The enhancing effect of fucoidan derived from Undaria pinnatifida on immunoglobulin production by mouse spleen lymphocytes

In this study, we revealed that a Mekabu (Udaria pinnantifida) extract enhanced immunoglobulin (Ig) production of mouse spleen lymphocytes. Furthermore, it was suggested that water-soluble and high molecular weight ingredients in the Mekabu extract have significant enhancing effect on Ig production. Therefore, fucoidan was estimated as the active component.     

Cysteine Residues in the Major Capsid Protein,Vp1, of the JC Virus Are Important for Protein Stability and Oligomer Formation

The capsid of the human polyomavirus JC virus (JCV) consists of 72 pentameric capsomeres of a major structural protein, Vp1. The cysteine residues of the related Vp1 of SV40 are known to contribute to Vp1 folding, pentamer formation, pentamer-pentamer contacts, and capsid stabilization. In light of the presence of a slight structural difference between JCV Vp1 and SV40 counterpart, the way the former folds could be either different from or similar to the latter. We found a difference: an important contribution of Vp1 cysteines to the formation of infectious virions, unique in JCV and absent in SV40. Having introduced amino acid substitution at each of six cysteines (C42, C80, C97, C200, C247, and C260) in JCV Vp1, we found that, when expressed in HeLa cells, the Vp1 level was decreased in C80A and C247A mutants, and remained normal in the other mutants. Additionally, the C80A and C247A Vp1-expressing cell extracts did not show the hemagglutination activity characteristic of JCV particles. The C80A and C247A mutant Vp1s were found to be less stable than the wild-type Vp1 in HeLa cells. When produced in a reconstituted in vitro protein translation system, these two mutant proteins were stable, suggesting that some cellular factors were responsible for their degradation. As determined by their sucrose gradient sedimentation profiles, in vitro translated C247A Vp1 formed pentamers, but in vitro translated C80A Vp1 was entirely monomeric. When individually incorporated into the JCV genome, the C80A and C247A mutants, but not the other Vp1 cysteine residues mutants, interfered with JCV infectivity. Furthermore, the C80A, but not the C247A, mutation prevented the nuclear localization of Vp1 in JCV genome transfected cells. These findings suggest that C80 of JCV Vp1 is required for Vp1 stability and pentamer formation, and C247 is involved in capsid assembly in the nucleus.     

Marked variations in patterns of cellulase activity against crystalline- vs. carboxymethyl-cellulose in the digestive systems of diverse, wood-feeding termites

Abstract.  Throughout the history of studies on cellulose digestion in termites, carboxymethyl-cellulose has been preferably used as a substrate for measuring cellulase activity in termites due to its high solubility. However, carboxymethyl-cellulose degradation is not directly related to digestibility of naturally occurring cellulose because many noncellulolytic organisms can also hydrolyse carboxymethyl-cellulose. To address this issue, a comparative study of microcrystalline cellulose digestion is performed in diverse xylophagous termites, using gut homogenates. For those termites harbouring gut flagellates , the majority of crystalline cellulose appears to be digested in the hindgut, both in the supernatant and the pellet. For Nasutitermes takasagoensis , a termite free of gut flagellates, crystalline cellulose is degraded primarily in the midgut supernatant, and partially in the pellet of the hindgut. The fungus-growing termite Odontotermes formosanus , which also does not possess intestinal flagellates, shows only a trace of crystalline cellulose hydrolysis throughout the gut. Comparison of levels of activity against crystalline cellulose with previously reported levels of activity against carboxymethyl-cellulose in the gut of each termite reveals significant differences between these activities. The results suggest that the hindgut flagellates produce commonly cellobiohydrolases in addition to endo-β-1,4-glucanases, which presumably act synergistically to digest cellulose. Preliminary evidence for the involvement of bacteria in the cellulose digestion of N. takasagoensis is also found.     

Electron Transfer between Plastocyanin and P700 in Various Types of Photosystem I Complex

Three types of PS I Chl-protein complex, PS I 180, PS I 65,and PS I 30, have been prepared and the kinetic properties ofthe transfer of electrons from plastocyanin to P700 in the PSI complexes with different sized antennae were examined. ThePS I 180 complex, which consists of 180 Chi per P700, showedthe almost same rate constant and effects of cations for thetransfer of electrons from plastocyanin to P700 as those obtainedwith PS I-enriched membrane fragments. The rate constant increasedwith the addition of low concentrations of monovalent and divalentcations, but decreased with high concentrations of cations.However, the rate was severely reduced in the case of the PSI 65 and PS I 30 complexes, and quite different effects of cationswere observed. Given the presence of additional 25- to 28-kDapolypeptides in the PS I 180 complex as compared to the PS I65 and PS I 30 complexes, we discuss a possible function forthese polypeptides in the regulation of the reaction betweenplastocyanin and P700. ¹This work was supported in part by a Grant-in-Aid for ScientificResearch from the Ministry of Education, Science and Cultureof Japan. (Received May 27, 1988; Accepted November 7, 1988)     

Hearing vulnerability after noise exposure in a mouse model of reactive oxygen species overproduction

Previous studies have convincingly argued that reactive oxygen species (ROS ) contribute to the development of several major types of sensorineural hearing loss, such as noise‐induced hearing loss (NIHL ), drug‐induced hearing loss, and age‐related hearing loss. However, the underlying molecular mechanisms induced by ROS in these pathologies remain unclear. To resolve this issue, we established an in vivo model of ROS overproduction by generating a transgenic (TG ) mouse line expressing the human NADPH oxidase 4 (NOX 4, NOX 4‐ TG mice), which is a constitutively active ROS ‐producing enzyme that does not require stimulation or an activator. Overproduction of ROS was detected at the cochlea of the inner ear in NOX 4 ‐TG mice, but they showed normal hearing function under baseline conditions. However, they demonstrated hearing function vulnerability, especially at high‐frequency sounds, upon exposure to intense noise, which was accompanied by loss of cochlear outer hair cells (OHC s). The vulnerability to loss of hearing function and OHC s was rescued by treatment with the antioxidant Tempol. Additionally, we found increased protein levels of the heat‐shock protein 47 (HSP 47) in models using HEK 293 cells, including H₂O₂ treatment and cells with stable and transient expression of NOX 4. Furthermore, the up‐regulated levels of Hsp47 were observed in both the cochlea and heart of NOX 4 ‐TG mice. Thus, antioxidant therapy is a promising approach for the treatment of NIHL . Hsp47 may be an endogenous antioxidant factor, compensating for the chronic ROS overexposure in vivo , and counteracting ROS ‐related hearing loss.

     

Non-random distribution of transduction termini in transductants from the integrated R plasmid, R100-1

Summary Tra ⁺and tra ^–derivatives of drug resistance plasmid, R100-1, were isolated by phage P1 from an Hfr donor with integrated R100-1 and then analyzed by complementation tests with tra ^–point mutants of Flac. Tra ⁺derivatives of R100-1 carrying tetracycline resistance alone and those carrying all six drug-resistance genes could support transfer of tra ^–point mutants of Flac except Flac traJ, whereas all of tra ^–derivatives of R100-1 failed to complement any one of tra ^–point mutants of Flac. This suggests that these tra ^–derivatives of R100-1 carrying tetracycline resistance gene are deleted for all the transfer genes impaired in the Flac point mutants tested. We assume a hot point, probably a specific base sequence similar to an IS element, at the left of the tetracycline gene (Fig. 1) becomes a transduction terminus in transduction of the integrated R100-1 by phage P1. Complementation analysis of tra ^–derivatives carrying five resistance genes except the tetracycline gene led us to a supposition that a gene(s), probably analogous to traJ of the F plasmid, is located on R100-1 near the tetracycline gene which plays an important regulatory role for self-transfer as well as for the complementation of tra ^–Flac mutants.     

A DPP-4 Inhibitor Suppresses Fibrosis and Inflammation on Experimental Autoimmune Myocarditis in Mice

Myocarditis is a critical inflammatory disorder which causes life-threatening conditions. No specific or effective treatment has been established. DPP-4 inhibitors have salutary effects not only on type 2 diabetes but also on certain cardiovascular diseases. However, the role of a DPP-4 inhibitor on myocarditis has not been investigated. To clarify the effects of a DPP-4 inhibitor on myocarditis, we used an experimental autoimmune myocarditis (EAM) model in Balb/c mice. EAM mice were assigned to the following groups: EAM mice group treated with a DPP-4 inhibitor (linagliptin) (n = 19) and those untreated (n = 22). Pathological analysis revealed that the myocardial fibrosis area ratio in the treated group was significantly lower than in the untreated group. RT-PCR analysis demonstrated that the levels of mRNA expression of IL-2, TNF-α, IL-1β and IL-6 were significantly lower in the treated group than in the untreated group. Lymphocyte proliferation assay showed that treatment with the DPP-4 inhibitor had no effect on antigen-induced spleen cell proliferation. Administration of the DPP-4 inhibitor remarkably suppressed cardiac fibrosis and reduced inflammatory cytokine gene expression in EAM mice. Thus, the agents present in DPP-4 inhibitors may be useful to treat and/or prevent clinical myocarditis.     

Simultaneous determination of isoflavones and bisphenol A in rat serum by high-performance liquid chromatography coupled with coulometric array detection

A method for simultaneous detection and quantification is presented to determine the presence of isoflavones and bisphenol A in a biological sample. A coulometric array detector was used with reversed-phase high-performance liquid chromatography (HPLC). Daidzein (1), glycitein (2), genistein (3) and their glucoside conjugates, daidzin (4), glycitin (5) and genistin (6), were measured as phytochemicals. Also assayed here was equol (7), a metabolite from compound 1, and bisphenol A (8), an industrial chemical that acts as an endocrine disrupter. All chemicals were simultaneously detected by using a 600-mV single detection voltage with high efficacy. A mixture of 1, 3 and 8 was orally administered to rats, and the levels of these three chemicals in the serum were clearly increased after a 4 kU beta-glucuronidase treatment. The levels of compounds 1 and 3 in the serum were detected at 1665 and 2040 ng/ml, while 8 was at a low level of 417 ng/ml. Compound 7 in the serum was not detected until after enzymatic hydrolysis (72 ng/ml). These results suggest that this analytical method would be useful for metabolic and pharmacokinetic studies on isoflavones and bisphenol A.     

A recurrent mutation in type II collagen gene causes Legg-Calvé-Perthes disease in a Japanese family

Legg-Calvé-Perthes disease (LCPD) is a common childhood hip disorder characterized by sequential stages of involvement of the capital femoral epiphyses, including subchondral fracture, fragmentation, re-ossification and healing with residual deformity. Most cases are sporadic, but familial cases have been described, with some families having multiple affected members. Genetic factors have been implicated in the etiology of LCPD, but the causal gene has not been identified. We have located a missense mutation (p.G1170S) in the type II collagen gene (COL2A1) in a Japanese family with an autosomal dominant hip disorder manifesting as LCPD and showing considerable intra-familial phenotypic variation. This is the first report of a mutation in hereditary LCPD. COL2A1 mutations may be more common in LCPD patients than currently thought, particularly in familial and/or bilateral cases.