Enrichment and identification of integral membrane proteins from barley aleurone layers by reversed-phase chromatography, SDS-PAGE, and LC-MS/MS

The plasma membrane of the cereal aleurone layer is the site of perception of germination signals and release of enzymes to the starchy endosperm. Analysis of membrane proteins is challenging due to their hydrophobicity and low abundance; thus, little is known about the membrane proteins involved in seed germination. A membrane fraction highly enriched for the plasma membrane H+-ATPase was prepared from barley aleurone layers by aqueous two-phase partitioning. Because detergent and salt washes did not efficiently remove soluble proteins from the membrane preparations, an alternative procedure was developed, comprising batch reversed-phase chromatography with stepwise elution of hydrophobic proteins by 2-propanol. Proteins in the most hydrophobic fraction were separated by SDS-PAGE and identified by LC-MS/MS and barley EST sequence database search. The method was efficient for enrichment of integral membrane proteins with relatively low levels of soluble contaminating proteins. Forty-six proteins associated with barley aleurone plasma membranes were identified, including proteins with more than 10 transmembrane domains. Among the identified proteins were two new isoforms of the plasma membrane H+-ATPase, two proteins possibly involved in ion-channel regulation, and two proteins of unknown function. This represents the first analysis of membrane proteins involved in seed germination using a proteomics approach.     

Effect of pentoxifylline on arachidonic acid metabolism,neutral lipid synthesis and accumulation during induction of the lipocyte phenotype by retinol in murine hepatic stellate cell

In liver fibrosis, the quiescent hepatic stellate cells (HSC) are activated to proliferate and express the activated myofibroblast phenotype, losing fat droplets and the stored vitamin A, and depositing more extracellular matrix. Therapeutic strategies for liver fibrosis are focused on HSC. Pentoxifylline (PTF), an analog of the methylxanthine, prevents the biochemical and histological changes associated with animal liver fibrosis. The aim of the present study was to investigate the phenotypic change of myofibroblasts into quiescent lipocytes by PTF and/or retinol, using a permanent cell line GRX that represents murine HSC. We studied the action of both drugs on the synthesis of neutral lipids, activity of phospholipase A₂ (PLA₂), release of arachidonic acid (AA) and prostaglandins synthesis. Accumulation and synthesis of neutral lipids was dependent upon association of retinol with PTF. PTF (0.5 mg/mL) alone did not induce lipid accumulation and synthesis, but in cells induced by physiologic concentration of retinol (1–2.5 M), it increased the quantity of stored lipids. Retinol and PTF (5 M and 0.1 mg/mL, respectively) had a synergistic effect on neutral lipid synthesis and accumulation. In higher PTF concentrations (0.5 and 0.7 mg/ml), the synthesis was stimulated but accumulation decreased. Membrane-associated PLA₂ activity decreased after PTF treatment, which increased the AA release 8 fold, and significantly increased the production of PGE₂, but not of PGF₂. However, when in presence of retinol, we observed a slightly higher increase in PGE₂ and PGF₂ production. In conclusion, PTF treatment generated an excess of free AA. We propose that retinol counteracts the action of PTF on the AA release and PGs production, even though both drugs stimulated the lipocyte induction in the HSC.     

Stroma-mediated granulocyte-macrophage colony-stimulating factor (GM-CSF) control of myelopoiesis: spatial organisation of intercellular interactions

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the major cytokines involved in control of haemopoiesis both in bone marrow and in extramedullar sites. Its biological activity depends upon the composition and physicochemical properties of the microenvironment provided by the supporting stroma. GM-CSF activity is modulated and controlled by the stromal heparan-sulphate proteoglycans, but their optimal interaction occurs only at low pH. We questioned whether the microenvironment organisation of the interface between stroma and haemopoietic cells provides such conditions. We studied myeloid progenitor proliferation in contact with bone marrow-derived and extramedullar stromas using electron microscopy and selective labelling of pericellular components. We present evidence that, upon interaction, the two cell types reorganise their interface both in shape and molecular composition. Haemopoietic cells extend projections that considerably increase the area of intercellular contact, and stromal cells form lamellipodia and carry out a redistribution of membrane-associated sialylated glycoconjugates and proteoglycans. Such rearrangements lead to extensive capping of negatively charged molecules at the interface between the supporting stroma and the haemopoietic cells, leading potentially to a local decrease in pH. Our results indicate that the distribution of negative charges at the cellular interface may be responsible for the selectivity of cell response to GM-CSF.Publication of the Millennium Institute for Tissue Bioengineering. The study was supported by PRONEX, CNPq and FINEP grants from the Brazilian Ministry of Science and Technology and a FAPERJ grant from the Rio de Janeiro State Government.     

Molecular cloning and 3D structure prediction of the first raw-starch-degrading glucoamylase without a separate starch-binding domain

Raw-starch-degrading glucoamylases have been known as multidomain enzymes consisting of a catalytic domain connected to a starch-binding domain (SBD) by an O-glycosylated linker region. A molecular genetics approach has been chosen to find structural differences between two related glucoamylases, raw-starch-degrading Glm and nondegrading Glu, from the yeasts Saccharomycopsis fibuligera IFO 0111 and HUT 7212, respectively. We have found that Glm and Glu show a high primary (77%) and tertiary structure similarity. Glm, although possessing a good ability for raw starch degradation, did not show consensus amino acid residues to any SBD found in glucoamylases or other amylolytic enzymes. Raw starch binding and digestion by Glm must thus depend on the existence of a site(s) lying within the intact protein which lacks a separate SBD. The enzyme represents a structurally new type of raw-starch-degrading glucoamylase.     

Two different clean-up procedures for liquid chromatographic determination of ochratoxin A in urine

This paper describes two different procedures for extraction of ochratoxin A (OTA) from urine samples: one using acidic chloroform-methanol mixture, followed by solid-phase extraction (SPE) clean-up and the other using commercial Chem Elut columns and a chloroform-formic acid mixture. The recovery of OTA using the procedure with silica gel columns was 82% with a R.S.D. < 8.4% and the detection and quantitation limits were 0.5 and 1.5 ng OTA/ml, respectively. The recovery of OTA in the second procedure with urine samples purified only on commercial Chem Elut columns was 95% with R.S.D. < 4.0%, and detection and quantitation limits 0.3 and 0.9 ng/ml, respectively. Both procedures of OTA extraction effectively eliminate interfering substances and give reliable and repeatable results. However, the procedure with Chem Elut columns gave higher recovery and lower detection and quantitation limits. It was successfully applied in determining OTA in human urine samples.     

The proteolytic stability of 'designed' beta-peptides containing alpha-peptide-bond mimics and of mixed alpha,beta-peptides: application to the construction of MHC-binding peptides

Whereas alpha-peptides are rapidly degraded in vivo and in vitro by a multitude of peptidases, substrates constructed entirely of or incorporating homologated alpha-amino acid (i.e., beta-amino acid) units exhibit a superior stability profile. Efforts made so far to proteolytically hydrolyze a beta-beta peptide bond have not proved fruitful; a study aimed at breaching this proteolytic stability is discussed here. A series of such bonds have been designed with side-chain groups similar in relative positions (constitution) and three-dimensional arrangements (configuration) as found about alpha-peptidic amide bonds. Increasing the prospect for degradation would permit the tuning of beta-peptide stability; here, however, no cleavage was observed (1, 2, 4-6, Table 1). Peptides comprised of alpha- and beta-amino acids (mixed alpha,beta-peptides, 8-11) are expected to benefit from both recognition by a natural receptor and a high level of proteolytic stability, ideal characteristics of pharmacologically active compounds. Beta3-peptides containing alpha-amino acid moieties at the N-terminus are degraded, albeit slowly, by several peptidases. Of particular interest is the ability of pronase to cleave an alpha-beta peptide bond, namely that of alphaAla-beta3 hAla. Significantly, successful hydrolysis is independent of the configuration of the beta-amino acid. Some of the alpha,beta-peptides discussed here are being investigated for their binding affinities to class I MHC proteins. The computer-programming steps required to prepare alpha,beta-peptides on an automated peptide synthesizer are presented.     

Lacustrine basin to delta evolution in the Zagorje Basin,a Pannonian sub-basin (Late Miocene: Pontian,NW Croatia)

Pontian deposits of the Zagorje Basin constitute a coarsening-upward succession, which reflects the infilling style of this Pannonian sub-basin, i.e. the progradation of clastic systems into the brackish lake. Six facies differentiated correspond to lake floor, channelled slope, distal and proximal pro-delta, distal and proximal mouth bars, and lagoon/bay to swamp and alluvial environments. The deposition in the mouth-bar area was dominated by frictional forces. The upper-stage plane-bed sands are the main mouth-bar facies, which is uncommon in the geological record. The main factors for the origin of such bars include an abundant sand supply by the high-energy fluvial system(s), low-energy of the receiving basin and shallow depositional depth. Prolonged, catastrophic floods generated sustained hyperpycnal flows, which bypassed the mouth-bar area and fed the slope/pro-delta. The high ratio between sediment supply and subsidence rate resulted in a fast moving progradational wave, which involved the entire SW Pannonian Basin, including the Zagorje Basin.     

Expression of catalytic subunit of bovine enterokinase in the filamentous fungus Aspergillus niger

The cDNA encoding for catalytic subunit of bovine enterokinase (EK(L)), to which the sequence for Kex2 protease cleavage site was inserted, was expressed in the protease deficient filamentous fungus Aspergillus niger AB1.13. Fungal transformants were obtained in which expression of the glucoamylase fusion gene resulted in secretion of the protein into growth medium. Fusion polypeptide was processed to mature EK(L) by endogenous Kex-2 like protease cleavage during secretory pathway. The highest quantity of EK(L), up to 5 mg l(-1), was obtained in soya milk medium. The secreted EK(L) was easily purified from other proteins found in A. niger culture supernatant, using ion exchange and affinity chromatography. The yield of the purified and highly active EK(L) was 1.9 mg l(-1) of culture.