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.     

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.     

GNIP, a novel protein that binds and activates glycogenin, the self-glucosylating initiator of glycogen biosynthesis

Glycogenin is a self-glucosylating protein involved in the initiation of glycogen biosynthesis. Self-glucosylation leads to the formation of an oligosaccharide chain, which, when long enough, supports the action of glycogen synthase to elongate it and form a mature glycogen molecule. To identify possible regulators of glycogenin, the yeast two-hybrid strategy was employed. By using rabbit skeletal muscle glycogenin as a bait, cDNAs encoding three different proteins were isolated from the human skeletal muscle cDNA library. Two of the cDNAs encoded glycogenin and glycogen synthase, respectively, proteins known to be interactors. The third cDNA encoded a polypeptide of unknown function and was designated GNIP (glycogenin interacting protein). Northern blot analysis revealed that GNIP mRNA is highly expressed in skeletal muscle. The gene for GNIP generates at least four isoforms by alternative splicing. The largest isoform GNIP1 contains, from NH(2)- to COOH-terminal, a RING finger, a B box, a putative coiled-coil region, and a B30.2-like motif. The previously identified protein TRIM7 (tripartite motif containing protein 7) is also derived from the GNIP gene and is composed of the RING finger, B box, and coiled-coil regions. The GNIP2 and GNIP3 isoforms consist of the coiled-coil region and B30.2-like domain. Physical interaction between GNIP2 and glycogenin was confirmed by co-immunoprecipitation, and in addition GNIP2 was shown to stimulate glycogenin self-glucosylation 3-4-fold. GNIPs may represent a novel participant in the initiation of glycogen synthesis.     

Increased vacuolar Na(+)/H(+) exchange activity in Salicornia bigelovii Torr. in response to NaCl

Shoots of the halophyte Salicornia bigelovii are larger and more succulent when grown in highly saline environments. This increased growth and water uptake has been correlated with a large and specific cellular accumulation of sodium. In glycophytes, sensitivity to salt has been associated with an inability to remove sodium ions effectively from the cytoplasm in order to protect salt-sensitive metabolic processes. Therefore, in Salicornia bigelovii efficient vacuolar sequestration of sodium may be part of the mechanism underlying salt tolerance. The ability to compartmentalize sodium may result from a stimulation of the proton pumps that provide the driving force for increased sodium transport into the vacuole via a Na(+)/H(+) exchanger. In current studies, increased vacuolar pyrophosphatase activity (hydrolysis of inorganic pyrophosphate and proton translocation) and protein accumulation were observed in Salicornia bigelovii grown in high concentrations of NaCl. Based on sodium-induced dissipation of a pyrophosphate-dependent pH gradient in vacuolar membrane vesicles, a Na(+)/H(+) exchange activity was identified and characterized. This activity is sodium concentration-dependent, specific for sodium and lithium, sensitive to methyl-isobutyl amiloride, and independent of an electrical potential. Vacuolar Na(+)/H(+) exchange activity varied as a function of plant growth in salt. The affinity of the transporter for Na(+) is almost three times higher in plants grown in high levels of salt (K(m)=3.8 and 11.5 mM for plants grown in high and low salt, respectively) suggesting a role for exchange activity in the salt adaptation of Salicornia bigelovii.     

The Endoplasmic reticulum-associated maize GL8 protein is a component of the acyl-coenzyme A elongase ivolved in the production of cuticular waxes

The gl8 gene is required for the normal accumulation of cuticular waxes on maize (Zea mays) seedling leaves. The predicted GL8 protein exhibits significant sequence similarity to a class of enzymes that catalyze the reduction of a ketone group to a hydroxyl group. Polyclonal antibodies raised against the recombinant Escherichia coli-expressed GL8 protein were used to investigate the function of this protein in planta. Subcellular fractionation experiments indicate that the GL8 protein is associated with the endoplasmic reticulum membranes. Furthermore, polyclonal antibodies raised against the partially purified leek (Allium porrum) microsomal acyl-coenzyme A (CoA) elongase can react with the E. coli-expressed GL8 protein. In addition, anti-GL8 immunoglobulin G inhibited the in vitro elongation of stearoyl-CoA by leek and maize microsomal acyl-CoA elongase. In combination, these findings indicate that the GL8 protein is a component of the acyl-CoA elongase. In addition, the finding that anti-GL8 immunoglobulin G did not significantly inhibit the 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA dehydrase, and (E) 2,3-enoyl-CoA reductase partial reactions of leek or maize acyl-CoA elongase lends further support to our previous hypothesis that the GL8 protein functions as a beta-ketoacyl reductase during the elongation of very long-chain fatty acids required for the production of cuticular waxes.     

Pharmacological comparison of a recombinant CB1 cannabinoid receptor with its G(alpha 16) fusion product

The pharmacology of G protein-coupled receptors is widely accepted to depend on the G protein subunit to which the agonist-stimulated receptor couples. In order to investigate whether CB(1) agonist-mediated signal transduction via an engineered G(alpha 16) system is different than that of the G(i/o) coupling normally preferred by the CB(1) receptor, we transfected the human recombinant CB(1) receptor (hCB(1)) or a fusion protein comprising the hCB(1) receptor and G(alpha 16) (hCB(1)-G(alpha 16)) into HEK293 cells. From competition binding studies, the rank order of ligand affinities at the hCB(1)-G(alpha 16) fusion protein was found to be similar to that for hCB(1): HU 210 > CP 55,940 > or = SR 141716A > WIN 55212-2 > anandamide > JWH 015. Agonists increased [(35)S]GTP gamma S binding or inhibited forskolin-stimulated cAMP, presumably by coupling to G(i/o), in cells expressing hCB(1) but not hCB(1)-G(alpha 16). However, an analogous rank order of potencies was observed for these agonists in their ability to evoke increases in intracellular calcium concentration in cells expressing hCB(1)-G(alpha 16) but not hCB(1). These data demonstrate that ligand affinities for the hCB(1) receptor are not affected by fusion to the G(alpha 16) subunit. Furthermore, there is essentially no difference in the function of the hCB(1) receptor when coupled to G(i/o) or G (alpha 16).