Focused Proteomics Analysis of Habu Snake (Protobothrops flavoviridis) Venom Using Antivenom-Based Affinity Chromatography Reveals Novel Myonecrosis-Enhancing Activity of Thrombin-Like Serine Proteases.

Snakebites are one of the major causes of death and long-term disability in the developing countries due to the presence of various bioactive peptides and proteins in snake venom. In Japan, the venom of the habu snake (Protobothrops flavoviridis) causes severe permanent damage due to its myonecrotic toxins. Antivenom immunoglobulins are an effective therapy for snakebites, and antivenom was recently developed with effective suppressive activity against myonecrosis induced by snake venom. To compare the properties of an antivenom having anti-myonecrotic activity with those of conventional antivenom with no anti-myonecrotic activity, this study applied focused proteomics analysis of habu venom proteins using 2D gel electrophoresis. As a target protein for antivenom immunoglobulins with anti-myonecrotic activity, we identified a thrombin-like serine protease, TLSP2 (TLf2), which was an inactive proteolytic isoform due to the replacement of the active site, His43 with Arg. Additionally, we identified the unique properties and a novel synergistic function of pseudoenzyme TLf2 as a myonecrosis-enhancing factor. To our knowledge, this is the first report of a function of a catalytically inactive snake serine protease.

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

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.

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

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.

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

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.

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

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 in vitro. 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.

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

Small serum proteins (SSPs) are low-molecular-weight proteins in snake serum with affinities for various venom proteins. Five SSPs, PfSSP-1 through PfSSP-5, have been reported in Protobothrops flavoviridis ("habu", Pf) serum so far. Recently, we reported that the five genes encoding these PfSSPs 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.

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

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.

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

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.

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

The nucleotide sequence of Protobothrops flavoviridis (Pf) 30534 bp genome segment which contains genes encoding small serum proteins (SSPs) was deciphered. The genome segment contained five SSP genes (PfSSPs), PfSSP-4, PfSSP-5, PfSSP-1, PfSSP-2, and PfSSP-3 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 P. flavoviridis and Ophiophagus hannah (Oh) showed that the nucleotide segment encompassing from PfSSP-1 to PfSSP-2 was inverted. The inactive form of PfSSP-1, named PfSSP-1δ(Ψ), found in the intergenic region (I-Reg) between PfSSP-5 and PfSSP-1 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 PfSSPs into two subgroups, the Long SSP subgroup of PfSSP-1, PfSSP-2 and PfSSP-5 or the Short SSP subgroup of PfSSP-3 and PfSSP-4 The mathematical analysis also showed that PfSSPs 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 SSPs of various snakes showed that the evolutionary emerging order of SSPs was as follows: SSP-5, SSP-4, SSP-2, SSP-1, and SSP-3 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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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

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.

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.

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.

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

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.

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

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.