Complete sequence and organization of pBtoxis,the toxin-coding plasmid of Bacillus thuringiensis subsp. israelensis

The entire 127,923-bp sequence of the toxin-encoding plasmid pBtoxis from Bacillus thuringiensis subsp. israelensis is presented and analyzed. In addition to the four known Cry and two known Cyt toxins, a third Cyt-type sequence was found with an additional C-terminal domain previously unseen in such proteins. Many plasmid-encoded genes could be involved in several functions other than toxin production. The most striking of these are several genes potentially affecting host sporulation and germination and a set of genes for the production and export of a peptide antibiotic.     

Ancient ontogenies: larval development of the Lower Cretaceous anuran Shomronella jordanica (Amphibia: Pipoidea)

The Lower Cretaceous anuran Shomronella jordanica (Pipoidea) is represented by an assemblage of nearly 300 tadpoles of different ages. The size of the assemblage allows a reconstruction of the larval ontogeny of this species. We describe the ossification sequence and growth rates of S. jordanica and present reconstructions of tadpoles at different stages. The ontogeny of the species seems to be similar to that of extant pipids in many aspects. Larvae are similar in shape and size to those of Xenopus laevis but lack the anterior barbels that are typical for all extant pipids. The ossification sequence is closer to that of pipids than to that of other anurans. We present evidence that suggests that this species was terrestrial as an adult, and we discuss the implications of these data for the evolution of anuran development.     

Casein kinase 2 specifically binds to and phosphorylates the carboxy termini of ENaC subunits.

A number of findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na+ channel (ENaC). A recent study has demonstrated that the C tails of the beta and gamma subunits of ENaC are subject to phosphorylation by at least three protein kinases [Shi, H., Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R. & Garty, H. (2002) J. Biol. Chem. 277, 13539-13547]. One of them was identified as ERK which phosphorylates betaT613 and gammaT623 and affects the channel interaction with Nedd4. The current study identifies a second protein kinase as casein kinase 2 (CK2), or CK-2-like kinase. It phosphorylates betaS631, a well-conserved serine on the beta subunit. Such phosphorylation is observed both in vitro using glutathione-S-transferase-ENaC fusion proteins and in vivo in ENaC-expressing Xenopus oocytes. The gamma subunit is weakly phosphorylated by this protein kinase on another residue (gammaT599), and the C tail of alpha is not significantly phosphorylated by this kinase. Thus, CK2 may be involved in the regulation of the epithelial Na+ channel.     

Advantage of Using Inosine at the 3′ Termini of 16S rRNA Gene Universal Primers for the Study of Microbial Diversity

To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3′ termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54°C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50°C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3′ termini in studying the microbial diversity of environmental samples.     

Retroviral transduction of interferon-γ cDNA into a nonimmunogenic murinefibrosarcoma: generation of T cells in draining lymph nodes capable of treating established parental metastatic tumor

Gene modification of tumor cells with the cDNA for interferon (IFN) has been shown to increase the immunogenicity of some tumor cells. In order to explore further the possible therapeutic relevance of these previous findings, two clones of the nonimmunogenic MCA-102 fibrosarcoma of C57BL/6 origin were retrovirally transduced with the cDNA encoding murine IFN: 102.4JK (4JK), a clone with relatively high major histocompatibility complex (MHC) class I expression, and 102.24JK (24JK), a clone with low expression of surface MHC class I molecules. Retroviral transduction of tumor cells with the cDNA encoding for IFN resulted in a substantial up-regulation of MHC class I surface expression in the 24JK clone but little change of class I in the 4JK clone. In an attempt to generate antitumor lymphocytes, these gene-modified cells were inoculated into mouse footpads and draining lymph nodes (DLN) were removed, dispersed, and cultured in vitro for 10 days with irradiated tumor cells and interleukin-2. DLN from mice bearing either unmodified tumor or tumor transduced with cDNA encoding neomycin resistance (Neo ^R) or IFN, were used to treat recipients harboring 3-day pulmonary metastases induced by the parental, unmodified tumor. Treatment with DLN cells obtained following the injection of 24JK tumor cells modified with the gene for IFN significantly reduced the number of pulmonary metastases in four separate experiments, compared to groups treated by DLN cells generated from inoculation of either the unmodified, parental 24JK clone or the same clone transduced with theNeo ^R gene only. In contrast, DLN cells induced either by IFN-transduced 4JK (high expression of MHC class I) or an unmodified 4JK tumor (moderate expression of MHC class I) had significant but equal therapeutic efficacy. Although the in vitro growth rate of tumor cell lines was unaffected by the insertion of the mouse IFN cDNA, their in vivo (s.c.) growth rates were significantly slower than those of the nontransduced tumors. Thus, after retroviral transduction of the murine IFN cDNA into a nonimmunogenic tumor with a very low level of surface expression of MHC class I, modified tumor cells could elicit therapeutic T cells from DLN capable of successfully treating established pulmonary metastases upon adoptive transfer. This strategy significantly confirms previous observations on the potential therapeutic effects of gene modification of tumor cells with IFN and extends the realm of therapeutic possibilities to include the use of DLN cells for the development of T-cell based immunotherapies against nonimmunogenic human tumors.     

Repeated Reserpine Increases Striatal Dopamine Receptor and Guanine Nucleotide Binding Protein RNA

Abstract: In the present study the effects of repeated administration of reserpine on striatal dopamine receptor and guanine nucleotide binding protein mRNAs were determined. Twenty-four hours after seven consecutive daily injections of reserpine—a treatment that is known to produce functional sensitization of D₁ and D₂ dopamine receptors—the level of striatal D₁ dopamine receptor mRNA was unchanged. However, the level of mRNA for the G protein G_sα was increased by 127%. After extended reserpine treatment for 14 days, levels of both striatal D₁ DA receptor and G_sα mRNAs were elevated by 99 and 78%, respectively. Seven days of reserpine treatment also increased levels of mRNA of the striatal D₂ dopamine receptor and of G proteins G_i2α and G_oα by 200, 79, and 32%, respectively. After 14 days of reserpine treatment the level of striatal D₂ dopamine receptor mRNA was increased by twofold. In contrast, levels of the mRNAs coding for the G proteins G_i2α and G_oα were unchanged. These data suggest that dopamine receptors and their respective G proteins play important roles in the development of sensitization of striatal dopamine receptors during repeated reserpine treatment. Furthermore, the persistent increase in level of striatal G_sα mRNA suggests that this G protein is necessary to maintain supersensitivity of the striatal D₁ dopamine receptor system following long-term dopamine depletion.     

Attenuation of water velocity and incident light as a function of shape parameters inGracilaria

Characteristic shape parameters, light intensities and relative water velocities were estimated in thalli of free-moving seaweeds, using threeGracilaria-like species:G. cornea, G. conferta andGracilariopsis lemaneiformis. Relative velocities over the branches were determined mostly by rotation of the algae in the water as opposed to linear translation. The tree major shape parameters of the thallus, weight, volumetric specific weight and areal specific weight, explain the differences in relative velocity. Relative velocities near the center of the thallus were about 50% to 80% of the external velocity. Light intensities at the vicinity of the center ofG. cornea ranged between 60% to 90% of the external light intensity. Light attenuation in the center was proportional to the weight of the thallus. The significance of light and velocity attenuation as a function of shape parameters is discussed.Author for correspondence     

Purification and properties of laccase from Botrytis cinerea

The partial purification of an extracellular laccase from Botrytis cinerea is described. Specificity of the enzyme, its K_m for a number of substrates and sensitivity to some inhibitors are described. The enzyme is a typical laccase but has an exceptionally low pI and great stability to acid pH. On gel electrophoresis two isoenzymes could be detected.