Pyruvate Induces Transient Tumor Hypoxia by Enhancing Mitochondrial Oxygen Consumption and Potentiates the Anti-Tumor Effect of a Hypoxia-Activated Prodrug TH-302

Background

TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302.

Methodology/Results

The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O₂), with minimal effect under aerobic conditions (21% O₂). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500–1500 mm³. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼550 mm³), significantly delayed tumor growth.

Conclusions/Significance

Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.     

Clonal Structure,Seed Set,and Self-Pollination Rate in Mass-Flowering Bamboo Species during Off-Year Flowering Events

Bamboos are typical examples of highly synchronized semelparous species. Their mass-flowering events occur at supra-annual intervals but they sometimes flower on a small scale in off-years. If some bamboo ramets (culms) of a genet flower and die in off-years, whereas other culms of the same genet do not flower synchronously, the genet can still survive blooming in an off-year and could participate in the next mass-flowering event. At genet level, the effect might be similar to that achieved by synchronously reproducing iteroparous plants. In addition, if multiple genets flower simultaneously in off-years, cross-pollination will be promoted. However, it is not known whether all the culms in a genet flower synchronously and whether multiple genets flower in off-years. We determined the clonal structure of three temperate dwarf bamboo species, i.e., Sasa senanensis, S. kurilensis, and S. palmata, at 24 off-year flowering sites and the surrounding areas in northern Japan using seven microsatellite markers. We also estimated seed set at seven of the sites and self-pollination rates at five sites to determine off-year reproductive success. Next, we investigated whether seed sets at the culm level were related to flowering area and/or number of flowering genets, using generalized linear mixed-effect models (GLMMs). Multiple genets flowered at 9/24 flowering sites. We found that 40/96 of the genets identified had some flowering culms. Non-flowering culms were present in 24/40 flowering genets. Seed set was in the range 2.2%–12.5% and the self-pollination rate was 96.3%. In the best GLMM, seed set increased with flowering area. Seeds were produced in off-years, but cross-pollination was rare in off-years. We suggest that some dwarf bamboos may exhibit iteroparity or imperfectly synchronized semelparity at the genet level, a characteristic similar to that of other reproductively synchronous plants. We also found synchronous flowering of a few genets even in off-years.     

Patient Age and the Prognosis of Idiopathic Membranous Nephropathy

Background

Idiopathic membranous nephropathy (IMN) is increasingly seen in older patients. However, differences in disease presentation and outcomes between older and younger IMN patients remain controversial. We compared patient characteristics between younger and older IMN patients.

Methods

We recruited 171 Japanese patients with IMN, including 90 (52.6%) patients <65 years old, 40 (23.4%) patients 65–70 years, and 41 (24.0%) patients ≥71 years. Clinical characteristics and outcomes were compared between younger and older IMN patients.

Results

During a median observation period of 37 months, 103 (60.2%) patients achieved complete proteinuria remission, which was not significantly associated with patient age (P = 0.831). However, 13 (7.6%) patients were hospitalized because of infection. Multivariate Cox proportional hazards models identified older age [adjusted hazard ratio (HR) = 3.11, 95% confidence interval (CI): 1.45–7.49, per 10 years; P = 0.003], prednisolone use (adjusted HR = 11.8, 95% CI: 1.59–242.5; P = 0.014), and cyclosporine used in combination with prednisolone (adjusted HR = 10.3, 95% CI: 1.59–204.4; P = 0.012) as significant predictors of infection. A <25% decrease in proteinuria at 1 month after immunosuppressive therapy initiation also predicted infection (adjusted HR = 6.72, 95% CI: 1.51–37.8; P = 0.012).

Conclusions

Younger and older IMN patients had similar renal outcomes. However, older patients were more likely to develop infection when using immunosuppressants. Patients with a poor response in the first month following the initiation of immunosuppressive therapy should be carefully monitored for infection and may require a faster prednisolone taper.     

Effects of Mycobacteria Major Secretion Protein,Ag85B,on Allergic Inflammation in the Lung

Many epidemiological studies have suggested that the recent increase in prevalence and severity of allergic diseases such as asthma is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccination. However, the underlying mechanisms by which mycobacterial components suppress allergic diseases are not yet fully understood. Here we showed the inhibitory mechanisms for development of allergic airway inflammation by using highly purified recombinant Ag85B (rAg85B), which is one of the major protein antigens secreted from M. tuberculosis. Ag85B is thought to be a single immunogenic protein that can elicit a strong Th1-type immune response in hosts infected with mycobacteria, including individuals vaccinated with BCG. Administration of rAg85B showed a strong inhibitory effect on the development of allergic airway inflammation with induction of Th1-response and IL-17and IL-22 production. Both cytokines induced by rAg85B were involved in the induction of Th17-related cytokine-production innate immune cells in the lung. Administration of neutralizing antibodies to IL-17 or IL-22 in rAg85B-treated mice revealed that IL-17 induced the infiltration of neutrophils in BAL fluid and that allergen-induced bronchial eosinophilia was inhibited by IL-22. Furthermore, enhancement of the expression of genes associated with tissue homeostasis and wound healing was observed in bronchial tissues after rAg85B administration in a Th17-related cytokine dependent manner. The results of this study provide evidence for the potential usefulness of rAg85B as a novel approach for anti-allergic effect and tissue repair other than the role as a conventional TB vaccine.     

Associated Factors of Atrophic Gastritis Diagnosed by Double-Contrast Upper Gastrointestinal Barium X-Ray Radiography: A Cross-Sectional Study Analyzing 6,901 Healthy Subjects in Japan

Background

Double-contrast upper gastrointestinal barium X-ray radiography (UGI-XR) is one of the most widely conducted gastric cancer screening methods. It has been executed to find gastric cancer, but has not been usually executed to detect premalignant atrophic mucosa of stomach. To understand the meaning of UGI-XR-based atrophic gastritis, we analyzed its association with several causative factors including Helicobacter pylori (HP) infection.

Methods

We evaluated 6,901 healthy adults in Japan. UGI-XR-based atrophic gastritis was diagnosed based on the irregular shape of areae gastricae and its expansion in the stomach.

Results

Of the 6,433 subjects with no history of HP eradication and free from gastric acid suppressants, 1,936 were diagnosed as UGI-XR-based atrophic gastritis (mild: 234, moderate: 822, severe: 880). These were univariately associated with serum HP IgG and serum pepsinogen I/II ratio with statistical significance. The multiple logistic analysis calculating standardized coefficients (β) and odds ratio (OR) demonstrated that serum HP IgG (β = 1.499, OR = 4.48), current smoking (β = 0.526, OR = 1.69), age (β = 0.401, OR = 1.49), low serum pepsinogen I/II ratio (β = 0.339, OR = 1.40), and male gender (β = 0.306, OR = 1.36) showed significant positive association with UGI-XR-based atrophic gastritis whereas drinking and body mass index did not. Among the age/sex/smoking/drinking-matched 227 pairs derived from chronically HP-infected and successfully HP-eradicated subjects, UGI-XR-based atrophic gastritis was detected in 99.1% of the former but in only 59.5% of the latter subjects (p<0.0001). Contrastively, UGI-XR-based atrophic gastritis was detected in 13 of 14 HP-positive proton pump inhibitor users (92.9%) and 33 of 34 HP-positive histamine H₂-receptor antagonist users (97.1%), which are not significantly different from gastric acid suppressant-free subjects.

Conclusions

The presence of UGI-XR-based atrophic gastritis is positively associated with Helicobacter pylori infection, current smoking, age, decreased serum pepsinogen I/II ratio, and male gender. Eradication of Helicobacter pylori seems to superficially improve UGI-XR-based atrophic gastritis whereas intake of gastric acid suppressants does not.     

Growth/differentiation factor-5 induces growth arrest and apoptosis in mouse B lineage cells with modulation by Smad

Bone morphogenetic proteins, including growth/differentiation factor-5 (GDF-5), are multifunctional cytokines. Recent studies of intracellular signal transduction mechanisms for the transforming growth factor-beta superfamily have focused on Smad proteins. However, scant attention has been given to the mechanism by which GDF-5 exerts its negative growth effect on immunological competent cells. In the present study, we demonstrated that GDF-5 induced cell cycle arrest in the G1 phase before the appearance of apoptosis in mouse B cell hybridoma HS-72 cells, while the ectopic expression of Smad6 and Smad7 in HS-72 cells suppressed the GDF-5-induced G1 cell cycle arrest by abolishing the expression of p21(CIP-1/WAF-1) and hypophosphorylation of retinoblastoma protein. Moreover, we found that Smad6 and Smad7 suppressed GDF-5-induced apoptosis in HS-72 cells. These findings indicated that Smad6 and Smad7 exhibit inhibitory effects toward GDF-5-mediated signaling in B lineage cells.     

Effect of protonation of the N-acetyl neuraminic acid residue of sialyl Lewis(X): a molecular orbital study with insights into its binding properties toward the carbohydrate recognition domain of E-selectin

Semiempirical molecular orbital (MO) calculations with an implicit treatment of the water environment were employed in order to assess whether the sialyl Lewis(X) (sLe(X)) tetrasaccharide binds to E-selectin in the anionic or neutral (i.e., protonated) state. The analysis of the frontier molecular orbitals, electrostatic potential surfaces, and conformational behavior of the sugar indicates that its neutral form possesses the necessary characteristics for binding. In particular, the LUMO level of the neutral sLe(X) molecule is localized both on the carboxylic group of the N-acetyl neuraminic acid (NeuNAc) residue and on the nearby glycosidic linkage. These two moieties interact with the Arg97 residue of E-selectin, as revealed by a recent crystal structure analysis of the E-selectin/sLe(X) complex. The energetics of this specific interaction was investigated with the aid of ab initio Hartree-Fock MO calculations, which resulted in a BSSE-corrected binding energy of 16.63 kcal/mol. Our observations could open up new perspectives in the design of sLe(X) mimics.     

Protein tyrosine phosphatase 1B deficiency potentiates PERK/eIF2α signaling in brown adipocytes

Background

Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and body mass, and has been implicated in endoplasmic reticulum (ER) stress. Herein, we assess the role of PTP1B in ER stress in brown adipocytes, which are key regulators of thermogenesis and metabolic response.

Methodology/Principal Findings

To determine the role of PTP1B in ER stress, we utilized brown adipose tissue (BAT) from mice with adipose-specific PTP1B deletion, and brown adipocytes deficient in PTP1B and reconstituted with PTP1B wild type (WT) or the substrate-trapping PTP1B D181A (D/A) mutant. PTP1B deficiency led to upregulation of PERK-eIF2α phosphorylation and IRE1α-XBP1 sub-arms of the unfolded protein response. In addition, PTP1B deficiency sensitized differentiated brown adipocytes to chemical-induced ER stress. Moreover, PERK activation and tyrosine phosphorylation were increased in BAT and adipocytes lacking PTP1B. Increased PERK activity resulted in the induction of eIF2α phosphorylation at Ser51 and better translatability of ATF4 mRNA in response to ER stress. At the molecular level, we demonstrate direct interaction between PTP1B and PERK and identify PERK Tyr615 as a mediator of this association.

Conclusions

Collectively, the data demonstrate that PTP1B is a physiologically-relevant modulator of ER stress in brown adipocytes and that PTP1B deficiency modulates PERK-eIF2α phosphorylation and protein synthesis.     

Small molecules with similar structures exhibit agonist, neutral antagonist or inverse agonist activity toward angiotensin II type 1 receptor

Small differences in the chemical structures of ligands can be responsible for agonism, neutral antagonism or inverse agonism toward a G-protein-coupled receptor (GPCR). Although each ligand may stabilize the receptor conformation in a different way, little is known about the precise conformational differences. We synthesized the angiotensin II type 1 receptor blocker (ARB) olmesartan, R239470 and R794847, which induced inverse agonism, antagonism and agonism, respectively, and then investigated the ligand-specific changes in the receptor conformation with respect to stabilization around transmembrane (TM)3. The results of substituted cysteine accessibility mapping studies support the novel concept that ligand-induced changes in the conformation of TM3 play a role in stabilizing GPCR. Although the agonist-, neutral antagonist and inverse agonist-binding sites in the AT(1) receptor are similar, each ligand induced specific conformational changes in TM3. In addition, all of the experimental data were obtained with functional receptors in a native membrane environment (in situ).     

Multiple SNPs in intron 41 of thyroglobulin gene are associated with autoimmune thyroid disease in the Japanese population

BACKGROUND: The etiology of the autoimmune thyroid diseases (AITDs), Graves' disease (GD) and Hashimoto's thyroiditis (HT), is largely unknown. However, genetic susceptibility is believed to play a major role. Two whole genome scans from Japan and from the US identified a locus on chromosome 8q24 that showed evidence for linkage with AITD and HT. Recent studies have demonstrated an association between thyroglobulin (Tg) polymorphisms and AITD in Caucasians, suggesting that Tg is a susceptibility gene on 8q24. OBJECTIVES: The objective of the study was to refine Tg association with AITD, by analyzing a panel of 25 SNPs across an extended 260 kb region of the Tg. METHODS: We studied 458 Japanese AITD patients (287 GD and 171 HT patients) and 221 matched Japanese control subjects in association studies. Case-control association studies were performed using 25 Tg single nucleotide polymorphisms (SNPs) chosen from a database of the Single Nucleotide Polymorphism Database (dbSNP). Haplotype analysis was undertaken using the computer program SNPAlyze version 7.0. PRINCIPAL FINDINGS AND CONCLUSIONS: In total, 5 SNPs revealed association with GD (P<0.05), with the strongest SNP associations at rs2256366 (P?=?0.002) and rs2687836 (P?=?0.0077), both located in intron 41 of the Tg gene. Because of the strong LD between these two strongest associated variants, we performed the haplotype analysis, and identified a major protective haplotype for GD (P?=?0.001). These results suggested that the Tg gene is involved in susceptibility for GD and AITD in the Japanese.