Superoxide production at phagosomal cup/phagosome through beta I protein kinase C during Fc gamma R-mediated phagocytosis in microglia

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a substrate for betaI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O(2)(-)) production was profoundly inhibited by G?6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O(2)(-) production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O(2)(-) production, and that O(2)(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O(2)(-) production during FcgammaR-mediated phagocytosis.     

Microtubule‐organizing centers of Aspergillus nidulans are anchored at septa by a disordered protein

Microtubule‐organizing centers (MTOCs) are large, multi‐subunit protein complexes. Schizosaccharomyces pombe harbors MTOCs at spindle pole bodies, transient MTOCs in the division plane (eMTOCs) and nuclear‐envelope associated MTOCs in interphase cells (iMTOCs). In the filamentous fungus Aspergillus nidulans SPBs and septum‐associated MTOCs were described. Although comparable to S. pombe eMTOCs, A. nidulans sMTOCS are permanent septum‐associated structures. The composition of sMTOCs is poorly understood and how they are targeted to septa was unknown. Here, we show that in A. nidulans several SPB outer plaque proteins also locate to sMTOCs while other SPB proteins do not, including SfiA, a protein required for SPB duplication in Saccharomyces cerevisiae and S. pombe and PcpA, the anchor for γ‐TuSCs at the SPB inner plaque. The A. nidulans disordered protein Spa18^Mto2 and the centrosomin‐domain containing protein ApsB^Mto1 were required for recruiting the γ‐TuRC component GcpC to sMTOCs and for seeding MT formation from septa. Testing different septum‐associated proteins for a role in sMTOC function, Spa10 was identified. It forms a septal pore disc structure, recruits Spa18 and ApsB to septa and is required for sMTOC activity. This is the first evidence for a septum‐specific protein, Spa10, as anchor for a specific class of MTOCs.     

Phylogenetic position of Rickettsia tsutsugamushi and the relationship among its antigenic variants by analyses of 16S rRNA gene sequences

Abstract The 16S rRNA gene sequences of Rickettsia tsutsugamushi and Rickettsia sibirica were determined by PCR and DNA sequencing. Phylogenetic analysis revealed that R. sibirica is positioned in a cluster of the genus Rickettsia with a similarity value of 98.1–99.6%, whereas R. tsutsugamushi is located apart from the cluster with a similarity value of 90.2–90.6%. This evidence suggests that R. tsutsugamushi should be excluded taxonomically from the genus Rickettsia . The phylogenetic classification of six antigenic variants in R. tsutsugamushi moderately reflected their antigenic relationship known in closely and distantly related strains.     

Antimicrobial substances from rhizomes of the giant knotweed Polygonum sachalinense against the fish pathogen Photobacterium damselae subsp. piscicida

The antimicrobial compounds against the fish pathogen Photobacterium damselae subsp. piscicida were isolated from Polygonum sachalinense rhizomes. The structures of the antimicrobial compounds 1 and 2 were determined by 1H and 13C NMR, 2D-NMR (COSY, HSQC, HMBC and ROESY) and FAB-MS to be phenylpropanoid glycosides, vanicoside A and B, respectively. Both compounds have feruloyl and p-coumaroyl groups bonded to a sucrose moiety in their structures. Vanicoside A also has an acetyl group in the sucrose moiety. The MIC values for vanicoside A and B against Ph. damselae subsp. piscicida DPp-1 were 32 and 64 microg/ml, respectively. The antimicrobial activities of these vanicosides were modest, in contrast to higher activities (MICs at < 4 microg/ml) of antibiotics, florphenicol, ampicillin and amoxicillin, which have been generally used for treating pasteurellosis. The activities of the vanicosides, however, were higher than those (MICs at 256 microg/ml) of ferulic acid and p-coumaric acid. It was suggested that the structure of phenylpropanoids esterified with sucrose was essential for higher antimicrobial activity of vanicosides and also acetylation of sucrose might affect the activity against the bacterium.     

Singlet oxygen inhibits the repair of photosystem II by suppressing the translation elongation of the D1 protein in Synechocystis sp. PCC 6803

Singlet oxygen, generated during photosynthesis, is a strong oxidant that can, potentially, damage various molecules of biological importance. We investigated the effects in vivo of singlet oxygen on the photodamage to photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. Increases in intracellular concentrations of singlet oxygen, caused by the presence of photosensitizers, such as rose bengal and ethyl eosin, stimulated the apparent photodamage to PSII. However, actual photodamage to PSII, as assessed in the presence of chloramphenicol, was unaffected by the production of singlet oxygen. These observations suggest that singlet oxygen produced by added photosensitizers acts by inhibiting the repair of photodamaged PSII. Labeling of proteins in vivo revealed that singlet oxygen inhibited the synthesis of proteins de novo and, in particular, the synthesis of the D1 protein. Northern blotting analysis indicated that the accumulation of psbA mRNAs, which encode the D1 protein, was unaffected by the production of singlet oxygen. Subcellular localization of polysomes with bound psbA mRNAs suggested that the primary target of singlet oxygen might be the elongation step of translation.     

Protein kinase A-regulated membrane trafficking of a green fluorescent protein-aquaporin 5 chimera in MDCK cells

The green fluorescent protein (GFP) of the jellyfish, Aeqorea victoria, was used as an autofluorescent tag to track the trafficking of aquaporin 5 (AQP5), an exocrine gland-type water channel. Two groups of chimeric proteins were constructed; one in which GFP was fused to the amino-terminus of AQP5 (GFP-AQP5) and the other, in which it was fused to the carboxyl terminus of it (AQP5-GFP). In each group, 2 chimeras were produced, a wild-type AQP5 with its normal sequence and a mutant AQP5 having a mutated amino acid at 259, i.e., GFP-AQP5-T259A and AQP5-GFP-T259A. They were used to transfect Madin-Darby canine kidney (MDCK) cells. The GFP-AQP5 chimera was localized in the intracellular vesicles, which trafficked to the plasma membrane in response to N(6), 2'-O-dibutyryladenosine 3', 5'-cyclic monophosphate (dbcAMP). Membrane trafficking was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquimolinesulfonamide (H-89) but not by palmitoyl-dl-carnitine chloride (PCC). In contrast, the AQP5-GFP chimera expressed in MDCK cells was localized constitutively on the plasma membrane. The cellular localization of the latter chimera was not affected by stimulation with dbcAMP in the presence or absence of H-89 or PCC. Replacement of Thr-259 with Ala-259 did not affect the dbcAMP-induced translocation of the chimeric protein, suggesting that phosphorylation of Thr-259 was not necessary for AQP5 trafficking under the present experimental conditions. Thus, the GFP-AQP5 chimera will be a useful tool to study AQP5 trafficking in vitro, whereas the constitutive membrane localization of the AQP5-GFP chimera suggests the importance of the carboxyl terminus of the AQP5 protein for its sorting, whether it is translocated to intracellular vesicles or to the plasma membrane.     

Adsorption Mechanism of Cr(VI) onto Coir Pith

Reductive adsorption of Cr(VI) on coir pith (hereafter CP) was examined as a function of pH, ionic strength, and temperature. The CP contains 1.33 meq g^{? 1} phenolic, 0.43 meq g^{? 1} of lactonic, and 0.35 meq g^{? 1} carboxylic sites. Thus the CP surface is enriched with electron-donating oxygen functionalities. As evidenced by infrared (IR) spectroscopy, the Cr(VI) → Cr(III) conversion is facilitated by CP sites that are enriched with O─ O functional groups. The adsorption of reduced Cr(VI) was found to occur via C─ O─ functional groups first forming innersphere complexes with the CP surface, yielding keto (> C═ O) groups on the CP surface. The reductive adsorption of Cr(VI) was almost completed within 3 to 4 h, and it was dependent on pH and background ionic strength, yielding the highest monolayer coverage (9.56E-7 mol m^{? 2}) at pH 3.7 in 0.1 M NaNO₃. The Γ_Cr(III) followed the order with respect to the ionic strength: Γ_{0.1 M} > Γ_{0.01 M} > Γ_{0.001 M}. The initial rate constant, k _i , increased with temperature as k _i ^{313 K} > k _i ^{303 K} > k _i ^{293 K} > k _i ^{283 K}.     

Rapid determination of tetracycline antibiotics in serum by reversed-phase high-performance liquid chromatography with fluorescence detection

A rapid and accurate determination of tetracycline antibiotics in human serum by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed, based on protein precipitation in serum. Various reagents for precipitation were investigated, and 24% trichloroacetic acid in methanolic solution gave the maximum recovery (at least 94.3%) and interference-free chromatograms of different three tetracyclines. At a concentration of 0.5 μg/ml, the precision (relative standard deviation) ranged from 1.12 to 1.94%. In the range 0.04–10.0 μg/ml for oxytetracycline and chlorotetracycline and 0.01–10.0 μg/ml for tetracycline, linear responses were observed. The detection limits of this method were 10–35 ng/ml for all three antibiotics. The proposed method was applied to the determination of serum concentrations in subjects receiving tetracycline antibiotics.