Bitterness Reduction of Citrus Fruits by β-Cyclodextrin

The levels of free amino acid, ammonia nitrogen and guanidino compounds were examined in renal failure rats induced by adenine. Among the essential amino acids in the serum, the marked reduction of lysine, valine, leucine and isoleucine was confirmed in the adenine-fed group as compared with the control group. Tyrosine and ornithine were also significantly reduced in the adenine-fed rats, while glycine, arginine and aspartic acid were significantly elevated. The urinary excretion of leucine, isoleucine and non-essential amino acids (glutamic acid, histidine, aspartic acid, citrulline, tyrosine, ornithine) was found to be high. On the other hand, adenine administered orally caused hyperammonemia. Furthermore, the results of the present study show that intake of adenine increased extraordinarily the level of guanidinosuccinic acid and methylguanidine in the serum, while the value of serum guanidinosuccinic acid and methylguanidine in rats fed on a control diet was not detectable.     

Expression of the selectable marker gene bsrm in BALB/MK cells induces apoptosis by overproduction of hydrogen peroxide.

Transduction of the retroviral vector LBmSN, which expresses the blasticidin S resistance gene bsrm in the murine keratinocyte cell line BALB/MK, induces death in these cells. Cell death is caused by a factor called DOKEB (death factor obtained from keratinocytes expressing bsrm), which is released before the cells' death. In this report we describe and discuss the purification and characterization of DOKEB. Our results were as follows. (i) The 5-day-old medium from the modified BALB/MK cells with LBmSN was used for purification and characterization by filtration and chromatography: DOKEB was a stable and highly hydrophilic compound, with a molecular mass less than that of 1 amino acid. (ii) The conditioned medium containing DOKEB was reactive against thiobarbituric acid and dichlorofluorescein diacetate. (iii) DOKEB activity was neutralized by the incubation of the conditioned medium with catalase. Therefore, our conclusion is that the BALB/MK cells expressing bsrm produce a large amount of hydrogen peroxide, which catalyzes the process of apoptosis of those cells.     

Crystal structure of Escherichia coli methionyl-tRNA synthetase highlights species-specific features

The 3D structure of monomeric C-truncated Escherichia coli methionyl-tRNA synthetase, a class 1 aminoacyl-tRNA synthetase, has been solved at 2.0 A resolution. Remarkably, the polypeptide connecting the two halves of the Rossmann fold exposes two identical knuckles related by a 2-fold axis but with zinc in the distal knuckle only. Examination of available MetRS orthologs reveals four classes according to the number and zinc content of the putative knuckles. Extreme cases are exemplified by the MetRS of eucaryotic or archaeal origin, where two knuckles and two metal ions are expected, and by the mitochondrial enzymes, which are predicted to have one knuckle without metal ion.     

Cellular expression of murine Ym1 and Ym2, chitinase family proteins,as revealed by in situ hybridization and immunohistochemistry

Ym is one of the chitinase family proteins, which are widely distributed in mammalian bodies and can bind glycosaminoglycans such as heparin/heparan sulfate. Ym1 is a macrophage protein produced in parasitic infections, while its isoform, Ym2, is upregulated in lung under allergic conditions. In the present study, we revealed the distinct cellular expression of Ym1 and Ym2 in normal mice by in situ hybridization and immunohistochemistry. Ym1 was principally expressed in the lung, spleen, and bone marrow, while Ym2 was found in the stomach. Ym1-expressing cells in the lung were alveolar macrophages, and the immunoreactivity for Ym1 was localized in rough endoplasmic reticulum. In the spleen, Ym1-expressing cells gathered in the red pulp and were electron microscopically identified as immature neutrophils. In the bone marrow, immature neutrophils were intensely immunoreactive, but lost this immunoreactivity with maturation. Moreover, needle-shaped crystals in the cytoplasm of macrophages, which formed erythroblastic islands, also showed intense Ym1 immunoreactivity. Ym2 expression was restricted to the stratified squamous epithelium in the junctional region between forestomach and glandular stomach. The function of Ym1 and Ym2 is still unclear; however, the distinct cellular localization under normal conditions suggests their important roles in hematopoiesis, tissue remodeling, or immune responses as an endogenous lectin.     

Cathepsin L is crucial for a Th1-type immune response during Leishmania major infection

Prior to the activation of CD4+ T cells, exogenous proteins are digested by endo/lysosomal enzymes in antigen-presenting cells (APCs) to produce antigenic peptides that are presented on MHC class II molecules. In the studies described here, the functional significance of cathepsin L for antigen processing and Th1/Th2 differentiation in experimental leishmaniasis was investigated. We first demonstrated that cathepsin L is one of the candidates for endo/lysosomal enzymes in the processing of soluble Leishmania antigen (SLA) by using CLIK148, a specific inhibitor of cathepsin L. Treatment of BALB/c or DBA/2 mice with CLIK148 exacerbated the disease by enhancing an SLA-specific Th2-type response such as IL-4 production. CLIK148 did not exert any direct influence on Leishmania major promastigotes themselves or on the course of L. major infection in SCID mice. Taken together, these findings suggest that treatment of host mice with CLIK148 affects the processing of SLA in APCs, resulting in the potentiation of Th2-type immune responses and thus leading to exacerbation of the disease. Furthermore, endo/lysosomal cathepsin L was found to be functionally distinct from previously described cathepsins B and D.     

Absence of superoxide dismutase activity in a soluble cellular isoform of prion protein produced by baculovirus expression system

A method for expression and purification of a soluble form of histidine (HIS)-tagged murine prion protein (bacMuPrP), which lacks the entire C-terminal cleavage and glycosyl phosphatidyl inositol (GPI) addition site, has been developed using a recombinant baculovirus expression system and purification with Ni-NTA agarose affinity chromatography. In mammalian sources, PrP(C) is attached to the cell membrane by a GPI anchor. However, in our system, bacMuPrP was secreted into the media, enabling its easy purification in abundance. Indirect immunofluorescence studies and immunoblot analysis localized not in cell membrane but in the perinuclear endoplasmic reticulum region in cells and is secreted into the media. Tunicamycin treatment revealed non-glycosylated proteins were secreted into the media, suggesting that glycosylation is not necessary for bacMuPrP secretion. Density-gradient sedimentation analysis demonstrated a sedimentation coefficient of secretory bacMuPrP as 2.3 S, indicating a monomeric form. Although affinity-purified PrP from mouse brain or recombinant prion protein (PrP) produced by Escherichia coli and refolded in the presence of copper has been reported to display superoxide dismutase (SOD) activity, bacMuPrP did not show SOD activity. These results suggest that bacMuPrP has a different biochemical and biophysical characterization from mammalian and bacterial-derived PrP. Furthermore, this simple expression system may provide an adequate source for structural, functional, and biochemical analyses of PrP.     

Multistep nucleus formation and a separate subunit contribution of the amyloidgenesis of heat-denatured monellin

Monellin (MN) is a sweet-tasting plant protein known to form fibrous aggregates in the heat-denatured state. Here the amyloid-type aggregation process of MN is extensively characterized. The amyloidgenesis was initiated in a highly denatured state of MN. A seeding effect of skipping a lag phase of the amyloid formation kinetics established a nucleation-dependent aggregation mechanism. A finely controlled experimental protocol revealed an additional prenucleus stage preceding the maturation of the nucleus, indicating that the initial lag phase is composed of multiple conformational events. The results obtained for the aggregation properties of the separate A and B subunit chains of MN and a recombinant single-chain MN suggest that the B chain exclusively contributed to the amyloid-type aggregation. These findings suggest a scheme for the amyloidgenesis of MN and their subunits, and provide a unique model of amyloidgenesis that is regulated by the subunit composition of protein.