Angiotensin-converting enzyme is a GPI-anchored protein releasing factor crucial for fertilization

The angiotensin-converting enzyme (ACE) is a key regulator of blood pressure. It is known to cleave small peptides, such as angiotensin I and bradykinin and changes their biological activities, leading to upregulation of blood pressure. Here we describe a new activity for ACE: a glycosylphosphatidylinositol (GPI)-anchored protein releasing activity (GPIase activity). Unlike its peptidase activity, GPIase activity is weakly inhibited by the tightly binding ACE inhibitor and not inactivated by substitutions of core amino acid residues for the peptidase activity, suggesting that the active site elements for GPIase differ from those for peptidase activity. ACE shed various GPI-anchored proteins from the cell surface, and the process was accelerated by the lipid raft disruptor filipin. The released products carried portions of the GPI anchor, indicating cleavage within the GPI moiety. Further analysis by high-performance liquid chromatography-mass spectrometry predicted the cleavage site at the mannose-mannose linkage. GPI-anchored proteins such as TESP5 and PH-20 were released from the sperm membrane of wild-type mice but not in Ace knockout sperm in vivo. Moreover, peptidase-inactivated E414D mutant ACE and also PI-PLC rescued the egg-binding deficiency of Ace knockout sperms, implying that ACE plays a crucial role in fertilization through this activity.     

A novel thermophilic pectate lyase containing two catalytic modules of Clostridium stercorarium

The Clostridium stercorarium F-9 pel9A gene encodes a pectate lyase Pel9A consisting of 1,240 amino acids with a molecular weight of 135,171. The mature form of Pel9A is a modular enzyme composed of two family-9 catalytic modules of polysaccharide lyases, CM9-1 and CM9-2, in order from the N terminus. Pel9A showed an overall sequence similarity to the hypothetical pectate lyase PelX of Bacillus halodurans (sequence identity 53%), and CM9-2 showed moderate sequence similarities to some pectate lyases of family 9. Sequence identity between CM9-1 and CM9-2 was 21.3%. The full-length Pel9A lacking the N-terminal signal peptide was expressed, purified, and characterized. The enzyme required Ca(2+) ion for its enzyme activity and showed high activity toward polygalacturonic acid but lower activity toward pectin, indicating that Pel9A is a pectate lyase. Immunological analysis using an antiserum raised against the purified enzyme indicated that Pel9A is constitutively synthesized by C. stercorarium F-9.     

Enhanced expression of PDX-1 and Ngn3 by exendin-4 during beta cell regeneration in STZ-treated mice

Progenitor cells exist in the adult pancreas and transform to endocrine cells in pathological conditions. To address the mechanism of beta cell regeneration, mice were treated with streptozotocin (STZ group) or streptozotocin and exendin-4 (STZ + Ex-4 group), and the expression of PDX-1, Ngn3, insulin, IRS-2, and Foxo1 was investigated. PDX-1 mRNA was upregulated biphasically and induction of Ngn3 mRNA occurred shortly after the first increase of PDX-1 expression, a pattern similar to that observed during embryogenesis. PDX-1-positive cells appeared only in islet-like cell clusters (ICCs) in STZ group, but they appeared both in ducts and ICCs in STZ + Ex-4 group. Ngn3-positive cells emerged in ICCs but not in ducts. Therefore, regeneration seemed to occur mainly from intra-islet stem/progenitor cells. Exendin-4 upregulated PDX-1 expression which paralleled increased IRS-2 expression and translocation of Foxo1 from nucleus to cytoplasm. Further analysis of beta cell regeneration should help in the design of novel therapy for diabetes.     

Decrease in glucose-stimulated insulin secretion following exposure to magnetic fields

We evaluated the effects of extremely low frequency magnetic field (ELFMF) on glucose-stimulated insulin secretion from HIT-T15 cells and investigated the mechanisms of these effects. We demonstrated that exposure to ELFMF at 5mT decreased glucose-stimulated insulin secretion by preventing the increases in cellular adenosine 5'-triphosphate/adenosine 5'-diphosphate, membrane depolarization, and cytosolic free calcium ion concentration. The glucose-induced upregulation of insulin mRNA expression was also attenuated by exposure to ELFMF, although cell viability was not affected. These findings demonstrate the potential of exposure to ELFMF for clinical use as a novel inhibitory method of insulin secretion.     

Quercetin appears in the lymph of unanesthetized rats as its phase II metabolites after administered into the stomach

Quercetin is a major flavonoid in plant foods and potentially has beneficial effects on disease prevention. The present work demonstrated that quercetin was transported into the lymph after being metabolized in the gastrointestinal mucosa of rats. Glucuronide/sulfate and methylated conjugates of quercetin appeared in the lymph, but not quercetin aglycone. The highest lymphatic concentration was found at as rapid as 30 min after administration, suggesting gastric absorption, whereas the mucosal glucuronidation activity was significantly higher in the duodenum and jejunum than in the stomach. This is the first report to show the lymphatic flavonoid transport pathway from the gastrointestinal tract.     

Role of charged and hydrophobic residues in the oligomerization of the PYRIN domain of ASC

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein composed of two homophilic protein-protein interaction domains, a PYRIN domain (PYD) and a caspase recruitment domain. PYD-dependent oligomerization of ASC is thought to play a crucial role in formation of a molecular platform, the inflammasome, which activates caspase-1. When expressed in cells, the PYD of ASC was shown to form cytoplasmic filaments through self-association. Over 70 single point mutants were analyzed for filament formation in cells expressing the mutant proteins. The set of mutations comprised every single amino acid residue with a charged side chain (Arg, Lys, Asp, and Glu) and a large hydrophobic side chain (Ile, Leu, Met, Phe, Pro, and Val). Filament formation of the ASC PYD was prevented by mutation of Lys21, Leu25, Lys26, Pro40, Arg41, Asp48, and Asp51 of helices 2, 3, and 4. These data identify a coherent interaction surface, establishing a molecular model of PYD-PYD complexes with an important role for charge-charge interactions.     

Triggering of TLR3 by polyI:C in human corneal epithelial cells to induce inflammatory cytokines

Epithelial cells of the ocular surface are key in the first-line defense as a part of the mucosal immune system against pathogens. We investigated whether polyI:C induces the production by human corneal epithelial cells (HCEC) of pro-inflammatory cytokines and IFN-beta, and whether Toll-like receptor (TLR)-3 expression is amplified by polyI:C. TLR3 was expressed on the surface of HCEC. Stimulation with polyI:C elicited the elevated production and mRNA expression of IL-6 and IL-8 in HCEC. While polyI:C induced IFN-beta, far stronger than human fibroblasts, and TLR3 gene expression in HCEC, LPS stimulation did not. Similarly, polyI:C, but not LPS, induced the gene expression of IkappaBalpha and MAIL, members of the IkappaB family, in HCEC. The innate immune response of HCEC is distinct from that of immune-competent cells, and we suggest that this is indicative of the symbiotic relationship between corneal epithelium and microbes inhabiting the ocular surface.     

Akt is a neutral amplifier for Th cell differentiation

Both CD28 and its relative, inducible costimulator (ICOS), have a binding motif for phosphatidylinositol 3-kinase (PI3K) in their cytoplasmic tail, and the binding of PI3K leads to activation of a serine/threonine kinase, Akt. The role of Akt in cytokine production and helper T (Th) cell differentiation remains obscure. In this study, we found that enforced expression of the constitutively active form (E40K) of Akt rendered CD4(+) T cells activated. Wild-type of Akt and E40K promoted Th1 cell differentiation in C57BL/6-derived and Th1-polarized BALB/c-derived CD4(+) T cells, while both promoted Th2 cell differentiation in BALB/c-derived and Th2-polarized C57BL/6 CD4(+) T cells. E40K also facilitated Th1 differentiation in CD4(+) T cells from IL-4-deficient mice with the BALB/c background. E40K up-regulated expression of NF-AT and c-Myb, which may be related to the augmentation of cytokine production by E40K. These findings indicate that the mechanism by which Akt augments cytokine production via CD28 and ICOS is Th cell type-specific and reflects the intracellular status affected by the cytokine milieu. We conclude that Akt is a neutral amplifier of T cell activation and Th differentiation.     

Carotenoid-induced cooperative formation of bacterial photosynthetic LH1 complex

A simple reconstitution technique has been developed and then applied to prepare a series of light-harvesting antenna 1 (LH1) complexes with a programmed carotenoid composition, not available from native photosynthetic membranes. The complexes were reconstituted with different C(40) carotenoids, having two structural parameters variable: the functional side groups and the number of conjugated C-C double bonds, systematically increasing from 9 to 13. The complexes, differing only in the type of carotenoid, bound to an otherwise identical bacteriochlorophyll-polypeptide matrix, can serve as a unique model system in which the relationship between the carotenoid character and the functioning of pigment-protein complexes can be investigated. The reconstituted LH1 complexes resemble the native antenna, isolated from wild-type Rhodospirillum rubrum, but their coloration is entirely determined by carotenoid. Along with the increase in its conjugation size, the carotenoid absorption transitions gradually shift to the red. Thus, the extension of the conjugation size of the antenna carotenoids provides a mechanism for the spectral tuning of light harvesting in the visible part of the spectrum. The carotenoids in the reconstitution system promote the LH1 formation and seem to bind and transfer the excitation energy specifically only to a species with characteristically red-shifted absorption and emission maxima, apparently, due to a cooperative effect. Monitoring the LH1 formation by steady-state absorption and fluorescence spectroscopies reveals that in the presence of carotenoids it proceeds without spectrally resolved intermediates, leading directly to B880. The effect of the carotenoid is enhanced when the pigment contains the hydroxy or methoxy side groups, implying that, in parallel to hydrophobic interactions and pi-pi stacking, other interactions are also involved in the formation and stabilization of LH1.