Phylogenetic relationships of ferns deduced from rbcL gene sequence

Part of the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) gene (rbcL) was sequenced from three fern species: Adiantum capillus-veneris, Botrypus strictus, and Osmunda cinnamomea var. fokiensis. This region included 1,333 base pairs, about 90% of the gene. Maximum likelihood analysis of the deduced amino acid sequences indicated that (1) Botrypus (Ophioglossaceae) clustered monophyletically with other ferns (Adiantum, Angiopteris, Osmunda); the closest relative to Botrypus among the three species was Osmunda, which did not support the hypothesis that the Ophioglossaceae are linked to the progymnosperm-seed plant lineage. (2) Eusporangiate ferns containing Botrypus (Ophioglossaceae) and Angiopteris (Marattiaceae) were a paraphyletic group. (3) Seed plants and the four fern species examined formed a monophyletic group, but ferns and bryophytes (liverwort) did not. Variations in rates of substitution for synonymous and nonsynonymous codons were found in fern lineages.Correspondence to: M. Hasebe     

Phylogenetic relationships in gnetophyta deduced fromrbcL gene sequences

Part of the large subunit of the ribulose-1,5-bisphosphate carboxylase gene (rbcL) was sequenced (1333 base pairs) from three species of gymnosperms:Ephedra sinica Gnetum parvifolium, Welwitschia mirabilis. Phylogenetic trees inferred from the neighbor joining, Wagner parsimony and maximum likelihood methods showed thatGnetum andWelwitschia were more closely related to each other than either is toEphedra within Gnetophyta, and the result supports previous cladistical analysis of morphological data.     

Intrageneric relationships of maple trees based on the chloroplast DNA restriction fragment length polymorphisms

A maple tree genus,Acer is the largest genus in broad-leaved deciduous trees and contains about 200 species. A delimitation of the genus is clear but the intrageneric classification was controversial because of homoplasies in morphological characters. In this study, a phylogenetic relationship inAcer was inferred based on chloroplast DNA restriction site polymorphisms with 17 restriction endonucleases and previously proposed intrageneric classifications were evaluated. The phylogenetic tree showed that (1) sectionsArguta, Cissifolia, Lithocarpa, Macrantha, Palmata, Spicata, Tataricum, Trifoliata sensu Ogata (1967: Bull. Tokyo Univ. Forests 63: 89–206) were monophyletic groups respectively, (2) sectionsCampestria, Goniocarpa, Platanoidea sensu Ogata (1967) were polyphyletic respectively, and (3) sectionsDistyla andParviflora formed a sister group. An average of estimated nucleotide substitution rates of Acer chloroplast DNA was calculated as 7.9×10⁻¹¹±1.4×10⁻¹¹ nucleotide substitutions par site par year, which coincides well with previously reported rates of perennial plants. Divergence eras of eastern Asia and North American species in both sectionsSpicata andRubra were estimated to be late Miocene. In consideration with previous data, multiple migrations and disjunctions are likely to have formed the eastern Asian and North American disjunct distribution.     

Pivotal role of Bcl-2 family proteins in the regulation of chondrocyte apoptosis

During endochondral ossification, chondrocytes undergo hypertrophic differentiation and die by apoptosis. The level of inorganic phosphate (P(i)) elevates at the site of cartilage mineralization, and when chondrocytes were treated with P(i), they underwent rapid apoptosis. Gene silencing of the proapoptotic Bcl-2 homology 3-only molecule bnip3 significantly suppressed P(i)-induced apoptosis. Conversely, overexpression of Bcl-xL suppressed, and its knockdown promoted, the apoptosis of chondrocytes. Bnip3 was associated with Bcl-xL in chondrocytes stimulated with P(i). Bcl-xL was expressed uniformly in the growth plate chondrocytes, whereas Bnip3 expression was exclusively localized in the hypertrophic chondrocytes. Finally, we generated chondrocyte-specific bcl-x knock-out mice using the Cre-loxP recombination system, and we provided evidence that the hypertrophic chondrocyte layer was shortened in those mice because of an increased apoptosis of prehypertrophic and hypertrophic chondrocytes, with the mice afflicted with dwarfism as a result. These results suggest the pivotal role of Bcl-2 family members in the regulation of chondrocyte apoptosis.     

Cloning and functional characterization of the cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus

A filamentous fungus Aspergillus terreus produces itaconic acid, which is predicted to be derived from cis-aconitic acid via catalysis by cis-aconitic acid decarboxylase (CAD) in the carbon metabolism of the fungus. To clarify the enzyme's function and a pathway for itaconic acid biosynthesis, we cloned a novel gene encoding the enzyme. The open reading frame of this gene (CAD1) consists of 1,529 bp encoding 490 amino acids and is interrupted by a single intron. Among the identified proteins in the database, the primary structure of the protein encoded by CAD1 shared high identity with the MmgE/PrpD family of proteins, including a number of 2-methylcitrate dehydratases of bacteria. The cloned gene excluding an intron was introduced into the expression plasmid pAUR-CAD1 controlled by the ADH1 promoter. The CAD activity in Saccharomyces cerevisiae was confirmed by directly detecting itaconic acid as a product from cis-aconitic acid as a substrate. This result reveals for the first time that this gene encodes CAD, which is essential for itaconic acid production in A. terreus.     

Infection with the human immunodeficiency virus (HIV) is associated with an in vivo increase in B lymphocyte activation and immaturity

The expression of phenotypic markers on B lymphocytes in patients with the acquired immune deficiency syndrome (AIDS), in human immunodeficiency virus (HIV) seropositive individuals, and in healthy seronegative donors was examined by two-color flow cytometry. Patients with AIDS and HIV-seropositive individuals showed an elevated percentage of B cells bearing an activation marker, the transferrin receptor, when compared with donors not infected with HIV. A decrease in the percentage of resting (Leu-8 positive) B cells was also seen in AIDS patients and HIV-seropositive individuals. An increased percentage of circulating, immature (CALLA-positive, CD10) B cells was seen in AIDS patients. These phenotypic changes were accompanied by an increased level of spontaneous IgG and IgM secretion, and increased cell size within the total B cell population and in some B cell subpopulations, in patients with AIDS and in HIV-seropositive people. These results demonstrate that phenotypic changes indicative of in vivo B cell activation and immaturity accompany the polyclonal production of Ig seen in HIV-infected individuals.     

Differential Changes in Neuronal Excitability in the Spinal Dorsal Horn After Spinal Nerve Ligation in Rats

Previous studies demonstrated that peripheral nerve injury induced excessive neuronal response and glial activation in the spinal cord dorsal horn, and such change has been proposed to reflect the development and maintenance of neuropathic pain states. The aim of this study was to examine neuronal excitability and glial activation in the spinal dorsal horn after peripheral nerve injury. We examined noxious heat stimulation-induced c-Fos protein-like immunoreactivity (Fos-LI) neuron profiles in fourth-to-sixth lumbar (L4–L6) level spinal dorsal horn neurons after fifth lumbar spinal nerve ligation (L5 SNL). Immunofluorescence labeling of OX-42 and GFAP was also performed in histological sections of the spinal cord. A significant increase in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L4 level was found at 3 days after SNL, but returned to a level similar to that in sham-operated controls by 14 days after injury. As expected, a decrease in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L5 level was found at 3 days after SNL. However, these profiles had reappeared in large numbers by 14 and 21 days after injury. Immunofluorescence labeling of OX-42 and GFAP indicated sequential activation of microglia and astrocytes in the spinal dorsal horn. We conclude that nerve injury causes differential changes in neuronal excitability in the spinal dorsal horn, which may coincide with glial activation. These changes may play a substantial role in the pathogenesis of neuropathic pain after peripheral nerve injury.