Spontaneous neutrophil apoptosis involves caspase 3-mediated activation of protein kinase C-delta

Neutrophils are short-lived leukocytes that die by apoptosis. Whereas stress-induced apoptosis is mediated by the p38 mitogen-activated protein (MAP) kinase pathway (Frasch, S. C., Nick, J. A., Fadok, V. A., Bratton, D. L., Worthen, G. S., and Henson, P. M. (1998) J. Biol. Chem. 273, 8389-8397), signals regulating spontaneous neutrophil apoptosis have not been fully determined. In this study we found increased activation of protein kinase C (PKC)-beta and -delta in neutrophils undergoing spontaneous apoptosis, but we show that only activation of PKC-delta was directly involved in the induction of apoptosis. PKC-delta can be proteolytically activated by caspase 3. We detected the 40-kDa caspase-generated fragment of PKC-delta in apoptotic neutrophils and showed that the caspase 3 inhibitor Asp-Glu-Val-Asp-fluoromethylketone prevented generation of the 40-kDa PKC-delta fragment and delayed neutrophil apoptosis. In a cell-free system, removal of PKC-delta by immunoprecipitation reduced DNA fragmentation, whereas loss of PKC-alpha, -beta, or -zeta had no significant effect. Rottlerin and LY379196 inhibit PKC-delta and PKC-beta, respectively. Only Rottlerin was able to delay neutrophil apoptosis. Inhibitors of MAP-ERK kinase 1 (PD98059) or p38 MAP kinase (SB202190) had no effect on neutrophil apoptosis, and activation of p42/44 and p38 MAP kinase did not increase in apoptotic neutrophils. We conclude that spontaneous neutrophil apoptosis involves activation of PKC-delta but is MAP kinase-independent.     

Anesthesia of polar bears using xylazine-zolazepam-tiletamine or zolazepam-tiletamine

Immobilization features and physiologic effects of combinations of xylazine-zolazepam-tiletamine (XZT) and zolazepam-tiletamine (ZT or Telazol) were compared in nine captive and 17 free-ranging polar bears (Ursus maritimus) between 1998 and 2001. Although induction time was similar between drugs, induction dosage and volume were less with XZT. Induction of immobilization with XZT was predictable and smooth, muscle relaxation was good, and all bears remained completely immobilized and unresponsive to stimuli throughout a 1 hr handling period. The combination XZT was safely tolerated at two to three times the recommended dosage of 5 mg/kg (i.e., xylazine at 2 mg/kg + Telazol at 3 mg/kg). Bears immobilized with XZT had slower pulse rates, higher mean arterial pressures, and lower arterial oxygen tensions than bears immobilized with ZT. Rectal temperature increased slowly over time (approximately 0.5 C per hr) following immobilization with XZT. Based on response to a painful stimulus (compression of a claw bed), XZT was a more effective analgesic than ZT. Although the immobilization effects of XZT could not be reversed with the alpha 2-antagonist drug tolazoline, they were reversed with yohimbine or atipamezole. However, the time to complete reversal of effects (i.e., standing and ambulatory) was highly variable among bears.     

Evolution and function of the sucrose-phosphate synthase gene families in wheat and other grasses

Suc-phosphate synthase (SPS) is a key regulatory enzyme in the pathway of Suc biosynthesis and has been linked to quantitative trait loci controlling plant growth and yield. In dicotyledonous plants there are three SPS gene families: A, B, and C. Here we report the finding of five families of SPS genes in wheat (Triticum aestivum) and other monocotyledonous plants from the family Poaceae (grasses). Three of these form separate subfamilies within the previously described A, B, and C gene families, but the other two form a novel and distinctive D family, which on present evidence is only found in the Poaceae. The D-type SPS proteins lack the phosphorylation sites associated with 14-3-3 protein binding and osmotic stress activation, and the linker region between the N-terminal catalytic glucosyltransferase domain and the C-terminal Suc-phosphatase-like domain is 80 to 90 amino acid residues shorter than in the A, B, or C types. The D family appears to have arisen after the divergence of mono- and dicotyledonous plants, with a later duplication event resulting in the two D-type subfamilies. Each of the SPS gene families in wheat showed different, but overlapping, spatial and temporal expression patterns, and in most organs at least two different SPS genes are expressed. Analysis of expressed sequence tags indicated similar expression patterns to wheat for each SPS gene family in barley (Hordeum vulgare) but not in more distantly related grasses. We identified an expressed sequence tag from rice (Oryza sativa) that appears to be derived from an endogenous antisense SPS gene, and this might account for the apparently low level of expression of the related OsSPS11 sense gene, adding to the already extensive list of mechanisms for regulating the activity of SPS in plants.     

Coadministration of DNA Encoding Interleukin-6 and Hemagglutinin Confers Protection from Influenza Virus Challenge in Mice

This study was conducted to investigate whether Accell gene gun coadministration of DNA encoding human interleukin-6 (IL-6) would enhance protective immune responses in mice to an equine influenza A virus hemagglutinin (HA) DNA vaccine. Mice that received HA DNA alone exhibited accelerated clearance of homologous challenge virus but were not protected from infection. In contrast, mice that received both HA and IL-6 DNA had no detectable virus in their lungs after challenge. These results strongly support the use of IL-6 as a cytokine adjuvant in DNA vaccination.     

Limited proteolysis of Escherichia coli cytidine 5'-triphosphate synthase. Identification of residues required for CTP formation and GTP-dependent activation of glutamine hydrolysis.

Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis. Lys187 is located at the CTP/UTP-binding site within the synthase domain, and cleavage at this site destroyed all synthase activity. Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP). The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP. K187A was able to form tetramers in the presence of UTP and ATP. Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer (GAT) domain. Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage. The R429A and R429A/K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH3-dependent CTP formation. For these mutants, the values of kcat/Km and kcat for glutamine-dependent CTP formation were reduced approximately 20-fold and approximately 10-fold, respectively, relative to wild-type enzyme; however, the value of Km for glutamine was not significantly altered. Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold. This suggests that Arg429 plays a role in both GTP-dependent activation and GTP binding.     

Urinary excretion of biologically active chorionic gonadotrophin by the pregnant marmoset (Callithrix jacchus jacchus).

No significant correlation exists between the amount of biologically active marmoset chorionic gonadotrophin (mCG) in urine and results obtained with an immunological pregnancy test. The pregnant marmoset excretes large amounts of oestrogenic steroids, which must be removed, to prevent the enhancement of the response of the bioassay for mCG. More than 99% of these unconjugated and conjugated urinary oestrogens can be removed by extraction with acetone and ether. mCG is excreted throughout pregnancy, maximum levels occurring between the 8th and 9th week of gestation. There is a considerable within- and between-animal variation in the amount of mCG excreted. However, the pattern of gonadotrophin excretion by the pregnant marmoset is similar to that of man and the apes but unlike that of baboons and macaques.     

Light meromyosin paracrystal formation

Studies of paracrystal formation by column purified light meromyosin (LMM) prepared in a variety of ways led to the following conclusions: (a) different portions of the myosin rod may be coded for different stagger relationships. This was concluded from observations that paracrystals with different axial repeat periodicities could be obtained either with LMM framents of different lengths prepared with the same enzyme, or with LMM fragments of identical lengths but prepared with different enzymes. (b) Paracrystals with a 14-nm axial repeat periodicity are most likely formed by the aggregation of sheets with a 44-nm axial repeat within the sheets which are staggered by 14 nm. All of the axial repeat patterns expected from one sheet or aggregates of more than one sheet, on this basis, were observed in the same electron micrograph. (c) C-protein binding probably occurs preferentially to LMM molecules related in some specific way. This was concluded from the observation that the same axial repeat pattern was obtained in paracrystals formed from different LMM preparations in the presence of C-protein, regardless of differences in the axial repeat obtained in the absence of C-protein. (d) Nucleic acid is responsible for the 43-nm axial repeat patterns observed in paracrystals formed by the ethanol-resistant fraction of LMM. In the absence of nuclei acid, paracrystals with a 14nm axial repeat are obtained. (e) The 43-nm axial repeat pattern observed with the ethanol-resistant fraction of LMM is different for LMM preparations obtained by trypsin and papain digestions.     

The pyrimidinergic P2Y6 receptor mediates a novel release of proinflammatory cytokines and chemokines in monocytic cells stimulated with UDP

The human P2Y6 receptor (hP2Y6) is a member of the G protein-coupled pyrimidinergic P2 receptor family that responds specifically to the extracellular nucleotide uridine diphosphate (UDP). Recently, the hP2Y6 receptor has been reported to mediate monocyte IL-8 production in response to UDP or lipopolysaccharide (LPS), but the role of hP2Y6 in regulating other pro-inflammatory cytokines or mediators is largely unknown. We demonstrate here that UDP specifically induces soluble TNF-alpha and IL-8 production in a promonocytic U937 cell line stably transfected with hP2Y6. However, we did not detect IL-1alpha, IL-1beta, IL-6, IL-10, IL-18, and PGE2 in the conditioned media from the same cell line. These results distinguish UDP/P2Y6 signaling from LPS signaling. Interestingly, UDP induces the production of IL-8, but not TNF-alpha, in human astrocytoma 1321N1 cell lines stably transfected with hP2Y6. Therefore, the immune effect of UDP/P2Y6 signaling on the production of proinflammatory cytokines is selective and dependent on cell types. We further identify that UDP can also induce the production of proinflammatory chemokines MCP-1 and IP-10 in hP2Y6 transfected promonocytic U937 cell lines, but not astrocytoma 1321N1 cell lines stably transfected with hP2Y6. From the Taqman analysis, UDP stimulation significantly upregulates the mRNA levels of IL-8, IP-10, and IL-1beta, but not TNF-alpha. Taken together, these new findings expand the pro-inflammatory biology of UDP mediated by the P2Y6 receptor.     

Requirement of cortical actin organization for bombesin, endothelin, and EGF receptor internalization

The role of actinorganization in occupancy-induced receptor internalization remainspoorly defined. Here we report that treatment of mouse Swiss 3T3 cellswith latrunculin A, a potent inhibitor of actin polymerization(including cortical actin), inhibited the internalization of theendogenous bombesin/gastrin-releasing peptide (GRP) receptor, as judgedby uptake of ¹²⁵I-labeled GRP or fluorescent Cy3-labeledbombesin. In contrast, cells pretreated with cytochalasin D showedminimal inhibition of bombesin/GRP receptor internalization. Similarly,pretreatment of Swiss 3T3 cells with the potent Rho-kinaseinhibitor HA-1077, at concentrations (10-20 µM) thatabrogated bombesin-mediated stress fiber formation, did notsignificantly alter receptor-mediated internalization of¹²⁵I-GRP. These results indicate that bombesin/GRP receptorinternalization depends on latrunculin A-sensitive cortical actinrather than on rapidly turning over actin stress fibers that aredisrupted by either cytochalasin D or HA-1077. The rates andtotal levels of internalization of the endogenously expressedendothelin A receptor and epidermal growth factor receptor were alsomarkedly reduced by latrunculin A in Swiss 3T3 cells. The potency oflatrunculin A for inhibiting G protein-coupled receptor endocytosis wascomparable to that for reducing internalization of the epidermal growthfactor tyrosine kinase receptor. We conclude that cortical actinstructures, disrupted by latrunculin A, are necessary foroccupancy-induced receptor internalization in animal cells.