Phosphorylation by Pho85 cyclin-dependent kinase acts as a signal for the down-regulation of the yeast sphingoid long-chain base kinase Lcb4 during the stationary phase

Sphingoid long-chain base 1-phosphates (LCBPs) act as bioactive lipid molecules in eukaryotic cells. In yeast, LCBPs are synthesized mainly by the long-chain base kinase Lcb4p. Until now, the regulatory mechanism for Lcb4p has been unclear. In the present study, we found that Lcb4p is post-translationally modified by phosphorylation. Using a protein kinase mutant yeast collection, we further demonstrated that the cyclin-dependent kinase Pho85p is involved in this phosphorylation. Pho85p functions in a number of cellular processes, especially in response to environmental changes. Two of 10 Pho85p cyclins, Pcl1p and Pcl2p had overlapping functions in the phosphorylation of Lcb4p. Site-directed mutagenesis identified the phosphorylation sites in Lcb4p as Ser(451) and Ser(455). Additionally, pulse-chase experiments revealed that Lcb4p is degraded via the ubiquitin-dependent pathway. The protein was stabilized in Deltapho85 cells, suggesting that phosphorylation acts as a signal for the degradation. Lcb4p is down-regulated in the stationary phase of cell growth, and both phosphorylation and ubiquitination appear to be important for this process. Moreover, we demonstrated that Lcb4p is delivered to the vacuole for degradation via the multivesicular body. Since forced accumulation of LCBPs results in prolonged growth during the stationary phase, down-regulation of Lcb4p may be physiologically important for proper cellular responses to nutrient deprivation.     

Human M-ficolin is a secretory protein that activates the lectin complement pathway

Three types of ficolins have been identified in humans: L-ficolin, M-ficolin, and H-ficolin. Similar to mannose-binding lectin, L-ficolin and H-ficolin are the recognition molecules in the lectin complement pathway. Another human ficolin, M-ficolin, is a nonserum ficolin that is expressed in leukocytes and lung; however, little is known about its physiologic roles. In this study, we report the characterization of M-ficolin in terms of its protein localization and lectin activity. M-ficolin was localized in secretory granules in the cytoplasm of neutrophils, monocytes, and type II alveolar epithelial cells in lung. M-ficolin precipitated with mannose-binding lectin-associated serine proteases (MASP)-1 and MASP-2 in a co-immunoprecipitation assay, indicating that M-ficolin forms complexes with MASP-1 and MASP-2. M-ficolin-MASP complexes activated complement on N-acetylglucosamine (GlcNAc)-coated microplates in a C4 deposition assay. M-ficolin bound to several neoglycoproteins bearing GlcNAc, N-acetylgalactosamine, and sialyl-N-acetyllactosamine, suggesting that M-ficolin can recognize the common carbohydrate residues found in microbes. Indeed, M-ficolin bound to Staphylococcus aureus through GlcNAc. These results indicate that M-ficolin, like its family members, functions as a recognition molecule of the lectin complement pathway and plays an important role in innate immunity.     

ATR-FTIR spectroscopy and isotope labeling of the PM intermediate of Paracoccus denitrificans cytochrome c oxidase

The structure of the P(M) intermediate of Paracoccus denitrificans cytochrome c oxidase was investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. Transitions from the oxidized to P(M) state were initiated by perfusion with CO/oxygen buffer, and the extent of conversion was quantitated by simultaneously monitoring visible absorption changes. In prior work, tentative assignments of bands were proposed for heme a(3), a change in the environment of the protonated state of a carboxylic acid, and a covalently linked histidine-tyrosine ligand to Cu(B) that has been found in the catalytic site. In this work, reduced minus oxidized difference spectra at pH 6.5 and 9.0 and P(M) minus oxidized difference spectra at pH 9.0 were compared in unlabeled, universally (15)N-labeled, and tyrosine-ring-d(4)-labeled proteins to improve these assignments. In the reduced minus oxidized difference spectrum, (15)N labeling resulted in large changes in the amide II region and a 9 cm(-1) downshift in a 1105 cm(-1) trough that is attributed to histidine. In contrast, changes induced by tyrosine-ring-d(4) labeling were barely detectable where the isotope-sensitive bands are expected. Both isotope substitutions had large effects on P(M) minus oxidized difference spectra. A prominent trough at 1542 cm(-1) was shifted to 1527 cm(-1) with (15)N labeling, and its magnitude was diminished with the appearance of a 1438 cm(-1) trough with tyrosine-ring-d(4) labeling. Both isotope substitutions also had large effects on a 1314 cm(-1) trough in the same spectra. These shifts indicate that the bands are linked to both a nitrogenous compound and a tyrosine, the most obvious candidate being the covalent histidine-tyrosine ligand of Cu(B). Comparison with model material data suggests that the tyrosine hydroxyl group is protonated when the binuclear center is oxidized but deprotonated in the P(M) intermediate. Positive bands at 1519 and 1570 cm(-1) were replaced with bands at 1504 and 1556 cm(-1), respectively, with tyrosine-ring-d(4) labeling, are characteristic of upsilon(7a)(C-O) and upsilon(C-C) bands of neutral phenolic radicals, and most likely reflect the formation of the neutral radical state of the histidine-tyrosine ligand in P(M).     

CTLA-4+CD8+ T cells that encounter B7-2+ iris pigment epithelial cells express their own B7-2 to achieve global suppression of T cell activation

Pigment epithelial (PE) cells cultured from the eye possess the novel property of suppressing TCR-dependent activation of T cells in vitro. Iris PE (IPE) cells accomplish this suppression by a direct cell contact mechanism in which B7-2 expressed by the PE cells interacts with CTLA-4 on responding T cells. Because CTLA-4 expression is constitutively expressed on a very small proportion of naive splenic T cells and since exposure of splenic T cells to IPE leads to global T cell suppression, we have inquired into the mechanism by which suppression is achieved. Using splenic T cells and IPE from donor mice with disrupted genes for CD80 (B7-1), CD86 (B7-2), CTLA-4, and/or CD28, we report that B7-2(+) IPE in the presence of anti-CD3 supported selectively the activation of CTLA-4(+) CD8(+) T cells that express their own B7-2 and secrete enhanced amounts of active TGFbeta. By contrast, activation of CTLA-4-negative T cells, especially CD4(+) cells, in these cultures was profoundly suppressed. Because global suppression of T cell activation in these cultures was obtained only when both IPE and T cells possessed B7-2 genes and expressed the costimulators as surface molecules, we propose that T cells activated in the presence of parenchymal cells from the eye (an immune privileged site) express B7-2 in a manner that equips them to suppress bystander T cells. Thus, B7-2 expression on T cells participates in their eventual ability to function as regulators in vitro.     

Marrow stromal cells and osteoclast precursors differentially contribute to TNF-alpha-induced osteoclastogenesis in vivo

The marrow stromal cell is the principal source of the key osteoclastogenic cytokine receptor activator of NF-kappaB (RANK) ligand (RANKL). To individualize the role of marrow stromal cells in varying states of TNF-alpha-driven osteoclast formation in vivo, we generated chimeric mice in which wild-type (WT) marrow, immunodepleted of T cells and stromal cells, is transplanted into lethally irradiated mice deleted of both the p55 and p75 TNFR. As control, similarly treated WT marrow was transplanted into WT mice. Each group was administered increasing doses of TNF-alpha. Exposure to high-dose cytokine ex vivo induces exuberant osteoclastogenesis irrespective of in vivo TNF-alpha treatment or whether the recipient animals possess TNF-alpha-responsive stromal cells. In contrast, the osteoclastogenic capacity of marrow treated with lower-dose TNF-alpha requires priming by TNFR-bearing stromal cells in vivo. Importantly, the osteoclastogenic contribution of cytokine responsive stromal cells in vivo diminishes as the dose of TNF-alpha increases. In keeping with this conclusion, mice with severe inflammatory arthritis develop profound osteoclastogenesis and bone erosion independent of stromal cell expression of TNFR. The direct induction of osteoclast recruitment by TNF-alpha is characterized by enhanced RANK expression and sensitization of precursor cells to RANKL. Thus, osteolysis attending relatively modest elevations in ambient TNF-alpha depends upon responsive stromal cells. Alternatively, in states of severe periarticular inflammation, TNF-alpha may fully exert its bone erosive effects by directly promoting the differentiation of osteoclast precursors independent of cytokine-responsive stromal cells and T lymphocytes.     

TAGE (toxic AGEs) theory in diabetic complications

Diabetic complication is a leading cause of acquired blindness, end-stage renal failure, a variety of neuropathies and accelerated atherosclerosis. Chronic hyperglycemia is initially involved in the pathogenesis of diabetic micro- and macro-vascular complications via various metabolic derangements. High glucose increased production of various types of advanced glycation end-products (AGEs). Recently, we found that glyceraldehyde-derived AGEs (AGE-2) play an important role in the pathogenesis of angiopathy in diabetic patients. There is considerable interest in receptor for AGEs (RAGE) found on many cell types, particularly those affected in diabetes. Recent studies suggest that interaction of AGE-2 (predominantly structure of toxic AGEs; TAGE) with RAGE alters intracellular signaling, gene expression, release of pro-inflamatory molecules and production of reactive oxygen species (ROS) that contribute towards the pathology of diabetic complications. We propose three pathways for the in vivo formation of AGE-2 precursor, glyceraldehyde, such as i) glycolytic pathway, ii) polyol pathway, and iii) fructose metabolic pathway. Glyceraldehyde can be transported or can leak passively across the plasma membrane. It can react non-enzymatically with proteins to lead to accelerated formation of TAGE at both intracellularly and extracellularly. In this review, we discuss the molecular mechanisms of diabetic complications, especially focusing on toxic AGEs (TAGE) and their receptor (RAGE) system.     

In vitro cytotoxicity assay to evaluate the toxicity of an electrophilic reactive metabolite using glutathione-depleted rat primary cultured hepatocytes

Glutathione plays an important role as not only a scavenger of reactive oxygen species but also in the conjugation or detoxification of electrophilic reactive metabolites, which has been thought to be one of the causes for idiosyncratic drug toxicity (IDT). Therefore, toxic responses to the reactive metabolites have been expected to be expressed more strongly in a glutathione-depleted condition. In the present study, we attempted to establish an in vitro cytotoxicity assay method to evaluate the toxicity of the reactive metabolite using rat primary cultured hepatocytes with cellular glutathione depletion by l-buthionine-S,R-sulfoximine. Also, we investigated whether the IDT risk is predictable by comparing the cytotoxic sensitivity between glutathione-depleted hepatocytes and untreated hepatocytes. Consequently, 10 drugs of 42 approved drugs, which were classified into 4 IDT categories (Withdrawn, Black box warning, Warning, and Safe), demonstrated higher cytotoxic sensitivity in the glutathione-depleted hepatocytes. Furthermore, a correlation was observed between the incidence of drugs with higher cytotoxic sensitivity in the glutathione-depleted hepatocytes and the IDT risk. The incidence was 50% in the Withdrawn category, 38% in the Black box warning category, 22% in the Warning category, and 8% in the Safe category. These results suggest that the IDT risk of some drugs may be predicted by comparing the cytotoxic sensitivity between them. Additionally, this method may be useful as a screening in the early stage of drug development where leads/candidates are optimized.     

Expression of foreign type I ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) stimulates photosynthesis in cyanobacterium Synechococcus PCC7942 cells

A reporter gene assay revealed that promoters derived from Synechococcus PCC7942 (S.7942) psbAI and Synechocystis PCC6803 (S.6803) psbAII were suitable for the expression of foreign ribulose-bisphosphate carboxylase (RuBisCO; EC 4.1.1.39) in S.7942 cells. Transformational vectors with a promoter and a foreign RuBisCO gene, cvrbc originated from Allochromatium vinosum, were constructed on a binary vector, pUC303, and introduced to S.7942 cells. When the cvrbc was expressed with the S.7942 psbAI promoter, the total RuBisCO activity increased 2.5- to 4-fold than that of the wild type cell. The S.6803 psbAII promoter increased the activity of the transformant 1.5–2 times of that of wild type cell. There was a significant increase in the rate of photosynthesis depending on the increase of RuBisCO activity. The maximum rate of photosynthesis of the transformant cell was 1.63 times higher than that of the wild type under the illumination of 400 μmol m⁻² s⁻¹, at 20 mM bicarbonate and at 30 °C. Although the photosynthesis of the higher plant is limited by the ability of photosystems under high irradiance and the high CO₂ concentration, that of the S.7942 cell is limited by the RuBisCO activity, even at high CO₂ concentrations and under high irradiance.     

Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression

CD82, also known as KAI1, was recently identified as a prostate cancer metastasis suppressor gene on human chromosome 11p1.2 (ref. 1). The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker CD82 (refs. 1,2). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers, whereas overexpression of CD82 specifically suppresses tumor metastasis in various animal models. To define the mechanism of action of KAI1, we used a yeast two-hybrid screen and identified an endothelial cell-surface protein, DARC (also known as gp-Fy), as an interacting partner of KAI1. Our results indicate that the cancer cells expressing KAI1 attach to vascular endothelial cells through direct interaction between KAI1 and DARC, and that this interaction leads to inhibition of tumor cell proliferation and induction of senescence by modulating the expression of TBX2 and p21. Furthermore, the metastasis-suppression activity of KAI1 was significantly compromised in DARC knockout mice, whereas KAI1 completely abrogated pulmonary metastasis in wild-type and heterozygous littermates. These results provide direct evidence that DARC is essential for the function of CD82 as a suppressor of metastasis.     

Maricurvus nonylphenolicus gen. nov., sp. nov., a nonylphenol-degrading bacterium isolated from seawater

A novel aerobic, Gram-negative bacterial strain, designated KU41E(T), which degrades p-n-nonylphenol, was isolated from seawater obtained from the coastal region of Ishigaki Island, Japan. Cells are motile, curved rods with a single polar flagellum. Strain KU41E(T) grew at 20-35 °C, pH 7.0-8.0, in the presence of 1.0-4.0% NaCl. The predominant respiratory lipoquinone was ubiquinone-8, and the major cellular fatty acids were summed feature 3 (C(15:0) iso 2OH and/or C(16:1) ω7c, 28.4%), C(18:1) ω7c (19.8%), and C(16:0) (17.0%). The DNA G + C content was 48.6 mol%. The 16S rRNA gene sequence analysis indicated that strain KU41E(T) is affiliated with the order Alteromonadales within the class Gammaproteobacteria and is most closely related to Pseudoteredinibacter isoporae SW-11(T) (93.6% similarity) and Teredinibacter turnerae T7902(T) (91.9% similarity). On the basis of physiological, chemotaxonomic, and phylogenetic data, strain KU41E(T) is suggested to represent a novel species of a new genus, for which the name Maricurvus nonylphenolicus gen. nov., sp. nov. is proposed. The type strain of M. nonylphenolicus is KU41E(T) (=JCM 17778(T)).