Monoclonal Suncus Antibodies: Generation of Fusion Partners to Produce Suncus-Suncus Hybridomas.

We used suncus (Suncus murinus; house musk shrew) to generate partner cells for cell fusion to produce suncus monoclonal antibodies. Suncus are insectivores that are genetically distant to rodents, and recognize antigens and epitopes that are not immunogenic in mice and rats, which are the animals most commonly used in basic life science research and from which monoclonal antibodies are usually produced. To date, monoclonal antibodies from suncus have not been generated due to the lack of a plasmacytoma fusion partner. To obtain suncus plasmacytoma cell lines suitable as a cell fusion partner, we injected suncus at both sides of the tail base with antigen emulsion, collected the lymph nodes and spleens, and cultured the cells to obtain immortalized lymphoid cell lines visually resembling mouse SP2/0-Ag14 myeloma cells. Three suncus immunized with the antigen provided 4 cell lines of suncus plasmacytoma, but they did not secrete immunoglobulins. Antibody-producing hybrid cells were generated from these cell lines using a cell fusion technique. Using one of the cell lines as a fusion partner, we obtained six lines of immunoglobulin-producing hybrid cells which secreted an unidentified monoclonal IgG. When these 6 lines were used as new fusion partners, we obtained several hybrid cell lines which secreted immunogen-specific monoclonal antibodies. These hybrid cells can be cloned and cryopreserved. We also obtained another good fusion partner which initially secreted antibody but later stopped doing so. These suncus-suncus hybrid cell lines will be useful for the production of suncus monoclonal antibodies.

Mechanism of ghrelin-induced gastric contractions in Suncus murinus (house musk shrew): involvement of intrinsic primary afferent neurons.

Here, we have reported that motilin can induce contractions in a dose-dependent manner in isolated Suncus murinus (house musk shrew) stomach. We have also shown that after pretreatment with a low dose of motilin (10(-10) M), ghrelin also induces gastric contractions at levels of 10(-10) M to 10(-7) M. However, the neural mechanism of ghrelin action in the stomach has not been fully revealed. In the present study, we studied the mechanism of ghrelin-induced contraction in vitro using a pharmacological method. The responses to ghrelin in the stomach were almost completely abolished by hexamethonium and were significantly suppressed by the administration of phentolamine, prazosin, ondansetron, and naloxone. Additionally, N-nitro-l-arginine methylester significantly potentiated the contractions. Importantly, the mucosa is essential for ghrelin-induced, but not motilin-induced, gastric contractions. To evaluate the involvement of intrinsic primary afferent neurons (IPANs), which are multiaxonal neurons that pass signals from the mucosa to the myenteric plexus, we examined the effect of the IPAN-related pathway on ghrelin-induced contractions and found that pretreatment with adenosine and tachykinergic receptor 3 antagonists (SR142801) significantly eliminated the contractions and GR113808 (5-hydroxytryptamine receptor 4 antagonist) almost completely eliminated it. The results indicate that ghrelin stimulates and modulates suncus gastric contractions through cholinergic, adrenergic, serotonergic, opioidergic neurons and nitric oxide synthases in the myenteric plexus. The mucosa is also important for ghrelin-induced gastric contractions, and IPANs may be the important interneurons that pass the signal from the mucosa to the myenteric plexus.

Sensitivity of liver injury in heterozygous Sod2 knockout mice treated with troglitazone or acetaminophen.

Recently, it was reported that the intraperitoneal administration of 30 mg/kg/day troglitazone to heterozygous superoxide dismutase 2 gene knockout (Sod2+/-) mice for twenty-eight days caused liver injury, manifested by increased serum ALT activity and hepatic necrosis. Therefore, we evaluated the reproducibility of troglitazone-induced liver injury in Sod2+/- mice, as well as their validity as an animal model with higher sensitivity to mitochondrial toxicity by single-dose treatment with acetaminophen in Sod2+/- mice. Although we conducted a repeated dose toxicity study in Sod2+/- mice treated orally with 300 mg/kg/day troglitazone for twenty-eight days, no hepatocellular necrosis was observed in our study. On the other hand, six hours and twenty-four hours after an administration of 300 mg/kg acetaminophen, plasma ALT activity was significantly increased in Sod2+/- mice, compared to wild-type mice. In particular, six hours after administration, hepatic centrilobular necrosis was observed only in Sod2+/- mice. These results suggest that Sod2+/- mice are valuable as an animal model with higher sensitivity to mitochondrial toxicity. On the other hand, it was suggested that the mitochondrial damage alone might not be the major cause of the troglitazone-induced idiosyncratic liver injury observed in humans.

Insertion of an intracisternal A particle retrotransposon element in plasma membrane calcium ATPase 2 gene attenuates its expression and produces an ataxic phenotype in joggle mutant mice.

Using forward genetic analysis, we identified the insertion of an intracisternal A particle (IAP) retrotransposon element in the plasma membrane calcium ATPase 2 gene (Pmca2/Atp2b2) in the joggle mouse, a novel mutant that displays ataxic gait by postnatal day 12. Expression of Pmca2 mRNA in the joggle mouse is only 5% of that in the wild type mouse. The insertion is located 15 bp downstream of the donor splice site of the exon containing the initiation codon. Chimeric mRNA composed of the 5'-region of Pmca2 and the IAP element were detected, indicating that some of the primary transcripts are terminated by polyadenylation signals in long terminal repeats of the IAP element. We also identified cryptic splice sites in the IAP element that are likely involved in aberrant splicing of the Pmca2 primary transcripts that leads to rapid degradation of mRNA through nonsense mediated mRNA decay. Ataxia was observed in compound heterozygous mice carrying the joggle mutation and the wriggle mutation, a previously reported missense Pmca2 mutant. Thus, we attributed ataxia in joggle mice to reduced expression of Pmca2, resulting from insertion of the IAP element.

Premolar and molar rotation in wild Japanese serow populations on Honshu Island, Japan.

The skulls of 1195 Japanese serow (Capricornis crispus) from six geographically distinct populations were examined for tooth rotation. Our objectives were to determine tooth rotation patterns in Japanese serows, investigate geographical variation in tooth rotation and examine whether space limitations in the jaw accounted for variations. We then sought to explain the origin of tooth rotation in the Japanese serow. Rotated teeth were found in 131 specimens (62 males, 57 females and 12 unknown gender), with no statistically significant difference between males and females (chi(2)=0.03, P=0.86, d.f.=1). Among the six populations, the frequency of tooth rotation varied from 3.7-32.1% (average 11.0%). Most tooth rotation occurred in the upper third and fourth premolars. The lingual cusp of anomalous teeth was rotated 30 degrees -90 degrees mesially or distally from the buccolingual tooth axis, and the rotation direction differed among populations. However, we found no difference in skull or tooth morphology between normal individuals and those with tooth rotation. Therefore, hereditary factors may be involved in Japanese serow tooth rotation. We concluded that genetic differentiation occurred in the past among local Japanese serow populations isolated in mountainous habitats. Gene frequencies were likely subject to random drift, especially during possible population bottlenecks, when genetic factors could most strongly affect the direction of rotation.

Analysis of dental anomalies in the Siberian mole, Talpa altaica (Insectivora, Talpidae).

We re-examined tooth variation in specimens of the Siberian mole, Talpa altaica, from the collection of the Siberian Zoological Museum and discuss the mechanisms of dental evolution. The number of teeth counted in 1789 specimens ranged from 34 to 47, and supernumerary, absent, and connate teeth were observed. The most frequent tooth anomaly was an absent tooth in the premolar region (200 maxillary first premolars and 190 mandibular third premolars), which does not support Fujita and Kirino's terminal reduction hypothesis in the mandible [Fujita T, Kirino T. Ha No Kaibougaku. 21st ed. Tokyo: Kanehara Publishers Inc.; 1976 (in Japanese)]. Supernumerary teeth were found in premolar rows and in the incisor and molar regions. An maxillary fourth molar, positioned distal to the normal third molar, was thought to result from a genetically programmed atavistic event during the natal stages. Connate teeth were observed only in the premolar rows and were thought to have developed with the fusion of two independent tooth germs. Connate premolars appeared to result from an expression of an incomplete division of tooth germ at an early developmental stage or a reunion of independent tooth germs, based on the morphological similarity of the normal and supernumerary premolars. These extraordinarily frequent tooth anomalies of T. altaica are of much interest both in terms of tooth development and classification.

Variations in the number of teeth in wild Japanese serow (Naemorhedus crispus).

Skulls of 755 Japanese serow (Naemorhedus crispus) were examined for numerical anomalies of teeth, and these were found in 30 (4.0%) specimens. In addition to supernumerary and congenital missing teeth, embedded teeth were also identified. Supernumerary teeth were found in 10 (1.3%) specimens, congenital missing teeth were found in nine (1.2%) specimens, and embedded teeth were found in 11 (1.5%) specimens. Supernumerary teeth were found among the incisors, the upper and lower premolars, and the lower molars. Based on the appearance of certain tooth classes, their size, and their form, atavism was considered to be a possible cause of the occurrence of some of these teeth. Congenital missing teeth were found at the loci of the lower incisors, canines and P(2). In Japanese serow, there appeared to be a tendency for congenitally missing P(2) as well as P(1). All embedded teeth were found at the locus of P(2). These teeth may have occurred as an intermediate state between normal and congenital missing teeth. Variations in the number of teeth in Japanese serow were concordant with their phylogenetic background. Moreover, although variations in the number of cheek teeth tended to be concentrated in the mesial region of the premolars, the tendency in the incisive region was for teeth to appear mesial as well as distal within an incisor-tooth sequence. These results, which are not in accordance with terminal reduction theory, indicate that the phenomenon of tooth degeneration does not only occur at the end of a tooth row.