Highly secreted tryptophanyl tRNA synthetase 1 as a potential theranostic target for hypercytokinemic severe sepsis.

Despite intensive clinical and scientific efforts, the mortality rate of sepsis remains high due to the lack of precise biomarkers for patient stratification and therapeutic guidance. Secreted human tryptophanyl-tRNA synthetase 1 (WARS1), an endogenous ligand for Toll-like receptor (TLR) 2 and TLR4 against infection, activates the genes that signify the hyperinflammatory sepsis phenotype. High plasma WARS1 levels stratified the early death of critically ill patients with sepsis, along with elevated levels of cytokines, chemokines, and lactate, as well as increased numbers of absolute neutrophils and monocytes, and higher Sequential Organ Failure Assessment (SOFA) scores. These symptoms were recapitulated in severely ill septic mice with hypercytokinemia. Further, injection of WARS1 into mildly septic mice worsened morbidity and mortality. We created an anti-human WARS1-neutralizing antibody that suppresses proinflammatory cytokine expression in marmosets with endotoxemia. Administration of this antibody into severe septic mice attenuated cytokine storm, organ failure, and early mortality. With antibiotics, the antibody almost completely prevented fatalities. These data imply that blood-circulating WARS1-guided anti-WARS1 therapy may provide a novel theranostic strategy for life-threatening systemic hyperinflammatory sepsis.

Tryptophan-dependent and -independent secretions of tryptophanyl- tRNA synthetase mediate innate inflammatory responses.

While cytoplasmic tryptophanyl-tRNA synthetase (WARS1) ligates tryptophan (Trp) to its cognate tRNAs for protein synthesis, it also plays a role as an innate immune activator in extracellular space. However, its secretion mechanism remains elusive. Here, we report that in response to stimuli, WARS1 can be secreted via two distinct pathways: via Trp-dependent secretion of naked protein and via Trp-independent plasma-membrane-derived vesicles (PMVs). In the direct pathway, Trp binding to WARS1 induces a "closed" conformation, generating a hydrophobic surface and basic pocket. The Trp-bound WARS1 then binds stable phosphatidylinositol (4,5)-biphosphate and inner plasma membrane leaflet, passing across the membrane. In the PMV-mediated secretion, WARS1 recruits calpain 2, which is activated by calcium. WARS1 released from PMVs induces inflammatory responses in vivo. These results provide insights into the secretion mechanisms of WARS1 and improve our understanding of how WARS1 is involved in the control of local and systemic inflammation upon infection.

Predictive modeling and probabilistic risk assessment of Clostridium perfringens in hamburgers and sandwiches.

This study aimed to develop a mathematical model for the survival of Clostridium perfringens in hamburgers and sandwiches and to evaluate their microbial risk. The primary model was developed in hamburgers using 4 strains of C. perfringens at 5, 10, 15, 25 and 37 °C, and the kinetic parameters of the primary model were fitted well with the Weibull model (R 2 ≥ 0.95). The secondary model was developed and validated in hamburgers and sandwiches using the Davey model, which was evaluated by B f , A f , and RMSE values within the acceptable range. A probabilistic risk model was developed and simulated using @Risk program to estimate the probability of infection (P inf ) of C. perfringens based on the data on prevalence (n = 100), time, temperature, and consumption of hamburgers and sandwiches (150.00 ± 20.96 g). Based on the simulation model, the mean C. perfringens exposure dose was 0.00976 CFU/g, and the estimated mean P inf was 1.78 × 10-13, which was very low in comparison with the current available data. The proposed model and the result can thus be useful to establish risk management options and microbial criteria for C. perfringens contamination in hamburgers and sandwiches. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-01000-z.

CagL polymorphisms between East Asian and Western Helicobacter pylori are associated with different abilities to induce IL-8 secretion.

Helicobacter pylori colonizes human gastric mucosa. Its infection is associated with gastric diseases including gastric cancer. CagA is one of the most important toxins produced by H. pylori. It is related to gastric cancer which can be injected into host cells via a type IV secretion system (T4SS). CagL is a structural component of T4SS apparatus, which triggers host cell signaling pathway. It has been reported that CagL polymorphisms may influence the severity of disease development. To explore the contribution of CagL polymorphisms between East Asian and Western H. pylori in pathogenesis, cagL gene in G27 H. pylori was swapped by K74 cagL which is identical to East Asian CagL consensus sequence and by Western 26695 H. pylori, resulting in G27 ΔcagL/cagLK74 and G27 ΔcagL/cagL26695, respectively. Intriguingly, G27 ΔcagL/cagLK74 showed significantly less ability of IL-8 induction than G27 ΔcagL/cagL26695 while displayed similar abilities of CagA phosphorylation, and cell elongation. Taken together, this study suggests that the CagL polymorphism may influence IL-8 induction, and K74 CagL has less ability to induce IL-8 secretion than G27 or 26695 CagL. Further research should address how the different capabilities of IL-8 induction between intraspecies-CagL are associated with the large differences of the incidence of gastric cancer between East Asian and Western countries.

Tryptophanyl-tRNA Synthetase 1 Signals Activate TREM-1 via TLR2 and TLR4.

Tryptophanyl-tRNA synthetase 1 (WARS1) is an endogenous ligand of mammalian Toll-like receptors (TLR) 2 and TLR4. Microarray data, using mRNA from WARS1-treated human peripheral blood mononuclear cells (PBMCs), had indicated WARS1 to mainly activate innate inflammatory responses. However, exact molecular mechanism remains to be understood. The triggering receptor expressed on myeloid cells (TREM)-1 is an amplifier of pro-inflammatory processes. We found WARS1 to significantly activate TREM-1 at both mRNA and protein levels, along with its cell surface expression and secretion in macrophages. WARS1 stimulated TREM-1 production via TLR2 and TLR4, mediated by both MyD88 and TRIF, since targeted deletion of TLR4, TLR2, MyD88, and TRIF mostly abrogated TREM-1 activation. Furthermore, WARS1 promoted TREM-1 downstream phosphorylation of DAP12, Syk, and AKT. Knockdown of TREM-1 and inhibition of Syk kinase significantly suppressed the activation of inflammatory signaling loop from MyD88 and TRIF, leading to p38 MAPK, ERK, and NF-κB inactivation. Finally, MyD88, TRIF, and TREM-1 signaling pathways were shown to be cooperatively involved in WARS1-triggered massive production of IL-6, TNF-α, IFN-ß, MIP-1α, MCP-1, and CXCL2, where activation of Syk kinase was crucial. Taken together, our data provided a new insight into WARS1's strategy to amplify innate inflammatory responses via TREM-1.

The presence of neutrophils causes RANKL expression in periodontal tissue, giving rise to osteoclast formation.

BACKGROUNDS AND OBJECTIVE: Increased neutrophil infiltration and osteoclast formation are key characteristics of periodontitis. The effect of these neutrophils on osteoclast formation in periodontitis remains unclear. Therefore, we investigated the effects of neutrophils on osteoclast formation in a neutrophil-deficient mouse model of periodontitis. METHODS: Anti-Ly6G antibody (Ab) was used for neutrophil depletion in two mouse models: periodontitis and air pouch. In the periodontitis experiments, mice were divided into PBS-administered control (C), control Ab-administered periodontitis (P), and anti-Ly6G Ab-administered periodontitis (P + Ly6G) groups. Periodontitis was induced by ligature of mandibular first molars. In the air pouch experiments, mice were divided into PBS-administered (C), LPS and control Ab-administered (LPS), and LPS and anti-Ly6G Ab-administered (LPS + Ly6G) groups. Neutrophil migration into air pouches was induced by LPS injection. Flow cytometry was used to examine CD11b+ Ly6G+ neutrophils in the blood of periodontitis mice and CD11b+ Ly6G+ RANKL+ neutrophils in exudates of air pouch mice. In periodontal tissue, Ly6G+ neutrophil and RANKL+ cell numbers in periodontal ligament and alveolar bone areas were estimated using immunohistochemistry, osteoclast numbers were measured using TRAP assay, and alveolar bone loss was determined by H&E staining. RESULTS: In blood, CD11b+ Ly6G+ neutrophils were found in greater percentage in the P group than in the C group on days 3 and 7. However, the percentage of neutrophils was lower in the P + Ly6G group than in the C and P groups. In periodontal tissue, the numbers of Ly6G+ neutrophils and RANKL+ cells were lower in the P + Ly6G group than in the P group on day 3. Ly6G+ neutrophil numbers decreased more in the P + Ly6G group than in the P group on day 7, but RANKL+ cell numbers did not decrease in the P + Ly6G group. In exudates, the number of CD11b+ Ly6G+ RANKL+ neutrophils was greater in the LPS group than in the C and LPS + Ly6G groups. On days 3 and 7, the numbers of osteoclasts and alveolar bone loss were greater in periodontal tissue in the P and P + Ly6G groups than in the C group. Interestingly, there were fewer osteoclasts in the P + Ly6G group than in the P group on day 3. CONCLUSION: Neutrophil deficiency caused a reduction in numbers of both RANKL+ cells and osteoclasts in periodontitis-induced tissues only on day 3. Furthermore, in the LPS-injected air pouch model, neutrophil deficiency reduced the influx of RANKL+ neutrophils. These findings suggest that the presence of neutrophils induces RANKL expression and could induce osteoclast formation in the early stages of periodontitis.

Evolutionary mechanism leading to the multi-cagA genotype in Helicobacter pylori.

Infection with CagA+ Helicobacter pylori strains is linked to an increased risk for gastric diseases, including gastric cancer. Recent evidence indicates that dynamic expansion and contraction of cagA copy number may serve as a novel mechanism to enhance disease development. Herein, comparative genomic analysis divided hpEurope into two groups: hpEurope/type-A and type-B. Only hpEurope/type-B displayed the multi-cagA genotype. Further analysis showed that cagPAI appears to have been independently introduced into two different H. pylori types, termed pre-type-A and pre-type-B, which consequently evolved to cagPAI type-A and type-B, respectively; importantly, all multi-cagA genotype strains displayed cagPAI type-B. Two direct cagA-flanking repeats of a genetic element termed CHA-ud were essential for the multi-cagA genotype in strain PMSS1 (hpEurope/type-B and cagPAI type-B). Furthermore, introduction of this genetic element into strain G27 (hpEurope/type-A and cagPAI type-A) was sufficient to generate the multi-cagA genotype. The critical steps in the evolution of the multi-cagA genotype involved creation of CHA-ud at cagA upstream in cagPAI type-B strains followed by its duplication to cagA downstream. En masse, elucidation of the mechanism by which H. pylori evolved to carry multiple copies of cagA helps to provide a better understanding of how this ancient pathogen interacts with its host.

Intermittent PTH administration improves alveolar bone formation in type 1 diabetic rats with periodontitis.

BACKGROUND: Periodontitis is an infectious disease that manifests as alveolar bone loss surrounding the roots of teeth. Diabetes aggravates periodontitis-induced alveolar bone loss via suppression of bone formation. Intermittent parathyroid hormone (PTH) administration displays an anabolic effect on bone. In this study, we investigated the effect of intermittent PTH administration on alveolar bone loss in type 1 diabetic rats with periodontitis. METHODS: Rats were divided into control (C), periodontitis (P), periodontitis treated with PTH (P + PTH), diabetes with periodontitis (DP), and diabetes with periodontitis treated with PTH (DP + PTH) groups. To induce type 1 diabetes, rats were injected with streptozotocin and periodontitis was induced bilaterally by applying ligatures to the mandibular first molars for 30 days. During the experimental period, the P + PTH and DP + PTH groups were subcutaneously injected with PTH (40 µg/kg) three times per week, whereas the C, P, and DP groups were injected with citrate buffer. To observe the mineralization of the alveolar bone, the DP and DP + PTH groups were injected with calcein on days 10 and 27, and with alizarin red on day 20. Thirty days after ligation, histological findings and fluorescence labeling were analyzed in the furcations of the mandibular first molars. Sclerostin-positive osteocytes were assessed by immunohistochemical analyses. RESULTS: The DP groups had smaller areas of alveolar bone than the other groups, and the DP + PTH group had a larger alveolar bone area than the DP group. The DP group had less osteoid formation than the C group, whereas the DP + PTH had greater osteoid formation than the DP group. Fluorescence labeling results revealed that the DP + PTH group had more mineral deposition on the alveolar bone than the DP group. The DP + PTH group exhibited lower percentage of sclerostin-positive osteocytes in alveolar bone than the DP group. CONCLUSIONS: Intermittent PTH administration diminishes alveolar bone loss and sclerostin expression in osteocytes, but increases osteoid formation and mineralization, suggesting that intermittent PTH administration attenuates diabetes-aggravated alveolar bone loss by the induction of bone formation. PTH-induced bone formation may be related to the regulation of osteocytic sclerostin expression in type 1 diabetic rats with periodontitis.

Tumor necrosis factor-α antagonist diminishes osteocytic RANKL and sclerostin expression in diabetes rats with periodontitis.

Type 1 diabetes with periodontitis shows elevated TNF-α expression. Tumor necrosis factor (TNF)-α stimulates the expression of receptor activator of nuclear factor-κB ligand (RANKL) and sclerostin. The objective of this study was to determine the effect of TNF-α expression of osteocytic RANKL and sclerostin in type 1 diabetes rats with periodontitis using infliximab (IFX), a TNF-α antagonist. Rats were divided into two timepoint groups: day 3 and day 20. Each timepoint group was then divided into four subgroups: 1) control (C, n = 6 for each time point); 2) periodontitis (P, n = 6 for each time point); 3) diabetes with periodontitis (DP, n = 8 for each time point); and 4) diabetes with periodontitis treated with IFX (DP+IFX, n = 8 for each time point). To induce type 1 diabetes, rats were injected with streptozotocin (50 mg/kg dissolved in 0.1 M citrate buffer). Periodontitis was then induced by ligature of the mandibular first molars at day 7 after STZ injection (day 0). IFX was administered once for the 3 day group (on day 0) and twice for the 20 day group (on days 7 and 14). The DP group showed greater alveolar bone loss than the P group on day 20 (P = 0.020). On day 3, higher osteoclast formation and RANKL-positive osteocytes in P group (P = 0.000 and P = 0.011, respectively) and DP group (P = 0.006 and P = 0.017, respectively) than those in C group were observed. However, there was no significant difference in osteoclast formation or RANKL-positive osteocytes between P and DP groups. The DP+IFX group exhibited lower alveolar bone loss (P = 0.041), osteoclast formation (P = 0.019), and RANKL-positive osteocytes (P = 0.009) than that of the DP group. On day 20, DP group showed a lower osteoid area (P = 0.001) and more sclerostin-positive osteocytes (P = 0.000) than P group. On days 3 and 20, the DP+IFX group showed more osteoid area (P = 0.048 and 0.040, respectively) but lower sclerostin-positive osteocytes (both P = 0.000) than DP group. Taken together, these results suggest that TNF-α antagonist can diminish osteocytic RANKL/sclerostin expression and osteoclast formation, eventually recovering osteoid formation. Therefore, TNF-α might mediate alveolar bone loss via inducing expression of osteocytic RANKL and sclerostin in type 1 diabetes rats with periodontitis.

Dynamic Expansion and Contraction of cagA Copy Number in Helicobacter pylori Impact Development of Gastric Disease.

Infection with Helicobacter pylori is a major risk factor for development of gastric disease, including gastric cancer. Patients infected with H. pylori strains that express CagA are at even greater risk of gastric carcinoma. Given the importance of CagA, this report describes a new molecular mechanism by which the cagA copy number dynamically expands and contracts in H. pylori Analysis of strain PMSS1 revealed a heterogeneous population in terms of numbers of cagA copies; strains carried from zero to four copies of cagA that were arranged as direct repeats within the chromosome. Each of the multiple copies of cagA was expressed and encoded functional CagA; strains with more cagA repeats exhibited higher levels of CagA expression and increased levels of delivery and phosphorylation of CagA within host cells. This concomitantly resulted in more virulent phenotypes as measured by cell elongation and interleukin-8 (IL-8) induction. Sequence analysis of the repeat region revealed three cagA homologous areas (CHAs) within the cagA repeats. Of these, CHA-ud flanked each of the cagA copies and is likely important for the dynamic variation of cagA copy numbers. Analysis of a large panel of clinical isolates showed that 7.5% of H. pylori strains isolated in the United States harbored multiple cagA repeats, while none of the tested Korean isolates carried more than one copy of cagA Finally, H. pylori strains carrying multiple cagA copies were differentially associated with gastric disease. Thus, the dynamic expansion and contraction of cagA copy numbers may serve as a novel mechanism by which H. pylori modulates gastric disease development.IMPORTANCE Severity of H. pylori-associated disease is directly associated with carriage of the CagA toxin. Though the sequences of the CagA protein can differ across strains, previous analyses showed that virtually all H. pylori strains carry one or no copies of cagA This study showed that H. pylori can carry multiple tandem copies of cagA that can change dynamically. Isolates harboring more cagA copies produced more CagA, thus enhancing toxicity to host cells. Analysis of 314 H. pylori clinical strains isolated from patients in South Korea and the United States showed that 7.5% of clinical strains in the United States carried multiple cagA copies whereas none of the South Korean strains did. This study demonstrated a novel molecular mechanism by which H. pylori dynamically modulates cagA copy number, which affects CagA expression and activity and may impact downstream development of gastric disease.

Helicobacter pylori-Induced HB-EGF Upregulates Gastrin Expression via the EGF Receptor, C-Raf, Mek1, and Erk2 in the MAPK Pathway.

Helicobacter pylori is associated with hypergastrinemia, which has been linked to the development of gastric diseases. Although the molecular mechanism is not fully understood, H. pylori is known to modulate the Erk pathway for induction of gastrin expression. Herein we found that an epidermal growth factor (EGF) receptor kinase inhibitor significantly blocked H. pylori-induced gastrin promoter activity, suggesting involvement of EGF receptor ligands. Indeed, H. pylori induced mRNA expression of EGF family members such as amphiregulin, EGF, heparin-binding EGF-like growth factor (HB-EGF), and transforming growth factor-α. Of these, specific siRNA targeting of HB-EGF significantly blocked H. pylori-induced gastrin expression. Moreover, H. pylori induced HB-EGF ectodomain shedding, which we found to be a critical process for H. pylori-induced gastrin expression. Thus, we demonstrate a novel role for human mature HB-EGF in stimulating gastrin promoter activity during H. pylori infection. Further investigation using specific siRNAs targeting each isoform of Raf, Mek, and Erk elucidated that the mechanism underlying H. pylori-induced gastrin expression can be delineated as the sequential activation of HB-EGF, the EGF receptor, C-Raf, Mek1, and the Erk2 molecules in the MAPK pathway. Surprisingly, whereas Erk2 acts as a potent activator of gastrin expression, siRNA knockdown of Erk1 induced gastrin promoter activity, suggesting that Erk1 typically acts as a repressor of gastrin expression. Elucidation of the mechanism of gastrin modulation by HB-EGF-mediated EGF receptor transactivation should facilitate the development of therapeutic strategies against H. pylori-related hypergastrinemia and consequently gastric disease development, including gastric cancers.

Helicobacter pylori outer membrane protein, HomC, shows geographic dependent polymorphism that is influenced by the Bab family.

The array of outer membrane proteins (OMPs) found in Helicobacter pylori provides a crucial component for persistent colonization within the gastric niche. Not only does H. pylori harbor a wide number of OMPs, but these OMPs often vary across strains; this likely contributes to immune evasion, adaptation during long term colonization, and potentially differential disease progression. Previous work from our group described OMP differences among the Bab family (babA, babB, and babC) and Hom family (homA and homB) from 80 American H. pylori clinical isolates (AH) and 80 South Korean H. pylori clinical isolates (KH). In the current study, we expanded our investigation to include the less well characterized Hom family member, HomC.Overall, we identified and genotyped three homC variants: homC S , homC L , and homC M , in both populations. Similar to other polymorphic genes, the KH group showed less overall diversity, with 97.5% of strains harboring homC L . In contrast, a more heterogeneous profile was observed in strains derived from an American population; we found nearly equal distribution of homC S and homC L . Further analysis of the AH group identified associations between homC polymorphism and bab genotype; in AH strains, there was a significant association between homC L and carriage of babA at locus A. Since babA is an important virulence factor for the development of severe gastric disease, these data may suggest that homC polymorphism plays a role in H. pylori pathogenesis.

Effect of Areca Nut on Helicobacter pylori-Induced Gastric Diseases in Mice.

Areca nut (AN) chewing is a habit in many countries in Central, Southern, and Southeast Asia. It is strongly associated with the occurrence of oral, pharyngeal, and esophageal cancer as well as systemic inflammation. However, the association between AN intake and the development of gastric lesions has not yet been identified. The aim of this study was to investigate the effect of AN on gastric diseases using a mouse model for Helicobacter pylori infection. We studied four groups of mice: those fed a normal diet (ND), those fed a diet containing 2.5% AN (AD), those fed ND and infected with H. pylori PMSS1 strain (ND/HP), and those fed AD and infected with H. pylori PMSS1 strain (AD/HP). Food intake and body weight were monitored weekly during the experiments. At 10 weeks, the mice were sacrificed, and the stomach weight, H. pylori colonization, and gastric inflammation were evaluated. The stomach weight had increased significantly in the ND/HP and AD/HP groups along with increases in H. pylori colonization; however, there was no significant difference between these two groups with respect to stomach weight and colonization. On histological grading, mononuclear cell infiltration was severer in the AD/HP group than in the ND/HP group. These data suggest that chronic gastric inflammation was aggravated by AN treatment in the mice with H. pylori-induced gastric lesions. Furthermore, as previously suggested, this animal model is useful to determine the effect of potential carcinogens on gastric lesions induced by H. pylori infection.

Crystal structure of CagL from Helicobacter pylori K74 strain.

Helicobacter pylori (Hp) CagL is a component of the type IV secretion system (T4SS) and interacts with integrin in host cells through its flexible RGD domain to translocate CagA. Differences in CagL amino acid polymorphisms between Western and East-Asian Hps are correlated with clinical outcome. CagL of East-Asian clinical Hp isolate K74 (CagL(K74)) contains multiple residue variations upstream of RGD motif and has different integrin binding affinities compared to those of CagL from Western Hp 26695. Here, we report the crystal structure of CagL(K74). The structure displayed a six-helix bundle including two short α-helices, and the RGD motif was found in the long rigid α2 helix flanked by the conserved protease-sensitive and RGD-helper sequences, as observed in CagL(26695). However, two additional salt bridges were found between the helices compared with the CagL(26695) structure, suggesting that the putative flexible region harboring the RGD motif may be more stable in this CagL variant.

Wogonin ameliorates hyperglycemia and dyslipidemia via PPARα activation in db/db mice.

BACKGROUND & AIMS: Wogonin is a flavonoid extracted from the root of Scutellaria baicalensis Gerogi. We evaluated the therapeutic effects of wogonin using db/db mice. METHODS: Mice received wogonin or vehicle by oral gavage for 2 weeks. Blood glucose, insulin, and cholesterol levels were measured, and liver morphology was observed with histopathological analysis. The mRNA expression levels of PPARα, PPARγ, and adiponectin in the liver and white adipose tissue (WAT) were determined by real-time PCR. Immunoblotting for AMPK and PPARγ, and adipocyte differentiation were investigated in vitro using 3T3-L1 cells. A luciferase assay was used to measure PPARα and PPARγ binding activity. RESULTS: The wogonin group showed decreased weight gain without a change in food intake and improved glucose tolerance. Serum insulin and cholesterol levels in the wogonin group were significantly decreased compared to those in the control group. The wogonin group also showed less accumulation of lipid droplets and glycogen in the liver. PPARα and PPARγ expression levels in the liver and WAT and adiponectin expression level in WAT in the wogonin group were higher than those in the control group. In 3T3-L1 cells, wogonin was shown to stimulate AMPK activation in a dose-dependent manner. The presence of wogonin did not affect adipocyte differentiation or PPARγ protein level during adipogenesis. Notably, wogonin enhanced PPARα but not PPARγ transactivation. CONCLUSIONS: These indicate that wogonin may have beneficial effects on glucose and lipid metabolism related to enhanced PPARα and adiponectin expression via AMPK activation. Importantly, wogonin did not cause deleterious effects, such as weight gain and fatty liver. Wogonin might be a useful therapeutic agent to treat type 2 diabetes.

Selective derivatization of nucleotide diphosphate (NDP)-4-keto sugars for electrospray ionization-mass spectrometry (ESI-MS).

Nucleotide diphosphate (NDP) sugars are widely present in antibiotics and glycoconjugates, such as protein- and lipid-linked oligosaccharides, where they act as substrates for glycosyltransferase in eukaryotes and prokaryotes. Among NDP sugars, NDP-4-keto sugars are key intermediates in the synthesis of structurally diverse NDP sugars with different functional groups. However, the structural identification of the NDP-4-keto sugars via mass spectrometry (electrospray ionization-mass spectrometry (ESI-MS)) continues to be a challenge because of the carbonyl group in these sugars interferes with ionization process. In this study, we evaluated various hydroxylamine compounds for the derivatization of NDP-4-keto sugars, so that the detection of the sugars by ESI-MS is more efficient. As a result, O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine was found to be the most effective tagging molecule for the detection of NDP-4-keto sugars without being interfered by original MS. This method can be used for identifying NDP-4-keto sugars such as thymidine diphosphate (TDP)-, adenosine diphosphate (ADP)-, uridine diphosphate (UDP)-, and cytosine diphosphate (CDP)-4-keto sugars as well as new NDP-4-keto-dehydratases.

Inactivation of phosphomannose isomerase gene abolishes sporulation and antibiotic production in Streptomyces coelicolor.

Phosphomannose isomerases (PMIs) in bacteria and fungi catalyze the reversible conversion of D-fructose-6-phosphate to D-mannose-6-phosphate during biosynthesis of GDP-mannose, which is the main intermediate in the mannosylation of important cell wall components, glycoproteins, and certain glycolipids. In the present study, the kinetic parameters of PMI from Streptomyces coelicolor were obtained, and its function on antibiotic production and sporulation was studied. manA (SCO3025) encoding PMI in S. coelicolor was deleted by insertional inactivation. Its mutant (S. coelicolor∆manA) was found to exhibit a bld-like phenotype. Additionally, S. coelicolor∆manA failed to produce the antibiotics actinorhodin and red tripyrolle undecylprodigiosin in liquid media. To identify the function of manA, the gene was cloned and expressed in Escherichia coli BL21 (DE3). The purified recombinant ManA exhibited PMI activity (K(cat)/K(m) (mM(-1) s(-1) = 0.41 for D-mannose-6-phosphate), but failed to show GDP-D-mannose pyrophosphorylase [GMP (ManC)] activity. Complementation analysis with manA from S. coelicolor or E. coli resulted in the recovery of bld-like phenotype of S. coelicolor∆manA. SCO3026, another ORF that encodes a protein with sequence similarity towards bifunctional PMI and GMP, was also tested for its ability to function as an alternate ManA. However, the purified protein of SCO3026 failed to exhibit both PMI and GMP activity. The present study shows that enzymes involved in carbohydrate metabolism could control cellular differentiation as well as the production of secondary metabolites.

Role of amphipathic helix of a herpesviral protein in membrane deformation and T cell receptor downregulation.

Lipid rafts are membrane microdomains that function as platforms for signal transduction and membrane trafficking. Tyrosine kinase interacting protein (Tip) of T lymphotropic Herpesvirus saimiri (HVS) is targeted to lipid rafts in T cells and downregulates TCR and CD4 surface expression. Here, we report that the membrane-proximal amphipathic helix preceding Tip's transmembrane (TM) domain mediates lipid raft localization and membrane deformation. In turn, this motif directs Tip's lysosomal trafficking and selective TCR downregulation. The amphipathic helix binds to the negatively charged lipids and induces liposome tubulation, the TM domain mediates oligomerization, and cooperation of the membrane-proximal helix with the TM domain is sufficient for localization to lipid rafts and lysosomal compartments, especially the mutivesicular bodies. These findings suggest that the membrane-proximal amphipathic helix and TM domain provide HVS Tip with the unique ability to deform the cellular membranes in lipid rafts and to downregulate TCRs potentially through MVB formation.

Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the alpha subunit of GS protein.

The serotonin type 6 (5-HT(6)) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G(S)). To identify the structural basis for the interaction of the 5-HT(6) receptor with the G(S) protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT(6) receptor and the alpha subunit of G(S) (Galpha(S)). The direct interaction of iL3 and Galpha(S) was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Galpha(S) were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10(-6)M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Galpha(S). The critical residues within the iL3 region for the interaction with Galpha(S) were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT(6) receptor mutants.