(GTPyS), to membrane G proteins were not different in NT and HT cell lines. However, PAF-and mastoparan-stimulated binding of GTPyS to G proteins, which was fully PTX-sensitive, was 2.5-fold higher in HT than NT cell lines. These data suggest an enhanced receptor-mediated activation of PTX-sensitive G proteins despite unchanged receptor and G protein expression. Thus, this study not only suggests that enhanced signal transduction and cell proliferation are abnormalities in a certain group of patients with essential hypertension but also explains these findings as a result of an enhanced G protein activation in this common disorder.

No abstract

Temperature gradient gel electrophoresis (TGGE) is well suited for fingerprinting bacterial communities by separating PCR-amplified fragments of 16S rRNA genes (16S ribosomal DNA [rDNA]). A strategy was developed and was generally applicable for linking 16S rDNA from community fingerprints to pure culture isolates from the same habitat. For this, digoxigenin-labeled polynucleotide probes were generated by PCR, using bands excised from TGGE community fingerprints as a template, and applied in hybridizations with dot blotted 16S rDNA amplified from bacterial isolates. Within 16S rDNA, the hypervariable V6 region, corresponding to positions 984 to 1047 (Escherichia coli 16S rDNA sequence), which is a subset of the region used for TGGE (positions 968 to 1401), best met the criteria of high phylogenetic variability, required for sufficient probe specificity, and closely flanking conserved priming sites for amplification. Removal of flanking conserved bases was necessary to enable the differentiation of closely related species. This was achieved by 5′ exonuclease digestion, terminated by phosphorothioate bonds which were synthesized into the primers. The remaining complementary strand was removed by single-strand-specific digestion. Standard hybridization with truncated probes allowed differentiation of bacteria which differed by only two bases within the probe target site and 1.2% within the complete 16S rDNA. However, a truncated probe, derived from an excised TGGE band of a rhizosphere community, hybridized with three phylogenetically related isolates with identical V6 sequences. Only one of the isolates comigrated with the excised band in TGGE, which was shown to be due to identical sequences, demonstrating the utility of a combined TGGE and V6 probe approach.

Proliferation and immunoglobulin secretion of B lymphocytes are regulated by specific antigens and numerous accessory immunomodulatory factors. Lysophosphatidic acid (LPA) is a glycerophospholipid mediator that is released from activated blood platelets, attains high levels in serum, and exerts potent stimulatory effects on, e.g., neutrophils, monocytes, and T lymphocytes. LPA is also generated by a secretory, cytokine-inducible phospholipase A2 present in high concentrations in inflammatory exudates and septic states. We investigated effects of LPA on human Epstein-Barr virus-immortalized B lymphoblasts, a model for immunoglobulin-secreting B cells. Intracellular Ca2+ was determined with fura 2 and the formation of inositol 1,4,5-trisphosphate by anion-exchange chromatography. LPA stimulated an increase in inositol 1,4,5-trisphosphate levels and induced a transient rise in intracellular free Ca2+concentration from 105 ± 17 to 226 ± 21 nM. This Ca2+ signal resulted from Ca2+ mobilization and Ca2+ influx and was subject to homologous desensitization. Pertussis toxin inhibited these responses by ∼70%. Furthermore, LPA stimulated a 27.5% increase in guanosine 5′- O-(3-thiotriphosphate) binding to permeabilized B lymphoblasts, which suggests the direct activation of pertussis toxin-sensitive G proteins by LPA. LPA stimulated a strong increase in the specific phosphorylation of the mitogen-activated protein kinase (immunoblot analysis) that was prevented by the MEK inhibitor PD-98059. Finally, LPA triggered a 2-fold increase in DNA synthesis ([3H]thymidine incorporation) and a 2-fold increase in B lymphoblast number and evoked a 20- to 50-fold increase in immunoglobulin formation. By RT-PCR we detected specific mRNA transcripts for the recently cloned human LPA receptor. Thus our data suggest that LPA behaves as a B cell growth factor.

Larval Drosophila are used as a genetically accessible study case in many areas of biological research. Here we report a fast, robust and user-friendly procedure for the whole-body multi-fluorescence imaging of Drosophila larvae; the protocol has been optimized specifically for larvae by systematically tackling the pitfalls associated with clearing this small but cuticularized organism. Tests on various fluorescent proteins reveal that the recently introduced monomeric infrared fluorescent protein (mIFP) is particularly suitable for our approach. This approach comprises an effective, low-cost clearing protocol with minimal handling time and reduced toxicity in the reagents employed. It combines a success rate high enough to allow for small-scale screening approaches and a resolution sufficient for cellularlevel analyses with light sheet and confocal microscopy. Given that publications and database documentations typically specify expression patterns of transgenic driver lines only within a given organ system of interest, the present procedure should be versatile enough to extend such documentation systematically to the whole body. As examples, the expression patterns of transgenic driver lines covering the majority of neurons, or subsets of chemosensory, central brain or motor neurons, are documented in the context of whole larval body volumes (using nsyb-Gal4, IR76b-Gal4, APL-Gal4 and mushroom body Kenyon cells, or OK371-Gal4, respectively). Notably, the presented protocol allows for triple-color fluorescence imaging with near-infrared, red and yellow fluorescent proteins.