Inhibition of Mouse Blastocyst Hatching by Subsite-Specific Trypsin Inhibitors,Peptidyl Argininals1

To explore the substrate or subsite specificity of a mouse hatching enzyme, effects of leupeptin [acetyl(P₄)-Leu(P₃)-Leu(P₂)-argininal(P₁)] and its analogs (peptidyl argininals) on mouse blastocyst hatching were investigated. The compounds containing benzyloxycarbonyl group (Z) in the P₄ position inhibited the hatching more strongly than those containing acetyl group or unprotected N-terminal amino acid. Among five Z-Leu-P₂-argininals, a derivative containing a P₂ Ser residue was the most potent inhibitor, and the derivatives containing Leu, Thr, Pro, and Gly in the P₂ position followed in this order. Then, we synthesized four Z-P₃-Ser-argininals and tested their effects on hatching. The result indicated that the compound with Phe residue in the P₃ position was the strongest inhibitor, and the Leu-, Pro-, and Ala-containing derivatives were ranked in this order. Thus, among Z-dipeptidyl-argininals tested, Z-Phe-Ser-argininal most potently inhibited the mouse embryonic hatching, suggesting the preference of the mouse hatching enzyme for Phe(P₃)-Ser(P₂)-Arg(P₁) sequence as a substrate.     

Characterization of the complex between mannose-binding lectin trimer and mannose-binding lectin-associated serine proteases

Mannose-binding lectin (MBL) is an oligomeric serum lectin involved in innate immunity. Human MBL is complexed with three types of serine proteases (MASP-1, MASP-2 and MASP-3) and two types of their truncated forms (sMAP and MAp44). When an MBL complex binds to carbohydrates of pathogens, the complement system is activated via the lectin pathway. Human MBL is a mixture of different sized oligomers that range mainly from trimers to hexamers. It has been suggested that different MBL oligomers may have distinct MASP compositions. In the present study, an MBL trimer (MBL-I) exclusive of other oligomers was isolated from human serum by chromatography. Immunoblot analysis of MBL-I revealed that it had been co-purified with MASP-1 and sMAP. This suggests that MASP-1 and sMAP are bound to each other in MBL-I. The MBL-I complex was found to activate C2, but to lack the ability to activate C4 due to the absence of MASP-2.     

No effects of pulsed electromagnetic fields on expression of cell adhesion molecules (integrin, CD44) and matrix metalloproteinase-2/9 in osteosarcoma cell lines

Pulsed electromagnetic fields (PEMF) could enhance the cytocidal effects of chemotherapeutic drugs on malignant tumor cell lines, but metastasis effects of PEMF on tumor cells have not been investigated. We investigated the effects of PEMF exposure on the expression levels of some metastasis-related molecules, including integrin α subunits (α1, α2, α3, α4, α5, α6, αv), integrin β subunits (β1, β2, β3, β4), CD44, and matrix metalloproteinase-2/9 (MMP-2/9) in four human osteosarcoma cell lines (HOS, MG-63, SAOS-2, NY) and two mouse osteosarcoma cell lines (DOS, LM8) by using FACScan analysis, gelatin zymography, and Western blot analysis. Our results indicate that PEMF exposure has no effect on the expression of some molecules that are associated with tumor cell invasion and metastasis, and therefore suggest that PEMF exposure may be safely applied to chemotherapy for osteosarcoma.     

Digesta Passage Time,Digestibility, and Total Gut Fill in Captive Japanese Macaques (<Emphasis Type="BoldItalic">Macaca fuscata</Emphasis>): Effects Food Type and Food Intake Level

Digestion is an important process in understanding the feeding ecology of animals. We examined digesta passage time, digestibility, and total gut fill in Japanese macaques (Macaca fuscata; n = 4) under 4 dietary conditions representing the seasonal and regional variations in the diets of wild populations to determine the effects of food type and food intake on these digestive features. Food type is associated with mean retention time (MRT), digestibility, and total gut fill. Dry matter intake (DMI) of food correlates positively with total gut fill but not with MRT or digestibility. Conversely, indigestible DMI affected MRT negatively. Thus, when Japanese macaques consume high-fiber foods, MRT becomes shorter and digestibility is lower than when eating low-fiber foods. Moreover, macaques experience increases in total gut fill when they consume high-fiber diets or a large amount of food. Japanese macaques may excrete difficult-to-digest food components quickly; they nevertheless buffer an increase in food intake by an increase in gut fill. Our study offers new insights into the relationship between feeding ecology and nutritional physiology in primates by simultaneously examining the effects of food type and intake level on MRT and digestibility.     

Molecular cloning and expression of long-wavelength-sensitive cone opsin in the brain of a tropical damselfish

Ovarian development of the sapphire devil, Chrysiptera cyanea, exhibits photoperiodism and is stimulated under long-day conditions. Previous studies suggest that red light is more effective than green and blue lights for inducing ovarian development. In addition, the extra-retinal photoreception involved in the ovarian development is suggested in this species. The present study aimed to clone the red-light-sensitive cone opsin (sdLWS) of this species, to demonstrate its expression in the brain, and to confirm the effectiveness of red light on the initiation of ovarian development. A phylogenetic analysis revealed that sdLWS belongs to the long-wavelength-sensitive opsin (LWS opsin) group, showing high identity (77–92%) with the LWS opsin of other fishes. RT-PCR showed that sdLWS is expressed in the eye, brain, and ovary. In situ hybridization indicated that sdLWS is expressed in the third ventricle periventricular area in the anterior hypothalamus. Exposing fish to long-day conditions of red light resulted in the appearance of vitellogenic oocytes in the ovary and an increase in the gonadosomatic index within 2 weeks, suggesting that fish immediately undergo oocyte development under red light conditions. These results indicate that sdLWS is a possible candidate of deep brain photoreceptor molecule involved in photoperiodic ovarian development in the sapphire devil.     

Detection of autoantibodies to citrullinated BiP in rheumatoid arthritis patients and pro-inflammatory role of citrullinated BiP in collagen-induced arthritis

Introduction

Anti-citrullinated protein/peptide antibodies (ACPAs) are highly specific to rheumatoid arthritis (RA) patients and are thought to have a close relationship with the pathogenesis of arthritis. Several proteins, including fibrinogen, vimentin, and alpha-enolase, were reported as ACPA-target antigens, and their importance in RA pathogenesis was widely proposed. We identified citrullinated immunoglobulin binding protein (citBiP) as another ACPA target in RA patients and examined its pro-inflammatory role in arthritis.

Methods

We measured the levels of anti-citBiP, anti-BiP, and anti-cyclic citrullinated peptide (CCP) antibodies in the serum of RA patients (n = 100), systemic lupus erythematosus (SLE) patients (n = 60), and healthy controls (n = 30) using ELISA and immunoblotting. Epitope mapping was performed using 27 citBiP-derived peptides. In the mouse study, after DBA/1J mice were immunized with BiP or citBiP, serum titers of ACPAs were measured by ELISA and immunohistochemistry. The development of collagen-induced arthritis (CIA) was observed in BiP- or citBiP-pre-immunized mice.

Results

The serum levels of anti-BiP and anti-citBiP antibodies were significantly increased in RA patients, although only anti-BiP antibodies were slightly increased in SLE patients. Interestingly, anti-citBiP antibody levels were higher than anti-BiP antibody levels in 72% of RA patients, whereas no significant increase in anti-citBiP antibody levels was detected in SLE patients and healthy controls. The serum levels of anti-CCP antibodies were correlated with those of anti-citBiP antibodies in RA patients (R² = 0.41). Several citrulline residues of citBiP were determined to be major epitopes of anti-citBiP antibodies, one of which showed cross-reactivity with CCP. Immunization of DBA/1J mice with citBiP induced several kinds of ACPAs, including anti-CCP and anti-citrullinated fibrinogen antibodies. Pre-immunization with citBiP exacerbated CIA, and anti-CCP antibody levels were increased in citBiP-pre-immunized CIA mice.

Conclusions

CitBiP is a newly described ACPA target that may play a pro-inflammatory role in arthritis.     

Incongruence among mitochondrial,chloroplast and nuclear gene trees in <Emphasis Type="Italic">Pinus</Emphasis> subgenus <Emphasis Type="Italic">Strobus</Emphasis> (Pinaceae)

Introgression has been considered to be one of main factors leading to phylogenetic incongruence among different datasets at lower taxonomic levels. In the plants of Pinaceae, the mtDNA, cpDNA, and nuclear DNA (nrDNA) may have different evolutionary histories through introgression because they are inherited maternally, paternally and biparentally, respectively. We compared mtDNA, cpDNA, and two low-copy nrDNA phylogenetic trees in the genus Pinus subgenus Strobus, in order to detect unknown past introgression events in this group. nrDNA trees were mostly congruent with the cpDNA tree, and supported the recent sectional and subsectional classification system. In contrast, mtDNA trees split the members of sect. Quinquefoliae into two groups that were not observed in the other gene trees. The factors constituting incongruence may be divided into the following two categories: the different splits within subsect. Strobus, and the non-monophyly of subsect. Gerardianae. The former was hypothesized to have been caused by the past introgression of cpDNA, mtDNA or both between Eurasian and North American species through Beringia. The latter was likely caused by the chimeric structure of the mtDNA sequence of P. bungeana, which might have originated through past hybridization, or through a horizontal transfer event and subsequent recombination. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis of riccardin C and its seven analogues. Part 1: The role of their phenolic hydroxy groups as LXRα agonists

Riccardin C, a nuclear receptor LXRα selective agonist, is an 18-membered macrocyclic bisbibenzyl isolated from several liverworts. Synthesis of riccardin C and its seven O-methylated derivatives was accomplished. The synthetic sequence highlights an intramolecular Suzuki–Miyaura coupling in the formation of the 18-membered biaryl linkage present in riccardin C. The structure–activity relationship of these compounds suggests that all of the phenolic hydroxy groups present in riccardin C are essential for the activation of LXRα.     

Bis(hetero)aryl derivatives as unique kinesin spindle protein inhibitors

Synthesis of 4-(4-tert-butylphenyl)pyridine analogues as kinesin spindle protein (KSP) inhibitors, SAR, cytotoxicity and mitotic arrest in HeLa cells are described. Interestingly, PVZB1194 showed potent KSP inhibition only in the presence of microtubules and distinct KSP localization from a known KSP inhibitor S-trytylcysteine analogue in mitosis. The observations would have resulted from a different molecular mechanism of KSP inhibition and suggest a novel biological regulation for KSP in mitosis.