Functional demonstration of the ability of a primary spermatogonium as a stem cell by tracing a single cell destiny in Xenopus laevis

In Xenopus, although primary spermatogonium (PG), the largest cell in the testis, is believed to be spermatogonial stem cell by histological observations, functional evidence has never been obtained. In the present study, we first indicated that culture of juvenile testis in a medium supplemented with follicle stimulating hormone resulted in no proliferation of PG. In this culture system, early secondary spermatogonia could undergo mitotic divisions with a concomitant decrease in their size, so that they became distinguishable in size from PG. Because the subcutaneous environment of juveniles permitted aggregates of the dissociated testicular cells to reconstruct the normal testis structure, we next inserted a genetically marked PG isolated from cultured testes into the aggregate and transplanted it subcutaneously. In this system, 73.9% of the aggregates contained a marked PG. When we observed the aggregates 12 weeks after transplantation, most aggregates (70.0%) contained marked PG that had self-renewed. Among these, fully growing aggregates contained many spermatogenic cells at the later developmental stage. These results suggested that isolated PG from the cultured testes had the ability as stem cells, and that purification of the spermatogenic stem cells became reliable in Xenopus.     

Immunoaffinity extraction of a peptide modified by a small molecule

We investigated the affinity extraction conditions required to isolate peptide fragments modified with small molecules using an antibody that has a high affinity for the target small molecule. Investigation of antibody conformation and the retention behavior of the modified peptides on an immunosorbent matrix demonstrated the importance in efficient extraction of both the dissociation of hydrophobic interactions and the breakdown of the antibody conformation. Hydrophobic interactions, which anchor the small ligand to the paratope, were retained even when the three-dimensional structure of the antibody disintegrated in an acidic solution. For efficient extraction of a target peptide modified by a small molecule, it is therefore important to use an acidic solvent containing an organic modifier such as methanol at a concentration greater than 40% (v/v). We demonstrated the feasibility of this immunoaffinity extraction by application of this procedure to the analysis of modified peptide fragments obtained from a digestion of human serum albumin. The peptide fragments were affinity labeled with chenodeoxycholyl adenylate for analysis of the chenodeoxycholate binding site. This purification method could isolate the low levels of modified peptide contained in the reaction mixture, despite the presence of appreciable quantities of unlabeled peptide fragments.     

Physical restriction of pods causes seed size reduction of a brassinosteroid-deficient faba bean (Vicia faba)

BACKGROUND AND AIMS: A brassinosteroid-deficient mutant faba bean (Vicia faba 'Rinrei') shows dwarfism in many organs including pods and seeds. 'Rinrei' has normal-sized seeds together with dwarf seeds, suggesting that dwarfism in the seed may be indirectly caused by brassinosteroid deficiency. The mechanism of seed size reduction in this mutant was investigated. METHODS: The associations between seed orientation in the pod, seed numbers per pod and pod lengths with seed sizes were analysed in 'Rinrei' and the wild-type plant. KEY RESULTS: 'Rinrei' seeds are tightly arranged in pods containing two or three seeds. Seed size decreased as the number of seeds per pod increased or as the length of the pod decreased. Where no physical restriction occurred between seeds in a pod, the wild-type faba bean seeds had a nearly constant size regardless of seed number per pod or pod length. 'Rinrei' seeds in pods containing single seeds were the same size as wild-type seeds. Brassinolide treatment increased the seed size and the length of pods containing three seeds in 'Rinrei'. CONCLUSION: Seed size of 'Rinrei' is mainly regulated through a reduction of pod length due to brassinosteroid deficiency; physical restriction within pods causes a reduction in seed size. These results suggest a possible mechanism for increasing faba bean yields to optimal levels.     

Inhibition of HIV-1 replication by vesicular stomatitis virus envelope glycoprotein pseudotyped baculovirus vector-transduced ribozyme in mammalian cells

The baculovirus has recently emerged as a promising vector for in vivo gene therapy. To investigate its potential as a delivery vector for an anti-virus ribozyme targeting HIV-1, we constructed recombinant baculovirus vectors bearing a ribozyme-synthesizing cassette driven by the tRNA(i)(Met) promoter with enhanced transduction efficiency by displaying vesicular stomatitis virus glycoprotein (VSV-G) on the viral envelope. Transduction of HeLa CD4(+) cells with a recombinant baculovirus delivering the HIV-1 U5 gene-specific ribozyme dramatically suppressed HIV-1 expression in this cell line. The VSV-G pseudotyped baculovirus vector-transduced ribozyme potently inhibited HIV-1 replication compared to a recombinant baculovirus vector-transduced ribozyme lacking VSV-G. The use of a baculovirus vector might be beneficial for application in gene therapy.     

Real-time monitoring of antibody secretion from hybridomas on a microchip by time-resolved luminescence anisotropy analysis

This article presents a real-time monitoring system for cellular analysis using micro total analysis systems technology. Time-resolved luminescence anisotropy analysis was adopted for real-time detection of small amounts of a target protein produced by a small number of cells. The system was tested by real-time monitoring of the antibody secretion by hybridomas. The cells were successfully cultivated in a micro-incubation chamber (240 nl) fabricated on a microchip. The quantification of the antibody was achieved using the Ru(II) complex-labeled Staphylococcus aureus protein A probe, which can bind specifically to the Fc region of the antibody. Using this system, we detected as little as 24 fmol of immunoglobulin G under physiological conditions without the bound/free separation protocol. We successfully achieved real-time and quantitative monitoring of small amounts of antibody production by approximately 200 hybridoma cells. This method could be applied to various cellular analyses using small numbers of cells.     

Identification of oxidized methionine sites in erythrocyte membrane protein by liquid chromatography/electrospray ionization mass spectrometry peptide mapping

In this study, we used peptide mapping combined with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI MS) to examine the methionine oxidation of band 3 of erythrocyte membrane protein. Initially, we identified the methionine sites oxidized by chloramine T (N-chloro-p-toluenesulfoamide), a hydrophilic reagent. There were three oxidized methionines (Met 559, Met 741, and Met 909) in band 3, and these methionines were located in a hydrophilic region determined by previous topological studies of band 3. In addition, we found that C12E8, a polyoxyethylene detergent, leads to the oxidation of methionines in a transmembrane segment in band 3, and this oxidation occurs in a C12E8 preincubation time-dependent manner. In a previous study, it was found that peroxides accumulate in a polyoxyethylene detergent. Thus, our method enabled the direct and quantitative detection of protein damage due to detergent peroxides. Furthermore, we examined methionine oxidation in the presence of 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) or diethyl pyrocarbonate (DEPC), which induced either an outward or an inward conformation in band 3, respectively. Our results indicated that the location of Met 741 was associated with the band 3 conformation induced by band 3-mediated anion transport. In conclusion, we found that methionine oxidation can be applied to examine membrane protein structures as follows: (1) for topological studies of membrane proteins, (2) for assessing the quality of proteins in detergent solubilization studies, and (3) for the detection of conformational changes in membrane proteins.     

Leptin activates AMP-activated protein kinase in hepatic cells via a JAK2-dependent pathway

AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis within the individual cell. Recent reports have suggested that leptin, an adipocyte-secreted hormone, phosphorylates AMPK in skeletal muscle directly. However, little is known about the interaction between leptin signaling and AMPK activation. Here, we report that the leptin-induced phosphorylation of AMPK was detected in Huh7 cells expressing long form leptin receptor (OBRb) as well as short form leptin receptor (OBRa). In addition, we demonstrate that AMPK activation does not require the phosphorylation of either Tyr-985 or Tyr-1138 within the OBRb and may occur via a STAT3-independent signaling pathway. We also show that Huh7 cells expressing OBRb and SOCS3 (inhibitor of JAK2) resulted in a marked reduction of AMPK activation in response to leptin. These findings suggest that the activation of JAK2, but not STAT3, may play a critical role in leptin-induced AMPK activation in Huh7 cells.     

The immunoglobulin-like domain is involved in interaction of Neuregulin1 with ErbB

Neuregulin1 (NRG1) is a growth factor that signals through the interaction of the epidermal growth factor (EGF)-like domain with ErbB receptors. An immunoglobulin (Ig)-like domain is contained together with EGF-like domain in the ectodomain of some isoforms generated by alternative splicing, but its role in NRG1 signaling remained unclear. In the present study, we identified a novel isoform of NRG1 containing an Ig-like domain conserved among species from adult Xenopus laevis, which is predominantly expressed in the testis and brain. We generated recombinant proteins for the whole ectodomain and EGF-like domain alone of the isoform to compare their effects on cell proliferation, and phosphorylation of and their association with ErbB receptor, demonstrating that the ectodomain had approximately 10(3)-fold higher abilities than the EGF-like domain. Therefore, the Ig-like domain is probably essential for efficient interaction of an EGF-like domain with ErbB receptors.