The ubiquitin C‐terminal hydrolase UCH‐L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton

Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C‐terminal hydrolase UCH‐L1 that promotes the invasion of epithelial cells by Listeria monocytogenes and Salmonella enterica. Knockdown of UCH‐L1 reduced the uptake of both bacteria, while expression of the catalytically active enzyme promoted efficient internalization in the UCH‐L1‐negative HeLa cell line. The entry of L. monocytogenes involves binding to the receptor tyrosine kinase Met, which leads to receptor phosphorylation and ubiquitination. UCH‐L1 controls the early membrane‐associated events of this triggering cascade since knockdown was associated with altered phosphorylation of the c‐cbl docking site on Tyr1003, reduced ubiquitination of the receptor and altered activation of downstream ERK1/2‐ and AKT‐dependent signalling in response to the natural ligand Hepatocyte Growth Factor (HGF). The regulation of cytoskeleton dynamics was further confirmed by the induction of actin stress fibres in HeLa expressing the active enzyme but not the catalytic mutant UCH‐L1_C90S. These findings highlight a previously unrecognized involvement of the ubiquitin cycle in bacterial entry. UCH‐L1 is highly expressed in malignant cells that may therefore be particularly susceptible to invasion by bacteria‐based drug delivery systems.     

Postnatal changes of some enzymatic activities of energy supplying metabolism in the cortex, inner and outer medulla of the rat kidney

1. In rat kidney cortex, outer and inner medulla the development of activities of seven enzymes was investigated during postnatal ontogeny (10, 20, 30, 60 and 90 days of age). The enzymes were selected in such a manner, as to characterize most of the main metabolic pathways of energy supplying metabolism: hexokinase (glucose phosphorylation, HK), glycerol-3-phosphate dehydrogenase (glycerolphosphate metabolism or shunt, GPDH), triose phosphate dehydrogenase (glycolytic carbohydrate breakdown, TPDH), lactate dehydrogenase (lactate metabolism, LDH), citrate synthase (tricarboxylic acid cycle, aerobic metabolism, CS), malate NAD dehydrogenase (tricarboxylic acid cycle, intra-extra mitochondrial hydrogen transport, MDH) and 3-hydroxyacyl-CoA-dehydrogenase (fatty acid catabolism, HOADH). 2. The renal cortex already differs metabolically from the medullar structures on the 10th day of life. It displays a high activity of aerobic breakdown of both fatty acids and carbohydrates. Its metabolic capacity further increases up to the 30th day of life. 3. The outer medullar structure is not grossly different from the inner medulla on the 10th day of life. Further it differentiates into a highly aerobic tissue mainly able to utilize carbohydrates. It can, however, to some extent, also utilize fatty acids aerobically and produce lactate from carbohydrates anaerobically. 4. The inner medullar structure is best equipped to utilize carbohydrates by anaerobic glycolysis, forming lactate. This feature is already pronounced on the 10th day of life, its capacity increases to some extent during postnatal development, being highest between the 10th and the 60th day of life.     

Inhibition of human neutrophil chemotaxis by the protein kinase inhibitor, 1-(5-isoquinolinesulfonyl) piperazine

The protein kinase inhibitor, 1-(5-isoquinolinesulfonyl) piperazine (C-I), inhibits superoxide release from human neutrophils (PMN) stimulated with phorbol myristate acetate or synthetic diacylglycerol, without inhibiting superoxide release from PMN stimulated with the chemoattractants C5a or N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). In this study, we investigated the effect of C-I on human PMN chemotaxis to C5a, f-Met-Leu-Phe, leukotriene B4 (LTB4), and fluoresceinated N-formyl-methionyl-leucyl-phenylalanine-lysine (f-Met-Leu-Phe-Lys-FITC). PMN, preincubated for 5 min at 37 degrees C with 0 to 200 microM C-I, were tested for their migratory responses to the chemoattractants. C-I (greater than or equal to 1 microM) significantly inhibited PMN chemotaxis to f-Met-Leu-Phe, f-Met-Leu-Phe-Lys-FITC, and C5a without affecting random migration. Maximal inhibition of chemotaxis to these attractants occurred with greater than or equal to 50 microM C-I, at which chemotaxis was inhibited by 80 to 95%. The C-I inhibition was reversible. In contrast, 200 microM C-I did not inhibit the number of PMN migrating to LTB4, although, the leading front of PMN migration to LTB4 was inhibited by C-I. C-I inhibited PMN orientation to C5a and f-Met-Leu-Phe without affecting orientation to LTB4. C-I did not inhibit the binding of radiolabeled f-Met-Leu-Phe or f-Met-Leu-Phe-Lys-FITC to PMN. These findings suggest that the chemotactic responses of PMN to f-Met-Leu-Phe and C5a involve a protein kinase-dependent reaction which is inhibited by C-I.     

Plant regeneration by organogenesis in Vitis rootstock species

A procedure for the regeneration of Vitis rootstocks plantlets by organogenesis from foliar tissues is described. Leaves from mature plants grown in growth chambers or from plantlets grown in tubes were wounded with a scalpel and cultured on a modified Murashige and Skoog liquid medium containing different concentrations of benzyl-aminopurine. The presence of benzyl-aminopurine is required for shoot formation. The age of the source explant, the composition of the culture medium and the culture temperature are important parameters of the regeneration process.Abbreviations BA 6-benzyl-aminopurine - MS Murashige and Skoog medium - MM modified Murashige and Skoog medium     

Selective priming of rate and duration of the respiratory burst of neutrophils by 1,2-diacyl and 1-O-alkyl-2-acyl diglycerides. Possible relation to effects on protein kinase C

Both 1,2-diacyl- and 1-O-alkyl-2-acyl-sn-glycerols are released during stimulation of human polymorphonuclear leukocytes (PMNL). 1,2-Diacylglycerols have received intense interest as intracellular "second messengers" due to their ability to activate protein kinase C (Ca2+ phospholipid-dependent enzyme). However, little is known about bioactivities of the alkylacylglycerols. This study compared the ability of 1,2-diacyl- and 1-O-alkyl-2-acylglycerols to modulate the respiratory burst of stimulated PMNL, a response which depends on the activation of an NADPH oxidase to generate bactericidal species of reduced oxygen. Direct stimulation by N-formyl-Met-Leu-Phe caused an abrupt release of H2O2 which ceased within 2.5 min. Preincubation with diacylglycerols (1-oleoyl-2-acetylglycerol,5-30 microM, and 1,2-dioctanoylglycerol,2-5 microM) caused a decrease in lag time, 3-fold increase in initial rate of H2O2 release, and marked prolongation of the response to N-formyl-Met-Leu-Phe (features characteristic of a priming effect). Preincubation with alkylacylglycerols (1-O-delta 9-octadecenyl-2-acetylglycerol, 5-30 microM, and 1-O-octyl-2-octanoylglycerol, 20-50 microM) primed initiation (shortened lag time and increased velocity) but, in contrast to diacylglycerols, did not alter duration of H2O2 release. While low concentrations of diacylglycerols (5-30 microM) primed PMNL, higher concentrations (greater than or equal to 70 microM) stimulated the cells directly. In contrast, higher (70-100 microM) concentrations of alkylacylglycerols did not prime the responses but, in fact, inhibited priming (especially of duration) induced by diacylglycerol. The high concentrations of alkylacylglycerol also inhibited direct stimulation induced by high concentrations of diacylglycerol. Direct stimulation by high concentrations of diacylglycerol probably involves activation of protein kinase C, whereas alkylacylglycerol was found to inhibit activation of protein kinase C by diacylglycerol in vitro. Thus, diacylglycerols are complete priming agonists, altering both rate and duration of the response. In contrast, alkylacylglycerols may have biphasic, concentration-related effects in modulation of functions of PMNL. At low concentrations, they may facilitate initiation of functional events; however, as their concentration increases, they may serve to terminate responses. The distinct priming effects of these diglycerides also reveal that priming can involve at least two distinct events: 1) initiation and 2) prolongation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fatty acid binding proteins in the three types of rat skeletal muscle

By means of Sephadex G-50 column chromatography, a Mr 12,000 fatty acid binding protein (FABP) was found to be present in all three types of skeletal muscle. FABP concentrations in muscle cytosols (105,000g supernatant) were fiber type specific with binding levels (expressed as pmole [14C]oleate bound/mg protein) of 70 +/- 7 in fast-twitch white (FTW) (heart FABP = 469 +/- 33). Cytosols from all three fiber types cross-reacted with antibody to pure heart FABP on Ouchterlony immunodiffusion analysis. FABP content, determined by radial immunodiffusion, followed the same pattern in the muscle types as that in the binding assay. The values (in micrograms/mg protein) were 3.3 +/- 0.1 in FTW, 17.0 +/- 0.4 in FTR, and 31.7 +/- 1.4 in STR fibers (heart = 55). Disc gel electrophoresis revealed a protein band in each fiber type that had migration identical to that of pure heart FABP and was proportional to the amounts determined by Sephadex G-50 chromatography and immunoassay. In addition, Western blots of tissue cytosols, using antibody to heart FABP, detected single protein bands identical in size to pure heart FABP in all three types of skeletal muscle. These results show the presence of a FABP in all skeletal muscle types with an immunologic and electrophoretic characterization identical to that of heart FABP.     

Symmetric infection of rotavirus on polarized human intestinal epithelial (Caco-2) cells.

When rotavirus infects the mature villus tip cells of the small intestine, it encounters a highly polarized epithelium. In order to understand this virus-cell interaction more completely, we utilized a cell culture-adapted rhesus rotavirus (RRV) to infect human intestinal (Caco-2) and Madin-Darby canine kidney (MDCK-1) polarized epithelial cells grown on a permeable support. Filter-grown Caco-2 cells and MDCK-1 cells, producing a transepithelial resistance of 300 to 500 and greater than 1,000 omega . cm2, respectively, were infected from either the apical or basolateral domain with RRV or Semliki Forest virus. Whereas Semliki Forest virus infection only occurred when input virions had access to the basolateral domain of MDCK-1 or Caco-2 cells, RRV infected MDCK-1 and Caco-2 monolayers in a symmetric manner. The effect of rotavirus infection on monolayer permeability was analyzed by measuring the transepithelial electrical resistance. Rotavirus infection on filter-grown Caco-2 cells caused a transmembrane leak at 18 h postinfection, before the development of the cytopathic effect (CPE) and extensive virus release. Electrical resistance was completely abolished between 24 and 36 h postinfection. Although no CPE could be detected on RRV-infected MDCK cells, the infection caused a transmembrane leak that totally abolished the electrical resistance at 18 to 24 h postinfection. Cell viability and the CPE analysis together with immunohistochemistry and immunofluorescence data indicated that the abolishment of resistance across the monolayer was due not to an effect on the plasma membrane of the cells but to an effect on the paracellular pathway limited by tight junctions. Attachment and penetration of rotavirus onto Caco-2 cells caused no measurable transmembrane leak during the first hour of infection.     

Phospholipase A2 activation in human neutrophils. Differential actions of diacylglycerols and alkylacylglycerols in priming cells for stimulation by N-formyl-Met-Leu-Phe

Both 1,2-diacyl- and 1-O-alkyl-2-acylglycerols are formed during stimulation of human neutrophils (PMN), and both can prime respiratory burst responses for stimulation by the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP); however, mechanisms of priming are unknown. Arachidonic acid (AA) release through phospholipase A2 activation and metabolism by 5-lipoxygenase are important activities of PMN during inflammation and could be involved in the process of primed stimulation. Therefore, we have examined the ability of diacyl- and alkylacylglycerols to act as priming agents for AA release and metabolism in human neutrophils. After prelabeling PMN phospholipids with [3H]AA, priming was tested by incubating human PMN with the diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), or its alkylacyl analog, 1-O-delta 9-octadecenyl-2-acetylglycerol (EAG) before stimulating with fMLP. fMLP (1 microM), OAG (20 microM), or EAG (20 microM) individually caused little or no release of labeled AA. However, after priming PMN with the same concentrations of either OAG or EAG, stimulation with 1 microM fMLP caused rapid (peak after 1 min) release of 6-8% of [3H]AA from cellular phospholipids; total release was similar with either diglyceride. Priming cells with OAG also enhanced conversion of released AA to leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) upon subsequent fMLP stimulation, but AA metabolites were not increased in EAG-primed PMN. If fMLP was replaced with the calcium ionophore A23187 (which directly causes release of AA and production of LTB4 and 5-HETE), priming by both diglycerides again enhanced release of [3H]AA, but only OAG priming increased lipoxygenase activity. Indeed, EAG pretreatment markedly reduced LTB4 and 5-HETE production. Thus, both diglycerides prime release of AA from membrane phospholipids but have opposite actions on the subsequent metabolism of AA.     

Stimulation of hexose transport by human polymorphonuclear leukocytes: a possible role for protein kinase C

The protein C kinase activators 1-O-oleoyl, 2-O-acetylglycerol, 12-O-tetradecanoyl phorbol-13-acetate, and mezerein, stimulated deoxyglucose uptake in human neutrophils. The responses were stimulus specific since no effect was noted with the diether analogues 1-O-hexadecyl-2-O-ethylglycerol, 1-O-palmitoyl-2-O-acetyl or 1-O-palmitoyl-3-O-acetyl diesters of propanediol, or with 1,2-diolein. Stimulation of deoxyglucose uptake had the characteristics of carrier facilitated hexose transport. Stimulated uptake of deoxy-glucose was inhibited by trifluoperazine (10-30 microM). Activation of protein kinase C therefore appears to trigger events involved in hexose transport.