On the prosthetic groups of the NiFe sulfhydrogenase from Pyrococcus furiosus: topology, structure, and temperature-dependent redox chemistry

 The sulfhydrogenase complex of Pyrococcus furiosus is an αβγδ heterotetramer with both hydrogenase activity (borne by the αδ subunits) and sulfur reductase activity (carried by the βγ subunits). The β-subunit contains at least two [4Fe-4S] cubanes and the γ-subunit contains one [2Fe-2S] cluster and one FAD molecule. The δ-subunit contains three [4Fe-4S] cubanes and the α-subunit carries the NiFe dinuclear center. Only three Fe/S signals are observed in EPR-monitored reduction by dithionite, NADPH, or internal substrate upon heating. All other clusters presumably have reduction potentials well below that of the H⁺/H₂ couple. Heat-induced reduction by internal substrate allows, for the first time, EPR monitoring of the NiFe center in a hyperthermophilic hydrogenase, which passes through a number of states, some of which are similar to states previously defined for mesophilic hydrogenases. The complexity of the observed transitions reflects a combination of temperature-dependent activation and temperature-dependent reduction potentials. Received: 10 December 1998 / Accepted: 16 February 1999     

Mycobacteria activate γδ T-cell anti-tumour responses via cytokines from type 1 myeloid dendritic cells: a mechanism of action for cancer immunotherapy

Attenuated and heat-killed mycobacteria display demonstrable activity against cancer in the clinic; however, the induced immune response is poorly characterised and potential biomarkers of response ill-defined. We investigated whether three mycobacterial preparations currently used in the clinic (BCG and heat-killed Mycobacterium vaccae and Mycobacterium obuense) can stimulate anti-tumour effector responses in human γδ T-cells. γδ T-cell responses were characterised by measuring cytokine production, expression of granzyme B and cytotoxicity against tumour target cells. Results show that γδ T-cells are activated by these mycobacterial preparations, as indicated by upregulation of activation marker expression and proliferation. Activated γδ T-cells display enhanced effector responses, as shown by upregulated granzyme B expression, production of the T_H1 cytokines IFN-γ and TNF-α, and enhanced degranulation in response to susceptible and zoledronic acid-treated resistant tumour cells. Moreover, γδ T-cell activation is induced by IL-12, IL-1β and TNF-α from circulating type 1 myeloid dendritic cells (DCs), but not from type 2 myeloid DCs or plasmacytoid DCs. Taken together, we show that BCG, M. vaccae and M. obuense induce γδ T-cell anti-tumour effector responses indirectly via a specific subset of circulating DCs and suggest a mechanism for the potential immunotherapeutic effects of BCG, M. vaccae and M. obuense in cancer.     

Phenotypic and functional alterations of Vγ2Vδ2 T cell subsets in patients with active nasopharyngeal carcinoma

Introduction  Human Vγ2Vδ2 T cells play important role in immunity to infection and cancer by monitoring self and foreign isoprenoid metabolites with their γδ T cell antigen receptors. Like CD4 and CD8 αβ T cells, adult peripheral Vγ2Vδ2 T cells represent a pool of heterogeneous cells with distinct functional capabilities. Purpose  The aim of this study was to characterize the phenotypes and functions of various Vγ2Vδ2 T cell subsets in patients with nasopharyngeal carcinoma (NPC). We sought to develop a better understanding of the role of these cells during the course of disease and to facilitate the development of immunotherapeutic strategies against NPC. Results  Although similar total percentages of peripheral blood Vγ2Vδ2 T cells were found in both NPC patients and normal donors, Vγ2Vδ2 T cells from NPC patients showed decreased cytotoxicity against tumor cells whereas Vγ2Vδ2 T cells from normal donors showed potent cytotoxicity. To investigate further, we compared the phenotypic characteristics of Vγ2Vδ2 T cells from 96 patients with NPC and 54 healthy controls. The fraction of late effector memory Vγ2Vδ2 T cells (T_{EM RA}) was significantly increased in NPC patients with corresponding decreases in the fraction of early memory Vγ2Vδ2 T cells (T_CM) compared with those in healthy controls. Moreover, T_{EM RA} and T_CM Vγ2Vδ2 cells from NPC patients produced significantly less IFN-γ and TNF-α, potentially contributing to their impaired cytotoxicity. Radiotherapy or concurrent chemo-radiotherapy further increased the T_{EM RA} Vγ2Vδ2 T cell population but did not correct the impaired production of IFN-γ and TNF-α observed for T_{EM RA} Vγ2Vδ2 T cells. Conclusion  We have identified distinct alterations in the Vγ2Vδ2 T cell subsets of patients with NPC. Moreover, the overall cellular effector function of γδ T cells is compromised in these patients. Our data suggest that the contribution of Vγ2Vδ2 T cells to control NPC may depend on the activation state and differentiation of these cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Salt stress induction of some key antioxidant enzymes and metabolites in eight Iranian wild almond species

The present work describes the changes in the activities of key antioxidant enzymes and the levels of some metabolites in relation to salt tolerance in eight wild almond species. All the species were exposed to four levels of NaCl (control, 40, 80 and 120 mM). Plant fresh biomass, α-, γ- and δ-tocopherol, total soluble proteins, malondialdehyde (MDA_eq), H₂O₂, total phenolics, and the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were analyzed in leaves of salt-stressed and non-stressed plants of the eight almond species. In all the species, salt stress significantly enhanced the activities of SOD and POD, levels of total phenolics and γ- and δ-tocopherols. High levels of salt stress significantly depressed the levels of total soluble proteins, MDA and CAT activity, while salt stress did not significantly affect leaf H₂O₂ contents. Regression analysis showed that the relationship between salt levels and total soluble proteins, CAT, γ-tocopherol, MDA_eq, SOD and POD were statistically significant. Principal component analysis discriminated the almond species based on their degree of tolerance/sensitivity to saline conditions: Prunus reuteri and P. glauca were ranked as salt tolerant, P. lycioides and P. scoparia as moderately tolerant, and P. communis, P. eleagnifolia, P. arabica and P. orientalis as salt sensitive. The results could be used for selecting salt tolerant genotypes to be used as rootstocks for almond cultivation.     

Diversity and evolution of T-cell receptor variable region genes in mammals and birds

 The receptor of a T lymphocyte (TCR) recognizes nonself antigens in the company of major histocompatibility complex (MHC) molecules presented to it by the antigen-presenting cell. The variable region of TCR is encoded by either a concatenation of variable region (TCR-V), diversity region (TCR-D), and joining region (TCR-J) genes, or a concatenation of TCR-V and TCR-J genes. The TCR-V genes exist as a multigene family in vertebrate species. Here we study the evolutionary relationships of TCR-V genes from humans, sheep, cattle, rabbits, mice, and chicken. These six species can be classified into two groups according to the frequency of γδ T-cells in their peripheral T-cell populations. The "γδ low" group of species includes humans and mice, in which γδ T-cells constitute very limited portion of the T-cell population. The "γδ high" group includes sheep, cattle, rabbits, and chicken, in which γδ T-cells comprise up to 60% of the T-cell population. Here, we compiled TCR-V sequences from the six species and conducted a phylogenetic analysis. We identified various TCR-V gene subgroups based on the analysis. We found that humans and mice have representatives from nearly all of the subgroups identified, while other species have lost subgroups to different extent. Therefore, the γδ low species have a high degree of diversity of TCR-V genes, while γδ high species all have limited diversity of TCR-V genes. This pattern is similar to that found for immunoglobulin variable region (IGV) genes. Received: 20 May 1999 / Revised: 13 July 1999     

Characterization of the γδ T cell response to acute leukemia

Background: Previous work from our center has suggested a correlation between increased donor-derived Vδ1+ γδ T cells and long-term relapse-free survival following bone marrow transplantation for leukemia. Questions remain, however, as to whether this observation can be explained by a γδ T cell-based immune response against primary leukemia. Methods: We examined γδ T cell receptor (TCR) phenotype, cell proliferation, and cytolytic activity following culture with irradiated primary leukemia blasts from a haploidentical first-degree relative. Subsequently, we also studied the γδ TCR phenotype and complimentarity determining region 3 (CDR3) cDNA sequences from 17 newly diagnosed leukemia patients. Results: In 17/28 (61%) of in vitro cultures, γδ T cells proliferated in culture with primary blasts. Vδ1+ T cells were proportionally increased in all cultures and were the predominant cell population in 6/17. In the 7 cultures where cytotoxicity could be assessed, 6 (86%) showed some degree of cytotoxicity to the primary leukemia. Vδ1+ T cells were also the predominant γδ T cell subtype in pre-treatment leukemia patients principally due to loss of Vδ2+ T cells rather than expansion of Vδ1+ cells. The Vδ1 CDR3-region cDNA sequence from these patients revealed exclusive use of the Jδ1 constant region and sequence conservation in 4/11 patients. Conclusions: γδ T cells exhibit an in vitro response to primary leukemia blasts that is manifested by proliferation, an increased proportion of Vδ1+ T cells, and cytotoxicity to the primary leukemia blasts. The Vδ1+ T cell population is also predominant in newly diagnosed leukemia patients likely due to a loss of circulating Vδ2+ T cells. A small proportion of newly diagnosed patients showed Vδ1 CDR3 region similarity. These findings suggest a role for γδ T cells in the immune response to leukemia.Paul F. Meeh and Michelle King are contributed equally to this work.     

Vγ9/Vδ2 T lymphocytes in Italian patients with Beh?et's disease: evidence for expansion, and tumour necrosis factor receptor II and interleukin-12 receptor β1 expression in active disease

Beh?et's disease is a multisystem disease in which there is evidence of immunological dysregulation. It has been proposed that γ/δ T cells are involved in its pathogenesis. The aim of the present study was to assess the capacity of γ/δ T cells with phenotype Vγ9/Vδ2, from a group of Italian patients with Beh?et's disease, to proliferate in the presence of various phosphoantigens and to express tumour necrosis factor (TNF) and IL-12 receptors. Twenty-five patients and 45 healthy individuals were studied. Vγ9/Vδ2 T cells were analyzed by fluorescence activated cell sorting, utilizing specific monoclonal antibodies. For the expansion of Vγ9/Vδ2 T cells, lymphocytes were cultured in the presence of various phosphoantigens. The expression of TNF receptor II and IL-12 receptor β₁ was evaluated with the simultaneous use of anti-TNF receptor II phycoerythrin-labelled (PE) or anti-IL-12 receptor β₁ PE and anti-Vδ2 T-cell receptor fluorescein isothiocyanate. There was a certain hierarchy in the response of Vγ9/Vδ2 T cells toward the different phosphoantigens, with the highest expansion factor obtained with dimethylallyl pyrophosphate and the lowest with xylose 1P. The expansion factor was fivefold greater in patients with active disease than in those with inactive disease or in control individuals. TNF receptor II and IL-12 receptor β₁ expressions were increased in both patients and control individuals. The proportion of Vγ9/Vδ2 T cells bearing these receptors was raised in active disease when Vγ9/Vδ2 T cells were cultured in the presence of dimethylallyl pyrophosphate. These results indicate that Vγ9/Vδ2 T cell activation is correlated with disease progression and probably involved in the pathogenesis.     

Role of adhesion molecules in recruitment of Vδ1 T cells from the peripheral blood to the tumor tissue of esophageal cancer patients

 The mechanism responsible for tissue specific localization of γδ T cell subsets is not well understood. In order to explain the sequestration of specific γδ T cell subsets in the peripheral blood and tumor tissue of patients with esophageal cancer, we examined the function and expression of adhesion molecules on these cells. A hierarchy in the expression of adhesion molecules was observed. In vitro activated γδ T cells showed dominant expression of LFA-1 (CD11a), VLA-α4 (CD49d), intermediate expression of VLA-α5 (CD49e) and L-selectin (CD62L), but low expression of CD44v6 and α_Eβ₇ (CD103). It was observed that the γδ T cells use LFA-1, L-selectin and CD44v6 to bind to squamous cell carcinoma (SCC) cells, whereas they adhere to fibroblast cells using LFA-1, VLA-α4 and VLA-α5. Vδ1 T cell subsets from the peripheral blood γδ T cells utilize a larger array of adhesion molecules, namely LFA-1, VLA-α4, VLA-α5, L-selectin and α_Eβ_7, to bind to SCC cells compared to the restricted usage of LFA-1, L-selectin and CD44v6 by the Vδ2 T cells. Flow cytometric analysis of tumor infiltrating lymphocytes from the esophageal tumors confirmed the selective accumulation of Vδ1⁺γδ T cells in the tumor compartment. It thus appears that adhesion molecules expressed on these lymphocytes play an important role in the recruitment and retention of Vδ1 T cells in the tumor milieu. Received: 27 November 2000 / Accepted: 1 March 2001     

Detection of intracellular cytokines during antioxidant supplementation in corticoid-dependent asthmatics and modulation of adhesion molecule expression on cultured endothelial cells

In this study, we report on the interferon-γ (IFN-γ) and interleukin-4 (IL-4) cytokine responses to phorbol myristate acetate (PMA)+ionomycin-stimulated CD3+ lymphocytes in asthmatic subjects when compared with normal donors. There was a significantly lower production of intracellular IFN-γ in asthmatic patients. No difference was found for IL-4 production between these two groups. After administration of a multivitamin-mineral supplement containing selenium, zinc, vitamin A, vitamin B₆, vitamin C, and vitamin E for 6 mo, a significant increase in the percentage of CD3+/IL-4 positive cells (p<0.05) was found. The induction of endothelial cell adhesion molecule (CAM) expression in cultured human umbilical vein endothelial cells (HUVEC) and whole-blood mixture was studied using flow cytometry. The ICAM-1 and VCAM-1 expressions were higher in the patients than in control donors (p<0.05). There is a correlation between the increased percentage of CD3+/IFN-γ positive cells and reduced endothelial ICAM-1 and VCAM-1 expression after 6 mo of intervention period. No apparent effect of supplementation on CAM expression was found, suggesting that these changes do not arise from an antioxidant mechanism. This newly developed whole-blood technique for the assessment of CAM expression can be of use for monitoring therapy in inflammatory diseases.     

The Polarity of Lipid-Exposed Residues Contributes to the Functional Differences between Torpedo and Muscle-Type Nicotinic Receptors

A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues αS287, αC412, βY441, γM299, γS460, δM293, δS297 and δN305 in the Torpedo AChR were replaced with those found in the muscle-type receptor. Mutant receptor expression was measured in Xenopus oocytes using [¹²⁵I]-α-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5- to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46% decrease in the ACh 50% inhibitory concentration (EC₅₀), while three mutant combinations resulted in 1.7- to 2-fold increases in ACh EC₅₀. Finally, seven mutant combinations resulted in a decrease in normalized, ACh-induced currents. Our results suggest that these residues, although remote from the ion channel pore, (1) contribute to ion channel gating, (2) may affect trafficking of AChR into specialized membrane domains and (3) account for the functional differences between Torpedo and muscle-type AChR. These findings emphasize the importance of the lipid-protein interface in the functional differences between the Torpedo and muscle-type AChRs.     

Low resolution structure of subunit b (b (22-156)) of Escherichia coli F(1)F(O) ATP synthase in solution and the b-delta assembly

The first low resolution solution structure of the soluble domain of subunit b (b _22–156) of the Escherichia coli F₁F_O ATPsynthase was determined from small-angle X-ray scattering data. The dimeric protein has a boomerang-like shape with a total length of 16.2 ± 0.3 nm. Fluorescence correlation spectroscopy (FCS) shows that the protein binds effectively to the subunit δ, confirming their described neighborhood. Using the recombinant C-terminal domain (δ_91–177) of subunit δ and the C-terminal peptides of subunit b, b _120–140 and b _140–156, FCS titration experiments were performed to assign the segments involved in δ–b assembly. These data identify the very C-terminal tail b _140–156 to interact with δ_91–177. The novel 3D structure of this peptide has been determined by NMR spectroscopy. The molecule adopts a stable helix formation in solution with a flexible tail between amino acid 140 to 145.     

Gamma /delta T cells provide innate immunity against renal cell carcinoma

Host immune function plays a certain role against the development of renal cell carcinomas (RCCs), but the mechanism is not entirely understood. Human gamma/delta (γ/δ) T cells defend the body against infection. In this study, we clarify the role of γ/δ T cells in the surveillance system against RCCs by analyzing the γ/δ T cells in peripheral blood mononucleocytes (PBMs) and tumor infiltrating lymphocytes (TILs) from 41 patients with RCCs. The results showed that the number of γ/δ T cells expressing Vγ2 and Vδ2 in variable elements of TCR was elevated in the PBMs in 10 patients, but not in any of 32 healthy individuals. The proportion of patients with an elevated number of γ/δ T cells (>10%) increased with cancer stage. The level of the γ/δ T cells decreased after surgery. The γ/δ T cells in the TILs were more activated than those in the PBMs. Evaluation of the junctional diversity of TCR Vγ2 and Vδ2 chains showed that the increased peripheral blood γ/δ T cells were oligoclonal rather than polyclonal. Taken together, our findings suggest that γ/δ T cells recognize certain RCC-related antigens and play a role in the surveillance system against RCCs. Received: 30 November 2000 / Accepted: 2 January 2001     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits from Spinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.     

Using yeast two-hybrid system to detect interactions of ATP synthase subunits fromSpinacia oleracea

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding α, β, γ, δ and ε subunits ofSpinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding β-galactosidase was detected. Of all the combinations, that of γ and ε subunit genes showed the highest level of reporter gene expression, while those of α and β, a and ε, β and ε and β and δ induced stable and significant reporter gene expression. The combination of δ and ε as well as that of δ and γ induced weak and unstable reporter gene expression. However, combinations of α and γ, β and γ and α and δ did not induce reporter gene expression. These results suggested that specific and strong interactions between γ and ε, α and β, α and ε, β and ε and β and δ subunits, and weak and transient interactions between δ and ε and δ and γ subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.     

Using the water signal to detect invisible exchanging protons in the catalytic triad of a serine protease

Chemical Exchange Saturation Transfer (CEST) is an MRI approach that can indirectly detect exchange broadened protons that are invisible in traditional NMR spectra. We modified the CEST pulse sequence for use on high-resolution spectrometers and developed a quantitative approach for measuring exchange rates based upon CEST spectra. This new methodology was applied to the rapidly exchanging Hδ1 and Hε2 protons of His57 in the catalytic triad of bovine chymotrypsinogen-A (bCT-A). CEST enabled observation of Hε2 at neutral pH values, and also allowed measurement of solvent exchange rates for His57-Hδ1 and His57-Hε2 across a wide pH range (3–10). Hδ1 exchange was only dependent upon the charge state of the His57 (k _ex,Im+ = 470 s⁻¹, k _ex,Im = 50 s⁻¹), while Hε2 exchange was found to be catalyzed by hydroxide ion and phosphate base ( k_{\textOH ^-} k_{{{\text{OH}}^{ - } }}  = 1.7 × 10¹⁰ M⁻¹ s⁻¹, k_{\textHPO4^{2 -}} k_{{{\text{HPO}}_{4}^{2 - } }}  = 1.7 × 10⁶ M⁻¹ s⁻¹), reflecting its greater exposure to solute catalysts. Concomitant with the disappearance of the Hε2 signal as the pH was increased above its pK _a, was the appearance of a novel signal (δ = 12 ppm), which we assigned to Hγ of the nearby Ser195 nucleophile, that is hydrogen bonded to Nε2 of neutral His57. The chemical shift of Hγ is about 7 ppm downfield from a typical hydroxyl proton, suggesting a highly polarized O–Hγ bond. The significant alkoxide character of Oγ indicates that Ser195 is preactivated for nucleophilic attack before substrate binding. CEST should be generally useful for mechanistic investigations of many enzymes with labile protons involved in active site chemistry.     

Intravesical administration of γδ T cells successfully prevents the growth of bladder cancer in the murine model

Background  Superficial bladder cancers are usually managed with transurethral resection followed by the intravesical administration of Bacillus Calmette-Guerin which requires major histocompatibility complex (MHC) class I expression on cancer cells. Since cancer cells often loose MHC expression, a novel immunotherapy such as MHC-unrestricted γδ T cell therapy is desired. Objective  To clarify the relationship between the expression of MHC class I and clinicopathological features in bladder cancer patients, and investigate the effects of the administration of intravesical γδ T cells on bladder cancer. Methods  Samples from 123 patients who had undergone either transurethral resection or radical cystectomies were examined for MHC expression and the relationship between this and the clinicopathological features was analyzed statistically. The in vitro and in vivo effects of γδ T cells expanded by zoledronic acid (ZOL) against several types of cancer cell line and an orthotopic bladder cancer murine model which was pretreated with ZOL were investigated. Results  MHC-diminished superficial bladder cancer was significantly more progressive than MHC-conservative bladder cancer (P = 0.047). In addition, there was a significant association between diminished MHC expression and poor disease free survival (P = 0.041) and overall survival (P = 0.018) after radical cystectomy. In vitro, all of the cell lines pretreated with 5-μM ZOL showed a marked increase in sensitivity to lysis by γδ T cells. Moreover, intravesical administration of γδ T cells with 5-μM ZOL significantly demonstrated antitumor activity against bladder cancer cells in the orthotopic murine model (P < 0.001), resulting in prolonged survival. Conclusion  The present murine model provides a potentially interesting option to develop immunotherapy using γδ T cells for bladder cancer in human. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. T. Yuasa and K. Sato contributed equally to the study.