Immune reactions in patients with superficial bladder cancer after intradermal and intravesical treatment with bacillus Calmette-Guérin

Summary The immune reactivity of patients with strongly recurrent superficial bladder cancer was followed after combined intravesical and intradermal bacillus Calmette-Guérin (BCG) immunotherapy. All patients in this study were previously treated without success with intravesical chemotherapy. The BCG treatment regimen consisted of weekly administrations with BCG (RIVM) for six consecutive weeks, both intravesically and intradermally. In this study, sera and peripheral blood leukocytes (PBL) of patients were tested serially. Besides BCG-antigen-specific reactions, e.g. skin reactivity to purified protein derivatives of Mycobacterium tuberculosis (PPD), antibody formation and antigen stimulation of PBL in vitro, non-antigen-specific immune reactivities were also measured, e.g. mitogen response and spontaneous cytotoxic activity of PBL. In addition the antibody response to bladder carcinoma antigens and the cytotoxic activity of PBL for the bladder carcinoma cell line T24 and the natural-killer-sensitive K562 cell line were investigated. The results obtained from the various assays were evaluated for their prognostic value in relation to the length of the tumor-free interval after the BCG treatment. Because sera and PBL were only obtained during the first 6 months after the BCG treatment, the immune reactivity was compared to the clinical results at that same time. At 6 months after therapy 12 out of 40 BCG-treated patients were tumor-free whereas 28 out of 40 showed a recurrence. Skin reactivity to tuberculin PPD was measured in 40 patients during a period of 3–6 months after therapy. Of patients who showed a recurrence of the tumor within 6 months, 48% of them showed a transient response or developed no response at all to PPD. In the group of patients with a longer tumor-free period (n=10), only one patient lost the response to tuberculin PPD. Although PBL of a limited number of patients were tested, it was observed that the cytotoxicity to the bladder carcinoma cell line T24, and the natural-killer-sensitive K562 cell line increased in a number of the patients (7 out of 14, and 9 out of 14 respectively). Reactivity of PBL to mitogens and subset distribution (ratio T-helper: T-suppressor/cytotoxic) were not influenced by the BCG treatment. Antibody response to mycobacterial antigen was detected in 9 out of 23 patients investigated. Of these 9 patients, 8 belonged to the group with a recurrence of the tumor within 6 months (n=17). There was no correlation between the skin reactivity and the antibody response to tuberculin PPD. Furthermore, none of the 25 patients showed an antibody response to bladder carcinoma antigens. Sera of bladder carcinoma patients (n=19) reduced the mitogen-induced proliferation of lymphocytes, compared to sera of healthy controls (n=13), indicating the presence of circulating suppressor factor(s). Our results indicate that the absence of a Mantoux conversion or the presence of transient reaction to tuberculin PPD were highly related (91%) to a relapse of the disease. On the other hand, the cytotoxic activity of PBL to T24 and K562 cell lines, or their reactivity to tuberculin PPD or mitogens, gives no predictive information about the clinical results (tumor-free interval) of the BCG therapy. Abbreviations used: BCG, bacillus Calmette-Guérin; NK, natural killer; PBL, peripheral blood leukocytes; PPD, purified protein derivative of Mycobacterium tuberculosis; ELISA, enzyme-linked immunosorbent assay     

Suppression of in vitro lymphocyte responsiveness to purified protein derivative by measles virus. A reexploration of the phenomenon.

Clinical measles and measles vaccination have classically been associated with transient in vivo impairment of delayed hypersensitivity-type responses, especially skin test reactivity to purified protein derivative (PPD). In vitro data appeared to substantiate this in vivo observation by the demonstration of suppression of lymphocyte responsiveness to PPD by measles. Utilizing a measles preparation which has been recently demonstrated to elicit specific blastogenesis of sensitized human lymphocytes in vitro, we have reexplored the question of in vitro suppression of lymphocyte responsiveness to PPD by this virus. In contrast to previous reports, this study demonstrates that the addition of both measles and PPD to lymphocyte cultures can have a variable effect on lymphocyte responsiveness to PPD alone. This effect varies from marked inhibition to enhancement beyond a summation effect. The response is different for each lymphocyte donor and is dose related but cannot be predicted on the basis of combinations of high or low concentrations of either antigen. Purified, attenuated measles virus (Enders' strain), which uniformly suppressed in vitro lymphocyte reactivity when tested alone also demonstrated a significant dose related enhancement of the response to PPD alone. The present data suggest a reconsideration of the supposed importance of transient diminution of skin test reactivity during measles infection or immunization.     

Tuberculin response of lymphocytes from human skin test nonreactors; evidence for in vitro primary sensitization of T lymphocytes.

Tuberculin-purified protein derivative (PPD) is a B-lymphocyte mitogen in a variety of experimental animals. Although peripheral blood mononuclear cells (PB MNC) from healthy human tuberculin responders consistently responded to PPD by increased incorporation of [³H]thymidine, cell fractionation studies showed this to be due to T-lymphocyte rather than B-cell blastogenesis. Moreover, utilizing thymidine suicide experiments, the T-lymphocyte response could be categorized as antigenic rather than nonspecific mitogenic reactivity. Kinetic studies revealed a delayed peak of PPD-induced thymidine incorporation in PB MNC from tuberculin skin test-negative as compared to skin test-positive donors. This suggested in vitro primary sensitization of T lymphocytes to PPD, which was corroborated in experiments demonstrating tuberculin reactivity of human umbilical-cord blood lymphocytes.     

Transfer factor—Transfer of tuberculin cutaneous sensitivity in an allogeneic and xenogeneic monkey model

Tuberculin cutaneous sensitivity can be transferred from rhesus monkeys, sensitized to tuberculin using BCG, to rhesus and cynomolgus recipients with viable or disrupted leucocytes, and with a dialysed lysate preparation from 3 × 10⁸ leucocytes. Dialysable transfer factor (TF) using the lyophilized aqueous dialysate of a leucocyte lysate prepared by the freeze-thaw method did not give an active material. Modifications to the preparative method, in that leucocytes from fresh blood were disrupted gently by mechanical shearing forces, the lysate dialysed against a balanced salt solution and injected without lyophilization, yielded an active preparation. Transferred tuberculin (PPD) skin reactivity, confirmed by biopsy, was always less than the reactivity of the donor monkey and lasted for approximately 2–3 months. Although donor monkeys showed good in vitro lymphocyte transformation response to antigen stimulation, recipient monkeys which became skin test positive did not have a concomitant blastogenic response to PPD.     

Immune reactivity in cattle with ocular squamous cell carcinoma after intralesional BCG immunotherapy

Summary Lymphocyte stimulation with Con A and specific immune reactivity to BCG (antibody formation to BCG and DTH reaction to PPD) were determined in BCG-treated, surgically treated and untreated cows with ocular squamous cell carcinoma. In tumor-bearing cows the Con A-induced proliferation of lymphocytes was reduced when compared to healthy controls. This suppression consisted of a reduced blastogenic response to Con A of lymphocytes from tumor-bearing cows, and the presence of a factor in the sera of these animals, as these sera suppressed the blastogenic response of lymphocytes from healthy cows. BCG had only a minor influence on the suppressive activity. Antibodies to BCG were demonstrated in 50% of the BCG-treated animals. The formation of antibodies was not influenced by intradermal injection of PPD of Mycobacterium bovis. Absorption of a BCG antibody containing serum with BOSCC tumor extracts did not reveal the existence of cross reacting antigens between BCG and BOSCC. Pretherapeutic and posttherapeutic Con A reactivity could not be correlated with clinical response. Of the 30 BCG treated cows 29 developed a positive DTH reaction to PPD. Correlation between clinical response and immune reactivity was seen only with regard to the DTH reaction to PPD: this reaction remained positive for a longer period after treatment in animals with a favorable clinical outcome than in nonresponding animals.Animals were maintained under the guidelines laid down by the Faculty of Veterinary Medicine, State University, Utrecht, The NetherlandsGrant recipient of the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation) Abbreviations used: BCG, Bacillus Calmette-Guerin; BOSCC, bovine ocular squamous cell carcinoma PBL peripheral blood leukocytes; PPD, purified protein derivative of Mycobacteria; DTH, delayed type hypersensitivity Con A, concanavalin A; PHA, phytohemagglutinin; PWM, pokeweed mitogen     

Interspecies transfer by "immune" RNA of lymphocyte proliferative response to specific antigen

Ribonucleic acid (RNA) extracted from the lymph nodes of BCG sensitized cattle transferred tuberculin sensitivity to normal guinea pig lymphocytes as indicated by increased incorporation in vitro of ³H-thymidine in response to Purified Protein Derivative (PPD). The RNA treated lymphocytes were unresponsive to a nonspecific antigen, histoplasmin. Ribonuclease treatment of the RNA abolished its ability to transfer tuberculin reactivity and RNA extracted from the lymph nodes of normal cattle was also ineffective.     

Potentiation of the in vitro specific cytotoxic response to syngeneic lymphoma cells by PPD-stimulated tuberculin sensitive cells.

Spleen cells from rats immunized with the syngeneic (C58NT)D Gross virus-induced lymphoma have been shown to differentiate into cytotoxic effector cells following secondary in vitro stimulation with tumor cells. In the studies presented here, we evaluated whether cells specifically responding to PPD would increase the development of specific cytotoxic reactivity by a second cell population primed to lymphoma antigen. Mixtures of (C58NT)D-primed and BCG-primed responding cells generated cytotoxic activity to syngeneic lymphoma cells following cocultivation with mitomycin C-treated stimulating (C58NT)D cells; the addition of PPD to these mixtures produced a significant increase in cytotoxicity. The increased antitumor response resulted from an increase in specific cytotoxic activity from primed precursor cells. Responding cells activated with PPD alone in the absence of lymphoma antigen showed no lytic activity. Optimal numbers of tuberculin sensitive cells and concentration of PPD were determined. Evaluation of the kinetics of the generation of the cytotoxic response indicated that the addition of BCG-primed ceils and PPD increased the magnitude of cytotoxicity but did not alter the time course of the generation of cytotoxic activity. The addition of tuberculin sensitive cells and PPD to the in vitro secondary immune response also led to augmentation of generation of cells with antitumor activity detectable in vivo.     

Monitoring CD27 expression to evaluate Mycobacterium tuberculosis activity in HIV-1 infected individuals in vivo

The level of bacterial activity is only poorly defined during asymptomatic Mycobacterium tuberculosis (MTB) infection. The objective was to study the capacity of a new biomarker, the expression of the T cell maturation marker CD27 on MTB-specific CD4 T cells, to identify active tuberculosis (TB) disease in subjects from a MTB and HIV endemic region. The frequency and CD27 expression of circulating MTB-specific CD4 T cells was determined in 96 study participants after stimulation with purified protein derivative (PPD) using intracellular cytokine staining for IFNgamma (IFNγ). Subjects were then stratified by their TB and HIV status. Within PPD responders, a CD27⁻ phenotype was associated with active TB in HIV⁻ (p = 0.0003) and HIV⁺ (p = 0.057) subjects, respectively. In addition, loss of CD27 expression preceded development of active TB in one HIV seroconverter. Interestingly, in contrast to HIV⁻ subjects, MTB-specific CD4 T cell populations from HIV⁺ TB-asymptomatic subjects were often dominated by CD27⁻ cells. These data indicate that down-regulation of CD27 on MTB-specific CD4 T cell could be used as a biomarker of active TB, potentially preceding clinical TB disease. Furthermore, these data are consistent with the hypothesis that late, chronic HIV infection is frequently associated with increased mycobacterial activity in vivo. The analysis of T cell maturation and activation markers might thus be a useful tool to monitor TB disease progression.     

Selective induction and inhibition of direct and lectin-dependent cell-mediated cytotoxic reactivity

The immune reactivity of mice (C57BL/6, H-2^b) which had been challenged with various numbers (10²–10⁸) of allogeneic tumor cells (P815, H-2^d) was assessed at various times after challenge. Challenge with a high dose (10⁸) of tumor cells resulted in the development of direct cytotoxicity (DCMC), lectin-dependent cytotoxicity (LDCC), delayed-type hypersensitivity (DTH), and antibody production, whereas challenge with lower doses (< 10⁶) of tumor cells favored development of DTH and LDCC with marginal or no DCMC or antibody production. Spleen cells from low-dose alloimmune animals failed to produce DCMC when cultured with P815 cells in vitro and were capable of nonspecifically suppressing the DCMC response of normal spleen cells in MLC. Treatment with cyclophosphamide (100 mg/kg) prior to alloimmunization did not alter the pattern of DTH and cytotoxic reactivity, although treatment after alloimmunization was immunosuppressive for all forms of reactivity. When low-dose challenge was followed by cyclophosphamide treatment and a subsequent high-dose challenge, selective inhibition of DTH, LDCC, and suppressor activity, but not DCMC, was observed. The data suggest that (a) the initial challenge dose plays a significant role in determining which effector and regulatory populations will be activated and what direction the expression of immune reactivity will take; (b) the activated responding populations of DTH, DCMC, and LDCC effector cells are sensitive to cyclophosphamide treatment, whereas the precursors of each are resistant to the effects of the drug; (c) low-dose alloimmunization may be used in combination with cyclophosphamide treatment to modulate DTH, DCMC, and LDCC reactivity in a selective manner; (d) the cytotoxic effector cells responding to highdose challenge and mediating DCMC and those responding to low-dose challenge and mediating LDCC appear to arise from distinct precursor populations.     

Lymphocyte suppression associated with alpha globulin. Changes in cellular immunity

Sensitization of guinea pigs to 2,4-dinitrofluorobenzene is accompanied by increases in alpha globulins determined electrophoretically. During sensitization, lymphocyte responses were measured in vitro by mitogen induced ³H-thymidine uptake in whole blood cultures and in vivo by dermal skin reactivity. Following 5 days of dinitrofluorobenzene sensitization alpha globulins were elevated and lymphocyte transformation to phytohemagglutinin, pokeweed mitogen, and concanavalin A was significantly suppressed. When the alpha globulins returned to normal levels following sensitization, lymphocyte responses returned to pretreatment values. Antigen induced lymphocyte responsiveness was also suppressed concomitant with elevations in alpha globulins. Tuberculin sensitive guinea pigs responded poorly to PPD in vitro and in vivo during DNFB sensitization. It is suggested that increases in alpha globulins detected during the development of cellular immunity are associated with immunosuppression.     

Molecular organization of the non-bilayer phospholipid arrangements that induce an autoimmune disease resembling human lupus in mice

Abstract

Non-bilayer phospholipid arrangements are three-dimensional structures that can form when anionic phospholipids with an intermediate form of the tubular hexagonal phase II (H_II), such as phosphatidic acid, phosphatidylserine or cardiolipin, are present in a bilayer of lipids. The drugs chlorpromazine and procainamide, which trigger a lupus-like disease in humans, can induce the formation of non-bilayer phospholipid arrangements, and we have previously shown that liposomes with non-bilayer arrangements induced by these drugs cause an autoimmune disease resembling human lupus in mice. Here we show that liposomes with non-bilayer phospholipid arrangements induced by Mn²⁺ cause a similar disease in mice. We extensively characterize the physical properties and immunological reactivity of liposomes made of the zwitterionic lipid phosphatidylcholine and a H_II-preferring lipid, in the absence or presence of Mn²⁺, chlorpromazine or procainamide. We use an hapten inhibition assay to define the epitope recognized by sera of mice with the disease, and by a monoclonal antibody that binds specifically to non-bilayer phospholipid arrangements, and we report that phosphorylcholine and glycerolphosphorylcholine, which form part of the polar region of phosphatidylcholine, are the only haptens that block the binding of the tested antibodies to non-bilayer arrangements. We propose a model in which the negatively charged H_II-preferring lipids form an inverted micelle by electrostatic interactions with the positive charge of Mn²⁺, chlorpromazine or procainamide; the inverted micelle is inserted into the bilayer of phosphatidylcholine, whose polar regions are exposed and become targets for antibody production. This model may be relevant in the pathogenesis of human lupus.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 56-62 (antigenic site 2) of myoglobin.

This work was carried out in order to study the effects of substitutions outside antigenic site 2 of sperm whale myoglobin (SpMb) on the reactivity of protein variants with antisite 2 monoclonal antibodies (mAbs). A synthetic peptide corresponding to region 56–62 (site 2) of SpMb was used as an immunogen in mice in its free form (i.e., without coupling to any carrier) to prepare a panel of mAbs whose predetermined specificity is directed, by design, against this region. The binding of three of these mAbs to eight Mbs from different species was studied. Myoglobins of Pacific common dolphin, finback whale, and horse, which have no substitutions within region 56–62 relative to SpMb, showed remarkable differences in their cross-reactivities and relative affinities with each of the mAbs. Myoglobins of badger, chicken, and dog, although they have an identical substitution within the site (Ala-57 to Gly), exhibited cross-reactivities with a given mAb that were affected differently. Echidna Mb, which has one replacement (Glu-59 to Ala) within region 56–62, displayed greatly reduced cross-reactivities and relative binding affinities. The results, especially those from Mbs that have no substitutions within the boundaries of site 2, clearly indicate that substitutions outside site 2 of Mb can exert drastic effects on the binding of the Mb variants with mAbs whose specificity was predesigned to be against the site. These indirect effects and their impact on site reactivity will completely explain previous findings on cross-reactivities of Mb variants with mAbs of unknown specificity and will rule out the postulations of discontinuous sites in Mb, which were based on the assumption that every substitution affecting reactivity is directly involved in binding to antibody.     

In vitro stimulation of human granulopoiesis by purified protein derivative (PPD)

Summary The effect of purified protein derivative (PPD) on human granulopoiesis was studied in an in vitro semisolid culture system of human bone marrow in which PPD was incorporated into the leukocyte feeder layers. We observed that preincubation of the feeder layers with PPD was necessary to induce a significant rise of agar culture colony-forming units (CFU-c) with a maximum of 3 days' preincubation and a dose of 200 g for 10 ⁶ leukocytes. A similar effect was obtained when a conditioned medium from PPD-stimulated leukocytes was used instead of feeder layers. We have found a significant correlation between the skin test response of the leukocyte donors to PPD and the colony-stimulating activity of their leukocytes exposed to PPD: these results suggest that PPD could stimulate human granulopoiesis by an indirect effect on CSF-producing mononuclear cells.     

Two new behavioral QTLs, Emo4 and Reb1, map to mouse Chromosome 1: Congenic strains and candidate gene identification studies

By use of newly developed subcongenic strains of mice from a parental B6.129-Il10^−/− knockout/congenic strain, we have narrowed the critical region for a new behavioral QTL, called Emo4, for open-field activity to a segment of Chromosome 1 between Erbb4 (68.4Mb) and B3gnt7 (86.2 Mb). We have also uncovered an additional QTL governing repetitive beam breaks in the open field. This QTL, called Reb1, maps to the interval between Asb1 (91.4 Mb) and NM_172851 (100.0 Mb) and is one of the first QTLs mapped for this type of behavior. Genome-wide microarray expression analyses were then undertaken to help to identify candidate genes that may be the cause of these genetic differences in open-field performance. In this effort, we analyzed global gene expression differences in the amygdalae by use of Affymetrix GeneChips between B6, B6.129-Il10^−/−, and B6.129R4. Several probe sets representing target Chr 1 genes were found that showed significantly differential expression in the subcongenic and congenic strains. Several candidate genes have been identified. One of these regions coincides with an homologous region in humans that has been associated with autism, a disease whose symptoms include repetitive actions. This study illustrates that the use of congenic strains combined with global gene expression analyses can produce a list of viable candidates. It further shows that caution should be observed when analyzing the effects of knockout/congenic strains because many of the gene expression differences in these comparisons could not be attributable to the ablated Il10 gene but rather to passenger gene effects.     

In aged mice,low surrogate light chain promotes pro‐B‐cell apoptotic resistance,compromises the PreBCR checkpoint,and favors generation of autoreactive,phosphorylcholine‐specific B cells

In aged mice, new B‐cell development is diminished and the antibody repertoire becomes more autoreactive. Our studies suggest that (i) apoptosis contributes to reduced B lymphopoiesis in old age and preferentially eliminates those B‐cell precursors with higher levels of the surrogate light chain (SLC) proteins (λ5/VpreB) and (ii) λ5^low B‐cell precursors generate new B cells which show increased reactivity to the self‐antigen/bacterial antigen phosphorylcholine (PC). Pro‐B cells in old bone marrow as well as pro‐B cells from young adult λ5‐deficient mice are resistant to cytokine‐induced apoptosis (TNFα; TGFβ), indicating that low λ5 expression in pro‐B cells is sufficient to cause increased survival. Transfer of TNFα‐producing ‘age‐associated B cells’ (ABC; CD21/35^? CD23^?) or follicular (FO) B cells from aged mice into RAG‐2 KO recipients led to preferential loss of λ5^high pro‐B cells, but retention of λ5^low, apoptosis‐resistant pro‐B cells. In old mice, there is increased reactivity to PC in both immature bone marrow B cells and mature splenic FO B cells. In young mice, absence of λ5 expression led to a similar increase in PC reactivity among bone marrow and splenic B cells. We propose that in old age, increased apoptosis, mediated in part by TNFα‐producing B cells, results in preferential loss of SLC^high pro‐B cells within the bone marrow. Further B‐cell development then occurs via an ‘SLC^low’ pathway that not only impairs B‐cell generation, but promotes autoreactivity within the naïve antibody repertoires in the bone marrow and periphery.     

Comparative analysis of collagen type II-specific immune responses during development of collagen-induced arthritis in two B10 mouse strains

Introduction

Immune responses against collagen type II (CII) are crucial for the development of collagen-induced arthritis (CIA). The aim of the present study was to evaluate and compare the CII-directed T cell and antibody specificity at different time points in the course of CIA using two mouse strains on the B10 genetic background - B10.Q, expressing A^q MHC class II molecules, and B10.DR4.Ncf1^*/*, expressing human rheumatoid arthritis-associated MHC II DR4 molecules (DRA*0101/DRB*0401).

Methods

B10.Q and B10.DR4.Ncf1^*/* mice were immunized with CII emulsified in adjuvant and development of CIA was assessed. T cells from draining lymph nodes were restimulated in vitro with CII peptides and interferon-gamma (IFN-γ) levels in culture supernatants were evaluated by ELISA. CII-specific antibody levels in serum samples were measured by ELISA.

Results

At four different CIA time points we analyzed T cell specificity to the immunodominant CII epitope 259-273 (CII259-273) and several posttranslationally modified forms of CII259-273 as well as antibody responses to three B cell immunodominant epitopes on CII (C1, U1, J1). Our data show that CII-specific T and B cell responses increase dramatically after disease onset in both strains and are sustained during the disease course. Concerning anti-CII antibody fine specificity, during all investigated stages of CIA the B10.Q mice responded predominantly to the C1 epitope, whereas the B10.DR4.Ncf1^*/* mice also recognized the U1 epitope. In the established disease phase, T cell reactivity toward the galactosylated CII259-273 peptide was similar between the DR4- and the A^q-expressing strains whereas the response to the non-modified CII peptide was dramatically enhanced in the DR4 mice compared with the B10.Q. In addition, we show that the difference in the transgenic DR4-restricted T cell specificity to CII259-273 is not dependent on the degree of glycosylation of the collagen used for immunization.

Conclusions

The present study provides important evaluation of CII-specific immune responses at different phases during CIA development as well as a comparative analysis between two CIA mouse models. We indicate significant differences in CII T cell and antibody specificities between the two strains and highlight a need for improved humanized B10.DR4 mouse model for rheumatoid arthritis.     

Myoglobin: a pseudo-enzymatic scavenger of nitric oxide

Myoglobin (Mb) is reported in biochemistry and physiology textbooks to act as an O₂ reservoir and to facilitate O₂ diffusion from capillaries to mitochondria, to sustain cellular respiration. Recently, it has been proposed that Mb is an intracellular scavenger of bioactive nitric oxide (NO), regulating its level in the skeletal and cardiac muscle and thereby protecting mitochondrial respiration, which is impaired by NO. This novel function of Mb is based on the rapid and irreversible reaction of ferrous oxygenated Mb (MbO₂) with NO yielding ferric oxidized Mb (metMb) and nitrate (NO₃^–). The efficiency of this process, which is postulated to depend on the superoxide (O₂^–) character acquired by O₂ once bound to the heme iron, may be enhanced by intramolecular diffusion of NO trapped momentarily into cavities of the protein matrix. O₂ can also react with ferrous nitrosylated Mb (MbNO), albeit very slowly, leading to metMb and NO₃^–. The O₂-dependent NO-detoxification process may be considered to be pseudo-enzymatic given that metMb obtained by the primary reaction of MbO₂ with NO is reduced back to ferrous Mb by a specific metMb-reductase, and can therefore repeat a cycle of NO conversion to harmless nitrate.     

Use of urease inhibitors to reduce ammonia loss following application of urea to flooded rice fields

Ammonia (NH₃) volatilization is an important mechanism for nitrogen (N) loss from flooded rice fields following the application of urea into the floodwater. One method of reducing losses is to use a urease inhibitor that retards the hydrolysis of urea by soil urease and allows the urea to diffuse deeper into the soil. The two chemicals that have shown most promise are phenylphosphorodiamidate [PPD] and N(n-butyl)thiophosphorictriamide [NBPT], but they seldom work effectively. PPD decomposes rapidly when the pH departs from neutrality, and NBPT must be converted to the oxygen analogue for it to be effective. Our field studies in Thailand show that the activity of PPD can be prolonged, and NH₃ loss markedly reduced, by controlling the floodwater pH with the algicide terbutryn. A mixture of NBPT and PPD in the presence of terbutryn was even more effective than PPD alone. It appears that during the time when the PPD was effective, NBPT was being converted to the oxygen analogue. The combined urease inhibitor-algicide treatment reduced NH₃ loss from 10 to 0.4 kg N ha^-1.     

Development and precise characterization of phospho-site-specific antibody of Ser(357) of IRS-1: elimination of cross reactivity with adjacent Ser(358)

Antibodies that recognize specifically phosphorylated sites on proteins are widely utilized for studying the regulation and biological function of phosphoproteins. The proposed strategy is a powerful, analytical tool allowing the generation of phospho-site specific antibodies albeit adjacent phosphorylation sites are present. Here, we demonstrate the assessment and elimination of cross reactivity of phospho-site-specific-Ser³⁵⁷ IRS-1 antibody. While determining the specificity of p-Ser³⁵⁷ antiserum we came across the cross reactivity of the antiserum with adjacent Ser³⁵⁸ which was successfully abolished by an improved immuno-purification method. The specificity of the purified antiserum was then verified by indirect ELISA, results of ELISA were also mirrored in the experiments carried out in BHK-IR cells using different mutants of IRS-1 carrying mutations at either Ser³⁵⁷/Ser³⁵⁸/Ser^357/358. Immuno-purified-p-Ser³⁵⁷ did not react with IRS-1 Ala³⁵⁷ and IRS-1 Ala^357/358. In conclusion, the present study describes generation and characterization of p-Ser³⁵⁷ IRS-1 antibody, which reacts with IRS-1 in site specific and phosphorylation state-dependent manner without showing cross reactivity to adjacent Ser³⁵⁸. This antibody can be effectively used to further clarify the inhibitory role of Ser³⁵⁷ in insulin signal transduction.