Analysis of the germination of spores of Bacillus subtilis with temperature sensitive spo mutations in the spoVA operon

A Bacillus subtilis strain with a base substitution in the ribosome-binding site of spoVAC was temperature sensitive (ts) in sporulation and spores prepared at the permissive temperature were ts in L-alanine-triggered germination, but not in germination with Ca2+-dipicolinic acid (DPA) or dodecylamine. Spores of a ts spo mutant with a missense mutation in the spoVAC coding region were not ts for germination with l-alanine, dodecylamine or Ca2+-DPA. These findings are discussed in light of the proposal that SpoVA proteins are involved not only in DPA uptake during sporulation, but also in DPA release during nutrient-mediated spore germination.     

Analysis of interactions between nutrient germinant receptors and SpoVA proteins of Bacillus subtilis spores

Yeast two-hybrid and Far Western analyses were used to detect interactions between Bacillus subtilis spores' nutrient germinant receptor proteins and proteins encoded by the spoVA operon, all of which are involved in spore germination and located in the spores' inner membrane. These analyses indicated that two subunits of the GerA nutrient germinant receptor interact, consistent with previous genetic data, and that some GerA proteins interact with SpoVAD and some with SpoVAE. SpoVA proteins appear to be involved in the release of the spore's dipicolinic acid during spore germination, an event triggered by the binding of nutrient germinants to their receptors. Consequently, these new findings suggest that nutrient germinant receptors physically contact SpoVA proteins, and presumably this is a route for signal transduction during spore germination.     

Bacterial expression and enzymatic activity analysis of ME1, a ribosome-inactivating protein from Mirabilis expansa

Ribosome-inactivating proteins (RIPs) are toxic proteins synthesized by many plants and some bacteria, that specifically depurinate the 28S RNA and thus interrupt protein translation. RIPs hold broad interest because of their potential use as plant defense factors against pathogens. However, study of the activity of type I RIPs has been hampered since their expression in Escherichia coli has typically been toxic to the model system. Mirabilis expansa, an Andean root crop, produces a type I RIP called ME1 in large quantities in its storage roots. In this study, the cDNA sequence of ME1 was used to successfully express the recombinant ME1 protein in E. coli. The production of recombinant ME1 in E. coli was confirmed by Western blot analysis using anti-ME1 antibodies. The studies with fluorescence-labeled ME1 showed that ME1 can enter bacteria and be distributed in the cytoplasm uniformly, indicating its ability to access the protein synthesis machinery of the bacteria. The recombinant enzyme was active and depurinated yeast ribosomes. However, both native and recombinant ME1 proteins failed to depurinate the E. coli ribosomes, explaining the non-toxicity of recombinant ME1 to E. coli. Structural modeling of ME1 showed that it has folding patterns similar to other RIPs, indicating that ME1 and PAP, which share a similar folding pattern, can show different substrate specificity towards E. coli ribosomes. The results presented here are very significant, as few reports are available in the area of bacterial interaction with type I RIPs.     

Localization of SpoVAD to the inner membrane of spores of Bacillus subtilis

The products of the hexacistronic spoVA operon of Bacillus subtilis may be involved in the transport of dipicolinic acid into the forespore during sporulation and its release during spore germination. The major hydrophilic coding region of B. subtilis spoVAD was cloned, the protein was expressed in Escherichia coli as a His tag fusion protein, and a rabbit antiserum was raised against the purified protein. Western blot analyses of fractions from B. subtilis spores showed that SpoVAD is an integral inner membrane protein present at levels >50-fold higher than those of the spore's nutrient germinant receptors that are also present in the inner membrane. SpoVAD also persisted in outgrowing spores.     

Immunohistochemical expression of rabphilin-3A-like (Noc2) in normal and tumor tissues of human endocrine pancreas

Involvement of rabphilin-3A-like (RPH3AL), or Noc2, the potential effector of Ras-associated binding proteins Rab3A and Rab27A in the regulation of exocytotic processes in the endocrine pancreas has been demonstrated in experimental models. Noc2 expression together with other regulatory molecules of the exocytotic machinery in human tissues, however, has not been studied. We evaluated immunohistochemical expression of the key molecules of the exocytotic machinery, Noc2, Rab3A, Rab27A, and RIM2, together with the characteristic islet cell hormones, insulin and glucagon in normal and endocrine tumor tissues of human pancreas. Normal pancreatic islets were stained for all of these proteins and showed strong cytoplasmic localization. A similar pattern of strong cytoplasmic expression of these proteins was observed in the majority of endocrine tumors. By contrast, the exocrine portions of normal appearing pancreas completely lacked Rab27A staining and showed decreased expression of the proteins, Noc2, Rab3A, and RIM2. The staining pattern of Noc2 and Rab27A was similar to the staining pattern of glucagon-producing cells within the islets. The concomitant expression of Noc2 with these molecules suggests that Noc2 may serve as an effector for Rab3A and Rab27A and that it is involved in the regulation of exocytosis of the endocrine pancreas in humans.     

Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera: Elateroidea) in southern and central Brazil

Bioluminescence in beetles is found mainly in the Elateroidea superfamily (Elateridae, Lampyridae and Phengodidae). The Neotropical region accounts for the richest diversity of bioluminescent species in the world with about 500 described species, most occurring in the Amazon, Atlantic rainforest and Cerrado (savanna) ecosystems in Brazil. The origin and evolution of bioluminescence, as well as the taxonomic status of several Neotropical taxa in these families remains unclear. In order to contribute to a better understanding of the phylogeny and evolution of bioluminescent Elateroidea we sequenced and analyzed sequences of mitochondrial NADH2 and the nuclear 28S genes and of the cloned luciferase sequences of Brazilian species belonging to the following genera: (Lampyridae) Macrolampis, Photuris, Amydetes, Bicellonycha, Aspisoma, Lucidota, Cratomorphus; (Elateridae) Conoderus, Pyrophorus, Hapsodrilus, Pyrearinus, Fulgeochlizus; and (Phengodidae) Pseudophengodes, Phrixothrix, Euryopa and Brasilocerus. Our study supports a closer phylogenetic relationship between Elateridae and Phengodidae as other molecular studies, in contrast with previous morphologic and molecular studies that clustered Lampyridae/Phengodidae. Molecular data also supported division of the Phengodinae subfamily into the tribes Phengodini and Mastinocerini. The position of the genus Amydetes supports the status of the Amydetinae as a subfamily. The genus Euryopa is included in the Mastinocerini tribe within the Phengodinae/Phengodidae. Copyright © 2013 John Wiley & Sons, Ltd.     

Evaluation of tumorigenic potential of high yielding cloned MDCK cells for live-attenuated influenza vaccine using in vitro growth characteristics,metastatic gene expression and in vivo nude mice model

Several mammalian cell lines, including Madin–Darby canine kidney (MDCK) cells have been approved by regulators for manufacturing of human vaccines. A new MDCK 9B9-1E4 cloned cell line has been created which is capable of producing live attenuated influenza vaccine (LAIV) with high yield. This cell line was shown to be non tumorigenic in eight week old adult athymic nude mouse model. This property is desirable for vaccine production and is unique to this cell line and is not known to be shared by other MDCK cell lines that are currently used for vaccine production. This significant difference in tumorigenic phenotype required further characterization of this cell line to ensure its safety for use in vaccine production. This is particularly important for LAIV production where it is not possible to incorporate a virus inactivation and/or removal step during manufacturing. Characterization of this cell line included extensive adventitious agent testing, tumorigenicity and oncogenicity assessment studies. Here, we describe the development of tumorigenic MDCK cell lines for use as positive controls and in vitro methods to aid in the evaluation of the tumorigenicity of MDCK 9B9-1E4 cloned cells. Tumorigenic MDCK cells were successfully generated following Hras and cMyc oncogene transfection of MDCK 9B9-1E4 cloned cells. In this study we demonstrate the lack of tumorigenic potential of the MDCK 9B9-1E4 cloned cell line in adult athymic nude mice model.     

Isolation and characterization of an RIP (ribosome-inactivating protein)-like protein from tobacco with dual enzymatic activity

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a protein termed tobacco RIP (TRIP) was isolated from tobacco (Nicotiana tabacum) leaves and purified using ion exchange and gel filtration chromatography in combination with yeast ribosome depurination assays. TRIP has a molecular mass of 26 kD as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed strong N-glycosidase activity as manifested by the depurination of yeast rRNA. Purified TRIP showed immunoreactivity with antibodies of RIPs from Mirabilis expansa. TRIP released fewer amounts of adenine residues from ribosomal (Artemia sp. and rat ribosomes) and non-ribosomal substrates (herring sperm DNA, rRNA, and tRNA) compared with other RIPs. TRIP inhibited translation in wheat (Triticum aestivum) germ more efficiently than in rabbit reticulocytes, showing an IC50 at 30 ng in the former system. Antimicrobial assays using highly purified TRIP (50 microg mL(-1)) conducted against various fungi and bacterial pathogens showed the strongest inhibitory activity against Trichoderma reesei and Pseudomonas solancearum. A 15-amino acid internal polypeptide sequence of TRIP was identical with the internal sequences of the iron-superoxide dismutase (Fe-SOD) from wild tobacco (Nicotiana plumbaginifolia), Arabidopsis, and potato (Solanum tuberosum). Purified TRIP showed SOD activity, and Escherichia coli Fe-SOD was observed to have RIP activity too. Thus, TRIP may be considered a dual activity enzyme showing RIP-like activity and Fe-SOD characteristics.