Identification and characterization of integrin-binding sialoprotein (IBSP) genes in reptile and amphibian

Integrin-binding sialoprotein (IBSP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family; and the whole SIBLING family is further included in a larger secretory calcium-binding phosphoprotein (SCPP) family. SIBLING proteins are known to construct a part of the non-collagenous extracellular matrices of calcified tissues, and considered to have arisen by duplication and subsequent divergent evolution of a single ancient gene. To understand the alterations of SIBLING molecules associated with the evolution of calcified tissues in vertebrates, we initiated a search for lower vertebrate orthologs of SIBLING genes. In the present study, an IBSP ortholog from a reptile (caiman) and two distinct orthologs from an amphibian (African clawed toad) were identified and characterized. As expected, the toad IBSP genes were transcribed only in calcified tissue (jaw and tibia), as also seen in mammals. The caiman, toad, avian, and mammalian IBSPs share several unique features specific for IBSP and apparently have similar properties. Furthermore, analysis of the sequences suggested that the IBSP molecule might have gradually intensified its functions related to calcification during its evolutionary process through tetrapods.     

Protective roles of redox-active protein thioredoxin-1 for severe acute pancreatitis

Severe acute pancreatitis is a disease with high mortality, and infiltration of inflammatory cells and reactive oxygen species have a crucial role in the pathophysiology of this disease. Thioredoxin-1 (TRX-1) is an endogenous redox-active multifunctional protein with antioxidant and anti-inflammatory effects. TRX-1 is induced in various inflammatory conditions and shows cytoprotective effects. The aim of the present study was to clarify the protective roles of TRX-1 in the host defense mechanism against severe acute pancreatitis. Experimental acute pancreatitis was induced by intraperitoneal administration of cerulein, a CCK analog, and aggravated by lipopolysaccharide injection in transgenic mice overexpressing human TRX-1 (hTRX-1) and control C57BL/6 mice. Transgenic overexpression of hTRX-1 strikingly attenuated the severity of experimental acute pancreatitis. TRX-1 overexpression suppressed neutrophil infiltration as determined by myeloperoxidase activity, oxidative stress as determined by malondialdehyde concentration, and cytoplasmic degradation of inhibitor of kappaB-alpha, thereby suppressing proinflammatory cytokines, tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6; a neutrophil chemoattractant, keratinocyte-derived chemokine; and inducible nitric oxide synthase in the pancreas. Administration of recombinant hTRX-1 also suppressed neutrophil infiltration, reduced the inflammation of the pancreas and the lung, and improved the mortality rate. The present study suggests that TRX-1 has potent antioxidant and anti-inflammatory actions in experimental acute pancreatitis and might be a new therapeutic strategy to improve the prognosis of severe acute pancreatitis.     

Disturbed activation of endoplasmic reticulum stress transducers by familial Alzheimer's disease-linked presenilin-1 mutations

Recent studies have shown independently that presenilin-1 (PS1) null mutants and familial Alzheimer's disease (FAD)-linked mutants should both down-regulate signaling of the unfolded protein response (UPR). However, it is difficult to accept that both mutants possess the same effects on the UPR. Furthermore, contrary to these observations, neither loss of PS1 and PS2 function nor expression of FAD-linked PS1 mutants were reported to have a discernable impact on the UPR. Therefore, re-examination and detailed analyses are needed to clarify the relationship between PS1 function and UPR signaling. Here, we report that PS1/PS2 null and dominant negative PS1 mutants, which are mutated at aspartate residue 257 or 385, did not affect signaling of the UPR. In contrast, FAD-linked PS1 mutants were confirmed to disturb UPR signaling by inhibiting activation of both Ire1alpha and ATF6, both of which are endoplasmic reticulum (ER) stress transducers in the UPR. Furthermore, PS1 mutants also disturbed activation of PERK (PKR-like ER kinase), which plays a crucial role in inhibiting translation during ER stress. Taken together, these observations suggested that PS1 mutations could affect signaling pathways controlled by each of the respective ER-stress transducers, possibly through a gain-of-function.     

Increased insulin sensitivity in Gsalpha knockout mice

The stimulatory guanine nucleotide-binding protein (G(s)) is required for hormone-stimulated cAMP generation. Gnas, the gene encoding the G(s) alpha-subunit, is imprinted, and targeted disruption of this gene in mice leads to distinct phenotypes in heterozygotes depending on whether the maternal (m-/+) or paternal (+/p-) allele is mutated. Notably, m-/+ mice become obese, whereas +/p- mice are thinner than normal. In this study we show that despite these opposite changes in energy metabolism, both m-/+ and +/p- mice have greater sensitivity to insulin, with low to normal fasting glucose levels, low fasting insulin levels, improved glucose tolerance, and exaggerated hypoglycemic response to administered insulin. The combination of increased insulin sensitivity with obesity in m-/+ mice is unusual, because obesity is typically associated with insulin resistance. In skeletal muscles isolated from both m-/+ and +/p- mice, the basal rate of 2-deoxyglucose uptake was normal, whereas the rate of 2-deoxyglucose uptake in response to maximal insulin stimulation was significantly increased. The similar changes in muscle sensitivity to insulin in m-/+ and +/p- mice may reflect the fact that muscle G(s)alpha expression is reduced by approximately 50% in both groups of mice. GLUT4 expression is unaffected in muscles from +/p- mice. Increased responsiveness to insulin is therefore the result of altered insulin signaling and/or GLUT4 translocation. This is the first direct demonstration in a genetically altered in vivo model that G(s)-coupled pathways negatively regulate insulin signaling.     

Serum amyloid P component is the Shiga toxin 2-neutralizing factor in human blood

It has been suggested that some factor present in human plasma binds to Shiga toxin 2 (Stx2) and neutralizes it in vitro (Bitzan, M., Klemt, M., Steffens, R., and Muller-Wiefel, D. E. (1993) Infection 21, 140-145). This factor does not exist in other species (Caprioli, A., Luzzi, I., Seganti, L., Marchetti, M., Karmali, M., Clarke, I., and Boyd, B. (1994) Recent Adv. VTEC Infect. 353-356). Because analysis of this factor is important to understanding the pathology induced by Shiga toxin-producing Escherichia coli, we purified this factor from human plasma and identified it. Purification was carried out by serially subjecting human plasma to Con A-Sepharose, DEAE-Sepharose, hydroxyapatite, and gel-filtration high performance liquid chromatography (HPLC), using Stx2-neutralizing activity as the indicator. The gel-filtration HPLC fraction yielded a single band on SDS-polyacrylamide gel electrophoresis. Twenty N-terminal amino acid residues of this fraction were analyzed and found to correspond perfectly to human serum amyloid P component (HuSAP). Because commercially available HuSAP also showed Stx2 binding and neutralizing activity, we identified this factor as HuSAP.     

The novel kappa-opioid receptor agonist TRK-820 suppresses the rewarding and locomotor-enhancing effects of morphine in mice

The effects of the novel kappa-opioid receptor agonist TRK-820 on the rewarding and locomotor-enhancing effects of morphine were investigated in mice. Morphine (1-5 mg/kg, s.c.) caused a dose-related preference for the drug-associated place. In contrast, TRK-820 (0.003-0.03 mg/kg, s.c.) did not produce a significant preference for either compartment of the test box. In combination studies, co-injection of TRK-820 (0.01 and 0.03 mg/kg, s.c.) with morphine significantly suppressed the morphine (5 mg/kg, s.c.)-induced place preference, and this effect of TRK-820 was antagonized by pretreatment with nor-BN1 (3 mg/kg, s.c.), a selective kappa-opioid receptor antagonist. TRK-820 also suppressed morphine-induced hyperlocomotion, and this suppression was also blocked by nor-BNI. These results suggest that TRK-820 suppresses the rewarding and locomotor-enhancing effects of morphine through the activation of kappa-opioid receptors. Thus, we propose that TRK-820 may be useful for controlling pain while reducing undesirable side-effects.     

Anthocyanins from red flower tea (Benibana-cha), Camellia sinensis

Three anthocyanins were isolated from the leaves of red flower tea (Benibana-cha), Camellia sinensis, and their structures were determined by means of chemical and spectroscopic analyses. Two are the anthocyanins, delphinidin and cyanidin 3-O-beta-D-galactosides, respectively. Whereas the third, delphinidin 3-0-beta-D-(6-(E)-p-coumaryl)galactopyranoside. The anthocyanins were also contained in the flowers of Benibana-cha in different compositions.     

Characterization of exo-(1,4)-alpha glucan lyase from red alga Gracilaria chorda. Activation, inactivation and the kinetic properties of the enzyme

Exo-(1,4)-alpha glucan lyase (GLase) was purified from a red alga Gracilaria chorda. The enzyme was activated 1.3-fold in the presence of Ca(2+) and Cl(-) ions. The ions also stabilized the enzyme increasing the temperature of its maximum activity from 45 degrees C to 50 degrees C. GLase was inactivated by chemical modification with carbodiimide and a carboxyl group of the enzyme was shown essential to the lyase activity. A tryptophanyl residue(s) was also shown to be important for the activity and was probably involved in substrate binding. K(m) values of the enzyme were 2.3 mM for maltose, 0.4 mM for maltotriose and 0.1 mM for maltooligosaccharides of degree of polymerization (dp) 4-7, and the k(0) values for the oligosaccharides were similar (42-53 s(-1)). The analysis of these kinetic parameters showed that the enzyme has four subsites to accommodate oligosaccharides. The subsite map of GLase was unique, since subsite 1 and subsite 2 have large positive and small negative affinities, respectively. The subsite map of this type has not been found in other enzymes with exo-action on alpha-1,4-glucan. The K(m) and k(0) values for the polysaccharides were lower (0.03 mM) and higher (60-100 s(-1)), respectively, suggesting the presence of another affinity site specific to the polysaccharides.