Sphingosine inhibits phosphatidate phosphohydrolase in human neutrophils by a protein kinase C-independent mechanism

Human neutrophils have been labeled in 1-O-alkyl-phosphatidylcholine with 3H in both the alkyl chain and the choline moiety. Upon stimulation of these labeled cells with formyl-Met-Leu-Phe, C5a, or phorbol 12-myristate 13-acetate, phospholipase D is activated to produce 1-O-[3H]alkylphosphatidic acid ([3H]alkyl-PA) and [3H]choline. The [3H]alkyl-PA is then dephosphorylated by phosphatidate phosphohydrolase (PPH) to produce 1-O-[3H]alkyldiglyceride ([3H]alkyl-DG). Sphingosine, a sphingoid base known to inhibit protein kinase C (PKC), causes a dose-dependent inhibition of [3H]alkyl-DG formation. This inhibition is accompanied by increased accumulation of [3H]alkyl-PA without alterations in [3H]choline formation. Studies using various other sphingoid bases demonstrate that a long hydrocarbon chain and an amino group are required for the inhibition of DG formation. These results suggest that sphingoid bases inhibit PPH activity without altering phospholipase D activation and that they exhibit a similar structure-activity relationship for both PPH and PKC. K252a, a PKC inhibitor which acts by competing for ATP binding sites, does not inhibit the formation of [3H]alkyl-DG, [3H]alkyl-PA, or [3H]choline at a concentration (3 microM) that completely blocks phorbol 12-myristate 13-acetate-induced protein phosphorylation. Moreover, in neutrophil homogenates, sphingosine but not octylamine, inhibits PPH activity in a dose-dependent manner. Thus sphingosine inhibits PPH activity by a PKC-independent mechanism, raising the possibility that sphingoid bases may play a role in regulating PPH-mediated lipid metabolism in stimulated cells.     

Parauronema virginianum n. g., n. sp., a Marine Hymenostome Ciliate

SYNOPSIS. Parauronema virginianum n. g., n. sp., a marine hymenostome ciliate is described from the Virginia coast. Structural studies were made on specimens treated with the Chatton-Lwoff silver impregnation technic and on animals observed with the phase microscope. Particular attention was given to the buccal ciliature and its importance to generic assignment in the order Hymenostomatida.     

A preliminary investigation of L-phenylalanine ammonialyase activity in asparagus: Distribution and response to storage, excision and incubation

The level of L-phenylalanine ammonia-lyase (PAL) (E.C. 4.3.1.5 [EC] )activity was greatest in the basal portion of freshly harvestedasparagus spears and decreased toward the tips. Basal disksdeveloped large increases in L-phenylalanine ammonia-lyase activityin response to excision and incubation. The level of PAL activitymeasured in basal tissue of intact spears increased with storage,while the ability of the same tissue to respond to excisionand incubation with increased PAL activity was lost. (Received March 23, 1971; )     

2-D DIGE analysis of the mitochondrial proteome from human skeletal muscle reveals time course-dependent remodelling in response to 14 consecutive days of endurance exercise training

Adaptation of skeletal muscle to repeated bouts of endurance exercise increases aerobic capacity and improves mitochondrial function. However, the adaptation of human skeletal muscle mitochondrial proteome to short‐term endurance exercise training has not been investigated. Eight sedentary males cycled for 60 min at 80% of peak oxygen consumption (VO_2peak) each day for 14 consecutive days, resulting in an increase in VO_2peak of 17.5±3.8% (p<0.01). Mitochondria‐enriched protein fractions from skeletal muscle biopsies taken from m. vastus lateralis at baseline, and on the morning following the 7th and 14th training sessions were subjected to 2‐D DIGE analysis with subsequent MS followed by database interrogation to identify the proteins of interest. Thirty‐one protein spots were differentially expressed after either 7 or 14 days of training (ANOVA, p<0.05). These proteins included subunits of the electron transport chain, enzymes of the tricarboxylic acid cycle, phosphotransfer enzymes, and regulatory factors in mitochondrial protein synthesis, oxygen transport, and antioxidant capacity. Several proteins demonstrated a time course‐dependent induction during training. Our results illustrate the phenomenon of skeletal muscle plasticity with the extensive remodelling of the mitochondrial proteome occurring after just 7 days of exercise training suggestive of enhanced capacity for adenosine triphosphate generation at a cellular level.     

Genetic and morphological variation in Viola suavis s.l. (Violaceae) in the western Balkan Peninsula: two endemic subspecies revealed

The Balkan Peninsula, with many endemic species, is known as one of the most important speciation and diversification centres in Europe. Here, we present a study of the western Balkan populations of the polymorphic European species, V. suavis s.l., which have been reported under the name V. adriatica, but their taxonomic status and position within the genus have remained uncertain. Viola suavis s.l. and nine close relatives sampled across Europe were subjected to molecular (sequencing of nuclear ribosomal internal transcribed spacers and amplified fragment length polymorphism), karyological and morphometric analyses. Our results revealed the presence of four allopatric, genetically and morphologically differentiated lineages within V. suavis s.l. in Europe, which are suggested here to be recognized at the subspecific rank. Populations from the western Balkans were segregated into two distinct entities: (1) those from north-western Croatia correspond to the previously recognized taxon, V. suavis subsp. adriatica and (2) those from southern Dalmatia (southern Croatia, southern Bosnia and Herzegovina, and south-western Montenegro) are described here as V. suavis subsp. austrodalmatica subsp. nov. The other two lineages of V. suavis s.l., which both harbour blue- and white-flowered morphotypes, occur in central and eastern Europe (V. suavis subsp. suavis) and in north-eastern Spain (plants provisionally treated as V. suavis ‘Spain’). The AFLP and morphological data indicate gene flow between the nominate subspecies and V. suavis subsp. adriatica in a few localities. The distribution of the two western Balkan subspecies is discussed and an identification key to the V. suavis subspecies in Europe is presented.     

Intestinal Intraepithelial Lymphocyte-Enterocyte Crosstalk Regulates Production of Bactericidal Angiogenin 4 by Paneth Cells upon Microbial Challenge

Antimicrobial proteins influence intestinal microbial ecology and limit proliferation of pathogens, yet the regulation of their expression has only been partially elucidated. Here, we have identified a putative pathway involving epithelial cells and intestinal intraepithelial lymphocytes (iIELs) that leads to antimicrobial protein (AMP) production by Paneth cells. Mice lacking γδ iIELs (TCRδ^-/-) express significantly reduced levels of the AMP angiogenin 4 (Ang4). These mice were also unable to up-regulate Ang4 production following oral challenge by Salmonella, leading to higher levels of mucosal invasion compared to their wild type counterparts during the first 2 hours post-challenge. The transfer of γδ iIELs from wild type (WT) mice to TCRδ^-/- mice restored Ang4 production and Salmonella invasion levels were reduced to those obtained in WT mice. The ability to restore Ang4 production in TCRδ^-/- mice was shown to be restricted to γδ iIELs expressing Vγ7-encoded TCRs. Using a novel intestinal crypt co-culture system we identified a putative pathway of Ang4 production initiated by exposure to Salmonella, intestinal commensals or microbial antigens that induced intestinal epithelial cells to produce cytokines including IL‑23 in a TLR-mediated manner. Exposure of TCR-Vγ7⁺ γδ iIELs to IL-23 promoted IL‑22 production, which triggered Paneth cells to secrete Ang4. These findings identify a novel role for γδ iIELs in mucosal defence through sensing immediate epithelial cell cytokine responses and influencing AMP production. This in turn can contribute to the maintenance of intestinal microbial homeostasis and epithelial barrier function, and limit pathogen invasion.