Role of TGF-ß and p38 MAPK in TSG-6 Expression in Adipose Tissue-Derived Stem Cells In Vitro and In Vivo.

Mesenchymal stem cells (MSCs) regulate immune cell activity by expressing tumor necrosis factor-α (TNF-α)-stimulated gene 6 (TSG-6) in inflammatory environments; however, whether anti-inflammatory responses affect TSG-6 expression in MSCs is not well understood. Therefore, we investigated whether transforming growth factor-ß (TGF-ß) regulates TSG-6 expression in adipose tissue-derived stem cells (ASCs) and whether effective immunosuppression can be achieved using ASCs and TGF-ß signaling inhibitor A83-01. TGF-ß significantly decreased TSG-6 expression in ASCs, but A83-01 and the p38 inhibitor SB202190 significantly increased it. However, in septic C57BL/6 mice, A83-01 further reduced the survival rate of the lipopolysaccharide (LPS)-treated group and ASC transplantation did not improve the severity induced by LPS. ASC transplantation alleviated the severity of sepsis induced by LPS+A83-01. In co-culture of macrophages and ASCs, A83-01 decreased TSG-6 expression whereas A83-01 and SB202190 reduced Cox-2 and IDO-2 expression in ASCs. These results suggest that TSG-6 expression in ASCs can be regulated by high concentrations of pro-inflammatory cytokines in vitro and in vivo, and that A83-01 and SB202190 can reduce the expression of immunomodulators in ASCs. Therefore, our data suggest that co-treatment of ASCs with TGF-ß or p38 inhibitors is not adequate to modulate inflammation.

Cultured human skeletal muscle satellite cells exhibit characteristics of mesenchymal stem cells and play anti-inflammatory roles through prostaglandin E2 and hepatocyte growth factors.

Skeletal muscle satellite cells (SkMSCs) play crucial roles in muscle fiber maintenance, repair, and remodeling; however, it remains unknown if these properties are preserved in cultured SkMSCs. In this study, we investigated the characteristics of cultured SkMSCs and their ability to regulate the activity of M1 macrophages. SkMSCs grew well with an average population doubling time of 26.26 ± 6.85 h during 10 passages (P). At P5, Pax7, MyoD, cluster of differentiation (CD)34, and CD56 were not expressed in SkMSCs, but the MSC markers CD73, CD105, and CD90 were expressed and the cells were differentiated into adipocytes and osteoblasts. When SkMSCs were cocultured with macrophages, interleukin (IL)-1ß secretion was decreased, prostaglandin (PG)E2 was produced in coculture, and cyclooxygenase-2 protein was induced in an SkMSC-dependent manner. Hepatocyte growth factor (HGF) was highly secreted by monocultured SkMSCs; interferon-γ and lipopolysaccharide reduced its expression level. However, HGF expression recovered when SkMSCs and macrophages were cocultured. Although exogenous PGE2 upregulated macrophage pro-IL-1ß expression, it suppressed the secretion of cleaved IL-1ß. In contrast, HGF decreased active IL-1ß secretion without affecting pro-IL-1ß expression. Co-treatment of macrophages with HGF and PGE2 reduced pro-IL-1ß expression level and active IL-1ß secretion. Our results suggest that SkMSCs lose their satellite cell properties during serial passaging but acquire mesenchymal stem cell properties including the ability to exert an anti-inflammatory response for macrophages through PGE2 and HGF.

DNA-binding Cell-penetrating Peptide-based TRAIL Over-expression in Adipose Tissue-derived Mesenchymal Stem Cells Inhibits Glioma U251MG Growth.

BACKGROUND/AIM: Genetic manipulation of stem cells using non-viral vectors is still limited due to low transfection efficiency. We investigated whether the DNA-binding cell-permeation peptides (CPP) can enhance the transfection efficiency of non-viral vectors in adipose tissue-derived mesenchymal stem cells (ASCs) and whether ASCs over-expressing TRAIL through CPP can inhibit the growth of glioma U251MG cells in vitro and in vivo. MATERIALS AND METHODS: ASCs were genetically engineered to over-express TRAIL by using CPP, pCMV3-TRAIL and lipid-based transfection reagents (X-tremeGENE). RESULTS: The transfection efficiency of ASCs increased by approximately 7% using CPP; 53.9% of ASCs were transfected and TRAIL expression in ASCs increased by approximately 3 times compared to X-tremeGENE alone. ASCs over-expressing TRAIL using CPP inhibited growth of glioma U251MG cells both in vitro and in the U251MG xenograft model. CONCLUSION: CPP can be used as an enhancer for genetically manipulating ASCs and tumor treatment.

TRAIL-overexpressing Adipose Tissue-derived Mesenchymal Stem Cells Efficiently Inhibit Tumor Growth in an H460 Xenograft Model.

BACKGROUND/AIM: Mesenchymal stem cell-based tumor therapy is still limited due to the insufficient secretion of effectors and discrepancies between their in vitro and in vivo efficacy. We investigated whether genetically engineered adipose tissue-derived mesenchymal stem cells (ASCs) overexpressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) had inhibitory effects on H460 tumor growth both in vitro and in an H460 xenograft model. MATERIALS AND METHODS: Genetically engineered adipose tissue-derived mesenchymal stem cells (ASCs) overexpressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were obtained from plasmid transfection with pCMV3-TRAIL and -interferon (IFN)-ß (producing ASC-TRAIL and ASC-IFN-ß, respectively). Death of H460 cells co-cultured with ASCs, ASC-TRAIL, and ASC-IFN-ß or exposed to their conditioned medium was evaluated via apoptosis and cytotoxicity assays. In addition, in an H460 xenograft model (n=10 per group), the antitumor potential of TRAIL-overexpressing, and IFN-ß-overexpressing ASCs was investigated. RESULTS: Conditioned medium obtained from ASC-IFN-ß increased apoptosis of H460 cells more than did ASC-TRAIL. Additionally, in H460 xenograft models, while native ASCs promoted tumor growth, ASC-TRAIL and ASC-IFN-ß both dramatically suppressed tumor growth. Interestingly, in the context of ASC-IFN-ß, tumors were detected only in 20% of nude mice, with smaller sizes and lower weights than those of the control group. CONCLUSION: TRAIL-overexpressing ASCs can be used to treat tumors; ASC-IFN-ß in particular secrete a higher level of TRAIL.

Ultrastructure of the fertilized egg envelope from Melanotaenia praecox, Melanotaeniidae, Teleostei.

We examined the morphology of fertilized egg and ultrastructures of fertilized egg envelopes of dwarf rainbowfish (Melanotaenia praecox) belong to Melanotaeniidae using light and electron microscopes. The fertilized eggs were spherical with adhesive filament, transparent, demersal, and had a narrow perivitelline space and small oil droplets. The size of fertilized egg was 1.02 ± 0.18 mm (n = 30), and there were two kinds of adhesive filament on the fertilized eggs. The long and thick (diameter 12.22 ± 0.52 µm, n = 20) adhesive filaments were only at the area of animal pole, and short and thin (diameter 1.99 ± 0.23 µm, n = 20) adhesive filaments were around the long filaments. A micropyle was conical shaped with adhesive filament and located near the animal pole of egg. The outer surface of fertilized egg was rough side. Also, the total thickness of the fertilized egg envelope was about 7.46 ± 0.41 µm (n = 20), the fertilized egg envelope consisted of two layers, an inner lamellae layer and an outer layer with high electron-density. And the inner layer was 8 layers. Collectively, these morphological characteristics and adhesive property of fertilized egg with adhesive filaments, and ultrastructures of micropyle, outer surface, and section of fertilized egg envelope are showed species specificity.

Interaction between the tumor microenvironment and resection margin in different gross types of hepatocellular carcinoma.

BACKGROUND AND AIM: There is no consensus regarding the safe resection margin in hepatocellular carcinoma (HCC). Several studies reported that different gross types require different resection margins. We investigated the changes in the tumor microenvironment (TME) in different gross types of HCC. METHODS: We selected tumor tissue and normal tissue 1 and 2 cm away from the HCC. We analyzed the expression status of TME genes and the correlation between TME genes and the effective resection margin. We further divided the patients into two groups: group 1 included expanding and vaguely nodular types, whereas group 2 included nodular with perinodular extension, multinodular confluent, and infiltrative types. RESULTS: Group 2 showed 27% and 45% 5-year disease-free survival (DFS) and overall survival (OS) rates, respectively. Group 2 was a significant prognostic factor for DFS and OS. In cases with a resection margin of less than 1 cm or more than 2 cm, there were no differences in recurrence and survival rate between the two groups. Group 1 patients who had a resection margin that ranged from 1 to 2 cm showed significantly better DFS and OS rates. ß-Catenin and matrix metalloproteinase 9 expression was significantly decreased and that of E-cadherin was significantly increased according to the resection margin in group 1. CONCLUSIONS: Patients with expanding and vaguely nodular HCC may safely undergo surgical resection with a narrow resection margin, and patients with the other gross types must undergo surgical resection with more than a 2-cm resection margin because of their TME conditions.

Increased Serum Angiopoietin-Like 6 Ahead of Metabolic Syndrome in a Prospective Cohort Study.

BACKGROUND: Despite being an anti-obesity hepatokine, the levels of serum angiopoietin-like 6 (ANGPTL6) are elevated in various metabolic diseases. Thus, ANGPTL6 expression may reflect metabolic burden and may have compensatory roles. This study investigated the association between serum ANGPTL6 levels and new-onset metabolic syndrome. METHODS: In total, 221 participants without metabolic syndrome were randomly selected from a rural cohort in Korea. Baseline serum ANGPTL6 levels were measured using an enzyme-linked immunosorbent assay. Anthropometric and biochemical markers were analyzed before and after follow-up examinations. RESULTS: During an average follow-up period of 2.75 (interquartile range, 0.76) years, 82 participants (37.1%) presented new-onset metabolic syndrome and had higher ANGPTL6 levels before onset than those without metabolic syndrome (48.03±18.84 ng/mL vs. 64.75±43.35 ng/mL, P=0.001). In the multivariable adjusted models, the odds ratio for the development of metabolic syndrome in the highest quartile of ANGPTL6 levels was 3.61 (95% confidence interval, 1.27 to 10.26). The use of ANGPTL6 levels in addition to the conventional components improved the prediction of new-onset metabolic syndrome (area under the receiver operating characteristic curve: 0.775 vs. 0.807, P=0.036). CONCLUSION: Increased serum ANGPTL6 levels precede the development of metabolic syndrome and its components, including low high density lipoprotein, high triglyceride, and high glucose levels, which have an independent predictive value for metabolic syndrome.

Comparative ultrastructure of the fertilized egg envelope in Corydoras adolfoi and Corydoras sterbai, Callichthyidae, Teleostei.

In teleost, the structural characteristics of fertilized egg and egg envelope are very important for classification of genus or species. The structures of fertilized egg and egg envelope from Corydoras adolfoi and Corydoras sterbai, Callichthyidae, Siluriformes in teleost were examined by scanning and transmission electron microscopes to confirm whether these morphological structures have specificities of species and family or not. The fertilized eggs of C. adolfoi and C. sterbai were non-transparent, spherical, demersal, and strong adhesive. There were no structural differences between two species through the light microscope. The size of the fertilized eggs of C. adolfoi was 1.95 ± 0.03 mm (n = 20), and that of C. sterbai was 1.92 ± 0.03 mm (n = 20). The perivitelline space was almost not developed in both species. In both species, the adhesive protuberances structures were on the outer surface of egg envelope. And fibrous structures were specially located at attachment part of spawning bed. And the egg envelope consisted of two layers, an inner lamellae layer and an outer strong adhesive layer with high electron dense protuberances structures in cross section. Consequentially, the fertilized eggs, outer surface on the egg envelope and cross section of egg envelope have identical structure. So, these structural characteristics of fertilized eggs and egg envelope show genus Corydoras specificity.

Serum leptin level is associated with phase angle in CKD5 patients not undergoing dialysis.

OBJECTIVE: Malnutrition is very complex in patients with end-stage renal disease (ESRD) and is associated with poor prognosis. This is because hemodynamic changes, hormonal changes, persistent inflammatory reactions, and fluid overloads are more complicated as uremia is worsening. Bio-impedance spectroscopy (BIS) is a useful method to estimate fluid balance (Overhydration/ extracellular water, OH/ECW) and nutritional status (Phase angle, PhA). We aimed to evaluate the volume and nutritional status by BIS and to investigate the relationship between the appetite regulating hormones and the parameters of BIS in patients with stage 5 chronic kidney disease not undergoing dialysis (CKD5-ND). METHODS: We enrolled a total of 91 CKD5-ND patients. We measured routine serum markers including albumin and NT-proBNP and the appetite regulating hormones, leptin and ghrelin. We defined poor nutritional status as a PhA < 4.5°, and proper nutritional status as a PhA ≥ 4.5°. We also evaluated each patient's nutritional status by assessing their geriatric nutritional risk index (GNRI) and their volume status by measuring NT-proBNP. RESULTS: Forty-one patients (45%) had poor nutritional status. Patients with a poor nutritional status had significantly higher OH/ECW (29.6 ± 12.7% vs. 6.2 ± 10.3%, p<0.001) and lower levels of leptin (3.8 ± 3.1 vs. 7.0 ± 6.2 ng/mL, p = 0.004) than those with proper nutritional status. PhA was associated with GNRI (r = 0.597, P<0.001) and NT-proBNP was associated with OH/ECW (r = 0.384, P<0.001). Leptin was negatively correlated with OH/ECW (r = -0.288, p = 0.006). In contrast, leptin was positively correlated with PhA (r = 0.263, p = 0.012). In multivariate logistic regression, high level of leptin (OR 7.00, 95% CI 1.74-28.10) was associated with proper nutrition, while an increased OH/ECW (OR 0.65, 95% CI 0.51-0.84) was associated with poor nutrition. CONCLUSIONS: Our study demonstrates that CKD5-ND patients with poor nutrition generally also suffer from excessive body fluid. Low leptin level suggests poor nutrition in CKD5-ND patients. PhA could be used as a nutritional index for ESRD patients.

Pancreatic radiation effect in apoptosis-related rectal radiation toxicity.

Pancreatic radiation effect (PRE) can be a component of gastrointestinal tract (GIT) radiotoxicity. This inter-organ correlation between the GIT and the pancreas was assessed through a rat model. Separate local irradiation to the abdomen and the pelvis was applied concurrently for 8-week-old male Sprague Dawley rats. Abdominal irradiation was categorized into pancreatic shield (PS) and non-pancreatic shield (NPS) irradiation. After 5 Gy and 15 Gy irradiation, the rectal mucosa was analyzed at the first week (early phase, Ep) and the 14th week (late phase, Lp). A slow gain in body weight was observed initially, particularly in the NPS group receiving a 15 Gy dose (P < 0.001). The large number of apoptotic bodies after 15 Gy at Ep decreased at Lp. At Ep for the 5-Gy group, the NPS group revealed more fibrotic change than the PS group (P = 0.002). Cleaved caspase-3 (CCP3) expression was greater at Lp, and the Ep-Lp increase was prominent in the NPS-15-Gy group (P = 0.010). At Lp, for 15 Gy irradiation, CCP3 was expressed more in the NPS group than in the PS group (P = 0.032). Despite no direct toxicity difference between the PS and NPS groups, small changes in parameters such as fibrosis or CCP3 expression suggest that pancreatic shielding does have an effect on the radiation response in the rectal mucosa, which suggests a need for a multi-organ effect-based approach in GIT radiotoxicity assessment.

Serum Fibroblast Growth Factor 21 and New-Onset Metabolic Syndrome: KoGES-ARIRANG Study.

PURPOSE: Fibroblast growth factor 21 (FGF21) is a crucial metabolic regulator, with multiple favorable effects on glucose homeostasis and lipid metabolism. Since serum FGF21 level has been implicated as a potential marker for the early identification of metabolic syndrome (MetS), we investigated the association between serum FGF21 level and the development of MetS in a population-based prospective study. MATERIALS AND METHODS: We conducted a prospective study of 221 randomly sampled adults without MetS from a general population-based cohort study who were examined from 2005-2008 (baseline) and from 2008-2011 (follow-up). Baseline serum FGF21 levels were analyzed using enzyme-linked immunosorbent assay. RESULTS: During the average 2.8-year follow-up period, 82 participants (36.6%) developed new-onset MetS. Serum FGF21 levels were significantly higher in patients with new-onset MetS than in those without MetS (209.56±226.80 vs. 110.09±81.10, p<0.01). In multivariate adjusted models, the odds for MetS development were greater in patients with serum FGF21 levels in the highest quartile, compared to those in the lowest quartile (3.84, 95% confidence interval: 1.59-9.28). CONCLUSION: Serum FGF21 level was an independent predictor for new-onset MetS in a population-based prospective study.

A PPAR-Gamma Agonist Rosiglitazone Suppresses Fibrotic Response in Human Pterygium Fibroblasts by Modulating the p38 MAPK Pathway.

Purpose: Fibroblast activation may play an important role in pterygium progression. Synthetic peroxisome proliferator-activated receptor γ (PPAR-γ) ligands have been shown to be effective antifibrotic agents against transforming growth factor ß1 (TGF-ß1) induced fibrosis in several tissues. We aimed to investigate the antifibrotic effects of the PPAR-γ ligand rosiglitazone in pterygium fibroblasts and the underlying mechanisms. Methods: Profibrotic activation was induced by TGF-ß1 in primary cultured human pterygium fibroblasts and the effect of rosiglitazone treatment on α-smooth muscle actin (α-SMA), and extra cellular matrix proteins synthesis was detected by western blotting, real-time PCR, immunostaining, and flow cytometry. Pharmaceutical inhibition of PPAR-γ receptor was used to determine the dependency or otherwise of rosiglitazone's action on PPAR-γ signaling. Major signaling pathways downstream of TGF-ß1 were investigated by western blotting to assess their possible association with rosiglitazone's effect. Cell viability and apoptosis were investigated to assess drug-induced cytotoxicity, and the effect of rosiglitazone treatment on cell migration was further determined. Results: α-SMA and fibronectin synthesis induced by TGF-ß1 were suppressed by rosiglitazone treatment in a dose-dependent manner. Rosiglitazone also inhibited intrinsic TGF-ß1 expression. Smad2/3, ERK1/2, and P38 pathways were activated in response to TGF-ß1. Rosiglitazone suppressed TGF-ß1-induced P38 MAPK activation, while ERK1/2 and Smad2/3 signaling remained unaffected. The observed antifibrotic effect of rosiglitazone was not affected by the PPAR-γ antagonist GW9662, indicating it is not PPAR-γ dependent. Rosiglitazone also inhibited the proliferation and migration of pterygium fibroblasts. Conclusions: Rosiglitazone suppresses TGF-ß1-induced myofibroblast activation and extra cellular matrix synthesis in pterygium fibroblasts at least partly through the modulation of the p38 MAPK pathway.

Adipose tissue-derived mesenchymal stem cells cultured at high density express IFN-ß and suppress the growth of MCF-7 human breast cancer cells.

Although it has been reported that mesenchymal stem cells (MSCs) suppress tumor growth in vitro and in vivo, little is known about the underlying molecular mechanisms. We found that type I interferon is expressed in adipose tissue-derived stem cells (ASCs) cultured at high density, and ASCs and their conditioned medium (ASC-CM) suppress the growth of MCF-7 cells in vitro. Growth inhibition was amplified by glucose deprivation that resulted from high density culture of ASCs after 3days. The cytotoxic effect of the ASC-CM obtained from high density culture of ASCs was neutralized by anti-IFN-ß antibody. STAT1 was phosphorylated in MCF-7 cells treated with ASC-CM, and JAK1/JAK2 inhibitor treatment decreased STAT1 phosphorylation. The cytotoxic effect of ASC-CM was reduced especially by JAK1 inhibitors in MCF-7 cells. Our findings suggest that ASCs cultured at high density express type I interferons, which suppresses tumor growth via STAT1 activation resulting from IFN-ß secretion in MCF-7 breast cancer cells.

Anti-obesity effect of alkaline reduced water in high fat-fed obese mice.

Whether or not alkaline reduced water (ARW) has a positive effect on obesity is unclear. This study aims to prove the positive effect of ARW in high-fat (HF) diet-induced obesity (DIO) in C57BL/6 mice model. Toward this, obesity was induced by feeding the C57BL/6 male mice with high-fat diet (w/w 45% fat) for 12 weeks. Thereafter, the animals were administered with either ARW or tap water. Next, the degree of adiposity and DIO-associated parameters were assessed: clinico-pathological parameters, biochemical measurements, histopathological analysis of liver, the expression of cholesterol metabolism-related genes in the liver, and serum levels of adipokine and cytokine. We found that ARW-fed mice significantly ameliorated adiposity: controlled body weight gain, reduced the accumulation of epididymal fats and decreased liver fats as compared to control mice. Accordingly, ARW coordinated the level of adiponectin and leptin. Further, mRNA expression of cytochrome P450 (CYP)7A1 was upregulated. In summary, our data shows that ARW intake inhibits the progression of HF-DIO in mice. This is the first note on anti-obesity effect of ARW, clinically implying the safer fluid remedy for obesity control.

Functional Expression of P2Y Receptors in WERI-Rb1 Retinoblastoma Cells.

P2Y receptors are metabotropic G-protein-coupled receptors, which are involved in many important biologic functions in the central nervous system including retina. Subtypes of P2Y receptors in retinal tissue vary according to the species and the cell types. We examined the molecular and pharmacologic profiles of P2Y purinoceptors in retinoblastoma cell, which has not been identified yet. To achieve this goal, we used Ca(2+) imaging technique and western blot analysis in WERI-Rb-1 cell, a human retinoblastoma cell line. ATP (10 µM) elicited strong but transient [Ca(2+)](i) increase in a concentration-dependent manner from more than 80% of the WERI-Rb-1 cells (n=46). Orders of potency of P2Y agonists in evoking [Ca(2+)](i) transients were 2MeS-ATP>ATP>>UTP=αß-MeATP, which was compatible with the subclass of P2Y(1) receptor. The [Ca(2+)](i) transients evoked by applications of 2MeS-ATP and/or ATP were also profoundly suppressed in the presence of P2Y(1) selective blocker (MRS 2179; 30 µM). P2Y(1) receptor expression in WERI-Rb-1 cells was also identified by using western blot. Taken together, P2Y(1) receptor is mainly expressed in a retinoblastoma cell, which elicits Ca(2+) release from internal Ca(2+) storage sites via the phospholipase C-mediated pathway. P2Y(1) receptor activation in retinoblastoma cell could be a useful model to investigate the role of purinergic [Ca(2+)](i) signaling in neural tissue as well as to find a novel therapeutic target to this lethal cancer.

Repression of human telomerase reverse transcriptase using artificial zinc finger transcription factors.

Telomerase activation is a key step in the development of human cancers. Expression of the catalytic subunit, human telomerase reverse transcriptase (hTERT), represents the limiting factor for telomerase activity. In this study, we have used artificial zinc finger protein (ZFP) transcription factors (TF) to repress the expression of hTERT in human cancer cell lines at the transcriptional level. We have constructed four-fingered ZFPs derived from the human genome which binds 12-bp recognition sequences within the promoter of the hTERT gene and fused them with a KRAB repressor domain to create a potent transcriptional repressor. Luciferase activity was decreased by >80% in all of the transcriptional repressors with luciferase reporter assay. When they were transfected into the telomerase-positive HEK293 cell line, a decrease of mRNA level and telomerase activity together with shortening of telomere length was observed. Actual growth of HEK293 cells was also inhibited by transfection of artificial ZFP-TFs. The repression was maintained for 100 days of culture. The repression of telomerase expression by artificial ZFP-TFs targeting the promoter region of the hTERT presents a new promising strategy for inhibiting the growth of human cancer cells.

Predictive factors for interferon and ribavirin combination therapy in patients with chronic hepatitis C.

AIM: To confirm the predictive factors for interferon (IFN)-alpha and ribavirin combination therapy for chronic hepatitis patients with hepatitis C virus (HCV) genotype 1b. METHODS: HCV RNA from 50 patients infected with HCV genotype 1b was studied by cloning and sequencing of interferon sensitivity determining region (ISDR), PKR-eIF2alpha phosphorylation homology domain (PePHD). Patients were treated with IFN-alpha and ribavirin for 6 mo and grouped by effectiveness of the therapy. A variety of factors were analyzed. RESULTS: Our data showed that age, HCV RNA titer, and ISDR type could be used as the predictive factors for combined IFN-alpha and ribavirin efficacy. Characteristically, mutations in PePHD appeared only when the combination therapy was effective. Other factors, such as sex and alanine aminotransferase (ALT) level, were not related to its efficacy. Adjusting for age and HCV RNA titer indicated that the ISDR type was the most potent predictive factor. CONCLUSION: HCV RNA ISDR type is an important factor for predicting efficacy of IFN-alpha and ribavirin combination therapy in Korean patients.

Calcium mobilization by activation of M(3)/M(5) muscarinic receptors in the human retinoblastoma.

Activation of muscarinic acetylcholine receptors (mAChR) is one of the most important signal transduction pathways in the human body. In this study, we investigated the role of mAChR activation in relation to its subtypes in human retinoblastoma cell-lines (WERI-Rb-1) using Ca(2+) measurement, real-time PCR, and Western Blot techniques. Acetylcholine (ACh) produced prominent [Ca(2+)](i) transients in a repeated manner in WERI-Rb-1 cells. The maximal amplitude of the [Ca(2+)](i) transient was almost completely suppressed by 97.3 +/- 0.8% after atropine (1 microM) pretreatment. Similar suppressions were noted after pretreatments with thapsigargin (1 microM), an ER Ca(2+)-ATPase (SERCA) inhibitor, whereas the ACh-induced [Ca(2+)](i) transient was not affected even in the absence of extracellular calcium. U-73122 (1 microM), a PLC inhibitor, and xestospongin C (2 microM), an IP(3)-receptor antagonist, elicited 11.5 +/- 2.9% and 17.8 +/- 1.9% suppressions, respectively. The 50% inhibitory concentration of (IC(50)) values for blockade of a 100 microM ACh response by pirenzepine and 4-DAMP were 315.8 and 9.1 nM, respectively. Moreover, both M(3) and M(5) mAChRs were prominent in quantitative real-time-PCR. Taken together, the M(3)/M(5) subtypes appear to be the major contributor, leading to intracellular calcium mobilization from the internal store via an IP(3)-dependent pathway in the undifferentiated retinoblastoma cells.

Characteristic of aromatic amino acid substitution at alpha 96 of hemoglobin.

Replacement of valine by tryptophan or tyrosine at position alpha96 of the alpha chain (alpha96Val), located in the alpha(1)beta(2) subunit interface of hemoglobin leads to low oxygen affinity hemoglobin, and has been suggested to be due to the extra stability introduced by an aromatic amino acid at the alpha96 position. The characteristic of aromatic amino acid substitution at the alpha96 of hemoglobin has been further investigated by producing double mutant r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp). r Hb (alpha42Tyr --> Phe) is known to exhibit almost no cooperativity in binding oxygen, and possesses high oxygen affinity due to the disruption of the hydrogen bond between alpha42Tyr and beta99Asp in thealpha(1)beta(2) subunit interface of deoxy Hb A. The second mutation, alpha96Val -->Trp, may compensate the functional defects of r Hb (alpha42Tyr --> Phe), if the stability due to the introduction of trypophan at the alpha 96 position is strong enough to overcome the defect of r Hb (alpha42Tyr --> Phe). Double mutant r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp) exhibited almost no cooperativity in binding oxygen and possessed high oxygen affinity, similarly to that of r Hb (alpha42Tyr --> Phe). (1)H NMR spectroscopic data of r Hb (alpha42Tyr --> Phe, alpha96Val --> Trp) also showed a very unstable deoxy-quaternary structure. The present investigation has demonstrated that the presence of the crucible hydrogen bond between alpha 42Tyr and beta 99Asp is essential for the novel oxygen binding properties of deoxy Hb (alpha96Val --> Trp) .