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MOLECULAR AND CELLULAR PATHOGENESIS
Helicobacter pylori Urease Suppresses Bactericidal Activity of Peroxynitrite via Carbon Dioxide Production
Hideo Kuwahara, Yoichi Miyamoto, Takaaki Akaike, Tatsuo Kubota, Tomohiro Sawa, Shinichiro Okamoto, Hiroshi Maeda
Hideo Kuwahara
Department of Microbiology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, 1 and Department of Medical Technology, Ginkyo College of Medical Science, 1-6-2 Okubo, Kumamoto 860-0083, 2 Japan
Yoichi Miyamoto
Department of Microbiology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, 1 and
Takaaki Akaike
Tatsuo Kubota
Tomohiro Sawa
Shinichiro Okamoto
Hiroshi Maeda
DOI: 10.1128/IAI.68.8.4378-4383.2000
Helicobacter pylori can produce a persistent infection in the human stomach, where chronic and active inflammation, including the infiltration of phagocytes such as neutrophils and monocytes, is induced. H. pylori may have a defense system against the antimicrobial actions of phagocytes. We studied the defense mechanism of H. pylori against host-derived peroxynitrite (ONOO−), a bactericidal metabolite of nitric oxide, focusing on the role of H. pylori urease, which produces CO2 and NH3 from urea and is known to be an essential factor for colonization. The viability of H. pylori decreased in a time-dependent manner with continuous exposure to 1 μM ONOO−, i.e., 0.2% of the initial bacteria remained after a 5-min treatment without urea. The bactericidal action of ONOO− against H. pyloriwas significantly attenuated by the addition of 10 mM urea, the substrate for urease, whereas ONOO−-induced killing of a urease-deficient mutant of H. pylori or Campylobacter jejuni, another microaerophilic bacterium lacking urease, was not affected by the addition of urea. Such a protective effect of urea was potentiated by supplementation with exogenous urease, and it was almost completely nullified by 10 μM flurofamide, a specific inhibitor of urease. The bactericidal action of ONOO− was also suppressed by the addition of 20 mM NaHCO3 but not by the addition of 20 mM NH3. In addition, the nitration ofl-tyrosine of H. pylori after treatment with ONOO− was significantly reduced by the addition of urea or NaHCO3, as assessed by high-performance liquid chromatography with electrochemical detection. These results suggest that H. pylori-associated urease functions to produce a potent ONOO− scavenger, CO2/HCO3 −, that defends the bacteria from ONOO− cytotoxicity. The protective effect of urease may thus facilitate sustained bacterial colonization in the infected gastric mucosa.
Nitric oxide (NO) is known to play an important role in host defense against a variety of microbes (1, 12, 15, 20, 36, 37), although NO itself does not show sufficient antimicrobial activity (24, 55). Some metabolites of NO, such as peroxynitrite (ONOO−), are considered to be responsible for the antimicrobial as well as the pathogenic effects of NO. NO and superoxide (O2 −) react in a diffusion-limited manner, forming ONOO− (5), a strong oxidant and nitrating agent (4, 5, 23) that exhibits potent bactericidal activity (22, 57) as well as cytotoxicity for mammalian cells in vitro and in vivo (4,5). It has been reported that both NO and O2 − that are simultaneously produced in local areas of infection are critically involved in antimicrobial defense in murine salmonellosis (Salmonella enterica serovar Typhimurium infection), possibly through formation of ONOO− (49).
Helicobacter pylori can infect human gastric mucosa chronically; such infection is known to be associated with gastritis, peptic gastric ulcer, duodenal ulcer, and an increased risk for gastric cancer (3, 6, 21, 45, 52). A unique feature of H. pylori infection is its persistence, which causes prolonged active inflammation, including infiltration of neutrophils and monocytes in gastric mucosa (11, 39). Increased expression of the inducible type of NO synthase (iNOS) (16-18, 30, 42, 47) and elevated formation of nitrotyrosine (17, 30) are also observed in the gastric mucosae of patients with H. pyloriinfection. However, the mechanism of the persistent infection ofH. pylori, despite the production of highly bactericidal ONOO− and other reactive nitrogen species, is not clear.
Several investigations have suggested a role for H. pyloriurease in the survival and pathogenesis of the bacteria (29, 31,35, 46). Urease catalyzes the hydrolysis of urea to form carbon dioxide (CO2) and ammonia (NH3). It is reported that urease functions in H. pylori infection to neutralize gastric acid by producing NH3 (31). Enhanced production of NH3 also may facilitate the formation of NH3-derived compounds, such as monochloramine, which shows cytotoxic effects on host cells (46). Enhancement of bacterial motility (35) and inhibition of phagocytic clearance of bacteria (29) were also reported as functions of urease. The pathogenic potential of urease is so far mainly attributed to NH3 produced by the enzymatic reaction. In contrast, little attention has been paid to the roles of CO2/HCO3 − produced in the same process. It is noteworthy that the chemical reactivity of ONOO− is reported to be modulated by CO2/HCO3 − (26, 28, 54). Specifically, ONOO− reacts rapidly with CO2, and through the formation of ONOOCO2 −, not only is isomerization of ONOO− to NO3 − accelerated (27, 50), but also the nitration potency of ONOO− is significantly enhanced and the oxidation potential is markedly attenuated (54, 56). For example, CO2/HCO3 − facilitates ONOO−-induced nitration of aromatic compounds, such as tyrosine and guanine (guanosine); however, it suppresses their oxidation (26, 54, 56). In addition, the in vitro bactericidal activity of ONOO− on Escherichia coli was reduced by the addition of NaHCO3 (22,57).
Therefore, the purpose of this study was to clarify the role of urease in persistent colonization of H. pylori, especially to examine the protective effects of CO2 produced by urease against the bactericidal activity of ONOO− in vitro.
Bacteria. H. pylori ATCC 43504 was obtained from the American Type Culture Collection (Manassas, Va.). H. pyloriHPK5 and its isogenic ureB mutant HPT209 (lacking urease), which was produced by allelic exchange mutagenesis, were generously provided by T. Nakazawa, Department of Microbiology, Yamaguchi University School of Medicine, Ube, Japan (35).Campylobacter jejuni isolated from a clinical source was also used in this study. These bacteria were routinely grown in brucella broth (Becton Dickinson & Co., Cockeysville, Md.) supplemented with 10% fetal calf serum (Intergen Co., Purchase, N.Y.) in the presence (H. pylori HPT209) or absence (H. pyloriATCC 43504, H. pylori HPK5, and C. jejuni) of 7 μg of kanamycin sulfate/ml under microaerobic conditions maintained in a GasPak jar (Becton Dickinson & Co.) with an H2- and CO2-generating agent, CampyPak (Becton Dickinson & Co.).
Reagents.ONOO− was prepared from nitrite and hydrogen peroxide in a quenched-flow reactor as previously described (5). The NO-liberating agent propylamine NONOate (CH3N[N(O)NO]−(CH2)3NH2 +CH3, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazine) (P-NONOate) which has a half-life of 7.6 min in aqueous solutions at a neutral pH under our experimental conditions, was obtained from Dojindo Laboratories, Kumamoto, Japan. Urea, NaHCO3, and an aqueous solution of ammonia (NH4OH) were from Wako Pure Chemicals Co. Ltd., Osaka, Japan. Urease from Bacillus pasteurii, 3-nitro-l-tyrosine (nitrotyrosine), andl-tyrosine were purchased from Sigma Chemical Co., St. Louis, Mo. Dihydrorhodamine 123 (DHR) was purchased from Molecular Probes, Inc., Eugene, Oreg. Two urease inhibitors,N-(diaminophosphinyl)-4-fluorobenzamide (flurofamide) and acetohydroxamic acid (AHX), were from ICN Biomedicals Inc., Aurora, Ohio, and Nacalai Tesque Inc., Kyoto, Japan, respectively. Pronase was obtained from Calbiochem-Novobiochem Co., La Jolla, Calif. All other reagents were from commercial sources.
Bactericidal assays.We used 0.5 M phosphate buffer (pH 7.6) containing 0.15 M NaCl (0.5 M phosphate-buffered saline [PBS]) for the bactericidal assays of ONOO− and other effector substances to minimize changes in pH during the reaction because of infusion of the alkaline solution of ONOO− (in 10 mM NaOH) and production of NH3 and HCO3 −, which might have affected the pH of the reaction mixture. Suspensions of H. pylori cultured for 36 to 48 h and of C. jejuni cultured for 24 h were washed with and resuspended in 0.5 M PBS immediately before use. Bacterial suspensions were kept on ice until use. Urease activity and bacterial motility and morphology were checked before each use. The constant-flux infusion method (7, 40) was used to treat the bacteria with steady concentrations of ONOO−. In the constant-flux infusion process, the effective and constant concentration of ONOO−is maintained by balancing infusion and decomposition of ONOO− in the system. The concentrations of ONOO− maintained constant were estimated by the DHR oxidation assay, as described earlier (8). Specifically, DHR (28 μM) was added to the reaction mixture of ONOO−without bacteria; simultaneously, the ONOO− infusion was stopped, and the amount of the oxidized product rhodamine was measured fluorometrically. The concentration of ONOO− was then estimated by using a standard curve of the amount of rhodamine generated as a function of ONOO−, which was prepared separately by reaction of DHR with a bolus of ONOO−injected into 0.5 M PBS. As a result, by infusion 10, 100, and 1,000 μM ONOO− in 10 mM NaOH into 0.5 M PBS (1.2 ml) at a flow rate of 240 μl/min, the concentrations of ONOO− remained constant at 0.3, 1, and 3 μM, respectively. H. pylori orC. jejuni (108 CFU/ml each) samples were treated constantly with 1 μM ONOO− by infusing 100 μM ONOO− in the absence or presence of urea, NaHCO3, or NH4OH as described above. Aliquots (120 μl) were removed from the reaction mixture at 30-s intervals and were immediately diluted with nutrient broth (Eiken Chemical Co. Ltd., Tokyo, Japan) and seeded on brucella agar plates containing 5% lysed horse blood (Nippon Bio-Test Laboratories Inc., Tokyo, Japan) for the colony-forming assay. After cultivation for 5 days (H. pylori) or 2 days (C. jejuni) under microaerobic conditions, the number of colonies formed was determined.
To examine the bactericidal effect of NO on H. pylori, P-NONOate at 1, 10, and 100 μM was added to the suspension ofH. pylori ATCC 43504 (108 CFU/ml) in 0.5 M PBS. After incubation for 3, 5, and 10 min, aliquots of the bacterial suspension were diluted with nutrient broth and seeded on brucella agar plates containing 5% lysed horse blood. Viable H. pyloriorganisms were quantified by the colony-forming assay, as just described.
Measurement of nitrotyrosine.Suspensions of H. pylori (5 × 108 CFU/ml) treated continuously with 1 μM ONOO− for 3 min in the presence or absence of 20 mM NaHCO3 or 10 mM urea, as described above, were centrifuged at 1,600 × g and then resuspended in 10 mM potassium phosphate buffer (pH 7.4) containing 0.15 M NaCl (250 μl). Aliquots (100 μl) were treated with 60 μg of pronase/ml for 18 h at 50°C. The bacterial suspension became clear and no precipitate was seen in the resultant reaction mixture even after centrifugation at 10,000 × g, indicating that the bacterial cells were completely digested by the pronase treatment. After filtration through a centrifugal filter unit (Ultrafree-MC with a 10,000 nominal molecular weight limit; Millipore Corp., Bedford, Mass.), the filtrate was processed by high-performance liquid chromatography (HPLC) coupled to electrochemical detection with 12 CoulArray electrode cells (ESA, Inc., Chelmsford, Mass.) (9, 19). Nitrotyrosine recovered from the bacterial cells was separated on a reverse-phase column (4.6 by 250 mm) (TSKgel ODS-80Ts; Tosoh Co., Tokyo, Japan) and eluted with 50 mM sodium acetate buffer (pH 4.7) containing 5% methanol at a flow rate of 0.8 ml/min. The CoulArray electrode array detector was operated with applied potentials at 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, and 750 mV. Peaks of nitrotyrosine and l-tyrosine were identified and quantified based on comigration with known concentrations of authentic standards and their electrochemical activation profiles. Identification of nitrotyrosine was confirmed by the disappearance of the peak after reduction of nitrotyrosine to aminotyrosine by 20 mM sodium dithionite. The amounts of nitrotyrosine and tyrosine were quantified from the peak areas obtained at 750 and 600 mV, respectively.
Statistical analysis.Statistical analyses were done with the two-tailed t test for unpaired data.
Bactericidal effect of ONOO− on H. pylori. It is now known that ONOO− is a key intermediate in the NO-dependent bactericidal effect. Induction of iNOS and formation of nitrotyrosine, an indicator of ONOO− formation, inH. pylori-infected stomach have also been documented (16-18, 30, 42, 47). Therefore, we examined the bactericidal activity of authentic ONOO− (5) by using the constant-flux infusion method (7, 40). The number of viable bacteria expressed as CFU declined after exposure to ONOO− in a dose- and time-dependent manner (Fig.1A). Products of ONOO−decomposition, mainly nitrate anion (28), showed no bactericidal activity against H. pylori (Fig. 1A).
(A) Bactericidal effect of ONOO− onH. pylori ATCC 43504. Decomposed ONOO−(equivalent to 1 mM ONOO−) (●) or ONOO− at 10 μM (◊), 100 μM (○), or 1 mM (□) in 10 mM NaOH was infused into 1.2 ml of bacterial suspension (108 CFU/ml) at a flow rate of 240 μl/min. Every 30 s, 120-μl aliquots were removed from the reaction mixture, and the number of viable bacteria was determined by the colony-forming assay. Concentrations of ONOO− in the reaction mixture during infusion of 10 μM, 100 μM, and 1 mM ONOO− were assumed to be maintained at constant 0.3, 1, and 3 μM concentrations, respectively. Data are means ± SD from three independent experiments. (B) Survival ofH. pylori after exposure to the NO-donor P-NONOate. H. pylori ATCC 43504 organisms (108 CFU/ml) were incubated for the indicated periods with 1 (○), 10 (□), or 100 (▵) μM P-NONOate, followed by colony-forming assay. (C and D) Effects of urea on bactericidal action of ONOO− onH. pylori ATCC 43504 (C) and C. jejuni (D). (E and F) Effects of urea on the bactericidal action of ONOO−on H. pylori HPK5 (E) and its isogenic ureBmutant, HPT209 (F). Bacteria (108 CFU/ml) were exposed to a constant concentration of ONOO− (1 μM) in the presence (●) or absence (○) of 10 mM urea for the indicated periods, and the number of viable bacteria at each time point was determined. Data from three independent experiments are expressed as means ± SD. ∗ and ∗∗, P < 0.05 and P < 0.01 versus control without urea, respectively.
The NO-liberating agent P-NONOate was examined by incubation withH. pylori ATCC 43504 (108 CFU/ml) in 0.5 M PBS (pH 7.6) for up to 10 min. P-NONOate at 1, 10, or 100 μM did not affect the viability of the bacteria (Fig. 1B). It is known that one molecule of P-NONOate releases two molecules of NO with a half-life of 7.6 min at a neutral pH (25, 40). Hence, an appreciable concentration of NO (up to 200 μM) did not kill this bacterium, nor was a sufficient amount of bactericidal metabolites of NO, such as ONOO−, formed during the reaction period. In any event, these results indicate that NO per se exhibits very little bactericidal action, which is a great contrast to ONOO−.
Effect of urea on bactericidal action of ONOO− onH. pylori and C. jejuni. As shown in Fig. 1C, in the presence of a physiological concentration (10 mM) of urea, survival of H. pylori was significantly increased. Because urea did not affect the decomposition rate of ONOO− at the pH range 7.0 to 10.0, as assessed by measuring absorbance at 302 nm (data not shown), direct detoxification of ONOO− by urea itself was not plausible. When clinically isolated C. jejuni, another microaerophilic bacteria lacking urease activity, was treated with ONOO− in the same experimental settings in the presence or absence of urea, the susceptibility of C. jejuni to ONOO− was not affected by the addition of urea (Fig. 1D), suggesting that the contribution of urease produced by H. pylori to the suppression of the cytotoxicity of ONOO− was required. To further verify this notion, the bactericidal action of ONOO− against H. pyloriHPK5 and its isogenic mutant HPT209, lacking urease, was examined with or without the addition of 10 mM urea. The strains showed similar sensitivities to ONOO− in the absence of urea (Fig. 1E and F). In contrast, urea attenuated the bactericidal effect of ONOO− on the wild-type strain, HPK5 (Fig. 1E), but it did not affect the bacterial killing of ONOO− for the mutant with the urease gene disruption, HPT209 (Fig. 1F). This result indicates again that urease activity is required for urea-dependent attenuation of the ONOO− cytotoxicity.
The role of urease was further investigated with two urease inhibitors. It has been reported that flurofamide is a specific inhibitor of extracellular urease and that AHX is effective on both intracellular and extracellular urease (35). In the presence of 10 μM flurofamide or 70 mM AHX, the protective effect of urea was almost completely nullified (Fig. 2A), while in the absence of urea, these urease inhibitors did not affect the bactericidal action of ONOO− on H. pylori (data not shown). Flurofamide seemed to be more effective than AHX, so extracellular urease localized on the surface of bacterial cells plays an important role in suppressing the bactericidal action of ONOO−. In contrast to urease inhibitors, the addition of urease derived from B. pasteurii augmented the protective effect of urea (Fig. 2A). These data indicate that the bactericidal effect of ONOO− against H. pylori is diminished by bacterial urease activity.
(A) Effects of urease inhibitors and additional urease on the bactericidal action of ONOO− on H. pylori. H. pylori ATCC 43504 organisms (108 CFU/ml) were exposed to a constant concentration of ONOO− (1 μM) for 3 min in the absence (a) or presence (b to e) of 10 mM urea. Reactions were performed in the presence of 10 μM flurofamide (c), 70 mM AHX (d), or 48 U of B. pasteurii urease per ml (e). (B) Effects of NaHCO3 and NH3 on bactericidal action of ONOO− on H. pylori. H. pylori ATCC 43504 organisms (108 CFU/ml) were exposed to a constant concentration of ONOO− (1 μM) for 3 min in the presence of 10 mM urea (b), 20 mM NH3 (c), 20 mM NaHCO3(d), or 10 mM urea plus 20 mM NaHCO3 (e) or in the absence of all compounds (a), and the colony-forming assay was performed. Data from three independent experiments are expressed as means ± SD. ∗, P < 0.01 versus a; †, P < 0.05 versus b; ∗∗, P < 0.005 versus b; and ‡, P < 0.005 versus d.
Effects of NaHCO3 and NH3 on bactericidal action of ONOO− on H. pylori. We examined the effects of the products of the urea-urease reaction, CO2and NH3, on the bactericidal activity of ONOO−. NaHCO3 (20 mM) suppressed bacterial killing by ONOO− to the same degree as 10 mM urea, whereas NH4OH (20 mM) did not (Fig. 2B). Furthermore, urea (10 mM) plus NaHCO3 (20 mM) showed an additive protective effect for the survival of H. pylori exposed to ONOO−(Fig. 2B), suggesting that urease increases bacterial survival in in vivo situations in which physiological concentrations of HCO3 − and urea are close to those used in this experiment, i.e., about 20 and 10 mM, respectively (38).
A change in the pH of the media might affect the chemical reactivity of ONOO− (26, 27, 56). In our experimental settings, however, NH3 released after urea hydrolysis byH. pylori urease did not alter the pH of the reaction mixture. The pH values of the suspension of 108 CFU ofH. pylori ATCC 43504 per ml in 0.5 M PBS after 0, 1, 2, 3, 4, and 5 min of infusion of 100 μM ONOO− at a flow rate of 240 μl/min in the absence of urea were 7.57 ± 0.01, 7.60 ± 0.01, 7.63 ± 0.01, 7.66 ± 0.02, 7.68 ± 0.02, and 7.69 ± 0.01, respectively, and those obtained in the presence of 10 mM urea were 7.57 ± 0.01, 7.60 ± 0.01, 7.63 ± 0.02, 7.65 ± 0.02, 7.67 ± 0.02, and 7.69 ± 0.01 (means ± standard deviations [SD] of three independent experiments). In addition, as shown in Fig. 1A, infusion of an alkaline solution alone (decomposed ONOO− in 10 mM NaOH) did not affect the viability of H. pylori. Also, NH3 per se had no appreciable effect on the bactericidal action of ONOO− (Fig. 2B). We therefore deduced that the protective effect of urease against ONOO− is dependent on its CO2 production but is not dependent on NH3release or the change in pH.
Nitrotyrosine formation in H. pylori after treatment with ONOO−.ONOO− is known to nitrate aromatic compounds, including tyrosine (4, 23). To assess the effect of urease activity on the chemical reactivity of ONOO− with the bacterial components, we quantified nitrotyrosine in H. pylori cells by using HPLC coupled to electrochemical detection with 12 electrodes (Fig.3A). The amount of nitrotyrosine in the bacterial cells exposed to 1 μM ONOO− for 3 min was 267 ± 22 pmol/108 CFU, or 7.48% ± 1.2% of the total tyrosine (Fig. 3B). Nitrotyrosine was not detected (less than 0.1 pmol/108 CFU) in the control bacterial cells (no exposure to ONOO−). In contrast, we could not detect any appreciable amount of nitrotyrosine in the bacterial cells treated with P-NONOate (data not shown), indicating that ONOO−, but not NO, exhibits a strong tyrosine-nitrating potential in H. pylori. The addition of 10 mM urea or 20 mM NaHCO3 to the reaction mixture of ONOO− lowered the formation of nitrotyrosine by 50% (Fig. 3B). Since CO2 accelerates decomposition of ONOO− (27, 50), it is plausible that CO2/HCO3 − added or formed by bacterial urease might increase the decomposition rate of ONOO− and thus suppress the reactivity of ONOO− with the bacteria.
Nitrotyrosine formation in H. pylori cells after treatment with ONOO−. 3-Nitro-l-tyrosine (3-Nitro-Tyr) was quantified by HPLC coupled to electrode detectors, with the pronase digests of non-ONOO−-treated H. pylori ATCC 43504 cells (a) or those exposed to 1 μM ONOO− (b to d) for 3 min in the absence (b) or presence of 10 mM urea (c) and 20 mM NaHCO3 (d). (A) Elution profile of the pronase digest of H. pylori exposed to 1 μM ONOO− in the absence of urea or NaHCO3. (B) The amounts of 3-Nitro-Tyr formed in H. pylori were expressed as the ratio of 3-Nitro-Tyr to total l-tyrosine (Tyr) recovered from bacterial cells. ND, 3-Nitro-Tyr was not detected (<0.1 pmol/108 CFU). ∗, P < 0.05 versus b. Data are means ± SD of three independent experiments.
H. pylori produces a large quantity of urease, which amounts to 5% of the total protein of the bacterium (14). Urease genes in the H. pylori genome are composed of two gene clusters: ureAB genes and ureIEFGH genes (10). Colonization of H. pylori mutants whoseureA, ureB, ureG, or ureIgene was disrupted in experimental animals was known to be suppressed (13, 44, 48, 53). In addition, proton pump inhibitors used for treatment of H. pylori infection inhibit bacterial urease in an irreversible fashion (33). All these studies imply that H. pylori urease is essential for H. pylori colonization in the stomach.
Several studies were carried out to elucidate the roles of H. pylori urease in bacterial colonization in the stomach. Neutralization of gastric acid with NH3 produced by the enzyme might allow the bacterium to survive in the acidic milieu (31). It is reported that the motility of H. pylori, which is known to be an important characteristic of the bacterium in the colonization of experimental animals, is enhanced by the urea-urease reaction, particularly in a viscous environment (35). Inhibition of neutrophil function by NH3was also proposed as a pathogenic mechanism of this enzyme (32).
In addition to these possible roles of urease, the results obtained in this study clearly demonstrate that H. pylori urease functions as a part of the defense system of the bacteria themselves against ONOO− (Fig. 1 and 2).
In previous work, elevated generation of ONOO− in vivo and its involvement in antimicrobial host defense were reported for a murine salmonellosis model. Results indicated that suppressing ONOO− generation by inhibiting either NO or O2 − production or by scavenging these radicals accelerated the growth of S. enterica serovar Typhimurium in the liver and further augmented its pathogenicity, as evidenced by the increased mortality of infected mice (49). It was thus suggested that ONOO− effectively clears bacteria from sites of infection in vivo (1, 49). In recent years, increased expression of iNOS mRNA and its product has been confirmed inH. pylori-infected gastric tissues of patients and experimental animals (16-18, 30, 42, 47). Formation of ONOO− and/or other reactive nitrogen species produced by the NO2 −-H2O2-myeloperoxidase system at sites of infection by H. pylori is also suggested by the immunohistochemical detection of nitrotyrosine (17, 30,51). Furthermore, it has recently been reported that not only phagocytic inflammatory cells but also H. pylori itself produce O2 − (34), which indicates that ONOO− may be formed in and around the bacteria in vivo, where production of NO and O2 − is simultaneously elevated as described above. Consequently, ONOO− may function as a major bactericidal effector forH. pylori in the stomach. In a separate experiment, however, no significant difference was found between the number of H. pylori organisms colonizing iNOS-knockout mice and that in wild-type mice (unpublished observation). In this context, it is quite reasonable that H. pylori has evolved with the system, such as urease, that is capable of detoxifying ONOO−, and hence steady and sustained colonization in the infected stomach is facilitated.
A high concentration of ONOO− was used in the present study so that we could obtain reproducible results and clearly demonstrate the bactericidal action of ONOO−. The bacteria were directly exposed to a 1 μM effective concentration of ONOO− in vitro, which is considered to be an extremely severe condition for the bacteria compared with the in vivo setting in infected foci containing various endogenous substances that affect the reactivity of ONOO− (2). Even under such conditions, the physiological concentration of urea increased the survival fractions of two strains of H. pylori (ATCC 43504 and HPK5) 3.7- to 8.4-fold after exposure to ONOO− for 5 min (Fig. 1C and E). Therefore, it is conceivable that the urease could function efficiently as a protective factor of H. pyloriagainst ONOO− produced in vivo.
Although it is reported that ONOO−-dependent nitration of aromatic compounds, including tyrosine, is enhanced in the presence of CO2 (26, 54), formation of nitrotyrosine inH. pylori was suppressed by the addition of urea or NaHCO3 (Fig. 3B). Recently, Romero et al. reported that CO2 shortened the half-life and the diffusion distance of ONOO− and hence inhibited the oxidation of oxyhemoglobin in red blood cells by ONOO− (43). Therefore, the results obtained in this study suggest that CO2 formed by bacterial urease inhibits the reactivity of ONOO− with the bacterial components and accelerates its decomposition outside the bacterial cells. It is of great importance, then, that H. pylori urease is localized not only in the cytoplasm but also on the surface of the bacteria (41). In our experimental settings, surface-bound urease seemed to play an important role in the decomposition of ONOO− (Fig. 2A).
In conclusion, urease of H. pylori plays a role in the defense against the toxicity of ONOO− via production of CO2, and it may confer the capacity for sustained infection in vivo. Improved understanding of the pathogenic role of urease, in view of a host-pathogen interaction, will help in the exploration of effective therapeutic treatments for H. pylori infection and its related gastric diseases, including gastric cancer.
We thank Judith B. Gandy for editing and Rie Yoshimoto for typing the manuscript.
This work was supported by a Grant-in-Aid for Scientific Research from Monbusho (Ministry of Education, Science and Culture) of Japan.
Editor: D. L. Burns
Received 3 January 2000.
Returned for modification 23 February 2000.
Accepted 8 May 2000.
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Infection and Immunity Aug 2000, 68 (8) 4378-4383; DOI: 10.1128/IAI.68.8.4378-4383.2000
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As suggested by herbal healers in Patalkot, crush fresh Ginger (10g) along with Black Pepper seeds (4 nos.) in 10 ml water, squeeze it. The filtrate should be applied over the painful body parts, two to three times a day. No harm in trying. Even modern researches indicates that Ginger may calm arthritis pain, possibly by lowering your prostaglandin levels. There are studies that indicate that Ginger could reduce pain and inflammation more effectively than non-steroidal anti-inflammatory drugs.


Attendance of a 90-minute sales presentation of Westgate Resorts is required.
A valid, active MAJOR CREDIT CARD will be required to check in at this resort.
Both husband and wife must be between 28 and 68 years of age (Head of household must be 28 +, other can be 25 +).
Married, or cohabiting couples must have a combined Gross Annual Income of at least $60,000. Combined Annual Gross Income is defined as the combined annual earned income on W-2 forms. Income does not include Child Support or Alimony or Social Security, Commissions or Investments.
If married, engaged or co-habiting, both persons must attend the sales presentation together.
Have not participated in a Westgate Resorts' sales presentation within the past 12 months or twice in a lifetime.
Debit or pre-paid cards are not accepted.
Failure to meet these requirements or complete our presentation will result in your credit card being charged the full retail rate of the resort (at the time of your stay) and the retail amount of any other incentive(s) you have received.
United States residents are qualified to tour except residents of Alaska, Hawaii, Maine, Washington, and Puerto Rico. In the State of Florida, residents of Dade, Duval, Orange, Osceola, Seminole and Lake Counties OR within a 60-mile radius of Orlando, are not eligible for the promotion.
Westgate Resorts' owners are not eligible for the promotion.
No groups and no more than one family. Two or more families or friends traveling together with the same promotion each, are not eligible.
Attendance of a 90-minute sales presentation of Westgate Resorts is required. This promotion does not include attraction tickets. THESE PACKAGES ARE PREPAID AND YOUR CREDIT CARD WILL BE CHARGED IN FULL WITHIN 24 HOURS OF MAKING THE RESERVATION REQUEST. All persons must be 28 years of age or older. If married, both husband and wife must attend the sales presentation together. Combined annual income must be at least $60,000 with a major credit card. Guests must present a major credit card and valid photo ID at the tour reception desk. Not eligible if you have previously toured Westgate Resorts in the past 12 months or twice in a lifetime. No "group" tours will be accepted, no more than two families occupying the same villa. If you have a reservation with us, we cannot offer another reservation to other friends or relatives of yours, but they can stay with you in your villa if the maximum occupancy allows it. All reservations are subject to availability and based on a first come, first served basis. Failure to attend the sales presentation, or failure to qualify for this promotion, will result in a charge to your credit or check card of the full ongoing regular rate at check-out. This vacation package is non-refundable; however, it can be rescheduled one time upon 72-hour advance notice and is subject to a $25 rescheduling fee. In the event, you cannot travel on your scheduled dates, you must give at least 72-hours advance notice of cancellation to avoid "no show" charges. Failure to give 72-hour notice, will result in forfeiture of your vacation package. Should you desire to re-schedule your arrival date, simply let us know prior to 72-hours in advance of this confirmed arrival date, and based upon availability, we will help you select other travel dates within the next six-month period. No refunds for early departures. Offer subject to change without notice. This offer is not transferable and is not valid in conjunction with any other offer. Accommodations price shown is good for 3 nights for two (2) adults and up to two (2) children, under the age of eighteen (18), in a studio villa during value season, unless otherwise noted. Reservations are first-come, first-served, based upon availability and inventory. Average promotional value for packages shown is between $33 and $119 per night based on travel season. Travel during peak season, holidays and special events are subject to a surcharge of $15 extra per additional night during peak season, and $30 extra per night during holiday season and special events. Up to two additional nights, and one and two bedroom units are available. In the State of Florida, residents of Dade, Duval, Orange, Osceola, Seminole and Lake Counties OR within a 60-mile radius of Orlando, are not eligible for the promotion.
Vacation package amenities include, where available, access to pools, paddle boats, mini golf, fitness center, bike rentals, putt-putt golf, sports court, sports equipment rental, movie theater, boarding pass printing, and in-room safe. Guest is responsible for any and all taxes, fees, and/or other government and airport-imposed charges, Wi-Fi Internet Access, local / long distance calls, tips, gratuities, meals, beverages, alcohol, transportation, package handling fee, spa services, and waterpark admittance.
OUR SERVICES ARE LIMITED TO MAKING THE RESERVATION ON THE HOTEL OR RESORT YOU SELECT, PAYING FOR SUCH ACCOMMODATIONS AND (IF INCLUDED IN THE PACKAGE) PAYING FOR THE ATTRACTION TICKETS SELECTED IN THE RESERVATION. ALL INCIDENTALS AND HOTEL/RESORT FEES ARE THE GUEST'S RESPONSIBILITY. TAXES ARE THE GUEST'S RESPONSIBILITY, UNLESS INDICATED IN THE PRICE DISCLOSURE. WE ARE NOT RESPONSIBLE FOR THE ACTUAL FULFILLMENT OF THE SERVICES FROM NEITHER THE HOTEL NOR RESORT SELECTED, AND ANY COMPLAINTS SHOULD BE ADDRESSED DIRECTLY WITH THE SERVICE PROVIDER (HOTEL OR RESORT'S FRONT DESK) NOT TO US.
IMPORTANT NOTE: Participants in the promotion who do not meet the qualifications set forth in the above Details of Participation, including those who fail to attend the sales presentation, will have charged to their credit or check card a penalty fee of $350 or the full regular rate of the accommodations at the time of check-out, whichever is greater. Participants with confirmed reservations who fail to check into their reserved accommodations on the first reserved date or who fail to reschedule their reservations AT LEAST 72 HOURS IN ADVANCE OF CHECK-IN DATE will forfeit the package purchase price. By making a reservation in response to this promotion, you are agreeing to this policy. Offer expires 12 months from the date of purchase.


Situs poker online – What to know?
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What you ought to mind while online gambling poker?
The excitement of poker is improving around the globe each day. It is actually a short keep an eye on to treasures as well as elegance. We have the ability to raise try and taking part in poker proficiently by speaking about some rules. One of the suggestions is to consider courses in relation to online gambling poker. We ought to revenue and re-study quantities in guides we definitely have the basic facts are determined nicely well before as consultant are prepared for in accomplishing good quality. Proclaiming plus making reference to will without a doubt assist us understand it far better. If anything drops smooth to see sensation suitable, we can easily very easily hold the motivation to figure out why it could possibly not.
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We need to produce a mentally accrued make a take note of difficult scenarios; therefore we could quickly evaluate them at yet another time. If we are taking part in on the net, we ought to look into the palm background also look at it once we are carried out acquiring a great time. Looking at and searching at palms and wrists and wrists much outside the dish meal table is really judi poker online very successful way to obtain info for comprehending, nonetheless we ought to right now see that. Extremely regularly, our company is moving too completely get check out increased inside of the transform and determine to mobile phone no matter. Wind up anybody was miscalculating his coronary heart set up in addition to our bank account kings withstood up; consequently we simply shrug plus keep on.
Nowadays, gambling with casinos is becoming more and more popular. Let us see what is casino, how gambling is performed in casinos and why gamblers go for casino gaming.
Casino is nothing but a place for playing gambling related games. Thus as a whole, we can denote a casino simply as a gambling house. The users can be able to perform a variety of gambling activities in these casinos, as per their wish. Apart from gambling, casinos are also noted for the hosting of various live entertainment shows like sports events and stand-up comedy shows and so on.
The word gambling refers to a risky game with a great potential for making money. We can also say it as a betting game with an expectation of getting a desired outcome, thereby making huge amount of money. Gambling is just like coin tossing. The user will get an unexpected amount of money, if he gets the desired result. On the contrary, there is also a chance of facing a great loss of money, if he gets the opposite result. Thus it is said that gambling usually has two faces (good and bad), as tossing a coin (head and tail).
Gambling was once considered as an illegal (un-reputable) activity, but in the today's modern world, it is being declared as a fund raising activity. Off course, it is facing a tremendous growth because of its money making possibilities. The funds raised through gambling are generally utilized for various health care activities and charity funding activities and so on.In addition to money making, gambling can also be performed for some other reasons like acquiring several material goods, time pass and excitement and for socialization purposes.
Risky stocking and trading activities and so on.
It is a risky process with a chance of even facing a huge loss.
All the casino gamblers should follow a common rule that they must be in an age between sixteen and twenty one, in order to perform gambling. All other age groups are strictly prohibited from playing pokerqq games in most countries.
It is being reported that by the year of 2011, United states of America (USA) had generated a higher revenue rate of around 58,030$M via casino gaming.
Among the wide array of games, the wagering course that may make us provide the most fun-packed fascinating encounter as well as get excited to the major could possibly be gambling establishment games. However in dominoqq games' condition, fortune symbolizes a crucial part in dissatisfaction and also the success from the game. Combined with lot of money numerous reasonable contemplating is enough to have the internet dominoqq online games. Nowadays on the web gambling establishment game titles are trending one of many people which is because of the reality that these particular online games are not as a result rigid to do when comparing on the online games in the genuine dominoqq facilities. These dominoqq services are certainly not therefore affordable to deal with. Plus when experiencing and playing the games you have to be careful to get the cash.
This is usually a factor that is annoying and you also can be diverted for the amusement applications that can be found in the internet dominoqq middle management. They will help you to spend funds far more other and over the games. And you also must follow the suggestions that happen to be advised concerning the men and women in the establishments. They can have the outfit rules. Most of these limits are prior inside the on the web dominoqq websites. You possibly will not be fault to execute any sort of game titles within the internet site. Therefore you could very well get games' variety get and also to try. Basically exactly the same online game will be the many types. These variants can be different throughout the recommendations. Therefore while you are prone to carry out the general game, in the beginning you have to look at suggestions in the games along with the situations. Only you may undoubtedly exactly execute using the total game without any problems.
Should you be not prepared to place cash into ion gambling establishment internet sites in evaluating the gambling establishment online games however, you have ample appeal to you can easily opt for the Amount Down payment Dominoqq entertainment web sites. This can let the online games to examine free of charge and you will definitely conduct using the tests while using reward that is provided for your consideration initially. This is the smart choice to discover the gambling establishment betting encounter. That will help you try it out should you be considered a new comer towards the internet dominoqq world. Once you have expertise you will spend the money for money and start to perform with all the game titles and natural stone the internet situs dominoqq terpercaya planet.


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Ross Prieto, Former Surfside Inspector: 5 Fast Facts You Need to Know
By Erin Laviola
Updated Aug 4, 2021 at 3:38pm
Getty/City of Surfside Ross Prieto was a former Surfside, Florida, building inspector.
Rosendo "Ross" Prieto previously served as a building official for the town of Surfside, Florida. The town received a report highlighting "major structural damage" at Champlain Towers South community in October 2018, according to government records.
Prieto downplayed the seriousness of the report, according to NPR. The outlet reports Prieto told members of the condo association just weeks later that the building was "in very good shape" and safe.
Prieto is no longer a Surfside employee, NPR reported. According to the Miami Herald, he left the position in 2020.
The Champlain Towers South collapsed early in the morning on June 24th. As of this writing, at least 32 people have been confirmed dead and more than 100 others remain unaccounted for, CNN reported.
1. The 2018 Structural Report Called for 'Extremely Expensive Repairs' to the Condo Tower
Town of Surfside, FloridaA consulting firm recommended extensive structural repairs to the condo tower during a 2018 review.
The Champlain Towers South Condominium Association hired Morabito Consultants in 2018 to analyze the condo for repairs. It was a standard procedure to get the building's "40-year Recertification," as required by the town of Surfside and Miami-Dade County, the firm explained in a news release.
Morabito's task was to "understand and document the extent of structural issues that require repair and/or remediation in the immediate and near future," according to the report that was made public by the city of Surfside. Morabito Consultants sent the report to the treasurer of the condo association on October 8, 2018.
The report listed several serious structural issues that needed to be repaired without delay. It also described flooding, water infiltration in several areas and cracks in the concrete. The firm cautioned that if the problems were ignored, the "concrete deterioration" would "expand exponentially." Morabito added that the repairs needed would be "extremely expensive." The AP reports the estimated cost for repairs was $9 million.
2. Prieto Met With the Condo Association in November 2018 & Said the Building Was 'In Very Good Shape'
Town of Surfside, FloridaRosendo Prieto met with the Champlain Tower South Condominium Association two days after receiving the structural report that outlined necessary repairs.
Prieto met with the Champlain Tower South Condominium Association on November 15, 2018, according to NPR. The outlet obtained the minutes from the meeting and reported that Prieto expressed optimism about the condition of the tower condo. The minutes of the meeting reportedly include that Prieto had reviewed the "structural engineer report" and that it "appears the building is in very good shape."
The town of Surfside released an email Prieto sent the next day. In the message, he didn't directly mention the structural report. He wrote the meeting at Champlain Tower South had gone "very well" and that the "response was very positive from everyone in the room."
Prieto added that "all main concerns over their forty-year recertification process were addressed. This particular building is not due to begin their forty-year until 2021 but they have decided to start the process early which I wholeheartedly endorse and wish that this trend would catch on with other properties."
NPR's Weekend Edition Sunday spoke with a Champlain Tower South resident, Susana Alvarez, who attended the meeting on November 15, 2018. She told the outlet that town officials reassured them their building was safe:
I want you to know that in 2018, we had a board meeting. And we sat there with the town of Surfside. And the town of Surfside said to us that the building was not in bad shape, that the building was not in bad shape. That is what they said, OK? The structural engineer has been around for a while. We took out $15 million to fix that building at his say-so. No one ever, ever, ever told us that this – that that building was in such bad shape – no one, no one.
It makes me think that the engineer did not properly do his work. I mean, this doesn't just happen. This was in a matter of seconds, out of nowhere.
3. An Email Released by the Town of Surfside Confirms Prieto Had Received the Structural Report But He Says He Doesn't Remember Receiving the Report
Town of Surfside, FloridaRosendo Prieto received the structural report, as confirmed by emails released by the town of Surfside.
Prieto received the structural report a few weeks after it was submitted. An email released by the town of Surfside shows he was sent the report on November 13, 2018. It's unclear when, or if, he read it.
Prieto told the Miami Herald he doesn't remember receiving the report or the email.
Morabito Consultants put out a statement following the deadly collapse. In the news release, the firm stressed that at the time of the disaster, "roof repairs were underway" at the building. But no work had been done to fix the concrete problems, which the firm had warned would worsen if they weren't addressed promptly.
Morabito Consultants was not involved in restoration. The statement explains, "Our firm exclusively provides engineering consulting services. We do not provide construction-related services, such as building repair and restoration contracting." The firm added, "We are deeply troubled by this building collapse and are working closely with the investigating authorities to understand why the structure failed. As we do so, we also continue to pray for all those impacted by this tragic event."
4. State Records Show Prieto Has Been a Licensed General Contractor & Inspector For Several Decades
Florida Department of Business and Professional RegulationRosendo Prieto has been a licensed building inspector for several years.
Prieto has been working in the construction industry for several decades. According to public records on the Florida Department of Business and Professional Regulation website, Prieto has been a licensed general contractor since at least 1991. He received a license as a certified roofing contractor in 1996.
Prieto has also been certified as an inspector since the mid-1990s. He received both "Standard Inspector" and "Standard Roofing Inspector" licenses in 1997. His current "Building Code Administrator" license has been active since November 2006.
Prieto was the assistant director of building and zoning for the village of Miami Shores, Florida, when the Biscayne Kennel Club collapsed in 1997 during a construction project, killing two workers. Prieto told The Associated Press after the deadly dog track collapse in 1998 that inspectors had visited the project several times and "everything was going according to plan." He added, "From what I hear, this is just a construction accident. Accidents can happen."
Prieto told the Sun-Sentinel he himself had visited the dog track project on inspections three or four times. "
5. Prieto Was Working as an Interim Building Official in Doral, Florida
Doral, Florida Rosendo "Ross" Prieto has been working as a Building Official in Doral.
Prieto was most recently working for the city of Doral, Florida, which is within Miami-Dade County. He was serving as an interim building official, CNN reported.
A county document listing "Building Officials" included Prieto's name for the city of Doral. The list was last updated on May 13, 2021.
A search of the city of Doral's website also brings up Prieto's name as the "Interim Building Official." But he is not included on the "Building Department" home page or on the "Building Department Personnel" page.
The Wall Street Journal reports Prieto has since been placed on leave from his current job.
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Rosendo "Ross" Prieto is the former Surfside, Florida, building inspector who told the Champlain Tower South condo association the building was safe in 2018.

redpajama_set_name: RedPajamaCommonCrawl


the dorsal stream: Happy Birthday, Bro!
I don't see my brother as much as I'd like, and we haven't watched The Sure Thing — together anyway — for ages. And it might be a little while until we get to see it again.
Happy Birthday, Bro: I'm talking to you cordless.


text:
RIP. Mike Dobbin...( a REAL Red ).
mufcpavlos
Discussion Starter • #1 • Feb 2, 2009
My friend called me yesterday and told me the bad news Mike Dobbin the secretary of the London's branch club died on thursday. I had the luck to meet him 3 years ago when i came for the first time in manchester. He was a great man! He spent his life for united. he travelled all over the world to follow united. Europe, USA, Africa, Asia everywhere united played he was there! He is part of the club's history!
Rest In Peace my friend Mike. UNITED UNITED UNITED!
SALFORD RED
Thanks for posting that Pav, only met Mike once many years ago, never knew him personally or anything but he's known by thousands of reds.
(taken from Red News, there's more but i think it would be disrespectful to Mike and Red News for me to copy/paste onto our forum here).
It is with great sadness - devastating news to all who knew him well - that we have to inform you that well known United fan Mike Dobbin passed away of cancer this morning.
A great United fan - in fact though he'd loathe such a tag put on him, I'd say he's probably United's greatest fan - having only missed something like 6 games in 30+ years and close to seeing United 2000 times in 40+ countries. Wherever United were playing, Mike was there. He was an inspiration; kind, helpful to all who wore a United scarf and all round decent fella. One of the good guys.
Mike was a RN reader, and we have known him for a long, long time, always smiling when we'd see him as we were selling as he approached with a plastic bag full of programmes and wearing a shirt from whichever exotic destination had played in last. We will miss that. Boy will we and so many of us will miss him. I don't think there's anyone with a bad word for him, always keen to help others, moving from Manchester to London and running the London Fan Club these past 30+ years, helping so many in the process. Unassuming, Mike never has bragged about his record of going to games, just you know that if United were playing, he'll be there.
He liked a nice pint and it was always great to share his company. Mike only found out he was seriously ill recently and the subsequent concern amongst matchgoing support was like nothing I've known before such was the care and love for a United supporting legend; an institution at United, full stop. There will never be another to match Mike at United.
Players come and go but the likes of Mike were there, game in, game out, all his life and on Saturday we'd like all Utd fans to take a moment to remember Mike Dobbin. RIP.
Our thoughts and best wishes go to his immediate family and all members of the United family who knew Mike. It is a sad day.
Details of Mike's funeral arrangements have been confirmed.
It will take place at 3pm on Friday February 6th (an appropriate date and time for a Red if ever there was one) at Blackley (aka Blakeley) Crematorium, Victoria Avenue, Blackley, Manchester, M9 8JP.
BIG RESPECT to a man who devoted so much of his life supporting United and helping fellow Reds. He knew the meaning of the word UNITED.
You're with The Babes now Mike. Rest peaceful.
I read on another forum that he watched us home and away for over five decades. That's a massive level of commitment from anybody. What makes it more special is that he was London based and travelled to Manchester for every home game. If anybody is deserving of the title 'Top Red' then it's Mike Dobbin. The club needs more supporters like him.
I think it's quite fitting that the last match he got to see us play was in Japan when we became champions of the world.
What zuco said, glad he got to see us world champions. Nice touch.
never even knew the guy personally, but thanks for the article......can tell straight away this guy was the best, a shame to hear of his passing, but you can bet he'll be watching still from afar.
Arse Bandito
never had the pleasure to meet him, even taking the london supporters bus, but rest in peace and best wishes at this time to all his family.
the ultimate red, you can tell.
i'm sure he'll be watching down on us with sir matt and the babes, again cheering us on to victory like he has done thousands of times before
Rooneys_Temper
TROLOLOLOL
RIP..............The Red Flag Will Be Flying High For You
Very dedicated to the cause a great fan .
hdcantona
Member of the Year 2012 & 2013
Rooneys_Temper said:
or at half - mast
Can't say i heard of him but after reading up on him even i find the news a bit sad.
Thankful for people like him
Rest well Mike
He sounds like a fantastic guy and a top, top fan.
Rest In Peace .
Lets hope he is looking on in heaven when we match the dippers 18 titles .
peterswellman
RIP fellow red.
Check the Milkman
I'm so gorgeous they want to arrest me
R.I.P Mike Dobbin
MUT all-time great
Sad indeed. Probably an unmatched level of commitment from what I've read, may he forever rest in peace.
Giggsy the Legend
RIP Mike Dobbin.
Rest In Peace, a true red devil
Ajant
R.I.P Mike Dobbin.
support us from the heavesns when we beat the dippers' 18 titles
mufc-ok Feb 3, 2009


Place of Supply under GST is explained thoroughly in this article! GST has been implemented but people are confusing in adapting its rules and necessary provisions. GST has merged 17 types of taxes in itself reducing the cascading effect or multiple taxations on a single income such as sales tax, service tax etc. GST structure is divided into four categories CGST, SGST, IGST, UTGST.
The levy of Tax under the GST act is based on two things from which one is,"Location of Supplier and another one is "Place of Supply".
Location of Supplier – It is the registered place of supplier's Business.
Place of Supply – In GST, it is the registered Place of recipient's business but there are certain rules which we will go to discuss in this article.
What is Place of Supply under GST?
In order to determine the nature of goods, it is important to understand the "Place of Supply" of such goods and services. The reason behind such is determining whether CGST and SGST or IGST will be applicable on such.
CGST and SGST are applicable on intrastate supplies while IGST is applicable on interstate supplies and this can be determined by the Place of Supply.
Q. What will Place of Supply where the supply involves a movement of goods, whether by the recipient or supplier or third person?
Ans. Place of Supply shall be determined by the location of the goods at the time of Final Delivery to the recipient.
Example – Mr. Raj, a trader of Rajasthan received an order to supply his goods to a dealer in Maharashtra. For this, Mr. Raj ordered his one of the branch in Banaras to supply goods to the dealer in Maharashtra. In such a case, goods have moved from Banaras to Maharashtra thus the place of supply will be Maharashtra and in such a case IGST will be applicable.
Q. What will Place of Supply where the supply of goods is made on the direction of third Party, it may be an agent or not?
Ans. Where the goods are delivered to the recipient or any other person on the direction of the third party, the Place of Supply shall be determined as the registered place of business of the third party, irrespective where the goods have been delivered.
Example – A dealer of Rajasthan deals with a dealer of Gujarat to supply the goods but the dealer of Gujarat directed the dealer of Rajasthan to supply the goods to Delhi at one of his customers. Here in the given case, although the goods have been moved to Delhi directly the place of Supply will be considered as the principal place of business of the third party i.e. Gujarat.
Q. What will be the place of Supply in GST where it does not involve the movement of goods?
Ans. Where the supply does not involve any movement of goods then place of supply shall be the location of goods at the time of delivery to the recipient.
Example – Mr. Arjun has a business establishment in Bangalore but purchased a workstation in Delhi from Ms. Sunita having the business establishment in Delhi. Here the supply of goods does not involve any movement as it as an immovable property. Hence Place of Supply in such a case shall be Delhi, the location of goods at the time of Final delivery. This will be considered as an intrastate Supply and thus attract CGST and SGST.
Q. What will be the place of Supply in case of installation or assembling of goods at the site?
Ans. Here the place of supply will be the place at which goods are assembled or installed at the site.
Example – Ms. Komal has appointed Ms. Seema having a business establishment in Lucknow for the installation of the lift in his workstation for his Lucknow branch. Then in such a case, Place of Supply shall be deemed to be the place where the goods are installed. Place of Supply in such a case will be Lucknow, thus CGST and SGST will be applicable.
Q. What will be the Place of Supply where the goods are being supplied on a board like a vehicle, vessel, train, aircraft, or motor etc?
Ans. In above case, Place of Supply shall be the location where the goods are boarded on the conveyance.
Example – Flight is ready to travel from Delhi to Goa. Food is served by the third agency to be served on the flight during the travel. Hence, the Place of Supply will be Delhi because the first location where the goods are boarded to served on the flight in Delhi.
Q. What will be the Place of Supply where the same could not be determined by Sub Section (2), (3),(4), and (5)?
Ans. In such a case, Place of Supply shall be determined in such a manner as may be prescribed.
Q. What will be the Place of Supply where the services are provided in respect of an immovable property?
Ans. In respect of immovable property, Place of Supply shall be the place where the immovable property is situated or supposed to be.
Example – Ms. Swati has given a contract of construction of workstation in Punjab to Mr. Vikas has a business establishment in Gujarat. In such a case, immovable property is supposed to be located in Punjab. Hence, the Place of Supply shall be Punjab.
Other Examples – Hotel Accommodation etc.
Q. What will be the place of Supply where the service required the physical presence of both the service provider and service receiver?
Ans. Where the service required the physical presence of both the service provider and service receiver, Place of Supply shall be placed where the services have been provided.
Example – Ms. Ruchi has provided beautician services to Ms. Poorva in Rajasthan. Here the service required the physical presence of both Ms. Ruchi and Ms. Poorva hence the place of supply shall be the place where the services are performed. In such a case, Place of Supply shall be Rajasthan.
Other Example – Catering, Restaurant, fitness, personal grooming, consultancy and health services etc.
Q. What will be the Place of Supply in respect of admission to an event or amusement park?
Ans. In respect of admission to an event or amusement park, the place of supply shall be the location where the event is held or amusement park is located.
Example – Mr. Arpit organized an event in Hyderabad. Here the Place of Supply shall be the location where the event has to take place i.e. Hyderabad.
Q. What will be the Place of Supply in respect of organization to an event or other ancillary activities?
Ans. The Place of Supply in case of the organization of an event if take place with the registered person then it shall be the location of such registered person.
But where the organization of an event takes place with the unregistered person, place of supply shall be the place where the event is held.
Q. What will be the Place of Supply in case of Transportation of goods?
Ans. The Place of Supply in case of Transportation of goods shall be the location of the recipient if he is registered. However, in case of the unregistered person, the Place of Supply shall be the location of goods where they have started for transportation.
Q. What will be the Place of Supply in case of Transportation of passengers?
Ans. Place of Supply shall be the location of the passenger if he is registered or the location where he embarks his journey if he is unregistered.
Q.What will be the Place of Supply in case services are given on a conveyance like a vessel, vehicle, train or aircraft?
Ans. Where the services are provided on a conveyance, the Place of Supply shall be the first schedule point of departure of that conveyance.
Q.What will be the Place of Supply in case of Telecommunication Services?
Ans. The Place of Supply in case of prepaid mobile or Internet connection shall be determined as the billing address of the recipient.
However, in case of installation of Fixed Leased line, Internet leased line, cable or dish antenna, Place of Supply shall be the place of their installation.
In case of telecom services provided through the internet banking or E-wallets like Paytm, Free recharge, the Place of Supply shall be determined as the address of the recipient available on the record of such companies.
Q.What will be Place of Supply in case of services provided to Banks or other Financial Institutions?
Ans. The Place of Supply shall be the location of the recipient of the service available in the records of Banks or other Financial Institutions (in case of account holders).
However, services provided by banks or financial institutions to the non account holders or whose address is not mentioned in its record Place of Supply shall be the location of the supplier of service.
Example – Individual, non account holder taken the service of Demand Draft from the Bank. In such a case, Under GST, Place of Supply shall be the location of Bank.
Q. What will be the Place of Supply in case of Insurance Services?
Ans. The Place of Supply in case of Insurance Services shall be the location of the recipient in case he is registered. And for the non registered persons, In GST, Place of Supply shall be the location of the recipient maintained in the records of the service provider.


We are holding this meeting to explore how reintegration programs contribute to the well-being of children coming out of armed groups and forces, and to hear more about their impact on their communities. Reintegration is an important step both in protection and prevention. By helping these children, we also help ensure they do not return to an armed existence and we help break the cycle of violence that is all too common in conflict situation.
As you know, I am just back from Myanmar. There I discussed this issue with our colleagues at UNICEF, who—despite intense challenges in other parts of the country—are doing an amazing job with the children being released by the Tatmadaw army. A large part of my engagement was advocating with the Government to finalize implementation of the Joint Action Plan to end and prevent the recruitment and use of children by the national army. Since 2012, over 800 children and young adults recruited as children have been released from the Tatmadaw. In accordance with the Action Plan, they received reintegration services provided by UNICEF, ILO and NGO partners. They have been provided with education and vocational training to find a place in a society where poverty has been an important driving factor for underage recruitment.
In Myanmar, there are currently 7 non-state parties to conflict listed by the Secretary-General for recruitment and use of children. Boys and girls who escape or are released from these armed groups face an uncertain future, with little or no opportunities to benefit from services to rebuild their lives.
Unfortunately, the challenges faced by the children of Myanmar are not unique. In every country on my agenda, we know of missed opportunities – due to lack of resources, capacity, or access – to help the most vulnerable recover from the trauma of war. These are also missed opportunities to prevent them from falling back into violence.
We all agree on the importance of reintegration, but, in a world faced with multiple and complex crises, we still struggle to find adequate resources for the short- and long-term support of children recovering from the trauma of war.
The mere fact that today's event is co-sponsored by so many Member States speaks volumes about our common desire to change this situation.
In a few minutes, you will hear Kabba Williams talk about his personal experience with reintegration programs in Sierra Leone. I know he will speak more eloquently than any of us could about the importance of providing education and vocational training to learn the skills needed to adapt to civilian life.
I would like to set the stage for him by highlighting other guiding principles for our action.
First and foremost, the best interest of the child must be at the heart of any program established for boys and girls. Working with communities is also key, to overcome stigma and to help children reunite with their families. The provision of physical and mental health support to overcome the long-term effects of war is the cornerstone of these efforts.
The specific needs of girls cannot be overlooked. Too often, they are hidden victims of conflict who suffer stigma, rejection by their families and communities, in addition to psychological and physical consequences of sexual violence and other abuse. Equally, boys may need specialized programs—so our call is for gender-sensitive programming to tailor assistance most appropriately to meet the needs of the children.
Allow me to speak about solutions.
Conflict has evolved—so it is time to reconceptualize reintegration and its role in preventing future conflict. I strongly believe that reintegration support should be fundamental to emergency, recovery and peacebuilding efforts. It should also be part of our work to prevent conflict. Currently, despite the best efforts of UNICEF, national authorities, NGOs and others, it is often a very small part in overall recovery efforts, and not at the core of restabilizing and rebuilding societies torn apart by war. One recent study by the Clingendael Institute of the Netherlands demonstrated that in the DDR triad, Demobilization and Disarmament received massively more funding than Reintegration. For children, this has particularly dire consequences.
We are all looking towards 2030 and how we can coalesce support around 16 Sustainable Development Goals. The theme of children and their well-being is at the center of many of them. By taking a harder look at the importance of reintegration programs in conflict and post-conflict settings, we will better understand how paying attention to this small but strategic piece of the puzzle will help us reach those Goals. An important element that I will be focusing on in the coming months and years is that of treating children involved as agents of change—Kabba here personifies that ethos and has an inspiring story to tell.
But the reality remains: year in, year out, thousands of boys and girls need support for reintegration in places like the Central African Republic, Somalia, South Sudan, Iraq, and many other challenging environments.
Providing adequate, meaningful and long-term services is a huge task. With resources currently available, this is sometimes a mission impossible. Most importantly, reintegration and rehabilitation efforts must be sustainable through time and cannot depend on just international efforts. Capacity building and resources for national sustainable reintegration and rehabilitation efforts must also be supported sooner rather than later.
To address this problem, my office is working on a collaborative project to support reintegration work in the field and establish a long-term, multi-year funding mechanism for the reintegration of children. This will include a specific focus on girls, and enhance existing work in psychosocial and education programming, as well as vocational training. This Global Reintegration Fund for Children is a mechanism that our colleagues at the UN could tap into, but its funding that would be also available for reintegration work by Member States recovery from conflict, NGOs and regional organizations.
We are working with the World Bank to set up a fund that will be available to support existing programming solutions on reintegration, as well as provide space to explore new innovations that can be accessible to all sectors engaged in this endeavor from UN agencies to national reintegration offices, from NGOs to regional organizations. During the initial 5 years, pilot programs will be identified and run in 2-3 countries on the CAAC agenda. The idea is to use best practices knowledge to create new elements for these programs to meet the needs of children in today's conflicts and to evaluate their impact based on a) sustainability and ownership; and b) non-voluntary re-recruitment for lack of options.
For this, it is fundamental to establish proof of concept that programming for these children has to be longer than the current norm of six months. It is widely acknowledged that longer programs would be the most beneficial, but we require additional evidence and experience in this regard. With the enormous body of knowledge currently available from UNICEF, War Child and others, combined with new information gleaned from the pilot projects, including application at national level, we may be able to create a new paradigm that the minimum program should be 3 years or 5 years or longer, and then seek the funding to support them ensuring national ownership and sustainability at the end. This could revolutionize our approach and give child protection actors access to funding to transform the lives of these children. It can also impact on the ground by making reintegration options more desirable to those children still engaged and by providing the assistance needed to ensure no reincidence.
Down the road, I would eventually like to establish a roster of child protection specialists ready to be deployed to support these programs when and where they are needed. Child protection officers and advisers are a crucial part of capacity both at emerging actions and in sustainable endeavors, they are often undervalued and under rated – this must stop.
I thank our Member State co-sponsors, especially Poland, and welcome today's discussions. I hope that we will come up with innovative solutions and commitments to use reintegration not only as a tool to rebuild lives, but also to rebuild communities, on a sustainable basis.
Less children should return to a life of violence simply because there is no other option. All children used and abused by, in and for armed conflict should be given a real chance to create their own future.


text:
367 Amicitia è un asteroide della fascia principale del diametro medio di circa 19,13 km. Scoperto nel 1893, presenta un'orbita caratterizzata da un semiasse maggiore pari a 2,2193109 UA e da un'eccentricità di 0,0959804, inclinata di 2,94322° rispetto all'eclittica.

Il suo nome è dedicato all'amicizia.

Note

Bibliografia

Altri progetti

Collegamenti esterni

Corpi celesti scoperti nel 1893
Amicitia

redpajama_set_name: RedPajamaWikipedia


Although he did not disclose its valuation, CEO and co-founderAndrew Rubin says it's was "a significant uptick from our series D" in 2017. At that time, Illumio had a valuation of $1.18 billion, according to Pitchbook.
Rubin recalls that when he first co-founded the company, the service Illumio provides — a specific type of cybersecurity tech, called segmentation — wasn't very well known. Illumio's brand of segmentation creates virtual "zones" in data centers and the cloud, so that that each applications can be individually isolated and secured.