Administration of Cripto in GRP78 overexpressed human MSCs enhances stem cell viability and angiogenesis during human MSC transplantation therapy.

OBJECTIVES: The purpose of this study was to explore the effectiveness of concurrent GRP78 overexpression combined with Cripto on hMSC proliferation and migration both in vitro and in vivo. Specifically, we explored whether the treatment enhances effectiveness of hMSC transplantation in ischaemic tissue. MATERIALS AND METHODS: Human MSCs obtained from human adipose tissue were cultured in α-minimum essential medium (Hyclone, Logan, UT, USA) supplemented with 10% (v/v) foetal bovine serum (Hyclone), 100 U mL-1 penicillin and 100 µg mL-1 streptomycin. Murine hindlimb ischaemic model was generated with 8-week-old male nude BALB/c mice (Biogenomics, Seoul, Korea) maintained under a 12-h light/dark cycle following the established protocol with minor modification. Cellular injection was performed no later than 3 hour after surgery. Lipofectamine transfection, single-cell cultivation assay, transwell assay, scratch wound-healing migration assay, immunohistochemistry and western blotting assays were performed. RESULTS: Overexpression of GRP78 along with Cripto enhanced hMSC proliferation, migration and invasion. It increased interaction of surface GRP78 receptor with Cripto via JAK2/STAT3 pathway. We confirmed our proposed mechanism by showing that treatment with GRP78 antibody blocks the enhancement in vitro. In vivo, we observed that Cripto induced by the hypoxic environment in hindlimb ischaemic model interacts with the overexpressed GRP78 and increases hMSC proliferation, migration and invasion potentials as well as angiogenesis around transplanted ischaemic site via cytokine secretions. CONCLUSIONS: These results demonstrate supporting evidences that GRP78-Cripto combination technique offers novel strategy to enhance MSC proliferation, migration and invasion potentials as well as angiogenesis around ischaemic site, ultimately facilitating MSC-based transplantation therapy in ischaemic conditions.

Role of HSPA1L as a cellular prion protein stabilizer in tumor progression via HIF-1α/GP78 axis.

The cellular prion protein (PrPC) is associated with metastasis, tumor progression and recurrence; however, the precise mechanisms underlying its action is not well understood. Our study found that PrPC degradation decreased tumor progression in colorectal cancer (CRC). In a CRC cell line and human CRC tissue exposed to hypoxia, induced heat-shock 70-kDa protein-1-like (HSPA1L) expression stabilized hypoxia-inducible factor-1α (HIF-1α) protein and promoted PrPC accumulation and tumorigenicity in vivo. PrPC was degraded via the proteasome pathway mediated by the ubiquitin-protein E3 ligase glycoprotein 78 (GP78), which interacts directly with PrPC. However, hypoxia-induced HSPA1L interacted with GP78 and inhibited its functions. HSPA1L knockdown facilitated the interaction of GP78 and PrPC, thereby increasing PrPC ubiquitination. Thus, GP78 was identified as the ubiquitinase for PrPC, thereby revealing an essential mechanism that controls PrPC levels in CRC. Our results suggest that the HSPA1L/HIF-1α/GP78 axis has a crucial role in PrPC accumulation during tumor progression.

Antiproteinuric Effects of Green Tea Extract on Tacrolimus-Induced Nephrotoxicity in Mice.

INTRODUCTION: It has been reported that proteinuria is an early predictive marker in detection of tacrolimus (TAC) nephrotoxicity. The aim of this study was to investigate the antiproteinuric effects of green tea extract (GTE) on TAC-induced acute nephrotoxicity in mice. METHODS: The mice (n = 20) were divided into 4 groups (n = 5 per group); control group mice were intraperitoneally (IP) injected with 0.9% saline, TAC group mice were IP injected with TAC 1 mg/kg, and inducible nitric oxide synthase (iNOS) inhibitor group mice were given in addition NG-nitro-L-arginine-methyl ester 12 mmol/L by subcutaneous injection. TAC-GTE group mice were given TAC by IP injection and GTE 100 mg/kg by subcutaneous injection. RESULTS: The 24-hour urine protein amounts were significantly increased in TAC group mice (36.1 ± 9.9 mg/d) compared with control group mice (13.3 ± 5.4 mg/d) and significantly decreased in TAC-GTE group mice (19.1 ± 6.9 mg/d, P < .01) compared with TAC group mice. The nitric oxide (NO) production by TAC was significantly suppressed by GTE and iNOS inhibitor injection. Renal tissue malondialdehyde (MDA) level was significantly increased in the TAC group compared with the control group and was significantly decreased in the TAC-GTE group compared with that of the TAC group. The antioxidant enzyme activities of superoxide dismutase and catalase were significantly suppressed in the TAC group compared with the control group and were restored in the GTE injection group. CONCLUSIONS: GTE treatment has beneficial antiproteinuric effects on TAC-induced acute renal injury in mice.

NF-kappa B activation pathway is essential for the chemokine expression in intestinal epithelial cells stimulated with Clostridium difficile toxin A.

Intestinal epithelial cells are known to upregulate the expression of several chemokines in response to stimulation with bacterial toxin. However, the cellular mechanisms of Clostridium difficile toxin A-induced mucosal inflammation have not yet been fully elucidated. In this study, we investigated whether nuclear factor-kappa B (NF-kappaB) could regulate chemokine expression in intestinal epithelial cells. Toxin A increased the levels of NF-kappaB complexes containing p65/p50 heterodimers and p65/p65 homodimers. Concurrently, toxin A decreased the levels of IkappaBalpha. Toxin A stimulation also increased the signals of phosphorylated IkappaB kinase (IKK)alpha/beta and NF-kappaB-inducing kinase (NIK). In the toxin A-stimulated HT-29 cells, the suppression of IKK or NIK inhibited the upregulation of downstream target genes of NF-kappaB such as IL-8 and monocyte-chemotactic protein (MCP)-1 and similarly, inhibition of NF-kappaB also downregulated the expression of IL-8, growth-related oncogene-alpha, and MCP-1. These results suggest that NF-kappaB signalling events may be involved in the inflammatory responses to toxin A produced by toxigenic C. difficile.

Protein kinase A regulates chondrogenesis of mesenchymal cells at the post-precartilage condensation stage via protein kinase C-alpha signaling.

Chondrogenesis of mesenchymal cells during in vitro micromass culture requires the generation of cyclic adenosine monophosphate (cAMP) and subsequent activation of cAMP-dependent protein kinase A (PKA). In this study, we investigated the regulatory activity of PKA during chondrogenesis of chick limb bud mesenchymal cells. PKA activity was high in 1-day and 2-day cultures, which was followed by a slight decrease in 4-day and 5-day old cultures. Inhibition of PKA blocked chondrogenesis. It did not affect precartilage condensation, but it blocked the progression from the precartilage condensation stage to cartilage nodule formation. The PKA inhibition-induced blockage of chondrogenesis was accompanied by an altered expression of N-cadherin. Although expression of N-cadherin was detected during the early period of chondrogenesis, it became reduced as chondrogenesis proceeded. Still, inhibition of PKA maintained expression of N-cadherin throughout the micromass culture period. The inhibition of PKA did not affect expression of protein kinase C-alpha (PKCalpha), PKCepsilon, PKCdelta, and PKClambda/iota, which are the isoforms expressed in differentiating mesenchymal cells. However, PKA inhibition completely blocked activation of PKCalpha. Because PKC activity regulates N-cadherin expression and chondrogenesis, the PKA-mediated regulation of PKCalpha appears to be responsible for the PKA regulation of N-cadherin expression and chondrogenesis. Taken together, our results suggest that PKA regulates chondrogenesis by activating PKCalpha at the stage of post-precartilage condensation.

Plasminogen activator inhibitor-1 4G/5G promoter polymorphism and coagulation factor VII Arg353-->Gln polymorphism in Korean patients with coronary artery disease.

An increased risk for arterial thrombosis is associated with high plasma levels of coagulation and fibrinolytic factors such as PAI-1 and FVII. In this study, the 4G/5G polymorphism in the promoter of PAI-1 gene and Arg353-->Gln polymorphism in the FVII gene were analysed in 139 normal adults and 158 patients with coronary artery disease (CAD), and their association with plasma lipid traits was investigated. There were no significant differences in the allele frequencies of PAI-1 and FVII polymorphisms between control and patient groups. The allelic distributions of both polymorphisms in Koreans were similar to those in Japanese but significantly different from those in Caucasians. In the CAD group, the 4G homozygotes of PAI-1 polymorphism showed significantly higher levels of total (p=0.0250) and LDL cholesterol (p=0.0335) with individuals having other genotypes. However, FVII polymorphism showed no association with lipid levels. In conclusion, the 4G/5G PAI-1 promoter polymorphism and Arg353-->Gln FVII polymorphism are not major genetic risk factors for CAD in Koreans. However, 4G allele of PAI-1 polymorphism revealed to be associated with the levels of cholesterol, especially LDL cholesterol levels in CAD patients.

Epidermal growth factor negatively regulates chondrogenesis of mesenchymal cells by modulating the protein kinase C-alpha, Erk-1, and p38 MAPK signaling pathways.

During limb development, epithelial cells in the apical ectodermal ridge keep the underlying mesenchymal cells in a proliferative state preventing differentiation by secreting signaling molecules such as epidermal growth factor (EGF). We investigated the molecular mechanism of the EGF effect on the regulation of micromass culture-induced chondrogenesis of chick limb bud mesenchymal cells as a model system. We found that expression and tyrosine phosphorylation of the EGF receptor was increased transiently during chondrogenesis. Exogenous EGF inhibited chondrogenic differentiation of mesenchymal cells, and this effect was reversed by the EGF receptor inhibitor AG1478. EGF treatment also inhibited the expression and activation of protein kinase C-alpha, whereas it activated Erk-1 and inhibited p38 mitogen-activated protein kinase, all of which appeared to be involved in the EGF-induced inhibition of chondrogenesis. Stimulation of the EGF receptor blocked precartilage condensation and altered the expression of cell adhesion molecules such as N-cadherin and integrins alpha(5) and beta(1). All these EGF effects were reversible by AG1478. The data indicate that EGF negatively regulate chondrogenesis of chick limb bud mesenchymal cells by inhibiting precartilage condensation and by modulating signaling pathways including those of protein kinase C-alpha, Erk-1, and p38 mitogen-activated protein kinase.

Opposing role of mitogen-activated protein kinase subtypes, erk-1/2 and p38, in the regulation of chondrogenesis of mesenchymes.

The present studies were performed to determine subtype-specific roles of mitogen-activated protein kinase in chondrogenesis. Erk-1/2 activities, downstream of protein kinase C, decreased as chondrogenesis proceeded, whereas p38 activities, independent of protein kinase C, continuously increased during chondrogenesis. Inhibition of Erk-1/2 with PD98059 enhanced chondrogenesis up to 1. 7-fold, whereas inhibition of p38 with SB203580 reduced it to about 30% of the control level. Inhibition of Erk-1/2 or p38 did not affect precartilage condensation. However, cartilage nodule formation was significantly blocked by the inhibition of p38, whereas Erk-1/2 inhibition did not affect it. Modulation of chondrogenesis by the inhibition of Erk-1/2 and p38 was accompanied by altered expression of adhesion molecules in an opposite way. Expression of N-cadherin was reduced as chondrogenesis proceeded. Inhibition of p38 caused sustained expression of N-cadherin, whereas Erk-1/2 inhibition accelerated the reduction of N-cadherin expression. Expression of integrin alpha5beta1 and fibronectin were found to transiently increase during chondrogenesis. Inhibition of p38 caused continuous increase of expression of these molecules, whereas Erk-1/2 inhibition accelerated the decrease of expression of these molecules at a later period of chondrogenesis. Because temporal expression of these adhesion molecules regulates chondrogenesis, the above results indicate that Erk-1/2 and p38 conversely regulate chondrogenesis at post-precartilage condensation stages by modulating expression of adhesion molecules.

The changes in electrolytes and acid-base balance after artificially induced acute diarrhea by laxatives.

Following the acute diarrhea in patients (n = 24) overnight with commonly used laxatives for bowel preparation, the changes in electrolytes and acid-base balance in blood and urine were investigated. Though no alterations of serum sodium or potassium concentrations were noted, mild but significant reduction of mean values (+/- SEM) of plasma pH and HCO3 after diarrhea when compared to those before it developed (pH, from 7.42 +/- 0.01 to 7.39 +/- 0.01, p < 0.01; HCO3, from 25.8 +/- 0.6 to 23.7 +/- 0.6 mEq/L, p < 0.05). However, significant reduction of concentration in spot urine sodium from 150 +/- 12.3 to 93 +/- 14 mEq/g of crea. (p < 0.01) and increase in spot urine potassium from 33 +/- 3.2 to 51 +/- 6.0 mEq/g of crea. (p < 0.05) following diarrhea were seen with significant reduction of urine pH from 6.67 +/- 0.21 to 5.5 +/- 0.13 (p < 0.001). Also, with this effective urinary acidification following diarrhea, a significant reduction of urinary anion gap as well as significant increment of spot urine ammonium was accompanied (anion gap, from 80.4 +/- 11.1 to 44 +/- 8.5 mEq/g of crea. p < 0.001; ammonium, from 87 +/- 18.5 to 229 +/- 37 mg/g of crea. p < 0.001) in addition to the significant inverse correlation between these changes in spot urine from basal levels in 24 study subjects (y = -1.13 x +61, r = 0.7, p < 0.001). In conclusion, we observed that the acute diarrhea with laxatives used for bowel preparation caused a mild degree of metabolic acidosis with no changes in blood electrolytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Right atrial mass associated with hepatoma--2 case reports.

Hepatoma has a tendency to spread into the venous system, but intracavitary cardiac extension or metastasis of hepatocellular carcinoma is an uncommon form of cardiac malignancy. When the carcinoma grows from the hepatic vein into the right atrium, the right atrial tumor thrombis may hinder the blood flow. Therefore, these patients have the risk of sudden death. In the past, antemortem diagnosis of right atrial tumor thrombi in patients with primary hepatocellular carcinoma was difficult. But, echocardiography allowed easy detection of the intracardiac tumor thrombi. We describe two cases of hepatocellular carcinoma with right atrial tumor thrombi. In these cases, the right atrial tumor thrombi was detected by two-dimensional echocardiography. Recently, successful surgical removal of the right atrial tumor thrombi are reported in several cases. We advocate performing echocardiographic examination in patients with hepatoma who have cardiac symptoms and signs.

[A numerical taxonomic study on heterophyid trematodes].

A numerical taxonomy was studied on a group of heterophyid trematodes and analysis was made on the following species: Metagonimus yokogawai (3 OTU, Operational Taxonomic Unit), Metagonimus Miyata Type (3 OTU), Metagonimus takahashii (2 OTU), Heterophyes dispar (2 OTU), Heterophyes heterophyes (1 OTU), Heterophyes nocens (2 OTU), Heterophyopsis continua (1 OTU), Pygidiopsis summa (3 OTU), Stellantchasmus falcatus (2 OTU) and Stictodora lari (2 OTU). Twenty-six morphological characters were measured and their values were expressed as relative ratios. Similarity and correlation matrix among each individuals were calculated. Clustering analysis by Ward's method and factor analysis were performed using the SAS (Statistical Analysis System) package. As a result, the groups belonging to the genus of Metagonimus were divided into three phenons (Metagonimus yokogawai, Metagonimus Miyata Type, M. takahashii), and Metagonimus Miyata Type was classified as the level of subspecies of M. takahashii. The groups belonging to the genus Heterophyes were clearly divided into three phenons (Heterophyes dispar, H. heterophyes, H. nocens), and H. nocens was classified as not a subspecies level of H. heterophyes but a distinct species. Other species were classified as distinct phenons. From these results, the application of numerical taxonomy on trematode classification is considered to be a great aid to determine the limit of taxa.