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Over 800 known carotenoids are synthesized from phytoene or 4,4'-diapophytoene (dehydrosqualene) characterized by three conjugated double bonds. In this paper, we report that carotenoid desaturase CrtN from Staphylococcus aureus and Methylomonas can accept oxidosqualene, which is the precursor for plant- or animal-type triterpenoids, yielding the yellow carotenoid pigments with 8, 9, or 10 conjugated double bonds. The resulting pathway is the second nonnatural route for carotenoid pigments and the first pathway for carotenoid pigments not biosynthesized via (diapo)phytoene., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Daisuke Umeno reports financial support was provided by Government of Japan Ministry of Education Culture Sports Science and Technology. Yusuke Otani reports financial support was provided by Government of Japan Ministry of Education Culture Sports Science and Technology., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Brown macroalgae is a promising marine biomass for the production of bioethanol and biodiesel fuels. Here we investigate the biochemical processes used by marine oleaginous yeast for assimilating the major carbohydrate found in brown macroalgae. Briefly, yeast Rhodosporidiobolus fluvialis strain Y2 was isolated from seawater and grown in minimal medium containing reduced sugar alcohol mannitol as the sole carbon source with a salinity comparable to seawater. Conditions limiting nitrogen were used to facilitate lipid synthesis. R. fluvialis Y2 yielded 55.1% (w/w) and 39.1% (w/w) of lipids, per dry cell weight, from mannitol in the absence and presence of salinity, respectively. Furthermore, mannitol, as a sugar source, led to an increase in the composition of polyunsaturated fatty acids, linoleic acid (C18:2) and linolenic acid (C18:3), compared to glucose. This suggests that oxidation of mannitol leads to the activation of NADH-dependent fatty acid desaturases in R. fluvialis Y2. Such fatty acid composition may contribute to the cold-flow properties of biodiesel fuels. Our results identified a salt-tolerant oleaginous yeast species with unique metabolic traits, demonstrating a key role as a decomposer in the global carbon cycle through marine ecosystems. This is the first study on mannitol-induced synthesis of lipids enriched with polyunsaturated fatty acids by marine yeast., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In the living cells, the majority of proteins does not work alone, but interact with other proteins or other biomolecules to maintain the cellular function, constituting a "protein community". Previous efforts on mass spectroscopy-based protein interaction networks, interactomes, have provided a picture on the protein community. However, these were static information after cells were disrupted. For a better understanding of the protein community in cells, it is important to know the properties of intracellular dynamics and interactions. Since hydrodynamic size and mobility of proteins are related into such properties, direct measurement of diffusional motion of proteins in single living cells will be helpful for uncovering the properties. Here we completed measurement of the diffusion and homo-oligomeric properties of 369 cytoplasmic GFP-fusion proteins in living yeast Saccharomyces cerevisiae cells using fluorescence correlation spectroscopy (FCS). The large-scale analysis showed that the motions of majority of proteins obeyed a two-component (i.e. slow and fast components) diffusion model. Remarkably, both of the two components diffused more slowly than expected monomeric states. In addition, further analysis suggested that more proteins existed as homo-oligomeric states in living cells than previously expected. Our study, which characterizes the dynamics of proteins in living cells on a large-scale, provided a global view on intracellular protein dynamics to understand the protein community., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Human mitochondrial NAD kinase is a crucial enzyme responsible for the synthesis of mitochondrial NADP(+). Despite its significance, little is known about the regulation of this enzyme in the mitochondria. Several putative and known phosphorylation sites within the protein have been found using phosphoproteomics, and here, we examined the effect of phosphomimetic mutations at six of these sites. The enzymatic activity was downregulated by a substitution of an Asp residue at Ser-289 and Ser-376, but not a substitution of Ala, suggesting that the phosphorylation of these residues downregulates the enzyme. Moreover, the activity was completely inhibited by substituting Ser-188 with an Asp, Glu, or in particular Ala, which highlights two possibilities: first, that Ser-188 is critical for catalytic activity, and second, that phosphorylation of Ser-188 inhibits the activity. Ser-188, Ser-289, and Ser-376 were found to be highly conserved in the primary structures of mitochondrial NAD kinase homologs in higher animals. Moreover, Ser-188 has been frequently detected in human and mouse phosphorylation site studies, whereas Ser-289 and Ser-376 have not. Taken together, this indicates that Ser-188 (and perhaps the other residues) is an important phosphorylation site that can downregulate the NAD kinase activity of this critical enzyme., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Plasmodium falciparum spends most of its asexual life cycle within human erythrocytes, where proliferation and maturation occur. Development into the mature forms of P. falciparum causes severe symptoms due to its distinctive sequestration capability. However, the physiological roles and the molecular mechanisms of signaling pathways that govern development are poorly understood. Our previous study showed that P. falciparum exhibits stage-specific spontaneous Calcium (Ca(2+)) oscillations in ring and early trophozoites, and the latter was essential for parasite development. In this study, we show that luzindole (LZ), a selective melatonin receptor antagonist, inhibits parasite growth. Analyses of development and morphology of LZ-treated P. falciparum revealed that LZ severely disrupted intraerythrocytic maturation, resulting in parasite death. When LZ was added at ring stage, the parasite could not undergo further development, whereas LZ added at the trophozoite stage inhibited development from early into late schizonts. Live-cell Ca(2+) imaging showed that LZ treatment completely abolished Ca(2+) oscillation in the ring forms while having little effect on early trophozoites. Further, the melatonin-induced cAMP increase observed at ring and late trophozoite stage was attenuated by LZ treatment. These suggest that a complex interplay between IP3-Ca(2+) and cAMP signaling pathways is involved in intraerythrocytic development of P. falciparum., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Prions are self-propagating amyloids. Yeast prion [PSI(+)] is a protein-based heritable element, in which amyloid aggregates of the Sup35 protein are transmitted to daughter cells. Hsp104, an ATP-dependent disaggregase, and other chaperones are essential to maintain [PSI(+)]. Although previous reports have demonstrated the physical interactions of Hsp104 and Sup35 amyloids, the mechanism how Hsp104 interacts with Sup35 amyloids remains to be elucidated. Here we investigated the interaction between Hsp104 and Sup35 in the lysates of [PSI(+)] cells using fluorescence cross-correlation spectroscopy (FCCS), which can analyze the codiffusion events of different fluorophores. FCCS analysis showed a strong interaction between Hsp104 and Sup35 in [PSI(+)] lysates, but not in [psi(-)] lysates, suggesting that Hsp104 recognizes the amyloid aggregates of Sup35. Although the interaction was retained in ATP-depleted [PSI(+)] lysates, addition of ATP or guanidine hydrochloride, which is an inhibitor of Hsp104, to [PSI(+)] lysates weakened the interaction., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The transcription factor Odd-skipped related 2 (Osr2) functions in craniofacial and limb developments in mammals. We previously found that Osr2 gene expression is regulated by intracellular transcription factors such as Runx2, and C/EBP, whereas it remains unclear if extracellular factors would functionally regulate the Osr2 expression. In this study, we showed that TGF-β down-regulated the Osr2 expression, which is involved in regulation of cellular migration and cell cycle. Furthermore, the down-regulation was found to be mediated by Smad3/Smad4 and p38/ATF2 signaling molecules. The Osr2 promoter was shown to possess Smad3/4 binding element and ATF2 binding element between -647 and -64 of promoter. TGF-β induced cellular migration in C3H10T1/2 cells and arrested cell cycle at G1 phase in NMuMG-Fucci cells. In contrast, the Osr2 reduced the migration and also stimulated the cell-cycle progression. These results suggest that Osr2 is involved in TGF-β regulating cell migration and cell cycle via a Smad3-ATF2 transcriptional complex mediating pathway., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cell proliferation is a biological process in which chromosomes replicate in one cell and equally divide into two daughter cells. Our previous findings suggested that Odd-skipped related 2 (Osr2) plays an important role in cellular quiescence and proliferation under epigenetic regulation. However, the mechanism used by Osr2 to establish and maintain proliferation is unknown. To examine the functional role of Osr2 in cell proliferation, we analyzed its downstream target genes using microarray analysis following adenovirus-induced overexpression of Osr2 as well as knockdown with Osr2 siRNA, which showed that Osr2 regulates a multitude of genes involved in proliferation and the cell cycle, as well as development. Additional proliferation assays also indicated that Osr2 likely functions to elicit cell proliferation. Together, these results suggest that Osr2 plays important roles in proliferation and development., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Cellular behavior and development are extensively altered during the transition from cell cycle into quiescence, though the mechanism involved in establishing and maintaining quiescence is largely unknown. We found that Odd-skipped related 2 (Osr2) was up-regulated during cellular quiescence by serum starvation as well as culturing to confluence. To investigate the regulatory mechanism of Osr2 under these conditions, we characterized the mouse Osr2 promoter. CpG islands in the flanking region of the transcription start site were predominantly methylated in exponentially growing cells, resulting in silencing of Osr2 expression. In addition, CpG demethylation in quiescence caused activation of Osr2 expression, while acetylation of the H3 and H4 histones during quiescence also led to an increase in Osr2 expression. These results suggest that epigenetically regulated Osr2 plays an important role in cellular quiescence and proliferation., ((c) 2010 Elsevier Inc. All rights reserved.)

The adipokines are linked not only to metabolic regulation, but also to immune responses. Adiponectin, but not leptin or resistin induced interleukin-8 production from rheumatoid synovial fibroblasts (RSF). The culture supernatant of RSF treated with adiponectin induced chemotaxis, although adiponectin itself had no such effect. Addition of antibody against adiponectin, and inhibition of adiponectin receptor gene decreased adiponectin-induced IL-8 production. Nuclear translocation of nuclear factor-kappa B was increased by adiponectin. The induction of interleukin-8 was inhibited by mitogen-activated protein kinase inhibitors. These findings suggest that adiponectin contributes to the pathogenesis of rheumatoid arthritis.

Odd-skipped related 2 (Osr2) is expressed in the domain of epithelial-mesenchymal interactions during tooth and kidney development. In this study, we report that alternative splicing of exon 4 resulted in two transcripts, Osr2A and Osr2B. These variants utilized a different splice acceptor, and Osr2B exhibited a frameshift followed by an early stop codon. Osr2A mRNA produced a protein that was 36 amino acids longer than Osr2B at the C-terminus and had five zinc-finger domains, whereas Osr2B had three zinc-finger motives. The 2 Osr2 variants were found in kidney, skeletal muscle, and testis, and Osr2B was predominantly transcribed in each. Flag-tagged variants showed the same localization in cell nuclei, however, Gal4 fusion proteins showed opposite transcriptional activities. Further, Flag-tagged variants also showed opposite transcriptional activities, though they were in contrast to the findings for the Gal4-fused variants. Our results indicate that Osr2 bifurcates its function by alternative splicing.

NAD kinase is a key enzyme in NADP biosynthesis. We solved the crystal structure of polyphosphate/ATP-NAD kinase from Mycobacterium tuberculosis (Ppnk) complexed with NAD (Ppnk-NAD) at 2.6A resolution using apo-Ppnk structure solved in this work, and revealed the details of the structure and NAD-binding site. Superimposition of tertiary structures of apo-Ppnk and Ppnk-NAD demonstrated a substantial conformational difference in a loop (Ppnk-flexible loop). As a quaternary structure, these Ppnk structures exhibited tetramer as in solution condition. Notably, the Ppnk-flexible loop was involved in the intersubunit contact and probably related to the NAD-binding of the other subunit. Furthermore, the two residues (Asp189, His226) substantially contributed to creating NAD-binding site on the other subunit. The two residues and the residues involved in NAD-binding were conserved. However, residues corresponding to the Ppnk-flexible loop were not conserved, making us to speculate that the Ppnk-flexible loop may be Ppnk-specific.

A mouse monoclonal antibody (2D7 mAb), which specifically bound to the alpha2 domain of HLA class I, rapidly induces cell aggregation accompanied by weak cytotoxicity against ARH-77 cells, suggesting that 2D7 mAb had a potential for agonist antibody. In order to enhance this cytotoxicity, 2D7 mAb was engineered to be a small bivalent antibody fragment, 2D7 diabody. The resultant 2D7 diabody showed a strong cytotoxicity against ARH-77 cells. As a notable characteristic feature, the lethal effect of 2D7 diabody was quite rapid, mediated by a caspase-independent death pathway. Furthermore, 2D7 diabody also showed cytotoxicity against several leukemia and lymphoma cell lines, and mitogen-activated peripheral blood mononuclear cells (PBMC), but not for normal resting PBMC and adherent cell lines such as HUVEC. These results suggest that 2D7 diabody could be expected as a novel therapeutic antibody for hematological malignancies as well as inflammatory diseases.

Bone marrow stromal cells (MSCs) can be expanded rapidly in vitro and have the potential to be differentiated into neuronal, glial and endodermal cell types. However, induction for differentiation does not always have stable result. We present a new method for efficient induction and acquisition of neural progenitors, neuronal- and glial-like cells from MSCs. We demonstrate that rat MSCs can be induced to neurospheres and most cells are positive for nestin, which is an early marker of neuronal progenitors. In addition, we had success in proliferation of these neurospheres with undifferentiated characteristics and finally we could obtain large numbers of neuronal and glial phenotypes. Many of the cells expressed beta-tubulin III when they were cultivated with our method. MSCs can become a valuable cell source as an autograft for clinical application involving regeneration of the central nervous system.

Biodegradable poly D,L-lactic acid (PLA, molecular weight: ca. 5000) microcapsules containing a CXCR4 antagonist (4F-benzoyl-TE14011) were prepared (4F-benzoyl-TE14011-PLA), and their anti-metastatic activity was evaluated in mice. A single subcutaneous administration of 4F-benzoyl-TE14011-PLA significantly reduced the number of colonies formed by pulmonary metastasis of B16-BL6 melanoma cells expressing CXCR4. The same dose of 4F-benzoyl-TE14011 in a single or a series of treatments affected little. The substance 4F-benzoyl-TE14011 dose-dependently suppressed B16-BL6 cell growth. In the cells cultured with SDF-1, a more potent suppression was observed. 4F-Benzoyl-TE14011 was rapidly released from 4F-benzoyl-TE14011-PLA for an initial period, both in vitro and in vivo. A steady release was thereafter observed. Therefore, this drug release profile might contribute to prevention of melanoma metastasis at the steps involving the migration and cell growth. These results also show that a sustained drug release formulation could be a useful drug delivery system for CXCR4 antagonists.

Ceramide, the backbone of sphingolipids, has been reported to be involved in various cellular responses including apoptosis. We recently established and characterized a C2-ceramide-resistant HL-60 subline designated HL-CR. HL-CR cells were resistant to not only ceramide but also anti-cancer drugs including daunorubicin, etoposide, and cytosine arabinoside. To elucidate the mechanisms by which HL-CR cells became resistant to various apoptosis-inducing stimuli, the levels of Bcl-2 family proteins, which play crucial roles in drug-induced apoptosis, were compared between HL-CR and parental HL-60 cells. Among Bcl-2 family members, Bax, a pro-apoptotic Bcl-2 family protein, was highly expressed in HL-60 but was hardly detected in HL-CR cells. Transient transfection of bax-expressing plasmid, but not the vector alone, induced apoptosis in HL-CR cells. These results suggest that reduced expression of Bax might play a role in resistance to various apoptosis-inducing stimuli in HL-CR cells.

Understanding of the molecular system for DNA delivery into eucaryotic cells, a key to human DNA therapy, remains obscure. To understand this system, we undertook a study using the Saccharomyces cerevisiae model into which DNA delivery is easily assessed through competence (transformability) and for which all nonessential gene mutants (about 5000 strains) are available. We analyzed the competence of each of these mutants and identified three low-competence mutants, i.e., sin3Delta, she4Delta, and arc18Delta, and three high-competence mutants, i.e., pde2Delta, spf1Delta, and pmr1Delta. Through further studies using the six mutants, we concluded that the Arp2/3 activation machinery involving the Myo3/5p, Vrp1p, Las17p, Pan1p, and Arp2/3 complex is crucial to delivery (competence), and that high cAMP enhances competence via protein kinase A installing Tpk3p. We also propose that DNA is taken up via an endocytosis-like event, being at least partially different from well-known endocytosis.

Local and systemic expression of insulin-like growth factor-I (IGF-I) during bone formation was studied using the rat bone marrow ablation model. The temporal expression pattern of IGF-I mRNA in rat femurs was examined. The IGF-I mRNA level was enhanced rapidly after ablation reaching a level threefold greater than basal by day 3 (P < 0.01) and declined to basal or below basal level by day 5. Histological analysis showed that IGF- I immunoreactivity was predominantly associated with the mesenchymal cells at the bone/connective tissue interface and osteoblastic cells at active sites of bone formation. Serum level of IGF-I increased 50 and 130%, respectively (P < 0.005), over the basal level at days 3 and 6. We also investigated the systemic expression of IGF-I in liver and kidney. In contrast, hepatic IGF-I gene expression decreased 37 and 48%, respectively, at days 3 and 6 after marrow ablation (P < 0.001). Kidney IGF-I mRNA levels also fell 13 and 27%, respectively, at days 3 and 6 (P < 0.005). The present findings suggest that locally produced IGF-I during bone formation may not only serve as an autocrine/paracrine factor but also influence systemic expression of IGF-I in other organs., (Copyright 2001 Academic Press.)

Glycation of plasma proteins may contribute to an excess risk of developing atherosclerosis in patients with diabetes mellitus. Although it is believed that high-density lipoprotein (HDL) is nonenzymatically glycosylated at an increased level in diabetic individuals, little is known about a possible linkage between glycated HDL and endothelium dysfunction in diabetes. This study set out to clarify whether glucose-modified HDL affects the function of endothelial cells by examining the apoptosis of cultured human aortic endothelial cells (HAECs) exposed to a glycated-oxidized HDL (gly-ox-HDL) prepared in vitro. Incubation of HAECs with 100 microg/ml of gly-ox-HDL for 48 h showed apoptotic features, such as cell shrinkage, membrane blebbing, and concentration and fragmentation of the nucleus, and the degree of apoptosis was dose-dependent on the glucose used in the preparation of gly-ox-HDL. Stimulation of HAECs with gly-ox-HDL elicited a marked increase in caspase 3 activity and the expressions of active caspase 3 and caspase 9, whereas concomitant treatment with a caspase 3 inhibitor significantly blocked gly-ox-HDL-induced apoptosis of HAECs. The release of cytochrome c into cytosols markedly increased in HAECs during the treatment with gly-ox-HDL. The increased expressions of Bax and Bad were detected in HAECs incubated for 24 h with gly-ox-HDL, but gly-ox-HDL failed to interfere with the expression of Bcl-2 and Bcl-x. Moreover, in vitro experiments with HDL (gly-HDL) glycated in the presence of 2 mM EDTA and Cu(2+)-oxidized HDL suggested that the apoptotic effect of gly-ox-HDL on endothelial cells might be due to an additional oxidative modification of gly-HDL. Taken altogether, additional oxidation of HDL under hyperglycemic conditions may induce endothelial apoptosis through a mitochondrial dysfunction, following the deterioration of vascular function., (Copyright 2001 Academic Press.)

An enzyme with both inorganic polyphosphate [poly(P)]- and ATP-dependent NAD kinase activities was isolated from Micrococcus flavus. The enzyme was a dimer consisting of 34 kDa subunits, and was named poly(P)/ATP-NAD kinase. Internal amino acid sequences of the enzyme showed homologies with some function-unknown proteins released on the GenBank database. Among such proteins, hypothetical Rv1695 protein (Accession No. Z98268-16), which was encoded by a gene named "Rv1695" on genomic DNA of Mycobacterium tuberculosis H37Rv, was proposed to be poly(P)-dependent NAD kinase. By cloning and expression in Escherichia coli, Rv1695 was shown to encode poly(P)/ATP-NAD kinase and named ppnk. The ppnk product, recombinant-poly(P)/ATP-NAD kinase (Ppnk) was purified and characterized. The enzyme was a tetramaer consisting of 35 kDa subunits when expressed in E. coli. Poly(P)/ATP-NAD kinases of M. flavus and Ppnk of M. tuberculosis H37Rv specifically and completely phosphorylated NAD by utilizing commercially available poly(P)s and nucleoside triphosphates as phosphoryl donors., (Copyright 2000 Academic Press.)

Smads are intracellular signaling mediators for TGF-beta superfamily. Smad1 and Smad5 are activated by BMP receptors. Here, we have cloned mouse Smad8 and functionally characterized its ability to transduce signals from BMP receptors. Constitutively active BMP type I receptors, ALK-3 and ALK-6, as well as ALK-2, were phosphorylated Smad8 and induced Smad8 interaction with Smad4. Nuclear translocation of Smad8 was stimulated by constitutively active BMP type I receptors. In contrast, constitutively active TGF-beta type I receptor, ALK-5, did not exhibit any action on Smad8. Smad8 and Smad4 cooperatively induced the promoter of Xvent2, a homeobox gene that responds specifically to BMP signaling. Dominant-negative Smad8 was shown to inhibit the increase of alkaline phosphatase activity induced by BMP-2 on pluripotent mesenchymal C3H10T1/2 and myoblastic C2C12 cell lines. The presence of Smad8 mRNA in mouse calvaria cells and osteoblasts suggests a role of Smad8 in the osteoblast differentiation and maturation., (Copyright 2000 Academic Press.)

In order to examine the involvement of leptin in the ossification of spinal ligaments (OSL), the present study examined (i) serum levels of leptin and insulin in OSL patients and controls, (ii) serum leptin levels in children of OSL females with severe obesity, (iii) the expression of leptin receptor mRNA in human spinal ligaments, and (iv) effects of leptin on cultured human ligament cells. In the OSL females, serum leptin levels were significantly higher than those of the control females, and the levels were positively correlated to the serum insulin levels, while in the control females, there was a tendency of inverse correlation. The daughters of OSL females with severe obesity also had high serum leptin levels, although they had not developed OSL. The expression of leptin receptor mRNA was confirmed in the ligaments, but leptin did not influence the alkaline phosphatase activity nor procollagen type I carboxyl-terminal peptide content of the ligament cells. These findings suggest that leptin is involved genetically and indirectly with the pathogenesis of OSL in female patients., (Copyright 2000 Academic Press.)

Growth/differentiation factor-5 (GDF-5) is a new member of the BMP family supposed to be involved in chondrogenesis. We cloned the human GDF-5 gene from lambda phage library and sequenced its 3.5-kb 5'-flanking region. The transcription start site was mapped by 5'-RACE to the sequence that coincides with the initiator element. Electrophoresis mobility shift assays (EMSA) demonstrated a zinc finger transcription factor, YY1, to bind to the sequence surrounding the transcription start site. To localize positive and negative regulatory elements in the GDF-5 5'-upstream region, we constructed a series of progressively deleted promoter-reporter plasmids. The transient transfection assay with human osteoblastic Hos cells indicated that a minimal enhancer element resides within -117 to -27 relative to the transcription initiation site. Since the GDF-5 promoter was active even in fibroblastic L cells, a mechanism governing its chondrocyte-restrictive expression needs to be explored., (Copyright 1999 Academic Press.)

It has been suggested the the interaction of Escherichia coli O157-derived verotoxins (VTs) with the vascular endothelium plays a central role in the pathogenesis of the thrombotic microangiopathy and ischemic lesions characteristic of hemolytic uremic syndrome (HUS) and E. coli O157-associated hemorrhagic colitis. Intravenous administration of both E. coli O157-derived VT1 and lipopolysaccharide (LPS) in the rat induced a synergistic increase in thiobarbituric acid (TBA) values in those animal's plasma, as compared with that injected with VT1 or LPS alone. We then hypothesized that an increase in lipid peroxidation in the rat plasma was due to an enhanced production of endothelial cell-derived reactive oxidant. Based on determination of rat sera and cultured human aortic endothelial cells (HAECs), VT1 had little if any effect on LPS-stimulated increase of nitric oxide and the resultant peroxynitrite generations. Both RT-PCR and Western blot studies of reactive oxygen species-related enzymes showed that VT1 markedly decreased the expression of catalase mRNA and protein in HAECs, but caused less alteration in the levels of Cu, Zn-superoxide dismutase, and NADPH oxidase mRNA. Further studies by spin trapping analysis using 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) revealed a time-dependent increase in hydroxyl radicals by VT1 in HAECs. The accumulated data thus suggest that bacterial VT1 reduces mainly catalase levels in endothelial cells, which is synergistically potentiated by LPS, and that the resulting hydroxyl radical participates in endothelium injury through a marked enhancement of lipid peroxidation, leading to HUS., (Copyright 1999 Academic Press.)

HM1.24 antigen has been identified as a surface molecule preferentially expressed on terminally differentiated B cells, and its overexpression is observed in multiple myeloma cells. The HM1.24 antigen is, therefore, expected as a most potent target molecule for antibody-based immunotherapy for multiple myeloma. Here, we have identified the cDNA for human HM1.24 antigen and also analyzed its gene structure including the promoter region. The HM1.24 antigen is a type II membrane glycoprotein, which has been reported as a bone marrow stromal cell surface antigen BST2, and may exist as a homodimer on myeloma cell surface. Although a reason for the overexpression in myeloma cells is not understood, very interestingly, the promoter region of the HM1.24 gene has a tandem repeat of three cis elements for a transcription factor, STAT3, which mediates interleukin-6 (IL-6) response gene expression. Since IL-6 is a differentiation factor for B cells, and known as a paracrine/autocrine growth factor for multiple myeloma cells, the expression of HM1.24 antigen may be regulated by the activation of STAT3. Importantly, a humanized anti-HM1.24 antibody effectively lysed the CHO transformants which expressed HM1.24 antigen as high as human multiple myeloma cells, but not the cells with lower antigen expression. This evaluation shows that ADCC heavily depends on the expression level of target antigens and, therefore, the immunotherapy targeting the HM1.24 antigen should have a promising potential in clinical use., (Copyright 1999 Academic Press.)

Effects of five kinds of dopamine agonists on the activity of type 2A protein phosphatase in rat brain were studied. Apomorphine and SKF-38393 reduced the enzyme activity considerably and their effects were further enhanced in the presence of 10 microM Mn2+. Also, 6,7-ADTN slightly inhibited the activity. The present results suggest that type 2A protein phosphatase in the brain is possibly involved in dopamine mediated protein phosphorylation functions.

Oncogenic transformation has been considered to be in part a consequence of the elevated levels of 1,2-diacylglycerol(DG), resulting in the permanent activation of protein kinase C. DG content in transformed cells with v-H-ras, c-K-ras and N-ras oncogene increased 1.5-fold compared to that in non-transformed NIH/3T3 cells. DG kinase activity of membrane fractions, which plays an important role in DG attenuation, was significantly lower in all ras-transformed cells. On the contrary, DG kinase activity in cytosol fractions in ras-transformed cells was found to be increased. DG kinase translocated very markedly from cytosol to membranes in non-transformed NIH/3T3 cells by the treatment of phospholipase C. On the other hand, translocation of DG kinase in ras-transformed cells was slight, though the formation of DG by the treatment of phospholipase C was almost same between ras-transformed and NIH/3T3 cells. These results strongly support the idea that the increased DG content in ras-transformed cells is, at least partly due to the defect of DG kinase translocation, which may lead to the sustained activation of protein kinase C.