Expression-level dependent activation of recombinant human parathyroid hormone/parathyroid hormone-related peptide receptor: effect of human parathyroid hormone (1-34), (1-31), and (28-48)

A stable recombinant chinese hamster ovary (CHO) cell model system expressing the human type-1 receptor for parathyroid hormone and parathyroid hormone-related peptide (hPTH-R) was established for the analysis of human PTH (hPTH) variants. The cell lines showed receptor expression in the range from 10(5) to I.9 x 10(6) receptors per cell. The affinity of the receptors for hPTH-(1-34) was independent of the receptor number per cell (Kd approximately = 8 nmol/1). The induction of cAMP by hPTH-(1-34) is maximal in clones expressing >2x10(5) receptors per cell and Ca++ signals were maximal in cell lines expressing >1.4x10(6) receptors per cell. Second messenger specific inhibitors demonstrated that PTH-induced increases in intracellular cAMP and Ca++ are independent and Ca++ ions are derived from intracellular stores. The cAMP-specific receptor activator hPTH-(1-31) showed also an increase in intracellular Ca++. Even in cell lines expressing more than 10(6) receptors per cell the Ca++/PKC specific activator hPTH-(28-48) did not activate hPTH-Rs. Based on these results, synthesis of further derivatives of PTH is required to identify pathway-specific ligands for the type-1 hPTH-R.     

Misexpression screen in Drosophila melanogaster aiming to reveal novel factors involved in formation of body parts

To identify novel factors that lead a fly imaginal disc to adopt its developmental fate, we carried out a modular dominant misexpression screen in imaginal discs. We have identified two factors that appear to change the fate of the respective body structure and appear to lead to the transformation of a body part. In one mutant line, notum tissue, normally derived from wing imaginal tissue, formed close to the site of the sternopleural bristles, which are leg disc derivatives. In the other line, the arista is transformed into a tubular structure, resembling an abnormal leg. We found that ectopic expression of abrupt was responsible for this potential transformation of the arista.     

Transformation of nuclear and plastomic plant genomes by biolistic particle bombardment

Microprojectile bombardment is a powerful method for the transformation of various organisms and tissues. For plants, the biolistic approach is primarily used for transformation of cereals and other monocotyledons, as well as for dicotyledonous plants shown to be recalcitrant to Agrobacterium-based transformation of organellar genomes, and transformation of plant and algal chloroplasts has recently been reported. In this protocol paper we provide methods for nuclear and plastomic transformation of plants using the biolistic technique.     

Hyperaccumulator Alyssum murale relies on a different metal storage mechanism for cobalt than for nickel

The nickel (Ni) hyperaccumulator Alyssum murale has been developed as a commercial crop for phytoremediation/phytomining Ni from metal-enriched soils. Here, metal co-tolerance, accumulation and localization were investigated for A. murale exposed to metal co-contaminants. A. murale was irrigated with Ni-enriched nutrient solutions containing basal or elevated concentrations of cobalt (Co) or zinc (Zn). Metal localization and elemental associations were investigated in situ with synchrotron X-ray microfluorescence (SXRF) and computed-microtomography (CMT). A. murale hyperaccumulated Ni and Co (> 1000 microg g(-1) dry weight) from mixed-metal systems. Zinc was not hyperaccumulated. Elevated Co or Zn concentrations did not alter Ni accumulation or localization. SXRF images showed uniform Ni distribution in leaves and preferential localization of Co near leaf tips/margins. CMT images revealed that leaf epidermal tissue was enriched with Ni but devoid of Co, that Co was localized in the apoplasm of leaf ground tissue and that Co was sequestered on leaf surfaces near the tips/margins. Cobalt-rich mineral precipitate(s) form on leaves of Co-treated A. murale. Specialized biochemical processes linked with Ni (hyper)tolerance in A. murale do not confer (hyper)tolerance to Co. A. murale relies on a different metal storage mechanism for Co (exocellular sequestration) than for Ni (vacuolar sequestration).     

Exoribonuclease R interacts with endoribonuclease E and an RNA helicase in the psychrotrophic bacterium Pseudomonas syringae Lz4W

Endoribonuclease E, a key enzyme involved in RNA decay and processing in bacteria, organizes a protein complex called degradosome. In Escherichia coli, Rhodobacter capsulatus, and Streptomyces coelicolor, RNase E interacts with the phosphate-dependent exoribonuclease polynucleotide phosphorylase, DEAD-box helicase(s), and additional factors in an RNA-degrading complex. To characterize the degradosome of the psychrotrophic bacterium Pseudomonas syringae Lz4W, RNase E was enriched by cation exchange chromatography and fractionation in a glycerol density gradient. Most surprisingly, the hydrolytic exoribonuclease RNase R was found to co-purify with RNase E. Co-immunoprecipitation and Ni(2+)-affinity pull-down experiments confirmed the specific interaction between RNase R and RNase E. Additionally, the DEAD-box helicase RhlE was identified as part of this protein complex. Fractions comprising the three proteins showed RNase E and RNase R activity and efficiently degraded a synthetic stem-loop containing RNA in the presence of ATP. The unexpected association of RNase R with RNase E and RhlE in an RNA-degrading complex indicates that the cold-adapted P. syringae has a degradosome of novel structure. The identification of RNase R instead of polynucleotide phosphorylase in this complex underlines the importance of the interaction between endo- and exoribonucleases for the bacterial RNA metabolism. The physical association of RNase E with an exoribonuclease and an RNA helicase apparently is a common theme in the composition of bacterial RNA-degrading complexes.     

Solar ultraviolet radiation-induced DNA damage in aquatic organisms: potential environmental impact

Continuing depletion of stratospheric ozone and subsequent increases in deleterious ultraviolet (UV) radiation at the Earth's surface have fueled the interest in its ecological consequences for aquatic ecosystems. The DNA is certainly one of the key targets for UV-induced damage in a variety of aquatic organisms. UV radiation induces two of the most abundant mutagenic and cytotoxic DNA lesions, cyclobutane pyrimidine dimers (CPDs) and pyrimidine pyrimidone photoproducts (6-4PPs) and their Dewar valence isomers. However, aquatic organisms have developed a number of repair and tolerance mechanisms to counteract the damaging effects of UV on DNA. Photoreactivation with the help of the enzyme photolyase is one of the most important and frequently occurring repair mechanisms in a variety of organisms. Excision repair, which can be distinguished into base excision repair (BER) and nucleotide excision repair (NER), also play an important role in DNA repair in several organisms with the help of a number of glycosylases and polymerases, respectively. In addition, mechanisms such as mutagenic repair or dimer bypass, recombinational repair, cell-cycle checkpoints, apoptosis and certain alternative repair pathways are also operative in various organisms. This review deals with the UV-induced DNA damage and repair in a number of aquatic organisms as well as methods of detecting DNA damage.     

Plasmids in the bacterial assemblage of a dystrophic Lake: Evidence for plasmid-encoded nickel resistance

Sixty-two aerobic bacterial strains isolated from the unproductive dystrophic Lake Skärshultsjön (South Sweden) were screened for plasmids. The lake is considered to be an extreme environment because of its high concentration of persistent but nontoxic humic compounds. One-third of the isolates harbored multiple plasmids usually of similar high molecular weights (>25 Mdal). The plasmid-bearing strains were members of the common aquatic taxaPseudomonas spp.,Acinetobacter sp.,Alcaligenes sp.,Aeromonas/Vibrio group, andEnterobacteriaceae (taxonomy is tentative). The majority of isolates displayed multiple resistance to antibiotics and heavy metals. Some of them were capable of degrading aromatic compounds. Three isolates were chosen for curing experiments. Only strain S-68, anAlcaligenes sp., could be cured of one of its two plasmids. It harbored the two cryptic plasmids pQQ32 and pQQ70 of 32 and ca. 70 Mdal, and the latter was segregated during ethidium bromide treatment. Parental strain S-68 was capable of degrading some of nonchlorinated phenolic compounds and displayed resistance to a broad spectrum of antibiotics and the heavy metals Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, and Hg²⁺. Derivative strain S-68-41 lost its resistance to nickel, suggesting segregated plasmid PQQ70 coded for nickel resistance. Transformation experiments to restore nickel resistance in the cured derivative strain were not successful.     

Ketamine-induced tongue protrusions in rats

1. The purpose of this study was to demonstrate that ketamine anesthesia (100 mg/kg) induces tongue protrusions (P) in addition to retrusions (R) and swallows (S) in adult rats. 2. These linguo-pharyngeal events occur alone or combined in various sequential patterns. 3. The SPR sequence is not the predominant pattern in all preparations suggesting profound disruption of physiological linkages by ketamine. 4. Haloperidol administration suppresses these events for 1-120 min depending on the dose (0.75-2.5 mg/kg). 5. Swallows are the least vulnerable to haloperidol. 6. This and previous findings provide further evidence that ketamine induced linguo-pharyngeal activity can serve as a model for acute or tardive dyskinesia better than stereotypies.     

Rho‐kinase inhibitor Y‐27632 increases cellular proliferation and migration in human foreskin fibroblast cells

The idea of direct differentiation of somatic cells into other differentiated cell types has attracted a great interest recently. Rho‐kinase inhibitor Y‐27632 (ROCKi) is a potential drug molecule, which has been reported to support the gene expressions typical for the chondrocytes, thus restricting their phenotypic conversion to fibroblastic cells upon the cellular expansion. In this study, we have investigated the short‐term biological responses of ROCKi to human primary foreskin fibroblasts. The fibroblast cells were exposed to 1 and 10 μM ROCKi treatments. A proteomics analysis revealed expression changes of 56 proteins, and a further protein pathway analysis suggested their association with the cell morphology, the organization, and the increased cellular movement and the proliferation. These functional responses were confirmed by a Cell‐IQ time‐lapse imaging analysis. Rho‐kinase inhibitor treatment increased the cellular proliferation up to twofold during the first 12 h, and a wound model based migration assay showed 50% faster filling of the mechanically generated wound area. Additionally, significantly less vinculin‐associated focal adhesions were present in the ROCKi‐treated cells. Despite the marked changes in the cell behavior, ROCKi was not able to induce the expression of the chondrocyte‐specific genes, such as procollagen α₁(II) and aggrecan.