Differential expression of phospholipases D1 and D2 in mouse tissues

The differential expression of phospholipase D (PLD) isozymes, which include PLD1 and PLD2, was examined in various murine tissues, including the cerebrum, cerebellum, heart, lung, liver, spleen, stomach, pancreas, ileum, colon, adrenal gland, kidneys, testes, ovaries, and uterus. In Western blot analysis, only PLD1 was detected in the heart and ovary, while only PLD2 was detected in the pancreas and ileum. Both PLD1 and PLD2 were strongly expressed in the cerebrum, cerebellum, and lung, and both were also expressed in the liver, spleen, stomach, colon, kidney, testes, and uterus. Immunohistochemistry showed intense PLD immunostaining in the cerebrum, cerebellum, lungs, intestines, and testis, and weak PLD immunostaining in the liver, kidneys, spleen, and heart. These findings suggest that PLD1 and PLD2 are differentially expressed in the various organs of mice, and that each PLD isozyme plays a distinct role in each organ.     

Synthesis of closo-Dodecaboryl Lipids and their Liposomal Formation for Boron Neutron Capture Therapy

High accumulation and selective delivery of boron into tumor tissues are the most important requirements to achieve efficient neutron capture therapy of cancers. We focused on liposomal boron delivery system to achieve a large amount of boron delivery to tumor. We succeeded in the synthesis of the double-tailed boron cluster lipids 4a–c and 5a–c, which has a B₁₂H₁₁S-moiety as a hydrophilic function, by S-alkylation of B₁₂H₁₁SH with bromoacetyl and chloroacetocarbamate derivatives of diacylglycerols. Size distribution of liposomes prepared from the boron cluster lipid 4b, dimyristoylphosphatidylcholine, polyethyleneglycol-conjugated distearoylphosphatidylethanolamine, and cholesterol was determined as 100 nm in diameter by an electrophoretic light scattering spectrophotometer. Calcein-encapsulation experiments revealed that these boronated liposomes are stable at 37 °C in fetal bovine serum solution for 24 h.     

Matrix metalloproteinase secretion by gastric epithelial cells is regulated by E prostaglandins and MAPKs

Because matrix metalloproteinases (MMPs) play roles in inflammatory tissue injury, we asked whether MMP secretion by gastric epithelial cells may contribute to gastric injury in response to signals involved in Helicobacter pylori-induced inflammation and/or cyclooxygenase inhibition. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and epidermal growth factor (EGF) stimulated gastric cell MMP-1 secretion, indicating that MMP-1 secretion occurs in inflammatory as well as non-inflammatory situations. MMP-1 secretion required activation of the MAPK Erk and subsequent protein synthesis but was down-regulated by the alternate MAPK, p38. In contrast, secretion of MMP-13 was stimulated by TNF-alpha/IL-1beta but not EGF and was Erk-independent and mediated by p38. MMP-13 secretion was more rapid (peak, 6 h) than MMP-1 (peak > or =30 h) and only partly depended on protein synthesis, suggesting initial release of a pre-existing MMP-13 pool. Therefore, MMP-1 and MMP-13 secretion are differentially regulated by MAPKs. MMP-1 secretion was regulated by E prostaglandins (PGEs) in an Erk-dependent manner. PGEs enhanced Erk activation and MMP-1 secretion in response to EGF but inhibited Erk and MMP-1 when TNF-alpha and IL-1beta were the stimuli, indicating that the effects of PGEs on gastric cell responses are context-dependent. These data show that secretion of MMPs is differentially regulated by MAPKs and suggest mechanisms through which H. pylori infection and/or cyclooxygenase inhibition may induce epithelial cell signaling to contribute to gastric ulcerogenesis.     

Synthesis and antifungal activity of noble 5-arylamino- and 6-arylthio-4,7-dioxobenzoselenazoles

5-Arylamino- and 6-arylthio-4,7-dioxobenzoselenazoles 4 and 5 were synthesized and tested for in vitro antifungal activity against Candida and Aspergillus species. 5-Arylamino-4,7-dioxobenzoselenazoles 4 showed, in general, more potent antifungal activity than 6-arylthio-4,7-dioxobenzoselenazoles 5. The results suggest that 5-arylamino-4,7-dioxobenzoselenazoles 4 would be potent antifungal agents.     

Carbohydrate analysis by a phenol-sulfuric acid method in microplate format

Among many colorimetric methods for carbohydrate analysis, the phenol-sulfuric acid method is the easiest and most reliable method. It has been used for measuring neutral sugars in oligosaccharides, proteoglycans, glycoproteins, and glycolipids. This method is used widely because of its sensitivity and simplicity. In its original form, it required 50-450 nmol of monosaccharides or equivalent for analysis and thus is inadequate for precious samples. A scaled-down version requiring only 10-80 nmol of sugars was reported previously. We have now modified and optimized this method to use 96-well microplates for high throughput, to gain greater sensitivity, and to economize the reagents. This modified and optimized method allows longer linear range (1-150 nmol for Man) and excellent sensitivity. Moreover, our method is more convenient, requiring neither shaking nor covering, and takes less than 15 min to complete. The speed and simplicity of this method would make it most suitable for analyses of large numbers of samples such as chromatographic fractions.     

Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis

Summary A major challenge in the widespread application of human embryonic stem (hES) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of <5% as reported by previous studies. This study characterized cell death within frozen–thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (≈98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37 °C incubator, there was observed to be a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed-down by keeping the freshly-thawed hES colonies at 4 °C, with >90% of cells remaining viable after 90 min of incubation at 4 °C. This effect was reversible upon re-exposing the cells to physiological temperature. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 °C. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay confirmed apoptosis-induced nuclear DNA fragmentation in frozen–thawed hES cells after incubation at 37 °C for 90 min. Expression of active caspase-3 enzyme, which is another prominent marker of apoptosis, was confirmed by immunocytochemical staining, while transmission electron microscopy showed typical ultrastructural features of apoptosis such as chromatin condensation and margination to the nuclear membrane. Hence, our results demonstrated that apoptosis instead of cellular necrosis, is the major mechanism of the loss of viability of cryopreserved hES cells during freeze–thawing with conventional slow-cooling protocols.     

Functional Contribution of Ca2+ and Mg2+ to the Intermolecular Interaction of Visinin-like Proteins

The interaction of human visinin-like protein 1 (VILIP1) and visinin-like protein 3 (VILIP3) with divalent cations (Mg²⁺, Ca²⁺, Sr²⁺ and Ba²⁺) was explored using circular dichroism and fluorescence measurement. These results showed that the four cations each induced a different subtle change in the conformation of VILIPs. Moreover, VILIP1 and VILIP3 bound with Ca²⁺ or Mg²⁺ in a cooperative manner. Studies on the truncated mutants showed that the intact EF-3 and EF-4 were essential for the binding of VILIP1 with Ca²⁺ and Mg²⁺. Pull-down assay revealed that Ca²⁺ and Mg²⁺ enhanced the intermolecular interaction of VILIPs, and led to the formation of homo- and hetero-oligomer of VILIPs. Together with previous findings that Ca²⁺-dependent localization of VILIPs may be involved in the regulation of distinct cascades and deprivation of Ca²⁺-binding capacity of VILIPs did not completely eliminate their activity, it is likely to reflect that Mg²⁺-bound VILIPs may play a role in regulating the biological function of VILIPs in response to a concentration fluctuation of Ca²⁺ in cells.     

Effects of i and i+3 residue identity on cis-trans isomerism of the aromatic(i+1)-prolyl(i+2) amide bond: implications for type VI beta-turn formation

Cis-trans isomerization of amide bonds plays critical roles in protein molecular recognition, protein folding, protein misfolding, and disease. Aromatic-proline sequences are particularly prone to exhibit cis amide bonds. The roles of residues adjacent to a tyrosine-proline residue pair on cis-trans isomerism were examined. A short series of peptides XYPZ was synthesized and cis-trans isomerism was analyzed. Based on these initial studies, a series of peptides XYPN, X = all 20 canonical amino acids, was synthesized and analyzed by NMR for i residue effects on cis-trans isomerization. The following effects were observed: (a) aromatic residues immediately preceding Tyr-Pro disfavor cis amide bonds, with K(trans/cis)= 5.7-8.0, W > Y > F; (b) proline residues preceding Tyr-Pro lead to multiple species, exhibiting cis-trans isomerization of either or both X-Pro amide bonds; and (c) other residues exhibit similar values of K(trans/cis) (= 2.9-4.2), with Thr and protonated His exhibiting the highest fraction cis. beta-Branched and short polar residues were somewhat more favorable in stabilizing the cis conformation. Phosphorylation of serine at the i position modestly increases the stability of the cis conformer. In addition, the effect of the i+3 residue was examined in a limited series of peptides TYPZ. NMR data indicated that aromatic residues, Pro, Asn, Ala, and Val at the i+3 residue all favor cis amide bonds, with aromatic residues and Asn favoring more compact phi at Tyr(cis) and Ala and Pro favoring more extended phi at Tyr(cis). D-Alanine at the i+3 position particularly disfavors cis amide bonds.     

Proteomic analysis of differential protein expression in atherosclerosis

Although recent studies have shown that several pro-inflammatory proteins can be used as biomarkers for atherosclerosis, the mechanism of atherogenesis is unclear and little information is available regarding proteins involved in development of the disease. Atherosclerotic tissue samples were collected from patients in order to identify the proteins involved in atherogenesis. The protein expression profile of atherosclerosis patients was analysed using two-dimensional electrophoresis-based proteomics. Thirty-nine proteins were detected that were differentially expressed in the atherosclerotic aorta compared with the normal aorta. Twenty-seven of these proteins were identified in the MS-FIT database. They are involved in a number of biological processes, including calcium-mediated processes, migration of vascular smooth muscle cells, matrix metalloproteinase activation and regulation of pro-inflammatory cytokines. Confirmation of differential protein expression was performed by Western blot analysis. Potential applications of the results include the identification and characterization of signalling pathways involved in atherogenesis, and further exploration of the role of selected identified proteins in atherosclerosis.