Soybean CCA1-like MYB transcription factor GmMYB133 modulates isoflavonoid biosynthesis.

MYB transcription factors play important roles in the regulation of phenylpropanoid biosynthesis. However, the knowledge regarding the roles of CCA1-like MYBs in phenylpropanoid pathway is limited in plants. Previously, we identified 54 CCA1-like proteins in soybean. In the study, a CCA1-like MYB (GmMYB133) was functionally characterized as a positive regulator in isoflavonoid synthesis. GmMYB133 encodes a 330 aa protein featured with one CCA1 conserved motif. Further analysis indicated that the expression pattern of GmMYB133 was near-perfectly correlated with isoflavonoid accumulation as soybean embryos develop. GmMYB133 over-expression promoted the expression of two key isoflavonoid biosynthetic genes (GmCHS8 and GmIFS2) and increased total isoflavonoid content in hairy roots. Protein-protein interaction assays indicated that GmMYB133 might form hetero- and homodimers with an isoflavonoid regulator GmMYB176 and itself, respectively, while the subcellular localization of GmMYB133 can be altered by its interaction with 14-3-3 protein. These findings provided new insights into the functional roles of CCA1-like MYB proteins in the regulation of phenylpropanoid pathway, and will contribute to the future genetic engineering in the improvement of soybean seed quality.

Genome-Wide Analysis of CCA1-Like Proteins in Soybean and Functional Characterization of GmMYB138a.

Plant CIRCADIAN CLOCK ASSOCIATED1 (CCA1)-like proteins are a class of single-repeat MYELOBLASTOSIS ONCOGENE (MYB) transcription factors generally featured by a highly conserved motif SHAQK(Y/F)F, which play important roles in multiple biological processes. Soybean is an important grain legume for seed protein and edible vegetable oil. However, essential understandings regarding CCA1-like proteins are very limited in soybean. In this study, 54 CCA1-like proteins were identified by data mining of soybean genome. Phylogenetic analysis indicated that soybean CCA1-like subfamily showed evolutionary conservation and diversification. These CCA1-like genes displayed tissue-specific expression patterns, and analysis of genomic organization and evolution revealed 23 duplicated gene pairs. Among them, GmMYB138a was chosen for further investigation. Our protein-protein interaction studies revealed that GmMYB138a, but not its alternatively spliced isoform, interacts with a 14-3-3 protein (GmSGF14l). Although GmMYB138a was predominately localized in nucleus, the resulting complex of GmMYB138a and GmSGF14l was almost evenly distributed in nucleus and cytoplasm, supporting that 14-3-3s interact with their clients to alter their subcellular localization. Additionally, qPCR analysis suggested that GmMYB138a and GmSGF14l synergistically or antagonistically respond to drought, cold and salt stresses. Our findings will contribute to future research in regard to functions of soybean CCA1-like subfamily, especially regulatory mechanisms of GmMYB138a in response to abiotic stresses.

Two genetic variants in telomerase-associated protein 1 are associated with stomach cancer risk.

This study examined the impact of two single-nucleotide polymorphisms (SNPs) in the telomerase-associated protein 1 (TEP1) gene on the risk of breast, colorectal, hepatocellular, lung and stomach cancer. A significantly increased stomach cancer risk associated with the GG genotype at rs1760893 (odds ratio (OR)=1.64, 95% confidence interval (CI)=1.23-2.20, P=0.004) or CC genotype at rs1713423 (OR=2.40, 95% CI=1.88-3.07, P<0.0001) was observed, compared with their wild-type counterpart. The GG genotype at rs1760893 was also associated with enhanced hepatocellular cancer susceptibility (OR=1.46, 95% CI=1.05-2.03, P=0.02). In classification and regression tree analysis, individuals carrying the CC genotype at rs1713423 had 2.69-fold increased risk of stomach cancer (95% CI=2.18-3.32, P<0.0001) compared with the TT and TC genotypes. The current results suggested that genetic variants at TEP1 SNPs rs1760893 and rs1713423 may be associated significantly with increased risk of stomach cancer.

Negative effect of cyclin D1 overexpression on recurrence-free survival in stage II-IIIA lung adenocarcinoma and its expression modulation by vorinostat in vitro.

BACKGROUND: This study was aimed at identifying prognostic biomarkers for stage II-IIIA non-small cell lung cancer (NSCLC) according to histology and at investigating the effect of vorinostat on the expression of these biomarkers. METHODS: Expression levels of cyclin D1, cyclin A2, cyclin E, and p16 proteins that are involved in the G1-to-S phase progression of cell cycle were analyzed using immunohistochemistry in formalin-fixed paraffin-embedded tissues from 372 samples of stage II-IIIA NSCLC. The effect of vorinostat on the expression of these proteins, impacts on cell cycle, and histone modification was explored in lung cancer cells. RESULTS: Abnormal expression of cyclin A2, cyclin D1, cyclin E, and p16 was found in 66, 47, 34, and 51 % of 372 cases, respectively. Amongst the four proteins, only cyclin D1 overexpression was significantly associated with poor recurrence-free survival (adjusted hazard ratio = 1.87; 95 % confidence interval = 1.12 - 2.69, P = 0.02) in adenocarcinoma but not in squamous cell carcinoma (P = 0.44). Vorinostat inhibited cell cycle progression to the S-phase and induced down-regulation of cyclin D1 in vitro. The down-regulation of cyclin D1 by vorinostat was comparable to a siRNA-mediated knockdown of cyclin D1 in A549 cells, but vorinostat in the presence of benzo[a]pyrene showed a differential effect in different lung cancer cell lines. Cyclin D1 down-regulation by vorinostat was associated with the accumulation of dimethyl-H3K9 at the promoter of the gene. CONCLUSIONS: The present study suggests that cyclin D1 may be an independent prognostic factor for recurrence-free survival in stage II-IIIA adenocarcinoma of lung and its expression may be modulated by vorinostat.

Achieving 2.2 GPa Ultra-High Strength in Low-Alloy Steel Using a Direct Quenching and Partitioning Process.

Advanced high-strength steels (AHSS) have a wide range of applications in equipment safety and lightweight design, and enhancing the strength of AHSS to the ultra-high level of 2 GPa is currently a key focus. In this study, a new process of thermo-mechanical control process followed by direct quenching and partitioning (TMCP-DQP) was developed based on Fe-0.4C-1Mn-0.6Si (wt.%) low-alloy steel, and the effects of microstructure evolution on mechanical properties under TMCP-DQP process and conventional hot rolled quenched and tempered process (HR-QT) were comparatively studied. The results show that the TMCP-DQP process not only shortened the processing steps but also achieved outstanding comprehensive mechanical properties. The TMCP-DQP steel exhibited a tensile strength of 2.23 GPa, accompanied by 11.9% elongation and a Brinell hardness of 624 HBW, with an impact toughness of 28.5 J at -20 °C. In contrast, the HR-QT steel exhibited tensile strengths ranging from 2.16 GPa to 1.7 GPa and elongations between 5.2% and 12.2%. The microstructure of TMCP-DQP steel primarily consisted of lath martensite, containing thin-film retained austenite (RA), nanoscale rod-shaped carbides, and a minor number of nanoscale twins. The volume fraction of RA reached 7.7%, with an average carbon content of 7.1 at.% measured by three-dimensional atom probe tomography (3DAP). Compared with the HR-QT process, the TMCP-DQP process resulted in a finer microstructure, with a prior austenite grain (PAG) size of 11.91 µm, forming packets and blocks with widths of 5.12 µm and 1.63 µm. The TMCP-DQP process achieved the ultra-high strength of low-alloy steel through the synergistic effects of grain refinement, dislocation strengthening, and precipitation strengthening. The dynamic partitioning stage stabilized the RA through carbon enrichment, while the relaxation stage reduced a small portion of the dislocations generated by thermal deformation, and the self-tempering stage eliminated internal stresses, all guaranteeing considerable ductility and toughness. The TMCP-DQP process may offer a means for industries to streamline their manufacturing processes and provide a technological reference for producing 2.2 GPa grade AHSS.

RNA-binding motif protein 24 regulates myogenin expression and promotes myogenic differentiation.

The formation of muscle fibers involves sequential expression of many proteins that regulate key steps during myoblast-to-myotube transition. Myogenin is a major player in the initiation and maintenance of myogenic differentiation in a mouse myoblast cell line, C2C12. RNA-binding proteins bind to specific target RNA sequences and regulate gene expression in a post-transcriptional manner. This study demonstrates that RNA-binding motif protein 24 (Rbm24) interacts with the 3'-untranslated region of myogenin mRNA and affects its half-life in C2C12 myogenesis. Knockdown of Rbm24 expression by RNA interference significantly decreased myogenin expression associated with the inhibition of myogenesis. In contrast, the overexpression of Rbm24 by stable transfection of a plasmid increased myogenin expression and had a positive effect on myogenic differentiation. Ectopic expression of myogenin was also able to restore myogenic differentiation in Rbm24-knockdown cells. Together, our results suggest that Rbm24 binds to myogenin mRNA and regulates its stability in C2C12 cells. Rbm24 plays a crucial role in myogenic differentiation at least in part through a myogenin-dependent post-transcriptional regulatory pathway.

RNA-binding proteins Rbm38 and Rbm24 regulate myogenic differentiation via p21-dependent and -independent regulatory pathways.

Skeletal muscle differentiation entails organized sequential events, including cell cycle arrest of proliferating myoblast cells and cell fusion, which lead to the formation of multinucleated myotubes. This process involves both transcriptional and post-transcriptional regulation of the gene expression of myogenic proteins, as well as cell-cycle related proteins. RNA-binding proteins bind to specific sequences of target RNA and regulate gene expression in a post-transcriptional manner. However, few tissue-specific RNA binding proteins have been identified. Herein, we report that the RNA binding proteins Rbm24 and Rbm38 were found to be preferentially expressed in muscle during differentiation in vitro. Further, knockdown of either by RNA interference suppressed cell-cycle arrest and delayed myogenic differentiation in C2C12 cells. In contrast, over-expression of Rbm24 or Rbm38 induced cell cycle arrest, and then had a positive effect on myogenic differentiation. Immunoprecipitation-RT-PCR analysis using tagged Rbm proteins indicated that Rbm38 binds to the p21 transcript in vivo. Consistent with this, differentiation of Rbm38 knockdown cells was rescued by over-expression of p21. Together, our results suggest that Rbm38 plays a crucial role in cell cycle arrest and myogenic differentiation via its binding to p21.

Characterization of the soybean R2R3-MYB transcription factor GmMYB81 and its functional roles under abiotic stresses.

R2R3-type MYBs are a key group of regulatory factors that control diverse developmental processes and stress tolerance in plants. Soybean is a major legume crop with the richness of seed protein and edible vegetable oil, and 244 R2R3-type MYBs have been identified in soybean. However, the knowledge regarding their functional roles has been greatly limited as yet. In this study, a novel R2R3-type MYB (GmMYB81) was functionally characterized in soybean, and it is closely related to two abiotic stress-associated regulators (AtMYB44 and AtMYB77). GmMYB81 transcripts not only differentially accumulated in soybean tissues and during embryo development, but also were significantly enhanced by drought, salt and cold stress. Histochemical GUS assay in Arabidopsis indicated that GmMYB81 promoter showed high activity in seedlings, rosette leaves, inflorescences, silique wall, mature anthers, roots, and germinating seeds. Further investigation indicated that over-expression of GmMYB81 in Arabidopsis caused auxin-associated phenotypes, including small flower and silique, more branch, and weakened apical dominance. Moreover, over-expression of GmMYB81 significantly elevated the rates of seed germination and green seedling under salt and drought stress, indicating that GmMYB81 might confer plant tolerance to salt and drought stress during seed germination. Additionally, protein interaction analysis showed that GmMYB81 interacts with the abiotic stress regulator GmSGF14l. Further observation indicated that they displayed similar expression patterns under drought and salt stress, suggesting GmMYB81 and GmSGF14l might cooperatively affect stress tolerance. These findings will facilitate future investigations of the regulatory mechanisms of GmMYB81 in response to plant stress tolerance, especially seed germination under abiotic stresses.

STAT6 signalling is important in CD8 T-cell activation and defence against Toxoplasma gondii infection in the brain.

Signal transducer and activator of transcription (STAT) 6 is a molecule involved in interleukin (IL)-4 and -13 signalling. We investigated the role of STAT6 signalling in Toxoplasma gondii-infected mice using STAT6-deficient (STAT6(-/-)) and wild-type (WT) mice. A significantly larger number of cysts were recovered from the brain in STAT6(-/-) than in WT mice on days 28 and 56 post-infection. CD8(+) T cells in cerebrospinal fluid and spleen stimulated with T. gondii antigen produced higher levels of interferon (IFN)-gamma in WT than in STAT6(-/-) mice. CD8(+) T-cell function, estimated by expression of CD25 and cytotoxic activity, was lower in STAT6(-/-) than in WT mice. Transfer of CD8(+) but not CD4(+) T cells, purified from infected WT mice, into STAT6(-/-) mice successfully prevented formation of cysts in the brain. However, transfer of naïve CD8(+) T cells from WT into STAT6(-/-) mice did not show either activation of CD8(+) T cells or a decrease in the number of cysts in the brain. Transfer of splenic adherent cells from WT into STAT6(-/-) mice induced activation of CD8(+) T cells and decreased the number of cysts in the brain. Expression of CD86 on splenic dendritic cells and IL-12 p40 production were weaker in STAT6(-/-) than in WT mice after T. gondii infection. These results indicate that STAT6 signalling is important in CD8(+) T-cell activation, possibly through regulation of antigen-presenting cells, which could suppress T. gondii infection in the brain.

Overexpression of ß-Catenin and Cyclin D1 is Associated with Poor Overall Survival in Patients with Stage IA-IIA Squamous Cell Lung Cancer Irrespective of Adjuvant Chemotherapy.

INTRODUCTION: This study was aimed at understanding the effect of ß-catenin and cyclin D1 on overall survival in patients with early-stage NSCLC and at evaluating if the prognostic effect can be modified by adjuvant chemotherapy. METHODS: We retrospectively analyzed the expression of ß-catenin and cyclin D1 using immunohistochemistry in formalin-fixed paraffin-embedded tissues from 576 patients with early-stage NSCLC. RESULTS: The median duration of follow-up was 5.1 years. Overexpression of ß-catenin and cyclin D1 was found in 56% and 50% of 576 cases, respectively. Overexpression of ß-catenin and cyclin D1 was significantly associated with poor overall survival (p = 0.003 and p = 0.0009, respectively; log rank test) in squamous cell carcinomas, not in adenocarcinomas. The prognostic significance of each protein in the squamous cell carcinomas was limited to stages IA, IB, and IIA. In addition, simultaneous overexpression of ß-catenin and cyclin D1 in the squamous cell carcinomas synergistically increased hazard ratios (HRs) 15.79 (95% confidence interval [CI] = 1.09-51.23; p =0.04) for stage IA, 10.30 (95% CI = 2.29-46.41; p = 0.002) for stage 1B, and 3.55 (95% CI = 1.22-10.36; p = 0.02) times for stage 2A compared to those without overexpression of the two proteins, after adjusting for confounding factors. In addition, the effect was not dependent on adjuvant chemotherapy. CONCLUSIONS: The present study suggests that simultaneous overexpression of ß-catenin and cyclin D1 may be associated with poor overall survival irrespective of platinum-based adjuvant chemotherapy in stage IA-IIA squamous cell carcinoma of the lung.

Overexpression of MAPK15 in gastric cancer is associated with copy number gain and contributes to the stability of c-Jun.

This study was aimed at understanding the functional and clinicopathological significance of MAPK15 alteration in gastric cancer. Genome-wide copy number alterations (CNAs) were first investigated in 40 gastric cancers using Agilent aCGH-244K or aCGH-400K, and copy number gains of MAPK15 found in aCGH were validated in another set of 48 gastric cancer tissues. The expression of MAPK15 was analyzed using immunohistochemistry in concurrent lesions of normal, adenoma, and carcinoma from additional 45 gastric cancer patients. The effects of MAPK15 on cell cycle, c-Jun phosphorylation, and mRNA stability were analyzed in gastric cancer cells. Copy number gains of MAPK15 were found in 15 (17%) of 88 tumor tissues. The mRNA levels of MAPK15 were relatively high in the gastric cancer tissues and gastric cancer cells with higher copy number gains than those without. Knockdown of MAPK15 using siRNA in gastric cancer cells significantly suppressed cell proliferation and resulted in cell cycle arrest at G1-S phase. Reduced c-Jun phosphorylation and c-Jun half-life were observed in MAPK15-knockdowned cells. In addition, transient transfection of MAPK15 into AGS gastric cancer cells with low copy number resulted in an increase of c-Jun phosphorylation and stability. The overexpression of MAPK15 occurred at a high frequency in carcinomas (37%) compared to concurrent normal tissues (2%) and adenomas (21%). In conclusion, the present study suggests that MAPK15 overexpression may contribute to the malignant transformation of gastric mucosa by prolonging the stability of c-Jun. And, patients with copy number gain of MAPK15 in normal or premalignant tissues of stomach may have a chance to progress to invasive cancer.

Identification of 14-3-3 Family in Common Bean and Their Response to Abiotic Stress.

14-3-3s are a class of conserved regulatory proteins ubiquitously found in eukaryotes, which play important roles in a variety of cellular processes including response to diverse stresses. Although much has been learned about 14-3-3s in several plant species, it remains unknown in common bean. In this study, 9 common bean 14-3-3s (PvGF14s) were identified by exhaustive data mining against the publicly available common bean genomic database. A phylogenetic analysis revealed that each predicted PvGF14 was clustered with two GmSGF14 paralogs from soybean. Both epsilon-like and non-epsilon classes of PvGF14s were found in common bean, and the PvGF14s belonging to each class exhibited similar gene structure. Among 9 PvGF14s, only 8 are transcribed in common bean. Expression patterns of PvGF14s varied depending on tissue type, developmental stage and exposure of plants to stress. A protein-protein interaction study revealed that PvGF14a forms dimer with itself and with other PvGF14 isoforms. This study provides a first comprehensive look at common bean 14-3-3 proteins, a family of proteins with diverse functions in many cellular processes, especially in response to stresses.

Copy Number Gains at 8q24 and 20q11-q13 in Gastric Cancer Are More Common in Intestinal-Type than Diffuse-Type.

The present study was aimed at discovering DNA copy number alterations (CNAs) involved in the carcinogenesis of stomach and at understanding their clinicopathological significances in the Korean population. DNA copy numbers were analyzed using Agilent 244K or 400K array comparative genomic hybridization (aCGH) in fresh-frozen tumor and matched normal tissues from 40 gastric cancer patients. Some of the detected CNA regions were validated using multiplex ligation-dependent probe amplification (MLPA) in six of the 40 patients and customized Agilent 60K aCGH in an independent set of 48 gastric cancers. The mRNA levels of genes at common CNA regions were analyzed using quantitative real-time PCR. Copy number gains were more common than losses across the entire genome in tumor tissues compared to matched normal tissues. The mean number of alterations per case was 64 for gains and 40 for losses, and the median aberration length was 44016 bp for gains and 4732 bp for losses. Copy number gains were frequently detected at 7p22.1 (20%), 8q24.21 (27%-30%), 8q24.3 (22%-48%), 13q34 (20%-31%), and 20q11-q13 (25%-30%), and losses at 3p14.2 (43%), 4q35.2 (27%), 6q26 (23%), and 17p13.3 (20%-23%). CNAs at 7p22.1, 13q34, and 17p13.3 have not been reported in other populations. Most of the copy number losses were associated with down-regulation of mRNA levels, but the correlation between copy number gains and mRNA expression levels varied in a gene-dependent manner. In addition, copy number gains tended to occur more commonly in intestinal-type cancers than in diffuse-type cancers. In conclusion, the present study suggests that copy number gains at 8q24 and 20q11-q13 and losses at 3p14.2 may be common events in gastric cancer but CNAs at 7p22.1, 13q34, and 17p13.3 may be Korean-specific.

RARß2 hypermethylation is associated with poor recurrence-free survival in never-smokers with adenocarcinoma of the lung.

BACKGROUND: This study was aimed at investigating if the effect of RARß2 hypermethylation on recurrence-free survival (RFS) in non-small cell lung cancer (NSCLC) depends on one's smoking status and specific interacting proteins. RESULTS: We retrospectively analyzed the expressions of five proteins using immunohistochemistry in archival formalin-fixed and paraffin-embedded tissues from 578 NSCLC patients who had undergone surgical resection from 1994 through 2004. Promoter methylation of RARß2 was assessed by bisulfite pyrosequencing. Recurrence was found in 268 (46%) of 578 NSCLCs with a median follow-up period of 4.8 years. Overexpression of ß-catenin, c-MET, cyclin D1, and EGFR occurred in 55%, 72%, 51%, and 41% of the patients, respectively. E-cadherin expression was negative in 62% of the patients, and RARß2 hypermethylation was found in 37%. The abnormal expression of c-MET (P = 0.002) and EGFR (P = 0.001) was found to be highly prevalent in never-smokers. RARß2 hypermethylation was significantly associated with poor recurrence-free survival (RFS) in 128 never-smokers with adenocarcinoma (P = 0.01) For parsimonious model building, the five proteins were clustered into three groups (ß-catenin and E-cadherin; c-MET; cyclin D1 and EGFR) by an unsupervised hierarchical clustering and were included in a multivariate analysis. Cox proportional hazard analysis showed that RARß2 hypermethylation was significantly associated with poor RFS in 128 never-smokers with adenocarcinoma (adjusted hazard ratio [HR] = 2.19, 95% confidence interval [CI] = 1.28 to 3.47; P = 0.009), after adjusting for interacting proteins. CONCLUSIONS: The present study suggests that RARß2 hypermethylation may be an independent prognostic factor of RFS in never-smokers with adenocarcinoma of the lung.

Antioxidative properties of tripeptide libraries prepared by the combinatorial chemistry.

Two series of combinatorial tripeptide libraries were constructed, based on an antioxidative peptide isolated from a soybean protein hydrolysate. One was a library of 108 peptides containing either His or Tyr residues. Another was a library of 114 peptides related to Pro-His-His, which had been identified as an active core of the antioxidative peptide. The antioxidative properties of these libraries were examined by several methods, such as the antioxidative activity against the peroxidation of linoleic acid, the reducing activity, the radical scavenging activity, and the peroxynitrite scavenging activity. Two Tyr-containg tripeptides showed higher activities than those of two His-containing tripeptides in the peroxidation of linoleic acid. Tyr-His-Tyr showed a strong synergistic effects with phenolic antioxidants. However, the tripeptide had only marginal reducing activity and a moderate peroxynitrite scavenging activity. Cysteine-containing tripeptides showed the strong peroxynitrite scavenging activity. Change of either the N-terminus or C-terminus of Pro-His-His to other amino acid residues did not significantly alter their antioxidative activity. Tripeptides containing Trp or Tyr residues at the C-terminus had strong radical scavenging activities, but very weak peroxynitrite scavenging activity. The present results allow us to understand why protein digests have such a variety of antioxidative properties.

Adrenomedullin reduces expression of adhesion molecules on lymphatic endothelial cells.

Adrenomedullin (AM) is a novel vasoactive peptide which regulates vascular tone and vascular endothelial cell growth. We recently reported that lymphatic endothelial cells (LECs) are also an attractive target of AM and concluded that AM is a potent mediator of lympangiogenesis. In the present study, we conducted a genome-wide analysis of genes that are regulated by AM in LECs. AM profoundly suppressed gene expression of cell adhesion receptors and inflammatory factors in LECs, such as intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), endothelial adhesion molecule-1 (E-selectin), interleukin-8, and chemokines, QRT-PCR and flow cytometry analysis showed that AM dose-dependently suppressed the TNF-a-induced mRNA and protein expression of ICAM-1 and VCAM-l. Treatment of LECs with a cell permeable cyclic adenosine monophosphate (cAMP) analog, 8-Br-cAMP, mimicked the suppressive effect of AM on the expression of adhesion molecules. Moreover, both AM and 8-Br-cAMP suppressed TNF-α-induced NF-κB activation in LECs, indicating that AM reduces expression of adhesion molecules in LECs via a cAMP/NF-kB dependent pathway. These results suggest that AM may have an important role in the regulation of the expression of adhesion molecules in lymphatic endothelium, which is critical in the control of immune and inflammatory responses.

Adrenomedullin induces lymphangiogenesis and ameliorates secondary lymphoedema.

AIMS: Adrenomedullin (AM) is a multifunctional peptide hormone that plays a significant role in vasodilation and angiogenesis. Lymphoedema is a common but refractory disorder that is difficult to be treated with conventional therapy. We therefore investigated whether AM promotes lymphangiogenesis and improves lymphoedema. METHODS AND RESULTS: The effects of AM on lymphatic endothelial cells (LEC) were investigated. AM promoted proliferation, migration, and network formation of cultured human lymphatic microvascular endothelial cells (HLMVEC). AM increased intracellular cyclic adenosine monophosphate (cAMP) level in HLMVEC. The cell proliferation induced by AM was inhibited by a cAMP antagonist and mitogen-activated protein kinase kinase (MEK) inhibitors. Phosphorylated extracellular signal-regulated kinase (ERK) in HLMVEC was increased by AM. Continuous administration of AM (0.05 microg/kg/min) to BALB/c mice with tail lymphoedema resulted in a decrease in lymphoedema thickness. AM treatment increased the number of lymphatic vessels and blood vessels in the injury site. CONCLUSION: AM promoted LEC proliferation at least in part through the cAMP/MEK/ERK pathway, and infusion of AM induced lymphangiogenesis and improved lymphoedema in mice.

Acid hydrolysis of protein in a microcapillary tube for the recovery of tryptophan.

The acid hydrolysis of proteins was miniaturized and simplified by employing microcapillary tubes (100 microl in volume) with 6 M HCl containing 1% 2-mercaptoethanol and 3% phenol for an amino acid compositional analysis. The method not only eliminated the laborious evacuation step for the hydrolysis tube but also decreased the destruction of tryptophan during hydrolysis. The recovery of tryptophan was 79% by acid hydrolysis at 145 degrees C for 4 h. Since the acid mixture could be removed under vacuum, the hydrolysate was subjected to an amino acid analysis without neutralization or dilution.