Purification and biochemical characterization of a novel ecto-apyrase, MP67, from Mimosa pudica.

We have previously reported the presence of an apyrase in Mimosa pudica. However, only limited information is available for this enzyme. Thus, in this study, the apyrase was purified to homogeneity. The purified enzyme had a molecular mass of around 67 kD and was able to hydrolyze both nucleotide triphosphate and nucleotide diphosphate as substrates. The ratio of ATP to ADP hydrolysis velocity of the purified protein was 0.01 in the presence of calcium ion, showing extremely high substrate specificity toward ADP. Thus, we designated this novel apyrase as MP67. A cDNA clone of MP67 was obtained using primers designed from the amino acid sequence of trypsin-digested fragments of the protein. In addition, rapid amplification of cDNA ends-polymerase chain reaction was performed to clone a conventional apyrase (MpAPY2). Comparison of the deduced amino acid sequences showed that MP67 is similar to ecto-apyrases; however, it was distinct from conventional apyrase based on phylogenetic classification. MP67 and MpAPY2 were expressed in Escherichia coli, and the recombinant proteins were purified. The recombinant MP67 showed high substrate specificity toward ADP rather than ATP. A polyclonal antibody raised against the recombinant MP67 was used to examine the tissue distribution and localization of native MP67 in the plant. The results showed that MP67 was ubiquitously distributed in various tissues, most abundantly in leaves, and was localized to plasma membranes. Thus, MP67 is a novel ecto-apyrase with extremely high substrate specificity for ADP.

Isolation of heat-tolerant myoglobin from Asian swamp eel Monopterus albus.

Myoglobin from Asian swamp eel Monopterus albus was purified from fish muscle using salt fractionation followed by column chromatography and molecular filtration. The purified Mb of 0.68 mg/g wet weight of muscle was determined for its molecular mass by MALDI-TOF-MS to be 15,525.18 Da. Using isoelectric focusing technique, the purified Mb showed two derivatives with pI of 6.40 and 7.12. Six peptide fragments of this protein identified by LC-MS/MS were homologous to Mbs of sea raven Hemitripterus americanus, yellowfin tuna Thunnus albacores, blue marlin Makaira nigicans, common carp Cyprinus carpio, and goldfish Carassius auratus. According to the Mb denaturation, the swamp eel Mb had thermal stability higher than walking catfish Clarias batrachus Mb and striped catfish Pangasius hypophthalmus Mb, between 30 and 60 (°)C. For the thermal stability of Mb, the swamp eel Mb showed a biphasic behavior due to the O(2) dissociation and the heme orientation disorder, with the lowest increase in both Kd(f) and Kd(s). The thermal sensitivity of swamp eel Mb was lower than those of the other Mbs for both of fast and slow reaction stages. These results suggest that the swamp eel Mb globin structure is thermally stable, which is consistent with heat-tolerant behavior of the swamp eel particularly in drought habitat.

High molecular weight lectin isolated from the mucus of the giant African snail Achatina fulica.

To understand better the host defense mechanisms of mollusks against pathogens, we examined the anti-microbial activity of mucus from the giant African snail Achatina fulica. Hemagglutination activity of the mucus secreted by the integument of snails inoculated with Escherichia coli was observed to increase and to cause hemagglutination of rabbit red blood cells. Purification of the snail mucus lectin by sequential column chromatography revealed that the relative molecular mass of the lectin was 350 kDa. The hemagglutination activity of the lectin was Ca(2+)-dependent and was inhibited by galactose. Growth arrest tests showed that the lectin did not inhibit bacterial growth, but did induce agglutination of gram-positive and gram-negative bacteria. Tissue distribution analyses using a polyclonal antibody revealed that the lectin was expressed in the tissues of the mantle collar. The lectin isolated from the mucus of the snail appeared to contribute to its innate immunity.

Specific knockdown of delta-sarcoglycan gene in C2C12 in vitro causes post-translational loss of other sarcoglycans without mechanical stress.

The precise role of delta-sarcoglycan (SG) that is constitutively expressed in skeletal muscle cells and may serve for maintaining the sarcolemmal integrity has not been identified. The delta-SG protein is at first among SG complex. To specifically identify the role in C(2)C(12) cells during the myogenesis, we screened several RNA interference (RNAi) candidates at first, and knocked down both levels of the mRNA and protein, employing adenovirus-mediated RNAi. We found no morphological alteration at both myoblast and myotube stages by suppression of delta-SG. The specific knockdown of delta-SG accompanied a concomitant decrease of alpha-, beta-, and gamma-SGs preserving normal levels of each transcript. As for the localization, alpha-, beta-, and gamma-SGs were weakly stained on the cell membrane in delta-SG knockdown cells, whereas each SG in control cell was localized both on the cell membrane and myoplasm abundantly. This enhanced post-translational loss would represent similitude of the progression of cardiomuscular diseases in vitro. Different from cardiac muscle cells, skeletal muscle cell culture without muscle contraction may imply that mechanical stress per se is not primarily involved in the progression of limb-girdle muscular dystrophy. Furthermore, we have observed translocation of calpain-2 to cell membrane in delta-SG knockdown cells, suggesting that Ca(2+)-sensitive proteases, calpains closely take part in post-translational proteolysis.

Phase I and II pharmacokinetic and pharmacodynamic study of the proteasome inhibitor bortezomib in Japanese patients with relapsed or refractory multiple myeloma.

The purpose of this phase I and II study was to evaluate the safety, pharmacokinetics, pharmacodynamics, and efficacy of bortezomib in Japanese patients with relapsed or refractory multiple myeloma. This was a dose-escalation study designed to determine the recommended dose for Japanese patients (phase I) and to investigate the antitumor activity and safety (phase II) of bortezomib administered on days 1, 4, 8, and 11 every 21 days. Thirty-four patients were enrolled. A dose-limiting toxicity was febrile neutropenia, which occurred in one of six patients in the highest-dose cohort in phase I and led to the selection of 1.3 mg/m(2) as the recommended dose. Adverse events >or= grade 3 were rare except for hematological toxicities, although there was one fatal case of interstitial lung disease. The overall response rate was 30% (95% confidence interval, 16-49%). Pharmacokinetic evaluation showed a biexponential decline, characterized by a rapid distribution followed by a longer elimination, after dose administration, whereas the area under the concentration-time curve increased proportionately with the dose. Bortezomib was effective in Japanese patients with relapsed or refractory multiple myeloma. A favorable tolerability profile was also seen, although the potential for pulmonary toxicity should be monitored closely. The pharmacokinetic and pharmacodynamic profiles of bortezomib in the present study warrant further investigations, including more relevant administration schedules.

Comparative expression and tissue distribution analyses of astacin-like squid metalloprotease in squid and cuttlefish.

Astacin-like squid metalloprotease (ALSM) is a member of the astacin family of metalloproteases. In the present study, we investigated the expression and tissue distribution of ALSM in bigfin reef squid (Sepioteuthis lessoniana) and golden cuttlefish (Sepia esculenta). Myosin heavy chain hydrolysis tests showed ALSM-I-like activity in both species. We isolated partial cDNA clones showing high sequence similarity to ALSM-I and -III, suggesting that ALSM is common to squid and cuttlefish. Phylogenetic analysis showed that ALSMs are classified into two clades: ALSM-I forms one clade, and ALSM-II and -III form the other. ALSM was expressed in several tissues in bigfin reef squid, though expression was confined to the liver in cuttlefish. ALSMs are distributed in digestive organs but not in mantle muscle of squid and cuttlefish. Immunofluorescence analysis further showed that cellular localization of ALSM is evident not only in hepatic cells but also in pancreatic cells of bigfin reef squid. Thus, ALSM is commonly expressed in squid and cuttlefish, but its expression levels and distribution are distinct.

Bitter taste and blood glucose are not involved in the suppressive effect of dietary histidine on food intake.

Histamine decreases food intake by activating histaminergic neurons in the hypothalamus. Histamine is synthesized by histidine decarboxylase (HDC) from histidine. The purpose of this three-part animal study was to clarify the mechanism underlying the suppressive effect of dietary histidine on food intake. In experiment 1, we attempted to distinguish palatability from a direct effect of dietary histidine because histidine tastes slightly bitter to humans. We measured food intake every hour for 24 h in rats fed with a histidine-enriched diet or one of various quinine diets (0.001-0.8% quinine), also bitter. In experiment 2, we measured changes in blood glucose levels in rats fed with a standard or histidine-enriched diet because blood glucose is known to decrease food intake. In experiment 3, we intraperitoneally injected fluoromethylhistidine (FMH), an antagonistic inhibitor of HDC, in rats fed with a histidine-enriched diet. In experiment 1, food intake was almost the same in rats fed with the histidine-enriched diet as that in rats fed with the 0.01% quinine diet until 6 h, but food intake was low in other groups compared with that in the histidine-enriched diet group. After 6 h, food intake did not increase in rats fed with the histidine-enriched diet. In experiment 2, the blood glucose level rose quickly and then began to decrease at approximately 2 h in both groups of rats. However, it decreased more dramatically in rats fed with the histamine-enriched diet and reaches a significant difference from the decrease in the standard-diet group by 6 h. In experiment 3, food intake increased significantly in FMH-injected rats fed with the histidine-enriched diet compared with in non-FMH injected rats. Our results suggest that dietary histidine suppresses food intake by activating histaminergic neurons in the hypothalamus, independently bitter taste and blood glucose level.

Water channel activities of Mimosa pudica plasma membrane intrinsic proteins are regulated by direct interaction and phosphorylation.

cDNAs encoding aquaporins PIP1;1, PIP2;1, and TIP1;1 were isolated from Mimosa pudica (Mp) cDNA library. MpPIP1;1 exhibited no water channel activity; however, it facilitated the water channel activity of MpPIP2;1 in a phosphorylation-dependent manner. Mutagenesis analysis revealed that Ser-131 of MpPIP1;1 was phosphorylated by PKA and that cooperative regulation of the water channel activity of MpPIP2;1 was regulated by phosphorylation of Ser-131 of MpPIP1;1. Immunoprecipitation analysis revealed that MpPIP1;1 binds directly to MpPIP2;1 in a phosphorylation-independent manner, suggesting that phosphorylation of Ser-131 of MpPIP1;1 is involved in regulation of the structure of the channel complex with MpMIP2;1 and thereby affects water channel activity.

Gender effects in dietary histidine-induced anorexia.

OBJECTIVE: Histamine, a derivative of histidine, decreases food intake by activation of histamine neurons. The aim of the present study was to clarify gender-related differences in food intake through the histidine-histamine neuron system. METHODS: Male, female, and ovariectomized rats were fed a histidine-enriched diet or a control diet with the cafeteria method. RESULTS: The suppressive effect of histidine on food intake was greater in female rats than in male rats, and the suppressive effect of histidine on food intake was less in ovariectomized rats than in female rats. CONCLUSION: Our results indicate that females are more sensitive than males to dietary histidine-induced anorexia.

Expression and tissue distribution of astacin-like squid metalloprotease (ALSM).

Astacin metalloprotease family members function in a wide variety of biologic events, including cell differentiation and morphogenesis during embryonic development and adult tissue differentiation. We previously isolated and characterized an astacin-like squid metalloprotease (ALSM). To elucidate the embryonic expression of ALSM, we performed immunohistochemical analysis with specific antibodies and examined the expression profiles of ALSM isoforms by in situ hybridization analysis. Tissue distribution and expression were also examined in adult spear squid. mRNA expression of ALSM isoforms I and III was first detected in newly hatched squid and was restricted to the liver. No mRNA signals were detected in other tissues even in adult squids. At the protein level, both isoforms were prominent in the liver of embryos and later in digestive organs of adult squid. Both isoforms were also detected in muscle tissues, including mantle and tentacle muscle. Staining for ALSM III was also identified in the iris and in tissues near the eye in squid embryos. However, no reactive bands were detected by immunoblotting of adult squid eyes. Thus, ALSM is initially expressed at the late stage of embryogenesis in spear squid, and expression is restricted to the liver. Thereafter, ALSM isoforms function in various tissues in an isoform-dependent manner.

Nucleotide sequence of phospholipase A(2) gene expressed in snake pancreas reveals the molecular evolution of toxic phospholipase A(2) genes.

We have cloned two phospholipase A(2) (PLA(2)) DNA complementary to RNA that contained nucleotide sequences encoding pancreas loop by the reverse transcription-polymerase chain reaction cloning procedure using messenger RNA isolated from Laticauda semifasciata pancreas. Additionally, a gene clone encoding PLA(2) with the pancreatic loop sequence was isolated from a L. semifasciata genomic library. Subsequent sequence analysis revealed that PLA(2) clones encoding group IB" PLA(2). Comparative analysis of group IA and IB" PLA(2) genes revealed that the exon-intron organization is conserved in the genes of both groups. The invaded sequences in the second intron were very similar to those of the L. semifasciata group IA gene. This observation suggested that the integration of the invaded sequences occurred before the divergence of groups IA and IB" during the evolution of PLA(2) gene. The comparative analysis revealed that the arising of group IA PLA(2) occurred by the deletion and substitution of nucleotide sequences in exon III region during the process of accelerated evolution.

Comparative analysis of gene expression mechanisms between group IA and IB phospholipase A2 genes from sea snake Laticauda semifasciata.

Phospholipase A(2) (PLA(2)) genes expressed in the venom glands of the sea snake, Laticauda semifasciata, were investigated. Both mRNAs, encoding group IA (without a pancreatic loop) and group IB (with pancreatic loop), were detected from venom glands by Northern blot hybridization analysis and RT-PCR. The results of quantitative PCR analysis indicated that the expression amount of group IA genes was around 100-300 times greater than that of group IB genes. Sequence analysis of 5'-upstream regions and a reporter gene assay of the genes (groups IA and IB) previously cloned showed that the functional sequence (411 bp) was inserted in the 5'-flanking region of the group IA PLA(2) genes. It seemed that the contribution of the inserted sequence to the amount of transcribed mRNAs was greater than that of number of genes present in the genome. Comparative analysis of the 5'-flanking sequences from several snake genes encoding toxic PLA(2)s revealed that this sequence was probably inserted into an ancestral gene of PLA(2) with a pancreatic loop. After the duplication of the gene, which contained the inserted sequence, the PLA(2) gene without a pancreatic loop evolved from one of the duplicate genes. This inserted sequence might determine the future of the genes expressed in the venom glands.

Antimicrobial action of achacin is mediated by L-amino acid oxidase activity.

Achacin is an antibacterial glycoprotein purified from the mucus of the giant snail, Achatina fulica Férussac, as a humoral defense factor. We showed that achacin has L-amino acid oxidase activity and can generate cytotoxic H(2)O(2); however, the concentration of H(2)O(2) was not sufficient to kill bacteria. The antibacterial activity of achacin was inhibited by various H(2)O(2) scavengers. Immunochemical analysis revealed that achacin was preferentially bound to growth-phase bacteria, accounting for the important role in growth-phase-dependent antibacterial activity of achacin. Achacin may act as an important defense molecule against invading bacteria.

Umbilical cord blood dendritic cells are a rich source of soluble HLA-DR: synergistic effect of exosomes and dendritic cells on autologous or allogeneic T-Cell proliferation.

In this study, we compared soluble HLA-DR (sHLA-DR) production in the culture supernatants of various cell sources [T and B cells, monocytes and dendritic cells (DCs) either from adult peripheral blood (PB) or umbilical cord blood (UCB)]. DCs produced the highest amount of sHLA-DR molecules as compared to other cell sources, with UCB DCs producing the highest amount. Different kinetics of sHLA-DR production were found between immature and mature UCB DCs (mDC, iDC) (derived either from CD34(+) or CD14(+) cells). Maximum production of sHLA-DR was observed in 72-hour culture supernatants of both CD34- and CD14-derived mDCs, whereas it peaked in the 24-hour culture supernatants from iDC. sHLA-DR molecules were pelleted after sequential centrifugation from UCB CD34(+) DCs and were found to contain both 36 kD alpha-chain and 29 kD beta-chain of HLA-DR, CD86, and Fas molecules. These sHLA-DR containing vesicles/exosomes alone evoked weak proliferative responses from autologous and allogeneic T cells, but the immune response was significantly increased when vesicles/exosomes were presented with DCs.

Human umbilical cord blood NK T cells kill tumors by multiple cytotoxic mechanisms.

Natural killer (NK) T cells are restricted by CD1d and play an important role in the rejection of malignant tumors, but how kill these tumors is unclear. To investigate this, we cultured Valpha24+CD4+ NK T cells in human umbilical cord blood, which was enriched by immunomagnetic beads. In short-term (4 h) cytotoxicity assays, the NK T cells could kill only those targets expressing CD1d. In longer cytotoxicity assays (20 h), however, the NK T cells were able to kill all the tumors, regardless of CD1d expression. When each of the perforin, Fas-FasL, and TNF-alpha cytotoxic mechanisms were blocked, it was apparent that perforin killing dominated in both the short- and long-term assays. In the short-term assay, perforin killing required that the targets expressed CD1d, but killing was more efficient if Fas was present because then the Fas-FasL mechanism was also used. Thus, cells that lacked Fas and CD1d and were not killed in the 4-h assay, were instead lysed in 20-h assay through a combination of perforin and TNF-alpha killing. NK T cells can kill tumor targets by perforin, Fas-FasL, and TNF-alpha mechanisms. TNF-alpha killing requires longer contact between effectors and targets, suggesting that TNF-alpha acts by enhancing perforin killing.

The in vitro generation of Ph1+ ALL-specific HLA-A24-restricted cytotoxic T lymphocytes using a synthetic 16 mer minor bcr-abl peptide.

Sixteen mer peptide, which spans the junctional region of the acute lymphoid leukemia (ALL)-specific minor bcr-abl fusion protein and contains a motif that can bind to human leukocyte antigen (HLA)-A24, was constructed. We tried to generate Philadelphia chromosome 1 (Ph1) positive ALL-specific cytotoxic T lymphocytes (CTLs) from eight normal HLA-A24+ individuals with peptide-pulsed autologous dendritic cells (DCs). CTLs could be generated from the mononuclear cells (MNCs) of a single donor, which could kill peptide-pulsed autologous DCs and two A24+ ALL lines, while an HLA-A24+ CML line was only weakly killed and unpulsed DCs or the control lines Daudi or K562 were not recognized. Those CTLs consisted predominantly of CD8+ T cells whose cytotoxicity could be neutralized by monoclonal antibodies to HLA-class I or HLA-A24, and also produced interferon (IFN)-gamma after being stimulated with peptide-pulsed DCs.

Cloning and biochemical characterization of astacin-like squid metalloprotease.

We have cloned four cDNAs encoding astacin-like squid metalloproteases (ALSMs)-I and -II from the Japanese common squid and ALSMs-I and -III from the spear squid. Analysis of the deduced amino acid sequences revealed that ALSMs possess a signal peptide and a pro-sequence followed by an astacin-like catalytic domain and an MAM (meprin, A5 protein, receptor protein-tyrosine phosphatase mu) domain. Phylogenetic analysis revealed that ALSM corresponds to a new cluster of astacins. To analyze the function of the MAM domain, wild-type ALSM and an MAM-truncated mutant were expressed in a baculovirus expression system. The expressed protein encoding full-length ALSM hydrolyzed myosin heavy chain as effectively as native ALSM, whereas the MAM-truncated mutant possessed no protease activity, suggesting that the MAM domain contributes to substrate recognition. ALSM has been isolated from squid liver and mantle muscle. However, analysis with a specific antibody generated against ALSM indicated the presence of ALSM in a wide variety of tissues. ALSM was located in the extracellular matrix of mantle muscle cells. Thus, ALSM is a secreted protease, as are other members of the astacin family. The extracellular localization raises the possibility of substrates other than myosin. The physiological role of ALSM remains unknown, at this time.

The generation of immunocompetent dendritic cells from CD34+ acute myeloid or lymphoid leukemia cells.

The ability of CD34+ leukemic cells to differentiate to dendritic cells (DCs) was investigated in 18 acute myeloid leukemia (AML) and 4 lymphoid leukemia (ALL) patients. The generation of DCs was determined by the expression of DC-associated CD1a or CD83 (more than 30%) with costimulatory molecules, by CD80 antigens (>20%), and by the exhibition of allostimulatory activity. In the AML patients, allostimulatory mature DCs were generated from 3 of 9 M0 or M1, 2 of 5 M2,2 of 4 M4 or M5, and 3 of 4 ALL (L2) cases. In total, DCs were more efficiently induced from cases expressing over 75% of CD34+ among whole bone marrow mononuclear cells (8 of 12), compared with those under 75% (2 of 10; P < .05). B-cell (CD19), natural killer (NK)-cell (CD56), or T-cell (CD7) lineage markers, which were aberrantly expressed on the blasts, were rarely found on leukemic DCs at the end of the culture period, and myeloid (CD13, CD33), not lymphoid (CD10), markers were shown on ALL-derived DCs. In Philadelphia chromosome-positive ALL or AML patients with t (8;21), DCs were confirmed to be of leukemic origin by fluorescence in situ hybridization analysis.

Clinical effects of infusing anti-Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocytes into patients with severe chronic active EBV infection.

Immune cell therapy with autologous Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocytes (CTLs) or lymphokine-activated killer (LAK) cells was performed in 2 adults with severe chronic active EBV infection (SCAEBV). The patient in case 1, who had complications of pancytopenia, high fever, and massive splenomegaly, was treated with 13 doses of LAK cell infusion followed by 4 doses of autologous CTL infusion. The patient in case 2, who had liver dysfunction due to natural killer cell-type infection, was treated with 4 doses of autologous CTL infusion. In case 1, the LAK cell infusions were effective in lowering the viral load and improving several biochemical parameters (lactate dehydrogenase, soluble interleukin 2 receptor) and resulted in complete amelioration of the high fever. Subsequent infusions of autologous CTLs reduced the viral load only temporarily and were accompanied by an increase in frequency of EBV-specific T-cells in the blood. However, the patient's main problem of pancytopenia was not resolved. In case 2, infusion of autologous CTLs did not improve the patient's hepatic dysfunction or viral load but caused a significant increase in autoantibody levels. Thus the effect of auto-CTL treatment was limited or deteriorative in SCAEBV patients.