Production of functional spermatids from mouse germline stem cells in ectopically reconstituted seminiferous tubules

Testicular germ cell transplantation into the seminiferous tubules is at present the only way to induce spermatogenesis from a given source of spermatogonial stem cells. Here we show an alternative method that harnesses the self-organizing ability of testicular somatic cells. The testicular cells of embryonic or neonatal mice or rats and of newborn pigs were dissociated into single cells. Each of them reorganized into a tubular structure following implantation into the subcutis of immunodeficient mice. When mouse germline stem (GS) cells derived from spermatogonial stem cells and expanded in culture were intermingled with testicular cells of rodents, they were integrated in the reconstituted tubules and differentiated beyond meiosis into spermatids. Normal offspring were produced by the microinjection of those spermatids into oocytes. This method could be applicable to various mammalian species and useful for producing functional gametes from GS cells in a xenoectopic environment.     

Bio-photosensor: Cyanobacterial photosystem I coupled with transistor via molecular wire

We report on the first successful output of electrons directly from photosystem I (PSI) of thermophilic cyanobacteria to the gate of a field-effect transistor (FET) by bypassing electron flow via a newly designed molecular wire, i.e., artificial vitamin K₁, and a gold nanoparticle; in short, this newly manufactured photosensor employs a bio-functional unit as the core of the device. Photo-electrons generated by the irradiation of molecular complexes composed of reconstituted PSI on the gate were found to control the FET. This PSI-bio-photosensor can be used to interpret gradation in images. This PSI-FET system is moreover sufficiently stable for use exceeding a period of 1 year.     

Long-distance effects of site-directed mutations on backbone conformation in bacteriorhodopsin from solid state NMR of [1-13C]Val-labeled proteins.

We have recorded 13C cross-polarization-magic angle spinning and dipolar decoupled-magic angle spinning NMR spectra of [1-13C]Val-labeled wild-type bacteriorhodopsin (bR), and the V49A, V199A, T46V, T46V/V49A, D96N, and D85N mutants, in order to study conformational changes of the backbone caused by site-directed mutations along the extracellular surface and the cytoplasmic half channel. On the basis of spectral changes in the V49A and V199A mutants, and upon specific cleavage by chymotrypsin, we assigned the three well-resolved 13C signals observed at 172.93, 172.00, and 171. 11 ppm to [1-13C]Val 69, Val 49, and Val 199, respectively. The local conformations of the backbone at these residues are revealed by the conformation-dependent 13C chemical shifts. We find that at the ambient temperature of these measurements Val 69 is not in a beta-sheet, in spite of previous observations by electron microscopy and x-ray diffraction at cryogenic temperatures, but in a flexible turn structure that undergoes conformational fluctuation. Results with the T46V mutant suggest that there is a long-distance effect on backbone conformation between Thr 46 and Val 49. From the spectra of the D85N and E204Q mutants there also appears to be coupling between Val 49 and Asp 85 and between Asp 85 and Glu 204, respectively. In addition, the T2 measurement indicates conformational interaction between Asp 96 and extracellular surface. The protonation of Asp 85 in the photocycle therefore might induce changes in conformation or dynamics, or both, throughout the protein, from the extracellular surface to the side chain of Asp 96.     

PCR-based method for identifying the S-genotypes of Japanese pear cultivars

 Japanese pear (Pyrus pyrifolia Nakai), a member of the Rosaceae, shows gametophytic self-incompatibility that is controlled by the S-locus. The S-genotype of Japanese pear cultivars is an important factor for crossing and breeding. We report a rapid reliable method to identify these S-genotypes. It consists of PCR amplification of the S-RNase gene from genomic DNA and subsequent digestion of the PCR fragments with S-allele-specific restriction endonucleases. Using this method, we determined the unknown S-genotypes of nine Japanese pear cultivars and selected self-compatible varieties from the offspring of the self-compatible cultivar, ‘Osa-Nijisseiki’. Received: 8 June 1998 / Accepted: 19 October 1998     

Augmentation and suppression of action potentials by estradiol in the myometrium of pregnant rat.

The purpose of this study was to investigate the actions of estradiol on spontaneous and evoked action potentials in the isolated longitudinal smooth muscle cells of the pregnant rat. Single cells were obtained by enzymatic digestion from pregnant rat longitudinal myometrium. Action potentials and currents were recorded by whole-cell current-clamp and voltage-clamp methods, respectively. The acute effects of 17beta-estradiol on action potentials and inward and outward currents were investigated. The following results were obtained. The average resting membrane potential of single myometrial cells was -54 mV (n = 40). In many cells, an electrical stimulation evoked a membrane depolarization, and action potentials were superimposed on the depolarization. In some cells, spontaneous action potentials were observed. Estradiol (30 microM) slightly depolarized the membrane (ca. 5 mV) and attenuated the generation of action potentials by reducing the frequency and amplitude of the spikes. Afterhyperpolarization was also attenuated by estradiol (30 microM). On the other hand, in 5 of 35 cells, estradiol increased the first spike amplitude and action potential duration, while frequency of the spike generation and afterhyperpolarization were inhibited. In voltage-clamped muscle cells, estradiol inhibited both inward and outward currents. Acute inhibition or augmentation of spike generation by estradiol is due to the balance of inhibition of inward and outward currents. Inhibition of both currents also prevented afterhyperpolarization, causing potential-dependent block of Ca spikes.     

Structure comparison between oxidized and reduced plastocyanin from a fern, Dryopteris crassirhizoma.

The X-ray crystal structures of oxidized and reduced plastocyanin obtained from the fern Dryopteris crassirhizoma have been determined at 1.7 and 1.8 A resolution, respectively. The fern plastocyanin is unique in the longer main chain composed of 102 amino acid residues and in the unusual pH dependence due to the pi-pi stacking interaction around the copper site [Kohzuma, T., et al. (1999) J. Biol. Chem. 274, 11817-11823]. Here we report the structural comparison between the fern plastocyanin and other plastocyanins from cyanobacteria, green algae, and other higher plants, together with the structural changes of fern plastocyanin upon reduction. Glu59 hydrogen bonds to the OH of Tyr83, which is thought to be a possible conduit for electrons, in the oxidized state. However, it moves away from Tyr83 upon reduction like poplar plastocyanin.     

Targeted disruption of the gene encoding the proteolipid subunit of mouse vacuolar H(+)-ATPase leads to early embryonic lethality.

Vacuolar H(+)-ATPase (V-ATPase) is responsible for acidification of intracellular compartments in eukaryotic cells. Its 16-kDa subunit (proteolipid, PL16) plays a central role in V-ATPase function, forming the principal channel via which protons are translocated. To elucidate physiological roles of V-ATPase in mammalian cell function and embryogenesis, we attempted to generate a PL16 null mutant mouse by gene-targeting. Mice heterozygous (PL16(+/-)) for the proteolipid mutation were intercrossed and their offspring were classified according to genotype. There were no homozygous (PL16(-/-)) pups among 69 neonates examined, but a few PL16(-/-) embryos were found during the pre-implantation stages of embryonic development, up to day 3.5 post-coitum. These results suggested that PL16 (and hence V-ATPase) may play an essential role in cell proliferation and viability during early embryogenesis. PL16(+/-) mice were indistinguishable from their wild-type littermates and displayed no discernible abnormalities, although the PL16 mRNA level in PL16(+/-) mice decreased to about one-half of wild-type levels.     

MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube.

The Mesp1 gene encodes the basic HLH protein MesP1 which is expressed in the mesodermal cell lineage during early gastrulation. Disruption of the Mesp1 gene leads to aberrant heart morphogenesis, resulting in cardia bifida. In order to study the defects in Mesp1-expressing cells during gastrulation and in the specification of mesodermal cell lineages, we introduced a (beta)-galactosidase gene (lacZ) under the control of the Mesp1 promoter by homologous recombination. The early expression pattern revealed by (beta)-gal staining in heterozygous embryos was almost identical to that observed by whole mount in situ hybridization. However, the (beta)-gal activity was retained longer than the mRNA signal, which enabled us to follow cell migration during gastrulation. In heterozygous embryos, the Mesp1-expressing cells migrated out from the primitive streak and were incorporated into the head mesenchyme and heart field. In contrast, Mesp1-expressing cells in the homozygous deficient embryos stayed in the primitive streak for a longer period of time before departure. The expression of FLK-1, an early marker of endothelial cell precursors including heart precursors, also accumulated abnormally in the posterior region in Mesp1-deficient embryos. In addition, using the Cre-loxP site-specific recombination system, we could determine the lineage of the Mesp1-expressing cells. The first mesodermal cells that ingressed through the primitive streak were incorporated as the mesodermal component of the amnion, and the next mesodermal population mainly contributed to the myocardium of the heart tube but not to the endocardium. These results strongly suggest that MesP1 is expressed in the heart tube precursor cells and is required for mesodermal cells to depart from the primitive streak and to generate a single heart tube.     

Identification of two discrete peptide: N-glycanases in Oryzias latipes during embryogenesis.

Two different types of peptide:N-glycanase (PNGase) were identified in developing embryos of medaka fish ( Oryzias latipes ). Because the optimum pH values for their activities were acidic and neutral, they were designated as acid PNGase M and neutral PNGase M, respectively. The acid PNGase M corresponded to the enzyme that had been partially purified from medaka embryos (Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1991) J. Biol. Chem., 266, 22110-22114). The apparent molecular weight of this enzyme was 150 K, and the optimal pH was 3.5-4.0, and the K m for L-hyosophorin was 44 microM. L-Hyosophorin is a cortical alveolus-derived glycononapeptide with a large N-linked glycan chain present in the perivitelline space of the developing embryo. Acid PNGase M was competitively inhibited by a free de-N-glycosylated nonapeptide derived from L-hyosophorin. This enzyme was expressed in ovaries and embryos at all developmental stages after gastrulation, but activity was not detected in embryos at developmental stages between fertilization and gastrula. Several independent lines of evidence suggested that acid PNGase M may be responsible for the unusual accumulation of free N-glycans derived from yolk glycoproteins (Iwasaki,M., Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1992) J. Biol. Chem., 267, 24287-24296). In contrast, the neutral PNGase M was expressed in blastoderms from the 4-8 cell stage and in cells up to early gastrula. The general significance of these findings is that they show a developmental stage-dependent expression of the two PNGase activities, and that expression of the neutral PNGase M activity occurs concomitantly with the de-N-glycosylation of L-hyosophorin. These data thus support our conclusion that the neutral PNGase M is responsible for the developmental-stage-related de-N-glycosylation of the L-hyosophorin.     

Characterization of a Neurospora crassa photolyase-deficient mutant generated by repeat induced point mutation of the phr gene.

We produced a photolyase-deficient mutant by repeat induced point mutation using the Neurospora crassa photolyase gene cloned previously. This mutation identified a new gene, phr, which was mapped on the right arm of linkage group I by both RFLP mapping and conventional mapping. To investigate the relationship between photoreactivation and dark repair processes, especially excision repair, double mutants of phr with representative repair-defective mutants of different types were constructed and tested for UV sensitivity and photoreactivation. The results show that the phr mutation has no influence on dark repair. Tests with CPD and TC(6-4) photoproduct-specific antibodies demonstrated that the phr mutant is defective in CPD photolyase and confirmed that there is no TC(6-4) photolyase activity in N. crassa. Furthermore, N. crassa photolyase is not a blue light receptor in the signal transduction that induces carotenoid biosynthesis.