Crystal structures of the Pyrococcus abyssi Sm core and its complex with RNA. Common features of RNA binding in archaea and eukarya

The Sm proteins are conserved in all three domains of life and are always associated with U-rich RNA sequences. Their proposed function is to mediate RNA-RNA interactions. We present here the crystal structures of Pyrococcus abyssi Sm protein (PA-Sm1) and its complex with a uridine heptamer. The overall structure of the protein complex, a heptameric ring with a central cavity, is similar to that proposed for the eukaryotic Sm core complex and found for other archaeal Sm proteins. RNA molecules bind to the protein at two different sites. They interact specifically inside the ring with three highly conserved residues, defining the uridine-binding pocket. In addition, nucleotides also interact on the surface formed by the N-terminal alpha-helix as well as a conserved aromatic residue in beta-strand 2 of the PA-Sm1 protein. The mutation of this conserved aromatic residue shows the importance of this second site for the discrimination between RNA sequences. Given the high structural homology between archaeal and eukaryotic Sm proteins, the PA-Sm1.RNA complex provides a model for how the small nuclear RNA contacts the Sm proteins in the Sm core. In addition, it suggests how Sm proteins might exert their function as modulators of RNA-RNA interactions.     

SDF-1alpha-induced intracellular calcium transient involves Rho GTPase signalling and is required for migration of hematopoietic progenitor cells

Signalling through the chemokine stromal derived factor (SDF)-1alpha and its receptor CXCR4 has been recognized as a key event in the migratory response of hematopoietic stem and progenitor cells (HPC). Small GTPases of the Rho/Rac family might be involved in SDF-1alpha signalling at several different levels. In the present study we report that two toxins from Clostridium species which inhibit the small GTPase Rho suppressed SDF-1alpha-induced generation of intracellular calcium transients in HPC. Chelation of intracellular Ca(2+) with BAPTA or depletion of intracellular Ca(2+) stores with thapsigargin demonstrated that calcium transients are essential for SDF-1alpha-induced chemotactic migration of HPC. Furthermore, transplantation of HPC pretreated with Ca(2+) flux inhibitors into mice revealed a suppression of HPC homing to the bone marrow and increased levels of cells remaining in the bloodstream or circulating to the spleen. Our data indicate that the small GTPase Rho is required for the induction of Ca(2+) transients in HPC, which in turn are necessary for the coordinated migratory response of HPC both in vitro and in vivo.     

Identity and expression pattern of chemosensory proteins in Heliothis virescens (Lepidoptera, Noctuidae)

Analyzing the chemosensory organs of the moth Heliothis virescens, three proteins belonging to the family of insect chemosensory proteins (CSPs) have been cloned; they are called HvirCSP1, HvirCSP2 and HvirCSP3. The HvirCSPs show about 50% identity between each other and 30–76% identity to CSPs from other species. Overall, they are rather hydrophilic proteins but include a conserved hydrophobic motif. Tissue distribution and temporal expression pattern during the last pupal stages were assessed by Northern blots. HvirCSP mRNAs were detected in various parts of the adult body with a particular high expression level in legs. The expression of HvirCSP1 in legs started early during adult development, in parallel with the appearance of the cuticle. HvirCSP1 mRNA was detectable five days before eclosion (day E-5), increased dramatically on day E-3 and remained at high level into adult life. The tissue distribution and the time course of appearance of HvirCSPs are in agreement with a possible role in contact chemosensation.     

Phylogeny of the major lineages of Membracoidea (Insecta: Hemiptera: Cicadomorpha) based on 28S rDNA sequences

Analysis of sequences from a 3.5-kb region of the nuclear ribosomal 28S DNA gene spanning divergent domains D2-D10 supports the hypothesis, based on fossil, biogeographic, and behavioral evidence, that treehoppers (Aetalionidae and Membracidae) are derived from leafhoppers (Cicadellidae). Maximum-parsimony analysis indicated that treehoppers are the sister group of a lineage comprising the currently recognized cicadellid subfamilies Agalliinae, Megophthalminae, Adelungiinae, and Ulopinae. Based on this phylogenetic estimate, the derivation of treehoppers approximately coincided with shifts in physiology and behavior, including loss of brochosome production and a reversal from active, jumping nymphs to sessile, nonjumping nymphs. Myerslopiidae, traditionally placed as a tribe of the cicadellid subfamily Ulopinae, represented a basal lineage distinct from other extant membracoids. The analysis recovered a large leafhopper lineage comprising a polyphyletic Deltocephalinae (sensu stricto) and its apparent derivatives Koebeliinae, Eupelicinae (polyphyletic), Selenocephalinae, and Penthimiinae. Clades comprising Macropsinae, Neocoelidiinae, Scarinae, Iassinae, Coelidiinae, Eurymelinae + Idiocerinae, Evacanthini + Pagaroniini, Aphrodinae + Ledrinae (in part), Stenocotini + Tartessinae, and Cicadellini + Proconiini were also recovered with moderate to high branch support. Cicadellinae (sensu lato), Ledrinae, Typhlocybinae, and Xestocephalinae were consistently polyphyletic on the most-parsimonious topologies, but constraining these groups to be monophyletic did not significantly increase the length of the cladograms. Relationships among the major lineages received low branch support, suggesting that more data are needed to provide a robust phylogenetic estimate.     

Tailor-made dyes for fluorescence correlation spectroscopy (FCS)

Two new fluorescent labels are presented that are optimized for excitation with He/Ne laser and red diode lasers. Application in FCS and labeling of proteins and oligomers are demonstrated. A strong rise of quantum yield and emission life time upon binding to biomolecules are characteristic features of the dyes.     

NIM1 overexpression in Arabidopsis potentiates plant disease resistance and results in enhanced effectiveness of fungicides

The NIM1 (for noninducible immunity, also known as NPR1) gene is required for the biological and chemical activation of systemic acquired resistance (SAR) in Arabidopsis. Overexpression of NIM1 in wild-type plants (hereafter referred to as NIM1 plants or lines) results in varying degrees of resistance to different pathogens. Experiments were performed to address the basis of the enhanced disease resistance responses seen in the NIM1 plants. The increased resistance observed in the NIM1 lines correlated with increased NIM1 protein levels and rapid induction of PR1 gene expression, a marker for SAR induction in Arabidopsis, following pathogen inoculation. Levels of salicylic acid (SA), an endogenous signaling molecule required for SAR induction, were not significantly increased compared with wild-type plants. SA was required for the enhanced resistance in NIM1 plants, however, suggesting that the effect of NIM1 overexpression is that plants are more responsive to SA or a SA-dependent signal. This hypothesis is supported by the heightened responsiveness that NIM1 lines exhibited to the SAR-inducing compound benzo(1,2,3)-thiadiazole-7-car-bothioic acid S-methyl ester. Furthermore, the increased efficacy of three fungicides was observed in the NIM1 plants, suggesting that a combination of transgenic and chemical approaches may lead to effective and durable disease-control strategies.     

LaPSvS1, a (1-->3)-beta-galactan sulfate and its effect on angiogenesis in vivo and in vitro

LaPSvS1, a highly sulfated branched (1-->3)-beta-galactan was prepared from the arabino-galactan from Larix decidua Miller by partial hydrolysis and subsequent sulfation with SO(3)-pyridine in DMF. The molecular weight was analyzed by GPC and the sulfate content was determined by ion chromatography. LaPSvS1 exhibited good antiangiogenic and antiinflammatory effects in two different modifications of the known CAM-assay. In vitro results obtained in the FGF-2-trypsin-assay and in fluorospectrometric experiments revealed that LaPSvS1 interacts with the fibroblast growth factor 2 system. This interaction is correlated with the in vivo effect of LaPSvS1 on FGF-2 induced angiogenesis.     

High diagnostic accuracy of cytologic smears of central nervous system tumors. A 15-year experience based on 4,172 patients

OBJECTIVE: To investigate the diagnostic accuracy and current role of intraoperative cytologic smears of central nervous system tumors. STUDY DESIGN: Retrospective analysis of 4,172 patients operated on during 1985-1999, with 3,541 intraoperative smears performed during open procedures and 631 during stereotactic biopsies. RESULTS: Complete correlation with the final diagnosis was achieved in a mean of 89.8% (range, 83-93.7% per year). Diagnostic accuracy increased to 95% on average (range, 91.5-96.7% per year) when cases of partial correlation, mainly due to grading deviations, were included. The most accurate intraoperative diagnoses were obtained in cases of meningioma (97.9%), metastasis (96.3%) and glioblastoma (95.7%). A significant reduction in diagnostic accuracy was observed in cases of oligodendroglioma (80.9%) and ependymoma (77.7%). Besides diagnosis and grading, smear cytology provided resection guidance in cases of well-delineated tumors. CONCLUSION: Intraoperative smears in neurosurgery are easy to obtain and inexpensive and have high diagnostic accuracy. In addition to stereotactic biopsy procedures, intraoperative smears permit reliable intraoperative guidance during lesion targeting and resection.     

A naturally occurring mutation in the SLC21A6 gene causing impaired membrane localization of the hepatocyte uptake transporter

The organic anion transporter SLC21A6 (also known as OATP2, OATP-C, or LST-1) is involved in the hepatocellular uptake of a variety of endogenous and xenobiotic substances and drugs. We analyzed 81 human liver samples by immunoblotting and found one with a strongly reduced amount of SLC21A6 protein suggesting mutations in the SLC21A6 gene. The SLC21A6 cDNA from this sample contained five base pair changes in one allele; three of the mutations resulted in amino acid substitutions designated SLC21A6-N130D, SLC21A6-P155T, and SLC21A6-L193R. The former two were polymorphisms (SLC21A6*1b and SLC21A6*4), whereas SLC21A6-L193R represents the first naturally occurring mutation identified in one allele of the SLC21A6 gene, which affects protein maturation and organic anion transport. We introduced each of the mutations into the SLC21A6 cDNA and established stably transfected MDCKII cells expressing the respective mutant SLC21A6 protein. Immunofluorescence microscopy and uptake measurements were used to study localization and transport properties of the mutated proteins. Both proteins carrying the polymorphisms were sorted to the lateral membrane like wild-type SLC21A6, but their transport properties for the substrates cholyltaurine and 17beta-glucuronosyl estradiol were altered. Importantly, most of the mutant protein SLC21A6-L193R was retained intracellularly, and this single amino acid exchange abolished transport function.