Inhibition of natural type I IFN-producing and dendritic cell development by a small molecule receptor tyrosine kinase inhibitor with Flt3 affinity

In vivo steady-state type I natural IFN-producing and dendritic cell (DC) development is largely dependent on Flt3 signaling. Natural IFN-producing and DC progenitors and their respective downstream cell populations express the flt3 receptor, and Flt3 ligand (Flt3L)(-/-) mice have reduced while Flt3L-injected mice develop markedly increased numbers of both cell types. In the present study, we show that SU11657, a small multitargeted receptor tyrosine kinase inhibitor with Flt3 affinity, suppressed in vitro natural IFN-producing and DC development in Flt3L-supplemented mouse whole bone marrow cell cultures in a dose-dependant manner, while DC development in GM-CSF-supplemented cultures was not affected. In vivo SU11657 application led to a significant decrease of both natural IFN-producing and DCs, comparable to the reduction observed in Flt3L(-/-) mice. Conversely, Flt3L plasma levels increased massively in inhibitor-treated animals, likely via a regulatory feedback loop, without being able to compensate for pharmacological Flt3 inhibition. No obvious toxicity was observed, and hemopoietic progenitor cell and stem cell function remained intact as assessed by myeloid colony-forming unit activity and in vivo bone marrow repopulation assays. Furthermore, upon treatment discontinuation, IFN-producing and DCs recovered to normal levels, proving that treatment effects were transient. Given the importance of IFN-producing and DCs in regulation of immune responses, these findings might lead to new pharmacological strategies in prevention and treatment of autoimmune diseases and complications of organ or blood cell transplantation.     

The targeted disruption of the MYPT1 gene results in embryonic lethality

Myosin phosphatase (MP) is a major phosphatase responsible for the dephosphorylation of the regulatory light chain of myosin II. MYPT1, a target subunit of smooth and nonmuscle MP, is responsible for activation and regulation of MP. To identity the physiological roles of MP, we have generated MYPT1-deficient mice by gene targeting. The heterozygous mice showed no changes in expression levels of MYPT1 and no distinct phenotype compared to wild-type mice was observed. None of the F2 mice were homozygous for the MYPT1 deletion, indicating that the targeted disruption of the MYPT1 gene resulted in embryonic lethality. The point of embryonic lethality is before 7.5 dpc. These findings indicate that MYPT1 is essential for mouse embryogenesis.     

Monoclonal Antibody #5-2-26 Recognizes the Phosphatase-Sensitive Epitope of Rabies Virus Nucleoprotein

We prepared monoclonal antibodies (MAbs) against the rabies virus N protein, among which one antibody (MAb 5-2-26) was shown to lack reactivity with the phosphatase-treated N protein. The MAb was able to recognize the sodium dodecyl sulfate (SDS)-denatured N protein. The MAb did not recognize the N-protein analogues produced in Escherichia coli (E. coli), indicating that the N-gene products were not normally processed in E. coli after translation. On the other hand, the MAb reacted normally with N-gene products produced in COS-7 cells, but not with those produced in the presence of K-252a (a protein kinase inhibitor of a broad spectrum). The MAb displayed weak cross-reactivity with the Triton-insoluble network structures composed of several components, while another phosphoprotein (M1) of the virus was not recognized at all. These results suggest that MAb 5-2-26 preferentially recognizes a phosphatase-sensitive linear epitope of N protein, which may enable further investigations to be conducted on the mechanism of N-protein phosphorylation and its role(s) in virus replication.     

Aggregation of Thy-1 Glycoprotein Induces Thymocyte Apoptosis through Activation of CPP32-like Proteases

Mouse thymocytes are known to undergo apoptosis by ligating some unique anti-Thy-1 monoclonal antibodies (mAbs), G7 and KT16. However, the precise mechanisms of Thy-1-mediated apoptosis are as yet unclear. We investigated Thy-1-mediated apoptosis using our previously generated anti-Thy-1 mAb, MCS-34, which was similar to G7 because both antibodies recognized both Thy-1.1 and Thy-1.2 and bound Thy-1A epitope. Unlike G7, MCS-34 alone could not induce apoptosis in thymocytes; however, it could induce apoptosis when it was cross-linked with second antibodies. Thus, MCS-34 could not aggregate by itself, but G7 could. In the course of investigating the apoptosis-related molecules that were involved in the thymocyte apoptosis induced by cross-linking of MCS-34 or by G7 ligation, we found that CPP32-like proteases were activated during the apoptosis. Furthermore, the expression of bcl-2 and bcl-X_Lproteins was decreased in these apoptosis processes. Whereas the ligation of MCS-34 alone could not generate apoptosis signals that led to the activation of CPP32-like proteases and the decrease in bcl-2 and bcl-X_Lexpression, the aggregation of Thy-1 glycoprotein might be crucial to signal thymocyte apoptosis. These results indicate that MCS-34 is a useful anti-Thy-1 mAb for analyzing the Thy-1-mediated signals since MCS-34 can control the level of apoptosis by using second antibodies.     

Structure of the Mutant Fibrillin-1 Gene in the Tight Skin (TSK) Mouse

Mice carrying the tight skin (TSK) mutation harbors a 3.0-kbgenomic duplication (exons 17–40) of the fibrillin-1 gene(Fbn-1) locatedon band F of chromosome 2 as TSK mutation. Wecloned and sequenced the mutated Fbn-1 gene, since it is believedto be responsible for TSK syndrome. Sequence analysis showednumerous amino aciddifferences in the 5' and 3' segments betweenthe TSK mutation and wild-type fbn-1 gene, but any amino aciddifference between the TSK mutation and C57BL/6 mice. (TSK andC57Bl/6 mice are genetically similar, differing only by TSKmutation.) Four amino acid differences were observed betweentwo copies of TSK's fbn-1 gene encoded by exons 17–40.Our results suggest that the majority of structural differencesoccurred in the N and C termini segments during strain divergenceand only a few after the duplication event.     

Large schools of Japanese sardines,Sardinops melanostictus, mate in single pair units at night

Synopsis We observed the behavior of the spawning group of Japanese sardine,Sardinops melanostictus, in the field at night, and also confirmed the spawning behavior in the darkness in captivity. The chasing and mating were done in a pair of 1 male and 1 female. The 1-to-1 mating was a definite finding irrespective of the group density in the field, the density in the tank in captivity, or the sex ratio. This maybe because there is little visual information during spawning at night.     

LIFE HISTORY OF FRASER'S DOLPHIN, LAGENODELPHIS HOSEI, BASED ON A SCHOOL CAPTURED OFF THE PACIFIC COAST OF JAPAN

Life history of Fraser's dolphin, a little known delphinid species, was examined based on 108 specimens from a school captured by the driving fishing method in Japan. The sex ratio was approximately 1:1, and mature dolphins of both sexes formed the bulk of the school. The oldest animals were two males and a female of 17.5 yr. Age and body length at sexual maturity were estimated at 7–10 yr and 220–230 cm in males and 5–8 yr and 210–220 cm in females. Mature males were larger in body length than mature females and showed apparent secondary sexual features: deepening of the tail stock and widening and darkening of the lateral dark stripe. The annual ovulation rate was 0.49. The estimated neonatal length (110 cm) predicts a gestation period of about 12.5 mo and calving peaks in spring and probably also in fall. The calving interval was estimated to be about 2 yr. These life history parameters are similar to those of the striped and pantropical spotted dolphins, except for longevity. The reproductive rate of this species may be lower than that of other pelagic delphinids, if the observed shorter longevity is real.