Experimental subcutaneous granulomas in sheep with Dermatophilus-like microorganisms from porcine tonsil

The pathogenicity of the Dermatophilus-like microorganisms from porcine tonsil and the light and electron microscopic findings were studied with adult ewes. The early lesions were abscessess and advanced ones were granulomas after the subcutaneous inoculation. The granulomas were composed of the central bacterial colonies and the layers of the neutrophils, epithelioid cells and giant cells, and peripheral connective tissues. Epithelioid cells and giant cells were identified by the large euchromatic nuclei, abundant organelles and interdigitation of the blunt pseudopods in the ultrastructure. The lesions were very similar to subcutaneous granulomas in sheep and cattle due to Dermatophilus congolensis (atypical dermatophilosis), actinomycosis and nocardiosis.     

Benzyladenine-induced changes in the translatable mRNA population in excised cucumber cotyledons

Benzyladenine-induced changes in the translatable mRNA population in excised cucumber cotyledons were studied. Poly (A)⁺ RNA was prepared from etiolated cotyledons incubated with or without benzyladenine (BA) for various periods in the dark. Using nonequilibrium pH gradient electrophoresis-SDS polyacrylamide gel electrophoresis and isoelectric focusing-SDS polyacrylamide gel electrophoresis, both basic and neutral proteins translated in vitro were separated. About 240 spots were detected and 16 of them changed within 6 h after BA application. Some spots changed quickly (within 1–2 h). Among them, three were repressed markedly     

Isolation and Characterization of a Cytokinin-Binding Protein from the Water-Soluble Fraction of Tobacco Leaves

Cytokinin-binding protein (CBPI) was purified from the watersoluble fraction of tobacco leaves by successive chromatographyon benzyladenine-linked (BA-linked) Sepharose 4B, TSK-Gel G3000SWXL,t-zeatin-linked Sepharose 6B and TSK-Gel G3000SWXL. CBPI wasobtained as a monomer with a molecular weight of 31 kDa. Ithas one cytokinin-binding site, which shows a high affinityfor BA (Kd=1.1x10^–7 M) and other cytokinins. Biologicallyactive cytokinins competed with BA for binding to this protein,while biologically inactive analogues of adenine did not. Inall cases, cytokinin-binding activity was assayed by equilibriumdialysis. ¹ Present address: National Institute for Basic Biology, Okazaki,444 Japan.     

Decreased Expression of Matrix Metalloproteinase-9 and Increased Expression of Tissue Inhibitors of Matrix Metalloproteinase-1 in Paratuberculosis-Infected Cattle in the ELISA-Negative Subclinical Stage

We investigated the gene expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitors of matrix metalloproteinases-1 (TIMP-1) in peripheral blood cells from infected cattle with Mycobacterium avium subsp. paratuberculosis (Map) in the ELISA-negative subclinical stage compared with uninfected control cattle. Significant decreased MMP-9 expression and increased TIMP-1 expression were found in peripheral blood cells from Map-infected cattle after stimulation with Map lysate and Map purified protein derivative (PPD) than in control cattle by real-time RT-PCR analysis. In contrast to the uninfected controls, the activity of MMP-9 was also decreased in peripheral blood cell culture supernatants from Map-infected cattle at 24 hr after Map lysate and MapPPD stimulation by gelatin zymography analysis. As a result, the MMP-9 may play an important role in the development of Mycobacterium avium subsp. paratuberculosis disease.     

Synthesis and properties of 4'-ThioDNA: unexpected RNA-like behavior of 4'-ThioDNA

The synthesis and properties of fully modified 4′-thioDNAs, oligonucleotides consisting of 2′-deoxy-4′-thionucleosides, were examined. In addition to the known literature properties (preferable hybridization with RNA and resistance to endonuclease hydrolysis), we also observed higher resistance of 4′-thioDNA to 3′-exonuclease cleavage. Furthermore, we found that fully modified 4′-thioDNAs behaved like RNA molecules in their hybridization properties and structural aspect, at least in the case of the 4′-thioDNA duplex. This observation was confirmed by experiments using groove binders, in which a 4′-thioDNA duplex interacts with an RNA major groove binder, lividomycin A, but not with DNA groove binders, to give an increase in its thermal stability. Since a 4′-thioDNA duplex competitively inhibited the hydrolysis of an RNA duplex by RNase V₁, it was not only the physical properties but also this biological data suggested that a 4′-thioDNA duplex has an RNA-like structure.     

Unexpected A-form formation of 4'-thioDNA in solution, revealed by NMR, and the implications as to the mechanism of nuclease resistance

Fully modified 4′-thioDNA, an oligonucleotide only comprising 2′-deoxy-4′-thionucleosides, exhibited resistance to an endonuclease, in addition to preferable hybridization with RNA. Therefore, 4′-thioDNA is promising for application as a functional oligonucleotide. Fully modified 4′-thioDNA was found to behave like an RNA molecule, but no details of its structure beyond the results of circular dichroism analysis are available. Here, we have determined the structure of fully modified 4′-thioDNA with the sequence of d(CGCGAATTCGCG) by NMR. Most sugars take on the C3′-endo conformation. The major groove is narrow and deep, while the minor groove is wide and shallow. Thus, fully modified 4′-thioDNA takes on the A-form characteristic of RNA, both locally and globally. The only structure reported for 4′-thioDNA showed that partially modified 4′-thioDNA that contained some 2′-deoxy-4′-thionucleosides took on the B-form in the crystalline form. We have determined the structure of 4′-thioDNA in solution for the first time, and demonstrated unexpected differences between the two structures. The origin of the formation of the A-form is discussed. The remarkable biochemical properties reported for fully modified 4′-thioDNA, including nuclease-resistance, are rationalized in the light of the elucidated structure.     

Localization of insulinoma associated protein 2, IA-2 in mouse neuroendocrine tissues using two novel monoclonal antibodies

AimsInsulinoma-associated protein 2 (IA-2) is a member of the protein tyrosine phosphatase family that is localized on the insulin granule membrane. IA-2 is also well known as one of the major autoantigens in Type 1 diabetes mellitus. IA-2 gene deficient mice were recently established and showed abnormalities in insulin secretion. Thus, detailed localization of IA-2 was studied using wild-type and IA-2 gene deficient mice.Main methodsTo localize IA-2 expression in mouse neuroendocrine tissues, monoclonal antibodies were generated against IA-2 and western blot and immunohistochemical analyses were carried out in IA-2^+/+ mice. IA-2^?/? mice served as a negative control.Key findingsWestern blot analysis revealed that the 65 kDa form of IA-2 was observed in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, pituitary gland, muscular layers of the stomach, small intestine, and colon. By immunohistochemical analysis, IA-2 was produced in endocrine cells in pancreatic islets, adrenal medullary cells, thyroid C-cells, Kulchitsky cells, and anterior, intermediate, and posterior pituitary cells. In addition, IA-2 was found in somatostatin-producing D-cells and other small populations of cells were scattered in the gastric corpus. IA-2 expression in neurites was confirmed by the immunostaining of IA-2 using primary cultured neurons from the small intestine and nerve growth factor (NGF)-differentiated PC12 cells.SignificanceThe IA-2 distribution in peripheral neurons appeared more intensely in neurites rather than in the cell bodies.