Immune defence mechanism against blood-borne Pasteurella multocida in turkeys

Humoral and cellular immune defence factors involved in controlling blood-borne Pasteurella multocida were investigated in turkeys by the passive transfer of immune serum or by the treatment with macrophage-activating agents. The treated and untreated birds were intravenously inoculated with a virulent strain of P multocida, and the viable bacteria in the blood, liver and spleen were counted. In untreated birds, the bacteria were rapidly removed from the blood, and the majority were recovered from the liver and spleen 120 minutes after inoculation. Neither the transfer of immune serum nor the treatment with macrophage-activating agents significantly influenced the clearance rate of bacteria from the blood. The number of bacteria recovered from the liver 120 minutes after inoculation was slightly lower in the birds treated with macrophage-activating agents and significantly lower in those given immune serum than in the untreated birds. None of the treatments, however, significantly changed the number of bacteria recovered from the spleen 120 minutes after inoculation. The results suggest that the phagocytes in the liver, but not in the spleen, play a crucial role in the intravascular defence against P multocida in the presence of specific antibodies.     

In situ hybridization for the detection of feline interleukin 1 alpha mRNA on the paraffin-embedded section using biotin-labeled probes.

In situ hybridization (ISH) technique with a biotin-labeled probe was established for detecting feline interleukin 1 (IL-1) alpha mRNA in necropsied specimens. Homology between human IL-1 alpha cDNA used as a probe and feline IL-1 alpha mRNA was confirmed by means of dot blot hybridization using the biotin-labeled probe. Hence, we tried by this biotinylated probe to detect mRNA of IL-1 alpha in paraffin-embedded sections. The following results were obtained for the routine procedures: 1) coating slides with poly-L-lysine and/or heating at 60 degrees C at least for 6 hours gave an excellent result for the adhesion of the tissue sections, 2) 10 micrograms/ml solution of proteinase K treatment for 30 minutes or 50 to 100 micrograms/ml solution of proteinase K treatment for 10 to 30 minutes at 37 degrees C gave the good results in the detection of ISH signal, 3) suitable denaturation time of probes at 70 to 90 degrees C was 5 to 15 minutes, and 4) effective hybridization was obtained by incubation for 24 hours at 4 degrees C, for 18 to 24 hours at 25 degrees C or for 5 to 24 hours at 37 degrees C.     

2b Protein is essential to induce a novel gradual cell death in Zucchini yellow mosaic virus-inoculated cucumber cotyledon co-infected with Cucumber mosaic virus

Cucumber cotyledons inoculated with Cucumber mosaic virus (CMV, Pepo strain) or Zucchini yellow mosaic virus (ZYMV, Z5-1 isolate) developed either mild chlorotic spots or no symptoms. Cotyledons treated with CMV plus ZYMV also developed mild chlorotic spots. However, plants ZYMV-inoculated cotyledons had veinal yellowing and gradual cell death by 20 days postinoculation (dpi) when co-inoculated with CMV on the other cotyledon. When analyzing this synergism, an enzyme-linked immunosorbent assay showed that CMV gradually increased in CMV-inoculated cotyledons of plants, with the other cotyledon mock- or ZYMV-inoculated. However, CMV significantly increased at 9 to 14 dpi in the ZYMV-inoculated cotyledons of plants co-infected with CMV. ZYMV similarly increased in cotyledon pairs of both co-infected and singly infected plants. Inoculation with PepoΔ2b, a modified Pepo-CMV that lacks translation of the 2b protein, revealed that PepoΔ2b without the 2b protein systemically infected cucumber but induced no symptoms on cotyledons or true leaves. Plants with a ZYMV-inoculated cotyledon and co-infected with PepoΔ2b did not undergo cell death; nevertheless, PepoΔ2b was at high levels comparable to levels of CMV in the ZYMV-inoculated cotyledon. The 2b protein thus seems essential for induction of the novel gradual cell death in ZYMV-inoculated cotyledons of cucumbers co-infected with CMV.     

Resistance of a soybean cell line to oxyfluorfen by overproduction of mitochondrial protoporphyrinogen oxidase

The diphenyl ether herbicide oxyfluorfen (2-chloro-4-trifluoromethylphenyl 3-ethoxy-4-nitrophenyl ether) inhibits protoporphyrinogen oxidase (Protox) which catalyzes the oxidation of protoporphyrinogen IX (Protogen) to protoporphyrin IX (Proto IX), the last step of the common pathway to chlorophyll and haeme biosynthesis. We have selected an oxyfluorfen-resistant soybean cell line by stepwise selection methods, and the resistance mechanism has been investigated. No growth inhibition was observed in resistant cells at a concentration of 10(-7) M oxyfluorfen, a concentration at which normal cells did not survive. While the degree of inhibition of total extractable Protox by oxyfluorfen was the same in both cell types, the enzyme activity in the mitochondrial fraction from non-treated resistant cells was about nine-fold higher than that from normal cells. Northern analysis of mitochondrial Protox revealed that the concentration of mitochondrial Protox mRNA was much higher in resistant cells than that in normal cells. There were no differences in the absorption and metabolic breakdown of oxyfluorfen. The growth of resistant cells was also insensitive to oxadiazon [5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2-(3H)- one], the other chemical class of Protox inhibitor. Therefore, the resistance of the selected soybean cell line to oxyfluorfen is probably mainly due to the overproduction of mitochondrial Protox.     

A case report of a renal mixed epithelial and stromal tumor in a heterozygous S1P2 receptor deficient mouse

We report a case of mixed epithelial and stromal tumor of the kidney (MESTK) in a 32-week-old heterozygous sphingosine 1-phosphate-2 (S1P2) receptor deficient female mouse. A white solid mass replacing the left kidney was observed at the left retroperitoneal wall. Histologically, the tumor mass consisted of dimorphic cellular components of epithelial and stromal cells. Epithelial cells formed various sized irregular-shaped tubular structures resembling renal tubules surrounded by stromal cells. Immunohistochemically, epithelial cells were positive for cytokeratin, while stromal cells showed positive immunoreactivity with alpha-smooth muscle actin as well as vimentin. Based on the morphological and immunohistochemical findings, this tumor was diagnosed as a MESTK.     

Expression of hedgehog family genes in the rat uterus during early pregnancy

Hedgehog (Hh) plays a pivotal role in various tissues during embryonic development, tissue homeostasis and tumorigenesis. In mammals, Hh exists in three homologs: Desert hedgehog (Dhh), Indian hedgehog (Ihh) and Sonic hedgehog (Shh). In this study, we cloned full-length cDNAs encoding Dhh and Ihh from the rat uterus. Their amino acid sequences have a high homology with those of the mouse and human. In addition, the changes of Hh gene expression in the rat uterus during early pregnancy were analyzed. The results showed that all three hedgehog mRNAs were detected in the rat uterus at the proestrus stage and during early pregnancy (1.5, 3.5, 5.5 and 7.5 days post coitus: dpc). Ihh mRNA expression varied and peaked at 3.5 dpc in the luminal and glandular epithelium. Expression was decreased on 5.5 dpc with the exception of sustained expression in the glandular epithelium. Despite such Ihh variability, the expressions of Dhh and Shh mRNA remained unchanged. This indicated that Ihh was mainly expressed in the rat uterus during early pregnancy. Moreover, the Hh target gene (glioma-associated oncogene homolog 1; Gli1) was also highly expressed at 3.5 dpc in the epithelium and periepithelial stroma in a manner similar to the temporal pattern of Ihh expression. This suggests that Ihh signaling axis play a role in the rat uterus during early pregnancy. In summary, our results elucidate that Ihh is a predominant Hh protein in the rat uterus during early pregnancy and that other Hhs have the potential to be expressed. This observation will help to elucidate the basic molecular mechanism of rat uterus during early pregnancy.     

Timing and uniformity of embryonic gene activation affect subsequent pre-implantation development of cloned bovine embryos

In the present study, we examined the timing of onset, intensity, and mosaicism of embryonic gene expression in bovine nuclear transfer (NT) embryos. The relationship between gene expression and early embryonic development was also examined. To monitor the gene expression of NT embryos, we produced NT embryos with bovine transfected fibroblasts carrying a firefly luciferase gene under the control of a chicken beta-actin promoter, an expression system that has previously been shown to be representative of embryonic gene expression in mice. Photon count imaging showed that luciferase luminescence began in NT embryos with fibroblasts 48 hours post fusion (hpf) and reached a plateau at the 4- to 8-cell stage at 60 hpf. Only 4- to 8-cell NT embryos luminescent by 60 hpf developed to the blastocyst stage. At 60 hpf, strongly luminescent embryos developed to the blastocyst stage at a higher rate (P<0.05) than embryos with weak or absent luminescence. However, embryos with mosaic luminescence developed at a much lower rate (P<0.05) than those with whole-embryo luminescence, even if the embryos exhibited strong luminescence. Our results indicate that precise and uniform embryonic gene expression at the 4- to 8-cell stage at 60 hpf may be closely related to development of bovine NT embryos to the blastocyst stage.     

H1foo is indispensable for meiotic maturation of the mouse oocyte

Oocyte-specific linker histone H1foo is localized in the oocyte nucleus, either diffusely or bound to chromatin, during the processes of meiotic maturation and fertilization. This expression pattern suggests that H1foo plays a key role in the control of gene expression and chromatin modification during oogenesis and early embryogenesis. To reveal the function of H1foo, we microinjected antisense morpholino oligonucleotides (MO) against H1foo into mouse germinal-vesicle stage oocytes. The rate of in vitro maturation of the antisense MO group was significantly lower than that of the control group. Eggs that failed to extrude a first polar body following injection of antisense MO arrested at metaphase I. Additionally, co-injection of in vitro synthesized H1foo mRNA along with antisense MO successfully rescued expression of H1foo and improved the in vitro maturation rate. There was no difference in the rate of parthenogenesis between the antisense MO and control groups. These results indicate that H1foo is essential for maturation of germinal vesicle-stage oocytes.