Insulin-mimetic action of conglutin-γ, a lupin seed protein, in mouse myoblasts.

BACKGROUND AND AIMS: Lupin seed is referred to as an antidiabetic product in traditional medicine. Conglutin-γ, a lupin seed glycoprotein, was found to cause a significant plasma glucose reduction when orally administered to rats in glucose overload trials. Conglutin-γ was identified as being responsible for the claimed biological activity, and the aim of this work was to envisage its hypothetical insulin-mimetic cellular mechanism of action. Insulin is responsible for proteosynthesis control through IRS/AKT/P70S6k/PHAS1 pathways modulation, glucose homeostasis through PKC/Flotillin-2/caveolin-3/Cbl activation and muscle differentiation/hypertrophy via muscle-specific MHC gene transcription control. METHODS AND RESULTS: To assess whether conglutin-γ modulates the same insulin-activated kinases, myoblastic C2C12 cells were incubated after 72 h of differentiation with 100 nM insulin or 0.5 mg/mL (∼10 µM) conglutin-γ. Metformin-stimulated cells were used as a positive control. The effect on the above mentioned pathways was evaluated after 5, 10, 20 and 30 min. In the control cells medium insulin, conglutin-γ and metformin were not added. We demonstrated that insulin or conglutin-γ cell stimulation resulted in the persistent activation of protein synthetic pathway kinases and increased glucose transport, glut4 translocation and muscle-specific gene transcription regulation. CONCLUSIONS: Our results indicate that conglutin-γ may regulate muscle energy metabolism, protein synthesis and MHC gene transcription through the modulation of the same insulin signalling pathway, suggesting the potential therapeutic use of this natural legume protein in the treatment of diabetes and other insulin-resistant conditions, as well as the potential conglutin-γ influence on muscle cells differentiation and regulation of muscle growth.

Assessment of the tolerance to lupine-enriched pasta in peanut-allergic children.

BACKGROUND: Reports of allergy to lupine derivatives (as de novo sensitization or cross-reactivity in subjects allergic to peanut) are increasing as their use in food products increases. OBJECTIVES: The aim of this study was to assess: (1) lupine tolerance in a group of children allergic to peanut, using lupine enriched-pasta instead of raw flour as has been done in previous clinical studies; (2) whether technological treatments of lupine modify its cross-reactivity or co-sensitization with peanut; (3) the role of lupine seed proteins in sensitization, and (4) to identify the eliciting doses (EDs) by using double-blind, placebo-controlled food challenges (DBPCFC). METHODS: Twelve patients with a history of clinical allergic reactions to peanut were evaluated by skin prick tests (SPTs), the ImmunoCAP test, immunoblotting, and DBPCFC. The 12 selected subjects were included in a trial where lupine-enriched pasta and placebo pasta were administered in a DBPCFC protocol. RESULTS: Positive clinical reactions were observed in two children, the EDs being 0.2 and 6.4 g of pasta, corresponding to 50 mg and 1.6 g of lupine proteins, respectively. Beta-conglutin was the protein most involved in SPT positivity. CONCLUSION: Lupine-enriched pasta can be tolerated by most subjects suffering from peanut allergy, but a sizeable minority (2/12 of them in this case) can develop potentially dangerous clinical reactions. Information about possible reactions to lupine derivatives by those allergic to peanuts must be included in the labelling of lupine-enriched products to protect consumers at risk.

AMS-100: The Next Generation Magnetic Spectrometer in Space - An International Science Platform for Physics and Astrophysics at Lagrange Point 2.

The next generation magnetic spectrometer in space, AMS-100, is designed to have a geometrical acceptance of 100 m2 sr and to be operated for at least ten years at the Sun-Earth Lagrange Point 2. Compared to existing experiments, it will improve the sensitivity for the observation of new phenomena in cosmic rays, and in particular in cosmic antimatter, by at least a factor of 1000. The magnet design is based on high temperature superconductor tapes, which allow the construction of a thin solenoid with a homogeneous magnetic field of 1 Tesla inside. The inner volume is instrumented with a silicon tracker reaching a maximum detectable rigidity of 100 TV and a calorimeter system that is 70 radiation lengths deep, equivalent to four nuclear interaction lengths, which extends the energy reach for cosmic-ray nuclei up to the PeV scale, i.e. beyond the cosmic-ray knee. Covering most of the sky continuously, AMS-100 will detect high-energy gamma rays in the calorimeter system and by pair conversion in the thin solenoid, reconstructed with excellent angular resolution in the silicon tracker.

Comparative effects of limited tryptic hydrolysis on physicochemical and structural features of seed 11S globulins.

The effect of the limited proteolysis by trypsin on selected seed storage 11S globulins (broad bean and pea legumins, glycinin and helianthinin) was studied by high-sensitive differential scanning calorimetry, fluorescence spectroscopy and analysis of proteolysis kinetics. Different behaviour of glycinin and helianthinin, on one hand, and broad bean and pea legumins, on the other, were observed: in the first group changes in the physicochemical characteristics of the proteins due to their limited proteolysis are more pronounced in comparison with the second one, in relation with the extent of primary structure modifications. The differences observed have been evaluated in relation with the amino acid sequence features of the four 11S globulin studied and agree with the literature data concerning the protein structural changes in the course of the limited proteolysis.

Cloning, sequencing and expression in the seeds and radicles of two Lupinus albus conglutin gamma genes.

Two genes encoding conglutin gamma have been isolated from a Lupinus albus genomic library and sequenced. The expression of conglutin gamma was studied by partial amino acid sequencing of the mature seed protein and by nucleotide sequencing of reverse transcriptase-polymerase chain reaction products from various tissues during the plant life cycle.

Retro-inverso peptide analogues of Trypanosoma cruzi B13 protein epitopes fail to be recognized by human sera and peripheral blood mononuclear cells.

Retro inverso (RI) analogues of antigenic synthetic peptides, which are made of D-amino acids with a reversed sequence, may mimic the side chain conformation of natural all-L peptides. RI analogues were cross-reactively recognized by antibodies and CD4+ T cells reactive against natural all-L synthetic peptides or native proteins in animal models. Since peptides containing D-amino acids are highly resistant to proteolytic digestion, cross-reactive RI analogues may be ideal for in vivo administration to humans as synthetic peptide vaccines or immunomodulators. B13 is an immunodominant tandemly repetitive protein from Trypanosoma cruzi, a protozoan parasite that is the causative antigen of Chagas' disease. In order to test whether RI peptides can be recognized by human antibody and T cells, we synthesized two all-L peptides containing the immunodominant B (S12) and T (S15.7) cell epitopes of B13 protein from T. cruzi and their retro (R, made of all-L amino acids with reversed sequence), inverso (I, made of all-D amino acids) and RI analogues. Recognition of peptides S12, S12-R, S12-I and S12-RI by anti-B13 antibodies in sera from T. cruzi-infected patients was tested in competitive ELISA assay with recombinant B13 protein as the solid phase antigen. Peptides S15.7 and its topological analogues were tested at the 10-50 microM range in proliferation assays on peripheral blood mononuclear cells (PBMC) from S15.7-responder individuals. The median percentage inhibition of B13 ELISA for peptide S12 was 94%, while those of the RI analogue or the other topological analogues were below 12%. While peptide S15.7 was recognized by PBMC from all subjects tested, none recognized the RI analogue of the S15.7 T cell epitope. Our results indicate that cross-reactivity with natural epitopes is not an universal property of RI analogues. This may limit the general applicability of the use of cross-reactive RI analogues as human vaccines and immunotherapeutic agents.

Interaction of metal ions with lupin seed conglutin gamma.

Various metal ions were capable of aggregating and precipitating conglutin gamma, an oligomeric glycoprotein purified from Lupinus albus seeds, at neutral pH values. The most effective metal ions, at 60-fold molar excess to the protein, were Zn2+, Hg2+ and Cu2+; a lower influence on the physical status of conglutin gamma was observed with Cr3+, Fe3+, Co2+, Ni2+, Cd2+, Sn2+, and Pb2+, while Mg2+, Ca2+ and Mn2+ had no effect at all. The insolubilisation of the protein with Zn2+, which is fully reversible, strictly depended on both metal concentration and pH. with middle points of the sharp transitions at three-fold molar excess and pH 6.5, respectively. Conglutin gamma is also fully retained on a metal affinity chromatography column at which Zn2+ and Ni2+ were complexed. A drop of pH below 6.0 and the use of chelating agents, such as EDTA and imidazole, fully desorbed the protein. A slightly lower binding to immobilised Cu2+ and Co2+ and no binding with Mg2+, Cd2+ and Mn2+ were observed. The role of the numerous histidine residues of conglutin gamma in the binding of Zn2+ is discussed.

Analytical and preparative isoelectric focusing of peptides in immobilized pH gradients.

A new method for peptide analysis and purification is described, based on isoelectric focusing in immobilized pH gradients. On the analytical scale, the peptide zones can now be revealed by any stain for primary and secondary amino groups (e.g. ninhydrin, fluorescamine, dansyl chloride) since the buffering species, unlike conventional carrier ampholytes, contain only carboxyl and tertiary amino groups. For preparative purposes, conditions have been described to remove most contaminants (e.g. unreacted monomers, non-cross-linked, short polyacrylamide chains) from the gel matrix before the electrophoretic run. However, ca. 2% of the gel dry mass is still present as extractable material. The focused peptides can be recovered in high yields (ca. 90%) with a fairly high degree of purity (75%), the contaminants being mostly components eluted from the polyacrylamide gel.

Thermal stabilities of lupin seed conglutin gamma protomers and tetramers.

Various experimental approaches have been used in this work to assess the thermal stabilities of lupin seed conglutin gamma at two pH values, 4.5 and 7.5, at which the protein exists as a protomer and a tetramer, respectively. The patterns of thermal unfolding at the two pH values differed significantly; the tetramer aggregated and became insoluble, whereas the protomer was still soluble after thermal treatment. Also, the midpoint transition temperatures were dramatically different, being 60.3 and 75.1 degrees C for the protomer and tetramer, respectively. The behavior of conglutin gamma at neutral pH was also affected by disulfide formation/interchange, in that some unfolded protein molecules became covalently stabilized. More detailed analyses by differential scanning calorimetry and indirect fluorescence measurements, using 8-anilino-1-naphthalenesulfonic acid as a probe, confirmed the remarkable differences observed in the thermal stabilities of the two protein forms and allowed models for their unfolding patterns to be drawn.

Observation of multiple volume reflection of ultrarelativistic protons by a sequence of several bent silicon crystals.

The interactions of 400 GeV protons with different sequences of bent silicon crystals have been investigated at the H8 beam line of the CERN Super Proton Synchrotron. The multiple volume reflection of the proton beam has been studied in detail on a five-crystal reflector measuring an angular beam deflection theta = 52.96 +/- 0.14 microrad. The efficiency was found larger than 80% for an angular acceptance at the reflector entrance of 70 microrad, with a maximal efficiency value of epsilon = 0.90 +/- 0.01 +/- 0.03.

Volume reflection dependence of 400 GeV/c protons on the bent crystal curvature.

The trend of volume reflection parameters (deflection angle and efficiency) in a bent (110) silicon crystal has been investigated as a function of the crystal curvature with 400 GeV/c protons on the H8 beam line at the CERN Super Proton Synchrotron. This Letter describes the analysis performed at six different curvatures showing that the optimal radius for volume reflection is approximately 10 times greater than the critical radius for channeling. A strong scattering of the beam by the planar potential is also observed for a bend radius close to the critical one.

The molecular basis for N-glycosylation in the 11S globulin (legumin) of lupin seed.

Ion exchange-HPLC under denaturing conditions was used to purify to homogeneity the major M(r) 44,000 alpha subunit of lupin seed (Lupinus albus, L.) 11S storage globulin (legumin). The carboxymethylated subunit was digested with trypsin and the peptide fragments separated by reverse phase HPLC. Only one glycosylated peptide reacting with concanavalin A was identified by dot-blotting. Its amino acid sequence allowed the location of this peptide within a highly conserved region in proximity to the N-terminus of the alpha subunits of the 11S globulins from other seeds. The unique presence of a serine residue in a sequence N-X-S of lupin 11S globulin, compared with all other 11S proteins, allows it to be the only protein of this class to bear covalently linked carbohydrate.

The saccharide chain of lupin seed conglutin gamma is not responsible for the protection of the native protein from degradation by trypsin, but facilitates the refolding of the acid-treated protein to the resistant conformation.

Native glycosylated and enzymically deglycosylated conglutin gamma (a lupin seed oligomeric protein) both showed an unusual resistance to tryptic degradation. The result of this treatment was that a single 40-residue peptide was cleaved from the N-terminus of conglutin gamma light subunit. Acid treatment of the two protein forms led to their substantial unfolding, as indicated by CD spectra. After this treatment, both polypeptides were completely degraded by trypsin after a few minutes of incubation. Conversely, trypsin pulse experiments run under renaturing conditions demonstrated a different refolding behaviour of the two proteins: the glycosylated form became resistant to trypsin after a 7-h renaturation, while the deglycosylated form required 42 h renaturation. These results were confirmed by CD spectra and reverse-phase HPLC analyses of the glycosylated and deglycosylated conglutin gamma forms. Therefore, it was concluded that the saccharide chain of conglutin gamma increased the rate of formation of a trypsin-resistant conformation upon refolding of the acid-treated protein, without playing any direct role in the protection of the native protein from proteolysis.

Effect of pH, aeration and sucrose feeding on the invertase activity of intact S. cerevisiae cells grown in sugarcane blackstrap molasses.

S. cerevisiae was grown in a blackstrap molasses containing medium in batch and fed-batch cultures. The following parameters were varied: pH (from 4.0 to 6.5), dissolved oxygen (DO) (from 0 to 5.0 mg O2 L-1) and sucrose feeding rate. When glucose concentration (S) was higher than 0.5 g L-1 a reduction in the specific invertase activity of intact cells (v) and an oscillatory behavior of v values during fermentation were observed. Both the invertase reduction and the oscillatory behavior of v values could be related to the glucose inhibitory effect on invertase biosynthesis. The best culture conditions for attaining S. cerevisiae cells suitable for invertase production were: temperature = 30 degrees C; pH = 5.0; DO = 3.3 mg O2 L-1; (S) = 0.5 g L-1 and sucrose added into the fermenter according to the equations: (V-Vo) = t2/16 or (V - Vo) = (Vf - Vo).(e0.6t-1)/10.

The legumin precursor from white lupin seed. Identity of the subunits, assembly and proteolysis.

The precursors of the legumin-like storage protein from developing white lupin seeds (35 days after flowering) are trimers composed of protomers of M(r) 72,000 or 67,000. Some subunits of these oligomers contain processed precursor polypeptides, namely alpha polypeptides of either 52,000 or 44,000 linked through disulphide bonds to a beta polypeptide of 21,000, typical of the mature legumin. The prolegumin is glycosylated. Legumin oligomers purified from the same seeds are both trimers and hexamers; some of their subunits are still made of precursor polypeptides. The hexamer contains less precursor polypeptide than the trimer. A low level or absence of precursor appears to be a condition of hexamer assembly. The heterogenous prolegumin and legumin oligomers represent intermediates in the processing of the prolegumin to mature legumin. Hydrophobic-interaction chromatography on TSK-phenyl-5PW and titration with the hydrophobic probe 8-anilino-1-naphthalenesulphonate indicate that the legumin is less hydrophobic than the prolegumin. This is attributed to structural rearrangements at processing of the propolypeptide, made evident by the behaviour in CD and by the second-derivative ultraviolet spectra of the two proteins. The total protein extract of developing cotyledons at 40 days after flowering contains endopeptidases, similar to those existing in the resting seeds, which cause a limited cascade degradation of the prolegumin and legumin.

Heat-induced synthesis and tunicamycin-sensitive secretion of the putative storage glycoprotein conglutin gamma from mature lupin seeds.

SDS/PAGE, immune blotting with specific antibodies and amino acid sequence analyses revealed that 90% of the protein released from Lupinus albus seeds incubated in water at 60 degrees C for about 3 h was conglutin gamma, a putative storage glycoprotein already present in the protein bodies of mature seeds. Incorporation of [14C]leucine into the protein demonstrated that conglutin gamma was newly synthesized during the treatment and the use of protein synthesis inhibitors ruled out the secretion of constitutive conglutin gamma. Synthesis and secretion took place only over a narrow temperature range, 57.5-62.5 degrees C, and in a short time interval, 135-180 min, of incubation of the seed. The amount of secreted conglutin gamma, i.e. 1 mg/seed, was about three times that present inside the treated or untreated seed. Secreted conglutin gamma contained covalently linked carbohydrate as well as the constitutive protein. Inhibition of the glycosylation by tunicamycin did not affect conglutin gamma synthesis, but prevented its secretion from the seed, as indicated by quantifying conglutin gamma remaining in the seed. An accumulation of the protein outside the protein bodies and at the cotyledonary cell periphery was shown in these samples by immunocytochemistry. Peptide mapping of the fragments obtained by incubation of constitutive and secreted conglutin gamma with trypsin and pepsin revealed no difference between the two proteins. Lupin seeds were still viable after the treatment. However no similarities between conglutin gamma and heat-shock proteins were observed either in the amino acid sequence or other molecular features.

Nutritional and technological characteristics of new broad bean flaked products.

The effects of the technological processes (soaking in water or alkaline solutions, drying, puree preparation) and the supplementation with maize flour on the nutritional value and on the organoleptic characteristics of broad bean (Vicia faba, L. major) flakes have been studied. Protein content was not affected by technological process. The addition of maize flour decreased the protein content of the final product depending on the amount of the maize flour added. Amino acid composition showed a decrease of tryptophan due to technological processing. Supplementation with maize flour improved the amino acid pattern and, except for tryptophan, the amount of essential amino acids in the flakes supplemented with 25% or more maize flour well compared with the provisional pattern by F.A.O. In vitro digestibility trials did not evidence significant changes due to technological processes or to integration of broad beans with maize flour. Broad bean toxic factors (vicine and convicine glycosides) were only slightly affected by the alkaline treatment of the flakes. Glycosides content decreased with the increasing supplementation with maize flour but the relationship was not linear. The organoleptic tests were positive for texture and taste, whereas the appearance of the products should be improved.

Thrombopoietin levels in patients with primary and reactive thrombocytosis.

Human c-mpl ligand or thrombopoietin (TPO) has been proved to be a critical cytokine in the physiological regulation of thrombopoiesis. Previous evidence suggested that TPO production is constitutive and TPO plasma levels are regulated by the platelet-megakaryocyte mass through c-mpl receptor-mediated uptake and metabolism. To evaluate whether this mechanism of TPO level regulation is also operative in subjects with an elevated platelet count, we evaluated serum TPO in 32 patients with thrombocytosis due to essential thrombocythaemia (ET) or polycythaemia vera (PV) and in 70 subjects with reactive thrombocytosis; 32 healthy subjects were also studied. TPO levels were significantly higher in the ET and PV groups (median 246.2 pg/ml, range 93.5-4596) and reactive thrombocytosis patients (median 287 pg/ml, range 82.7-1960.0) than in normal subjects (median 156.7 pg/ml, range 62.2-352.7). No significant difference was found between the two groups of patients, indicating that serum TPO levels cannot differentiate between primary and reactive thrombocytosis. No significant correlation was found between platelet count and TPO levels in either ET-PV or reactive thrombocytosis, whereas a trend toward a correlation between acute-phase reactants and TPO was observed in patients with reactive thrombocytosis. These results indicate that TPO clearance by platelets-megakaryocytes is not fully operative or is not the only mechanism of TPO level regulation in primitive and reactive thrombocytosis.