Identification of the B Subtype of γ-Phospholipase A2 Inhibitor from Protobothrops flavoviridis Serum and Molecular Evolution of Snake Serum Phospholipase A2 Inhibitors

A cDNA encoding a novel phospholipase A₂ (PLA₂) inhibitor (PLI) was isolated from a Protobothrops flavoviridis snake (Tokunoshima island, Japan) liver cDNA library. This cDNA encoded a signal peptide of 19 amino acids followed by a mature protein of 181 amino acids. Its N-terminal amino acid sequence was completely in accord with that of a PLI, named PLI-II, previously found in P. flavoviridis serum. PLI-II showed a high similarity in sequence to the B subtype of γPLI, denoted γPLI-B, isolated from Agkistrodon blomhoffii siniticus serum. Thus, PLI-II is P. flavoviridis serum γPLI-B. Since PLI-I, previously isolated from P. flavoviridis serum, can be assigned as γPLI-A, P. flavoviridis serum contains both A and B subtypes of γPLI. Phylogenetic analysis of γPLIs from the sera of various kinds of snakes, Elapinae, Colubrinae, Laticaudinae, Acanthophiinae, Crotalinae, and Pythonidae, based on the amino acid sequences revealed that A and B subtypes of γPLIs are clearly separated from each other. It was also found that phylogenetic topologies of γPLIs are in good agreement with speciation processes of snakes. The BLAST search followed by analyses with particular Internet search engines of proteins with Cys/loop frameworks similar to those of PLI-II and PLI-I revealed that γPLI-Bs, including PLI-II and PLI-II-like proteins from mammalian sources, form a novel PLI-II family which possesses the common Cys/loop frameworks in the anterior and posterior three-finger motifs in the molecules. Several lines of evidence suggest that PLI-II is evolutionarily ancestral to PLI-I. The nucleotide sequence reported in this paper is available from the GenBank/EMBL/DDBJ databases under accession number AB290845.     

Optimization of γ-polyglutamic acid production byBacillus subtilis ZJU-7 using a surface-response methodology

The components of the media used to elicit the biosynthesis of poly-γ-glutamic acid (γ-PGA) byBacillus subtilis ZJU-7 were investigated, particularly the carbon and nitrogen sources. Of the 7 carbon sources investigated, sucrose induced the highest rate of γ-PGA productivity; among the nitrogen sources, tryptone had the best effect for γ-PGA production. A 2⁶⁻² fractional factorial design was used to screen factors that influence γ-PGA production significantly, and a central composite design was finally adopted to formulate the optimal medium. γ-PGA productivity improved approximately 2-fold when the optimal medium was used compared with the original nonoptimized medium, and volumetric productivity reached a maximum of 58.2 g/L after a 24-h cultivation period.     

Effect of interferon-γ on the susceptibility to Fas (CD95/APO-1)-mediated cell death in human hepatoma cells

Many tumors, including hepatocellular carcinomas (HCCs), resist Fas-mediated cell death, which is one of the effector mechanisms in the host's anti-tumor response; however, this resistance can be abolished by interferon-γ (IFN-γ). IFN-γ may sensitize Fas-mediated cell death in several ways, but the exact mechanism in HCCs is uncertain. In this study, we thoroughly investigated the effect of IFN-γ on the susceptibility of one human normal liver cell line and 12 HCC cell lines to Fas-mediated cell death. We also investigated the effect of IFN-γ on the expression of various apoptosis-related genes such as the Fas/TNF-related genes, the bcl-2 family, and the caspase family of genes. Although most cell lines showed considerable constitutive expression of Fas, all tested cell lines resisted Fas-mediated cell death without IFN-γ. When cells were pretreated with IFN-γ, only three cell lines were made significantly susceptible to Fas-mediated cell death (SNU-354, SNU-387 and SNU-423); the other 10 cell lines were not affected. IFN-γ increased the mRNA expression of Fas, TRAIL and caspase-1, and surface Fas was also increased. The strongly sensitized cell lines (SNU-354, SNU-387 and SNU-423) showed a particularly potent increment in surface Fas after IFN-γ treatment (increase in surface Fas >1.7-fold). This result enabled us to conclude that a potent increment of surface Fas expression is a major sensitizing mechanism of IFN-γ. We conclude that IFN-γ cannot play a sensitizing role in most HCC cell lines and that IFN-γ makes HCC cells susceptible to Fas-mediated cell death through a marked up-regulation of surface Fas in some HCC cells. Received: 3 August 2000 / Accepted: 24 November 2000     

Resistance to the proapoptotic effects of interferon-gamma on melanoma cells used in patient-specific dendritic cell immunotherapy is associated with improved overall survival

The use of whole cell tumor vaccines and various means of loading antigen onto dendritic cells have been under investigation for over a decade. Induction of apoptosis and the exposure of immune-stimulating proteins are thought to be beneficial for the use in immunotherapy protocols, but conclusive evidence in the clinical setting has been lacking. Incubation of melanoma cell lines with interferon-gamma (IFN-γ) increased phosphatidylserine and calreticulin exposure, but not in the IFN-γ-resistant cell line Lu-1205. Short-term autologous melanoma cell lines used for loading dendritic cells for immunotherapy showed differential response to the pro-apoptotic effects of IFN-γ. These IFN-γ-treated tumor cells (TCs) were irradiated and used for loading antigen for dendritic cell therapy. A log-rank comparison of survival for patients whose TCs were found to be either sensitive (upregulated phosphatidylserine and calreticulin) or insensitive to IFN-γ revealed a strongly significant correlation to progression-free (p = 0.003) and overall survival (p = 0.002) favorably in those patients whose cell lines were resistant to the proapoptotic effect of IFN-γ. These results suggest that the use of IFN-γ in anti-melanoma dendritic cell-based immunotherapy may only be beneficial when the cells do not undergo apoptosis in response to IFN-γ and support the contention that the use of some apoptotic cells in vaccines may be detrimental.     

Enhanced CD95-mediated apoptosis contributes to radiation hypersensitivity of <Emphasis Type="Italic">NBS</Emphasis> lymphoblasts

The molecular causes for enhanced radiosensitivity of Nijmegen Breakage Syndrome cells are unclear, especially as repair of DNA damage is hardly impeded in these cells. We clearly demonstrate that radiation hypersensitivity is accompanied by enhanced γ-radiation-induced apoptosis in NBS1 deficient lymphoblastoid cell lines. Differences in the apoptotic behavior of NBS1 ^−/− and NBS1 ^+/− cells are not due to an altered p53 stabilization or phosphorylation in NBS1 ^−/− cells. γ-radiation-induced caspase-8 activity is increased and visualization of CD95 clustering by laser scanning microscopy shows a significant higher activation of the death receptor in NBS1 ^−/− cells. Further investigation of the molecular mechanisms reveals a role for reactive oxygen species-triggered activation of CD95. These results demonstrate that NBS1 suppresses the CD95 death receptor-dependent apoptotic pathway after γ-irradiation and evidence is given that this is achieved by regulation of the PI3-K/AKT survival pathway.     

Pioglitazone, a synthetic ligand for PPARγ, induces apoptosis in RB-deficient human colorectal cancer cells

No published data are available about the expression of peroxisome proliferator-activated receptor γ (PPARγ) and the role of PPARγ in retinoblastoma protein (RB)-deficient human colorectal cancer (CRC) cells (SNU-C4 and SNU-C2A). Our aim was to investigate whether PPARγ is expressed in SNU-C4 and SNU-C2A cells and to elucidate possible molecular mechanisms underlying the effect of pioglitazone, a synthetic ligand for PPARγ, on cell growth in these cell lines. RT-PCR and Western blot analysis showed that both human CRC cell lines expressed PPARγ mRNA and protein. Pioglitazone inhibited the cell growth of both cell lines through G2/M phase block and apoptosis. In addition, pioglitazone caused a down-regulation of the X chromosome-linked inhibitor of apoptosis (XIAP), Bcl-2, and cyclooxygenase-2 (COX-2) under conditions leading to PPARγ down-regulation. These results suggest that pioglitazone may have therapeutic relevance or significance in the treatment of human CRC, and the down-regulation of XIAP, Bcl-2, and COX-2 may contribute to pioglitazone-induced apoptosis in these and other RB-deficient cell lines and tumors.     

Nitration of γ-tocopherol in plant tissues

Nitration of γ-tocopherol has been suggested to be an important mechanism for the regulation and detoxification of reactive nitrogen oxide species in animal tissues. To investigate whether this reaction does also occur in plants, reversed phase high-performance liquid chromatography (HPLC) and mass spectrometry (LC-MS) were used for analysis of 5-nitro-γ-tocopherol (5-NγT) in leaves and seeds. 5-nitro-γ-tocopherol (5-NγT) could be detected in an in vitro system where it was most likely generated by the reaction of γ-tocopherol with a nitric oxide radical. In vivo 5-NγT was identified in leaves of the Arabidopsis mutant line (vte4), which has insertion in the gene encoding γ-tocopherol methyltransferase and consequently lacks α-tocopherol and accumulates high levels of γ-tocopherol. Quantification of NO_x in leaves revealed that the vte4 mutant in comparison to wild type and the mutant vte1, which does not contain any tocopherol, has a reduced NO_x concentration. The level of 5-NγT in leaves of the vte4 mutant was shown to depend on the developmental stage and on the duration of light exposure. 5-NγT was also detectable in germinating seeds of Brassica napus, Nicotiana tabacum and Arabidopsis thaliana. These seeds have in common high γ-tocopherol contents. The rate of germination at two days after imbibition inversely correlated with the γ-tocopherol content of the seeds. The result suggests that γ-tocopherol or its respective derivative, 5-NγT, may prolong early development by reducing the level of NO_x.     

Regulation of proliferation of UM-SCV-1A and UM-SCV-6 vulvar carcinoma cells by cytokines

 The biology and pathogenesis of vulvar carcinoma are poorly understood at present. In order to understand this disease better, we have used recently developed squamous cell carcinoma lines of the vulva as models. Two cell lines originating from two individuals (UM-SCV-1A and UM-SCV-6) were cultured in vitro in 10% fetal calf serum. The effects of interleukins 10 and 13, interferons α and γ, granulocyte/macrophage-growth-stimulating factor (GM-CSF), tumor necrosis factor α (TNFα), and transforming growth factor β (TGFβ) on the proliferation of the cells was investigated by using radioactively labelled uridine as tracer. In addition, an investigation on the molecular structure of extracted cellular DNA was carried out to investigate whether programmed cell death (apoptosis) would be inducible by any of the factors. In UM-SCV-1A cells, interleukin-10 (IL-10) and interleukin-13 (IL-13) caused an approximately 12-fold decrease in DNA synthesis in cells cultured for 72 h (P<0.001), while GM-CSF had no significant effect. TGFβ showed a significant inhibitory effect on deoxyuridine incorporation (P<0.001), which was 2.0- and 4.2-fold at 48 h and 72 h, respectively. TFGα showed a 1.2-fold inhibitory effect on DNA synthesis at 48 h (P<0.01) and a 1.5-fold inhibition at 72 h (P<0.05). Interferon γ (IFNγ) showed an inhibitory effect on DNA synthesis (1.3-fold; P<0.01). In UM-SCV-6 cells, both IL-10 and IL-13 showed inhibitory effects on deoxyuridine incorporation (1.3- and 1.4-fold at 48 h, respectively; P<0.001) that were even more pronounced at 72 h (2.4- and 2.5-fold respectively; P<0.001). IFNγ caused a 3.6-fold inhibition of DNA synthesis by UM-SCV-6 cells at 72 h (P<0.001). Both TFGβ and TNFα inhibited uridine incorporation (3.0- and 1.6-fold at 48 h, respectively; 2.7-fold at 72 h for both factors). GM-CSF inihibited DNA synthesis by UM-SCV-6 cells 1.3- 2.0-fold at 48 h and 72 h, respectively. In dose/response analyses, the effect of INFα on DNA synthesis was inhibitory in both cell lines at 48 h, while stimulatory effects were observed at 72 h. Electrophoretic analyses of DNA isolated from cells cultured in the presence or absence of different factors did not reveal DNA fragmentation. All cytokines, with the exception of IFNα, showed inhibitory effects on DNA synthesis by vulvar carcinoma cells. Of the factors studied, the recently described interleukins 10 and 13 showed potent inhibition of cell growth, encouraging further investigation on the molecular mechanisms of the observed inhibition. Apoptosis does not seem to be induced in the two vulvar carcinoma cell lines by any of the cytokines studied. Received: 26 March 1996 / Accepted: 5 December 1996     

Engineering tocopherol biosynthetic pathway in lettuce

In order to increase tocopherol content, genes encoding Arabidopsis homogentisate phytyltransferase (HPT) and γ-tocopherol methyltransferase (γ-TMT) were constitutively over-expressed in lettuce (Lactuca sativa L. var. logifolia), alone or in combination. Over-expression of hpt could increase total tocopherol content, while over-expression of γ-tmt could shift tocopherol composition in favor of α-tocopherol. Transgenic lettuce lines expressing both hpt and γ-tmt produced significantly higher amount of tocopherol and elevated α-/γ-tocopherol ratio compared with non-transgenic control and transgenic lines harboring a single gene (hpt or γ-tmt). The best line produced eight times more tocopherol than the non-transgenic control and more than twice than hpt single-gene transgenic line.     

γ<Subscript>1</Subscript>-Dependent Down-regulation of Recombinant Voltage-gated Ca<Superscript>2+</Superscript> Channels

(1) Voltage-gated Ca²⁺ (Ca_V) channels are multi-subunit membrane complexes that allow depolarization-induced Ca²⁺ influx into cells. The skeletal muscle L-type Ca_V channels consist of an ion-conducting Ca_V1.1 subunit and auxiliary α₂δ−1, β₁ and γ₁ subunits. This complex serves both as a Ca_V channel and as a voltage sensor for excitation–contraction coupling. (2) Though much is known about the mechanisms by which the α₂δ−1 and β₁ subunits regulate Ca_V channel function, there is far less information on the γ₁ subunit. Previously, we characterized the interaction of γ₁ with the other components of the skeletal Ca_V channel complex, and showed that heterologous expression of this auxiliary subunit decreases Ca²⁺ current density in myotubes from γ₁ null mice. (3) In the current report, using Western blotting we show that the expression of the Ca_V1.1 protein is significantly lower when it is heterologously co-expressed with γ₁. Consistent with this, patch-clamp recordings showed that transient transfection of γ₁ drastically inhibited macroscopic currents through recombinant N-type (Ca_V2.2/α₂δ−1/β₃) channels expressed in HEK-293 cells. (4) These findings provide evidence that co-expression of the auxiliary γ₁ subunit results in a decreased expression of the ion-conducting subunit, which may help to explain the reduction in Ca²⁺ current density following γ₁ transfection.     

γ-Glutamylcysteine synthetase and γ-glutamyl transferase as differential enzymatic sources of γ-glutamylpeptides in mice

Some γ-glutamylpeptides in blood plasma are putative biomarkers for pathological conditions of the liver. γ-Glutamyltransferase (GGT) and γ-glutamylcysteine synthetase (γ-GCS) are two such potential enzymes that are responsible for the production of γ-glutamylpeptides. GGT produces γ-glutamylpeptides by transferring the γ-glutamyl moiety from glutathione to an amino acid or a peptide. γ-GCS normally catalyzes the production of γ-glutamylcysteine from glutamate and cysteine in the glutathione-synthesizing reaction, but other amino acids can also serve as an acceptor of a γ-glutamyl group, thus resulting in the formation of a variety of γ-glutamylpeptides. Based on liquid chromatography–mass spectrometry analyses, we observed differences in the distribution of γ-glutamylpeptides between the liver and kidney and were able to measure the activities of γ-GCS as well as the GGT reactions by quantifying the resulting γ-glutamylpeptides. The enzymatic characterization of γ-GCS in liver homogenates indicated that several γ-glutamylpeptides including γ-glutamyltaurine are actually produced. Cys showed the lowest Km value (0.06 mM) while other amino acids had much higher Km values (ranging from 21 to 1800 mM). The moderate Km values for these amino acids suggest that they were not the preferred amino acids in this conversion but were utilized as acceptor substrates for the production of the corresponding γ-glutamylpeptides by the γ-GCS reaction under Cys-deficient conditions. Thus, the production of these γ-glutamylpeptides by γ-GCS is directly correlated with a low Cys content, suggesting that their measurement in blood plasma could be useful for predicting the presymptomatic disease state of the liver with a defect in GSH redox balance.     

Differential blocking actions of 4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) and γ-hexachlorocyclohexane (γ-HCH) on γ-aminobutyric acid- and glutamate-induced responses of American cockroach neurons

4′-Ethynyl-4-n-propylbicycloorthobenzoate (EBOB) has been employed extensively as a radioligand in binding assays to evaluate the pharmacology of γ-aminobutyric acid (GABA)-gated Cl⁻ channels (GABARs) of insects and mammals, and γ-hexachlorocyclohexane (γ-HCH) was used as an insecticide targeting insect GABARs. Since recent studies have shown that not only GABARs but also glutamate-gated chloride channels (GluCls) are blocked by picrotoxinin, dieldrin and fipronil, the actions of EBOB and γ-HCH on native GABARs and GluCls of terminal abdominal ganglion neurons in American cockroach (Periplaneta americana) were tested using patch-clamp electrophysiology. A marked run-down of the GABA- and glutamate-induced responses of the cockroach neurons occurred, when a standard pipette solution was employed, but addition of pyruvate to the solution permitted stable recordings of these responses. With this solution, EBOB and γ-HCH were found to block not only the GABA- but also glutamate-gated responses, with the actions augmented by repeated co-application with the agonists. It was also found that prolonged pre-application of EBOB and γ-HCH prior to co-application with GABA and glutamate resulted in enhanced blocking actions, indicating resting-state actions of the blockers. The blocking actions of EBOB and γ-HCH on the GABA- and glutamate-induced responses were compared by determining IC₅₀ values under steady state condition. The IC₅₀ values for the actions of EBOB on GABAR and GluCls differed less than those of γ-HCH.     

cDNA cloning and functional characterization of Xenopus laevis DNase γ

We report here the cDNA cloning and functional analysis of Xenopus DNase γ (xDNase γ). Two forms of cDNAs are isolated from adult spleen: one composing a 933 bp open reading frame for the enzymatically active xDNase γ protein, and the other encoding an inactive short alternative form. Northern blot analysis revealed that the xDNase γ mRNA is expressed in spleen, liver, testis, and ovary. xDNase γ expression is scarcely detected in the tail muscle of tadpoles; however, it increases during metamorphosis and reaches a maximum during the late metamorphic climax. The ectopic expression of xDNase γ results in the appearance of extensive DNA fragmentation in C2C12 myoblasts after the induction of apoptosis. In contrast, Xenopus DNase I fails to induce apoptotic DNA ladder formation under the same conditions. Our results suggest a possible involvement of xDNase γ in apoptosis during amphibian metamorphosis. The nucleotide sequence of Xenopus DNase γ has been submitted to DDBJ/EMBL/GenBank database under the accession number AF059612     

The PH domain of Ras-GAP is sufficient forin Vitro binding toβγ subunits of heterotrimeric G proteins

Summary 1. The noncatalytic domain of Ras-GAP can affect signaling through G protein-coupled receptors by a poorly understood mechanism. 2. In this study, fusion proteins containing elements of the noncatalytic domain ofras-GAP were examined for their ability to bindβγ subunits of heterotrimeric G proteins and phosphotyrosine-containing polypeptides. 3. Our results demonstrate that purifiedβγ dimers associated with bacterially expressed GAP proteins and that this association does not require SH2 or SH3 domains but is dependent on the presence of the GAP pleckstrin-homology (PH) domain. In contrast, only the SH2 domains are necessary for binding to tyrosine phosphorylated proteins. 4. These findings raise the possibility that heterotrimeric G proteins might affect functioning ofras-like proteins throughβγ subunits acting on their regulatory molecules.     

Evolutionary Relationship Between Translation Initiation Factor eIF-2γ and Selenocysteine-Specific Elongation Factor SELB: Change of Function in Translation Factors

Eubacterial and eukaryotic translation initiation systems have very little in common, and therefore the evolutionary events that gave rise to these two disparate systems are difficult to ascertain. One common feature is the presence of initiation, elongation, and release factors belonging to a large GTPase superfamily. One of these initiation factors, the γ subunit of initiation factor 2 (eIF-2γ), is found only in eukaryotes and archaebacteria. We have sequenced eIF-2γ gene fragments from representative diplomonads, parabasalia, and microsporidia and used these new sequences together with new archaebacterial homologues to examine the phylogenetic position of eIF-2γ within the GTPase superfamily. The archaebacterial and eukaryotic eIF-2γ proteins are found to be very closely related, and are in turn related to SELB, the selenocysteine-specific elongation factor from eubacteria. The overall topology of the GTPase tree further suggests that the eIF-2γ/SELB group may represent an ancient subfamily of GTPases that diverged prior to the last common ancestor of extant life. Received: 2 January 1998 / Accepted: 1 June 1998     

Expression of maize γ-zein and β-zein genes in transgenic Nicotiana tabacum and Lotus corniculatus

Accumulation of zeins, the endosperm storage proteins of maize, in a heterologous plant expression system was attempted. Plants of Nicotiana tabacum and Lotus corniculatus were transformed by Agrobacterium with binary vectors harbouring genes that code for γ-zein and β-zein, two zeins rich in sulphur amino acids. Adding the ER retention signal KDEL to the C-terminal domain modified the zein polypeptides. Significant levels of γ-zein:KDEL and β-zein:KDEL were detected in primary transformants of tobacco. Moreover, the two zeins colocalized in leaf protein bodies of γ-/β-zein:KDEL plants derived from a cross between two primary transformants. Coexpression of γ-zein:KDEL and β-zein:KDEL could be a useful strategy to obtain genotypes of forage legumes which are able to accumulate sulphur amino acids to high levels. As a first step, L. corniculatus plants expressing γ-zein:KDEL in the leaves were obtained. This revised version was published online in June 2006 with corrections to the Cover Date.     

A Combined Refolding Technique for Recombinant Human Interferon-γ Inclusion Bodies by Ion-exchange Chromatography with a Urea Gradient

Summary A refolding strategy was described for on-column refolding of recombinant human interferon-γ (rhIFN-γ) inclusion bodies by ion-exchange chromatography (IEC). The rhIFN-γ was expressed in E. colias inclusion bodies. Triton X-100 was used first to wash the rhIFN-γ inclusion bodies before chromatographic refolding. The refolding process was performed by gradually decreasing the concentration of urea in the column after the denatured rhIFN-γ protein had bound onto the ion-exchange gel SP-Sepharose Fast Flow. The refolding and purification process for the denatured rhIFN-γ was carried through simultaneously and the purity of the refolded rhIFN-γ was up to 95%. The effects of protein loading, flow rate, urea gradient length and final urea concentration on the refolding were investigated in detail. Under the optimum conditions, the specific activity of rhIFN-γ was up to 7.5 × 10⁵ IU mg⁻¹and active protein recovery was up to 54%.     

Potent isozyme-selective inhibition of human glutathione S-transferase A1-1 by a novel glutathione S-conjugate

Summary. Elevated levels of glutathione S-transferases (GSTs) are among the factors associated with an increased resistance of tumors to a variety of antineoplastic drugs. Hence a major advancement to overcome GST-mediated detoxification of antineoplastic drugs is the development of GST inhibitors. Two such agents have been synthesized and tested on the human Alpha, Mu and Pi GST classes, which are the most representative targets for inhibitor design. The novel fluorescent glutathione S-conjugate L-γ-glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (4) has been found to be a highly potent inhibitor of human GSTA1-1 in vitro (IC₅₀=0.11±0.01 μM). The peptide is also able to inhibit GSTP1-1 and GSTM2-2 isoenzymes efficiently. The backbone-modified analog L-γ-(γ-oxa)glutamyl-(S-9-fluorenylmethyl)-L-cysteinyl-glycine (6), containing an urethanic junction as isosteric replacement of the γ-glutamyl-cysteine peptide bond, has been developed as γ-glutamyl transpeptidase-resistant mimic of 4 and evaluated in the same inhibition tests. The pseudopeptide 6 was shown to inhibit the GSTA1-1 protein, albeit to a lesser extent than the lead compound, with no effect on the activity of the isoenzymes belonging to the Mu and Pi classes. The comparative loss in biological activity consequent to the isosteric change confirms that the γ-glutamyl moiety plays an important role in modulating the affinity of the ligands addressed to interact with GSH-dependent proteins. The new specific inhibitors may have a potential in counteracting tumor-protective effects depending upon GSTA1-1 activity.