Postnatally disturbed pancreatic islet cell distribution in human islet amyloid polypeptide transgenic mice

OBJECTIVE: Islet amyloid polypeptide (IAPP)/amylin is produced by the pancreatic islet beta-cells, which also produce insulin. To study potential functions of IAPP, we have generated transgenic mice overexpressing human IAPP (hIAPP) in the beta-cells. These mice show a diabetic phenotype when challenged with an oral glucose load. In this study, we examined the islet cytoarchitecture in the hIAPP mice by examining islet cell distribution in the neonatal period, as well as 1, 3 and 6 months after birth. RESULTS: Neonatal transgenic mice exhibited normal islet cell distribution with beta-cells constituting the central islet portion, whereas glucagon and somatostatin-producing cells constituted the peripheral zone. In contrast, in hIAPP transgenic mice at the age of 1 month, the glucagon-immunoreactive (IR) cells were dispersed throughout the islets. Furthermore, at the age of 3 and 6 months, the islet organisation was similarly severely disturbed as at 1 month. Expression of both endogenous mouse IAPP and transgenic hIAPP was clearly higher in 6-month-old mice as compared to newborns, as revealed by mRNA in situ hybridisation. CONCLUSIONS: Mice transgenic for hIAPP have islets with disrupted islet cytoarchitecture in the postnatal period, particularly affecting the distribution of glucagon-IR cells. This islet cellular phenotype of hIAPP transgenic mice is similar to that of other mouse models of experimental diabetes and might contribute to the impaired glucose homeostasis.     

Dual benefit of reduced Cx43 on atherosclerosis in LDL receptor-deficient mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR(-/-)) mice were intercrossed with mice heterozygous for Cx43 (Cx43(+/-) mice). Male mice with normal (Cx43(+/+)LDLR(-/-)) or reduced (Cx43(+/-)LDLR(-/-)) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43(+/-)LDLR(-/-) mice compared with Cx43(+/+)LDLR(-/-) littermate controls. In addition, atheroma in Cx43(+/-)LDLR(-/-) mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye

Ultrasensitive detection of minute amounts of phosphorylated proteins and peptides is a key requirement for unraveling many of the most important signal transduction pathways in mammalian systems. Protein microarrays are potentially useful tools for sensitive screening of global protein expression and post-translational modifications, such as phosphorylation. However, the analysis of signaling pathways has been hampered by a lack of reagents capable of conveniently detecting the targets of protein kinases. Historically, phosphorylation detection methods have relied upon either radioisotopes ((gamma-(32)P)ATP(gamma-(33)P)ATP labeling) or phosphoamino acid-selective antibodies. Both of these methods suffer from relatively well-known shortcomings. In this study, a small molecule fluorophore phosphosensor technology is described, referred to as Pro-Q Diamond dye, which is capable of ultrasensitive global detection and quantitation of phosphorylated amino acid residues in peptides and proteins displayed on microarrays. The utility of the fluorescent Pro-Q Diamond phosphosensor dye technology is demonstrated using phosphoproteins and phosphopeptides as well as with protein kinase reactions performed in miniaturized microarray assay format. Instead of applying a phosphoamino acid-selective antibody labeled with a fluorescent or enzymatic tag for detection, a small, fluorescent probe is employed as a universal sensor of phosphorylation status. The detection limit for phosphoproteins on a variety of different commercially available protein array substrates was found to be 312-625 fg, depending upon the number of phosphate residues. Characterization of the enzymatic phosphorylation of immobilized peptide targets with Pro-Q Diamond dye readily permits differentiation between specific and non-specific peptide labeling at picogram to subpicogram levels of detection sensitivity.     

Establishment of a two-dimensional electrophoresis map for Neospora caninum tachyzoites by proteomics

Expressed proteins and antigens from Neospora caninum tachyzoites were studied by two-dimensional gel electrophoresis and immunoblot analysis combined with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Thirty-one spots corresponding to 20 different proteins were identified from N. caninum tachyzoites by peptide mass fingerprinting. Six proteins were identified from a N. caninum database (NTPase, 14-3-3 protein homologue, NcMIC1, NCDG1, NcGRA1 and NcGRA2), and 11 proteins were identified in closely related species using the T. gondii database (HSP70, HSP60, pyruvate kinase, tubulin alpha- and beta-chain, putative protein disulfide isomerase, enolase, actin, fructose-1,6-bisphosphatase, lactate dehydrogenase and glyceradehyde-3-phosphate dehydrogenase). One hundred and two antigen spots were observed using pH 4-7 IPG strips on immunoblot profiles. Among them, 17 spots corresponding to 11 antigenic proteins were identified from a N. caninum protein map. This study involved the construction of in-depth protein maps for N. caninum tachyzoites, which will be of value for studies of its pathogenesis, drug and vaccine development, and phylogenetic studies.     

Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis

Very little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis-associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis-related function of candidate protein interleukin-18 (IL-18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach was used to compare the protein expression profiles between PLA801C and PLA801D sublines. Eleven proteins were identified and further verified by one-dimensional Western blotting, Northern blot and/or semiquantitative reverse transciptase polymerase chain reaction analysis. Compared with those in poorly metastatic PLA801C subline, cytokeratin 18, tissue transglutaminase, Rho GDP-dissociation inhibitor 1, tropomyosin, fibroblast type, IL-18 and annexin I were significantly up-regulated, while protein disulfide isomerase, heat shock protein 60, peroxiredoxin 1, chlorine intracellular channel protein 1 (CLI1) and creatine kinase, B chain were significantly down-regulated in the highly metastatic PLA801D subline. Intriguingly, all the identified candidate proteins except for CLI1 have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was present in highly metastatic PLA801D but absent in poorly metastatic PLA801C, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense/IL-18 antisense into PLA801C/PLA801D sublines simultaneously. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro and down-regulated E-cadherin expression of PLA801C transfectants, while IL-18 antisense remarkably decreased cell invasion potency in vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play a role in metastasis by inhibiting E-cadherin expression.     

The solvent in CNBr cleavage reactions determines the fragmentation efficiency of ketosteroid isomerase fusion proteins used in the production of recombinant peptides

Abnormal fragmentation during cyanogen bromide polypeptide cleavage rarely occurs, although parallel side reactions are known to typically accompany normal cleavage. We have observed that cyanogen bromide cleavage of highly hydrophobic fusion proteins utilized for production of recombinant peptides results in almost complete abolishment of the expected reaction products when the reaction is carried out in 70% trifluoroacetic acid. On the basis of mass spectrometric analysis of the reaction products, we have identified a number of fragments whose origin can be attributed to incomplete fragmentation of the fusion protein, and to unspecific degradation affecting the carrier protein. Substituting the solvent in the reaction media with 70% formic acid or with a matrix composed of 6M guanidinium hydrochloride in 0.1M HCl, however, was found to alleviate polypeptide cleavage. We have attributed the poor yields of the CNBr cleavage carried out in 70% TFA to the increased hydrophobicity of our particular fusion proteins, and to the poor solubilizing ability of this reaction medium. We propose the utilization of chaotropic agents in the presence of diluted acids as the preferred cyanogen bromide cleavage medium of fusion proteins in order to maximize cleavage efficiency of hydrophobic sequences and to prevent deleterious degradation and structural modifications of the target peptides.     

Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1 families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c--both tagged and detagged--from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies.