Takeshi Kuraishi

Chimeric VEGF-ENZ7/PlGF Specifically Binding to VEGFR-2 Accelerates Skin Wound Healing via Enhancement of Neovascularization

Objective—VEGF-ENZ7/PlGF molecules composed of Orf virus-derived VEGF-ENZ7 and human PlGF1 were previously proven to be potent angiogenic factors stimulating angiogenesis without significant enhancement of vascular leakage and inflammation in vivo. For its future clinical application, there is a pressing need to better understand the beneficial effects of VEGF-ENZ7/PlGF during wound healing in adulthood. Methods and Results—In this study, several angiogenic factors were administrated to skin punched wounds of both wild-type and diabetic mice. The treatment with VEGF-ENZ7/PlGF accelerated wound closure accompanied with enhanced angiogenesis, the process was occurring slightly faster than that in VEGF-A164 group. Moreover, the macrophage infiltration and lymphangiogenesis level in healed wounds were strikingly lower in VEGF-ENZ7/PlGF group than VEGF-A164 group, suggesting that the increased inflammation was the key issue preventing speedy wound healing of VEGF-A164–treated skin. Considering clinical safety, we further examined the antigenicity of chimeric VEGF-ENZ7/PlGF. Compared with the original VEGF-ENZ7, the immunogenicity of VEGF-ENZ7/PlGF molecules was markedly decreased in mice and squirrel monkeys with the increase of PlGF1 humanized ratio. Conclusion—These results indicate that VEGF-ENZ7/PlGF molecules are superior to VEGF-A for the acceleration of either normal or delayed skin wound healing and might be regarded as potential drugs in therapeutic angiogenesis.

The poly(A) tail length of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of milk.

The length of casein mRNA from the lactating mouse mammary gland, as assessed on Northern blots, is shorter after weaning, but is elongated following the removal of milk. In order to investigate this phenomenon, the molecular structures of beta- and gamma-casein mRNAs were analysed. The coding and non-coding regions of the two forms were the same length, but the long form of casein mRNA had a longer poly(A) tail than the short form (P<0.05). In order to examine the stability of casein mRNA under identical conditions, casein mRNAs with the long and short poly(A) tails were incubated in the rabbit reticulocyte lysate (RRL) cell-free translation system. Casein mRNA with the long poly(A) tail had a longer half-life than that with the short tail (P<0.05). The beta- and gamma-casein mRNAs were first degraded into 0.92 and 0.81 kb fragments respectively. With undegraded mRNA, the poly(A) tail shortening by exoribonuclease was not observed until the end of the incubation. Northern blot analysis showed that casein mRNA with the long poly(A) tail was protected efficiently from endoribonucleases. We conclude that the length of the poly(A) tail of casein mRNA in the lactating mammary gland changes depending upon the accumulation and removal of the glands milk, and we show that the longer poly(A) tail potentially protects the mRNA from degradation by endoribonucleases.

Measles virus selectively blind to signaling lymphocyte activation molecule as a novel oncolytic virus for breast cancer treatment

Oncolytic viruses hold much promise as novel therapeutic agents that can be combined with conventional therapeutic modalities. Measles virus (MV) is known to enter cells using the signaling lymphocyte activation molecule (SLAM), which is expressed on cells of the immune system. Although human breast cancer cell lines do not express SLAM, we found that a wild-type MV (HL strain) efficiently infected various breast cancer cell lines, causing cell death. Based on this finding, we used reverse genetics to generate a recombinant MV selectively unable to use SLAM (rMV-SLAMblind). The rMV-SLAMblind lacked infectivity for SLAM-positive lymphoid cells, while retaining oncolytic activity against breast cancer cells. We showed that, unlike the MV vaccine strains, rMV-SLAMblind used PVRL4 (polio virus receptor-related 4) as a receptor to infect breast cancer cells and not the ubiquitously expressed CD46. Consistent with this, rMV-SLAMblind infected CD46-positive primary normal human cells at a much-reduced level, whereas a vaccine strain of the Edmonston lineage (rMV-Edmonston) efficiently infected and killed them. The rMV-SLAMblind showed antitumor activity against human breast cancer xenografts in immunodeficient mice. The oncolytic activity of rMV-SLAMblind was significantly greater than that of rMV-Edmonston. To assess the in vivo safety, three monkeys seronegative for MV were inoculated with rMV-SLAMblind, and no clinical symptoms were documented. On the basis of these results, rMV-SLAMblind could be a promising candidate as a novel oncolytic virus for breast cancer treatment.

Acute neuropathogenicity with experimental infection of equine herpesvirus 9 in common marmosets (Callithrix jacchus).

Background  Equine herpesvirus 9 (EHV‐9) is a new neurotropic equine herpesvirus which induced encephalitis in a variety of animals. However, there was no information on the susceptibility of EHV‐9 in primates.

The casein mRNA decay changes in parallel with the poly(A) tail length in the mouse mammary gland

Using beta- and gamma-casein mRNAs, the relationship between poly(A) tail length and half-life of mRNA is determined in the mouse mammary gland during pregnancy and lactation. beta- and gamma-Casein mRNAs increase before and after parturition, respectively. The poly(A) tail as well as the half-life of casein mRNA becomes longer upon the active casein mRNA synthesis. The poly(A) tail is shortened gradually as lactation progresses. The half-life of mRNA decreases approximately from 20 h at early to 4 h at late lactation. Northern blot analysis reveals that nuclear RNA has the same poly(A) tail length as casein mRNA in the cytoplasm does. Thus, the mammary gland changes the poly(A) tail length of casein mRNA. The poly(A) tail length changes in parallel with the level of poly(A) polymerase (PAP) mRNA during pregnancy and lactation, suggesting that the mammary gland determines the poly(A) tail length of casein mRNA through the change in the PAP gene expression. As the half-life of casein mRNA is related with the degree of polyadenylation, we conclude that the poly(A) tail elongation and shortening is a mechanism in regulating the mRNA decay.

Identification and Evolution of Venom Phospholipase A2 Inhibitors from Protobothrops elegans Serum

The cDNAs encoding venom phospholipase A2 (PLA2) 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-Aa and PeαPLI-Ab, 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 PLA2s. 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 PLA2 isozymes.

Morphological characterization of spermatozoa of the night monkey

Abstract The morphology, sizes and abnormality rates of spermatozoa in the night monkey were revealed in the present paper. Motile spermatozoa of three males, 7, 8 and 12–13 years old, were squeezed from the ducts of the cauda epididymis after cutting the ducts in cryopreservation media. The morphology of the spermatozoa and abnormalities in them were observed, and the sizes of the spermatozoa were measured in smear samples. The spermatozoa of the night monkey had heads with rounded and thick shapes. Measurement of the spermatozoa revealed that the average widths and lengths of the heads, average lengths of the middle pieces, and average total lengths from the head to tail tip were 4.7 ± 0.8, µm and 2.8 ± 0.4 µm, 6.6 ± 2.2, µm and 55.1 ± 6.2 µm, respectively (average ± SD). The total rates of abnormal spermatozoa were different among the 7-, 8- and 12–13- year-old night monkeys, 41.8%, 24.0% and 36.5%, respectively. Freezing of semen was also attempted using the procedure contained in a commercial kit for the mouse. Although the motility of the spermatozoa from the night monkeys was poor in fresh samples, the motility of their spermatozoa frozen-thawed with the commercial kit was similar to that before freezing.

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

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.

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

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

Dermatitis Associated with Infestation of a Trombiculid Mite, Leptotrombidium miyajimai, in an Amami Rabbit (Pentalagus furnessi)

Abstract 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.