NT Activity Research Articles

Specific spoilage bacteria in cold chain logistics: A study on ATP-related compounds degradation in large yellow croaker (Larimichthys crocea)

This study evaluated the contribution of three spoilage bacteria (Shewanella putrefaciens (S. putrefaciens), Pseudomonas fluorescens (P. fluorescens), and Aeromonas salmonicida (A. salmonicida)) to the degradation of adenosine triphosphate (ATP)-related compounds in fish juice and fillet systems of large yellow croaker during cold chain logistics. The contribution of spoilage bacteria was explored by determining the microbial counts, ATP-related compounds, and associated enzyme activities. The results revealed that the S. putrefaciens, P. fluorescens, and A. salmonicida have the highest activities in 5′-nucleotidase (5′-NT), acid phosphatase (ACP), and alkaline phosphatase (AKP), with 49.78, 162.24, and 142.3 U/gprot, respectively. The rapid degradation of inosine monophosphate (IMP) occurs with increasing 5′-NT activity. It indicates that S. putrefaciens, P. fluorescens, and A. salmonicida mainly secreted 5′-NT, ACP, and AKP to degrade IMP, respectively, and that 5′-NT was the key enzyme in degrading IMP. All three are notable producers of purine nucleoside phosphorylase (PNP) and xanthine oxidase (XOD). The results showed that S. putrefaciens is the primary bacterium in the pre-degradation phase of ATP-related compounds, and A. salmonicida presumably dominates in the post-degradation phase. This study offers critical insights into the research on spoilage mechanisms of marine fish throughout the cold chain logistics.

New Method for 5'-Nucleotidase Preparation and Evaluation of Its Catalytic Activity.

In this study, we established a new methodology for preparing 5'-nucleotidase (5'-NT) with the aim of enhancing our understanding of its enzyme activity and laying a basis for regulating the content of umami-enhancing nucleotides in pork. 5'-NT was prepared with Sephadex gel filtration and reverse-phase high-performance liquid chromatography, and its enzymatic properties and catalytic activity were evaluated. The results show that the molecular weight of the prepared 5'-NT was 57 kDa, the optimal catalytic temperature was 40 °C, and the optimal pH was 8. Zn2+, and sucrose showed inhibitory effects on the activity of 5'-NT, while K+, Na+, Ca2+, Mg2+, glucose, fructose, and trehalose promoted the activity of the studied compound. The prepared 5'-NT exhibited higher catalytic activity and selectivity against IMP compared with its commercial counterpart, while its catalytic activity against XMP was not significant (p > 0.05). In brief, we established a new methodology for preparing 5'-NT, enhancing our understanding of its enzyme activity and providing a solid basis for regulating the content of umami-enhancing nucleotides in pork through the control of endogenous 5'-NT activity.

Probiotic-Derived Ecto-5'-Nucleotidase Produces Anti-Inflammatory Adenosine Metabolites in Treg-Deficient Scurfy Mice.

Probiotic Limosilactobacillus reuteri DSM 17938 (DSM 17938) prolongs the survival of Treg-deficient scurfy (SF) mice and reduces multiorgan inflammation by a process requiring adenosine receptor 2A (A2A) on T cells. We hypothesized that L. reuteri-derived ecto-5'-nucleotidase (ecto-5'NT) activity acts to generate adenosine, which may be a central mediator for L. reuteri protection in SF mice. We evaluated DSM 17938-5'NT activity and the associated adenosine and inosine levels in plasma, gut, and liver of SF mice. We examined orally fed DSM 17938, DSM 17938Δ5NT (with a deleted 5'NT gene), and DSM 32846 (BG-R46) (a naturally selected strain derived from DSM 17938). Results showed that DSM 17938 and BG-R46 produced adenosine while "exhausting" AMP, whereas DSM 17938∆5NT did not generate adenosine in culture. Plasma 5'NT activity was increased by DSM 17938 or BG-R46, but not by DSM 17938Δ5NT in SF mice. BG-R46 increased both adenosine and inosine levels in the cecum of SF mice. DSM 17938 increased adenosine levels, whereas BG-R46 increased inosine levels in the liver. DSM 17938Δ5NT did not significantly change the levels of adenosine or inosine in the GI tract or the liver of SF mice. Although regulatory CD73+CD8+ T cells were decreased in spleen and blood of SF mice, these regulatory T cells could be increased by orally feeding DSM 17938 or BG-R46, but not DSM 17938Δ5NT. In conclusion, probiotic-5'NT may be a central mediator of DSM 17938 protection against autoimmunity. Optimal 5'NT activity from various probiotic strains could be beneficial in treating Treg-associated immune disorders in humans.

Effects of curcumin‐based photodynamic treatment combined with low‐temperature storage on shelf life and purine content of Litopenaeus vannamei

In this study, the effects of photodynamic treatment on the shelf life and purine content of Litopenaeus vannamei under low-temperature storage conditions were investigated. Indicators of shelf life were detected to evaluate the shelf life, and the change of purine content during storage detected by HPLC. The results showed that compared with cold storage, the shelf life of shrimp was extended from 3 days to 8 days with the treatment of microcrystalline storage. Moreover, the shelf life of shrimp was extended to 12 days when stored with the photodynamic treatment combined with microcrystalline storage (PDT + MT). The content of hypoxanthine decreased significantly after 24 h storage, which may be related to the inhibition of 5′-NT, PNP, ADA, and XOD activities. This study provided scientific support for the popularization and application of photodynamic technology in the storage of shrimp. Novelty impact statement Photodynamic technology is helpful to prolong the shelf life of shrimp during low-temperature. Photodynamic combined with low-temperature can affect purine content by affecting purine metabolism enzymes.

Effect of Maternal Marginal Zinc Deficiency on Development, Redox Status, and Gene Expression Related to Oxidation and Apoptosis in an Avian Embryo Model.

Maternal severe zinc (Zn) deficiency resulted in growth retardation and high mortality during embryonic development in human. Therefore, this study is aimed at evaluating the effect of maternal marginal Zn deficiency on the development and redox status to avoid severe Zn deficiency using an avian model. A total of 324 laying duck breeders at 214 days old were randomly allotted into 3 dietary Zn levels with 6 replicates of 18 ducks per replicate. The birds were fed experimental diets including 3 dietary supplemental Zn levels of 0 mg/kg (maternal Zn-deficient group, 29.2 mg Zn/kg diet), 60 mg/kg (maternal Zn-adequate group), and 120 mg/kg (maternal Zn-high group) for 6 weeks. Dietary Zn levels had on effect on egg production and fertility (P > 0.05), whereas dietary Zn deficiency decreased breeder plasma Zn concentration and erythrocytic alkaline phosphatase activity at week 6 and inhibited erythrocytic 5′-nucleotidase (5′-NT) activity at weeks 2, 4, and 6 (P < 0.05), indicating that marginal Zn-deficient status occurred after Zn depletion. Maternal marginal Zn deficiency increased embryonic mortality and contents of superoxide anion radical, MDA, and PPC and reduced MT content and CuZnSOD activity in duck embryonic livers on E29. The MDA content was positively correlated with embryonic mortality. Maternal marginal Zn deficiency increased BCL2-associated X protein and Caspase-9 mRNA expressions as well as decreased B-cell lymphoma-2 and MT1 mRNA and signal AKT1 and ERK1 protein expressions (P < 0.05). Breeder plasma Zn concentration and erythrocytic 5′-NT activities at week 6 were positively correlated with GSH-Px activity and GPx, MT1, and BCL2 mRNA expressions in embryonic livers on E29. In conclusion, erythrocytic 5′-NT activity could be more rapid and reliable to monitor marginal Zn-deficient status. Marginal Zn deficiency impaired hatchability and antioxidant defense system and then induced oxidative damage and apoptosis in the embryonic liver, contributing to the greater loss of duck embryonic death.

Sequence-Activity Relationships for the Snf7 Insecticidal dsRNA in Chrysomelidae.

The responsiveness of insects to oral delivery of insecticidal dsRNA has been shown to be dependent on dsRNA length and sequence match. Previous work with the western corn rootworm (WCR, Diabrotica virgifera virgifera; Coleoptera: Chrysomelidae) demonstrated that at least one ≥21 nt match must be present in the DvSnf7 dsRNA of approximately ≥60 base-pairs (bp) for activity. Further data is needed on the activity of <21 nt matches along with characterization of relationship between activity and the number of ≥21 nt matches. To characterize the sequence–activity relationship for insecticidal dsRNA further, the activity of orthologous Snf7 dsRNAs with 19, 20, and 21 nt contiguous matches against WCR was compared. Neither 19 nor 20 nt sequence matches were active, supporting that a ≥21 nt sequence match is required for activity. The relationship between the number of 21 nt matches with activity of Snf7 dsRNA orthologs from several Chrysomelid species was characterized using WCR and Colorado potato beetle (CPB, Leptinotarsa decemlineata; Coleoptera Chrysomelidae). For WCR, there was a strong relationship between an increasing number of 21 nt matches and increased activity (i.e., lower LC50 values). A similar relationship was observed for CPB with an exception for a single ortholog, which may be related to the exceptionally high rate of polymorphisms in CPB. Overall, these results demonstrate a general relationship between the number of 21 nt matches and activity, and this relationship could be used to inform a testing and assessment plan for an ecological risk assessment for an insecticidal dsRNA.

Association of human cytomegalovirus (HCMV) neutralizing antibodies with antibodies to the HCMV glycoprotein complexes

BackgroundHuman cytomegalovirus (HCMV) causes asymptomatic infections, but also causes congenital infections when women were infected with HCMV during pregnancy, and life-threatening diseases in immunocompromised patients. To better understand the mechanism of the neutralization activity against HCMV, the association of HCMV NT antibody titers was assessed with the antibody titers against each glycoprotein complex (gc) of HCMV.MethodsSera collected from 78 healthy adult volunteers were used. HCMV Merlin strain and HCMV clinical isolate strain 1612 were used in the NT assay with the plaque reduction assay, in which both the MRC-5 fibroblasts cells and the RPE-1 epithelial cells were used. Glycoprotein complex of gB, gH/gL complexes (gH/gL/gO and gH/gL/UL128–131A [PC]) and gM/gN were selected as target glycoproteins. 293FT cells expressed with gB, gM/gN, gH/gL/gO, or PC, were prepared and used for the measurement of the antibody titers against each gc in an indirect immunofluorescence assay (IIFA). The correlation between the IIFA titers to each gc and the HCMV-NT titers was evaluated.ResultsThere were no significant correlations between gB-specific IIFA titers and the HCMV-NT titers in epithelial cells or between gM/gN complex-specific IIFA titers and the HCMV-NT titers. On the other hand, there was a statistically significant positive correlation between the IIFA titers to gH/gL complexes and HCMV-NT titers.ConclusionsThe data suggest that the gH/gL complexes might be the major target to induce NT activity against HCMV.

Determination of nucleotide and enzyme degradation in haddock (Melanogrammus aeglefinus) and herring (Clupea harengus) after high pressure processing.

BackgroundThe degradation of nucleotides and their enzymes had been widely used to evaluate fish freshness. Immediately after fish death, adenosine triphosphate (ATP) degrades into inosine-5-monophosphate (IMP) via adenosine-5-diphosphate (ADP) and adenosine-5-monophosphate (AMP). IMP degradation continues to produce inosine (ino) and hypoxanthine (Hx) and further deteriorates the fish by producing xanthine and uric acid. The dephosphorylation of IMP to Ino is carried out by the enzyme 5′-nucleotidase (5′-NT), whereas the degradation of Ino to Hx is carried out by the enzyme nucleoside phosphorylase (NP). This study assesses the application of high pressure processing (HPP) in two species of fishes; haddock (Melanogrammus aeglefinus) and herring (Clupea harengus) as a means to extend the shelf-life by slowing down the rate of nucleotides degradation.MethodsHaddock (Melanogrammus aeglefinus) and herring (Clupea harengus) fillets were subjected to HPP at 200, 250 and 300 MPa for 1 and 3 min before being stored for 14 days. In addition, 5′-NT and NP enzyme activities were determined on both fish species that were subjected to 100–600 MPa for 1 and 3 min.ResultsAdenosine triphosphate, ADP and AMP in both haddock and herring were lower at higher pressure levels. Inosine (Ino) increased (p < 0.05) after treatment at higher pressures in both species. Hx in herring decreased significantly (p < 0.05) at higher pressures but not in haddock. K values are the ratio of Ino and Hx to all nucleotides. K values in haddock were not significantly (p > 0.05) affected by the pressure treatment. H values are ratio of Hx to the sum of IMP, Ino and Hx. H values in haddock were significantly decreased (p < 0.05) with increasing pressure level. F values are ratio of IMP to the sum of IMP, Ino and Hx. F values showed no significant effects (p > 0.05) after pressure treatment. Furthermore, K values in control herring were significantly higher (p < 0.05) than those of the pressure-treated samples. H values in herring decreased significantly (p < 0.05) with increasing pressure level. F values in herring showed no significant effects (p > 0.05) after pressure treatment. Pressure treatment brought a significant decrease (p < 0.05) in protein content in both haddock and herring. 5′-NT activity was 20–35 fold higher compared to NP activity in haddock and 15–44 fold higher than NP activity in herring. 5′-NT and NP activities decreased significantly with increasing pressure level in both species.DiscussionHigh pressure processing effectively slows down the conversion of Ino to Hx, delaying the undesirable flavour that develops in spoiling fish. The autolytic conversion of IMP to Ino by endogenous 5′-NT predominates in the earliest stages of storage is an autolytic process. However, both bacterial and endogenous NP enzymes are probably responsible for the gradual accumulation of Hx in fish. K values are recommended as a useful measurement of fish freshness.

NEUROTOXIC EFFECT OF TITANIUM DIOXIDE NANOPARTICLES: BIOCHEMICAL AND PATHOLOGICAL APPROACH IN MALE WISTAR RATS

Objective: Titanium dioxide nanoparticles (TiO2 NPs) are widely used in pharmaceutical, cosmeceutical, biomedical and industrial applications. The adverse effects of TiO2 NPs are also increasing alarmingly. The purpose of this study is to investigate the toxicity of TiO2 NPs on biochemical and histological changes in different regions of brain in adult male Wistar rats.Methods: Two different doses of TiO2 NPs (50 mg/kg b. w and 100 mg/kg b. w) administered orally for 14 d along with one control group, each group consisting of six animals. Standard biochemical methods were adopted for the estimation of enzymes alkaline phosphatase, 5’ nucleotidase, ATPases and gamma-glutamyl transpeptidase. Trace elements calcium, sodium, potassium and magnesium as well as metals like iron, zinc and copper were also estimated.Results: When compared with the control group, the enzymes ATPases, ALP, 5’-NT and GGT activities were significantly decreased in both the TiO2 NPs treated groups. Ca, Na, Fe, Cu and TiO2 contents were significantly increased in both the experimental groups, while the K, Mg and Zn contents decreased. However, the changes in the parameters studied were more in 100 mg treated group (p&lt;0.001) when compared to the 50 mg treated group (p&lt;0.05and p&lt;0.01). Moreover, it is also evident that different regions responded differently due to TiO2 NPs exposure. The changes were maximum in the cerebral hemisphere (p&lt;0.001) followed by medulla oblongata (p&lt;0.001) and cerebrum (p&lt;0.05).Conclusion: The results clearly imply that TiO2 NPs could impair the electrochemical gradient, ionic homeostasis and membrane stability in different regions of the rat brain.

Identification and functional characterization of the MdHB-1 gene promoter sequence from Malus×domestica

AbstractHomeobox 1 in Malus×domestica (MdHB-1) is a transcription factor that belongs to homeodomain-leucine zipper I (HD-Zip I) protein subfamily. According to previous reports, MdHB-1 could regulate ethylene synthesis by binding with the MdACO1 promoter, but other functions of MdHB-1 are still unknown. To reveal more clues concerning the characters of the MdHB-1 gene promoter and the functions of MdHB-1, the promoter region of MdHB-1 was cloned from the Royal Gala apple genome and recombined with the β-glucuronidase (GUS) gene in this study. This research was conducted in Nicotiana tabacum and supported by Agrobacterium-mediated transient transformation and bioinformatics analysis. Deletion analysis of the MdHB-1 promoter showed that the GUS gene could be activated by serially deleted promoters, and the activity promoted by 680 nucleotides (nt) was the lowest. The region, which is 266 nt upstream of the initiation code (ATG), was effective for GUS expression. Meanwhile, the activity of the MdHB-1 promoter (−1 057 nt), which was stronger than MdHB-1 promoter (−1 057 to −266 nt) and lack the 5′-untranslated region (5′-UTR), showed that 5′-UTR may have a positive effect on gene transcription. After the sequence analysis, the cis-acting elements that respond to hormones and environmental stresses were identified in the promoter region. The MdHB-1 promoter (1 057 nt) activity in Nicotiana tabacum was positively induced by ethrel and darkness, and it was suppressed by gibberellic acid (GA), whereas abscisic acid (ABA), salicylic acid (SA), wounding, and Pseudomonas syringae pv. tomato (DC3000) treatments revealed a slight auxo-action. These results reveal that the MdHB-1 promoter receive internal or external signals, and MdHB-1 may refer to many biological activities in apple, such as its stress response, development, and ripening.

Serum activities of adenosine deaminase, dipeptidyl peptidase IV and prolyl endopeptidase in patients with fibromyalgia: diagnostic implications.

Fibromyalgia (FM) is a chronic pain syndrome with number of symptoms that present challenge in terms of diagnosis and treatment. Patients with FM show abnormal profile of purines in plasma. In this work, we measured serum activities of enzymes involved in purine metabolism, namely total adenosine deaminase (ADE) and its isoforms (ADE1 and ADE2), ecto-ATPase, and 5'-nucleotidase (5'-NT). We also measured activity of dipeptidyl peptidase IV (DPPIV) and prolyl endopeptidase (PEP). Spectrophotometric and fluorometric methods were used for enzyme activity determinations. Enzyme activities were measured in sera of 24 patients with FM that were not undergoing pharmacological treatment during the study. Control group comprised 32 healthy control subjects. Significantly higher activities of total ADE (P=0.025) and ADE2 (P=0.011) were observed in FM patients, while no significant differences in ADE1, ecto-ATPase, and 5'-NT activities (P>0.05) were found when compared to healthy controls. Moreover, increase in the activity of DPPIV (P=0.015) and lower activity of PEP (P=0.011) were also found in the FM group. ROC analysis pointed to different diagnostic sensitivities/specificities for individual enzyme activities measured as follows: ADE (50.0/87.5), ADE2 (41.7/90.6), DPPIV (62.5/71.9), and PEP (83.3/62.5). ADE2 and PEP were shown to be independent predictors of FM, while combination of the two gives AUC of 0.786 (95% confidence interval of 0.656-0.885, P<0.05). Our results are showing that serum activities of ADE2 and PEP could be useful as biomarkers for FM diagnosis. However, relatively low diagnostic sensitivity of ADE2 and specificity of PEP must be taken into account.

Inhibitory effects promoted by 5′-nucleotides on the ecto-3′-nucleotidase activity of Leishmania amazonensis

The protozoan parasite Leishmania amazonensis is the etiological agent of cutaneous leishmaniasis. During its life cycle, the flagellated metacyclic promastigote forms are transmitted to vertebrate hosts by sandfly bites, and they develop into amastigotes inside macrophages, where they multiply. L. amazonensis possesses a bifunctional enzyme, called 3′-nucleotidase/nuclease (3′NT/NU), which is able to hydrolyze extracellular 3′-monophosphorylated nucleosides and nucleic acids. 3′NT/NU plays an important role in the generation of extracellular adenosine and has been described as a key enzyme in the acquisition of purines by trypanosomatids. Furthermore, it has been observed that 3′NT/NU also plays a valuable role in the establishment of parasitic infection. In this context, this study aimed to investigate the modulation of the 3′-nucleotidase (3′NT) activity of L. amazonensis by several nucleotides. It was observed that 3′NT activity is inhibited by micromolar concentrations of guanosine and guanine nucleotides. The inhibition promoted by 5′-GMP on the 3′NT activity of L. amazonensis is reversible and uncompetitive because the addition of the inhibitor decreased the kinetic parameters Km and Vmax. Finally, we found that the addition of 5′-GMP is able to reverse the stimulation promoted by 3′-AMP in a macrophage-parasite interaction assay. The determination of compounds that can inhibit the 3′NT activity of Leishmania is very important because this enzyme does not occur in mammals, making it a potential therapeutic target.

Vaccines Bearing Engineered HIV-1 gp41 Transmembrane and Cytoplasmic-tail Domains Elicit Rare Reactivities Targeting the C-terminal Subdomain of the MPER

Abstract Numerous vaccine strategies have failed to induce neutralizing (Nt) antibodies (Abs) against the HIV-1 gp41 MPER, the target of several broadly NtAbs. Factors contributing to this include an incomplete understanding of native MPER structure, and restricted/transient exposure of key Nt sites. Here, we describe novel MPER DNA and MPER-peptide liposome vaccines (PLVs) that best elicit Abs against N- and C-terminal (N- &amp; C-) subdomains of the MPER; these subdomains are targeted by the 2F5 and 4E10/10E8 NtAbs, respectively. DNA-prime, PLV-boost immunization of rabbits and guinea pigs (GPs) with vaccines that optimize exposure of the N-terminal subdomain, elicited low-level 2F5-like reactivities that were moderately elevated with boosting. Co-immunization with the vaccines increased durability of these responses, with preliminary results showing moderate Nt activity from GPs (but not rabbits; however, GP pre-immune sera exhibited high Nt background). DNA and PLV vaccines were optimized to expose both the N- and C-subdomains. DNA vaccines were engineered to (i) encode an N-terminal coiled-coil to support a trimeric structure, (ii) express the native gp41 TMD, and (iii) truncate the cytoplasmic tail. Lipids and peptides for PLVs were designed to optimally expose the C-subdomain. Prime-boost and co-immunization of rabbits with the DNA vaccines and mixed N- and C-subdomain PLVs elicited Abs targeting both Nt sites of the MPER, but no Nt activity. Similar immunizations are underway in GPs. Together, our results illustrate the importance of (i) the TMD in eliciting Abs against the C-subdomain, (ii) co-immunization, and (iii) perhaps of GPs as a model, for eliciting NtAb responses.

Localized thermosphere ionization events during the high‐speed stream interval of 29 April to 5 May 2011

AbstractWe analyze localized ionospheric‐thermospheric (IT) events in response to external driving by a high‐speed stream (HSS) during the ascending phase of the Solar Cycle 24. The HSS event occurred from ~ 29 April to 5 May, 2011. The HSS itself (and not the associated corotating interaction region) caused a moderate geomagnetic storm with peak SYM‐H = −55 nT and prolonged auroral activity. We analyze TIMED (Thermosphere‐Ionosphere‐Mesosphere Energetics and Dynamics)/SABER (Sounding of the Atmosphere using Broadband Emission Radiometry) measurements of nitric oxide (NO) cooling emission during the interval as a measure of thermospheric response to auroral heating. We identify several local cooling emission (LCE) events in high to subauroral latitudes. Individual cooling emission profiles during these LCE events are enhanced at ionospheric E layer altitudes. For the first time, we present electron density profiles in the vicinity of the LCE events using collocated COSMIC (Constellation Observing System for Meteorology, Ionosphere and Climate) radio occultation (RO) measurements. Measurements at local nighttime show the formation of an enhanced E layer (about 2.5 times increase over the undisturbed value) at ≥100 km altitude. Daytime electron density profiles show relatively smaller enhancements in the E layer. We suggest that the IT response is due to additional ionization caused by medium energy electron (&gt;10 keV) precipitation into the subauroral to high‐latitude atmosphere associated with geomagnetic activity during the HSS event.

5′-Nucleotidase from Vipera lebetina venom

5′-Nucleotidase (5′-NT) is widely represented in animal tissues (CD73) as well as in almost all snake venoms. In the present study, a 5′-NT isoform has been isolated from Vipera lebetina venom. The homodimeric isoform consists of monomers with molecular masses of 60 kDa. The enzyme is thermolabile and has pH optimum at 7.5. The 5′-NT activity is inhibited by metal ions Fe3+, Cu2+ and Zn2+, enhanced by Mn2+ while Mg2+ and Ca2+ have no remarkable effect. In addition to 120-kDa protein there are higher molecular forms of 5′-NT present in the V. lebetina venom. The cloning and sequencing of the 5′-NT coding cDNA resulted in 5′-truncated construct. MALDI-TOF and Orbitrap mass-spectrometry of the tryptic peptides confirmed the translated N-terminally truncated protein sequence concordance to the 5′-NT isolated from the venom. The isolated protein strongly inhibited ADP- or collagen-induced platelet aggregation.

Static magnetic field inhibits 5′ nucleotidase activity in cancerous and non-cancerous human gastric tissues

The aim of the present study is to investigate possible effects of static magnetic field (SMF) on 5′ nucleotidase (5′NT-CD73) and xanthine oxidase (XO) activities in cancerous and non-cancerous human gastric tissues in order to contribute to the elucidation of the anticancer activity of SMF. Cancerous and non-cancerous human gastric tissues removed from patients by surgical operations were used in the studies. SMF was created using two static magnets. Before and after treatment with SMF, 5′NT and XO activities in the tissue samples were measured. 5′NT activity was found to be lowered, but no significant change was observed in XO activity in the gastric tissues treated with the SMF. Our results suggest that SMF inhibits 5′NT enzyme in gastric tissues significantly. It is supposed that in addition to other proposed mechanisms, inhibition of purine catabolic activity due to inhibition of some key enzymes in the DNA turn-over like 5′NT might also play part in the anticancer activity of SMF.

Virus-like particle secretion and genotype-dependent immunogenicity of dengue virus serotype 2 DNA vaccine.

Dengue virus (DENV), composed of four distinct serotypes, is the most important and rapidly emerging arthropod-borne pathogen and imposes substantial economic and public health burdens. We constructed candidate vaccines containing the DNA of five of the genotypes of dengue virus serotype 2 (DENV-2) and evaluated the immunogenicity, the neutralizing (Nt) activity of the elicited antibodies, and the protective efficacy elicited in mice immunized with the vaccine candidates. We observed a significant correlation between the level of in vitro virus-like particle secretion, the elicited antibody response, and the protective efficacy of the vaccines containing the DNA of the different DENV genotypes in immunized mice. However, higher total IgG antibody levels did not always translate into higher Nt antibodies against homologous and heterologous viruses. We also found that, in contrast to previous reports, more than 50% of total IgG targeted ectodomain III (EDIII) of the E protein, and a substantial fraction of this population was interdomain highly neutralizing flavivirus subgroup-cross-reactive antibodies, such as monoclonal antibody 1B7-5. In addition, the lack of a critical epitope(s) in the Sylvatic genotype virus recognized by interdomain antibodies could be the major cause of the poor protection of mice vaccinated with the Asian 1 genotype vaccine (pVD2-Asian 1) from lethal challenge with virus of the Sylvatic genotype. In conclusion, although the pVD2-Asian 1 vaccine was immunogenic, elicited sufficient titers of Nt antibodies against all DENV-2 genotypes, and provided 100% protection against challenge with virus of the homologous Asian 1 genotype and virus of the heterologous Cosmopolitan genotype, it is critical to monitor the potential emergence of Sylvatic genotype viruses, since vaccine candidates under development may not protect vaccinated humans from these viruses. Five genotype-specific dengue virus serotype 2 (DENV-2) DNA vaccine candidates were evaluated for their immunogenicity, homologous and heterologous neutralizing (Nt) antibody titers, and cross-genotype protection in a murine model. The immunity elicited by our prototype vaccine candidate (Asian 1 genotype strain 16681) in mice was protective against viruses of other genotypes but not against virus of the Sylvatic genotype, whose emergence and potential risk after introduction into the human population have previously been demonstrated. The underlying mechanism of a lack of protection elicited by the prototype vaccine may at least be contributed by the absence of a flavivirus subgroup-cross-reactive, highly neutralizing monoclonal antibody 1B7-5-like epitope in DENV-2 of the Sylvatic genotype. The DENV DNA vaccine directs the synthesis and assembly of virus-like particles (VLPs) and induces immune responses similar to those elicited by live-attenuated vaccines, and its flexibility permits the fast deployment of vaccine to combat emerging viruses, such as Sylvatic genotype viruses. The enhanced VLP secretion obtained by replacement of ectodomain I-II (EDI-II) of the Cosmopolitan genotype vaccine construct (VD2-Cosmopolitan) with the Asian 1 EDI-II elicited significantly higher total IgG and Nt antibody titers and suggests a novel approach to enhance the immunogenicity of the DNA vaccine. A DENV vaccine capable of eliciting protective immunity against viruses of existing and emerging genotypes should be the focus of future DENV vaccine development.

Chemical Constituents of Apocynum lancifolium Flowers

are known in China as Luobuma and are used in Chinese folk and contemporary medicineas a medicinal tea and effective treatment for hypertension and neurasthenia and possess hypolipidemic, hepatotropic, antioxida nt,antiviral, and nephritic activity. The flowers are popular in Japan as a health drink and are sold as rejuvenating food additi vesin the battle with hypercholesterolemia and as antihypertensive sedative agents [2–5].Ground air-dried flowers of

5&amp;#39;-Nucleotidases, Nucleosides and their Distribution in the Brain: Pathological and Therapeutic Implications

Elements of the nucleoside system (nucleoside levels, 5'-nucleotidases (5'NTs) and other nucleoside metabolic enzymes, nucleoside transporters and nucleoside receptors) are unevenly distributed in the brain, suggesting that nucleosides have region-specific functions in the human brain. Indeed, adenosine (Ado) and non-Ado nucleosides, such as guanosine (Guo), inosine (Ino) and uridine (Urd), modulate both physiological and pathophysiological processes in the brain, such as sleep, pain, memory, depression, schizophrenia, epilepsy, Huntington's disease, Alzheimer's disease and Parkinson's disease. Interactions have been demonstrated in the nucleoside system between nucleoside levels and the activities of nucleoside metabolic enzymes, nucleoside transporters and Ado receptors in the human brain. Alterations in the nucleoside system may induce pathological changes, resulting in central nervous system (CNS) diseases. Moreover, several CNS diseases such as epilepsy may be treated by modulation of the nucleoside system, which is best achieved by modulating 5'NTs, as 'NTs exhibit numerous functions in the CNS, including intracellular and extracellular formation of nucleosides, termination of nucleoside triphosphate signaling, cell adhesion, synaptogenesis and cell proliferation. Thus, modulation of 5'NT activity may be a promising new therapeutic tool for treating several CNS diseases. The present article describes the regionally different activities of the nucleoside system, demonstrates the associations between these activities and 5'NT activity and discusses the therapeutic implications of these associations.

Soluble Ecto-5′-nucleotidase (5′-NT), Alkaline Phosphatase, and Adenosine Deaminase (ADA1) Activities in Neonatal Blood Favor Elevated Extracellular Adenosine

Extracellular adenosine, a key regulator of physiology and immune cell function that is found at elevated levels in neonatal blood, is generated by phosphohydrolysis of adenine nucleotides released from cells and catabolized by deamination to inosine. Generation of adenosine monophosphate (AMP) in blood is driven by cell-associated enzymes, whereas conversion of AMP to adenosine is largely mediated by soluble enzymes. The identities of the enzymes responsible for these activities in whole blood of neonates have been defined in this study and contrasted to adult blood. We demonstrate that soluble 5'-nucleotidase (5'-NT) and alkaline phosphatase (AP) mediate conversion of AMP to adenosine, whereas soluble adenosine deaminase (ADA) catabolizes adenosine to inosine. Newborn blood plasma demonstrates substantially higher adenosine-generating 5'-NT and AP activity and lower adenosine-metabolizing ADA activity than adult plasma. In addition to a role in soluble purine metabolism, abundant AP expressed on the surface of circulating neonatal neutrophils is the dominant AMPase on these cells. Plasma samples from infant observational cohorts reveal a relative plasma ADA deficiency at birth, followed by a gradual maturation of plasma ADA through infancy. The robust adenosine-generating capacity of neonates appears functionally relevant because supplementation with AMP inhibited whereas selective pharmacologic inhibition of 5'-NT enhanced Toll-like receptor-mediated TNF-α production in neonatal whole blood. Overall, we have characterized previously unrecognized age-dependent expression patterns of plasma purine-metabolizing enzymes that result in elevated plasma concentrations of anti-inflammatory adenosine in newborns. Targeted manipulation of purine-metabolizing enzymes may benefit this vulnerable population.