Loss of S100A9 (MRP14) results in reduced interleukin-8-induced CD11b surface expression,a polarized microfilament system,and diminished responsiveness to chemoattractants in vitro

The S100A9 (MRP14) protein is abundantly expressed in myeloid cells and has been associated with various inflammatory diseases. The S100A9-deficient mice described here were viable, fertile, and generally of healthy appearance. The myelopoietic potential of the S100A9-null bone marrow was normal. S100A8, the heterodimerization partner of S100A9 was not detectable in peripheral blood cells, suggesting that even a deficiency in both S100A8 and S100A9 proteins was compatible with viable and mature neutrophils. Surprisingly, the invasion of S100A9-deficient leukocytes into the peritoneum and into the skin in vivo was indistinguishable from that in wild-type mice. However, stimulation of S100A9-deficient neutrophils with interleukin-8 in vitro failed to provoke an up-regulation of CD11b. Migration upon a chemotactic stimulus through an endothelial monolayer was markedly diminished in S100A9-deficient neutrophils. Attenuated chemokinesis of the S100A9-deficient neutrophils was observed by using a three-dimensional collagen matrix migration assay. The altered migratory behavior was associated with a microfilament system that was highly polarized in unstimulated S100A9-deficient neutrophils. Our data suggest that loss of the calcium-binding S100A9 protein reduces the responsiveness of the neutrophils upon chemoattractant stimuli at least in vitro. Alternative pathways for neutrophil emigration may be responsible for the lack of any effect in the two in vivo models we have investigated so far.     

A novel GlcNAcalpha1-HPO3-6Gal(1-1)ceramide antigen and alkylated inositol-phosphoglycerolipids expressed by the liver fluke Fasciola hepatica

The acidic (glyco)lipids of the parasitic liver fluke Fasciola hepatica exhibited two different phosphate-containing species, designated AL-I and AL-II, which were analyzed by MALDI-TOF MS, ESI MS, NMR, methylation analysis, and combined GC-MS in conjunction with HF treatment. AL-I was structurally determined as 1-O-hexadecyl-sn-glycerol-3-phosphoinositol, an ether bond variant of lysophosphatidylinositol. The structure of AL-II was shown to be GlcNAcalpha1-HPO3-6Gal(1-1)ceramide. Ceramide analysis revealed as major components 2-hydroxyoctadecanoic acid [18:0(2-OH)] together with C18- and C20-phytosphingosines. AL-II was apparently highly antigenic and strongly recognized by both animal- and human-F. hepatica infection sera. Furthermore, inhibition ELISAs revealed that the unusual antigenic determinant GlcNAcalpha1-HPO3- phosphate might have a potential in the serodiagnosis of F. hepatica infections.     

Hepatic neovascularization after partial portal vein ligation: novel mechanism of chronic regulation of blood flow

The present study was undertaken to investigate hepatic microcirculatory response following partial portal vein ligation (PPVL) in rats. Portal pressure was markedly increased 2-6 wk after PPVL, but no significant reduction in sinusoidal perfusion and hepatocellular injury were detected. However, marked neovascularization was observed in PPVL rats using intravital microscopy and scanning electron microscopy (SEM). Extremely high red blood cell velocity (2,000-4,900 microm/s) was seen in these vessels. Injection of fluorescein sodium via the carotid artery revealed that the neovessels originated from the hepatic arterial vasculature. This was further confirmed by clamping the common hepatic artery and phenylephrine injection from the carotid artery. These vessels maintained sufficient flow after massive sinusoidal shutdown elicited by the portal infusion of endothelin receptor B agonist IRL-1620. SEM also showed extensive neovascularization at the hilum. Additionally, clamping the portal vein decreased sinusoidal perfusion only by 9.5% in PPVL, whereas a 71.2% decrease was observed in sham. These results strongly suggest that the liver maintains its microcirculatory flow by vascular remodeling from the hepatic arterial vasculature following PPVL.     

Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity

A Drosophila homolog of human Down syndrome cell adhesion molecule (DSCAM), an immunoglobulin superfamily member, was isolated by its affinity to Dock, an SH3/SH2 adaptor protein required for axon guidance. Dscam binds directly to both Dock's SH2 and SH3 domains. Genetic studies revealed that Dscam, Dock and Pak, a serine/threonine kinase, act together to direct pathfinding of Bolwig's nerve, containing a subclass of sensory axons, to an intermediate target in the embryo. Dscam also is required for the formation of axon pathways in the embryonic central nervous system. cDNA and genomic analyses reveal the existence of multiple forms of Dscam with a conserved architecture containing variable Ig and transmembrane domains. Alternative splicing can potentially generate more than 38,000 Dscam isoforms. This molecular diversity may contribute to the specificity of neuronal connectivity.     

Cleavage of polypeptide chain initiation factor eIF4GI during apoptosis in lymphoma cells: characterisation of an internal fragment generated by caspase-3-mediated cleavage

Polypeptide chain initiation factor eIF4GI undergoes caspase-mediated degradation during apoptosis to give characteristic fragments. The most prominent of these has an estimated mass of approximately 76 kDa (Middle-Fragment of Apoptotic cleavage of eIF4G; M-FAG). Subcellular fractionation of the BJAB lymphoma cell line after induction of apoptosis indicates that M-FAG occurs in both ribosome-bound and soluble forms. Affinity chromatography on m7GTP-Sepharose shows that M-FAG retains the ability of eIF4GI to associate with both the mRNA cap-binding protein eIF4E and initiation factor eIF4A and that the ribosome-bound form of M-FAG is also present as a complex with eIF4E and eIF4A. These data suggest that the binding sites for eIF4E, eIF4A and eIF3 on eIF4GI are retained in the caspase-generated fragment. M-FAG is also a substrate for cleavage by the Foot-and-Mouth-Disease Virus-encoded L protease. These properties, together with the pattern of recognition by a panel of antibodies, define the origin of the apoptotic cleavage fragment. N-terminal sequencing of the products of caspase-3-mediated eIF4GI cleavage has identified the major cleavage sites. The pattern of eIF4GI degradation and the possible roles of the individual cleavage products in cells undergoing apoptosis are discussed.     

G Proteins Modulate D2 Receptor-Coupled K(ATP) Channels in Rat Dopaminergic Terminals

The presynaptic dopamine (DA) D2 receptor-mediated regulation of ATP-sensitive potassium (K⁺ _ATP) channels was examined in slices of the rat caudate-putamen. When slices were incubated with the specific D2 receptor antagonist (–)-sulpiride (SLP), a concentration-dependent increase of extracellular DA release was observed. SLP-induced enhancement was completely antagonized by coincubation with the K⁺ _ATP channel opener diazoxide (DIA). Treatment of slices with the D2 receptor agonist quinpirole (QUI) almost completely inhibited DA outflow induced by the K⁺ _ATP channel blocker butanedione-monoxime (BDM). Coincubation of SLP and guanosine triphosphate (GTP) or its non-hydrolizable analogue guanylyl-5-imidodiphosphate [Gpp(NH)p], significantly reduced the SLP-induced effect on DA levels. Furthermore, we observed that BDM-induced DA outflow was markedly inhibited by G protein activators suggesting an additional receptor-independent regulation of K⁺ _ATP channel gating. Our results suggest that PTX-sensitive G proteins are involved in the signal transduction between D2 receptors and K⁺ _ATP channels. Furthermore, K⁺ _ATP channels can be modulated in a receptor-independent mechanism by G protein activators.     

Differential requirements for caspase-8 activity in the mechanism of phosphorylation of eIF2alpha, cleavage of eIF4GI and signaling events associated with the inhibition of protein synthesis in apoptotic Jurkat T cells

Previously we have reported that induction of apoptosis in Jurkat cells results in an inhibition of overall protein synthesis with the selective and rapid cleavage of eukaryotic initiation factor (eIF) 4GI. For the cleavage of eIF4GI, caspase-3 activity is both necessary and sufficient in vivo, in a process which does not require signaling through the p38 MAP kinase pathway. We now show that activation of the Fas/CD95 receptor promotes an early, transient increase in the level of eIF2alpha phosphorylation, which is temporally correlated with the onset of the inhibition of translation. This is associated with a modest increase in the autophosphorylation of the protein kinase activated by double-stranded RNA. Using a Jurkat cell line that is deficient in caspase-8 and resistant to anti-Fas-induced apoptosis, we show that whilst the cleavage of eIF4GI is caspase-8-dependent, the enhancement of eIF2alpha phosphorylation does not require caspase-8 activity and occurs prior to the cleavage of eIF4GI. In addition, activation of the Fas/CD95 receptor results in the caspase-8-dependent dephosphorylation and degradation of p70(S6K), the enhanced binding of 4E-BP1 to eIF4E, and, at later times, the cleavage of eIF2alpha. These data suggest that apoptosis impinges upon the activity of several polypeptides which are central to the regulation of protein synthesis and that multiple signaling pathways are involved in vivo.     

Chemical shift as a probe of molecular interfaces: NMR studies of DNA binding by the three amino-terminal zinc finger domains from transcription factor IIIA

We report the NMR resonance assignments for a macromolecular protein/DNA complex containing the three amino-terminal zinc fingers (92 amino acid residues) of Xenopus laevis TFIIIA (termed zf1-3) bound to the physiological DNA target (15 base pairs), and for the free DNA. Comparisons are made of the chemical shifts of protein backbone¹ H^N, ¹⁵N,¹³ C and¹³ C and DNA base and sugar protons of the free and bound species. Chemical shift changes are analyzed in the context of the structures of the zf1-3/DNA complex to assess the utility of chemical shift change as a probe of molecular interfaces. Chemical shift perturbations that occur upon binding in the zf1-3/DNA complex do not correspond directly to the structural interface, but rather arise from a number of direct and indirect structural and dynamic effects.     

Copurification of P6, MRP8, and MRP14 from Human Granulocytes and Separation of Individual Proteins

A method is described for purification of P6, MRP8, and MRP14, three calcium-binding proteins assigned to the S100 protein family. The purification procedure included preparation of human granulocytes, ammonium sulfate precipitation, and anion-exchange chromatography and resulted in the copurification of P6, MRP8, and MRP14. Individual proteins were separated by either preparative isoelectric focusing or preparative SDS–PAGE. The procedure was carried out in the course of 4 days and yielded several milligrams of essentially pure P6, MRP8, and MRP14 in either native or denatured form.     

Alkyne chemistry in crop protection

An overview is given of the significance of alkyne derivatives in crop protection chemistry. The main herbicidally, fungicidally, insecticidally and acaricidally active alkyne classes are presented, together with their synthetic routes, their mode of action and their biological efficacies. Also the importance of alkynes as intermediates in the preparation of agrochemicals is demonstrated.