Adipose triglyceride lipase regulates eicosanoid production in activated human mast cells

Human mast cells (MCs) contain TG-rich cytoplasmic lipid droplets (LDs) with high arachidonic acid (AA) content. Here, we investigated the functional role of adipose TG lipase (ATGL) in TG hydrolysis and the ensuing release of AA as substrate for eicosanoid generation by activated human primary MCs in culture. Silencing of ATGL in MCs by siRNAs induced the accumulation of neutral lipids in LDs. IgE-dependent activation of MCs triggered the secretion of the two major eicosanoids, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The immediate release of PGD2 from the activated MCs was solely dependent on cyclooxygenase (COX) 1, while during the delayed phase of lipid mediator production, the inducible COX-2 also contributed to its release. Importantly, when ATGL-silenced MCs were activated, the secretion of both PGD2 and LTC4 was significantly reduced. Interestingly, the inhibitory effect on the release of LTC4 was even more pronounced in ATGL-silenced MCs than in cytosolic phospholipase A₂-silenced MCs. These data show that ATGL hydrolyzes AA-containing TGs present in human MC LDs and define ATGL as a novel regulator of the substrate availability of AA for eicosanoid generation upon MC activation.     

Cloning, sequencing and mutational analysis of the cytochrome c552 gene (cycB) from Bradyrhizobium japonicum strain 110

We report the cloning and nucleotide sequence analysis of the cytochrome c552 gene (cycB) of Bradyrhizobium japonicum strain 110. The gene was identified with help of an oligonucleotide that was designed on the basis of the amino acid sequence determined for purified cytochrome c552 of B. japonicum strain CC705. The cycB gene product has an N-terminal 23-amino acid signal peptide that is missing in the mature cytochrome c552 protein. A B. japonicum cycB insertion mutant was constructed which had no observable phenotypic defects in denitrification and symbiotic nitrogen fixation. Thus, the function of c552 remains unknown.     

Biochemical Study on the Critical Period for Treatment of the Mottled Brindled Mouse

Hemizygous mottled brindled mice (Mobr/y mice) were treated by subcutaneous injection of copper and were decapitated on postnatal day 14. Cytochrome c oxidase (COX) activity of the brain mitochondria in the mice given 10 micrograms of copper/g on day 4 or 7 showed significant increases compared with that of untreated Mobr/y animals, and these mice had no neurological symptoms. Mice given 10 micrograms of copper/g on day 12 showed neither increases in COX activity nor clinical improvement. The brain levels of copper, noradrenaline, and dopamine in the mice treated on day 12 were the same as those in animals treated on day 4 or 7. The in vitro activities of dopamine-beta-hydroxylase of the brain were also the same among the treated mice, irrespective of the date of treatment. The results indicate that delays in copper treatment produce irreversible changes in COX activity of the brain and lead to clinical unresponsiveness to treatment.     

Mechanics of the cellular actin cortex: From signalling to shape change

The actin cortex is a thin layer of actin, myosin and actin-binding proteins that underlies the membrane of most animal cells. It is highly dynamic and can undergo remodelling on timescales of tens of seconds, thanks to protein turnover and myosin-mediated contractions. The cortex enables cells to resist external mechanical stresses, controls cell shape and allows cells to exert forces on their neighbours. Thus, its mechanical properties are the key to its physiological function. Here, we give an overview of how cortex composition, structure and dynamics control cortex mechanics and cell shape. We use mitosis as an example to illustrate how global and local regulation of cortex mechanics gives rise to a complex series of cell shape changes.     

The presence of 12β-deoxydecarbamoylsaxitoxin in the Japanese toxic dinoflagellate Alexandrium determined by simultaneous analysis for paralytic shellfish toxins using HILIC-LC–MS/MS

A differential screening study using high-resolution (HR)-hydrophilic interaction chromatography (HILIC)-electrospray ionization (ESI)–quadrupole time-of-flight mass spectrometry (Q-TOF MS) was conducted to identify saxitoxin (STX) analogues in the marine dinoflagellate toxic sub-clone Alexandrium tamarense Axat-2 and the non-toxic sub-clone UAT-014-009 derived from the same Japanese isolate. One unknown compound was identified only in the toxic sub-clone and was found to have the molecular formula C₉H₁₆N₆O₂. This structure differed from that of decarbamoyl STX (dcSTX; C₉H₁₆N₆O₃) by the loss of a single oxygen. A 12-deoxy-dcSTX standard (a mixture of 12α- and β-deoxy-dcSTX) was chemically prepared from dcSTX by reduction with sodium borohydride. The unknown compound in the toxic strain of A. tamarense was identified as 12β-deoxy-dcSTX by comparison of its HR-HILIC-LC–MS retention time and HR–MS/MS spectrum with those of the chemically prepared standard, and the identification was confirmed by high-sensitivity HPLC analysis with post-column fluorescent derivatization. Moreover, two Japanese isolates of A. catenella showing toxin profiles different from that of A. tamarense were also found to contain 12β-deoxy-dcSTX. Previously, 12β-deoxy-dcSTX was isolated from the freshwater cyanobacterium Lyngbya wollei, which produces a unique set of STX analogues. This study is the first evidence of the presence of 12β-deoxy-dcSTX in marine dinoflagellates.