2-Hydroxyisoflavanone dehydratase is a critical determinant of isoflavone productivity in hairy root cultures of Lotus japonicus

Hairy root cultures of a model legume, Lotus japonicus, were established to characterize two heterologous cDNAs encoding enzymes involved in isoflavone biosynthesis, i.e. licorice 2-hydroxyisoflavanone synthase (IFS) and soybean 2-hydroxyisoflavanone dehydratase (HID) catalyzing sequential reactions to yield isoflavones. While the control and the IFS overexpressor did not accumulate detectable isoflavones, the HID overexpressors did accumulate daidzein and genistein, showing that HID is a critical determinant of isoflavone productivity. Production of coumestrol in all the genotypes and isoliquiritigenin/liquiritigenin in IFS + HID-overexpressing lines was also noted. These results provide insight into the regulatory mechanism that controls isoflavonoid biosynthesis.     

Transport of camptothecin in hairy roots of Ophiorrhiza pumila

We have investigated the subcellular accumulation and transport of camptothecin (CPT), a monoterpene indole alkaloid, in hairy roots of Ophiorrhiza pumila. This hairy root produces high amounts of CPT and excretes it into the culture medium. When the hairy roots were exposed to UV radiation, autofluorescence emitted from CPT showed subcellular localization of CPT in the vacuole. Treatment with several inhibitors suggested that CPT excretion is a transporter-independent passive transport controlled by the concentration gradient of the compound. Interestingly, the hairy roots treated with brefeldin A, a vesicle transport inhibitor, showed increased CPT excretion. This could be explained by an increased transport rate of CPT from the endoplasmic reticulum (ER) to the cytoplasm when transport of CPT to the vacuole is blocked. The much higher concentration of CPT in the cytoplasm resulted in the increased excretion rate. This result indicates that CPT is biosynthesized at the ER and transported to accumulate in the vacuole by the same machinery that is used for vacuolar protein sorting. How O. pumila is insensitive to CPT is discussed.     

Asymmetric self-renewal and commitment of satellite stem cells in muscle

Satellite cells play a central role in mediating the growth and regeneration of skeletal muscle. However, whether satellite cells are stem cells, committed progenitors, or dedifferentiated myoblasts has remained unclear. Using Myf5-Cre and ROSA26-YFP Cre-reporter alleles, we observed that in vivo 10% of sublaminar Pax7-expressing satellite cells have never expressed Myf5. Moreover, we found that Pax7(+)/Myf5(-) satellite cells gave rise to Pax7(+)/Myf5(+) satellite cells through apical-basal oriented divisions that asymmetrically generated a basal Pax7(+)/Myf5(-) and an apical Pax7(+)/Myf5(+) cells. Prospective isolation and transplantation into muscle revealed that whereas Pax7(+)/Myf5(+) cells exhibited precocious differentiation, Pax7(+)/Myf5(-) cells extensively contributed to the satellite cell reservoir throughout the injected muscle. Therefore, we conclude that satellite cells are a heterogeneous population composed of stem cells and committed progenitors. These results provide critical insights into satellite cell biology and open new avenues for therapeutic treatment of neuromuscular diseases.     

Birth of a marmoset following injection of elongated spermatid from a prepubertal male

The common marmoset is a small nonhuman primate in which the application of transgenesis and genetic knockout techniques allows the generation of gene‐modified models of human diseases. However, its longer generation time than that of rodents is a major obstacle to the widespread use of gene‐modified marmosets for biomedical research. In this study, we examined the feasibility of shortening the generation time by using prepubertal marmoset males as gamete donors. We collected late round stage spermatids (Steps 5–7), elongated spermatids, and testicular spermatozoa from the testis of a prepubertal 11‐month‐old male marmoset and injected them into in vitro‐matured oocytes. After 7 days in culture, two embryos from elongated spermatid injection and two embryos from sperm injection were transferred into two separate recipient females. The recipient female that received elongated spermatid injection‐derived embryos became pregnant and gave birth to one female infant. This is the first demonstration that a spermatid from a prepubertal male primate can support full‐term development. Using this method, we can expect to obtain offspring of gene‐modified males 6 months to a year earlier than with natural mating     

Increased CD11b+ Gr‐1+ cell population in the placenta after infection with Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan pathogen that can cross the placenta, resulting in congenital toxoplasmosis with severe fetal brain abnormalities. The molecular mechanisms of immune responses against T. gondii infection in the placenta have largely remained unclear. An analytical method for characterizing phenotypes of immune cells in the placenta by flow cytometry was established and it was found that numbers of CD11b⁺ Gr‐1⁺ cells in the placenta increased significantly after T. gondii infection. These results suggest that innate immune responses play an important role in immunity against T. gondii infection via the feto‐maternal interface.     

Genetic analysis of L123 of the tRNA-mimicking eukaryote release factor eRF1, an amino acid residue critical for discrimination of stop codons

In eukaryotes, the tRNA-mimicking polypeptide-chain release factor, eRF1, decodes stop codons on the ribosome in a complex with eRF3; this complex exhibits striking structural similarity to the tRNA–eEF1A–GTP complex. Although amino acid residues or motifs of eRF1 that are critical for stop codon discrimination have been identified, the details of the molecular mechanisms involved in the function of the ribosomal decoding site remain obscure. Here, we report analyses of the position-123 amino acid of eRF1 (L123 in Saccharomyces cerevisiae eRF1), a residue that is phylogenetically conserved among species with canonical and variant genetic codes. In vivo readthrough efficiency analysis and genetic growth complementation analysis of the residue-123 systematic mutants suggested that this amino acid functions in stop codon discrimination in a manner coupled with eRF3 binding, and distinctive from previously reported adjacent residues. Furthermore, aminoglycoside antibiotic sensitivity analysis and ribosomal docking modeling of eRF1 in a quasi-A/T state suggested a functional interaction between the side chain of L123 and ribosomal residues critical for codon recognition in the decoding site, as a molecular explanation for coupling with eRF3. Our results provide insights into the molecular mechanisms underlying stop codon discrimination by a tRNA-mimicking protein on the ribosome.     

Discovery,Primary, and Crystal Structures and Capacitation-related Properties of a Prostate-derived Heparin-binding Protein WGA16 from Boar Sperm

Mammalian sperm acquire fertility through a functional maturation process called capacitation, where sperm membrane molecules are drastically remodeled. In this study, we found that a wheat germ agglutinin (WGA)-reactive protein on lipid rafts, named WGA16, is removed from the sperm surface on capacitation. WGA16 is a prostate-derived seminal plasma protein that has never been reported and is deposited on the sperm surface in the male reproductive tract. Based on protein and cDNA sequences for purified WGA16, it is a homologue of human zymogen granule protein 16 (ZG16) belonging to the Jacalin-related lectin (JRL) family in crystal and primary structures. A glycan array shows that WGA16 binds heparin through a basic patch containing Lys-53/Lys-73 residues but not the conventional lectin domain of the JRL family. WGA16 is glycosylated, contrary to other ZG16 members, and comparative mass spectrometry clearly shows its unique N-glycosylation profile among seminal plasma proteins. It has exposed GlcNAc and GalNAc residues without additional Gal residues. The GlcNAc/GalNAc residues can work as binding ligands for a sperm surface galactosyltransferase, which actually galactosylates WGA16 in situ in the presence of UDP-Gal. Interestingly, surface removal of WGA16 is experimentally induced by either UDP-Gal or heparin. In the crystal structure, N-glycosylated sites and a potential heparin-binding site face opposite sides. This geography of two functional sites suggest that WGA16 is deposited on the sperm surface through interaction between its N-glycans and the surface galactosyltransferase, whereas its heparin-binding domain may be involved in binding to sulfated glycosaminoglycans in the female tract, enabling removal of WGA16 from the sperm surface.     

Maladjustment to Academic Life and Employment Anxiety in University Students with Irritable Bowel Syndrome

The present study tested our hypothesis that university students with irritable bowel syndrome (IBS) may experience less satisfactory academic lives than those of students without IBS. We also verified the hypothesis that university students with IBS might have higher employment anxiety than students without IBS might. We conducted a cross-sectional study of 1,686 university students. Presence or absence of IBS was assessed via the Rome III Questionnaire. Two original items were used to evaluate academic life. The prevalence rates of IBS with diarrhea, IBS with constipation, mixed IBS, and unsubtyped IBS in the study population were 5%, 2%, 10%, and 3%, respectively. Regarding academic life, the proportions of participants who experienced maladjustment and employment anxiety were 29% and 50%, respectively. After adjusting for age, sex, and faculty, the odds ratios for maladjustment and employment anxiety were significantly higher in students who screened positively, relative to those who screened negatively, for IBS (OR, 1.62; 95% CI, 1.24–2.21; OR, 2.16; 95% CI, 1.68–2.81, respectively). In conclusion, maladjustment and anxiety over future employment were higher in university students with IBS relative to those without.     

Establishment of an Animal Model of Bisphosphonate-Related Osteonecrosis of the Jaws in Spontaneously Diabetic Torii Rats

Background

We evaluated the side effects of bisphosphonate (BP) on tooth extraction socket healing in spontaneously diabetic Torii (SDT) rats, an established model of non-obese type 2 diabetes mellitus, to develop an animal model of BP-related osteonecrosis of the jaws (BRONJ).

Materials and Methods

Male Sprague-Dawley (SD) rats and SDT rats were randomly assigned to the zoledronic acid (ZOL)-treated groups (SD/ZOL or SDT/ZOL) or to the control groups (SD/control or SDT/control). Rats in the SD/ZOL or SDT/ZOL groups received an intravenous bolus injection of ZOL (35 μg/kg) every 2 weeks. Each group consisted of 6 rats each. Twenty-one weeks after ZOL treatment began, the left maxillary molars were extracted. The rats were euthanized at 2, 4, or 8 weeks after tooth extraction, and the total maxillae were harvested for histological and histochemical studies.

Results

In the oral cavity, bone exposure persisted at the tooth extraction site in all rats of the SDT/ZOL group until 8 weeks after tooth extraction. In contrast, there was no bone exposure in SD/control or SDT/control groups, and only 1 of 6 rats in the SD/ZOL group showed bone exposure. Histologically, necrotic bone areas with empty lacunae, microbial colonies, and less invasion by inflammatory cells were observed. The number of tartrate-resistant acid phosphatase-positive osteoclasts was lower in the SDT/ZOL group than in the SD/control group. The mineral apposition rate was significantly lower in the SDT/ZOL group compared with the SD/control group.

Conclusions

This study demonstrated the development of BRONJ-like lesions in rats and suggested that low bone turnover with less inflammatory cell infiltration plays an important role in the development of BRONJ.