Sequence analysis of cohesive ends of the actinophage RP3 genome and construction of a transducible shuttle vector

Abstract The sequence of the cohesive ends of actinophage RP3 DNA has been determined. As with all other phages of Gram-positive bacteria that have been studied sofar, RP3 DNA has 3'-protruding ends. A shuttle cosmid has been constructed containing this cos area which can be efficiently transduced by phage RP3 to host cells of Streptomyces rimosus .     

Prevalence of Colonization Factor Antigens (CFAs) and Adherence to HeLa Cells in Enterotoxigenic Escherichia coli Isolated from Feces of Children in São Paulo

Fifty-eight enterotoxigenic Escherichia coli (ETEC) strains, isolated from children with and without diarrhea in Sao Paulo, were examined for the presence of colonization factor antigens (CFAs) and their ability to adhere to HeLa cells. Antisera to CFA/I, the coli surface (CS) antigens CS1CS3, CS2CS3, and CS2 of CFA/II, CFA/III, and CS5CS6 and CS6 of CFA/IV were used. CFAs were identified in 43% of the ETEC strains: 40% of the strains with CFAs harbored CFA/I, 24% carried CFA/II (CS1CS3), 24% carried CFA/IV (CS6), and 12% carried CFA/IV (CS5CS6). CFAs occurred mainly among ETEC strains producing only heat-stable (ST-I) enterotoxin and in strains also producing heat-labile toxin (LT-I). No ETEC strains tested expressed CFA/III. A marked change in serotypes of ST-I-producing strains was found in Sao Paulo between 1979 and 1990. Adherence to HeLa cells was detected in 14% of the ETEC strains. All of them had a diffuse adherence pattern and produced only ST-I, and 88% carried CS6 antigen.     

Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and β-1,3-glucanase in transgenic plants

The Nicotiana tabacum ap24 gene encoding a protein with antifungal activity toward Phytophthora infestans has been characterized. Analysis of cDNA clones revealed that at least three ap24-like genes are induced in tobacco upon infection with tobacco mosaic virus. Amino acid sequencing of the purified protein showed that AP24 is synthesized as a preproprotein from which an amino-terminal signal peptide and a carboxyl-terminal propeptide (CTPP) are cleaved off during post-translational processing. The functional role of the CTPP was investigated by expressing chimeric genes encoding either wild-type AP24 or a mutant protein lacking the CTPP. Plants expressing the wild-type construct resulted in proteins properly sorted to the vacuole. In contrast, the proteins produced in plants expressing the mutant construct were secreted extracellularly, indicating that the CTPP is necessary for targeting of AP24 to the vacuoles. Similar results were obtained for vacuolar chitinases and -1,3-glucanases of tobacco. The extracellularly targeted mutant proteins were shown to have retained their biological activity. Together, these results suggest that within all vacuolar pathogenesis-related proteins the targeting information resides in a short carboxyl-terminal propeptide which is removed during or after transport to the plant vacuole.     

Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene.

A bacterium, Pseudomonas sp. strain C1S1, able to grow on 2,4,6-trinitrotoluene (TNT), 2,4- and 2,6-dinitrotoluene, and 2-nitrotoluene as N sources, was isolated. The bacterium grew at 30 degrees C with fructose as a C source and accumulated nitrite. Through batch culture enrichment, we isolated a derivative strain, called Pseudomonas sp. clone A, which grew faster on TNT and did not accumulate nitrite in the culture medium. Use of TNT by these two strains as an N source involved the successive removal of nitro groups to yield 2,4- and 2,6-dinitrotoluene, 2-nitrotoluene, and toluene. Transfer of the Pseudomonas putida TOL plasmid pWW0-Km to Pseudomonas sp. clone A allowed the transconjugant bacteria to grow on TNT as the sole C and N source. All bacteria in this study, in addition to removing nitro groups from TNT, reduced nitro groups on the aromatic ring via hydroxylamine to amino derivatives. Azoxy dimers probably resulting from the condensation of partially reduced TNT derivatives were also found.     

Genetic analysis of the virD operon of Agrobacterium tumefaciens: a search for functions involved in transport of T-DNA into the plant cell nucleus and in T-DNA integration.

The transferred DNA (T-DNA) is transported from Agrobacterium tumefaciens to the nucleus and is stably integrated into the genome of many plant species. It has been proposed that the VirD2 protein, tightly attached to the T-DNA, pilots the T-DNA into the plant cell nucleus and that it is involved in integration. Using agroinfection and beta-glucuronidase expression as two different very sensitive transient assays for T-DNA transfer, together with assays for stable integration, we have shown that the C-terminal half of the VirD2 protein and the VirD3 protein are not involved in T-DNA integration. However, the bipartite nuclear localization signal, which is located within the C terminus of the VirD2 protein and which has previously been shown to be able to target a foreign protein into the plant cell nucleus, was shown to be required for efficient T-DNA transfer. virD4 mutants were shown by agroinfection to be completely inactive in T-DNA transfer.     

Melatonin controls cell proliferation and modulates mitochondrial physiology in pancreatic stellate cells

Journal of Physiology and Biochemistry - We have investigated the effects of melatonin on major pathways related with cellular proliferation and energetic metabolism in pancreatic stellate cells....     

Octopamine receptor subtypes and their modes of action

Octopamine receptor subclasses were first proposed to explain differences in the pharmacological profiles of a range of physiological responses to octopamine obtained in the extensor-tibiae neuromuscular preparation of the locust. Thus, OCTOPAMINE₁ receptors which inhibit an endogenous myogenic rhythm, increase intracellular calcium levels. Also OCTOPAMINE₂ receptors which modulate neuromuscular transmission in this preparation, increase the level of adenylate cyclase activity. The current status of this classification is reviewed by examining the pharmacology of responses to octopamine in a range of preparations. It is concluded that the distinction between OCTOPAMINE₁ and OCTOPAMINE₂ receptor types is still valid, but that OCTOPAMINE₂ receptors exhibit some tissue specific variations. Studies on a clonedDrosophila octopamine/tyramine (phenolamine) receptor are discussed and illustrate many of the difficulties presently encountered in making a definitive classification of octopamine receptors. These include the possibilities that single receptors may activate multiple second messenger systems and that different agonists may differentially couple the same receptor to different second messenger systems.     

Transformation of pecan and regeneration of transgenic plants

A gene transfer system developed for walnut (Juglans regia L.) was successfully applied to pecan (Carya illinoensis [Wang] K. Koch). Repetitively embryogenic somatic embryos derived from open-pollinated seed of Elliott, Wichita, and Schley were co-cultivated with Agrobacterium strain EHA 101/pCGN 7001, which contains marker genes for beta-glucuronidase activity and resistance to kanamycin. Several modifications of the standard walnut transformation techniques were tested, including a lower concentration of kanamycin and a modified induction medium, but these treatments had no measurable effect on efficiency of transformation. Nineteen of the 764 viable inoculated embryos produced transgenic subclones; 13 of these were from the line Elliott6, 3 from Schley5/3, and 3 from Wichita9. Transgenic embryos of Wichita9 germinated most readily and three subclones were successfully micropropagated. Three transgenic plants of one of these subclones were obtained by grafting the tissue cultured shoots to seedling pecan rootstock in the greenhouse. Gene insertion, initially detected by GUS activity, was confirmed by detection of integrated T-DNA sequences using Southern analysis.