Photoperiod- and temperature-dependent regulation of pupal beige/black polymorphism in the small copper butterfly, Lycaena phlaeas daimio Seitz

The small copper butterfly, Lycaena phlaeas daimio, has pupal beige/black polymorphism, the development of which is found to be controlled in an apparent association with the development of adult seasonal polymorphism (spring and summer morphs) by photoperiod and temperature in the larval stages. That is, the pupae of beige and black types developed under long-day and short-day conditions tend to develop into brown-winged and red-winged adults, respectively. In addition, a large proportion of long-day pharate pupae chilled at 4 degrees C for 5 days were observed to develop into pupae whose head-thoracic complexes and abdomens were judged to be of the black and intermediate types, respectively. They developed into adults with redder wings as compared to those obtained from unchilled pupae. The results indicate that the physiological mechanism underlying the photoperiodic control of the development of adult seasonal polymorphism may also play a significant role in the determination of pupal beige/black polymorphism in L. phlaeas daimio. Furthermore, cuticle melanization was found to be induced in the head-thoracic complexes of pupae by chilling of the pharate pupae. Melanization of pupal cuticle seems to occur in a close association with the development of reddish-winged adults.     

Changes of protein profiles during pollen development in<Emphasis Type="Italic"> Lilium longiflorum</Emphasis>

Pollen proteins of Lilium longiflorum were examined at different developmental stages (young, mature and cultured) using two-dimensional differential gel electrophoresis. Quantitative changes of six proteins (MP1–MP6) during pollen development were observed in the acidic and low molecular weight region. After water absorption on the culture medium, the quantities of all six proteins were drastically changed. Mass spectrometric analysis revealed that MP2, MP3, MP4 and MP6 are late embryogenesis abundant (LEA) (D-7) protein, profilin 3, profilin 1 and enolase, respectively. The remaining two proteins (MP1 and MP5) could not be identified by mass spectrometric analysis. Immunogold electron microscopic examination showed the presence of these proteins in different regions: MP1 around lipid bodies, suggesting possible involvement in lipid metabolism, MP4 near actin in the cytoplasm, indicating the possibility of its interaction with actin in the regulatory pathways of pollen, and MP2 and MP6 in the cytoplasm.     

Identification of two novel mutations in adenine phosphoribosyltransferase gene in patients with 2,8-dihydroxyadenine urolithiasis

Five mutations in the adenine phosphoribosyltransferase (APRT) gene have been described in Japanese patients with APRT deficiency. We investigated the APRT gene from three patients with APRT deficiency and two novel mutations, G133D and V84M, were determined.     

Development of improved Sindbis virus-based DNA expression vector

We have constructed an improved DNA expression vector based on the Sindbis virus. Several DNA-based Sindbis virus vectors were constructed to investigate the efficiency of transgene expression. These vectors, when transfected into mammalian cells, have been used to express heterologous genes. A recombinant genome of Sindbis plasmid DNA, in which the structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes, was placed under the control of a simian virus (SV 40) promoter with a hepatitis delta virus (HDV) antigenomic ribozyme and a polyadenylation signal. Transfection of mammalian cells with this Sindbis-based plasmid vector, pSin-SV40-HDV-SV40pA, resulted in transient high-level expression of the beta-galactosidase reporter gene. The expression level of beta-galactosidase from pSin-SV40-HDV-SV40pA was more than 16-fold higher than that of pSin-Lux originally reported by Herweijer et al. In vivo expression was also detected after injection of plasmid DNA into mouse quadriceps. In vivo expression was transient and undetectable after day 14. Furthermore, we demonstrate that the transfection of cells with this Sindbis virus vector results in apoptotic death on glioma cells. We have demonstrated a high-level expression of the exogenous beta-galactosidase gene from the pSin-SV40-HDV-SV40pA construct using a Sindbis replication system.     

Spectrometric analysis of degradation of a physiological substrate sigma32 by Escherichia coli AAA protease FtsH

We have established a fluorescence polarization assay system by which degradation of sigma32, a physiological substrate, by FtsH can be monitored spectrometrically. Using the system, it was found that an FtsH hexamer degrades approximately 0.5 molecules of Cy3-sigma32 per min at 42 degrees C and hydrolyzes approximately 140 ATP molecules during the degradation of a single molecule of Cy3-sigma32. Evidence also suggests that degradation of sigma32 proceeds from the N-terminus to the C-terminus. Although FtsH does not have a robust enough unfoldase activity to unfold a tightly folded proteins such as green fluorescent protein, it can unfold proteins with lower [Formula: see text] s such as glutathione S-transferase (Tm = 52 degrees C).     

Proliferation of donor mitochondrial DNA in nuclear transfer calves (Bos taurus) derived from cumulus cells

In embryos derived by nuclear-transfer (NT), fusion of donor cell and recipient oocyte caused mitochondrial heteroplasmy. Previous studies from other laboratories have reported either elimination or maintenance of donor-derived mitochondrial DNA (mtDNA) from somatic cells in cloned animals. Here we examined the distribution of donor mtDNA in NT embryos and calves derived from somatic cells. Donor mitochondria were clearly observed by fluorescence labeling in the cytoplasm of NT embryos immediately after fusion; however, fluorescence diminished to undetectable levels at 24 hr after nuclear transfer. By PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, donor mtDNAs were not detected in the NT embryos immediately after fusion (less than 3-4%). In contrast, three of nine NT calves exhibited heteroplasmy with donor cell mtDNA populations ranging from 6 to 40%. These results provide the first evidence of a significant replicative advantage of donor mtDNAs to recipient mtDNAs during the course of embryogenesis in NT calves from somatic cells.     

Microbial colonization drives lymphocyte accumulation and differentiation in the follicle-associated epithelium of Peyer's patches

Peyer's patches (PPs) are lined by follicle-associated epithelium (FAE) with Ag-transporting M cells. To investigate the spatial relationships of B cells, T cells, and dendritic cells (DCs) in PPs during microbial colonization, their in situ redistribution was examined in germfree (GF) rats exposed to a conventional pathogen-free microflora (conventionalized, CV). Although occasional B and T cells occurred in the FAE of GF rats, it contained mainly immature DCs (CD4(+)CD86(-)), whereas mature DCs (CD86(high)) were seen in the interfollicular zones even under GF conditions. In CV rats, DCs had disappeared from the FAE, which instead contained clusters by B and T cells associated with induction of putative M cell pockets. CD86 was seen neither in the FAE nor in the follicles under GF conditions, but it became apparent on intraepithelial B cells 5 wk after colonization. The level of CD86 on these B cells was comparable to that on germinal center B cells, although the B cell follicles did not show direct contact with the M cell areas. B cells in the follicular mantles acquired Bcl-2 after 12 wk in CV rats, whereas B cells in the FAE did not express Bcl-2 at a substantial level throughout the experimental period. The cellular redistribution patterns and phenotypic characteristics observed after colonization suggested that immature DCs, but not B cells, are involved in Ag presentation during primary immune responses against intestinal bacteria. However, the spatial cellular relationships sequentially being established among DCs, B cells, and T cells in PPs, are most likely important for the induction of post-germinal center B cells subsequently residing within the M cell pockets.     

Region of heat-stable enterotoxin II of Escherichia coli involved in translocation across the outer membrane

Heat-stable enterotoxin II of Escherichia coli (STII) is synthesized as a precursor form consisting of pre- and mature regions. The pre-region is cleaved off from the mature region during translocation across the inner membrane, and the mature region emerges in the periplasm. The mature region, composed of 48 amino acid residues, is processed in the periplasm by DsbA to form an intramolecular disulfide bond between Cys-10 and Cys-48 and between Cys-21 and Cys-36. STII formed with these disulfide bonds is efficiently secreted out of the cell through the secretory system, including TolC. However, it remains unknown which regions of STII are involved in interaction with TolC. In this study, we mutated the STII gene and examined the secretion of these STIIs into the culture supernatant. A deletion of the part covering from amino acid residue 37 to the carboxy terminal end did not markedly reduce the efficiency of secretion of STII into the culture supernatant. On the other hand, the efficiency of secretion of the peptide covering from the amino terminal end to position 18 to the culture supernatant was significantly low. These observations indicated that the central region of STII from amino acid residue 19 to that at position 36 is involved in the secretion of STII into the milieu. The experiment using a dsbA-deficient strain of E. coli showed that the disulfide bond between Cys-21 and Cys-36 by DsbA is necessary for STII to adapt to the structure that can cross the outer membrane.