A surfactant-based dressing can reduce the appearance of Pseudomonas aeruginosa pigments and uncover the dermal extracellular matrix in an ex vivo porcine skin wound model.

From previous studies, we have shown that viable colony forming units of bacteria and bacterial biofilms are reduced after sequential treatment with a surfactant-based dressing. Here, we sought to test the impact on visible bacterial pigments and the ultrastructural impact following the sequential treatment of the same surfactant-based dressing. Mature Pseudomonas aeruginosa biofilms were grown on ex vivo porcine skin explants, and an imaging-based analysis was used to compare the skin with and without a concentrated surfactant. In explants naturally tinted by bacterial chromophores, wiping alone had no effect, while the use of a surfactant-based dressing reduced coloration. Similarly, daily wiping led to increased immunohistochemical staining for P. aeruginosa antigens, but not in the surfactant group. Confocal immunofluorescent imaging revealed limited bacterial penetration and coating of the dermis and loose pieces of sloughing material. Ultrastructural analysis confirmed that the biofilms were masking the extracellular matrix (ECM), but the surfactant could remove them, re-exposing the ECM. The masking of the ECM may provide another non-inflammatory explanation for delayed healing, as the ECM is no longer accessible for wound cell locomotion. The use of a poloxamer-based surfactant appears to be an effective way to remove bacterial chromophores and the biofilm coating the ECM fibres.

Identification and Characterization of Antibiotic-Resistant, Gram-Negative Bacteria Isolated from Korean Fresh Produce and Agricultural Environment.

The consumption of fresh produce and fruits has increased over the last few years as a result of increasing consumer awareness of healthy lifestyles. Several studies have shown that fresh produces and fruits could be potential sources of human pathogens and antibiotic-resistant bacteria. In this study, 248 strains were isolated from lettuce and surrounding soil samples, and 202 single isolates selected by the random amplified polymorphic DNA (RAPD) fingerprinting method were further characterized. From 202 strains, 184 (91.2%) could be identified based on 16S rRNA gene sequencing, while 18 isolates (8.9%) could not be unequivocally identified. A total of 133 (69.3%) and 105 (54.7%) strains showed a resistance phenotype to ampicillin and cefoxitin, respectively, while resistance to gentamicin, tobramycin, ciprofloxacin, and tetracycline occurred only at low incidences. A closer investigation of selected strains by whole genome sequencing showed that seven of the fifteen sequenced strains did not possess any genes related to acquired antibiotic resistance. In addition, only one strain possessed potentially transferable antibiotic resistance genes together with plasmid-related sequences. Therefore, this study indicates that there is a low possibility of transferring antibiotic resistance by potential pathogenic enterobacteria via fresh produce in Korea. However, with regards to public health and consumer safety, fresh produce should nevertheless be continuously monitored to detect the occurrence of foodborne pathogens and to hinder the transfer of antibiotic resistance genes potentially present in these bacteria.

TGFß4 alleviates the phenotype of Charcot-Marie-Tooth disease type 1A.

The duplication of the peripheral myelin protein 22 (PMP22) gene causes a demyelinating type of neuropathy, commonly known as Charcot-Marie-Tooth disease type 1A (CMT1A). Development of effective drugs for CMT1A still remains as an unmet medical need. In the present study, we assessed the role of the transforming growth factor beta 4 (TGFß4)/Nodal axis in the pathogenesis of CMT1A. First, we identified PMP22 overexpression-induced Nodal expression in Schwann cells, which might be one of the downstream effectors in CMT1A. Administration of Nodal protein at the developmental stage of peripheral nerves induced the demyelinating phenotype in vivo. Second, we further isolated TGFß4 as an antagonist that could abolish Nodal-induced demyelination. Finally, we developed a recombinant TGFß4-fragment crystallizable (Fc) fusion protein, CX201, and demonstrated that its application had promyelinating efficacy in Schwann cells. CX201 administration improved the demyelinating phenotypes of CMT1A mouse models at both pre-symptomatic and post-symptomatic stages. These results suggest that the TGFß4/Nodal axis plays a crucial role in the pathogenesis of CMT1A and might be a potential therapeutic target for CMT1A.

Ellagic Acid Prevents Particulate Matter-Induced Pulmonary Inflammation and Hyperactivity in Mice: A Pilot Study.

The inhalation of fine particulate matter (PM) is a significant health-related environmental issue. Previously, we demonstrated that repeated PM exposure causes hyperlocomotive activity in mice, as well as inflammatory and hypoxic responses in their lungs. In this study, we evaluated the potential efficacy of ellagic acid (EA), a natural polyphenolic compound, against PM-induced pulmonary and behavioral abnormalities in mice. Four treatment groups were assigned in this study (n = 8): control (CON), particulate-matter-instilled (PMI), low-dose EA with PMI (EL + PMI), and high-dose EA with PMI (EH + PMI). EA (20 and 100 mg/kg body weight for low dose and high dose, respectively) was orally administered for 14 days in C57BL/6 mice, and after the eighth day, PM (5 mg/kg) was intratracheally instilled for 7 consecutive days. PM exposure induced inflammatory cell infiltration in the lungs following EA pretreatment. Moreover, PM exposure induced inflammatory protein expression in the bronchoalveolar lavage fluid and the expression of inflammatory (tumor necrosis factor alpha (Tnfα), interleukin (Il)-1b, and Il-6) and hypoxic (vascular endothelial growth factor alpha (Vegfα), ankyrin repeat domain 37 (Ankrd37)) response genes. However, EA pretreatment markedly prevented the induction of expression of inflammatory and hypoxic response genes in the lungs. Furthermore, PM exposure significantly triggered hyperactivity by increasing the total moving distance with an increase in moving speed in the open field test. On the contrary, EA pretreatment significantly prevented PM-induced hyperactivity. In conclusion, dietary intervention with EA may be a potential strategy to prevent PM-induced pathology and activity.

Particulate Matter Elevates Ocular Inflammation and Endoplasmic Reticulum Stress in Human Retinal Pigmented Epithelium Cells.

Because of their exposure to air, eyes can come into contact with air pollutants such as particulate matter (PM), which may cause severe ocular pathologies. Prolonged ocular PM exposure may increase inflammation and endoplasmic reticulum stress in the retina. Herein, we investigated whether PM exposure induces ocular inflammation and endoplasmic reticulum (ER) stress-related cellular responses in human retinal epithelium-19 (ARPE-19) cells. To understand how PM promotes ocular inflammation, we monitored the activation of the mitogen-activated protein kinase (MAPK)/nuclear factor kappa beta (NFκB) axis and the expression of key inflammatory mRNAs. We also measured the upregulation of signature components for the ER-related unfolded protein response (UPR) pathways, as well as intracellular calcium ([Ca2+]i) levels, as readouts for ER stress induction following PM exposure. Ocular PM exposure significantly elevated the expression of multiple cytokine mRNAs and increased phosphorylation levels of NFκB-MAPK axis in a PM dose-dependent manner. Moreover, incubation with PM significantly increased [Ca2+]i levels and the expression of UPR-related proteins, which indicated ER stress resulting from cell hypoxia, and upregulation of hypoxic adaptation mechanisms such as the ER-associated UPR pathways. Our study demonstrated that ocular PM exposure increased inflammation in ARPE-19 cells, by activating the MAPK/NFκB axis and cytokine mRNA expression, while also inducing ER stress and stress adaptation responses. These findings may provide helpful insight into clinical and non-clinical research examining the role of PM exposure in ocular pathophysiology and delineating its underlying molecular mechanisms.

Dietary Intervention with Quercetin Attenuates Diesel Exhaust Particle-Instilled Pulmonary Inflammation and Behavioral Abnormalities in Mice.

Exposure to diesel exhaust particles (DEPs) is inevitable and closely linked with increased health hazards, causing pulmonary abnormalities by increasing inflammation, hypoxia, and so on. Moreover, long-term exposure to DEPs may trigger whole-body toxicity with behavioral alterations. Therefore, nutritional intervention with natural components may be desirable to prevent and/or ameliorate DEP-inducible pathophysiology in mammals. Quercetin has been demonstrated to reduce metabolic complications by possessing antioxidative, anti-inflammatory, and antimutagenic effects. In this study, we investigated the effects of quercetin on pulmonary inflammation and behavioral alteration in male C57BL/6 mice against DEP instillation. The experimental mice were separated into four treatment groups (n = 8 per group), which include: vehicle control, DEP instillation, dietary intervention with a low dose of quercetin (20 mg/kg) for 14 days with DEP instillation for 7 days, or dietary intervention with a high dose of quercetin (100 mg/kg) for 14 days with DEP instillation for 7 days. Compared with the DEP-instilled group, dietary intervention with quercetin significantly attenuated eosinophils in the bronchoalveolar lavage fluid analysis, pulmonary cytokine, and hypoxic mRNA expressions regardless of quercetin concentrations. DEP instillation triggered hyperactivities in the experimental mice, while quercetin pretreatment successfully normalized DEP-inducible abnormalities regardless of the dosage. Therefore, dietary intervention with quercetin may be an applicable means to prevent DEP-triggered pulmonary and behavioral abnormalities.

Determining MMP-2 and MMP-9 reductive activities of bovine and equine amniotic membranes homogenates using fluorescence resonance energy transfer.

INTRODUCTION: Matrix metalloproteinases (MMPs)-2 and -9 are present in corneal ulcers, and an imbalance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) leads to further corneal degradation. Amniotic membrane homogenate (AMH) has proteolytic properties beneficial for corneal healing, but it is unknown whether AMH possesses TIMPs or effectively inhibits MMP-2 and MMP-9 activity. OBJECTIVE: To determine if bovine and equine AMH reduce in vitro MMP-2 and MMP-9 activities associated with the presence of TIMPs. PROCEDURES: Undiluted and diluted twofold series (0-fold to 16-fold dilutions) of equine amniotic membrane homogenates (EAMH, n = 8) and bovine amniotic membrane homogenates (BAMH, n = 8) were subjected to fluorescence resonance energy transfer, and the fluorescence emitted was recorded over time. Average fluorescence was calculated versus recombinant concentration. Enzyme-linked immunosorbent assays for TIMPs 1-4 were applied to quantify TIMPs in the samples. RESULTS: AMH from both species were able to inhibit MMP-2 and MMP-9 activities in vitro, and the inhibition efficacy decreased gradually with dilution. BAMH was significantly more effective than EAMH at inhibiting MMP-2 and MMP-9 in vitro. TIMPs -2 and -3 were present in EAMH and BAMH. TIMP-1 was detected only in BAMH, and TIMP-4 was not detected in any samples. CONCLUSION: Both EAMH and BAMH directly inhibited MMP-2 and MMP-9 in vitro without dilution, and BAMH showed better inhibition of MMP-2 and MMP-9 before and after dilution compared to EAMH.

An Exploratory Study of the Relationships Between Diesel Engine Exhaust Particle Inhalation, Pulmonary Inflammation and Anxious Behavior.

Recent technical developments brought negative side effects such as air pollution and large-scale fires, increasingly exposing people to diesel engine exhaust particles (DEP). Testing how DEP inhalation triggers pathophysiology in animal models could be useful in determining how it affects humans. To this end, the aim of this study was to investigate the effects of pulmonary exposure to DEP for seven consecutive days in experimental male C5BL6/N mice. Twenty-four C5BL6/N mice were treated with one of the three test materials: distilled water for control, a low DEP exposure (5 mg/kg), or a high DEP exposure (15 mg/kg). Exposure to DEP induced decreased body weight; however, it gradually increased pulmonary weight in a DEP-dose-dependent manner. DEP exposure significantly elevated soot accumulation in the lungs, with the alteration of pulmonary homeostasis. It also elevated infiltrated immune cells, thus significantly increasing inflammatory cytokine mRNA and protein production in the lungs and broncho-alveolar lavage fluid, respectively. Pulmonary DEP exposure also altered behavioral responses in the open field test (OFT). Low exposure elevated moving distance and speed, while significantly decreasing the number of trials to enter the central zone. Different concentrations of DEP resulted in different behavioral changes; however, while anxiety levels increased, their degree was independent of DEP concentrations. Results suggest that DEP exposure may possess pro-inflammatory responses in the lungs and trigger anxiety.

Testing the influence of surfactant-based wound dressings on proteinase activity.

Proteinases are enzymes that can digest other proteins. In chronic wounds, a sub-class of these enzymes with the ability to degrade the extracellular matrix (matrix metalloproteinases, MMPs) have been found to both inhibit healing and to be able to aid in enzymatically debriding a wound. Enzymatic debridement using the enzymes present in a wound is generally called autolytic debridement. Clinicians seeking to employ autolytic debridement typically use occlusive materials such as medical honey, alginate dressings and other occlusive dressings. A relatively new class of gel dressings comprised of surfactants are now available for clinical use. A variety of surfactants are used in the study of MMP biochemistry. Surfactants can deactivate MMPs or can enhance their activity, depending on the surfactant. In order to begin to understand how the MMPs found in chronic wounds would respond to these new dressings, we tested a serial dilution series of two of the currently available surfactant-based dressings to determine their effects on four separate MMPs. The dose-response versus MMP activity of bacterial collagenase, host-derived MMP-8 and MMPs-2 and -9 was assessed using a simple mix-and-read fluorescent peptide activity assay. The enzyme's native activity in the absence of the gel was used to compare against the surfactant-treated samples. We found that the surfactant affected the proteinase activity differently for each enzyme. The activity of the bacterial collagenase was increased at low concentrations but slightly inhibited as the concentrations increased. The host MMP-8 collagenase responded similarly in that it was inhibited at higher concentrations. Interestingly, both MMP gelatinases presented with substantially increased activities, with MMP-2 increased to 200% of native activity, while MMP-9 presented with an increase of 300% activity over the same concentration range. MMPs appear to respond to a surfactant-based gel dressing differentially, with the MMP most commonly elevated in chronic wounds having the highest boost to activity. In wounds with elevated MMPs, our data suggest that the use of these surfactant-based dressings would be expected to enhance the activity of MMPs 2 and 9 gelatinases while simultaneously inhibiting MMP-8 collagenase. Hypothetically, this imbalanced effect would support a protection of the native dermal collagen and removal of denatured materials. However, the demonstration of these anticipated consequences is still being investigated.

Differentiation of RotaTeq® vaccine strains from wild-type strains using NSP3 gene in reverse transcription polymerase chain reaction assay.

RotaTeq® is a live attenuated human-bovine reassortant vaccine against rotaviruses that is used worldwide. However, shedding of the virus used in RotaTeq® has been detected in the feces of children following vaccination by the oral route, possibly affecting community immunity. Therefore, a simple and efficient method to discriminate between virulent and RotaTeq® vaccine strains is required. In this study, a novel one-step multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay targeting the NSP3 gene was developed to detect RotaTeq® vaccine strains in fecal samples. RotaTeq® vaccine viruses were successfully distinguished from known wild-type rotavirus genotypes. In addition, the developed assay was able to detect rotaviruses in clinical stool samples obtained from South Korea during the 2011-2013 rotavirus seasons. Of the 1106 stool specimens from children with acute gastroenteritis that were screened, 286 rotaviruses were genotyped. RotaTeq® vaccine strains were identified in 39 samples (13.6%). The novel RT-PCR assay that was developed could be used to detect and discriminate between RotaTeq® vaccine strains that are shed in fecal matter, and to estimate the quantification of virus that has been shed after vaccination.

Identification of agents effective against multiple toxins and viruses by host-oriented cell targeting.

A longstanding and still-increasing threat to the effective treatment of infectious diseases is resistance to antimicrobial countermeasures. Potentially, the targeting of host proteins and pathways essential for the detrimental effects of pathogens offers an approach that may discover broad-spectrum anti-pathogen countermeasures and circumvent the effects of pathogen mutations leading to resistance. Here we report implementation of a strategy for discovering broad-spectrum host-oriented therapies against multiple pathogenic agents by multiplex screening of drugs for protection against the detrimental effects of multiple pathogens, identification of host cell pathways inhibited by the drug, and screening for effects of the agent on other pathogens exploiting the same pathway. We show that a clinically used antimalarial drug, Amodiaquine, discovered by this strategy, protects host cells against infection by multiple toxins and viruses by inhibiting host cathepsin B. Our results reveal the practicality of discovering broadly acting anti-pathogen countermeasures that target host proteins exploited by pathogens.

The Effect of Polyphenols Isolated from Cynanchi wilfordii Radix with Anti-inflammatory, Antioxidant, and Anti-bacterial Activity.

Recently, Cynanchi wilfordii Radix has gained wide use in Asian countries as a functional food effective for relieving fatigue, osteoporosis, and constipation, particularly in menopausal disorders. However, its anti-inflammatory and anti-microbial activities have not been explored in detail to date. The anti-inflammatory, antioxidant, and anti-bacterial properties of the Cynanchi wilfordii Radix extracts obtained with water, methanol, ethanol, and acetone were compared. All 4 polyphenol-containing extracts exhibited anti-inflammatory and antioxidant effects. The ethanol extract was found to elicit the most potent reduction of nitric oxide (NO), prostaglandin E2 (PGE2), and cytokine (IL-1ß, IL-6, IL-10, and TNF-α) levels, as well as inhibit the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a concentration-dependent manner. The evaluation of antioxidant activity also revealed the ethanol extract to have the highest free radical scavenging activity, measured as 85.3±0.4%, which is equivalent to 99.9% of the activity of α -tocopherol. In the assessment of anti-bacterial activity, only ethanol extract was found to inhibit the growth of the Bacillus species Bacillus cereus and Bacillus anthracis. These results show that polyphenols of Cynanchi wilfordii Radix have anti-inflammatory, antioxidant, and anti-bacterial properties that can be exploited and further improved for use as a supplementary functional food, in cosmetics, and for pharmaceutical purposes.

Middle East Respiratory Syndrome Infection Control and Prevention Guideline for Healthcare Facilities.

Middle East Respiratory Syndrome (MERS) is an acute viral respiratory illness with high mortality caused by a new strain of betacoronavirus (MERS-CoV). Since the report of the first patient in Saudi Arabia in 2012, large-scale outbreaks through hospital-acquired infection and inter-hospital transmission have been reported. Most of the patients reported in South Korea were also infected in hospital settings. Therefore, to eliminate the spread of MERS-CoV, infection prevention and control measures should be implemented with rigor. The present guideline has been drafted on the basis of the experiences of infection control in the South Korean hospitals involved in the recent MERS outbreak and on domestic and international infection prevention and control guidelines. To ensure efficient MERS-CoV infection prevention and control, care should be taken to provide comprehensive infection control measures including contact control, hand hygiene, personal protective equipment, disinfection, and environmental cleaning.

Characterization of RotaTeq® vaccine-derived rotaviruses in South Korean infants with rotavirus gastroenteritis.

Genotyping of human rotaviruses was performed in 191 rotavirus-positive fecal samples collected from infants with acute gastroenteritis, 3 years after the introduction of two rotavirus vaccines in South Korea. Among these samples, the most prevalent rotavirus genotype was G3P[8] (30.9%), followed by G1P[8] (27.7%), G4P[6] (15.2%), and G9P[8] (5.8%). Sequence analysis identified RotaTeq® vaccine-derived strains in 12 samples (6.3%), comprising 11 G1P[8] human-bovine double reassortant rotaviruses and 1 G1P[5] human-bovine single reassortant rotavirus. It is of note that cross-reactivity between the current G4-specific typing primer and RotaTeq®-specific G1 genotypes was found. A trace of the clinical and environmental routes of the rotavirus vaccine strains revealed unexpected complexity, and the diagnostic protocol for rotaviruses may require modification by using either another typing primer set or nucleotide sequence analysis.

A systematic review of genetic diversity of human rotavirus circulating in South Korea.

Rotavirus infections continue to be the leading cause of severe diarrhea in young Korean children. Rotavirus data acquired from uninterrupted surveillance studies between 1989 and 2009 in South Korea were analyzed to better understand the genetic diversity and evolution. The relationship between rotaviruses and the currently licensed rotavirus vaccine viruses was also examined. The most prevalent rotavirus strains, with genotype G1P[8], followed by G3P[8], G4P[6], and G2P[4], accounted for approximately 76.7% of the total identified strains, and more recently, rotavirus G9P[8] has significance increased to be the fifth most common genotype. Phylogenetic analyses underscored the heterogeneity between viral populations within each genotype, with different lineages and sub-lineages. Although the currently licensed rotavirus vaccines are effective, safe, and economical, additional data from rotavirus monitoring is necessary to evaluate the efficacy of these vaccines for their sustained use in South Korea. The present study provides comprehensive and up-to-date information regarding the epidemiology, genetic diversity, and evolution of the circulating rotaviruses in South Korea.

Whole-genome analysis of a rare human Korean G3P rotavirus strain suggests a complex evolutionary origin potentially involving reassortment events between feline and bovine rotaviruses.

A rare human rotavirus, G3P[9] strain RVA/Human-tc/KOR/CAU12-2-51/2013/G3P[9], was isolated from the stool of a 9-year-old female hospitalized with acute watery diarrhea in August 2012 in South Korea using a cell culture system, and its genome was analyzed. The complete genomic constellation of the CAU12-2-51 strain revealed a novel genotype constellation for human rotavirus, G3-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3. Phylogenetic analysis revealed that the CAU12-2-51 strain originated from feline- and bovine-like reassortment strains. The genes encoding VP4, VP7, NSP1, NSP3, NSP4, and NSP5 were related to human/feline-like and feline rotavirus strains, whereas the remaining five genes encoding VP1, VP2, VP3, VP6, and NSP2 were related to the human/bovine-like and bovine rotavirus strains. This novel strain was identified for the first time, providing evidence of feline/bovine-to-human transmission of rotavirus. The data presented herein provide information regarding rotavirus diversity and evolution.

Bidirectional effect of Wnt signaling antagonist DKK1 on the modulation of anthrax toxin uptake.

LRP6, a co-receptor for the morphogen Wnt, aids endocytosis of anthrax complexes. Here we report that Dickkopf1 (DKK1) protein, a secreted LRP6 ligand and antagonist, is also a modulator of anthrax toxin sensitivity. shRNA-mediated gene silencing or TALEN-mediated gene knockout of DKK1 reduced sensitivity of cells to PA-dependent hybrid toxins. However, unlike the solely inhibitory effect on Wnt signaling, the effects of DKK1 overexpression on anthrax toxicity were bidirectional, depending on its endogenous expression and cell context. Fluorescence microscopy and biochemical analyses showed that DKK1 facilitates internalization of anthrax toxins and their receptors, an event mediated by DKK1-LRP6-Kremen2 complex. Monoclonal antibodies against DKK1 provided dose-dependent protection to macrophages from killing by anthrax lethal toxin (LT). Our discovery that DKK1 forms ternary structure with LRP6 and Kremen2 in promoting PA-mediated toxin internalization provides a paradigm for bacterial exploitation of mechanisms that host cells use to internalize signaling proteins.

Calpain-dependent cytoskeletal rearrangement exploited for anthrax toxin endocytosis.

The protective antigen component of Bacillus anthracis toxins can interact with at least three distinct proteins on the host cell surface, capillary morphogenesis gene 2 (CMG2), tumor endothelial marker 8, and ß1-integrin, and, with the assistance of other host proteins, enters targeted cells by receptor-mediated endocytosis. Using an antisense-based phenotypic screen, we discovered the role of calpains in this process. We show that functions of a ubiquitous Ca(2+)-dependent cysteine protease, calpain-2, and of the calpain substrate talin-1 are exploited for association of anthrax toxin and its principal receptor, CMG2, with higher-order actin filaments and consequently for toxin entry into host cells. Down-regulated expression of calpain-2 or talin-1, or pharmacological interference with calpain action, did not affect toxin binding but reduced endocytosis and increased the survival of cells exposed to anthrax lethal toxin. Adventitious expression of wild-type talin-1 promoted toxin endocytosis and lethality, whereas expression of a talin-1 mutant (L432G) that is insensitive to calpain cleavage did not. Disruption of talin-1, which links integrin-containing focal adhesion complexes to the actin cytoskeleton, facilitated association of toxin bound to its principal cell-surface receptor, CMG2, with higher-order actin filaments undergoing dynamic disassembly and reassembly during endocytosis. Our results reveal a mechanism by which a bacterial toxin uses constitutively occurring calpain-mediated cytoskeletal rearrangement for internalization.

Heterodimeric integrin complexes containing beta1-integrin promote internalization and lethality of anthrax toxin.

To kill macrophages, the lethal factor component of Bacillus anthracis toxin binds to a carrier protein (PA), which then interacts with the CMG2 receptor protein on the cell surface and is endocytosed into the cytoplasm. CMG2, as well as TEM8, a second PA receptor not present on macrophages, contain a von Willebrand A domain that is crucial for toxin binding. Here we report that integrin beta1, another cell surface von Willebrand A domain protein, can mediate and potentiate anthrax toxin endocytosis. By using microarray-based analysis to globally correlate gene expression profiles with toxin sensitivity, we associated toxin effects with the integrin-activating proteins osteopontin and CD44. Further study showed that PA binds to alpha4beta1- and alpha5beta1-integrin complexes, leading to their conjoint endocytosis, and also interacts-weakly relative to CMG2 but comparably to TEM8--with purified alpha5beta1 complex in vitro. Monoclonal antibody directed against beta1-integrin or its alpha integrin partners reduced PA/integrin endocytosis and anthrax toxin lethality, and hyaluronic acid--which interferes with CD44-mediated integrin activation--had similar effects. Remarkably, whereas deficiency of CMG2 protected macrophages from rapid killing by large toxin doses (>50 ng/mL), by 24 h the toxin-treated cells were dead. Such late killing of CMG2-deficient cells by high dose toxin as well as the late death observed during exposure of CMG2-producing macrophages to low-dose toxin (<1 ng/mL), was dependent on integrin function. Effects of inactivating both CMG2 and integrin were synergistic. Collectively, our findings argue strongly that beta1-integrin can both potentiate CMG2-mediated endocytosis and serve independently as a low-affinity PA receptor.

Methylglyoxal accumulation by glutathione depletion leads to cell cycle arrest in Dictyostelium.

Reduced glutathione (GSH) serves as a primary redox buffer and its depletion causes growth inhibition or apoptosis in many organisms. In Dictyostelium discoideum, the null mutant (gcsA(-)) of gcsA encoding gamma-glutamylcysteine synthetase shows growth arrest and developmental defect when GSH is depleted. To investigate the mechanism by which GSH depletion induces growth arrest, a proteomic analysis was performed and aldose reductase (AlrA) was identified as the most prominently induced protein in gcsA(-) cells. Induction of AlrA was dependent on GSH concentration and was repressed by GSH but not effectively by either the reducing agent such as dithiothreitol or overexpression of superoxide dismutase. Methylglyoxal (MG), a toxic alpha-ketoaldehyde, strongly induced alrA expression and AlrA catalysed MG reduction efficiently. The alrA knockdown gcsA(-) cells (gcsA(-)/alrA(as)) exhibited more decreased growth rate than gcsA(-) cells, whereas the gcsA(-) cells overexpressing alrA (gcsA(-)/alrA(oe)) showed the recovery of growth rate. Interestingly, intracellular MG levels were significantly augmented in gcsA(-)/alrA(as) cells compared with gcsA(-) cells following GSH depletion. By contrast, gcsA(-)/alrA(oe) cells showed repression of MG induction. Furthermore, MG treatment inhibited growth of wild-type KAx3 cells, inducing G1 phase arrest. Thus, our findings suggest that MG accumulated by GSH depletion inhibits cell growth in Dictyostelium.