Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs

Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV–LACK, rCDV–TSA, and rCDV–LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV–LACK showed markedly smaller nodules without ulceration. Although the rCDV–TSA- and rCDV–LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV–LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.     

Expression of <Emphasis Type="Italic">Ruminococcus albus</Emphasis> Xylanase Gene (<Emphasis Type="Italic">xyn</Emphasis>A) in <Emphasis Type="Italic">Streptococcus bovis</Emphasis> 12-U-1

The objective of this study was to ligate the xylanase gene A (xynA) isolated from Ruminococcus albus 7 into the promoter and signal-peptide region of the lichenase [β-(1,3-1,4)-glucanase] gene of Streptococcus bovis JB1. This fusion gene was inserted into the pSBE11 vector, and the resulting recombinant, plasmid pXA, was used to transform S. bovis 12-U-1 cells. The transformant, S. bovis 12UXA, secreted the xylanase, which was stable against freeze-thaw treatment and long-time incubation at 37°C. The introduction of pXA and production of xylanase did not affect cell growth, and the xylanase produced degraded xylan from oat-spelt and birchwood. Received: 24 June 2002 / Accepted: 7 October 2002     

Arf GTPase-activating protein ASAP1 interacts with Rab11 effector FIP3 and regulates pericentrosomal localization of transferrin receptor-positive recycling endosome

ADP-ribosylation factors (Arfs) and Arf GTPase-activating proteins (GAPs) are key regulators of membrane trafficking and the actin cytoskeleton. The Arf GAP ASAP1 contains an N-terminal BAR domain, which can induce membrane tubulation. Here, we report that the BAR domain of ASAP1 can also function as a protein binding site. Two-hybrid screening identified FIP3, which is a putative Arf6- and Rab11-effector, as a candidate ASAP1 BAR domain-binding protein. Both coimmunoprecipitation and in vitro pulldown assays confirmed that ASAP1 directly binds to FIP3 through its BAR domain. ASAP1 formed a ternary complex with Rab11 through FIP3. FIP3 binding to the BAR domain stimulated ASAP1 GAP activity against Arf1, but not Arf6. ASAP1 colocalized with FIP3 in the pericentrosomal endocytic recycling compartment. Depletion of ASAP1 or FIP3 by small interfering RNA changed the localization of transferrin receptor, which is a marker of the recycling endosome, in HeLa cells. The depletion also altered the trafficking of endocytosed transferrin. These results support the conclusion that ASAP1, like FIP3, functions as a component of the endocytic recycling compartment.     

Identification of minimal sequence for HIV-1 fusion inhibitors

Emergence of multi-drug resistant HIV-1 is a serious problem for AIDS treatment. Recently, the virus-cell membrane fusion process has been identified as a promising target for the development of novel drugs against these resistant variants. In this study, we identified a 29-residue peptide fusion inhibitor, SC29EK, which shows activity comparable to the previously reported inhibitor SC35EK. Some residues in SC29EK not required for interaction with virus gp41 heptad repeat 1 (HR1) were replaced with a non-proteinogenic amino acid, 2-aminoisobutyric acid (Aib), to stabilize the alpha-helix structure and to provide resistance to peptidases.     

Syntheses of water-soluble [60]fullerene derivatives and their enhancing effect on neurite outgrowth in NGF-treated PC12 cells

Water-soluble [60]fullerene (C₆₀) derivatives were synthesized to examine their bioactivities. PC12 cells were used as a model of nerve cells and the bioactivities of synthesized C₆₀ derivatives together with some reported ones were tested. Among the compounds tested, C₆₀/(γ-CyD)₂, C₆₀-bis(γ-CyD) (5) containing C₆₀-mono(γ-CyD) (5′), and C₆₀/PVP were sufficiently soluble in water and showed an enhancing effect on the neurite outgrowth of NGF-treated PC12 cells.     

Landscape genetic structure of <Emphasis Type="Italic">Betula maximowicziana</Emphasis> in the Chichibu mountain range,central Japan

We evaluated the genetic structure of 16 Betula maximowicziana populations in the Chichibu mountain range, central Japan, located within a 25-km radius; all but two populations were at altitudes of 1,100–1,400 m. The results indicate the effects of geographic topology on the landscape genetic structure of the populations and should facilitate the development of local-scale strategies to conserve and manage them. Analyses involving 11 nuclear simple sequence repeat loci showed that most populations had similar intrapopulation genetic diversity parameters. Population differentiation (F _ST = 0.021, G′_ST = 0.033) parameters for the populations examined were low but were relatively high compared to those obtained in a previous study covering populations in a much larger area with a radius of approximately 1,000 km (F _ST = 0.062, G′_ST = 0.102). Three populations (Iriyama, Kanayamasawa, and Nishizawa) were differentiated from the other populations by Monmonier’s and spatial analysis of molecular variance algorithms or by STRUCTURE analysis. Since a high mountain ridge (nearly 2,000 m) separates the Kanayamasawa and Nishizawa populations from the other 14 populations and the Kanayamasawa and Nishizawa populations are themselves separated by another mountain ridge, the genetic structure appears to be partly due to mountain ridges acting as genetic barriers and restricting gene flow. However, the Iriyama population is genetically different but not separated by any clear geographic barrier. These results show that the landscape genetic structure is complex in the mountain range and we need to pay attention, within landscape genetic studies and conservation programs, to geographic barriers and local population differentiation.     

Growth and morphological development of laboratory-reared larvae and juveniles of the Laotioan indigenous cyprinid Hypsibarbus malcolmi

The morphological development, including the pigmentation, body proportions, fins, and survival rate for 30 days after hatching, of laboratory-reared larval and juvenile Hypsibarbus malcolmi is described. Body lengths (BL) of larvae and juveniles were 2.0 ± 0.2 (mean ± SD) mm at 1 h after hatching (day 0) and 9.2 ± 0.6 mm on day 16, reaching 12.1 ± 0.9 mm on day 30. Yolk volume decreased linearly, with the yolk being completely absorbed by day 3 in all preflexion larvae (all specimens >3.2 mm BL). Feeding was observed on day 2 in fish which had rapidly undergone complete yolk absorption following mouth and anus opening on day 1, and on day 3 in all remaining fish. Myomere numbers were 20–21 + 11–12 = 31–33, although they were not clearly visible in juveniles. Melanophores were few on the body during days 0–2, but increased with growth and covered the entire upper dorsal body surface during the juvenile stage. Body proportions tended to become constant in juveniles. Notochord flexion began in larvae >5.2 mm BL on day 8, and was completed in larvae >8.4 mm BL on day 14. Specimens with full fin ray complements were initially observed on day 22 (10.4 mm BL in juveniles). All specimens >11.5 mm BL had attained the juvenile stage. A high survival rate of 92.7% was estimated on day 30.     

Horse domestication and conservation genetics of Przewalski's horse inferred from sex chromosomal and autosomal sequences

Despite their ability to interbreed and produce fertile offspring,there is continued disagreement about the genetic relationshipof the domestic horse (Equus caballus) to its endangered wildrelative, Przewalski's horse (Equus przewalskii). Analyses havediffered as to whether or not Przewalski's horse is placed phylogeneticallyas a separate sister group to domestic horses. Because Przewalski'shorse and domestic horse are so closely related, genetic datacan also be used to infer domestication-specific differencesbetween the two. To investigate the genetic relationship ofPrzewalski's horse to the domestic horse and to address whetherevolution of the domestic horse is driven by males or females,five homologous introns (a total of 3 kb) were sequenced onthe X and Y chromosomes in two Przewalski's horses and threebreeds of domestic horses: Arabian horse, Mongolian domestichorse, and Dartmoor pony. Five autosomal introns (a total of6 kb) were sequenced for these horses as well. The sequencesof sex chromosomal and autosomal introns were used to determinenucleotide diversity and the forces driving evolution in thesespecies. As a result, X chromosomal and autosomal data do notplace Przewalski's horses in a separate clade within phylogenetictrees for horses, suggesting a close relationship between domesticand Przewalski's horses. It was also found that there was alack of nucleotide diversity on the Y chromosome and highernucleotide diversity than expected on the X chromosome in domestichorses as compared with the Y chromosome and autosomes. Thissupports the hypothesis that very few male horses along withnumerous female horses founded the various domestic horse breeds.Patterns of nucleotide diversity among different types of chromosomeswere distinct for Przewalski's in contrast to domestic horses,supporting unique evolutionary histories of the two species.