3'-Sialyllactose protects against LPS-induced endothelial dysfunction by inhibiting superoxide-mediated ERK1/2/STAT1 activation and HMGB1/RAGE axis.

AIM: Endothelial hyperpermeability is an early stage of endothelial dysfunction associated with the progression and development of atherosclerosis. 3'-Sialyllactose (3'-SL) is the most abundant compound in human milk oligosaccharides, and it has the potential to regulate endothelial dysfunction. This study investigated the beneficial effects of 3'-SL on lipopolysaccharide (LPS)-induced endothelial dysfunction in vitro and in vivo. MAIN METHODS: We established LPS-induced endothelial dysfunction models in both cultured bovine aortic endothelial cells (BAECs) and mouse models to determine the effects of 3'-SL. Western blotting, qRT-PCR analysis, immunofluorescence staining, and en face staining were employed to clarify underlying mechanisms. Superoxide production was measured by 2',7'-dichlorofluorescin diacetate, and dihydroethidium staining. KEY FINDINGS: LPS significantly decreased cell viability, whereas 3'-SL treatment mitigated these effects via inhibiting ERK1/2 activation. Mechanistically, 3'-SL ameliorated LPS-induced ROS accumulation leading to ERK1/2 activation-mediated STAT1 phosphorylation and subsequent inhibition of downstream transcriptional target genes, including VCAM-1, TNF-α, IL-1ß, and MCP-1. Interestingly, LPS-induced ERK1/2/STAT1 activation leads to the HMGB1 release from the nucleus into the extracellular space, where it binds to RAGE, while 3'-SL suppressed EC hyperpermeability by suppressing the HMGB1/RAGE axis. This interaction also led to VE-cadherin endothelial junction disassembly and endothelial cell monolayer disruption through ERK1/2/STAT1 modulation. In mouse endothelium, en face staining revealed that 3'-SL abolished LPS-stimulated ROS production and VCAM-1 overexpression. SIGNIFICANCE: Our findings suggest that 3'-SL inhibits LPS-induced endothelial hyperpermeability by suppressing superoxide-mediated ERK1/2/STAT1 activation and HMGB1/RAGE axis. Therefore, 3'-SL may be a potential therapeutic agent for preventing the progression of atherosclerosis.

Human milk oligosaccharides 3'-sialyllactose and 6'-sialyllactose attenuate LPS-induced lung injury by inhibiting STAT1 and NF-κB signaling pathways.

Acute lung injury (ALI) is the leading cause of respiratory diseases induced by uncontrolled inflammation and cell death. Lipopolysaccharide (LPS) is a major trigger of ALI in the progression through macrophage differentiation and the accelerated release of pro-inflammatory cytokines. The present study aimed to investigate the protective effects of human milk oligosaccharides, specifically 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), on LPS-induced ALI and elucidate their underlying signaling pathways. The inhibitory effects of 3'-SL and 6'-SL on inflammation were evaluated using LPS-treated RAW 264.7 macrophages. To establish the ALI model, mice were treated with 10 mg/kg LPS for 24 h. Histological changes in the lung tissues were assessed using hematoxylin and eosin staining and immunofluorescence. LPS causes thickening of the alveolar wall infiltration of immune cells in lung tissues and increased serum levels of TNF-α, IL-1ß, and GM-CSF. However, these effects were significantly alleviated by 100 mg/kg of 3'-SL and 6'-SL. Consistent with the inhibitory effects of 3'-SL and 6'-SL on LPS-induced pro-inflammatory cytokine secretion in serum, 3'-SL and 6'-SL suppressed mRNA expression of TNF-α, IL-1ß, MCP-1, iNOS, and COX2 in LPS-induced RAW 264.7 cells. Mechanistically, 3'-SL and 6'-SL abolished LPS-mediated phosphorylation of NF-κB and STAT1. Interestingly, fludarabine treatment, a STAT1 inhibitor, did not affect LPS-mediated NF-κB phosphorylation. In summary, 3'-SL and 6'-SL protect LPS-induced macrophage activation and ALI through the STAT1 and NF-κB signaling pathways.

Pharmacokinetics and clinical efficacy of 6'-sialyllactose in patients with GNE myopathy: Randomized pilot trial.

GNE myopathy, caused by biallelic mutations in the GNE gene, is characterized by initial ankle dorsiflexor weakness and rimmed vacuoles in the muscle histopathology, resulting in reduced sialic acid production. Sialyllactose is a source of sialic acid. We performed a pilot clinical trial to analyze the pharmacokinetic properties of 6'-sialyllactose (6SL) and evaluated the safety, and efficacy of oral 6SL in patients with GNE myopathy. Ten participants were in the pharmacokinetic study, and 20 in the subsequent clinical trial. For the pharmacokinetic study, participants were administered either 3 g (low-dose) or 6 g (high-dose) of 6SL in a single dose. Plasma concentrations of 6SL, sialic acid, and sialic acid levels on the surface of red blood cells were periodically assessed in blood samples. Patients were randomly allocated to test (low- and high-dose groups) or placebo groups for the trial. Motor function, ambulation, plasma 6SL and sialic acid concentrations, GNE myopathy-functional activity scale scores, and MRI findings were assessed. 6SL was well tolerated, except for self-limited gastrointestinal discomfort. Free sialic acid in both low- and high-dose groups significantly increased at 6 and 12 weeks, but not in the placebo group. In the high-dose group, proximal limb powers improved with daily 6SL. Considering the fat fraction on muscle MRI, results in the high-dose group were superior to those in the low-dose group. 6SL may be a good candidate for GNE myopathy therapeutics as it induces an increase or reduces the decrease in limb muscle power, attenuates muscle degeneration, and improves the biochemical properties of sialic acid.

Temporal quantitative profiling of sialyllactoses and sialic acids after oral administration of sialyllactose to mini-pigs with osteoarthritis.

Sialyllactose (SL) is the most abundant acidic oligosaccharide in human breast milk and plays a primary role in various biological processes. Recently, SL has attracted attention as an excellent dietary supplement for arthritis because it is effective in cartilage protection and treatment. Despite the superior function of SL, there are few pharmacological studies of SL according to blood concentrations in arthritis models. In this study, we investigated quantitative changes in SL and sialic acids in the plasma obtained from mini-pigs with osteoarthritis throughout exogenous administration of SL using liquid chromatography-multiple reaction monitoring mass spectrometry. Plasma concentrations of SL and sialic acids in the SL-fed group showed a significant difference compared to the control group. Mini pigs were fed only Neu5Ac bound to SL, but the concentration patterns of the two types of sialic acid, Neu5Ac and Neu5Gc, were similar. In addition, the relative mRNA expression level of matrix metalloproteinases (MMPs), which is known as a critical factor in cartilage matrix degradation, was remarkably decreased in the synovial membrane of the SL-fed group. Consequently, the temporal quantitative profiling suggests that dietary SL can be metabolized and utilized in the body and may protect against cartilage degradation by suppressing MMP expression during osteoarthritis progression.

6'-Sialylactose abolished lipopolysaccharide-induced inflammation and hyper-permeability in endothelial cells.

Disruption of the endothelial barrier function and reduction in cell migration leads to endothelial dysfunction. One of the most abundant human milk oligosaccharides, 6'-sialylactose (6'-SL), is reported to exert various biological functions related to inflammatory responses. In this study, we evaluated the effects of 6'-SL on lipopolysaccharide (LPS)-induced inflammation caused by endothelial barrier damage. Our results showed that LPS at 500 ng/mL strongly not only abolished cell migration but also hyperactivated MAPK and NF-κB pathways. 6'-SL suppressed LPS-induced endothelial inflammation via ERK1/2, p38, and JNK MAPK pathways. 6'-SL supported endothelial junctions by upregulating PECAM-1 expression and mRNA levels of tight junctions, such as ZO-1 and occludin, which were downregulated by LPS stimulation. It significantly inhibited the nuclear translocation of NF-κB, along with the downregulation of inflammatory cytokines, including TNF-α, IL-1ß, MCP-1, VCAM-1, and ICAM-1. Furthermore, 6'-SL abolished NF-κB-mediated STAT3 in controlling endothelial migration and hyperpermeability via downregulating STAT3 activation and nuclear translocation. Finally, LPS induced over-expression of VCAM-1 and ZO-1 disassembly in both atheroprone and atheroprotective areas of mouse aorta, which were reversed by 6'-SL treatment. Altogether, our findings suggest that 6'-SL is a potent therapeutic agent for modulating inflammatory responses and endothelial hyperpermeability.

Inhibitory effects of 6'-sialyllactose on angiotensin II-induced proliferation, migration, and osteogenic switching in vascular smooth muscle cells.

Excessive production and migration of vascular smooth muscle cells (VSMCs) are associated with vascular remodeling that causes vascular diseases, such as restenosis and hypertension. Angiotensin II (Ang II) stimulation is a key factor in inducing abnormal VSMC function. This study aimed to investigate the effects of 6'-sialyllactose (6'SL), a human milk oligosaccharide, on Ang II-stimulated cell proliferation, migration and osteogenic switching in rat aortic smooth muscle cells (RASMCs) and human aortic smooth muscle cells (HASMCs). Compared with the control group, Ang II increased cell proliferation by activating MAPKs, including ERK1/2/p90RSK/Akt/mTOR and JNK pathways. However, 6'SL reversed Ang II-stimulated cell proliferation and the ERK1/2/p90RSK/Akt/mTOR pathways in RASMCs and HASMCs. Moreover, 6'SL suppressed Ang II-stimulated cell cycle progression from G0/G1 to S and G2/M phases in RASMCs. Furthermore, 6'SL effectively inhibited cell migration by downregulating NF-κB-mediated MMP2/9 and VCAM-1 expression levels. Interestingly, in RASMCs, 6'SL attenuated Ang II-induced osteogenic switching by reducing the production of p90RSK-mediated c-fos and JNK-mediated c-jun, leading to the downregulation of AP-1-mediated osteopontin production. Taken together, our data suggest that 6'SL inhibits Ang II-induced VSMC proliferation and migration by abolishing the ERK1/2/p90RSK-mediated Akt and NF-κB signaling pathways, respectively, and osteogenic switching by suppressing p90RSK- and JNK-mediated AP-1 activity.

Therapeutic effect of intra-articular injected 3'-sialyllactose on a minipig model of rheumatoid arthritis induced by collagen.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disease of joint, but there is no known cure. 3'-sialyllactose (3'-SL) is an oligosaccharide that is abundant in breast milk of mammals, and has anti-inflammatory properties. However, the efficacy of 3'-SL on RA remains unclear. The objective of the present study was to evaluate the therapeutic effect of 3'-SL after it was directly injected into the knee joint cavity of a RA minipig model. RESULTS: Minipig RA model was induced by intra-articular injection of bovine type II collagen emulsified with complete or incomplete Freund's adjuvant into left knee joint. In clinical assessment, lameness and swelling of the hindlimb and increased knee joint width were observed in all animals. After the onset of arthritis, 3'-SL (0, 2, 10, and 50 mg/kg) was directly administered to the left knee joint cavity once a week for 4 weeks. Compared to the vehicle control group, no significant difference in macroscopic observation of the synovial pathology or the expression of inflammation-related genes (IL-1ß, TNF-α, and COX2) in the synovial membrane of the knee joint was found. In microscopic observation, cell cloning of the articular cartilage was significantly reduced in proportion to the concentration of 3'-SL administered. CONCLUSIONS: Our results suggest that intra-articular injected 3'-SL had a therapeutic effect on collagen-induced arthritis at the cellular level with potential as a medication for RA.

3'-Sialyllactose Protects SW1353 Chondrocytic Cells From Interleukin-1ß-Induced Oxidative Stress and Inflammation.

Osteoarthritis (OA) is a major degenerative joint disease. Oxidative stress and inflammation play key roles in the pathogenesis of OA. 3'-Sialyllactose (3'-SL) is derived from human milk and is known to regulate a variety of biological functions related to immune homeostasis. This study aimed to investigate the therapeutic mechanisms of 3'-SL in interleukin-1ß (IL-1ß)-treated SW1353 chondrocytic cells. 3'-SL potently suppressed IL-1ß-induced oxidative stress by increasing the levels of enzymatic antioxidants. 3'-SL significantly reversed the IL-1ß mediated expression levels of reactive oxygen species in IL-1ß-stimulated chondrocytic cells. In addition, 3'-SL could reverse the increased levels of inflammatory markers such as nitrite, prostaglandin E2, inducible nitric oxide synthase, cyclooxygenase-2, IL-1ß, and IL-6 in IL-1ß-stimulated chondrocytic cells. Moreover, 3'-SL significantly inhibited the apoptotic process, as indicated by the downregulation of the pro-apoptotic protein Bax, upregulation of the anti-apoptotic protein Bcl-2 expression, and significant reduction in the number of TUNEL-positive cells in the IL-1ß-treated chondrocytic cells. Furthermore, 3'-SL reversed cartilage destruction by decreasing the release of matrix metalloproteinases (MMP), such as MMP1, MMP3, and MMP13. In contrast, 3'-SL significantly increased the expression levels of matrix synthesis proteins, such as collagen II and aggrecan, in IL-1ß-treated chondrocytic cells. 3'-SL dramatically suppressed the activation of mitogen-activated protein kinases (MAPK) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways, which are related to the pathogenesis of OA. Taken together, our data suggest that 3'-SL alleviates IL-1ß-induced OA pathogenesis via inhibition of activated MAPK and PI3K/AKT/NF-κB signaling cascades with the downregulation of oxidative stress and inflammation. Therefore, 3'-SL has the potential to be used as a natural compound for OA therapy owing to its ability to activate the antioxidant defense system and suppress inflammatory responses.

Simultaneous Quantification of 3'- and 6'-Sialyllactose in Rat Plasma Using Liquid Chromatography-Tandem Mass Spectrometry and Its Application to a Pharmacokinetic Study.

Sialyllactose (SL), an acidic oligosaccharide, has immune-protective effects against pathogens and helps with the development of the immune system and intestinal microorganisms. To elucidate the pharmacokinetic characterization after oral administration to rats, the simultaneous quantification method for 3'-SL and 6'-SL in rat plasma was validated, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an electrospray ionization (ESI) mode. Several types of columns [C18, amide, and hydrophilic interaction liquid chromatography (HILIC) phase] were used to separate the peaks of 3'-SL and 6'-SL, which improved chromatographic selectivity. Ultimately, the HILIC phase column had a good peak shape and quick resolution, with a mobile phase comprising ammonium acetate buffer and acetonitrile obtained by gradient elution. In addition, the simultaneous quantification of 3'-SL and 6'-SL in rat plasma samples were adequately applied to pharmacokinetic study.

Development and validation of a bioanalytical method of analyzing 3'- and 6'-sialyllactose using liquid chromatography-tandem mass spectrometry in minipig plasma and its application in a pharmacokinetic study.

Sialyllactose (SL) is an acidic oligosaccharide, consisting of a combination of sialic acid and lactose. It is found in human milk. It has immune-protective effects against pathogens in newborns and helps with the development of the immune system and intestinal microorganisms. We developed and validated a method by which 3'-SL and 6'-SL levels were simultaneously analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS), and evaluated the pharmacokinetics of the materials after systemic delivery to minipigs. To improve chromatographic selectivity, several types of columns (C18, amide, and HILIC phase) were used to separate the peaks of 3'-SL and 6'-SL. Ultimately the HILIC phase column was selected, as it had a good peak shape and quick resolution. The mobile phase comprised ammonium acetate buffer and acetonitrile with gradient elution. MS was performed in the negative ion and multiple reaction monitoring modes. Plasma samples were prepared using the protein precipitation method with methanol. A surrogate matrix was used for quantification because SLs are endogenous plasma compounds. The method developed was validated according to U.S. Food and Drug Administration guidance. A pharmacokinetic study was performed with intravenous administration of 3'-SL and 6'-SL in minipigs (Sus scrofa/Yucatan). The concentrations of 3'-SL and 6'-SL were readily measurable in the plasma samples, which suggests that the method adequately determined systemic exposure in minipigs.

Discrimination between target and non-target interactions on the viral surface by merging fluorescence emission into Rayleigh scattering.

Direct and quantitative determination of antibodies or cellular receptors dynamically binding to the surface of viral particles is the key issue for predicting the efficacy of therapeutic materials or host susceptibility to a new emerging pathogen. However, targeted visualization of infectious viruses is still highly challenging owing to their nanoscopic sizes and uncontrollable nonspecific interactions with loading molecules responsible for false signals. Here we present a multimodal single-molecule and single-particle (SMSP) visualization capable of simultaneously yet independently tracking Rayleigh scattering and fluorescence that, respectively, are generated from viruses (approximately 100 nm) and labeled interacting molecules. By analyzing real-time trajectories of fluorescent antibodies against a virus surface protein with reference to single virus-derived Rayleigh scattering, we determined heterogeneous binding stoichiometry of virus-antibody couplings irrespective of the nonspecific binder population. Therefore, our multimodal (or multi-level) SMSP assay visually identifies and selectively quantifies specific interactions between them with single binding event accuracy. As a 'specific-binding quantifier' to assess variable host susceptibility to a virus, it was further applied for distinguishing ratiometric bindings and spontaneous dissociation kinetics of synthesized isomeric receptors to influenza virus. The present framework could offer a solid analytical foundation for the development of a direct-acting antiviral agent inhibiting an integral viral enveloped protein and for nanobiological investigation for dissecting spatiotemporal nanoparticle-molecule interactions, which have been scarcely explored compared to those among plasmonic nanoparticles or among molecules only.

Toxicological evaluation of 6'-sialyllactose (6'-SL) sodium salt.

We performed a series of toxicity studies on the safety of 6'-sialyllactose (6'-SL) sodium salt as a food ingredient. 6'-SL sodium salt, up to a maximum dose of 5000 µg/plate, did not increase the number of revertant colonies in five strains of Salmonella typhimurium in the presence or absence of S9 metabolic activation. A chromosomal aberration assay (using Chinese hamster lung cells) found no clastogenic effects at any concentration of 6'-SL sodium salt in the presence or absence of S9 metabolic activation. An in vivo bone marrow micronucleus test in Kunming mice showed no clastogenic activities with 6'-SL sodium salt doses up to 2000 mg/kg body weight (bw). In an acute toxicity study, the mean lethal dose of 6'-SL sodium salt was greater than 20 g/kg bw in rats. In a 13-week subchronic toxicity investigation, no effects were found at doses up to 5.0 g/kg bw of 6'-SL sodium salt in food consumption, body weight, clinical signs, blood biochemistry and hematology, urinalysis, or ophthalmic and histological macroscopic examination of organs. The no-observed-adverse-effect level (NOAEL) was 5.0 g/kg bw/day in rats.

Development of a guinea pig cutaneous radiation injury model using low penetrating X-rays.

PURPOSE: A guinea pig skin model was developed to determine the dose-dependent response to soft X-ray radiation into the dermis. MATERIALS AND METHODS: X-ray exposure (50 kVp) was defined to a 4.0 × 4.0 cm area on the lateral surface of a guinea pig using lead shielding. Guinea pigs were exposed to a single fraction of X-ray irradiation ranging from 25-79 Gy via an XRAD320ix Biological Irradiator with the collimator removed. Gross skin changes were measured using clinical assessments defined by the Kumar scale. Skin contracture was assessed, as well as histological evaluations. RESULTS: Loss of dermal integrity was shown after a single dose of soft X-ray radiation at or above 32 Gy with the central 2.0 × 2.0 cm of the exposed site being the most affected. Hallmarks of the skin injury included moist desquamation, ulceration and wound contracture, as well as alterations in epithelium, dermis, muscle and adipose. Changes in the skin were time- and radiation dose-dependent. Full-thickness injury occurred without animal mortality or gross changes in the underlying organs. CONCLUSIONS: The guinea pig is an appropriate small animal model for the short-term screening of countermeasures for cutaneous radiation injury (CRI).

The Effect of Reduced Dietary Fat and Soy Supplementation on Circulating Adipocytokines in Postmenopausal Women: A Randomized Controlled 2-Month Trial.

The reduced risk of breast cancer observed in Asia has been linked with diets rich in soy foods, and observational studies suggest that regular soy food intake is related to lower circulating levels of some inflammatory markers which have been implicated in breast cancer risk. However, short-term intervention studies with soy-based diets in small numbers of women have shown few significant changes in adipocytokine levels. This 8-wk dietary intervention study in 57 healthy postmenopausal women investigated whether soy food supplementation (50 mg isoflavones or 15 g soy protein in the form of tofu) or a very low-fat diet (11.3% of total energy), similar to the traditional Asian diet, is associated with beneficial effects on serum levels of the following adipocytokines: TNF-α, IL-6, adiponectin, and resistin. We found no statistically significant changes in the levels of these adipocytokines in association with the very low-fat diet or soy supplementation. Only the change in TNF-α levels between the very low-fat and control diet groups had borderline statistical significance. We conclude that ingestion of a very low-fat diet or a soy food supplemented diet for 8 wk does not significantly alter important circulating adipocytokines.

Percutaneous progesterone delivery via cream or gel application in postmenopausal women: a randomized cross-over study of progesterone levels in serum, whole blood, saliva, and capillary blood.

OBJECTIVE: This study aims to investigate the distribution of progesterone in venous whole blood, venous serum, fingertip capillary blood, and saliva after its topical application in both cream and gel formulations. METHODS: Ten postmenopausal women were randomized to receive 80 mg of progesterone cream or gel applied daily for 14 days, crossing over after a 14-day washout. On the last day of each treatment period, venous blood, fingertip capillary blood, and saliva were sampled frequently for 24 hours after the final application. RESULTS: After progesterone cream or gel application, serum progesterone levels rose gradually, reaching a peak at 9 and 8 hours, respectively; AUC(0-24) h was significantly higher with cream (12.39 vs 8.32 ng h mL(-1), P = 0.0391). Whole venous blood levels followed a pattern similar to that of serum but were considerably lower. Saliva progesterone showed a peak at 1 and 6 hours after cream and gel application, respectively, and C(max) was comparable with cream and gel. Saliva AUC(0-24) h was substantially higher than the corresponding area under the curve for serum or whole blood but did not differ significantly by delivery method (39.02 and 58.37 ng h mL(-1), P = 0.69). In capillary blood, C(max) was reached at the same time (8 h) and was similar with both formulations; AUC(0-24) h was also similar with both formulations (1,056 ng h mL(-1) for cream and 999 ng h mL(-1) for gel) but was dramatically higher than the corresponding areas under the curve for venous serum and whole blood. CONCLUSIONS: After application of topical progesterone, saliva and capillary blood levels are approximately 10-fold and 100-fold greater, respectively, than those seen in serum or whole blood. High capillary blood and saliva levels indicate high absorption and transport of progesterone to tissues. Reliance on serum levels of progesterone for monitoring topical dose could lead to underestimation of tissue levels and consequent overdose.

Fission yeast LAMMER kinase Lkh1 regulates the cell cycle by phosphorylating the CDK-inhibitor Rum1.

In eukaryotes, LAMMER kinases are involved in various cellular events, including the cell cycle. However, no attempt has been made to investigate the mechanisms that underlie the involvement of LAMMER kinase. In this study, we performed a functional analysis of LAMMER kinase using the fission yeast, Schizosaccharomyces pombe. FACS analyses revealed that deletion of the gene that encodes the LAMMER kinase Lkh1 made mutant cells pass through the G1/S phase faster than their wild-type counterparts. Co-immunoprecipitation and an in vitro kinase assay also revealed that Lkh1 can interact with and phosphorylate Rum1 to activate this molecule as a cyclin-dependent kinase inhibitor, which blocks cell cycle progression from the G1 phase to the S phase. Peptide mass fingerprinting and kinase assay with Rum1(T110A) confirmed T110 as the Lkh1-dependent phosphorylation residue. In this report we present for the first time a positive acting mechanism that is responsible for the CKI activity of Rum1, in which the LAMMER kinase-mediated phosphorylation of Rum1 is involved.

The fission yeast GATA factor, Gaf1, modulates sexual development via direct down-regulation of ste11+ expression in response to nitrogen starvation.

Gaf1 is the first GATA family zinc-finger transcription factor identified in Schizosaccharomyces pombe. Here, we report that Gaf1 functions as a negatively acting transcription factor of ste11(+), delaying the entrance of cells exposed to transient nitrogen starvation into the meiotic cycle. gaf1Δ strains exhibited accelerated G(1)-arrest upon nitrogen starvation. Moreover, gaf1Δ mutation caused increased mating and sporulation frequency under both nitrogen-starved and unstarved conditions, while overexpression of gaf1(+) led to a significant impairment of sporulation. By microarray analysis, we found that approximately 63% (116 genes) of the 183 genes up-regulated in unstarved gaf1Δ cells were nitrogen starvation-responsive genes, and furthermore that 25 genes among the genes up-regulated by gaf1Δ mutation are Ste11 targets (e.g., gpa1(+), ste4(+), spk1(+), ste11(+), and mei2(+)). The phenotype caused by gaf1Δ mutation was masked by ste11Δ mutation, indicating that ste11(+) is epistatic to gaf1(+) with respect to sporulation efficiency, and accordingly that gaf1(+) functions upstream of ste11(+) in the signaling pathway governing sexual development. gaf1Δ strains showed accelerated ste11(+) expression under nitrogen starvation and increased ste11(+) expression even under normal conditions. Electrophoretic mobility shift assay analysis demonstrated that Gaf1 specifically binds to the canonical GATA motif (5'-HGATAR-3') spanning from -371 to -366 in ste11(+) promoter. Consequently, Gaf1 provides the prime example for negative regulation of ste11(+) transcription through direct binding to a cis-acting motif of its promoter.

Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe.

We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome providing a tool for studying eukaryotic biology. Comprehensive gene dispensability comparisons with budding yeast--the only other eukaryote for which a comprehensive knockout library exists--revealed that 83% of single-copy orthologs in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than nonessential genes to be present in a single copy, to be broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth.

Cl- -channel is essential for LDL-induced cell proliferation via the activation of Erk1/2 and PI3k/Akt and the upregulation of Egr-1 in human aortic smooth muscle cells.

Low-density lipoprotein (LDL) induces cell proliferation in human aortic smooth muscle cells (hAoSMCs), which may be involved in atherogenesis and intimal hyperplasia. Recent studies have demonstrated that Cl- channels are related to vessel cell proliferation induced by a variety of stimuli. In this study, we investigated a potential role of Cl-channels in the signaling pathway of LDL effects on hAoSMC proliferation with a focus on the activation of Erk1/2-PI3K/Akt and the subsequent upregulation of Egr-1. Cl- channel blockers, DIDS, but neither NPPB nor Furosemide, completely abolished the LDL-induced DNA synthesis and cell proliferation. Moreover, DIDS, but not NPPB, significantly decreased LDL-stimulated Cl- concentration, as judged by flow cytometry analysis using MQAE as a Cl- -detection dye. DIDS pretreatment completely abolished the activation of Erk1/2 and PI3K/Akt in a dose-dependent manner that is the hallmark of LDL activation, as judged by Western blot and proliferation assays. Moreover, pretreatment with DIDS (Cl- channel blockers) but not LY294002 (PI3K inhibitors) completely abolished the LDL-induced upregulation of Egr-1 to the same extent as PD98059 (MEK inhibitors to inhibit Erk), as judged by Western blot and luciferase reporter assays. This is the first report, to our knowledge, that DIDS-sensitive Cl- channels play a key role in the LDL-induced cell proliferation of hAoSMCs via the activation of Erk1/2 and PI3K/Akt and the upregulation of Egr-1.

Genome-wide identification of haploinsufficiency in fission yeast.

Abnormal phenotypes resulting from haploinsufficiency (HI) are due to the loss of one allele. Recent studies in budding yeast have shown that HI originates from insufficient protein levels or from a stoichiometric imbalance between subunits of protein complexes. In humans, however, HI often involves transcription factors. Therefore, the species differences in HI and the molecular mechanisms of species-specific HI remain under investigation. In this study, HI in fission yeast was systematically surveyed. HI in fission yeast affected genes related to signaling and to basic cellular processes, as observed in budding yeast. These results suggest that there are species differences in HI and that the HI that occurs in fission yeast is intermediate to and HI in budding yeast and humans.