled using membrane-impermeable reagents while the cells are intact rather than after cell lysis. Consequently, only extracellular oligosaccharides are labeled and subsequently captured. Utilizing information regarding the glycosylation site then allows for a rapid elimination of nonspecifically captured proteins (i.e. non-cell surface proteins) during the data analysis process, a feature that makes this approach unique to methods where no label or tag is used. Additionally, the CSC technology provides information about glycosylation site occupancy (i.e. whether a potential N-linked glycosylation site is actually glycosylated), which is important for determining the protein orientation within the membrane and, therefore, antigen selection and antibody design.To uncover information about the cell surface of myoblasts and to identify potential markers of myoblast differentiation, we used the CSC technology on the mouse myoblast C2C12 cell line model system (30, 31). This adherent cell line derived from satellite cells has routinely been used as a model for skeletal muscle development (e.g. Refs. 1, 32, and 33), skeletal muscle differentiation (e.g. Refs. 34–36), and studying muscular dystrophy (e.g. Refs. 37–39). Additionally, these cells have been used in cell-based therapies (e.g. Refs. 40–42). Using the CSC technology, 128 cell surface N-linked glycoproteins were identified, including several that were found to change in overall abundance as the myoblasts differentiate toward myotubes. The current data also confirmed the occupancy of 235 N-linked glycosites of which 226 were previously unconfirmed. The new information provided by the current study is expected to facilitate the development of useful tools for studying the differentiation of myoblasts toward myotubes.     

Therapeutic efficacy of theophylline in chronic lymphocytic leukemia

Theophylline, a methylxanthine commonly used as a treatment for asthma, has been shown to induce apoptosis in chronic lymphocytic leukemia (CLL) cells bothin vitro andin vivo. We have treated three advanced CLL patients with theophylline, and seen responses in two. The clinical courses of the responders are presented, and the literature concerning theophylline as therapy for CLL is reviewed.