Publications by authors named "Shosaku Hattori"

40 Publications

Successful blastocyst production by intracytoplasmic injection of sperm after in vitro maturation of follicular oocytes obtained from immature female squirrel monkeys (Saimiri boliviensis).

J Reprod Dev 2021 Aug 9;67(4):265-272. Epub 2021 Jul 9.

Graduate School of Agricultural Science, Utsunomiya University, Tochigi 321-8505, Japan.

Advanced reproductive technologies are being applied for the propagation of squirrel monkeys, to ensure their preservation as a genetic resource and the effective use of their gametes in the future. In the present study, oocytes and spermatozoa were collected from live squirrel monkeys, following which piezo intracytoplasmic sperm injection (ICSI) was performed using these gametes. Follicular development was induced by administering equine chorionic gonadotropin (eCG) containing inhibin antiserum to an immature squirrel monkey female. The unilateral ovary was excised after the administration of human chorionic gonadotropin (hCG), to induce ovulation, following which the larger developed follicular oocytes were collected. Follicular oocytes were prepared for ICSI using sperm from the epididymal tail of a unilateral testis extracted from a mature male. The embryos were continuously incubated in CMRL 1066 medium supplemented with 10% (v/v) fetal bovine serum. Embryo culture was performed with cumulus cells. Two experiments of ICSI carried out with three females resulted in 14 mature oocytes from the 49 cumulus-oocyte complexes collected and five embryos, three of which developed into blastocysts. These blastocysts were vitrified, thawed, and transferred to recipient monkeys, but no pregnancies resulted. In conclusion, the present study is the first to successfully produce ICSI-derived blastocysts from MII oocytes obtained by means of hormone administration (a combination of eCG+inhibin antiserum and hCG) and in vitro maturation in immature squirrel monkeys.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1262/jrd.2021-018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8423609PMC
August 2021

The Heterochromatin Block That Functions as a Rod Cell Microlens in Owl Monkeys Formed within a 15-Myr Time Span.

Genome Biol Evol 2021 03;13(3)

Primate Research Institute, Kyoto University, Inuyama, Japan.

In rod cells of many nocturnal mammals, heterochromatin localizes to the central region of the nucleus and serves as a lens to send light efficiently to the photoreceptor region. The genus Aotus (owl monkeys) is commonly considered to have undergone a shift from diurnal to nocturnal lifestyle. We recently demonstrated that rod cells of the Aotus species Aotus azarae possess a heterochromatin block at the center of its nucleus. The purpose of the present study was to estimate the time span in which the formation of the heterochromatin block took place. We performed three-dimensional hybridization analysis of the rod cell of another species, Aotus lemurinus. This analysis revealed the presence of a heterochromatin block that consisted of the same DNA components as those in A. azarae. These results indicate that the formation was complete at or before the separation of the two species. Based on the commonly accepted evolutionary history of New World monkeys and specifically of owl monkeys, the time span for the entire formation process was estimated to be 15 Myr at most.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/gbe/evab021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7991628PMC
March 2021

Recombinant SLAMblind Measles Virus Is a Promising Candidate for Nectin-4-Positive Triple Negative Breast Cancer Therapy.

Mol Ther Oncolytics 2020 Dec 30;19:127-135. Epub 2020 Sep 30.

Laboratory Animal Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

One of the most refractory breast cancer types is triple negative (TN) breast cancer, in which cells are resistant to both hormone and Herceptin treatments and, thus, often cause recurrence and metastasis. Effective treatments are needed to treat TN breast cancer. We previously demonstrated that rMV-SLAMblind, a recombinant measles virus, showed anti-tumor activity against breast cancer cells. Here, we examined whether rMV-SLAMblind is effective for treating TN breast cancer. Nectin-4, a receptor for rMV-SLAMblind, was expressed on the surface of 75% of the analyzed TN breast cancer cell lines. rMV-SLAMblind infected the nectin-4-expressing TN breast cancer cell lines, and significantly decreased the viability in half of the analyzed cell lines . Additionally, intratumoral injection of rMV-SLAMblind suppressed tumor growth in xenografts of MDA-MB-468 and HCC70 cells. To assess treatment for metastatic breast cancer, we performed intravenous administration of the luciferase-expressing-rMV-SLAMblind to MDA xenografted mice. Virus replicated in the tumor and resulted in significant suppression of the tumor growth. The safety of the virus was tested by its intravenous injection into healthy cynomolgus monkeys, which did not cause any measles-like symptoms. These results suggest that rMV-SLAMblind is a promising candidate as a therapeutic agent for treating metastatic and/or TN type breast cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omto.2020.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7585052PMC
December 2020

Postnatal testicular development and actin appearance in the seminiferous epithelium of the Habu, Trimeresurus flavoviridis.

Anat Histol Embryol 2021 Mar 26;50(2):417-421. Epub 2020 Oct 26.

Department of Veterinary Anatomy, The University of Tokyo, Tokyo, Japan.

The postnatal testicular development and actin distribution in the seminiferous epithelium were examined by light microscopy, using the testes of the Habu (Trimeresurus flavoviridis; snake) from 0-year-old to 3-year-old. At 0-year-old (about 1 month after birth), the testis was quite small in size, and the seminiferous epithelium was composed of only Sertoli cells and large spermatogonia. Actin immunoreactivity was observed in the peritubular myoid cells, but could not be detected in the seminiferous epithelium. At 1-year-old (about 10 months after birth), the testicular size increased to a great degree. In the seminiferous epithelium, spermatocytes newly appeared. Actin could still not be detected in the seminiferous epithelium. At 2-year-old (about 1 year and 10 months after birth), the testes continued to develop in size. In the seminiferous epithelium, elongate spermatids and round spermatids were frequently seen, in addition to Sertoli cells, spermatogonia and spermatocytes. Thus, active spermatogenesis was clearly recognized at this age. Moreover, the actin distribution in the seminiferous epithelium was observed at the site between Sertoli cells and spermatids, as well as that at adult stage. The immunoreactivity of actin in the peritubular myoid cells gradually increased from 0-year-old to 2-year-old. Conclusively, it seems likely that spermatogenesis in the Habu initiates at 2-year-old, accompanying with the appearance of actin in the seminiferous epithelium.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/ahe.12628DOI Listing
March 2021

Discovery of the Gene Encoding a Novel Small Serum Protein (SSP) of and the Evolution of SSPs.

Toxins (Basel) 2020 03 12;12(3). Epub 2020 Mar 12.

Department of Applied Life Science, Faculty of Bioscience and Biotechnology, Sojo University, Kumamoto 860-0082, Japan.

Small serum proteins (SSPs) are low-molecular-weight proteins in snake serum with affinities for various venom proteins. Five SSPs, SSP-1 through SSP-5, have been reported in ("habu", ) serum so far. Recently, we reported that the five genes encoding these SSPs are arranged in tandem on a single chromosome. However, the physiological functions and evolutionary origins of the five SSPs remain poorly understood. In a detailed analysis of the habu draft genome, we found a gene encoding a novel SSP, SSP-6. Structural analysis of the genes encoding SSPs and their genomic arrangement revealed the following: (1) SSP-6 forms a third SSP subgroup; (2) SSP-5 and SSP-6 were present in all snake genomes before the divergence of non-venomous and venomous snakes, while SSP-4 was acquired only by venomous snakes; (3) the composition of paralogous SSP genes in snake genomes seems to reflect snake habitat differences; and (4) the evolutionary emergence of SSP genes is probably related to the physiological functions of SSPs, with an initial snake repertoire of SSP-6 and SSP-5. SSP-4 and its derivative, SSP-3, as well as SSP-1 and SSP-2, appear to be venom-related and were acquired later.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/toxins12030177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150969PMC
March 2020

Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells.

Biochem Biophys Res Commun 2020 01 18;521(1):24-30. Epub 2019 Oct 18.

Laboratory of Veterinary Developmental Biology, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Japan. Electronic address:

Background: Cell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells.

Methods: Fused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells.

Results: Colonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells.

Conclusions: Our results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2019.10.026DOI Listing
January 2020

Distribution of actin filaments in the seminiferous epithelium of the Habu, Trimeresurus flavoviridis.

Anat Histol Embryol 2019 Sep 7;48(5):505-507. Epub 2019 Aug 7.

Department of Veterinary Anatomy, The University of Tokyo, Tokyo, Japan.

The distribution of actin filaments was examined in the seminiferous epithelium of the Habu (Trimeresurus flavoviridis; snake), by transmission electron microscopy and fluorescence histochemistry. By transmission electron microscopy, actin filaments were clearly found only at the site between Sertoli cell and spermatid without a lattice-like structure. Fluorescence histochemistry showed a weak labelling of actin filaments in the seminiferous epithelium, whereas these findings seem to be common among reptiles, they are different from those in mammals. Additionally, the bundles of actin filaments adjacent to the plasma membrane of Sertoli cells, appeared in other reptiles, were not observed in the Habu.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/ahe.12475DOI Listing
September 2019

Unique structure (construction and configuration) and evolution of the array of small serum protein genes of snake.

Biosci Rep 2019 07 5;39(7). Epub 2019 Jul 5.

Department of Applied Life Science, Faculty of Bioscience and Biotechnology, Sojo University, Kumamoto 860-0082, Japan.

The nucleotide sequence of () 30534 bp genome segment which contains genes encoding small serum proteins (SSPs) was deciphered. The genome segment contained five SSP genes (), , and in this order and had characteristic configuration and constructions of the particular nucleotide sequences inserted. Comparison between the configurations of the inserted chicken repeat-1 (CR1) fragments of and () showed that the nucleotide segment encompassing from to was inverted. The inactive form of , named , found in the intergenic region (I-Reg) between and had also been destroyed by insertions of the plural long interspersed nuclear elements (LINEs) and DNA transposons. The L2 LINE inserted into the third intron or the particular repetitive sequences inserted into the second intron structurally divided five s into two subgroups, the Long SSP subgroup of and or the Short SSP subgroup of and The mathematical analysis also showed that s of the Long SSP subgroup evolved alternately in an accelerated and neutral manner, whereas those of the Short SSP subgroup evolved in an accelerated manner. Moreover, the ortholog analysis of of various snakes showed that the evolutionary emerging order of s was as follows: , and The unique interpretation about accelerated evolution and the novel idea that the transposable elements such as LINEs and DNA transposons are involved in maintaining the host genome besides its own transposition natures were proposed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BSR20190560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609765PMC
July 2019

Morphological analyses of the retinal photoreceptor cells in the nocturnally adapted owl monkeys.

J Vet Med Sci 2018 Mar 26;80(3):413-420. Epub 2018 Jan 26.

Laboratory of Basic Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi 753-8515, Japan.

Owl monkeys are the only one species possessing the nocturnal lifestyles among the simian monkeys. Their eyes and retinas have been interested associating with the nocturnal adaptation. We examined the cellular specificity and electroretinogram (ERG) reactivity in the retina of the owl monkeys by comparison with the squirrel monkeys, taxonomically close-species and expressing diurnal behavior. Owl monkeys did not have clear structure of the foveal pit by the funduscope, whereas the retinal wholemount specimens indicated a small-condensed spot of the ganglion cells. There were abundant numbers of the rod photoreceptor cells in owl monkeys than those of the squirrel monkeys. However, the owl monkeys' retina did not possess superiority for rod cell-reactivity in the scotopic ERG responses. Scanning electron microscopic observation revealed that the rod cells in owl monkeys' retina had very small-sized inner and outer segments as compared with squirrel monkeys. Owl monkeys showed typical nocturnal traits such as rod-cell dominance. However, the individual photoreceptor cells seemed to be functionally weak for visual capacity, caused from the morphological immaturity at the inner and outer segments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1292/jvms.17-0418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880819PMC
March 2018

Efficacy of recombinant measles virus expressing highly pathogenic avian influenza virus (HPAIV) antigen against HPAIV infection in monkeys.

Sci Rep 2017 09 20;7(1):12017. Epub 2017 Sep 20.

Laboratory Animal Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.

Highly pathogenic avian influenza virus (HPAIV) is a serious threat not only to domestic fowls but also to humans. Vaccines inducing long-lasting immunity against HPAIV are required. In the present study, we generated recombinant measles virus (MV) expressing the hemagglutinin protein of HPAIV without the multibasic site necessary for its pathogenicity in chickens using the backbone of an MV vaccine strain (rMV-Ed-H5HA) or a wild-type MV-derived mutant (rMV-HL-Vko-H5HA). We examined protective efficacy of the candidate vaccines in the monkey infection model by the challenge with a HPAIV (H5N1). Cynomolgus monkeys inoculated with the candidate vaccines produced both anti-H5 HA and anti-MV antibodies. They recovered earlier from influenza symptoms than unvaccinated monkeys after the challenge with the HPAIV strain. Chest radiography and histopathological analyses confirmed less severe pneumonia in the vaccinated monkeys. Vaccination tended to suppress viral shedding and reduced the interleukin-6 levels in the lungs. Furthermore, the vaccination with rMV-Ed-H5HA of monkeys with pre-existing anti-MV immunity induced the production of anti-H5 HA antibodies. These results suggest that both candidate vaccines effectively reduce disease severity in naïve hosts, and that rMV-Ed-H5HA is a particularly good candidate vaccine against HPAIV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-08326-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607339PMC
September 2017

The taxonomic position and the unexpected divergence of the Habu viper, Protobothrops among Japanese subtropical islands.

Mol Phylogenet Evol 2016 08 28;101:91-100. Epub 2016 Apr 28.

Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.

There are four Habu species currently recognized in Japan: Protobothrops flavoviridis from the Amami Islands and the Okinawa Islands, P. tokarensis from the Tokara Islands, P. elegans from the Yaeyama Islands and Ovophis okinabvensis from the Amami Islands and the Okinawa Islands. To clarify their taxonomic positions, we determined the complete mitochondria genome sequence (approx. 17kb) from two specimens from two different islands each for P. flavoviridis, P. tokarensis and P. elegans as well as one specimen of O. okinavensis and reconstructed the molecular phylogeny of Protobothrops using the published sequences of related species. The maximum likelihood tree showed four major species groups within Protbothrops: Group I consisting of P. cornutus, P. dabieshanensis, P. jerdonii and P. xiangchengensis; Group II consisting of P. flavoviridis and P. tokarensis; Group III consisting of P. maolensis, P. mucrosquamatus and P. elegans; Group IV consisting of P. himalayanus and P. kaubacki. Since we observed an unexpected divergence and the paraphyly of the two samples of P. flavoviridis collected from different islands, Amami-Oshima and Okinawajima within the Group II, we expanded the analysis by increasing the number of P. flavoviridis and P. tokarensis collected from 10 islands: Amami-Oshima (5 specimens), Kakeromajima (4) and Tokunoshima (4) from the Amami Islands, Okinawajima (4), Iheyajima (4), Iejima (4), Tokashikijima (4) and Kumejima (4) from the Okinawa Islands, Kodakarajima (P. tokarensis) (4) and Takarajima (P. tokarensis) (4) from the Tokara Islands. The maximum likelihood tree of the 44 samples replicated the significant divergence of P. flavoviridis between the Amami Clade including Amami-Oshima, Kakeromajima and Tokunoshima and the Okinawa Clade including Okinawajima, Iheyajima, Iejima, Tokashikijima and Kumejima. The Amami Clade also include all specimens from the Tokara Islands currently known as an independent species, P. tokarensis, suggesting the paraphyly of the taxon, P. flavoviridis. In contrast, we observed a distinct lineage of the two specimens from the Yaeyama Islands, supporting the validity of the taxon, P. elegans as an independent species. By MCMC method, we estimated the divergence time between the Amami Clade and the Okinawa Clade to be 6.51MYA, suggesting that the vicariance of the two clades preceded the geological separation of the Amami Islands and the Okinawa Islands (∼1.5MYA). As expected from the limited mobility of terrestrial reptiles including snakes, we observed high genetic divergence in Habu mtDNA among Japanese subtropical island populations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ympev.2016.04.027DOI Listing
August 2016

Amyloidosis enhancing activity of bovine amyloid A fibrils in C3H/HeN mice and cynomolgus monkeys (Macaca fascicularis).

J Med Primatol 2016 06 12;45(3):112-7. Epub 2016 Apr 12.

Department of Veterinary Medicine, Gifu University, Gifu, Japan.

Background: In experimentally induced cases of AA amyloidosis, the development of disease is enhanced by the administration of homogenous or heterogeneous amyloid fibrils. In recent years, cross-species transmission of animal amyloidosis into human has become of particular concern.

Methods: Cynomolgus monkeys (Macaca fascicularis) and C3H/HeN mice were inoculated with bovine amyloid fibrils under acute inflammation.

Results: Amyloid A deposits were not detected in any of the monkeys, but mild-to-severe AA deposits were found in all mice.

Conclusions: These results suggest that unlike in rodents, cross-species transmission of AA amyloidosis is less likely to develop, at least during acute inflammation, in primates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jmp.12213DOI Listing
June 2016

Intracytoplasmic sperm injection into oocytes matured in vitro and early embryonic development in the owl monkey ().

Reprod Med Biol 2016 07 16;15(3):183-186. Epub 2015 Dec 16.

United Graduate School of Agricultural Science Tokyo University of Agriculture and Technology 183-8509 Fuchu-shi Japan.

Purpose: We explored the possibility of employing intracytoplasmic sperm injection (ICSI), involving oocytes and sperm of owl monkeys, to increase the availability of this species for investigations relating to malaria, etc., by increasing the number of animals in our laboratory.

Methods: Two owl monkeys (a female and a male), raised at the Amami Laboratory of the University of Tokyo, were used. Follicular oocytes surrounded with cumulus cells were cultured in vitro for approximately 25 h and cumulus cells were removed with 0.1 % hyaluronidase. Because of the poor motility of caudal epididymal sperm, sperm were injected without adding polyvinylpyrrolidone to immobilize them. The ICSI procedure was performed by an individual with considerable experience of human ICSI.

Results: We were able to produce two owl monkey embryos using ICSI of oocytes that matured to MII stage. Both embryos reached the 10-cell stage at 98 h after ICSI and showed signs of compaction, but failed to cleave further.

Conclusions: Although we successfully produced owl monkey embryos after ICSI, the embryos did not develop to the blastocyst stage. Many parameters need to be studied further, including superovulation, selection of culture media, and selection of good quality sperm in order to achieve successful ICSI in the owl monkey.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12522-015-0229-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5715853PMC
July 2016

Interisland variegation of venom [Lys(49)]phospholipase A2 isozyme genes in Protobothrops genus snakes in the southwestern islands of Japan.

Toxicon 2015 Dec 31;107(Pt B):210-6. Epub 2015 Aug 31.

Department of Applied Life Science, Faculty of Bioscience, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan.

Protobothrops tokarensis (Pt), a Crotalinae snake, inhabits only Takarajima and Kodakarajima islands of the Tokara Islands located in the immediate north of Amami-Oshima island of Japan. Kodakarajima P. tokarensis venom gland cDNA library gave four types of phospholipase A2 (PLA2) cDNAs encoding neutral [Asp(49)]PLA2, basic [Asp(49)]PLA2, highly basic [Asp(49)]PLA2, and [Lys(49)]PLA2. As the amino acid sequences encoded by their open reading frames (ORFs) were identical to those of PLA2, PLA-B, PLA-N, and BPI (a [Lys(49)]PLA2), respectively, from Amami-Oshima P. flavoviridis (Pf) venom, they were named PtPLA2, PtPLA-B, PtPLA-N, and PtBPI. Chromatography of P. tokarensis venom gave three PLA2 isozymes, PtPLA2, PtPLA-B, and PtBPI. However, BPII and BPIII ([Lys(49)]PLA2s) expressed in Amami-Oshima P. flavoviridis venom were not found in P. tokarensis venom. Genomic polymerase chain reaction (PCR) for P. tokarensis liver DNAs with the unique primers gave PtBPI gene. Notably it was found that LINE (long interspersed nuclear element)-1 fragment is inserted into second intron of PtBPI gene. The LINE-1 fragment may prevent duplication of PtBPI gene and thus formation of plural [Lys(49)]PLA2 genes in P. tokarensis genome. The interisland variegation of venom [Lys(49)]PLA2 isozyme genes in Protobothrops genus snakes in the southwestern islands of Japan is discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.toxicon.2015.08.024DOI Listing
December 2015

Histocytological specificities of adrenal cortex in the New World Monkeys, Aotus lemurinus and Saimiri boliviensis.

J Vet Med Sci 2016 Jan 28;78(1):161-5. Epub 2015 Aug 28.

Laboratory of Basic Veterinary Science, The United Graduate School of Veterinary Science, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.

The New World monkey Aotus spp. (night monkeys) are expected for use of valuable experimental animal with the close species of Saimiri spp. (squirrel monkeys). Saimiri is known to show spontaneous hypercortisolemia, although few reports in Aotus. We compared basic states of blood steroid hormones and histological structure of the adrenal glands in two monkeys. Serum cortisol and ACTH levels were statistically lower in Aotus than Saimiri. Conversely, Aotus adrenocortical area showed significant enlargement, especially at the zona fasciculata. Electron microscopic observation at Aotus fasciculata cells revealed notable accumulation of large lipid droplets and irregular shapes of the mitochondrial cristae. These results suggest potential differences in cellular activities for steroidogenesis between Aotus and Saimiri and experimental usefulness in adrenocortical physiology and pathological models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1292/jvms.15-0290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751139PMC
January 2016

The finding of a group IIE phospholipase A2 gene in a specified segment of Protobothrops flavoviridis genome and its possible evolutionary relationship to group IIA phospholipase A2 genes.

Toxins (Basel) 2014 Dec 18;6(12):3471-87. Epub 2014 Dec 18.

Department of Applied Life Science, Faculty of Bioscience and Biotechnology, Sojo University, Kumamoto 860-0082, Japan.

The genes encoding group IIE phospholipase A2, abbreviated as IIE PLA2, and its 5' and 3' flanking regions of Crotalinae snakes such as Protobothrops flavoviridis, P. tokarensis, P. elegans, and Ovophis okinavensis, were found and sequenced. The genes consisted of four exons and three introns and coded for 22 or 24 amino acid residues of the signal peptides and 134 amino acid residues of the mature proteins. These IIE PLA2s show high similarity to those from mammals and Colubridae snakes. The high expression level of IIE PLA2s in Crotalinae venom glands suggests that they should work as venomous proteins. The blast analysis indicated that the gene encoding OTUD3, which is ovarian tumor domain-containing protein 3, is located in the 3' downstream of IIE PLA2 gene. Moreover, a group IIA PLA2 gene was found in the 5' upstream of IIE PLA2 gene linked to the OTUD3 gene (OTUD3) in the P. flavoviridis genome. It became evident that the specified arrangement of IIA PLA2 gene, IIE PLA2 gene, and OTUD3 in this order is common in the genomes of humans to snakes. The present finding that the genes encoding various secretory PLA2s form a cluster in the genomes of humans to birds is closely related to the previous finding that six venom PLA2 isozyme genes are densely clustered in the so-called NIS-1 fragment of the P. flavoviridis genome. It is also suggested that venom IIA PLA2 genes may be evolutionarily derived from the IIE PLA2 gene.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/toxins6123471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280545PMC
December 2014

Epithelium specific ETS transcription factor, ESE-3, of Protobothrops flavoviridis snake venom gland transactivates the promoters of venom phospholipase A2 isozyme genes.

Toxicon 2014 Dec 22;92:133-9. Epub 2014 Oct 22.

Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto 860-0082, Japan. Electronic address:

Protobothrops flavoviridis (habu) (Crotalinae, Viperidae) is a Japanese venomous snake, and its venom contains the enzymes with a variety of physiological activities. The phospholipases A2 (PLA2s) are the major components and exert various toxic effects. They are expressed abundantly in the venom gland. It is thought that the venom gland-specific transcription factors play a key role for activation of PLA2 genes specifically expressed in the venom gland. Thus, the full-length cDNA library for P. flavoviridis venom gland after milking of the venom was made to explore the transcription factors therein. As a result, three cDNAs encoding epithelium-specific ETS transcription factors (ESE)-1, -2, and -3 were obtained. Among them, ESE-3 was specifically expressed in the venom gland and activated the proximal promoters of venom PLA2 genes, which are possibly regarded as the representatives of the venom gland-specific protein genes in P. flavoviridis. Interestingly, the binding specificity of ESE-3 to the ETS binding motif located near TATA box is well correlated with transcriptional activities for the venom PLA2 genes. This is the first report that venom gland-specific transcription factor could actually activate the promoters of the venom protein genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.toxicon.2014.10.001DOI Listing
December 2014

Failure of heterogeneous amyloid-enhancing factor in geriatric squirrel monkeys (Saimiri boliviensis).

J Med Primatol 2014 Dec 19;43(6):488-91. Epub 2014 Jul 19.

Laboratory of Veterinary Toxicology, Tokyo University of Agriculture and Technology, Tokyo, Japan.

Background: Cross-species transmission of AA amyloidosis between primates and other animals has not been previously reported.

Methods: Eight geriatric squirrel monkeys were intravenously administered chimpanzee, bovine, or chicken amyloid fibrils and simultaneously received inflammatory stimulation.

Results: AA amyloid deposition was not detected in any of the monkeys histopathologically or immunohistochemically.

Conclusions: These results suggest that heterogeneous AA amyloidosis may not be easily transmitted into primates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jmp.12136DOI Listing
December 2014

Dermatitis associated with infestation of a trombiculid mite, Leptotrombidium miyajimai, in an Amami rabbit (Pentalagus furnessi).

J Wildl Dis 2014 Apr 31;50(2):416-8. Epub 2014 Jan 31.

1  Laboratory of Veterinary Pathology, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan.

Severe dermatitis caused by trombiculid mite infestation was observed in an Amami rabbit (Pentalagus furnessi). The mite was identified as Leptotrombidium miyajimai. This is the first report of trombiculid mite-associated cutaneous lesions in Amami rabbits and also the first direct evidence of L. miyajimai parasitism of this host species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7589/2013-10-257DOI Listing
April 2014

Experimental infection of macaques with a wild water bird-derived highly pathogenic avian influenza virus (H5N1).

PLoS One 2013 18;8(12):e83551. Epub 2013 Dec 18.

Laboratory Animal Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Highly pathogenic avian influenza virus (HPAIV) continues to threaten human health. Non-human primate infection models of human influenza are desired. To establish an animal infection model with more natural transmission and to determine the pathogenicity of HPAIV isolated from a wild water bird in primates, we administered a Japanese isolate of HPAIV (A/whooper swan/Hokkaido/1/2008, H5N1 clade 2.3.2.1) to rhesus and cynomolgus monkeys, in droplet form, via the intratracheal route. Infection of the lower and upper respiratory tracts and viral shedding were observed in both macaques. Inoculation of rhesus monkeys with higher doses of the isolate resulted in stronger clinical symptoms of influenza. Our results demonstrate that HPAIV isolated from a water bird in Japan is pathogenic in monkeys by experimental inoculation, and provide a new method for HPAIV infection of non-human primate hosts, a good animal model for investigation of HPAIV pathogenicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0083551PLOS
October 2014

Suppression of severe lesions, myonecrosis and hemorrhage, caused by Protobothrops flavoviridis venom with its serum proteins.

Toxicon 2013 Dec 15;76:197-205. Epub 2013 Oct 15.

Department of Applied Life Science, Faculty of Bioscience and Biotechnology, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan. Electronic address:

Protobothrops flavoviridis serum proteins precipitated with ammonium sulfate were chromatographed on a DEAE-Toyopearl 650M column at pH 7.5 with stepwise increase or with linear gradient of NaCl concentration. Peaks 3 and 4 serum proteins, obtained by linear gradient elution and named Fr(de3) and Fr(de4), contained Habu serum factors (HSF) and phospholipase A2 (PLA2) inhibitors (PfPLI), respectively. The serum proteins eluted at 0.2 M NaCl by stepwise elution, named Fr(0.2NaCl), effectively suppressed myonecrosis and hemorrhage caused by P. flavoviridis venom in rat or mouse thigh muscles. The Fr(0.2NaCl) were fractionated by HPLC and the fractions, after SDS-PAGE, underwent far-western blot analysis with PLA2 ([Asp(49)]PLA2) and BPI ([Lys(49)]PLA2) as the probes. Four PfPLIs, namely, PfαPLI-A, PfαPLI-B, PfγPLI-A and PfγPLI-B, were identified together with their selective binding specificities to PLA2 species. In addition, a new 9 kDa protein, which is specifically bound to BPI, was found. Suppression of P. flavoviridis venom-induced severe lesions, such as myonecrosis, hemorrhage and edema, with its serum proteins was histopathologically observed in the present work for the first time. The cooperative use of P. flavoviridis antivenom and its serum proteins as medication for P. flavoviridis snake bites is discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.toxicon.2013.10.007DOI Listing
December 2013

Infectivity of Cryptosporidium andersoni and Cryptosporidium muris to normal and immunosuppressive cynomolgus monkeys.

J Vet Med Sci 2014 Mar 16;76(2):169-72. Epub 2013 Oct 16.

Drug Developmental Research Laboratories, Shionogi & Co., Ltd., 3-1-1, Futaba-Cho, Toyonaka, Osaka 561-0825, Japan.

Cryptosporidium andersoni and Cryptosporidium muris infections have been found in the mice and/or cattle. The oocysts of C. andersoni and C. muris have been sporadically detected in human feces, but the infectious capacity and features have been unknown, because of the scarcity of reports involving human infections. To assess the infectivity and the clinical and pathological features of C. andersoni and C. muris in primates, an experimental infectious study was conducted using cynomolgus monkeys. The monkeys were orally inoculated with oocysts of two different C. andersoni Kawatabi types and C. muris RN-66 under normal and immunosuppressive conditions. The feces of the monkeys were monitored for about 40 days after the administration of oocysts using the flotation method, but no shedding oocysts were observed under either both normal or immunosuppressive conditions. Gross and histopathological examinations were performed on the immunosuppressive monkeys, but these revealed no evidence of Cryptosporidium infections, even though the monkeys were subjected to immunosuppressive conditions. It is hypothesized that C. andersoni and C. muris pose little danger of infection in primates even under immunosuppressive conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1292/jvms.13-0350DOI Listing
March 2014

Structural characteristics and evolution of the Protobothrops elegans pancreatic phospholipase A2 gene in contrast with those of Protobothrops genus venom phospholipase A2 genes.

Biosci Biotechnol Biochem 2013 7;77(1):97-102. Epub 2013 Jan 7.

Department of Applied Life Science, Faculty of Bioscience and Biotechnology, Sojo University, Kumamoto, Japan.

The nucleotide sequence of the gene encoding Protobothrops elegans (Crotalinae) pancreatic phospholipase A(2) (PLA(2)), abbreviated PePancPLA(2), was determined by means of inverted PCR techniques. Since its deduced amino acid sequence contains a pancreatic loop and shows high similarity to that of Laticauda semifasciata (Elapinae) group IB pancreatic PLA(2), PePancPLA(2) is classified into group IB PLA(2). The nucleotide sequences of the PePancPLA(2) gene, the L. semifasciata group IB pancreatic PLA(2) gene, and the L. semifasciata group IA venom PLA(2) gene are similar to one another but greatly dissimilar to those of Protobothrops genus (Crotalinae) group II venom PLA(2) genes, suggesting that the Elapinae group IB PLA(2) gene and the group IA PLA(2) gene appeared after Elapinae was established, and that the Crotalinae group II venom PLA(2) genes came into existence before Elapinae and Crotalinae diverged. A phylogenetic analysis of their amino acid sequences confirms this.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1271/bbb.120595DOI Listing
July 2013

Phylogenetic relationships of three species within the family Heligmonellidae (Nematoda; Heligmosomoidea) from Japanese rodents and a lagomorph based on the sequences of ribosomal DNA internal transcribed spacers, ITS-1 and ITS-2.

Jpn J Vet Res 2012 Feb;60(1):15-21

Unit of Veterinary Parasitology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan.

Nematodes of the family Heligmonellidae (Heligmosomoidea; Trichostrongylina) reside in the digestive tracts of rodents and lagomorphs. Although this family contains large numbers of genera and species, genetic information on the Heligmonellidae is very limited. We collected and isolated adult worms of three species in Japan that belong to the family Heligmonellidae, namely Heligmonoides speciosus (Konno, 1963) Durette-Desset, 1970 (Hs) from Apodemus argenteus, Orientostrongylus ezoensis Tada, 1975 (Oe) from Rattus norvegicus and Lagostrongylus leporis (Schulz, 1931) (Ll) from Pentalagus furnessi, and sequenced the entire internal transcribed spacer regions, ITS-1 and ITS-2 of ribosomal DNA. ITS-1 of Hs, Oe and Ll was 426, 468 and 449 bp in length, and had a G+C content of about 41, 41 and 37 %, respectively. ITS-2 of Hs, Oe and Ll was 297, 319 and 276 bp in length and had a G+C content of about 38, 40 and 28%, respectively. The data of Hs, Oe and Ll were compared with those of two other known species within the family Heligmonellidae, Calorinensis minutus (Dujardin, 1845) (Cm) and Nippostrogylus brasiliensis (Travassos, 1914) (Nb), and with those of two species of Heligmosomidae (Heligmosomoidea), Heligmosomoides polygyrus bakeri and Ohbayashinema erbaevae. Phylogenetic analysis placed Hs, Oe and Ll in the same clade with Cm and Nb, forming a Heligmonellidae branch in both ITS-1 and ITS-2, separate from the Heligmosomoidea branch. These results demonstrated that the ITS-1 and ITS-2 sequences are useful for differentiating the Heligmonellidae nematode species. This study is the first to describe the ITS-1 and ITS-2 sequences of Hs, Oe and Ll.
View Article and Find Full Text PDF

Download full-text PDF

Source
February 2012

Structural characteristics and evolution of a novel venom phospholipase A2 gene from Protobothrops flavoviridis.

Biosci Biotechnol Biochem 2012 ;76(3):551-8

Department of Applied Life Science, Faculty of Bioscience and Biotechnology, Sojo University, Ikeda, Kumamoto, Japan.

A novel phospholipase A(2) (PLA(2)) gene, named PfPLA 6, was found in a 6,328-bp NIS-1(5')-a segment in the Protobothrops flavoviridis (Habu, Crotalinae) genome. A comparison of the aligned nucleotide sequences of Viperidae (Viperinae and Crotalinae) venom PLA(2) genes, including PfPLA 6, revealed the deletion of a 12-bp segment called S1EX 1 and a 55-bp segment called S2EX 1 in exon 1 and the interposition of a 219-bp segment called SINT 2 (SINE) in intron 2. A classification of Viperidae PLA(2) genes based on these structural modes indicated that the A-type genes (without SINE), including PfPLA 6, are evolutionarily ancestral to the B-type (Viperinae) and C-type (Crotalinae) PLA(2) genes (both with SINE). Since PfPLA 6 is a pseudogene, an active prototype of PfPLA 6 can be assumed to be the ancestral PLA(2) gene. Putative evolutionary processes from this A-type prototype PLA(2) gene to descendent PLA(2) genes are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1271/bbb.110848DOI Listing
July 2012

Identification and evolution of venom phospholipase A2 inhibitors from Protobothrops elegans serum.

Biosci Biotechnol Biochem 2011 7;75(3):480-8. Epub 2011 Mar 7.

Department of Applied Life Science, Faculty of Bioscience and Biotechnology, Sojo University, Ikeda, Kumamoto, Japan.

The cDNAs encoding venom phospholipase A(2) (PLA(2)) inhibitors (PLIs), named Protobothrops elegans (Pe)γPLI-A, PeγPLI-B, PeαPLI-A, and PeαPLI-B, were cloned from the P. elegans liver cDNA library. They were further divided into several constituents due to nucleotide substitutions in their open reading frames. For PeαPLI-A, two constituents, PeαPLI-A(a) and PeαPLI-A(b), were identified due to three nonsynonymous substitutions in exon 3. Far-western blot and mass-spectrometry analysis of the P. elegans serum proteins showed the presence of γPLIs, and αPLIs, which can bind venom PLA(2)s. In αPLIs from Protobothrops sera, A or B subtype-specific amino acid substitutions are concentrated only in exon 3. A comparison of γPLIs showed that γPLI-As are conserved and γPLI-Bs diversified. Mathematical analysis of the nucleotide sequences of Protobothrops γPLI-B cDNAs revealed that the particular loops in the three-finger motifs diversified by accelerated evolution. Such evolutionary features should have made serum PLIs acquire their respective inhibitory activities to adapt to venom PLA(2) isozymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1271/bbb.100676DOI Listing
August 2011

Cynomolgus monkeys (Macaca fascicularis) may not become infected with equine herpesvirus 9.

J Med Primatol 2011 Feb;40(1):18-20

Laboratory of Veterinary Pathology, Department of Veterinary Medicine, Gifu University, Gifu, Japan.

Background: It was suggested that Equine herpesvirus 9 (EHV-9) could be transmitted to higher non-human primates.

Methods: Four cynomolgus monkeys (Macaca fascicularis) were inoculated with EHV-9 by the nasal route.

Results: No abnormalities were observed pathologically, immunohistochemically, and genetically.

Conclusions: These findings indicate that cynomolgus monkeys are not susceptible to EHV-9.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1600-0684.2010.00438.xDOI Listing
February 2011

Unique structural characteristics and evolution of a cluster of venom phospholipase A2 isozyme genes of Protobothrops flavoviridis snake.

Gene 2010 Aug 18;461(1-2):15-25. Epub 2010 Apr 18.

Department of Applied Life Science, Faculty of Bioscience and Biotechnology, Sojo University, Kumamoto 860-0082, Japan.

Protobothrops flavoviridis (Crotalinae) venom gland phospholipase A(2) (PLA(2)) isozyme genes have evolved in an accelerated manner to acquire diverse physiological activities in their products. For elucidation of the multiplication mechanism of PLA(2) genes, a 25,026 bp genome segment harboring five PLA(2) isozyme genes was obtained from Amami-Oshima P. flavoviridis liver and sequenced. The gene PfPLA 2 encoded [Lys(49)]PLA(2) called BPII, the gene PfPLA 4 neurotoxic [Asp(49)]PLA(2) called PLA-N, the gene PfPLA 5 basic [Asp(49)]PLA(2) called PLA-B, and PfPLA 1(psi) and PfPLA 3(psi) were the inactivated genes. The 5' truncated reverse transcriptase (RT) elements, whose intact forms constitute long interspersed nuclear elements (LINEs), were found in close proximity to the 3' end of PLA(2) genes and named PLA(2) gene-coupled RT fragments (PcRTFs). The facts that PcRTFs have the stem-loop and repetitive sequence in the 3' untranslated region (UTR) which is characteristic of CR1 LINEs suggest that PcRTFs are the debris of P. flavoviridis ancestral CR1 LINEs, denoted as PfCR1s. Since the associated pairs of PLA(2) genes and PcRTFs are arranged in tandem in the 25,026 bp segment, it is thought that an ancestral PLA(2) gene-PfCR1 unit (PfPLA-PfCR1) which was produced by retrotransposition of PfCR1 by itself to the 3' end of PLA(2) gene duplicated several times to form a multimer of PfPLA-PfCR1, a cluster of PLA(2) genes, in the period after Crotalinae and Viperinae snakes branched off. Recombinational hot spot of a 37bp segment, named Scomb, was found in the region 548 bp upstream from the TATA box of PLA(2) genes. Thus, it could be assumed that multiplication of PfPLA-PfCR1 occurred by unequal crossing over of the segment, -Scomb-PfPLA-PfCR1-Scomb-. The PfCR1 moieties were afterward disrupted in the 5' portion to PcRTFs. The detection of two types of PcRTFs different in length which were produced by elimination of two definitive sequences in PfCR1 moiety possibly by gene conversion clearly supports such process but not multiplication of the PLA(2) gene-PcRTF unit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.gene.2010.04.001DOI Listing
August 2010

Island specific expression of a novel [Lys(49)]phospholipase A(2) (BPIII) in Protobothrops flavoviridis venom in Amami-Oshima, Japan.

Toxicon 2009 Sep 20;54(4):399-407. Epub 2009 May 20.

Department of Bioscience and Biotechnology, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan.

In search of the transcripts expressed in Protobothrops flavoviridis venom gland, 466 expressed sequence tags (ESTs) were generated from the venom gland cDNA library of P. flavoviridis in Amami-Oshima, Japan. The sequencing of randomly selected cDNA clones followed by identification in similarity search against existing databases led to the finding of a novel lysine-49-phospholipase A(2) ([Lys(49)]PLA(2)) clone. It coded for one amino acid-substituted BPII homologue or two amino acids-substituted BPI homologue in which BPII and BPI are [Lys(49)]PLA(2)s contained in Amami-Oshima and Tokunoshima P. flavoviridis venoms. This isozyme, named BPIII, was isolated from Amami-Oshima P. flavoviridis venom. BPIII gave a specific [M+2H](2+) peak of m/z 736.3 on mass spectrometry (MS) analysis after S-carboxamidomethylation and trypsin digestion when compared with BPII. It became evident from MS analysis after S-carboxamidomethylation and trypsin digestion of the mixed protein peaks ranging from BPI to BPII obtained by fractionation on a carboxymethyl cellulose column of Amami-Oshima and Tokunoshima P. flavoviridis venoms that BPIII protein is contained in Amami-Oshima P. flavoviridis venom but not in Tokunoshima P. flavoviridis venom. It is for the first time that a protein present in Amami-Oshima P. flavoviridis venom is not found in Tokunoshima P. flavoviridis venom.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.toxicon.2009.05.003DOI Listing
September 2009

Identification of the B subtype of gamma-phospholipase A2 inhibitor from Protobothrops flavoviridis serum and molecular evolution of snake serum phospholipase A2 inhibitors.

J Mol Evol 2008 Mar 4;66(3):298-307. Epub 2008 Mar 4.

Department of Applied Life Science, Faculty of Bioscience and Biotechnology, Sojo University, 4-22-1 Ikeda, Kumamoto, 860-0082, Japan.

A cDNA encoding a novel phospholipase A(2) (PLA(2)) inhibitor (PLI) was isolated from a Protobothrops flavoviridis snake (Tokunoshima island, Japan) liver cDNA library. This cDNA encoded a signal peptide of 19 amino acids followed by a mature protein of 181 amino acids. Its N-terminal amino acid sequence was completely in accord with that of a PLI, named PLI-II, previously found in P. flavoviridis serum. PLI-II showed a high similarity in sequence to the B subtype of gammaPLI, denoted gammaPLI-B, isolated from Agkistrodon blomhoffii siniticus serum. Thus, PLI-II is P. flavoviridis serum gammaPLI-B. Since PLI-I, previously isolated from P. flavoviridis serum, can be assigned as gammaPLI-A, P. flavoviridis serum contains both A and B subtypes of gammaPLI. Phylogenetic analysis of gammaPLIs from the sera of various kinds of snakes, Elapinae, Colubrinae, Laticaudinae, Acanthophiinae, Crotalinae, and Pythonidae, based on the amino acid sequences revealed that A and B subtypes of gammaPLIs are clearly separated from each other. It was also found that phylogenetic topologies of gammaPLIs are in good agreement with speciation processes of snakes. The BLAST search followed by analyses with particular Internet search engines of proteins with Cys/loop frameworks similar to those of PLI-II and PLI-I revealed that gammaPLI-Bs, including PLI-II and PLI-II-like proteins from mammalian sources, form a novel PLI-II family which possesses the common Cys/loop frameworks in the anterior and posterior three-finger motifs in the molecules. Several lines of evidence suggest that PLI-II is evolutionarily ancestral to PLI-I.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00239-008-9089-1DOI Listing
March 2008
-->