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1.
We have synthesized several ganglio-oligosaccharide structures using glycosyltransferases from Campylobacter jejuni. The enzymes, alpha-(2-->3/8)-sialyltransferase (Cst-II), beta-(1-->4)-N-acetylgalactosaminyltransferase (CgtA), and beta-(1-->3)-galactosyltransferase (CgtB), were produced in large-scale fermentation from Escherichia coli and further characterized based on their acceptor specificities. 2-Azidoethyl-glycosides corresponding to the oligosaccharides of GD3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GM2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GD2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), and GM1 (beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) were synthesized in high yields (gram-scale). In addition, a mammalian alpha-(2-->3)-sialyltransferase (ST3Gal I) was used to sialylate GM1 and generate GD1a (alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) oligosaccharide. We also cloned and expressed a rat UDP-N-acetylglucosamine-4'epimerase (GalNAcE) in E. coli AD202 cells for cost saving in situ conversion of less expensive UDP-GlcNAc to UDP-GalNAc.  相似文献   

2.
Reversible phosphorylations play a critical role in most biological pathways. Hence, in signaling studies great effort has been put into identification of a maximum number of phosphosites per experiment. Mass spectrometry (MS)-based phosphoproteomics approaches have been proven to be an ideal analytical method for mapping of phosphosites. However, because of sample complexity, fractionation of phosphopeptides prior to MS analysis is a crucial step. In the current study, we compare the chromatographic strategies electrostatic repulsion-hydrophilic interaction chromatography (ERLIC), hydrophilic interaction liquid chromatography (HILIC), and strong cation exchange chromatography (SCX) for their fractionation behavior of phosphopeptides. In addition, we investigate the use of repetitive TiO(2)-based enrichment steps for a maximum identification of phosphopeptides. On the basis of our results, SCX yields the highest number of identified phosphopeptides, whereas ERLIC is optimal for the identification of multiphosphorylated peptides. Consecutive incubations of fractions and flow-through by TiO(2) beads enrich qualitatively different sets of phosphopeptides, increasing the number of identified phosphopeptides per analysis.  相似文献   

3.
Macrophages treated with IFN-gamma and IL-2 before exposure to parasites develop the ability to resist infection with amastigotes of Leishmania major. In this cooperative interaction of cytokines, IL-2 can be replaced with any of several mAb directed against the beta chain of the IL-2 receptor, but not by antibodies to a number of other cell receptors or antigens. Thus, antibodies to the IL-2 receptor act as agonists of IL-2 in the induction of a biologic activity in macrophages, and macrophages, a nonproliferating cell type, respond to signals transmitted through the beta chain of the IL-2 receptor.  相似文献   

4.
We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (Tm) and structural analysis (CD spectra) of 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.  相似文献   

5.
Human umbilical blood vessels have the ability to close spontaneously following delivery at term. It has been suggested that prostaglandins may have a possible physiological role in its closure. This study investigates the effects of 6 naturally occurring prostaglandins (A1, A2, B1, B2, E2, F2a) on the umbilical blood vessels. Umbilical cords were collected from cases of normal spontaneous vaginal deliveries and cesarian section at term. A total of 41 strips of umbilical arteries and 26 strips of umbilical veins from 24 cords were used. A 4-point bioassay method was used to compare the potency of prostaglandins A1, A2, B1 and F2a with PGE2. The effect of Polyphloretin Phosphate (PPP) on prostaglandin-induced contractions was studied on umbilical artery strips from 12 cords. The 6 prostaglandins exerted a stimulant effect on the isolated strips of human umbilical arteries. Prostaglandin B2 was the most potent compound on the umbilical vein, followed by PGA2. PPP in the concentration range of 10 to 40 mcg/ml completely eliminated the responses of PGE2, F2a, A1, A2, and B1. Responses to PGB2 were considerably but not completely abolished. PPP (up to 40 mcg/ml) did not affect contractions induced by 5-hydroxytryptamine, suggesting the presence of discrete receptor sites in the blood vessels for different pharmacologically active compounds. This is the first report of the constrictor effect of PGA and PGB compounds. These naturally occuring prostaglandins with high potencies (compared with other prostaglandins and other vasoactive substances) may play a role in spontaneous closure of umbilical vessels. PGE1, E2, F1 and F2a are found in umbilical blood vessels obtained at term.  相似文献   

6.
SUCLA2-related mitochondrial DNA (mtDNA) depletion syndrome is a result of mutations in the β subunit of the ADP-dependent isoform of the Krebs cycle succinyl-CoA synthase (SCS). The mechanism of tissue specificity and mtDNA depletion is elusive but complementation by the GDP-dependent isoform encoded by SUCLG2, and the association with mitochondrial nucleoside diphosphate kinase (NDPK), is a plausible link. We have investigated this relationship by studying SUCLA2 deficient fibroblasts derived from patients and detected normal mtDNA content and normal NDPK activity. However, knockdown of SUCLG2 by shRNA in both patient and control fibroblasts resulted in a significant decrease in mtDNA amount, decreased NDPK and cytochrome c oxidase activities, and a marked growth impairment. This suggests that, SUCLG2, to a higher degree than SUCLA2, is crucial for mtDNA maintenance and that mitochondrial NDPK is involved. Although results pertain to a cell culture system, the findings might explain the pathomechanism and tissue specificity in mtDNA depletion caused by defective SUCLA2.  相似文献   

7.
2-Amino-alpha-carboline [26148-68-5] which was isolated from a pyrolysate of soybean globulin and which was mutagenic to Salmonella typhimurium in the presence of a rat-liver microsomal fraction (S9 mix), was converted into non-mutagenic 2-hydroxy-alpha-carboline by treatment with nitrite in acidic conditions. However, on prolonged treatment with nitrite and acid, 2-hydroxy-alpha-carboline was further converted into a new mutagen which did not require S9 mix for exhibition of the mutagenicity. This direct-acting mutagen was found to be 2-hydroxy-3-nitroso-alpha-carboline by mass and proton magnetic resonance spectroscopies.  相似文献   

8.
Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine. Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate. When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation. Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin. These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway.  相似文献   

9.
10.
Unambiguous assignments of the purine ring protons in the NMR spectra of adenine (Ia) and its 9-substituted derivatives (Ib-f) have been made by comparison with those of the isotopically labeled adenines VIIIa-f. 9-Alkyl-2-deuterioadenines (VIIIb-d), adenosine-2-d (VIIIe), and 2'-deoxyadenosine-2-d (VIIIf) were synthesized from the 9-substituted adenines Ib-f through cyclization of the monocyclic intermediates VIb-f with formic acid-d2 or 1-(formyl-d)-2(1H)-pyridone. Hydrolysis of VIIIe with 0.5 N aq. HCl gave adenine-2-d (VIIIa) in 77% yield.  相似文献   

11.
The Ca2+-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D2 receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D2 receptor, within an Arg-rich epitope that is also involved in the binding to Gi/o proteins and to the adenosine A2A receptor, with the formation of A2A-D2 receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A2A receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A2A-D2 receptor oligomerization. BRET competition experiments indicated that, in the A2A-D2 receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A2A receptor. Furthermore, Ca2+ was found to induce conformational changes in the CaM-A2A-D2 receptor oligomer and to selectively modulate A2A and D2 receptor-mediated MAPK signaling in the A2A-D2 receptor heteromer. These results may have implications for basal ganglia disorders, since A2A-D2 receptor heteromers are being considered as a target for anti-parkinsonian agents.G-protein-coupled receptors are able to form homo- and hetero-oligomers with unique biochemical and functional characteristics (17), and they are easily detected in vitro by using biophysical techniques (810). Heteromers of adenosine A2A and dopamine D2 receptors were one of the first G-protein-coupled receptor heteromers to be described (11). A close physical interaction between both receptors was shown using co-immunoprecipitation and co-localization assays (11) and fluorescence and bioluminescence resonance energy transfer (FRET2 or BRET) techniques (1214). At the biochemical level, two types of antagonistic A2A-D2 receptor interactions have been discovered that may explain the A2A-D2 receptor interactions described both at the neuronal and behavioral level (11, 1518). First, by means of an allosteric interaction in the receptor heteromer, stimulation of A2A receptor decreases the affinity of D2 receptor for their agonists (12). Second, the stimulation of the Gi/o-protein-coupled D2 receptor inhibits the cAMP accumulation induced by the stimulation of the Gs/olf-protein-coupled A2A receptor (11, 17, 18). In view of the well known role of dopamine in Parkinson disease, schizophrenia, and drug addiction, it has been suggested that the A2A-D2 receptor interactions in the central nervous system may provide new therapeutic approaches to combat these disorders (16, 19).An epitope-epitope electrostatic interaction between an Arg-rich epitope of the N terminus of the third intracellular loop (3IL) of the D2 receptor and an epitope containing a phosphorylated Ser localized in the distal part of the C terminus of the A2A receptor is involved in A2A-D2 receptor heteromer interface (14, 20, 21). The same Arg-rich epitope of the D2 receptor is able to interact with CaM (2225). In the absence of phosphorylated residues, adjacent aspartates or glutamates, which are abundant in CaM, may also form non-covalent complexes with Arg-rich epitopes (26). Therefore, CaM can potentially convey a Ca2+ signal to the D2 receptor through direct binding to the 3IL of the D2 receptor (22). Mass spectrometry data have shown that bovine CaM can form multiple non-covalent complexes with an Arg-rich peptide corresponding to the N-terminal region of the 3IL of the D2 receptor (VLRRRRKRVN) (24) as well as a peptide from the proximal C terminus of the A2A receptor (24). This epitope, whose sequence is 291RIREFRQTFR300 in the human A2A receptor, also contains several Arg residues. Since the suspected interaction between the A2A receptor and CaM was awaiting confirmation by assays using complete proteins, the present study was undertaken to demonstrate the existence of interactions between the A2A receptor and CaM both in a recombinant protein expression cell system and in the brain. A proteomics approach was used for the discovery of protein-protein interactions between the A2A receptor and CaM in rat brain, whereas BRET in transfected cells demonstrated a direct interaction between CaM and this receptor. Furthermore, by using BRET and sequential resonance energy transfer (SRET) techniques and analyzing MAPK signaling in transfected cells, evidence was obtained for CaM-A2A-D2 receptor oligomerization and a selective Ca2+-mediated modulation of A2A and D2 receptor function in the A2A-D2 receptor heteromer.  相似文献   

12.
13.

Background & Aims

Inflammation is a major risk factor for development of colorectal cancer (CRC). Prostaglandin synthase cyclooxygenase-2 (COX-2) encoded by the PTGS2 gene is the rate limiting enzyme in prostaglandin synthesis and therefore plays a distinct role as regulator of inflammation.

Methods

PTGS2 mRNA levels were determined in intestinal tissues from 85 intestinal adenoma cases, 115 CRC cases, and 17 healthy controls. The functional PTGS2 polymorphisms A-1195G (rs689466), G-765C (rs20417), T8473C (rs5275) were assessed in 200 CRC cases, 991 adenoma cases and 399 controls from the Norwegian KAM cohort.

Results

PTGS2 mRNA levels were higher in mild/moderate adenoma tissue compared to morphologically normal tissue from the same individual (P<0.0001) and (P<0.035) and compared to mucosa from healthy individuals (P<0.0039) and (P<0.0027), respectively. In CRC patients, PTGS2 mRNA levels were 8–9 times higher both in morphologically normal tissue and in cancer tissue, compared to healthy individuals (P<0.0001). PTGS2 A-1195G variant allele carriers were at reduced risk of CRC (odds ratio (OR) = 0.52, 95% confidence interval (95% CI): 0.28–0.99, P = 0.047). Homozygous carriers of the haplotype encompassing the A-1195G and G-765C wild type alleles and the T8473C variant allele (PTGS2 AGC) were at increased risk of CRC as compared to homozygous carriers of the PTGS2 AGT (A-1195G, G-765C, T8473C) haplotype (OR = 5.37, 95% CI: 1.40–20.5, P = 0.014). No association between the investigated polymorphisms and PTGS2 mRNA levels could be detected.

Conclusion

High intestinal PTGS2 mRNA level is an early event in colorectal cancer development as it occurs already in mild/moderate dysplasia. PTGS2 polymorphisms that have been associated with altered PTGS2 mRNA levels/COX-2 activity in some studies, although not the present study, were associated with colorectal cancer risk. Thus, both PTGS2 polymorphisms and PTGS2 mRNA levels may provide information regarding CRC risk.  相似文献   

14.
Biosynthetic relationship among aflatoxins B1, B2, M1, and M2.   总被引:7,自引:6,他引:1       下载免费PDF全文
Aflatoxins are a family of toxic, acetate-derived decaketides that arise biosynthetically through polyhydroxyanthraquinone intermediates. Most studies have assumed that aflatoxin B1 is the biosynthetic precursor of the other aflatoxins. We used a strain of Aspergillus flavus which accumulates aflatoxin B2 to investigate the later stages of aflatoxin biosynthesis. This strain produced aflatoxins B2 and M2 but no detectable aflatoxin B1 when grown over 12 days in a low-salt, defined growth medium containing asparagine. Addition of dichlorvos to this growth medium inhibited aflatoxin production with concomitant accumulation of versiconal hemiacetal acetate. When mycelial pellets were grown for 24, 48, and 72 h in growth medium and then transferred to a replacement medium, only aflatoxin B2 and M2 were recovered after 96 h of incubation. Addition of sterigmatocystin to the replacement medium led to the recovery of higher levels of aflatoxins B2 and M2 than were detected in control cultures, as well as to the formation of aflatoxins B1 and M1 and O-methylsterigmatocystin. These results support the hypothesis that aflatoxins B1 and B2 can arise independently via a branched pathway.  相似文献   

15.
The levels of fructose 2,6-P2 and 6-phosphofructo 2-kinase have been found to be decreased in the liver of both ketotic and non-ketotic diabetic rats, a good correlation between fall of hepatic fructose 2,6-P2, ketonemia and glycemia being observed. The "total" 6-phosphofructo 2-kinase activity and the "active" (non-phosphorylated) from of the enzyme were decreased to a different extent, resulting in a fall of the "active"/"total" activity ratio. Hepatic levels of glucose 1,6-P2 were lowered only in ketotic diabetes. Insulin treatment normalized all the values studied. Insulin administration to control rats decreased the hepatic levels of fructose 2,6-P2 and did not affect glucose 1,6-P2 levels. It also decreased the "active" form of 6-phosphofructo 2-kinase, without significantly altering the "total" activity.  相似文献   

16.
17.
We report the synthesis of a series of novel 2'-deoxy-2',2'-difluoro-5-halouridines and their corresponding phosphoramidate ProTides. All compounds were evaluated for antiviral activity and for cellular toxicity. Interestingly, 2'-deoxy-2',2'-difluoro-5-iodo- and -5-bromo-uridines showed selective activity against feline herpes virus replication in cell culture due to a specific recognition (activation) by the virus-encoded thymidine kinase.  相似文献   

18.
Soil cores (35 cm long, 7 cm diameter) from the Macaulay Land Use Research Institute's Sourhope Research Station in the Scottish Borders were kept and monitored at constant temperature (18± 1°C) for gas production using a 1.6 mm diameter stainless steel probe fitted with a membrane inlet and connected to a quadrupole mass spectrometer. This provided a novel method for on-line, real time monitoring of soil gas dynamics. In closed-system headspace experiments, O2 and CO2 (measured at m/z values 32 and 44, respectively) showed anti-phase diurnal fluctuations in low-intensity simulated daylight and under a light-dark (LD, 12:12 h) regime. O2 increased during periods of illumination and decreased in the dark. The inverse was true for CO2 production. Ar (m/z = 40) concentration and temperature (°C) remained constant throughout the experiments. The same phase-related oscillations, in CO2 and O2 concentrations, were observed at 2 and 5 cm depth in soil cores. The O2 concentration did not oscillate diurnally at 10 cm depth. In below-ground experiments, CH4 (m/z = 15) concentration showed diurnal cycles at 2, 5 and 10 cm depth. The CH4 production had the same diurnal phase cycle as CO2 but with lower amplitude. Evidence of below-ground diurnal oscillations in N2 (m/z = 28) concentration was provided at 5 cm depth. The scale of production and consumption of gases associated with soil-atmosphere interactions and below-ground processes, are shown to be a multifaceted output of several variables. These include light, circadian-controlled physiological rhythms of plants and microbes, and the interactions between these organisms.  相似文献   

19.
20.
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