Improvement of Sperm Motility of Sex-Reversed Male Rainbow Trout, Oncorhynchus mykiss, by Incubation in High-pH Artificial Seminal Plasma

Changes in the motility time of spermatozoa collected from the testes and the sperm duct of normal and sex-reversed male (XX) rainbow trout in physiological balanced salt solution were examined after incubation in artificial seminal plasmas of various pHs. Although untreated spermatozoa from the sperm duct retained motility for 60–90 s in the balanced salt solution, the spermatozoa collected from the testes were immotile. During the incubation in artificial seminal plasma of pH 7.0, the spermatozoa from the sperm duct hardly moved, similar to the testicular spermatozoa in the balanced salt solution. By suspending and incubating the testicular spermatozoa in artificial seminal plasma of pH 9.9 for 2 h at 4°C, the percentage of motile spermatozoa increased from 0–5% to 80%. The spermatozoa remained motile for at least 2 min after long-term incubation (12 h). When the full-sib eggs were inseminated with untreated testicular spermatozoa or testicular sperm treated for 2 h at high pH, the percentage survival increased from 5.5% to 53.8% at the eyed stage due to the high-pH treatment. The incubation of the spermatozoa in high-pH artificial seminal plasma improved the motility of the spermatozoa from the testes of the sex-reversed male that had lost its sperm duct. By this treatment, it is possible to markedly increase the mass production efficiency of all-female or all-female triploid sterile progenies.     

Investigation of Proteomic Profiles of Lamina of Ecklonia kurome (Laminariales): Homology-Based Cross-Species Protein Identification and Analysis of the Post-translational Processing of Vanadium-Dependent Bromoperoxidases Using MALDI-TOF/TOF

Proteomic profiles of the lamina of Ecklonia kurome Okamura, one of the Japanese dominant laminarialean kelps, were investigated by two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF. Due to the absence of E. kurome DNA or protein databases, homology-based cross-species protein identification was performed using a combination of three database-searching algorithms, Mascot peptide mass fingerprinting, Mascot MS/MS ion search, and mass spectrometry-based BLAST. Proteins were extracted from the lamina by an ethanol/phenol method and subjected to 2-DE (pI 4–7, 10 % polyacrylamide gel). More than 700 spots were detected in the 2-DE gel with CBB, and 93 spots (24 proteins) were successfully identified by MALDI-TOF/TOF and the cross-species database searching. The identified proteins mainly consisted of cytoplasmic carbohydrate metabolic enzymes, chloroplast proteins involved in photosynthesis, and haloperoxidases. Interestingly, vanadium-dependent bromoperoxidases (vBPO), which is thought to be involved in halogen uptake, synthesis of halogenated products, and detoxification of reactive oxygen species, were separated into at least 23 different spots. By comparing mass spectra, amino acid sequences predicted from tandem mass spectra and haloperoxidase activities of the vBPOs, we found that (1) at least two types of vBPOs were expressed in the lamina of E. kurome and (2) two pro-vBPOs might be activated by specific cleavage at N- and C-terminal regions.     

Selective tumoricidal effect of soluble proteoglucan extracted from the basidiomycete, Agaricus blazei Murill, mediated via natural killer cell activation and apoptosis

 We have isolated a novel type of natural tumoricidal product from the basidiomycete strain, Agaricus blazei Murill. Using the double-grafted tumor system in Balb/c mice, treatment of the primary tumor with an acid-treated fraction (ATF) obtained from the fruit bodies resulted in infiltration of the distant tumor by natural killer (NK) cells with marked tumoricidal activity. As shown by electrophoresis and DNA fragmentation assay, the ATF also directly inhibited tumor cell growth in vitro by inducing apoptotic processing; this apoptotic effect was also demonstrated by increased expression of the Apo2.7 antigen on the mitochondrial membranes of tumor cells, as shown by flow-cytometric analysis. The ATF had no effect on normal mouse splenic or interleukin-2-treated splenic mononuclear cells, indicating that it is selectively cytotoxic for the tumor cells. Cell-cycle analysis demonstrated that ATF induced the loss of S phase in MethA tumor cells, but did not affect normal splenic mononuclear cells, which were mainly in the G0G1 phase. Various chromatofocussing purification steps and NMR analysis showed the tumoricidal activity to be chiefly present in fractions containing (1→4)-α-D-glucan and (1→6)-β-D-glucan, present in a ratio of approximately 1:2 in the ATF (molecular mass 170 kDa), while the final purified fraction, HM3-G (molecular mass 380 kDa), with the highest tumoricidal activity, consisted of more than 90% glucose, the main component being (1→4)-α-D-glucan with (1→6)-β branching, in the ratio of approximately 4:1. Received: 27 August 1997 / Accepted 22 December 1997     

N-glycan structures of a recombinant mouse soluble Fcγ receptor II

N-glycans of a recombinant mouse soluble Fc receptor II (sFcRII) expressed in baby hamster kidney cells were released from glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a three-dimensional high-performance liquid chromatography (HPLC) mapping technique on three kinds of HPLC columns [Takahashi, et al. (1995) Anal. Biochem. 226: 139–46]. Eighteen different major N-glycan structures were identified, of which six were neutral (45%), five mono-sialyl (49%), one di-sialyl (4.6%), five tri-sialyl (1.1%), and one tetra-sialyl (0.3%). All N-glycan structures determined were complex type with fucosylation at the N-acetylglucosamine residue of the reducing end, and N-acetylneuraminic acid, when present, was -(2,3)-linked. The existence of a unique structure containing both N-acetylgalactosamine and -(2,3)-N-acetylneuraminic acid residues at the reducing ends, as below, was confirmed by MALDI-TOF mass spectrometry.     

Improvement in 5'-position-selective glucosylation of pyridoxine by Verticillium dahliae TPU 4900

Optimization of culture and reaction conditions for 5'-position-selective transglucosylation to pyridoxine by Verticillium dahliae TPU 4900 was investigated. V. dahliae TPU 4900 had high transglucosylation activity when grown with soluble starch as a carbon source and organic nitrogens such as Esusan meat as a nitrogen source at 15-20 degrees C. Both the yield of pyridoxine 5'-alpha-D-glucoside (PN-5'-alpha-G) and the 5'-position-selectivity reached a maximum when an intact-cell reaction was done at 50-60 degrees C and pH 7 with additions of dextrin. The transglucosylation activity in culture broth was 71 times with the optimization of culture conditions that under the conditions used for screening. The productivity of PN-5'-alpha-G synthesis was 6.9 times that under the initial conditions when the reaction conditions of intact cells were optimized. From 1000 mM (206 g/L) pyridoxine hydrochloride, PN-5'-alpha-G was synthesized to the concentration of 300 mM (98.4 g/L as PN-5'-alpha-G) with 5'-selectivity of 85% in 53 h by intact cells of V. dahliae TPU 4900.     

Maillard reaction-like lysine modification by a lipid peroxidation product: immunochemical detection of protein-bound 2-hydroxyheptanal in vivo

2-Hydroxyheptanal (2-HH) is one of the reactive aldehyde species generated during the peroxidation of n-6 polyunsaturated fatty acids, such as linoleic and arachidonic acids. Analogous to the Maillard reaction of reducing sugars, 2-HH readily reacts with lysine epsilon-amino groups. In the present study, to define the occurrence of the Maillard reaction-like lysine modification by 2-HH in vivo, we raised a monoclonal antibody directed to a trihydropyridinone (THPO) structure, 1-alkyl-4-butyl-5-pentyl-1,2,6-trihydropyridin-3-one, formed from 2-HH and lysine, and examined the presence of the antigenic structure in the human atherosclerotic aorta. Mice were immunized with the 2-HH-modified keyhole limpet hemocyanin (KLH) as the immunogen. Using a THPO-carrier protein conjugate, we screened the hybridomas and finally obtained a clone that produced the monoclonal antibody 3C8 (mAb3C8). The antibody strongly recognized bovine serum albumin (BSA) treated with 2-HH, but showed no cross-reactivity with BSAs modified with other related aldehydes. By using this antibody, it was revealed that the antigenic structure was indeed present in atherosclerotic lesions of the human aorta.     

Chromatographic and mass spectrometric analysis of glutathione in biological samples

Biological thiol compounds are classified into high-molecular-mass protein thiols and low-molecular-mass free thiols. Endogenous low-molecular-mass thiol compounds, namely, reduced glutathione (GSH) and its corresponding disulfide, glutathione disulfide (GSSG), are very important molecules that participate in a variety of physiological and pathological processes. GSH plays an essential role in protecting cells from oxidative and nitrosative stress and GSSG can be converted into the reduced form by action of glutathione reductase. Measurement of GSH and GSSG is a useful indicator of oxidative stress and disease risk. Many publications have reported successful determination of GSH and GSSG in biological samples. In this article, we review newly developed techniques, such as liquid chromatography coupled with mass spectrometry and tandem mass spectrometry, for identifying GSH bound to proteins, or for localizing GSH in bound or free forms at specific sites in organs and in cellular locations.     

Genetic diversity of an endangered aquatic plant, Potamogeton lucens subspecies sinicus

The genetic diversity of the species, Potamogeton lucens subsp. sinicus var. teganumensis, which is critically endangered in Japan, was investigated. This species now occurs in only two known localities in Japan. One is a native population (Oitoike population), but the other (Teganuma-Okahotto population) is found in a small artificial pond that was dug in 1998. It is considered that the Teganuma-Okahotto population grew from a soil seed bank. Based on RAPD variation, we compared the genetic diversity of the two populations of P. lucens var. teganumensis in Japan and one population of P. lucens subsp. sinicus var. sinicus in China. The Teganuma-Okahotto population showed RAPD variation, suggesting that it may be derived from more than one seed buried in old sediments. This population also had the highest value of Shannon's Information Index among the three study populations. This finding suggests that seeds buried in sediments can contain genetic variability, and may be used to conserve the genetic diversity of rare and endangered plants.     

Urinary yttrium excretion and effects of yttrium chloride on renal function in rats

Evaluation of yttrium exposure in biological samples has not been fully examined. To evaluate yttrium nephrotoxicity, yttrium chloride was orally administered to male Wistar rats and the urine volume (UV) and N-acetyl-beta-D-glucosaminidase (NAG) and creatinine excretion (Crt) were measured in 24-h urine samples. The urinary yttrium concentration and excretion rate were determined by inductively coupled plasma-argon emission spectrometry (ICP-AES). Large significant decreases of UV (>30%) and Crt (>10%) were observed at yttrium doses of 58.3-116.7 mg per rat, but no significant NAG changes was observed. This response pattern shows that a high yttrium dosage alters glomerular function rather than the proximal convoluted tubules. A urinary yttrium excretion rate of 0.216% and good dose-dependent urinary excretion (r=0.77) were confirmed. These results suggest that urinary yttrium is a suitable indicator of occupational and environmental exposure to this element, an increasingly important health issue because recent technological advances present significant potential risks of exposure to rare earth elements. We propose that the ICP-AES analytical method and animal experimental model described in this study will be a valuable tool for future research on the toxicology of rare earth elements.