Ghrelin system in pancreatic AR42J cells: its ligand stimulation evokes calcium signalling through ghrelin receptors

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor. The presence of ghrelin in pancreatic islet cells has been previously reported and it is known to increase the [Ca2+]i in (-cells, affecting insulin secretion. However, evidence for the existence of the ghrelin system and its calcium signalling pathway in the exocrine pancreas remains unclear. Thus this study aims, first, to investigate the expression of ghrelin and its receptor in pancreatic AR42J cells and, secondly, to elucidate its calcium signalling pathway. Our results showed that ghrelin and ghrelin receptor were consistently expressed in AR42J cells. Moreover, fluorescence imaging showed that cholecystokinin-8, ghrelin and growth hormone-releasing hexapeptide stimulate [Ca2+]i in AR42J cells in a dose-dependent manner. Ghrelin and the hexapeptide produced a biphasic elevation in [Ca2+]i with an initial transient increase, followed by a sustained plateau. In the presence of (D-Lys3)-GHRP-6, the [Ca2+]i evoked by ghrelin was suppressed. In the absence of extracellular Ca2+, the transient phase of the ghrelin response was maintained but greatly diminished while the plateau phase was completely abolished. Pretreatment with 2-aminoethoxydiphenyl borate and xestospongin C abolished the transient phase and inhibited the sustained phase of the ghrelin response. The stimulatory effect of ghrelin was also blocked by nifedipine. These results indicate that ligand stimulation of the ghrelin receptor could lead to a biphasic [Ca2+]i mobilization in these cells. These data suggests the presence of a ghrelin system in pancreatic AR42J cells. In addition, its roles in exocrine function are implicated in the pancreas.     

Kinetic resolution of ibuprofen catalyzed by Candida rugosa lipase in ionic liquids

Candida rugosa lipase-catalyzed esterification of ibuprofen with 1-propanol was conducted in seven ionic liquids and the results were compared with those in isooctane. Although the enzyme showed comparable or higher activity in some ionic liquids compared to that in isooctane, only in the case of [BMIM]PF6 was the enantioselectivity (E = 24.1) almost twice that (E = 13.0) of isooctane. In another six ionic liquids the enzyme enantioselectivity was much poorer (E = 1.1-6.4). At the same conversion of 30%, E of [BMIM]PF6 is more than triple that of isooctane. The lipase stability in [BMIM]PF6 was improved by 25% of that in isooctane. It was concluded that [BMIM]PF6 could be applied to substitute the conventional organic solvent (isooctane) in the esterification of ibuprofen.     

The ClpP double ring tetradecameric protease exhibits plastic ring-ring interactions, and the N termini of its subunits form flexible loops that are essential for ClpXP and ClpAP complex formation

ClpP is a conserved serine-protease with two heptameric rings that enclose a large chamber containing the protease active sites. Each ClpP subunit can be divided into a handle region, which mediates ring-ring interactions, and a head domain. ClpP associates with the hexameric ATPases ClpX and ClpA, which can unfold and translocate substrate proteins through the ClpP axial pores into the protease lumen for degradation. We have determined the x-ray structure of Streptococcus pneumoniae ClpP(A153P) at 2.5 A resolution. The structure revealed two novel features of ClpP which are essential for ClpXP and ClpAP functional activities. First, the Ala --> Pro mutation disrupts the handle region, resulting in an altered ring-ring dimerization interface, which, in conjunction with biochemical data, demonstrates the unusual plasticity of this region. Second, the structure shows the existence of a flexible N-terminal loop in each ClpP subunit. The loops line the axial pores in the ClpP tetradecamer and then protrude from the protease apical surface. The sequence of the N-terminal loop is highly conserved in ClpP across all kingdoms of life. These loops are essential determinants for complex formation between ClpP and ClpX/ClpA. Mutation of several amino acid residues in this loop or the truncation of the loop impairs ClpXP and ClpAP complex formation and prevents the coupling between ClpX/ClpA and ClpP activities.     

Disruption of Sertoli-germ cell adhesion function in the seminiferous epithelium of the rat testis can be limited to adherens junctions without affecting the blood-testis barrier integrity: an in vivo study using an androgen suppression model

During spermatogenesis, both adherens junctions (AJ) (such as ectoplasmic specialization (ES), a testis-specific AJ type at the Sertoli cell-spermatid interface (apical ES) or Sertoli-Sertoli cell interface (basal ES) in the apical compartment and BTB, respectively) and tight junctions (TJ) undergo extensive restructuring to permit germ cells to move across the blood-testis barrier (BTB) as well as the seminiferous epithelium from the basal compartment to the luminal edge to permit fully developed spermatids (spermatozoa) to be sloughed at spermiation. However, the integrity of the BTB cannot be compromised throughout spermatogenesis so that postmeiotic germ cell-specific antigens can be sequestered from the systemic circulation at all times. We thus hypothesize that AJ disruption in the seminiferous epithelium unlike other epithelia, can occur without compromising the BTB-barrier, even though these junctions, namely TJ and basal ES, co-exist side-by-side in the BTB. Using an intratesticular androgen suppression-induced germ cell loss model, we have shown that the disruption of AJs indeed was limited to the Sertoli-germ cell interface without perturbing the BTB. The testis apparently is using a unique physiological mechanism to induce the production of both TJ- and AJ-integral membrane proteins and their associated adaptors to maintain BTB integrity yet permitting a transient loss of cell adhesion function by dissociating N-cadherin from beta-catenin at the apical and basal ES. The enhanced production of TJ proteins, such as occludin and ZO-1, at the BTB site can supersede the transient loss of cadherin-catenin function at the basal ES. This thus allows germ cell depletion from the epithelium without compromising BTB integrity. It is plausible that the testis is using this novel mechanism to facilitate the movement of preleptotene and leptotene spermatocytes across the BTB at late stage VIII through early stage IX of the epithelial cycle in the rat while maintaining the BTB immunological barrier function.     

Interplay between site-specific mutations and cyclic nucleotides in modulating DNA recognition by Escherichia coli cyclic AMP receptor protein

Mutagenesis of various amino acids in Escherichia coli cyclic AMP receptor protein (CRP) has been shown to modulate protein compressibility and dynamics [Gekko et al. (2004) Biochemistry 43, 3844-3852]. Cooperativity of cAMP binding to CRP and the apparent DNA binding affinity are perturbed [Lin and Lee (2002) Biochemistry 41, 11857-11867]. The aim of this study is to explore the effects of mutation on the surface chemistry of CRP and to define the consequences of these changes in affecting specific DNA sequence recognition by CRP. Furthermore, the role of the interplay between mutation and specific identity of the bound cyclic nucleotide in this DNA recognition was explored. In the current study, effects of eight site-specific mutations (K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L) on DNA recognition of four sequences (Class I (site PI of lac), Class II (site PI of gal), and synthetic sequences that are hybrids of Classes I and II sites) modulated by three different cyclic nucleotides (cAMP, cCMP, and cGMP) were investigated. All mutations altered the surface chemistry of CRP as evidenced by the change in elution properties of these proteins from different matrixes. While T127L, S62F, K52N, and H159L exhibited unexpected behavior under combinations of specific experimental conditions, such as the identity of bound cyclic nucleotide and DNA sequence, in general, results showed that the affinities of CRP for DNA were sequence-dependent, increasing in the order of lacgal26 < gal26 < lac26 < gallac26 for all the mutants in the presence of 200 microM cAMP. The apparent association constants significantly increased in the order of no cyclic nucleotide approximately cGMP < cCMP < cAMP for all the examined DNA sequences. Linear correlation between the DeltaG for CRP-DNA complex formation and the cooperativity energy for cAMP binding was observed with gallac26, gal26, and lacgal26; however, the slope of this linear correlation is DNA sequence dependent. Structural information was presented to rationalize the interplay between CRP sequence and cyclic nucleotides in defining the recognition of DNA sequences.     

Protection of Xenopus laevis embryos against alcohol-induced delayed gut maturation and growth retardation by peroxiredoxin 5 and catalase

Accumulated evidence indicates that maternal alcohol consumption causes fetal enteric damage and growth retardation. In this study, we investigated the underlying molecular mechanisms in a Xenopus model of fetal alcohol exposure. We established a condition of transient alcohol exposure that produces tadpoles with delayed gut maturation and decreased body length. We then investigated the roles of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by microinjecting plasmids expressing catalase and peroxiredoxin 5 (PRDX5) into two-cell stage embryos. Finally, the effects of these enzymes on the expression of key gut developmental genes were determined by animal cap explant assay. We showed that exposure of Xenopus embryos to 0.5% alcohol from stage 13 to stage 22 produced tadpoles with delayed gut maturation, reduced growth, and down-regulation in several gut developmental genes, with VegT, Pax6 and Sox17 most vulnerable. We further demonstrated that microinjection of catalase attenuated alcohol-induced ROS production and restored the expression of VegT and Pax6, but protected the embryos from delayed gut development and retarded growth only partially. By contrast, microinjection of PRDX5 reduced both ROS and RNS production, and prevented the gut and growth defects, and restored VegT, Pax6 and Sox17 gene expression. A positive correlation was found between delayed gut maturation and reduced body length. These results indicate the crucial roles of both the ROS-Pax6 and RNS-Sox17 signaling axes in alcohol-induced fetal gut defects and growth retardation. In addition, they suggest strongly a cause-and-effect relationship between alcohol-induced delayed gut maturation and growth retardation.