Crystals of P2 myelin protein in lipid-bound form

The P2 protein of peripheral nervous system myelin induces experimental allergic neuritis in rats, a model of Guillain-Barré syndrome in humans. Previous purification procedures have used acid extraction to obtain the protein in lipid-free form (LF-P2). Here, we have purified the P2 protein in lipid-bound form (LB-P2) by extracting myelin with the detergent CHAPS, followed by Cu(2+)-affinity column chromatography. All myelin lipids were present in the preparation as shown by high-performance thin-layer chromatography and mass spectrometry. The LB-P2 preparation, which differs from LF-P2 in solubility and in the secondary-structure composition, was dialyzed to remove unbound lipids and excess detergent and crystallized using the hanging-drop vapor diffusion technique. Crystals of lipid-bound P2 appeared usually very reproducibly within 2 weeks at pH 5.7 in polyethylene glycol 6000 (PEG6000) at concentrations of 20-30% (w/v), and larger crystals were obtained by additional sitting-drop crystallization. X-ray diffraction showed reflections up to 2.7A. The crystallization conditions (25-30% PEG6000, pH 5.0) and the unit cell dimensions (a = 94.5A, b = 94.5A, c=74.2A, alpha = beta = 90 degrees, gamma = 120 degrees ) of LB-P2 were different from those earlier described for LF-P2 (10% PEG4000, pH 3, and unit cell dimensions a = 91.8A, b = 99.5A, c = 56.5A, alpha = beta = gamma = 90.0 degrees ). It is important that P2 has been crystallized with specifically bound lipids; therefore, solving this new crystal structure will reveal details of this protein's behavior and role in the myelin sheath.     

Requirement for down-regulation of the CCAAT-binding activity of the NF-Y transcription factor during skeletal muscle differentiation

NF-Y is composed of three subunits, NF-YA, NF-YB, and NF-YC, all required for DNA binding. All subunits are expressed in proliferating skeletal muscle cells, whereas NF-YA alone is undetectable in terminally differentiated cells in vitro. By immunohistochemistry, we show that the NF-YA protein is not expressed in the nuclei of skeletal and cardiac muscle cells in vivo. By chromatin immunoprecipitation experiments, we demonstrate herein that NF-Y does not bind to the CCAAT boxes of target promoters in differentiated muscle cells. Consistent with this, the activity of these promoters is down-regulated in differentiated muscle cells. Finally, forced expression of the NF-YA protein in cells committed to differentiate leads to an impairment in the down-regulation of cyclin A, cyclin B1, and cdk1 expression and is accompanied by a delay in myogenin expression. Thus, our results indicate that the suppression of NF-Y function is of crucial importance for the inhibition of several cell cycle genes and the induction of the early muscle-specific program in postmitotic muscle cells.     

The two variants of oxysterol binding protein-related protein-1 display different tissue expression patterns,have different intracellular localization,and are functionally distinct

Oxysterol binding protein (OSBP) homologs comprise a family of 12 proteins in humans (Jaworski et al., 2001; Lehto et al., 2001). Two variants of OSBP-related protein (ORP) 1 have been identified: a short one that consists of the carboxy-terminal ligand binding domain only (ORP1S, 437 aa) and a longer N-terminally extended form (ORP1L, 950 aa) encompassing three ankyrin repeats and a pleckstrin homology domain (PHD). We now report that the two mRNAs show marked differences in tissue expression. ORP1S predominates in skeletal muscle and heart, whereas ORP1L is the most abundant form in brain and lung. On differentiation of primary human monocytes into macrophages, both ORP1S and ORP1L mRNAs were induced, the up-regulation of ORP1L being >100-fold. The intracellular localization of the two ORP1 variants was found to be different. Whereas ORP1S is largely cytosolic, the ORP1L variant localizes to late endosomes. A significant amount of ORP1S but only little ORP1L was found in the nucleus. The ORP1L ankyrin repeat region (aa 1-237) was found to localize to late endosomes such as the full-length protein. This localization was even more pronounced for a fragment that additionally includes the PHD (aa 1-408). The amino-terminal region of ORP1L consisting of the ankyrin repeat and PHDs is therefore likely to be responsible for the targeting of ORP1L to late endosomes. Interestingly, overexpression of ORP1L was found to enhance the LXRalpha-mediated transactivation of a reporter gene, whereas ORP1S failed to influence this process. The results suggest that the two forms of ORP1 are functionally distinct and that ORP1L is involved in control of cellular lipid metabolism.     

TGF-beta 1 modulation of IGF-I signaling pathway in rat thyroid epithelial cells

Transforming growth factor beta 1 (TGF-beta 1) and insulin-like growth factor I (IGF-I) have contrasting effects on cell cycle regulation in thyroid cells and TGF-beta 1 induces a dramatic decrease in IGF-I-induced cell proliferation. The aim of the present study was to investigate the molecular mechanism of cross-talk between TGF-beta 1 and IGF-I in FRTL-5 cells. TGF-beta 1 affected IGF-I-stimulated insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with Grb2 protein. Moreover, TGF-beta 1 decreased the IGF-I-induced tyrosine phosphorylation of the adaptor protein CrkII and its association with the IGF-I receptor. These results were accompanied by TGF-beta 1 inhibition of IGF-I-stimulated mitogen-activated protein kinase phosphorylation and activation. Conversely, TGF-beta 1 did not alter IGF-I-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity, IGF-I-induced tyrosine phosphorylation of Shc, and its binding to Grb2. Taken together, these findings provide a molecular basis for the growth-inhibitory action of TGF-beta 1 on the IGF-I-induced mitogenic effect.     

Different activity of glyoxalase system enzymes in specimens of Sparus auratus exposed to sublethal copper concentrations

The present study regards possible changes in the activity of glyoxalase system enzymes (glyoxalase I, GI, and glyoxalase II, GII) in tissues (brain, liver and white muscle) of the mediterranean bony fish Sparus auratus after a 20 days exposure to sublethal concentrations (0.1 or 0.5 ppm) of Cu in the marine water and on control untreated animals. The experiments also included measurements of copper concentration in the tissues, as well as of lactate dehydrogenase (LDH) activity, to evaluate possible Cu accumulation and changes in glycolytic activity respectively. Cu accumulation only occurs in the liver. GI, GII and LDH activities kept unchanged in the brain after copper exposure. GI activity in liver and muscle of copper-exposed animals decreases probably for a slackening in the glycolytic rate, as suggested by the lowering of LDH activity. GII activity remains unchanged or increases (liver extract, 0.5 ppm of Cu), maybe to safeguard enough cellular levels of GSH.     

Nitric oxide and Mycobacterium leprae pathogenicity

Leprosy is an old, still dreaded infectious disease caused by the obligate intracellular bacterium Mycobacterium leprae. During the infectious process, M. leprae is faced with the host macrophagic environment, where the oxidative stress and NO release, combined with low pH, low pO2, and high pCO2, contribute to limit the growth of the bacilli. Comparative genomics has unraveled massive gene decay in M. leprae, linking the strictly parasitic lifestyle with the reductive genome evolution. Compared with Mycobacterium tuberculosis and Mycobacterium bovis, the leprosy bacillus has lost most of the genes involved in the detoxification of reactive oxygen and nitrogen species. The very low reactivity of the unique truncated hemoglobin retained by M. leprae could account for the susceptibility of this exceptionally slow-growing microbe to NO.     

Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction

Experimental proteome analysis was combined with a genome-wide prediction screen to characterize the protein content of the thylakoid lumen of Arabidopsis chloroplasts. Soluble thylakoid proteins were separated by two-dimensional electrophoresis and identified by mass spectrometry. The identities of 81 proteins were established, and N termini were sequenced to validate localization prediction. Gene annotation of the identified proteins was corrected by experimental data, and an interesting case of alternative splicing was discovered. Expression of a surprising number of paralogs was detected. Expression of five isomerases of different classes suggests strong (un)folding activity in the thylakoid lumen. These isomerases possibly are connected to a network of peripheral and lumenal proteins involved in antioxidative response, including peroxiredoxins, m-type thioredoxins, and a lumenal ascorbate peroxidase. Characteristics of the experimentally identified lumenal proteins and their orthologs were used for a genome-wide prediction of the lumenal proteome. Lumenal proteins with a typical twin-arginine translocation motif were predicted with good accuracy and sensitivity and included additional isomerases and proteases. Thus, prime functions of the lumenal proteome include assistance in the folding and proteolysis of thylakoid proteins as well as protection against oxidative stress. Many of the predicted lumenal proteins must be present at concentrations at least 10,000-fold lower than proteins of the photosynthetic apparatus.     

Antimicrobial activity of crude extracts from plant parts and corresponding calli of Bixa orellana L

Ethanol extracts from the different parts of B. orellana showed differential antimicrobial activity. It was found that the extracts of in vitro leaves showed maximum activity against Bacillus pumilus followed by the extracts from the roots and hypocotyls. The callus derived from different explants too showed antimicrobial activity. The leaf callus showed maximum activity. The zone of inhibition for the diluted extracts of in vitro hypocotyls and roots and their corresponding calli showed minimum zone of inhibition at concentration 24 mg/ml, whereas the diluted extract of in vitro leaves and leaf derived callus showed minimum zone of inhibition at 16 mg/ml.