Group VIB Ca2+-independent phospholipase A2gamma promotes cellular membrane hydrolysis and prostaglandin production in a manner distinct from other intracellular phospholipases A2

Although group VIA Ca2+-independent phospholipase A2beta (iPLA2beta) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2gamma. Lentiviral transfection of iPLA2gamma into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell death-associated release of arachidonic acid (AA), which was converted to prostaglandin E2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA2gamma small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA2gamma. iPLA2gamma protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA2gamma and other intracellular PLA2 enzymes acted on different phospholipid subclasses. Transfection of iPLA2gamma into HCA-7 cells also led to increased AA release and prostaglandin E2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA2gamma in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA2beta and iPLA2gamma in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA2gamma to tumorigenesis.     

Characterization of archaeal group II chaperonin-ADP-metal fluoride complexes: implications that group II chaperonins operate as a "two-stroke engine"

Group II chaperonins, found in Archaea and in the eukaryotic cytosol, act independently of a cofactor corresponding to GroES of group I chaperonins. Instead, the helical protrusion at the tip of the apical domain forms a built-in lid of the central cavity. Although many studies on the lid's conformation have been carried out, the conformation in each step of the ATPase cycle remains obscure. To clarify this issue, we examined the effects of ADP-aluminum fluoride (AlFx) and ADP-beryllium fluoride (BeFx) complexes on alpha-chaperonin from the hyperthermophilic archaeum, Thermococcus sp. strain KS-1. Biochemical assays, electron microscopic observations, and small angle x-ray scattering measurements demonstrate that alpha-chaperonin incubated with ADP and BeFx exists in an asymmetric conformation; one ring is open, and the other is closed. The result indicates that alpha-chaperonin also shares the inherent functional asymmetry of bacterial and eukaryotic cytosolic chaperonins. Most interestingly, addition of ADP and BeFx induced alpha-chaperonin to encapsulate unfolded proteins in the closed ring but did not trigger their folding. Moreover, alpha-chaperonin incubated with ATP and AlFx or BeFx adopted a symmetric closed conformation, and its functional turnover was inhibited. These forms are supposed to be intermediates during the reaction cycle of group II chaperonins.     

Cell migration and invasion assays

The processes of cell migration and invasion are integral to embryonic development and the functioning of adult organisms. Deregulation of these processes contributes to numerous diseases. Ras GTPases and in particular members of the Rho subfamily of GTPases play critical roles in cell migration and invasion. Here, we provide a collection of protocols to assay these functions. We describe two cell migration assays. The monolayer wound healing assay is very easy to implement, whereas the microliter-scale migration assay allows examination of cell behavior on defined extracellular matrices. We also describe two methods that allow the quantification of tumor cell invasion, a versatile transwell Matrigel invasion assay and an organotypic assay that examines the invasion of glioma cells through a rat brain slice.     

Spindle checkpoint signaling requires the mis6 kinetochore subcomplex, which interacts with mad2 and mitotic spindles

The spindle checkpoint coordinates cell cycle progression and chromosome segregation by inhibiting anaphase promoting complex/cyclosome until all kinetochores interact with the spindle properly. During early mitosis, the spindle checkpoint proteins, such as Mad2 and Bub1, accumulate at kinetochores that do not associate with the spindle. Here, we assess the requirement of various kinetochore components for the accumulation of Mad2 and Bub1 on the kinetochore in fission yeast and show that the necessity of the Mis6-complex and the Nuf2-complex is an evolutionarily conserved feature in the loading of Mad2 onto the kinetochore. Furthermore, we demonstrated that Nuf2 is required for maintaining the Mis6-complex on the kinetochore during mitosis. The Mis6-complex physically interacts with Mad2 under the condition that the Mad2-dependent checkpoint is activated. Ectopically expressed N-terminal fragments of Mis6 localize along the mitotic spindle, highlighting the potential binding ability of Mis6 not only to the centromeric chromatin but also to the spindle microtubules. We propose that the Mis6-complex, in collaboration with the Nuf2-complex, monitors the spindle-kinetochore attachment state and acts as a platform for Mad2 to accumulate at unattached kinetochores.     

Quinolactacide, a new quinolone insecticide from Penicillium citrinum Thom F 1539

By a screening program searching for new pesticides from fungal sources, an insecticidal compound was isolated from Penicillium citrinum F 1539. The compound, named quinolactacide, was novel and showed 88% mortality against green peach aphids (Myzus persicae) at 250 ppm. Its structure was determined by spectroscopic techniques.     

Chiral recognition of cyclic alpha-hydroxyketones by CD-sensitive zinc tetraphenylporphyrin tweezer

A combined chemical/chiroptical microscale protocol for the determination of absolute configurations of cyclic alpha-hydroxyketones is described. The hydroxyl group in cyclic alpha-hydroxyketones is converted into (3-aminopropylamino)acetate (NH2CH2CH2CH2NHCH2COOR), or more generally, according to a newly developed protocol, into (3-hydroxypropylamino)acetate group (HOCH2CH2CH2NHCH2COOR). The resultant conjugated compound forms a 1:1 host-guest complex with a dimeric zinc porphyrin tweezer, which exhibits exciton-coupled bisignate CD spectrum centered around the 420-nm porphyrin Soret band due to induced helicity between the two porphyrins in the complex. The absolute configurations of the alpha-stereogenic center is then determined by comparison of the sign of the observed CD exciton couplet of the complex with that of the preferred porphyrin twist predicted by the Merck Molecular Force Field (MMFFs) method.     

Effects of boron deficiency in cell suspension cultures of Populus alba L.

Cell suspension cultures of Populus alba L. (original cells) require at least 10 M boron for appropriate growth. Using original cells we established a cell line, T-5B, which can grow in a medium containing low levels of boron (5 M). The level of boron localized in the cell walls of T-5B cells was one-half that found in the cell walls of original cells maintained in medium containing 100 M boron, and the level of the rhamnogalacturonan II dimer, cross-linked by a borate ester, also decreased in the former. The sugar composition of whole cell walls of the T-5B cell line was similar that of the original cells, however pectic polysaccharides composed of arabinose or galacturonic acid were easily extracted from T-5B cell walls with 50 mM trans-1,2-cyclohexanediamine-N,N,N,N-tetraacetic acid. Our results suggest that boron deficiency causes a weakening of the interaction among pectic polysaccharides due to a decrease in boron-rhamnogalacturonanII cross-linkage.     

ATP-induced structural change of dephosphocoenzyme A kinase from Thermus thermophilus HB8

Dephosphocoenzyme A kinase (DCK) catalyzes phosphorylation in the final step of coenzyme A (CoA) biosynthesis. In this phosphorylation process, domain movements play a very important role. To reveal the structural changes induced by ligand binding, we determined the crystal structure of DCK from Thermus thermophilus HB8 by the multiwavelength anomalous dispersion method at 2.8 A. The crystal structure includes three independent protein molecules in the asymmetric unit: One is a liganded form and the others are unliganded. The topology shows a canonical nucleotide-binding protein possessing the P-loop motif. A structure homology search by DALI revealed the similarity of the DCKs from T. thermophilus HB8, Haemophilus influenzae, and Escherichia coli. Structural comparisons between the liganded and unliganded forms of DCK from T. thermophilus HB8 indicated domain movements induced by adenosine triphosphate (ATP) binding. For the domain movements, proline residues confer flexibility at the domain linkages. In particular, Pro91 plays an important role in moving the CoA domain.