Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata lines improved in somatic embryogenesis

We describe a simple and efficient protocol for regeneration-transformation of two diploid Medicago lines: the annual M. truncatula R108-1(c3) and the perennial M. sativa ssp. falcata (L.) Arcangeli PI.564263 selected previously as highly embryogenic genotypes. Here, embryo regeneration of R108-1 to complete plants was further improved by three successive in vitro regeneration cycles resulting in the line R108-1(c3). Agrobacterium tumefaciens-mediated transformation of leaf explants was carried out with promoter-gus constructs of two early nodulins (MsEnod12A and MsEnod12B) and one late nodulin (Srglb3). The transgenic plants thus produced on all explants within 3–4 months remained diploid and were fertile. This protocol appears to be the most efficient and fastest reported so far for leguminous plants. Received: 18 March 1997 / Revision received: 25 June 1997 / Accepted: 15 July 1997     

大鼠脑室内注射氨甲酰胆碱对肾钠,钾,水排出的影响

在麻醉大鼠侧脑室注射胆碱能激动剂氨甲酰胆碱（ＣＢＣ）引起显著的促钠排泄、促钾排泄和利尿反应（Ｐ＜０．０５）,其中促钠排泄反应与剂量之间呈量效关系（ｒ＝０．９９９７,Ｐ＜０．０５）。由脑室注射ＣＢＣ（２．７４×１０－３μｍｏｌ）引起的上述反应可以被胆碱能Ｍ受体阻断剂阿托品或Ｎ受体阻断剂六甲双胺预处理完全阻断（Ｐ＜０．０５）。同样,ＣＢＣ的肾脏效应也可被肾上腺素能α受体阻断剂酚妥拉明预处理所部分阻断（Ｐ＜０．０５）。上述结果表明脑室注射ＣＢＣ引起的促钠排泄、促钾排泄和利尿反应是刺激了脑胆碱能Ｍ或Ｎ受体,有部分效应可能继发刺激去甲肾上腺素能α受体。     

Adherence to human small intestines of capsulated Vibrio cholerae O139

Abstract Capsulated cells of V. cholerae O139 adhered to formalis-fixed or native mucosa of the small intestines from an adult and a child. The primary adherence target was mucus. Capsulated O139 cells adhered better to the antigen sampling cells (M cells) of ileal Peyer's patch than to the absorptive cells. O139 cells on the mucosa appeared as small aggregates. Similar organisms were found on the mucosa of duodenal biopsy samples from patients infected with V. cholerae O139. The findings indicated that capsulated cells of V. cholerae O139 tend to autoagglutinate and contribute to the effective adherence to the intestinal mucosa.     

Revision of the puella group of the genus Coenagrion Kirby, 1890 (Odonata,Zygoptera), with emphasis on morphologies contributing to reproductive isolation

The puella group of the zygopteran genus Coenagrion Kirby, 1890 is revised. The group, as treated here, includes C. puella (L., 1758), C. syriacum (Morton, 1924), C. ponticum (Bartenef, 1929) and C. intermedium (Lohmann, 1990). All four species are redescribed on the basis of a scanning electron microscopical (S.E.M.) analysis of morphological characters involved in copulation. Full species rank is attributed to intermedium. A key to males and females of the four species is included. Reproductive isolation is discussed in the genus Coenagrion.     

Diosmetin inhibits the growth and invasion of gastric cancer by interfering with M2 phenotype macrophage polarization

Overturning M2 phenotype macrophage polarization is a promising therapeutic strategy for gastric cancer (GC). Diosmetin (DIO) is a natural flavonoid with antitumor effect. The aim of this study was to investigate the effect of DIO on polarization of M2 phenotype macrophages in GC. THP-1 cells were induced to M2 phenotype macrophages and co-cultured with AGS cells. The effects of DIO were determined by flow cytometry, qRT-PCR, CCK-8, Transwell, and western blot. To explore the mechanisms, THP-1 cells were transfected with adenoviral vectors containing tumor necrosis factor receptor-associated factor 2 (TRAF2) or si-TRAF2. DIO (0, 5, 10, and 20 μM) restrained the M2 phenotype macrophage polarization. In addition, DIO (20 μM) reversed the increased viability and invasion of AGS cells induced by the co-culture of M2 macrophages. Mechanistically, TRAF2 knockdown inhibited the effect of M2 phenotype macrophages on AGS cells' growth and invasion. Furthermore, DIO (20 μM) was found to decrease TRAF2/NF-κB activity in GC cells. However, TRAF2 overexpressed reversed the inhibitory effect of DIO on the co-culture system. The in vivo study confirmed that DIO treatment (50 mg/kg) could repress the growth of GC. DIO treatment markedly reduced the expressions of Ki-67 and N-cadherin, and decreased the protein levels of TRAF2 and p-NF-κB/NF-κB. In conclusion, DIO inhibited the growth and invasion of GC cells by interfering with M2 phenotype macrophage polarization through repression of the TRAF2/NF-κB signaling pathway.     

Insecticidal Potential of α-Pinene and β-Caryophyllene against <i>Myzus persicae</i> and Their Impacts on Gene Expression

Myzus persicae (M. persicae) is now considered a threat to agricultural crops due to economic losses. Numerous synthetic insecticides applied every year against M. persicae, are reported to be unsafe for environment, humans, and beneficial insects. Furthermore, several species of Myzus have been found to develop resistance due to over application of these insecticides. Therefore, it is required to find some novel insecticide that would be safe for the environment as well as for humans. In the current study, two major pure constituents α-pinene and β-caryophyllene were evaluated for their insecticidal potential against M. persicae using a fumigant toxicity assay. Furthermore, impact of α-pinene and β-caryophyllene on expression of five different genes, e.g., HSP 60, FPPS I, OSD, TOL and ANT responsible for reproduction, dispersion, and growth of M. persicae has also been investigated. To perform fumigant toxicity assay, five different concentrations (3.5, 4, 4.5, 5 and 6 μL L⁻¹) of α-pinene and β-caryophyllene were prepared. Lethal concentration (LC) was calculated, and gene expression studies were executed through qRT PCR at LC₃₀ of α-pinene and β-caryophyllene. Both constituents demonstrated excellent fumigant toxicity effects against M. persicae at all five concentrations. However, α-pinene shows significantly better results (98%) as compared to β-caryophyllene (80%) after 72 h at 6 μL L⁻¹ of dose. The highest upregulation in expression was demonstrated at LC₃₀ dose of α-pinene in five in three out of five genes understudy (TOL, ANT, and FPPS I). Conversely, two genes HSP 60 and OSD demonstrated downregulation at LC₃₀ dose of β-caryophyllene. Conclusively, our results highlighted the promising insecticidal potential of both compounds α-pinene and β-caryophylleneby interfering with the reproduction and development related processes in M. persicae, allowing us to recommend the phytoconstituents under investigation as an ecofriendly alternative to synthetic insecticides.     

Coexistence of Mugil cephalus and M. curema in a coastal lagoon in the Gulf of Mexico

Mugil cephalus and M. curema coexist in Tamiahua Lagoon, with no difference in diet or digestive system, but with a separation of 3 months in reproductive timing.     

Developmental,hormonal, and pathogenesis-related regulation of the tobacco class I β-1,3-glucanase B promoter

The class I -1,3-glucanases are antifungal vacuolar proteins implicated in plant defense that show developmental, hormonal, and pathogenesis-related regulation. The tobacco enzymes are encoded by a small gene family with members derived from ancestors related to the present-day species Nicotiana sylvestris and N. tomentosiformis. We studied the expression in transgenic tobacco plants of a chimeric -glucuronidase (GUS) reporter gene fused to 1.6 kb of upstream sequence of the tobacco class I -1,3-glucanase B (GLB) gene, which is of N. tomentosiformis origin. Expression of the GUS reporter gene and the accumulation of class I -1,3-glucanase and its mRNA showed very similar patterns of regulation. In young seedlings the reporter gene was expressed in the roots. In mature tobacco plants it was preferentially expressed in lower leaves and roots and was induced in leaves by ethylene treatment and by infection with tobacco mosaic virus (TMV). Furthermore, it was down-regulated in cultured leaf discs by combinations of the hormones auxin and cytokinin. Histological studies of GUS activity showed that the GLB promoter shows highly localized expression in roots of seedlings. It is also expressed in a ring of cells around necrotic lesions induced by TMV infection, but not in cells immediately adjacent to the lesions or in the lesions themselves. The results of deletion analyses suggest that multiple positive and negative elements in the GLB promoter regulate its activity. The region from –1452 to –1193 containing two copies of the heptanucleotide AGCCGCC, which is highly conserved in plant-stress and defense-related genes, is necessary for high level expression in leaves. Additional regions important for organ-specific and regulated expression were: –568 to –402 for ethylene induction of leaves; –402 to –211 for expression in lower leaves and cultured leaf discs and for TMV induction of leaves; and –211 to –60 for expression in roots.     

Cytokeratin 18 is an M-cell marker in porcine Peyer's patches

The intermediate filaments of the dome epithelium of porcine Peyer's patches were studied by immunohistochemistry. The labelling patterns of monospecific antibodies directed against cytokeratins 8, 18 and 19 differed considerably. About 40% of the dome epithelial cells were intensely labelled by three different anti-cytokeratin 18 antibodies, indicating that large amounts of cytokeratin 18 are present in these cells. In order to verify that these cytokeratin-18-immunoreactive cells were M-cells, uptake studies using fluorescein-labelled yeast particles were performed. Numerous yeast particles were found exclusively in dome epithelial cells that were highly positive for cytokeratin 18, thus representing M-cells. In contrast, the content of cytokeratin 19 in M-cells was lower than that in neighbouring enterocytes. The labelling intensity of cytokeratin 8 did not differ between M-cells and enterocytes. In addition, the absence of vimentin and desmin from the dome epithelium of porcine Peyer's patches was demonstrated. The results show (1) that porcine M-cells differ from enterocytes in the composition of their cytoskeleton, (2) that cytokeratin 18 is a useful marker for detecting porcine M-cells and (3) that this marker directly correlas with M-cell function.