Morphogenic activity of fibroblast growth factor-2 on primary neural precursor cells in three-dimensional culture

Mouse neural precursor cells (NPC) were dissociated from fetal heads at the 10th day of gestation. When clumps of NPC were cultured in collagen gel, they grew and reorganized neural tube-like structures in medium containing fetal calf serum at 10% and supplemented with insulin, transferrin, cholera toxin and selenite. However, dissociated NPC died when they were cultured in collagen gel at low density in the same medium. Addition of fibroblast growth factor-2 (FGF-2) to this culture stimulated growth of NPC and formation of neural tube-like structures. The requirement for FGF-2 disappeared in high seeding density culture: they grew and formed neural tube-like structures without FGF-2. The structures formed in collagen gel were immunohistochemically positive against anti-FGF-2 antibody. The results show that the three-dimensional culture system provides a useful tool to study the roles of FGF-2 in morphogenesis of the central nervous system.     

Evidence from Crosslinking for Nearest-Neighbor Relationships among the Three Extrinsic Proteins of Spinach Photosystem II Complexes that are Associated with Oxygen Evolution

Treatment of oxygen-evolving Photosystem II complexes, whichlack light-harvesting chlorophyll a/b proteins, with a seriesof disuccinimidyl esters with different chain lengths yieldeda crosslinked product which consisted of one molecule each ofthe extrinsic 33 kDa and 23 kDa proteins. In addition, crosslinkingbetween the 33 kDa protein and the chlorophyll-carrying 47 kDaprotein and between the 23 kDa and 17 kDa proteins was confirmed.Thus, the three extrinsic proteins are closely associated witheach other to form a complex which is attached to the PS IIreaction center complexes. (Received December 1, 1989; Accepted May 2, 1990)     

Novel frame-shift mutation in Slc5a2 encoding SGLT2 in a strain of senescence-accelerated mouse SAMP10

The senescence-accelerated mouse prone10 (SAMP10) strain, a model of aging, exhibits cognitive impairments and cerebral atrophy. We noticed that SAMP10/TaSlc mice, a SAMP10 substrain, have developed persistent glucosuria over the past few years. In the present study, we characterized SAMP10/TaSlc mice and further identified a spontaneous mutation in the Slc5a2 gene encoding sodium-glucose co-transporter (SGLT) 2. The mean concentration of urine glucose was high in SAMP10/TaSlc mice and increased further with advancing age, whereas other strains of senescence-accelerated mice, including SAMP1/SkuSlc, SAMP6/TaSlc and SAMP8/TaSlc or normal aging control SAMR1/TaSlc mice, exhibited no detectable glucose in urine. SAMP10/TaSlc mice consumed increasing amounts of food and water compared to SAMR1/TaSlc mice, suggesting the compensation of polyuria and the loss of glucose. Oral glucose tolerance tests showed decreased glucose reabsorption in the kidney of SAMP10/TaSlc mice. In addition, blood glucose levels decreased in an age-dependent fashion. The kidney was innately larger than that of control mice with no histological alterations. We examined the expression levels of glucose transporters in the kidney. Among SGLT1, SGLT2, glucose transporter (GLUT) 1 and GLUT2, we found a significant decrease only in the level of SGLT2. DNA sequencing of SGLT2 in SAMP10/TaSlc mice revealed a single nucleotide deletion of guanine at 1236, which resulted in a frameshift mutation that produced a truncated protein. We designate this strain as SAMP10/TaSlc-Slc5a2^slc (SAMP10-ΔSglt2). Recently, SGLT2 inhibitors have been demonstrated to be effective for the treatment of patients with type 2 diabetes (T2D). SAMP10-ΔSglt2 mice may serve as a unique preclinical model to study the link between aging-related neurodegenerative disorders and T2D.     

Up-regulation of L-type high voltage-gated calcium channel subunits by sustained exposure to 1,4- and 1,5-benzodiazepines in cerebrocortical neurons

The aim of this study is to examine how sustained exposure to two 1,4-benzodiazepines (BZDs) with different action period, diazepam and brotizolam, and a 1,5-BZD, clobazam, affects L-type high voltage-gated calcium channel (HVCC) functions and its mechanisms using primary cultures of mouse cerebral cortical neurons. The sustained exposure to these three BZDs increased [⁴⁵Ca²⁺] influx, which was due to the enhanced [⁴⁵Ca²⁺] entry through L-type HVCCs but not through of Cav2.1 and Cav2.2. Increase in [³H]diltiazem binding after the exposure to these three BZDs was due to the increase in the binding sites of [³H]diltiazem. Western blot analysis showed increase of Cav1.2 and Cav1.3 in association with the increased expression of α2/δ1 subunit. Similar changes in [³H]diltiazem binding and L-type HVCC subunit expression were found in the cerebral cortex from mouse with BZD physical dependence. These results indicate that BZDs examined here have the potential to increase L-type HVCC functions mediated via the enhanced expression of not only Cav1.2 and Cav1.3 but also α2/δ1 subunit after their sustained exposure, which may participate in the development of physical dependence by these BZDs.     

Infectivity of different genotypes of human Blastocystis hominis isolates in chickens and rats

Most Blastocystis hominis isolates from humans are believed to be potentially zoonotic. This is because B. hominis isolates found in a variety of other host species have been found to have identical or relatively similar genotypes to those found in human isolates. However, the transmission of human B. hominis isolates to other animals has not been confirmed experimentally. In this study, the infectivity associated with several unique human Blastocystis genotypes (subtypes 2, 3, 4 and 7) was therefore investigated by infecting chickens and rats with two isolates of each subtype experimentally. The results showed that one isolate of subtype 4 and one isolate of subtype 7 was capable of infecting both chickens and rats, while two isolates of subtype 2, another isolate of subtype 4, and another isolate of subtype 7 could only infect chickens. Conversely, two isolates of subtype 3 failed to infect either of the animals. These results confirmed that several genotypes from human isolates could infect chickens and/or rats, indicating that chickens and rats are suitable experimental animal models for studying the zoonotic potential of human Blastocystis isolates.     

Estrogen receptors in medaka (Oryzias latipes) and estrogenic environmental contaminants: an in vitro-in vivo correlation

In many vertebrates, estrogens are necessary to promote the growth and differentiation of the female reproductive system during development, and have important reproductive roles in both males and females. Medaka (Oryzias latipes) has three estrogen receptor (ER) subtypes, ERα, ERβ1 and ERβ2. To evaluate the three medaka ER (mER)-ligand interactions, we applied the ERE-luciferase reporter assay system to characterize each ER subtype. In this transient transfection assay system using mammalian cells, the mER proteins displayed estrogen-dependent activation. 17β-Estradiol (E(2)) and op'-DDT showed high activation irrespective of ERs. Endosulfan also exhibited activation; with less/no transactivity measured using other pesticides, i.e., heptachlor, carbendazim, deltamethrin, acephate, dimethoate and amitraz. It was generally observed that ERβ2 had higher activation potential than ERα and ERβ1. To understand the molecular mechanism of estrogen action via ER, we also conducted E(2) treatment where we observed a trigger in ERβ2 expression upon E(2) exposure. The present data suggest that ERβ2 is essential for female gonad maintenance. The data were supported by induction of vitellogenin (VTG) mRNA in the liver and reduced VTG receptor mRNA expression in the gonad of both sexes. The present work will provide a basic tool allowing future studies to examine the receptor-ligand interactions and endocrine disrupting mechanisms, and also expands our knowledge of estrogen action on reproductive development in medaka.     

Plasmin: its role in the extracellular processing of progalanin in tumor tissue

Galanin is a neuropeptide that is widely distributed in the central and peripheral nervous systems. In a previous study, we showed that a small cell lung carcinoma (SCLC) cell line, SBC-3A, released progalanin but not galanin, and that progalanin was then converted to galanin(1-20), the active form. Because the galanin(1-20) had undergone hydrolysis at Arg and Lys residues, the protease concerned was surmised to have a trypsin-like activity. The present study was performed to identify the trypsin-like protease which had previously been found to activate progalanin in this tumor tissue. The protease was isolated using chromatography and electrophoresis, and identified in tumor extracts from SBC-3A tumor-bearing mice; the major protease was found to be plasmin. We next confirmed that extracellular processing of progalanin occurs in SCLC tumor tissue (tumors produced by the implantation of SBC-3A cells into mice), and in two types of breast tumor tissue (obtained by implantation into mice of BT-549 and MDA-MB-436 cells). In cell culture, processed forms of progalanin were undetectable in SBC-3A, BT-549 or MDA-MB-436 cells. Conversely, gel filtration chromatography analysis of tumor extracts from SBC-3A, BT-549 and MDA-MB-436-bearing mice, revealed that galanin-like immunoreactivity (galanin-LI) in these tumor extracts was due to the presence of progalanin (14 kDa) and galanin(1-20) (2 kDa). Moreover, trypsin-like protease activity was elevated, and plasmin was expressed abundantly in SBC-3A, BT-549 and MDA-MB-436 tumors in mice. In addition, tranexamic acid, a plasmin inhibitor, inhibited progalanin conversion to galanin(1-20). The present study revealed that plasmin was present in tumor tissue, and that it was responsible for processing progalanin to galanin(1-20) in the extracellular environment.     

Novel high-affinity Aβ-binding peptides identified by an advanced in vitro evolution, progressive library method

Recent studies have been supporting that the generation of Aβ42 oligomers is responsible for Alzheimer's disease. Therefore, those peptides which bind to Aβ42 are scientifically interesting and can be possible candidates for the diagnosis and therapy of Alzheimer's disease. A systemic in vitro evolution, developed recently and the designated progressive library method (PLM), was applied to obtain Ab42-binding aptamers peptides. As a result, high affinity peptide aptamers made of 8 or 9 amino acids could be identified by this approach, endorsing the methodological effectiveness. Namely, the selection products from the secondary library of diversified peptides, which was constructed based on the information obtained from the primary library selection, were confirmed to be superior to those selected from the primary library as had been reported previously. The affinities of those peptides measured by SPR (surface plasmon resonance) were comparable to or higher than that of those peptides so far reported (K(d) of 10??). The other peptides selected were confirmed of their binding by a novel mode of gel shift assay (fluorescence enhancement caused by the binding). Thus, novel Aβ42-binding peptides with high affinity were provided for the future Alzheimer's disease study. The demonstration of the effectiveness of the systemic in vitro evolution of PLM is very encouraging for the study of identifying novel functional peptides.