Bcl-2 protects cells from cytokine-induced nitric-oxide-dependent apoptosis

 Cytokine-mediated cell death in tumor cells can be achieved through endogenous nitric oxide (NO) from within tumor cells or exogenous NO from either activated macrophages or endothelial cells. The purpose of this study was to determine the role of Bcl-2 in NO-mediated apoptosis. The incubation of murine L929 and NIH3T3 cells with interleukin-1α (IL-1α) and interferon γ (IFNγ) induced high endogenous NO production only in the L929 cells that also underwent apoptosis. NIH3T3 cells were not resistant to NO-mediated apoptosis. In fact, the incubation of L929 and NIH3T3 cells with exogenous NO derived from NO donors, sodium nitroprusside, or S-nitroso-N-acetyl-DL-penicillamine (SNAP) induced death, characterized by typical apoptotic morphology and DNA fragmentation, in both cell types, but to a higher degree in NIH3T3 cells than in the L929 cells. We then measured the effect of Bcl-2 expression on exogenous NO-induced apoptosis. At both the mRNA and protein levels, L929 fibroblasts expressed higher levels of endogenous mouse Bcl-2 than did NIH3T3 cells. At the same time, L929 cells were much more resistant to exogenous NO-induced cell death than were NIH3T3 cells. The inverse correlation between mouse Bcl-2 expression and sensitivity to exogenous NO-mediated cell death was also found in the murine K-1735 melanoma C-23 and X-21 clonal populations. Transfection of both NIH3T3 cells and L929 cells with the human bcl-2 gene led to resistance to both exogenous and endogenous NO-mediated apoptosis. These data demonstrate that NO-mediated apoptosis can be suppressed by expression of Bcl-2, suggesting that abnormal expression of Bcl-2 may influence the efficacy of tumor immunotherapy. Received: 28 June 1998 / Accepted: 23 August 1996     

Interleukin-15 effectively potentiates the in vitro tumor-specific activity and proliferation of peripheral blood γδT cells isolated from glioblastoma patients

γδT cells play a regulatory role in both primary and metastatic tumor growth in humans. The mechanisms responsible for the activation and proliferation of circulating γδT cells should be fully understood prior to their adoptive transfer to cancer patients. We have examined in vitro functional effects of interleukin-15 (IL-15) on highly purified γδT cells isolated from glioblastoma patients. γδT cells constitutively express the heterotrimeric IL-2 receptor (IL-2R) αβγ, but the levels of IL-2Rβ or γ expression were not increased by incubation with saturating amounts of IL-15. IL-15 was shown to induce a maximal γδT cell proliferation, although at much higher concentrations (at least 2000 U/ml) than IL-2 (100 U/ml). Submaximal concentrations of IL-15 plus low concentrations of IL-2 produced an additive proliferative response. In contrast to the IL-2-induced response, this activity was completely or partially abrogated by anti-IL-2Rβ, or anti-IL-2Rγ antibodies, but not by anti-IL-2Rα antibodies. Incubation of γδT cells in the presence of IL-15 resulted not only in the appearance of NK and LAK activity, but also in specific autologous tumor cell killing activity, an additive effect being seen with IL-15 and IL-2. This IL-15-induced tumor-specific activity could be significantly blocked by anti-IL-2Rγ and anti-IL-2R-β mAb, but not by anti-IL-2Rα mAb. Thus, in contrast to IL-2, IL-15 activates tumor-specific γδT cells through the components of IL-2Rβ and IL-2Rγ, but not IL-2Rα. These enhanced in vitro tumor-specific and proliferative responses of γδT cells seen with IL-15 suggest a rational adjuvant imunotherapeutic use of γδT cells in cancer patients. Received: 23 January 1998 / Accepted: 20 May 1998     

Effects of Z-FA.FMK on d-galactosamine/tumor necrosis factor-alpha-induced kidney injury and oxidative stress in mice

Background The aim of this investigation was to demonstrate that benzyloxicarbonyl-l-phenylalanyl-alanine-fluoromethylketone (Z-FA.FMK), which is a pharmacological inhibitor of cathepsin B, has protective role on the kidney injury that occurs together with liver injury. Methods BALB/c male mice used in this study were divided into four groups. The first group was given physiologic saline only, the second group was administered Z-FA.FMK alone, the third group received d-galactosamine and tumor necrosis factor-alpha (d-GalN/TNF-α), and the fourth group was given both d-GalN/TNF-α and Z-FA.FMK. One hour after administration of 8 mg/kg Z-FA.FMK by intravenous injection, d-GalN (700 mg/kg) and TNF-α (15 μg/kg) were given by intraperitoneal injection. Results In the group given d-GalN/TNF-α, the following results were found: severe degenerative morphological changes in the kidney tissue, a significant increase in the number of activated caspase-3-positive tubular epithelial cell, an insignificant increase in the number of proliferating cell nuclear antigen (PCNA)-positive tubular epithelial cell, a decrease in the kidney glutathione (GSH) levels, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, an increase in the kidney lipid peroxidation (LPO) levels, lactate dehydrogenase (LDH) activity, serum aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities, uric acid and urea levels. In contrast, in the group given d-GalN/TNF-α and Z-FA.FMK, a significant decrease in the d-GalN/TNF-α-induced degenerative changes, a decrease in the number of activated caspase-3-positive tubular epithelial cell, a insignificant decrease in the number of PCNA-positive tubular epithelial cell, an increase in the kidney GSH levels, CAT, SOD and GPx activities, a decrease in the kidney LPO levels, LDH activity, serum AST and ALT activities, uric acid and urea levels were determined. Conclusion These results suggest that pretreatment with Z-FA.FMK markedly lessens the degree of impairment seen in d-GalN/TNF-α-induced kidney injury, which occurred together with liver injury in mice.     

Fidelity of a BAC-EGFP transgene in reporting dynamic expression of IL-7Rα in T cells

Interleukin-7 receptor α chain (IL-7Rα)-derived signals are critical for normal T cell development, mature T cell homeostasis, and longevity of memory T cells. IL-7Rα expression in T cells is dynamically regulated at different developmental and antigen-responding stages. However, the molecular mechanism underlying the dynamic regulation is not completely understood. Here we describe generation of a bacterial artificial chromosome (BAC)-based reporter transgenic mouse strain, which contains 210 kb DNA sequence flanking the Il7r locus. We used in vitro validated EGFP reporter and insulator sequences to facilitate the reporter transgene expression. Consistent with endogenous IL-7Rα expression, the BAC transgene was expressed in mature T cells, a portion of natural killer cells but not in mature B cells. In the thymus, the EGFP reporter and endogenous IL-7Rα showed synchronized silencing in CD4⁺CD8⁺ double positive stage, were both upregulated in CD4⁺ or CD8⁺ single positive thymocytes, and both continued to be co-expressed in na?ve T cells in the periphery. Upon encountering antigen, the antigen-specific effector CD8⁺ T cells downregulated both endogenous IL-7Rα and the EGFP reporter, which were upregulated in synchrony in antigen-specific memory CD8 T cells. These results indicate that the BAC-EGFP transgene reports endogenous IL-7Rα regulation with high fidelity, and further suggest that the 210 kb sequence flanking the Il7r locus contains sufficient genetic information to regulate its expression changes in T lineage cells. Our approach thus represents a critical initial step towards systematic dissection of the cis regulatory elements controlling dynamic IL-7Rα regulation during T cell development and cellular immune responses.     

Lipopolysaccharide Up-regulates IL-6Rα Expression in Cultured Leptomeningeal Cells via Activation of ERK1/2 Pathway

To clarify the response of leptomeningeal cells to immune stimulation, the effect of lipopolysaccharide (LPS) on expression of IL-6 receptors in the cultured leptomeningeal cells was investigated. The results showed that the expression of IL-6Rα was invisible in the purified leptomeningeal cells while it was seen in the cells when they were co-cultured with astrocytes. On the other hand, GP130 was moderately expressed in both conditions. Following incubation with different doses of LPS, IL-6Rα expression in purified leptomeningeal cells was increased in a time- and dose-dependent manner, while GP130 level remained unchanged. Concomitantly, phosphorylated ERK1/2 level was increased following LPS stimulation and its inhibition by PD98059 attenuated the LPS-induced increase of IL-6Rα expression. These data indicate that leptomeningeal cells can respond to immunogenic stimuli as manifested by expression of cytokine receptors. Moreover, ERK1/2 pathway seems to be involved in the process of LPS-induced IL-6Rα up-regulation in leptomeningeal cells.     

Lymphocyte subsets and T(h)1/T(h)2 immune responses in patients with adenocarcinoma of the oesophagus or oesophagogastric junction: relation to pTNM stage and clinical outcome

Introduction Recent studies have indicated that the cytokines produced by CD4⁺ T helper type 1 (T_h1) and type 2 (T_h2) cells are critically important in antitumour immunity and perhaps clinical outcome. From this perspective, we investigated the immunocompetence of patients with previously untreated cancer of the oesophagus or oesophagogastric junction (OGJ) in relation to stage of disease and postoperative survival.Methods Blood samples were taken prior to surgery from 32 patients with adenocarcinoma of the oesophagus or OGJ. Ten healthy volunteers served as normal controls. T-cell and monocyte subpopulations were determined using flow cytometry. Monocyte as well as T_h1- and T_h2-lymphocyte cytokine levels were assessed in stimulated whole blood cultures.Results Absolute T-cell and monocyte (subset) counts as well as monocyte cytokine levels were similar among patients and controls. Production of T_h1-type cytokines was higher in patients than in controls (IFN-, p=0.01; IL-2, p=0.05), whereas T_h2-type cytokine levels were comparable (IL-4, p=0.5; IL-13, p=0.3). T-cell CD4⁺/CD8⁺ ratios decreased as pTNM stage worsened (stage I/II vs stage III/IV, p=0.009). Of all measured immunological parameters, only IL-2 production significantly affected both overall survival (p=0.015) and disease-free survival (p=0.0062). High IL-2 levels corresponded with a favourable prognosis.Conclusions Patients awaiting surgery for adenocarcinoma of the oesophagus or oesophagogastric junction demonstrated a shift in the T_h1/T_h2 balance—in favour of T_h1—compared with healthy volunteers. The ability of T cells to produce IL-2 was related to survival indicating a crucial role of T_h1-type cells in antitumour immunosurveillance.     

A purified protein from Salmonella typhimurium inhibits proliferation of murine splenic anti-CD3 antibody-activated T-lymphocytes

Abstract In a previous study, we observed that the purified substance Salmonella typhimurium -derived inhibitor of T-cell proliferation (STI) had an immunosuppressive effect, demonstrated as the suppression of mitogenic lectin-induced proliferation of murine spleen cells. In the present study, we confirmed the immunosuppressive effect of STI, which suppressed the proliferation of murine splenic T-lymphocytes activated with the anti-CD3 antibody (Ab) and phorbol 12-myristate-13 acetate (PMA) and this phenomenon was accompanied by augmentation of interferon-γ (IFN-γ) secretion and inhibition of interleukin-2 (IL-2) secretion. Furthermore, the augmentation of IFN-γ secretion caused IL-2 receptor α chain (IL-2R α) over expression on T-cells. However, the addition of an anti-IFN-γ Ab and recombinant IL-2 (rIL-2) did not reverse the suppressed T-cell proliferation, although the level of IL-2R α expression on T-cells recovered to around normal. Furthermore, Western blotting using an anti-phosphotyrosine Ab showed that IL-2R-mediated tyrosine phosphorylation of protein substrates in T-cells was inhibited by incubation with STI for 48 h and this inhibition was not reversed by adding the anti-IFN-γ Ab and rIL-2. These results suggest that STI-induced suppression of T-cell proliferation involves a defect in IL-2R function and/or IL-2 signaling pathway in T-cells.     

Interleukin-2 enhances the growth of human melanoma cells derived form primary but not from metastatic tumours

Previously, we demonstrated that in vitro treatment of B16F10 murine melanoma cells with interleukin-2 (IL-2) enhances proliferation and metastasis. To further investigate the role played by IL-2 in human melanomas, we studied the expression of IL-2/IL-2 receptor and the effect of IL-2 on the proliferation of melanoma cell lines derived from primary (A375 and RMS cell lines) and metastatic (Hs294T cell line) tumours. We found a constitutive expression of cytoplasmic IL-2 and alpha, beta and gamma-subunits of the IL-2R on the surface of the three melanoma cell lines. The presence of IL-2 in the culture increased the proliferation rate in A375 and RMS cell lines, but no effect was observed in Hs294T metastatic cells. Biologically active IL-2 could be found in the supernatant of the three melanoma cell lines, particularly in A375 and RMS cells, in which an inhibition of the proliferation rate was observed when IL-2 was blocked. Moreover, the combination of anti-IL-2R beta and anti-IL-2R gamma blocking antibodies induced a significant down-regulation of cell proliferation in the three melanoma cell lines, and the combination of anti-IL-2R alpha, anti-IL-2R beta and anti-IL-2R gamma blocking antibodies inhibited IL-2-mediated growth stimulation in A375 and Hs294T cell lines. In RMS cells, a more significant effect was observed when only IL-2R gamma was blocked. Finally, exogenous IL-2 modulated the IL-2 endogenously produced by melanoma cells. These data show that IL-2 may modulate the growth of melanoma cells through autocrine or/and paracrine mechanisms.     

Human lymphokine-activated killer (LAK) cells: III. Effect ofl-phenylalanine methyl ester on LAK cell activation from human peripheral blood mononuclear cells: Possible protease involvement of monocytes,natural killer cells and LAK cells

Summary We have shown that depletion of monocytes from human peripheral blood mononuclear cells (PBMC) byl-phenylalanine methyl ester (PheOMe) enhanced lymphokine-activated killer cell (LAK) generation by recombinant interleukin-2 (rIL-2) at high cell density. In this study, we have investigated the mechanism of action of PheOMe on LAK activation by using trypsin, chymotrypsin, tosylphenylalaninechloromethanol (TPCK, a chymotrypsin inhibitor), tosyl-l-lysinechloromethane (TLCK, a trypsin inhibitor), phenylalaninol (PheOH), and benzamidine. PBMC were treated with 1–5 mM PheOMe for 40 min at room temperature in combination with the various agents, washed and assessed for their effects on natural killer (NK) activity against K562 cells and monocyte depletion. The treated cells were then cultured with or without rIL-2 for 3 days. LAK cytotoxicity was assayed against⁵¹Cr-labeled K562 and Raji tumor target cells. TPCK at 10 µg/ml partially inhibited depletion of monocytes by PheOMe. TLCK did not prevent depletion of monocytes nor inhibition of NK activity induced by PheOMe. TPCK and TLCK inhibited NK activity by themselves. TPCK but not TLCK inhibited rIL-2 induction of LAK cells. On the other hand, PheOH and benzamidine (analogs of PheOMe) lacked any effect on monocyte depletion but abrogated the inhibitory effect of PheOMe on NK activity. They had no effect on rIL-2 activation of LAK activity enhanced by PheOMe. Trypsin potentiated the inhibitory effect of PheOMe on NK activity and monocyte depletion. Trypsin partially inhibited IL-2 activation of LAK activity enhanced by PheOMe. Chymotrypsin had little effect on NK activity but prevented the inhibitory effect of PheOMe on NK activity. It had little effect on monocyte depletion induced by PheOMe. PheOMe was hydrolysed by monocytes and chymotrypsin to Phe and methanol as determined by HPLC. TPCK inhibited hydrolysis of PheOMe by monocytes. Our data suggest that the effects of PheOMe on monocytes, NK cells and LAK activation involve protease activities of monocytes.     

Superiority of the ear pinna over a subcutaneous tumour inoculation site for induction of a Th1-type cytokine response

 This study examines whether a correlation may be found between Th1- or Th2-type cytokine responses and resistance or susceptibility to tumour growth. Cytokine profiles were investigated in a well-defined mouse tumour model in which the injection site and the genetic background determine the phenotype of either tumour resistance or tumour susceptibility. DBA/2-derived ESb lymphoma variant cells with high metastatic capacity were inoculated into syngeneic mice either s.c., where they grow and metastasize, or into the ear pinna (i.e.), where they do not grow because of induction of protective immunity. Alternatively, the tumour cells were injected s.c. or i.e. into allogeneic B10.D2 mice, which are resistant to the tumour although they are identical at the MHC locus. Between 1 and 10 days after tumour cell injection the spleen-derived mRNA was tested for cytokine gene expression or the spleen cells were analysed by FACScan for T cell activation. The strongest cytokine response was observed in i.e. inoculated B10.D2 mice. This was characterized by an early (days 2–3) peak of interferon γ (INF-γ), interleukin-2 (IL-2), IL-2 receptor α (IL-2Rα) and IL-4. The cytokine mRNA response of i.e. inoculated DBA/2 mice was quite similar except that no IFN-γ could be detected. In s.c. inoculated B10.D2 mice, the IL-2, IL-2Rα and IFN-γ responses were weaker than after i.e. injection while the IL-4 response was comparable. The most striking difference between these cytokine profiles from tumour-resistant mice and those of s.c. inoculated tumour-susceptible DBA/2 mice was a delay in the latter in the IL-2, IL-2Rα and IFN-γ responses and the observation that the IL-4 response was not down-regulated. The persisting IL-4 response could down-regulate a Th1-type response and thereby explain tumour susceptibility as a consequence of host conditioning. Received: 4 September 1997 / Accepted: 2 October 1997     

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[³H]glucosamine, [³⁵S]sulfate, and L-[¹⁴C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-(3) and β-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[³H]glucosamine, but not of [³⁵S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[¹⁴C]leucine incorporation into total protein synthesis. These results suggest that analogs 3–5 exhibit an inhibitory effect on D-[³H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[³H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-α-(6) and β-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs, namely to ∼7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (∼40 kDa) than for control cultures (∼77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D -[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.     

IL-1-induced ERK1/2 activation up-regulates p21 protein by inhibition of degradation via ubiquitin-independent pathway in human melanoma cells A375

IL-1 inhibits the proliferation of human melanoma cells A375 by arresting the cell cycle at G0/G1 phase, which accompanies the increase of p21^Waf1/Cip1 (p21) protein. Here, we demonstrate that IL-1 induces the stabilization of p21 protein via ERK1/2 pathway. The degradation of p21 was inhibited by IL-1, however the ubiquitination level of p21 was not affected. In addition, the degradation of non-ubiquitinated form of lysine less mutant p21-K6R was also inhibited by IL-1, suggesting that IL-1 stabilized p21 protein via ubiquitin-independent pathway. Furthermore, the inhibition of p21 protein degradation was prevented by a selective inhibitor of ERK1/2 pathway, PD98059. These results suggest that IL-1-induced ERK1/2 activation leads to the up-regulation of p21 by inhibiting degradation via ubiquitin-independent pathway in human melanoma cells A375.     

Both B151-T cell replacing factor 1 and IL-5 regulate Ig secretion and IL-2 receptor expression on a cloned B lymphoma line

In the present study, we have demonstrated that both B151-T cell-replacing factor 1 and rIL-5 are responsible for the activity to partially induce CL-3 cells into IgM-synthesizing cells and also to synergize with IL-2 to augment IL-2R expression on and IgM synthesis in CL-3 cells. These actions of rIL-5 on a homogeneous cloned line (BCL1-CL-3 cells) allow us to identify and characterize the two alternated B cell developmental pathways. One is an IL-2-independent, IL-5-driven differentiation pathway without preceding up-regulated IL-2R expression, and the other is an IL-5 plus IL-2-dependent augmented differentiation pathway with preceding up-regulated IL-2R expression. We have also demonstrated the functional difference of two distinct B cell growth-promoting factors, B cell-stimulating factor 1 (rIL-4) and rIL-5. CL-3 cells are equally stimulated to grow by rIL-4 and rIL-5, whereas only rIL-5 can render CL-3 cells responsive to rIL-2, indicating that these two lymphokines affect B cells in a strikingly different manner.     

Synthesis of Guaiacol-α-D-glucoside and Curcumin-bis-α-D-glucoside by an Amyloglucosidase from Rhizopus

Guaiacol (2-methoxyphenol) and curcumin [1E,6E-1,7-di(4-hydroxy-3-methoxy-phenyl)-1,6-heptadiene-3,5-dione] were converted into their corresponding glucosides using glucose and an amyloglucosidase from Rhizopus. Guaiacol-α-D-glucoside yields ranged from 3 to 52% with the highest at pH 7.0. Curcumin-bis-α-D-glucoside yields ranged from 3 to 48% with the highest at pH 4.0 with 50% (w/w D-glucose) of enzyme. The phenolic hydroxyl group of guaiacol and both phenolic hydroxyl groups of curcumin were glucosylated at the C1 carbon of α-D-glucose indicating that the enzymatic reaction is stereospecific. Both guaiacol-α-D-glucoside and curcumin-bis-α-D-glucosides had antioxidant activities.     

Four glycoside hydrolases are differentially modulated by auxins, cytokinins, abscisic acid and gibberellic acid in apple fruit callus cultures

α-l-Arabinofuranosidase, α- and β-d-xylosidase, and β-d-glucosidase activity was detected in the soluble fraction (S-F) extracted with water and in the NaCl-released fraction (NaCl-F) extracted with a high-salt concentration buffer from apple callus cultures. The activity was found to be differentially modulated by the addition of various plant growth regulators (PGRs) to calluses that had lost their requirement for specific PGRs (“habituation” phenomenon). α-l-Arabinofuranosidase activity was 93%, 130%, 126% and 186% higher in the NaCl-F from IAA-, IBA-, ABA- and GA₃-treated callus than in that extracted from untreated callus while S-F α-l-arabinofuranosidase activity was only 71%, 24%, 55% and 66% higher, respectively. α-d-Xylosidase displayed low activity levels in both S-F and NaCl-F but 2iP-treated callus showed higher α-d-xylosidase activity in both fractions than the control. 2,4-D increased α-d-xylosidase activity by 110% in the NaCl-F but decreased it by 40% in the S-F. β-d-Xylosidase activity increased by 99% in S-F from 2iP-treated callus but slightly decreased in the NaCl-F. In GA₃-treated callus, NaCl-F β-d-xylosidase activity increased by 188%. S-F and NaCl-F from Picloram-treated callus showed undetectable or only slightly noticeable α-l-arabinofuranosidase, α-d-xylosidase and β-d-xylosidase activity. Interestingly, β-d-glucosidase activity rose 28-fold in the S-F extracted from Picloram-treated callus. β-d-glucosidase was the only enzyme assayed that greatly increased its NaCl-F activity after 10 subcultures, and the addition of any PGR to the callus culture –except for Picloram and ABA– decreased its activity, suggesting that this enzyme may be associated with certain stress conditions, such as PGR starvation or Picloram addition. This is the first report on glycoside hydrolases from fruit callus as modulated by different PGRs.     

A novel GH43 α-l-arabinofuranosidase from Humicola insolens: mode of action and synergy with GH51 α-l-arabinofuranosidases on wheat arabinoxylan

A novel α-l-arabinofuranosidase (α-AraF) belonging to glycoside hydrolase (GH) family 43 was cloned from Humicola insolens and expressed in Aspergillus oryzae. ¹H-NMR analysis revealed that the novel GH43 enzyme selectively hydrolysed (1→3)-α-l-arabinofuranosyl residues of doubly substituted xylopyranosyl residues in arabinoxylan and in arabinoxylan-derived oligosaccharides. The optimal activity of the cloned enzyme was at pH 6.7 and 53 °C. Two other novel α-l-arabinofuranosidases (α-AraFs), both belonging to GH family 51, were cloned from H. insolens and from the white-rot basidiomycete Meripilus giganteus. Both GH51 enzymes catalysed removal of (1→2) and (1→3)-α-l-arabinofuranosyl residues from singly substituted xylopyranosyls in arabinoxylan; the highest arabinose yields were obtained with the M. giganteus enzyme. Combinations (50:50) of the GH43 α-AraF from H. insolens and the GH51 α-AraFs from either M. giganteus or H. insolens resulted in a synergistic increase in arabinose release from water-soluble wheat arabinoxylan in extended reactions at pH 6 and 40 °C. This synergistic interaction between GH43 and GH51 α-AraFs was also evident when a GH43 α-AraF from a Bifidobacterium sp. was supplemented in combination with either of the GH51 enzymes. The synergistic effect is presumed to be a result of the GH51 α-AraFs being able to catalyse the removal of single-sitting (1→2)–α-l-arabinofuranosyls that resulted after the GH43 enzyme had catalysed the removal of (1→3)–α-l-arabinofuranosyl residues on doubly substituted xylopyranosyls in the wheat arabinoxylan.     

Comparison between crude Interleukin-2 and recombinant Interleukin-2 in maintaining killing activity of cultured lymphocytes

Peripheral blood lymphocytes, regional lymph node lymphocytes or malignant effusion lymphocytes from cancer patients were incubated with crude IL-2 (cIL-2) for 13 days. These effectors, which frequently expressed IL-2 receptor (IL-2R), proliferated well and possessed augmented killing activity against fresh autologous tumor cells and K562. However, when recombinant IL-2 (rIL-2) was added for the last 4 days of culture instead of cIL-2, IL-2R expression and killing activity against fresh autologous tumor cells decreased significantly (P<0.05). Phenotypic analysis indicated that cIL-2 significantly promoted the expansion of the cytotoxic population (CD8⁺ .11b^–)(P<0.05). The decreases in killing activity and IL-2R expression were restored by 0.004% PHA plus rIL-2, but not in the presence of rIFN-, rIL-1, rIL-l, rIL-4 or rIL-6. PHA-free cIL-2 maintained killing activity, but not IL-2R expression.We conclude that some factors in cIL-2 and a low dose of PHA-P are necessary for the maintenance of killing activity and IL-2R expression of cultured lymphocytes in the late phase of culture.     

Immunogenicity of recombinant human interleukin-2: Biological features and clinical relevance

The immunogenicity of recombinant interleukin-2 (rIL-2, EuroCetus, Amsterdam, Netherlands) was studied in seventy-six patients receiving different subcutaneous immunotherapy regimens. Patients presented with progressive metastatic renal cell carcinoma, malignant melanoma, colorectal cancer, B-cell lymphoma, and Hodgkin's disease. An enzyme immunoassay (EIA) was employed to screen patients for development of non-neutralizing antibodies against rIL-2, antibody specificity was confirmed by a standard Western blot. Neutralizing serum activity against rIL-2 was detected using a standard CTLL mouse proliferation assay. Additionally, serum levels of soluble interleukin-2 receptors and lymphocyte subsets expressing the CD56 natural killer (NK) associated antigen were measured.In a proportion of approximately 35% to 90% of the patients treated, non-neutralizing antibodies against rIL-2 could be detected after all treatment courses were evaluated. Antibodies were of the IgG, IgM, IgA and IgD subtypes. None of the 76 patients exhibited serum neutralizing activity after one treatment course. Five patients exhibited neutralizing anti-rIL-2 serum activity after two or more treatment courses of systemic rIL-2. In three of these patients, antibodies neutralized both recombinant and natural IL-2. Patients developing neutralizing anti-rIL-2 antibodies, exhibited significantly lower serum sIL-2 receptor levels upon the emergence of serum neutralizing activity than patients without antibody. Additionally, NK cell associated CD56 positivity was significantly lower in patients who exhibited neutralizing anti-rIL-2 serum activity than in patients who did not. A significant decrease in levels of soluble IL-2 receptors and CD56 NK cell positivity was observed, when comparing values prior to and after onset of serum neutralizing activity against rIL-2. However, while emergence of neutralizing antibodies to rIL-2 diminished rIL-2 induced biological activation, it did not coincide with abrogation of treatment response.Abbreviations rIL-2 recombinant interleukin-2 - EIA enzyme immuno assay - rIFN-2 recombinant interferon- 2     

Transglucosylation of ascorbic acid to ascorbic acid 2-glucoside by a recombinant sucrose phosphorylase from <Emphasis Type="Italic">Bifidobacterium longum</Emphasis>

A novel transglycosylation reaction from sucrose to l-ascorbic acid by a recombinant sucrose phosphorylase from Bifidobacterium longum was used to produce a stable l-ascorbic acid derivative. The major product was detected by HPLC, and confirmed to be 2-O-α-d-glucopyranosyl-l-ascorbic acid by LC-MS/MS analysis.