Efficient Antibody Production upon Suppression of O Mannosylation in the Yeast Ogataea minuta

When antibodies were expressed in the methylotrophic yeast Ogataea minuta, we found that abnormal O mannosylation occurred in the secreted antibody. Yeast-specific O mannosylation is initiated by the addition of mannose at serine (Ser) or threonine (Thr) residues in the endoplasmic reticulum via protein O mannosyltransferase (Pmt) activity. To suppress the addition of O-linked sugar chains on antibodies, we examined the possibility of inhibiting Pmt activity by the addition of a Pmt inhibitor during cultivation. The Pmt inhibitor was found to partially suppress the O mannosylation on the antibodies. Surprisingly, the suppression of O mannosylation was associated with an increased amount of assembled antibody (H2L2) and enhanced the antigen-binding activity of the secreted antibody. In this study, we demonstrated the expression of human antibody in O. minuta and elucidated the relationship between O mannosylation and antibody production in yeast.     

Induction of hepatocyte growth factor expression by maleic acid in human fibroblasts through MAPK activation

Carboxylic acids have various biological activities and play critical roles in cellular metabolic pathways such as the tricarboxylic acid (TCA) cycle. It has been shown that some carboxylic acids induce cell proliferation and production of cytokines or growth factors. However, there have been no reports on effects of carboxylic acids on hepatocyte growth factor (HGF) expression. In this study, we found that only maleic acid among various carboxylic acids examined markedly induced HGF production from human dermal fibroblasts. Maleic acid also induced HGF production from human lung fibroblasts and neuroblastoma cells. The stimulatory effect was accompanied by upregulation of HGF gene expression. Increase in phosphorylation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) but not in phosphorylation of p38 was observed from 6 h and up to 24 h after maleic acid addition. The ERK kinase inhibitor PD98059 and the JNK inhibitor SP600125 potently inhibited maleic acid-induced HGF production, while the p38 inhibitor SB203580 did not significantly inhibit the production. The protein synthesis inhibitor cycloheximide completely inhibited upregulation of HGF mRNA induced by maleic acid but superinduced HGF mRNA expression upregulated by 12-O-tetradecanoylphorbol 13-acetate (TPA). These results suggest that maleic acid indirectly induced HGF expression from human dermal fibroblasts through activation of ERK and JNK and that de novo protein synthesis is required for maleic acid-induced upregulation of HGF mRNA.     

Growth-related changes in histology and immunolocalization of steroid hormone receptors in gonads of the immature male green turtle (Chelonia mydas)

Studies on the population dynamics of sea turtles require histological evaluation of the ontogenetic development and the activity of the gonads for reproduction. To investigate the growth-related changes of gonads in the immature male green turtle (Chelonia mydas), the histological changes of testes and epididymides and the localization of the androgen receptor, estrogen receptor alpha, estrogen receptor beta, and progesterone receptor were examined. The testes were categorized histologically into six developmental stages, and a scarce relationship between straight carapace length and gonadal development was confirmed based on the histological analysis. Several kinds of steroid hormone receptors were examined to show distributions in both testes and epididymides, for which their immunoreactivities were enhanced according to the developmental stage of the testes. These results suggest that straight carapace length is not an adequate indicator of maturity determination, whereas histological and immunohistochemical evaluations are useful in identifying the growth stages of green turtles, owing to the higher sensitivity to steroid hormones that appear during growth.     

Alternate muscle activity observed between knee extensor synergists during low-level sustained contractions.

To determine quantitatively the features of alternate muscle activity between knee extensor synergists during low-level prolonged contraction, a surface electromyogram (EMG) was recorded from the rectus femoris (RF), vastus lateralis (VL), and vastus medialis (VM) in 11 subjects during isometric knee extension exercise at 2.5% of maximal voluntary contraction (MVC) for 60 min (experiment 1). Furthermore, to examine the relation between alternate muscle activity and contraction levels, six of the subjects also performed sustained knee extension at 5.0, 7.5, and 10.0% of MVC (experiment 2). Alternate muscle activity among the three muscles was assessed by quantitative analysis on the basis of the rate of integrated EMG sequences. In experiment 1, the number of alternations was significantly higher between RF and either VL or VM than between VL and VM. Moreover, the frequency of alternate muscle activity increased with time. In experiment 2, alternating muscle activity was found during contractions at 2.5 and 5.0% of MVC, although not at 7.5 and 10.0% of MVC, and the number of alternations was higher at 2.5 than at 5.0% of MVC. Thus the findings of the present study demonstrated that alternate muscle activity in the quadriceps muscle 1) appears only between biarticular RF muscle and monoarticular vasti muscles (VL and VM), and its frequency of alternations progressively increases with time, and 2) emerges under sustained contraction with force production levels < or =5.0% of MVC.     

Possible involvement of phospholipase A2 and cyclooxygenase in stimulatory action of L-histidine on protein synthesis in L6 myotubes

Effects of L-histidine and related compounds on protein synthesiswere studied in cultured L6 myotubes. L-Histidine specifically stimulated protein synthesis, whereas D-histidine, histamine, L-arginine and L-lysine did not. Inhibitors of phospholipase A₂, phospholipase C and cyclooxygenase intercepted the stimulatory action of L-histidine on protein synthesis, while inhibitors of protein kinase C and 5-lipoxygenase did not. These results suggest an involvement of phospholipase A₂ and cyclooxygenase in the stimulatory action of L-histidine on protein synthesis in L6 myotubes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis

Gibberellins (GAs) are diterpene plant hormones essential for many developmental processes. Although the GA biosynthesis pathway has been well studied, our knowledge on its early stage is still limited. There are two possible routes for the biosynthesis of isoprenoids leading to GAs, the mevalonate (MVA) pathway in the cytosol and the methylerythritol phosphate (MEP) pathway in plastids. To distinguish these possibilities, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C-labeling was achieved by blocking the endogenous pathway chemically or genetically during the feed of a (13)C-labeled precursor specific to the MVA or MEP pathways. Gas chromatography-mass spectrometry analyses demonstrated that both MVA and MEP pathways can contribute to the biosyntheses of GAs and campesterol, a cytosolic sterol, in Arabidopsis seedlings. While GAs are predominantly synthesized through the MEP pathway, the MVA pathway plays a major role in the biosynthesis of campesterol. Consistent with some crossover between the two pathways, phenotypic defects caused by the block of the MVA and MEP pathways were partially rescued by exogenous application of the MEP and MVA precursors, respectively. We also provide evidence to suggest that the MVA pathway still contributes to GA biosynthesis when this pathway is limiting.     

Land abandonment and changes in snow cover period accelerate range expansions of sika deer

Ongoing climate change and land‐use change have the potential to substantially alter the distribution of large herbivores. This may result in drastic changes in ecosystems by changing plant–herbivore interactions. Here, we developed a model explaining sika deer persistence and colonization between 25 years in terms of neighborhood occupancy and habitat suitability. We used climatic, land‐use, and topographic variables to calculate the habitat suitability and evaluated the contributions of the variables to past range changes of sika deer. We used this model to predict the changes in the range of sika deer over the next 100 years under four scenario groups with the combination of land‐use change and climate change. Our results showed that both climate change and land‐use change had affected the range of sika deer in the past 25 years. Habitat suitability increased in northern or mountainous regions, which account for 71.6% of Japan, in line with a decrease in the snow cover period. Habitat suitability decreased in suburban areas, which account for 28.4% of Japan, corresponding to land‐use changes related to urbanization. In the next 100 years, the decrease in snow cover period and the increase in land abandonment were predicted to accelerate the range expansion of sika deer. Comparison of these two driving factors revealed that climate change will contribute more to range expansion, particularly from the 2070s onward. In scenarios that assumed the influence of both climate change and land‐use change, the total sika deer range increased by between +4.6% and +11.9% from the baseline scenario. Climate change and land‐use change will require additional efforts for future management of sika deer, particularly in the long term.     

Biochemical Characterization of Casein Kinases II (CK-II) from Cultured Cells of the Liverwort, Marchantia polymorpha

Monomeric and oligomeric forms of CK-II have been purified froma 1.0 M KC1 extract of the liverwort, Marchantia polymorpha,by means of heparin-agarose column chromatography and gel filtrationon Superose 6HR (HPLC). It was found that (i) a monomeric kinase(approximately 38 kDa) is the main form of CK-II in the cells;and (ii) the enzymatic properties of oligomeric kinase (approximately140 kDa), which cross-reacts with anti-serum against DrosophilaCK-IIß, are similar to those of CK-II (₂ß₂)in various animal cells. (Received November 10, 1992; Accepted February 18, 1993)