Involvement of tumor necrosis factor-related apoptosis-inducing ligand in NK cell-mediated and IFN-gamma-dependent suppression of subcutaneous tumor growth

Natural killer (NK) cells and interferon- (IFN) gamma have been implicated in immune surveillance against tumor development. Here we show tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is a type II membrane protein belonging to the TNF family and plays a critical role in the NK cell-mediated and IFN-gamma-dependent suppression of subcutaneous growth of TRAIL-sensitive tumors. Administration of a neutralizing monoclonal antibody against TRAIL promoted outgrowth of subcutaneously inoculated TRAIL-sensitive tumors (L929, LB27.4, and Renca) but not TRAIL-resistant tumors (P815 and B16). Such a protective effect of TRAIL against TRAIL-sensitive tumors was abrogated in NK cell-depleted or IFN-gamma-deficient mice. These results suggested a substantial role of TRAIL as the effector molecule that eliminates subcutaneously developing TRAIL-sensitive tumors.     

Efficient production of desulfurizing cells with the aid of expert system

An expert system was used to achieve the high production of desulfurizing cells of Rhodococcus erythropolis KA 2-5-1. By adding a proper amount of sulfur containing component with the aid of the expert system, we could avoid excess feeding which resulted in the lowering of desulfurizing activity and starvation which caused serious damage to cell growth. In order to determine the addition amount by the expert system, the data of the amount of chemical elements contained in the cells were used as a reference for comparison with the medium components present. Culture experiments were carried out using a 5l jar fermentor with several kinds of media whose components were determined based on the inferred results with the aid of the expert system. We could restrict the amount of the sulfur component addition so that sulfur was a growth-limiting factor; in contrast, the amounts of other elements were sufficient for growth.As a result, the maximum specific production rate of 2-hydroxy biphenyl (2HBP) and the maximum cell concentration were 20mmolkg-drycells(-1)h(-1) and of 45g-drycellsl(-1), respectively. At 100h of cultivation, 1g/l of dibenzothiophene (DBT) was converted to 2HBP within 20h, i.e., 10mmolkg-drycells(-1)h(-1) of specific desulfurization activity, and the specific activity remained stable for a long period in the culture experiment.     

Cloning and sequence analysis of cDNA coding for a lectin from Helianthus tuberosus callus and its jasmonate-induced expression

Two lectins (designated as HTA I and HTA II) that seemed to be isolectins were found in Helianthus tuberosus callus. cDNA encoding HTA I was isolated from a ZAP Express expression library by immunoselection by using the anti-HTA antiserum. The sequence of this cDNA consisted of 432 bp nucleotides coding for a polypeptide of 143 amino acid residues (Mr, 15,314). When introduced into E. coli, the cDNA directed the synthesis of active HTA I as indicated by the hemagglutination activity. The deduced amino acid sequence showed homology with some lectins and jasmonate-induced proteins. When callus was cultured in the presence of methyl jasmonate (MeJA), the hemagglutination activity increased in a dose-dependent manner. The levels of expression of the HTA protein and of the corresponding mRNA also increased in the treated callus. In view of these results, HTA I is considered to be a jasmonate-induced protein.     

Apoptosis induced by nicotinamide-related compounds and quinolinic acid in HL-60 cells

In our previous paper, we have reported that niacin-related compounds, particularly picolinic acid, dipicolinic acid, and isonicotinamide, induced apoptosis in HL-60 cells but that niacin did not. Moreover, picolinamide, N1-methylnicotinamide, 6-aminonicotinamide, quinolinic acid, and cinchomeronic acid also had the function of DNA fragmentation in HL-60 cells analyzed by flow cytometry, the ratio of DNA fragmentation finally being about 40% after treatment with these compounds at 10 mM for 24 h. In this study, we found that these compounds also induced apoptosis in HL-60 cells. The wide-spectrum caspase inhibitors prevented DNA fragmentation induced by these compounds. Interestingly, 6-aminonicotinamide induced apoptosis at a comparatively low concentration, while picolinic acid, dipicolinic acid, and isonicotinamide did not at 1 mM. Our results suggest that both NAD metabolism and NAD biosynthesis may be related to the process of apoptosis induced by niacin-related compounds.     

A simple and rapid method for the detection of poly(ADP-ribose) by flow cytometry

Measurement of poly(ADP-ribose) levels was performed by a new method using a monoclonal antibody against poly(ADP-ribose) and flow cytometry from small amount of cultured cells without the need for isolation of poly(ADP-ribose) polymer. The increase of poly(ADP-ribose)-associated fluorescence intensity was observed in individual human leukemic HL-60 cells when treated with the carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and was blocked by the treatment with 3-aminobenzamide before MNNG treatment. It is easy and rapid to detect the time-dependent change of poly(ADP-ribose) levels in HL-60 cells after MNNG treatment. We easily found that the increase of the poly(ADP-ribose) level in nicotinic acid-treated lymphocytes after MNNG treatment was observed, but not in nicotinamide-treated lymphocytes. We investigated the change of poly(ADP-ribose) levels especially in the early phase of apoptosis. Our method is simple and rapid. It is suggested that the investigation of poly(ADP-ribosyl)ation in various fields is possible by using this new method.     

Proteases are associated with a minor fucoxanthin chlorophyll a/c-binding protein from the diatom,Chaetoceros gracilis

We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a/c‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.     

Evolutionary appearance of the plasma membrane H+-ATPase containing a penultimate threonine in the bryophyte

The plasma membrane H⁺-ATPase provides the driving force for solute transport via an electrochemical gradient of H⁺ across the plasma membrane, and regulates pH homeostasis and membrane potential in plant cells. However, the plasma membrane H⁺-ATPase in non-vascular plant bryophyte is largely unknown. Here, we show that the moss Physcomitrella patens, which is known as a model bryophyte, expresses both the penultimate Thr-containing H⁺-ATPase (pT H⁺-ATPase) and non-pT H⁺-ATPase as in the green algae, and that pT H⁺-ATPase is regulated by phosphorylation of its penultimate Thr. A search in the P. patens genome database revealed seven H⁺-ATPase genes, designated PpHA (Physcomitrella patens H⁺-ATPase). Six isoforms are the pT H⁺-ATPase; a remaining isoform is non-pT H⁺-ATPase. An apparent 95-kD protein was recognized by anti-H⁺-ATPase antibodies against an isoform of Arabidopsis thaliana and was phosphorylated on the penultimate Thr in response to a fungal toxin fusicoccin and light in protonemata, indicating that the 95-kD protein contains pT H⁺-ATPase. Furthermore, we could not detect the pT H⁺-ATPase in the charophyte alga Chara braunii, which is the closest relative of the land plants, by immunological methods. These results strongly suggest the pT H⁺-ATPase most likely appeared for the first time in bryophyte.     

Temperature and pressure denaturation of chignolin: Folding and unfolding simulation by multibaric-multithermal molecular dynamics method

A multibaric‐multithermal molecular dynamics (MD) simulation of a 10‐residue protein, chignolin, was performed. All‐atom model with the Amber parm99SB force field was used for the protein and the TIP3P model was used for the explicit water molecules. This MD simulation covered wide ranges of temperature between 260 and 560 K and pressure between 0.1 and 600 MPa and sampled many conformations without getting trapped in local‐minimum free‐energy states. Folding events to the native β‐hairpin structure occurred five times and unfolding events were observed four times. As the temperature and/or pressure increases, fraction of folded chignolin decreases. The partial molar enthalpy change ΔH and partial molar volume change ΔV of unfolding were calculated as ΔH = 24.1 ± 4.9 kJ/mol and ΔV = ?5.6 ± 1.5 cm³/mol, respectively. These values agree well with recent experimental results. Illustrating typical local‐minimum free‐energy conformations, folding and unfolding pathways were revealed. When chignolin unfolds from the β‐hairpin structure, only the C terminus or both C and N termini open first. It may undergo an α‐helix or 3₁₀‐helix structure and finally unfolds to the extended structure. Difference of the mechanism between temperature denaturation and pressure denaturation is also discussed. Temperature denaturation is caused by making the protein transferred to a higher entropy state and making it move around more with larger space. The reason for pressure denaturation is that water molecules approach the hydrophobic residues, which are not well hydrated at the folded state, and some hydrophobic contacts are broken. Proteins 2012;. © 2012 Wiley Periodicals, Inc.