Development of a monoclonal antibody for the detection of anti-canine CD20 chimeric antigen receptor expression on canine CD20 chimeric antigen receptor-transduced T cells

Chimeric antigen receptor (CAR) CAR-T cell therapy targeting CD20 can be a novel adoptive cell therapy for canine patients with B-cell malignancy. After injection of the CAR-T cells in vivo, monitoring circulating CAR-T cells is essential to prove in vivo persistence of CAR-T cells. In this study, we developed a novel monoclonal antibody against canine CD20 CAR, whose single-chain variable fragment was derived from the our previously reported anti-canine CD20 therapeutic antibody. Furthermore, we proved that this monoclonal antibody can detect therapeutic anti-canine CD20 chimeric antibody in the serum from healthy beagle dogs injected with the therapeutic antibody for safety study. This monoclonal antibody is a useful tool for monitoring both canine CD20-CAR-T cells and anti-canine CD20 therapeutic antibody for canine lymphoma.     

Somatic cell and innate immune responses in mammary glands of lactating cows to intramammary infusion of Bifidobacterium breve at pre-drying off period

Intramammary infusion of Bifidobacterium breve (B. breve)-induced somatic cell (SC) counts, chemiluminescent response (CL), lactoferrin (LF) concentrations and mastitis-causing pathogens from quarters with subclinical mastitis were measured to evaluate innate immune response of mammary glands in dairy cows at 3 to 4 weeks before drying off. SC counts in 7 quarters of 7 control cows and 5 quarters of 6 cows with mastitis increased markedly on day 1 and SC values in control cows were significantly (P<0.05) increased and returned to pre-infusion levels on day 5 after B. breve-infusion. CL values in both groups increased markedly on day 1 and then decreased after B. breve-infusion; however, CL values in cows with mastitis did not return to normal levels on day 5 and at postpartum. The CL values were highly correlated with their SC counts in milk from both groups. LF concentrations increased toward day 3 after B. breve-infusion and were higher in cows with mastitis. B. breve-infusion eliminated 16.6% (1/6) of pathogens from 6 quarters with chronic subclinical mastitis. B. breve-induced SC responses in quarters from 3 cows with mastitis showed characteristic patterns of recovery, persistent and new infections. B. breve-induced SC counts in quarters from the cows in the pre-drying off were lower (25.7–70.6%) than those of the cows in mid-lactation. The intrinsic innate immune response in cows on pre-drying off may be decreased and appears to be insufficient to eliminate pathogens from mammary gland in the pre-drying off.     

First report of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma malaysianum’ associated with <Emphasis Type="Italic">Elaeocarpus</Emphasis> yellows of <Emphasis Type="Italic">Elaeocarpus zollingeri</Emphasis>

“Elaeocarpus yellows” (ELY) is a widely reported phytoplasma disease of Elaeocarpus zollingeri trees in Japan. The phytoplasma associated with ELY (ELY phytoplasma) had not been identified at the species level because its 16S rRNA sequence had yet to be reported. Here, we report the results of a sequence analysis based on 16S rRNA and secA gene sequences, which showed that the ELY phytoplasma is related to ‘Candidatus Phytoplasma malaysianum’. To our knowledge, this is the first report showing the occurrence of ‘Ca. P. malaysianum’ outside Malaysia and the infection of E. zollingeri by the phytoplasma.     

Curious Oviposition Behavior in Phyllium westwoodii (Phasmatodea: Phylliidae): Preliminary Observations

We report that in a leaf insect, Phyllium westwoodii Wood-Mason (Phasmatodea: Phylliidae), two differing apertures can be used for oviposition, the color of eggs being affected by which aperture is used. Eggs which are forcibly propelled from the internal space within the valvulae of the abdomen are brown, whereas white eggs emerge slowly from the opening between the eighth sternite and the valvulae, and are deposited close to the ventral surface of the female. This unusual oviposition system does not appear to have been previously reported in phasmatids or in other insects.     

Cytosolic glutamine synthetase is present in the phloem sap of rice (Oryza sativa L.)

The presence of glutamine synthetase (GS) in the rice sieve tube was examined. Proteins in the rice phloem sap from leaf sheaths were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis, transferred to polyvinylidine difluoride membranes and immunoblotted with anti-GS1 antibody. A cross-reacting band, thought to be GS1, was detected in the phloem sap. Moreover, the phloem sap contained a significant amount of GS transferase activity. Previous studies have shown that the concentrations of substrates and cofactors in the rice phloem sap are sufficient for cytosolic GS reaction. These data suggest that physiologically active GS1 is present in rice phloem sap, which might convert glutamate to glutamine in vivo .     

Survey of the Occurrence of Equine Deep Digital Flexor Tendon Contraction (Clubfoot) in the Main Thoroughbred Breeding Area in Japan

At 114 ranches in the Hidaka District of Hokkaido, a total of 1,118 Thoroughbred foals that were born from January to June 2003 were examined at different times from May to September of the same year to ascertain the occurrence of clubfoot. Clubfoot was seen in a total of 179 foals (16%) at 89 ranches (78%). Also, 124 of the 179 foals (69.3%) had clubfoot of grade II or higher, and it was found that grade I was likely to be overlooked or rapidly advance. In the present study, the occurrence of clubfoot was higher than expected in the investigated region. Hence, it is necessary to establish accurate diagnostic techniques and clubfoot guidelines to minimize the impact.     

Proper reprogramming of imprinted and non‐imprinted genes in cloned cattle gametogenesis

Epigenetic abnormalities in cloned animals are caused by incomplete reprogramming of the donor nucleus during the nuclear transfer step (first reprogramming). However, during the second reprogramming step that occurs only in the germline cells, epigenetic errors not corrected during the first step are repaired. Consequently, epigenetic abnormalities in the somatic cells of cloned animals should be erased in their spermatozoa or oocytes. This is supported by the fact that offspring from cloned animals do not exhibit defects at birth or during postnatal development. To test this hypothesis in cloned cattle, we compared the DNA methylation level of two imprinted genes (H19 and PEG3) and three non‐imprinted genes (XIST, OCT4 and NANOG) and two repetitive elements (Satellite I and Satellite II) in blood and sperm DNAs from cloned and non‐cloned bulls. We found no differences between cloned and non‐cloned bulls. We also analyzed the DNA methylation levels of four repetitive elements (Satellite I, Satellite II, Alpha‐satellite and Art2) in oocytes recovered from cloned and non‐cloned cows. Again, no significant differences were observed between clones and non‐clones. These results suggested that imprinted and non‐imprinted genes and repetitive elements were properly reprogramed during gametogenesis in cloned cattle; therefore, they contributed to the soundness of cloned cattle offspring.     

Changes in muscle fiber type and expression of mRNA of myosin heavy chain isoforms in porcine muscle during pre‐ and postnatal development

The purpose of this study is to elucidate developmental changes in muscle fiber type in the pig during pre‐ and postnatal development. For this purpose, we performed a histochemical analysis for myosin adenosine triphosphatase activity to assess muscle fiber type and determined abundances of messenger RNA (mRNA) of myosin heavy chain (MHC) isoforms. Samples of Longissimus dorsi (LD) muscle were taken from fetuses on day 90 of the fetal stage. Further, samples of LD, Rhomboideus and Biceps femoris (B. femoris) muscles were taken from pigs when they were 1, 12, 26, 45 or 75 days old. Expression of MHC 2b mRNA in the LD and the B. femoris muscles rapidly and considerably increased from the late fetal stage to the early postnatal stage and this increase was associated with the development of type 2b fibers at least in the LD muscle. As shown by the rapid and considerable changes in expression of MHC 2b mRNA, it seems that a certain plasticity of muscle fiber type still remains in this developmental stage.     

Development of Novel Water-extraction System with Thermoelectric Module Using Solar and Wind Power in Arid Land

This study aimed to develop a water-extraction system which could produce the fresh water from the air in arid regions and which used renewable energies as the electric power source. In this paper, the experiments for water extraction from the air were carried out by using the novel multi-stage water-extraction device with Peltier deices for two cases of temperature and related humidity of the air. One was the case where the temperature and the related humidity of the air were constant, and the other was the case where they were simulated the variation of the temperature and related humidity of the air in a day of summer and spring in Loess Plateau, China. The effects of the temperature and related humidity of the atmospheric air and supply the electric power to Peltier devices on performance of water production of the device were investigated and reported.     

Detection of Thiophanate-methyl-resistant Strains in Diplocarpon mail, Causal Fungus of Apple Blotch

Isolates of Diplocarpon mali, causal fungus of apple blotch, collected from four prefectures in Japan in 1997–1998 were tested for sensitivity to thiophanate-methyl. Results from mycelial growth tests showed that MIC values of the fungicide were 0.19 μg/ml against all isolates from Akita, Nagano and Saga prefectures but 100 or 200 μg/ml against all isolates from the Tokusa area in Yamaguchi prefecture. Detached apple leaves sprayed with the fungicide developed severe symptoms when inoculated with the isolate from Tokusa, but developed no symptoms with the isolate from Nagano. These results are the first confirmation of thiophanate-methyl-resistant strains in D. mali. Received 1 September 1999/ Accepted in revised form 15 October 1999