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.     

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.     

Determination of the Optimal Concentration of Valproic Acid in Patients with Epilepsy: A Population Pharmacokinetic-Pharmacodynamic Analysis

Valproic acid (VPA) is one of the most widely prescribed antiepileptic drugs for the treatment of epileptic seizures. Although it is well known that the doses of VPA and its plasma concentrations are highly correlated, the plasma concentrations do not correlate well with the therapeutic effects of the VPA. In this study, we developed a population-based pharmacokinetic (PK)-pharmacodynamic (PD) model to determine the optimal concentration of VPA according to the clinical characteristics of each patient. This retrospective study included 77 VPA-treated Japanese patients with epilepsy. A nonlinear mixed-effects model best represented the relationship between the trough concentrations of VPA at steady-state and an over 50% reduction in seizure frequency. The model was fitted using a logistic regression model, in which the logit function of the probability was a linear function of the predicted trough concentration of VPA. The model showed that the age, seizure locus, the sodium channel neuronal type I alpha subunit rs3812718 polymorphism and co-administration of carbamazepine, clonazepam, phenytoin or topiramate were associated with an over 50% reduction in the seizure frequency. We plotted the receiver operating characteristic (ROC) curve for the logit(Pr) value of the model and the presence or absence of a more than 50% reduction in seizure frequency, and the areas under the curves with the 95% confidence interval from the ROC curve were 0.823 with 0.793–0.853. A logit(Pr) value of 0.1 was considered the optimal cut-off point (sensitivity = 71.8% and specificity = 80.4%), and we calculated the optimal trough concentration of VPA for each patient. Such parameters may be useful to determine the recommended therapeutic concentration of VPA for each patient, and the procedure may contribute to the further development of personalized pharmacological therapy for epilepsy.     

Comparative gene expression profiles in pancreatic islets associated with agouti yellow mutation and PACAP overexpression in mice

In diabetes mellitus, pituitary adenylate cyclase-activating polypeptide (PACAP) has insulinotropic and glucose-lowering properties. We previously demonstrated that transgenic mice overexpressing PACAP in pancreatic β-cells (PACAP-Tg) show attenuated pancreatic islet hyperplasia and hyperinsulinemia in type 2 diabetic models. To explore the underlying mechanisms, here we crossed PACAP-Tg mice with lethal yellow agouti (KKAy) diabetic mice, and performed gene chip analysis of laser capture microdissected pancreatic islets from four F₁ offspring genotypes (wild-type, PACAP-Tg, KKAy, and PACAP-Tg:KKAy). We identified 1371 probes with >16-fold differences between at least one pair of genotypes, and classified the probes into five clusters with characteristic expression patterns. Gene ontology enrichment analysis showed that genes involved in the terms ribosome and intracellular organelles such as ribonucleoprotein complex, mitochondrion, and chromosome organization were significantly enriched in clusters characterized by up-regulated genes in PACAP-Tg:KKAy mice compared with KKAy mice. These results may provide insight into the mechanisms of diabetes that accompany islet hyperplasia and amelioration by PACAP.     

pH‐responsive fluorescence chemical sensor constituted by conjugated polymers containing pyridine rings

Poly(p‐pyridinium phenylene ethynylene)s (PPyPE) functionalized with alternating donor–acceptor repeat units were synthesized by a Pd‐catalyzed Sonogashira coupling reaction between diethynyl monomer and di‐iodopyridine for use as a pH‐responsive fluorescence chemical sensor. The synthesized PPyPE, containing pyridine units, was characterized by FT‐IR, ¹H and ¹³C NMR, UV–visible and fluorescence spectroscopies. We investigated the relationship between changes of optical properties and protonation/deprotonation of PPyPE containing pyridine units in solution. Addition of HCl decreased and red‐shifted the fluorescence intensity of the conjugated polymers that contained pyridine rings; fluorescence intensity of the polymers increased upon addition of NaOH solution. The synthesized PPyPE was found to be an effective and reusable chemical sensor for pH sensing. Copyright © 2015 John Wiley & Sons, Ltd.     

EphA4 Regulates the Balance between Self-Renewal and Differentiation of Radial Glial Cells and Intermediate Neuronal Precursors in Cooperation with FGF Signaling

In mouse cerebral corticogenesis, neurons are generated from radial glial cells (RGCs) or from their immediate progeny, intermediate neuronal precursors (INPs). The balance between self-renewal of these neuronal precursors and specification of cell fate is critical for proper cortical development, but the signaling mechanisms that regulate this progression are poorly understood. EphA4, a member of the receptor tyrosine kinase superfamily, is expressed in RGCs during embryogenesis. To illuminate the function of EphA4 in RGC cell fate determination during early corticogenesis, we deleted Epha4 in cortical cells at E11.5 or E13.5. Loss of EphA4 at both stages led to precocious in vivo RGC differentiation toward neurogenesis. Cortical cells isolated at E14.5 and E15.5 from both deletion mutants showed reduced capacity for neurosphere formation with greater differentiation toward neurons. They also exhibited lower phosphorylation of ERK and FRS2α in the presence of FGF. The size of the cerebral cortex at P0 was smaller than that of controls when Epha4 was deleted at E11.5 but not when it was deleted at E13.5, although the cortical layers were formed normally in both mutants. The number of PAX6-positive RGCs decreased at later developmental stages only in the E11.5 Epha4 deletion mutant. These results suggest that EphA4, in cooperation with an FGF signal, contributes to the maintenance of RGC self-renewal and repression of RGC differentiation through the neuronal lineage. This function of EphA4 is especially critical and uncompensated in early stages of corticogenesis, and thus deletion at E11.5 reduces the size of the neonatal cortex.     

The M-Ras-RA-GEF-2-Rap1 pathway mediates tumor necrosis factor-alpha dependent regulation of integrin activation in splenocytes

The Rap1 small GTPase has been implicated in regulation of integrin-mediated leukocyte adhesion downstream of various chemokines and cytokines in many aspects of inflammatory and immune responses. However, the mechanism for Rap1 regulation in the adhesion signaling remains unclear. RA-GEF-2 is a member of the multiple-member family of guanine nucleotide exchange factors (GEFs) for Rap1 and characterized by the possession of a Ras/Rap1-associating domain, interacting with M-Ras-GTP as an effector, in addition to the GEF catalytic domain. Here, we show that RA-GEF-2 is specifically responsible for the activation of Rap1 that mediates tumor necrosis factor-alpha (TNF-alpha)-triggered integrin activation. In BAF3 hematopoietic cells, activated M-Ras potently induced lymphocyte function-associated antigen 1 (LFA-1)-mediated cell aggregation. This activation was totally abrogated by knockdown of RA-GEF-2 or Rap1. TNF-alpha treatment activated LFA-1 in a manner dependent on M-Ras, RA-GEF-2, and Rap1 and induced activation of M-Ras and Rap1 in the plasma membrane, which was accompanied by recruitment of RA-GEF-2. Finally, we demonstrated that M-Ras and RA-GEF-2 were indeed involved in TNF-alpha-stimulated and Rap1-mediated LFA-1 activation in splenocytes by using mice deficient in RA-GEF-2. These findings proved a crucial role of the cross-talk between two Ras-family GTPases M-Ras and Rap1, mediated by RA-GEF-2, in adhesion signaling.