Comparison of barley malt α-amylase isozymes 1 and 2: construction of cDNA hybrids by in vivo recombination and their expression in yeast

Germinating barley produces two α-amylase isozymes, AMY1 and AMY2, having 80% amino acid (aa) sequence identity and differing with respect to a number of functional properties. Recombinant AMY1 (re-AMYI) and AMY2 (re-AMY2) are produced in yeast, but whereas all re-AMYI is secreted, re-AMY2 accumulates within the cell and only traces are secreted. Expression of AMY1::AMY2 hybrid cDNAs may provide a means of understanding the difference in secretion efficiency between the two isozymes. Here, the efficient homologous recombination system of the yeast, Saccharomyces cerevisiae, was used to generate hybrids of barley AMY with the N-terminal portion derived from AMY1, including the signal peptide (SP), and the C-terminal portion from AMY2. Hybrid cDNAs were thus generated that encode either the SP alone, or the SP followed by the N-terminal 21, 26, 53, 67 or 90 aa from AMY1 and the complementary C-terminal sequences from AMY2. Larger amounts of re-AMY are secreted by hybrids containing, in addition to the SP, 53 or more aa of AMY1. In contrast, only traces of re-AMY are secreted for hybrids having 26 or fewer aa of AMY1. In this case, re-AMY hybrid accumulates intracellularly. Transformants secreting hybrid enzymes also accumulated some re-AMY within the cell. The AMY1 SP, therefore, does not ensure re-AMY2 secretion and a certain portion of the N-terminal sequence of AMY1 is required for secretion of a re-AMYI::AMY2 hybrid.     

Ribonuclease insolubilization using diazotized silk

Ribonuclease (pancreatic) (EC 3.1.27.5) and aspartate aminotransferase (l-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) have been immobilized on woven silk using the diazo method; the optimum conditions for coupling the enzyme to woven silk were established. The stability of each silk-enzyme system was examined. The kinetic constants and the effect of pH on the reaction rate of free and immobilized enzymes were compared.     

Sodium-coupled amino acid and sugar transport byNecturus small intestine

Summary Necturus small intestine actively absorbs sugars and amino acids by Na-coupled mechanisms that result in increases in the transepithelial electrical potential difference ( ^ms ) and the short-circuit current (I _sc) which can be attributed entirely to an increase in the rate of active Na absorption. Studies employing conventional microelectrodes indicate that the addition of alanine or galactose to the mucosal solution is followed by a biphasic response. Initially, there is a rapid depolarization of the electrical potential difference across the apical membrane ( ^ms ) which reverses polarity (i.e. cell interior becomes positive with respect to the mucosal solution) and a marked decrease in the ratio of the effective resistance of the mucosal membrane to that of the serosal membrane (R ^m /R ^s ); these events do not appear to be dependent on the availability of metabolic energy. These initial, rapid events are followed by a slow increase in (R ^m /R ^s ) toward control values which is paralleled by a repolarization of ^ms and increases in ^ms andI _sc; this slow series of events is dependent upon the availability of metabolic energy.The results of these studies indicate that: (i) the Na-coupled mechanisms that mediate the entry of sugars and amino acids across the apical membrane are rheogenic (conductive) and result in a decrease inR ^m and a depolarization of ^ms ; and (ii) the subsequent increase in (R ^m /R ^s ) and repolarization of ^ms are the results of a decrease inR ^s which is associated with an increase in the activity of the Na pump at the basolateral membrane.The physiologic implications of these findings are discussed and an equivalent electrical circuit model for rheogenic Na-coupled solute transport processes is analyzed.     

The interplay between viscoelastic and thermodynamic properties determines the birefringence of F-actin gels

F-actin gels of increasing concentrations (25-300 microM) display in vitro a progressive onset of birefringence due to orientational ordering of actin filaments. At F-actin concentrations <100 microM, this birefringence can be erased and restored at will by sonication and gentle flow, respectively. Hence, the orientational ordering does not result from a thermodynamic transition to a nematic phase but instead is due to mechanical stresses stored in the gels. In contrast, at F-actin concentrations > or =100 microM, gels display spontaneous birefringence recovery, at rest, which is the sign of true nematic ordering, in good agreement with statistical physics models of the isotropic/nematic transition. Well-aligned samples of F-actin gels could be produced and their small-angle x-ray scattering patterns are quite anisotropic. These patterns show no sign of filament positional short-range order and could be modeled by averaging the form factor with the Maier-Saupe nematic distribution function. The derived nematic order parameter S of the gels ranged from S = 0.7 at 300 microM to S = 0.4 at 25 microM. Both birefringence and small-angle x-ray scattering data indicate that, even in absence of cross-linking proteins, spontaneous cooperative alignment of actin filaments may arise in motile regions of living cells where F-actin concentrations can reach values of a few 100 microM.     

Towards a spin coupling model for the Mn4 cluster in Photosystem II

The X-band EPR spectra of the IR sensitive untreated PSII and of MeOH- and NH(3)-treated PSII from spinach in the S(2)-state are simulated with collinear and rhombic g- and Mn-hyperfine tensors. The obtained principal values indicate a 1Mn(III)3Mn(IV) composition for the Mn(4) cluster. The four isotropic components of the Mn-hyperfine tensors are found in good agreement with the previously published values determined from EPR and (55)Mn-ENDOR data. Assuming intrinsic isotropic components of the Mn-hyperfine interactions identical to those of the Mn-catalase, spin density values are calculated. A Y-shape 4J-coupling scheme is explored to reproduce the spin densities for the untreated PSII. All the required criteria such as a S=1/2 ground state with a low lying excited spin state (30 cm(-1)) and an easy conversion to a S=5/2 system responsible for the g=4.1 EPR signal are shown to be satisfied with four antiferromagnetic interactions lying between -290 and -130 cm(-1).     

The Small GTPase RalA controls exocytosis of large dense core secretory granules by interacting with ARF6-dependent phospholipase D1

RalA and RalB constitute a family of highly similar Ras-related GTPases widely distributed in different tissues. Recently, active forms of Ral proteins have been shown to bind to the exocyst complex, implicating them in the regulation of cellular secretion. Since RalA is present on the plasma membrane in neuroendocrine chromaffin and PC12 cells, we investigated the potential role of RalA in calcium-regulated exocytotic secretion. We show here that endogenous RalA is activated during exocytosis. Expression of the constitutively active RalA (G23V) mutant enhances secretagogue-evoked secretion from PC12 cells. Conversely, expression of the constitutively inactive GDP-bound RalA (G26A) or silencing of the RalA gene by RNA interference led to a strong impairment of the exocytotic response. RalA was found to co-localize with phospholipase D1 (PLD1) at the plasma membrane in PC12 cells. We demonstrate that cell stimulation triggers a direct interaction between RalA and ARF6-activated PLD1. Moreover, reduction of endogenous RalA expression level interfered with the activation of PLD1 observed in secretagogue-stimulated cells. Finally, using various RalA mutants selectively impaired in their ability to activate downstream effectors, we show that PLD1 activation is essential for the activation of secretion by GTP-loaded RalA. Together, these results provide evidence that RalA is a positive regulator of calcium-evoked exocytosis of large dense core secretory granules and suggest that stimulation of PLD1 and consequent changes in plasma membrane phospholipid composition is the major function RalA undertakes in calcium-regulated exocytosis.     

Role of phosphoinositide signaling in the control of insulin exocytosis

Phosphoinositides (PI) are important signaling molecules involved in the regulation of vesicular trafficking. We found that phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2)] increase the secretory response triggered by 10 mum Ca(2+) in streptolysin-O-permeabilized insulin-secreting INS-1E cells. In addition, nutrient-induced exocytosis was diminished in intact cells expressing constructs that sequester PI(4,5)P(2) and in cells transfected with constructs that reduce by RNA interference the level of two enzymes involved in PI(4,5)P(2) production, type III PI4-kinase beta and type I phosphatidylinositol 4-bisphosphate 5-kinase-gamma. To clarify the mechanism of action of PI, we investigated the involvement in the regulation of insulin exocytosis of three potential PI targets, phospholipase D1, the Ca(2+)-dependent activator protein for secretion 1, and Munc18-interacting protein 1. Transfection of insulin-secreting cells with plasmids that direct the synthesis of small interfering RNAs capable of reducing the endogenous levels of these proteins inhibited hormone release elicited by glucose- and cAMP-elevating agents without affecting basal release. Our data indicate that the production of PI(4,5)P(2) is necessary for proper control of beta-cell secretion and suggest that at least part of the effect of PI on insulin exocytosis could be exerted through the activation of phospholipase D1, Ca(2+)-dependent activator protein for secretion 1, and Munc18-interacting protein 1.     

Design of new anti-tumor agents interrupting deregulated signaling pathways induced by tyrosine kinase proteins. Inhibition of protein-protein interaction involving Grb2

Cellular signaling pathways induced by growth-factor receptors are frequently deregulated in cancer. Anti-tumor agents that inhibit their enzymatic tyrosine kinase activity have been designed and are now used in human chemotherapy. We propose here an alternative way to interrupt over-expressed signaling by inhibiting protein-protein interactions that involve either the over-expressed proteins or proteins located downstream. The adaptor protein Grb2 over-expressed in connection with HER2/ErbB2/neu in Ras signaling pathway was chosen as a target. Peptides with very high affinity for Grb2 were rationally designed from structural data. Their capacity to interrupt the signaling pathway, their anti-proliferative activity as well as their potential anti-tumor properties are described.     

TccP is an enterohaemorrhagic Escherichia coli O157:H7 type III effector protein that couples Tir to the actin-cytoskeleton

Subversion of host cell actin microfilaments is the hallmark of enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli infections. Both pathogens translocate the trans-membrane receptor protein-translocated intimin receptor (Tir), which links the extracellular bacterium to the cell cytoskeleton. While both converge on neural Wiskott-Aldrich syndrome protein (N-WASP), Tir-mediated actin accretion by EPEC and EHEC differ in that Tir(EPEC) requires both tyrosine phosphorylation and the host adaptor protein Nck, whereas Tir(EHEC) is not phosphorylated and utilizes an unidentified linker. Here we report the identification of Tir-cytoskeleton coupling protein (TccP), a novel EHEC effector that displays an Nck-like coupling activity following translocation into host cells. A tccP mutant did not affect Tir translocation and focusing but failed to recruit alpha-actinin, Arp3, N-WASP and actin to the site of bacterial adhesion. When expressed in EPEC, bacterial-derived TccP restored actin polymerization activity following infection of an Nck-deficient cell line. TccP has a similar biological activity on infected human intestinal explants ex vivo. Purified TccP activates N-WASP stimulating, in the presence of Arp2/3, actin polymerization in vitro. These results show that EHEC translocates both its own receptor (Tir) and an Nck-like protein (TccP) to facilitate actin polymerization.