[Bone marrow mesenchymal stem cells interactions with hepatocytes and hepatic stellate cells on different stiff substrates].

Objective: To explore the effect of substrate mechanical microenvironment and cell-cell interaction on differentiation of bone marrow mesenchymal stem cells (BMSCs), intrahepatic cellular function and phenotype. Methods: Bone marrow mesenchymal stem cells (BMSCs)-hepatocytes (HCs) and BMSCs-hepatic stellate cells (HSCs) were co-cultured on polyvinyl alcohol (PVA) hydrogel substrates at different stiffness (4.50 ± 0.47 kPa, 19.00 ± 3.51 kPa and 37.00 ± 2.09 kPa) by non-contact co-culture method. Furthermore, the effect of substrate mechanical microenvironment on BMSCs, HCs and HSCs and the activation and proliferation of HCs under different co-cultured condition was studied. A Student's t-test was used to compare the two groups. Results: The expression ofα-smooth muscle actin (α-SMA) and collagenα1- I (Col1A1) in BMSCs and HSCs cultured on its own increased with increase of substrate stiffness. After 72 h, the expression of albumin (ALB) of HCs on three stiff substrates was significantly higher than that of 24 and 48 h. Moreover, the expression of ALB of HCs increased with the increase of substrate stiffness. During the co-culture of BMSCs and HSCs, BMSCs of all three stiffness substrates promoted the expression ofα-SMA, Col1A1 in HSCs, but reduced the expression of PPARγin HSCs cells, thererby promoted the activation of HSCs, with apparent stiffness at 37 kPa. HSCs promoted the expression of ABL in BMSCs at three stiff substrates, but inhibited the expression of alpha-SMA and Col1A1 in BMSCs at 37 kPa, suggesting that co-culture had inhibited the differentiation of BMSCs myofibroblasts, and promoted the differentiation of hepatocyte-like cells, especially at high stiff substrates. In the co-culture of BMSCs and hepatic parenchymal cells, BMSCs had promoted the proliferation of hepatic parenchymal cells at 4.5 kPa. Further, hepatic parenchymal cells had inhibited the expression ofα-SMA in BMSCs, and promoted the expression of Alb, with inhibition of BMSCs differentiation towards myofibroblasts. Conclusion: The differentiation of BMSCs affects the substrate mechanical microenvironment, co-culture of HCs and HSCs. Simultaneously, affecting the function of hepatocytes in relation to the mechanical state of the substrates.

[The relationship between occupational wood dust exposure and nasal cancer].

Nasal cancer has not been included in the current list of legal occupational diseases in China. There is also a lack of systematic and in-depth study on the relationship between nasal cancer and occupational exposure factors in China. In September 2018, the department for work and pensions of UK released the latest edition of the "List of diseases covered by industrial injuries disablement benefit", which lists nasal cancer and nasopharyngeal carcinoma associated with wood dust exposure on the UK's occupational disease list. In order to better protect the health of workers, the relationship between occupational wood dust exposure and nasal cancer is reviewed, which provides a reference for further revision and improvement of occupational disease catalogue.

[The expression and significance of microtubule - driven protein KIF2A in epithelial ovarian cancer].

Objective: To investigate the protein and mRNA expression of KIF2A in ovarian cancer, and to investigate the migration and invasion ability changes in ovarian cancer cell line HO-8910 transfected by KIF2A-siRNA. Methods: Immunohistochemical method was used to detect the expression of KIF2A in 30 cases of ovarian cancer and 20 cases of ovarian normal tissues. The expression of KIF2A mRNA was detected by RT-PCR. The mRNA and protein expression of KIF2A in cell line HO-8910 was detected by RT-PCR and Western blot after transfected by KIF2A-siRNA in vitro. After the transfection, the cell migration and invasion ability were observed by scratch test and transwell experiments. Result: The expression of KIF2A mRNA and protein in HO-8910 was significantly lower than that in normal ovarian tissue (P<0.05). The capacities of migration and invasion of HO-8910 was suppressed notably after the knockdown of KIF2A (P<0.05). Conclusion: KIF2A gene expression was increased in ovarian cancer, and knockdown of KIF2A gene can inhibit the migration and invasion of ovarian cancer cells. It suggested that KIF2A gene may be a new target for the development of ovarian cancer.

Increased risk of breast cancer in individuals carrying the TNRC9 rs3803662 C>T polymorphism: a meta-analysis of case-control studies.

Currently, the relationship between the trinucleotide repeat containing 9 (TNRC9) rs3803662 C>T polymorphism and risk of breast cancer (BC) is uncertain. Here, we attempted to obtain a more accurate assessment of this association by conducting a meta-analysis of all eligible case-control investigations, comprising 44,820 cases and 58,316 controls. A comprehensive search was performed to identify all suitable studies involving the TNRC9 rs3803662 polymorphism and BC risk. Pooled odds ratios (ORs) and 95% confidence intervals (95%CIs) were estimated using fixed- or random-effect models. Heterogeneity, publication bias, and sensitivity analyses were also carried out. We found that the variant T allele of rs3803662 C>T greatly increases BC risk (CT vs CC: OR = 1.14, 95%CI = 1.07-1.22, P < 0.001; TT vs CC: OR = 1.38, 95%CI = 1.25-1.53, P < 0.001; CT/TT vs CC: OR = 1.19, 95%CI = 1.11-1.28, P < 0.001; TT vs CT/CC: OR = 1.28, 95%CI = 1.19-1.38, P < 0.001). Stratified analysis based on ethnicity also revealed a markedly increased risk in Asian and Caucasian populations. Moreover, studies with hospital-based control groups showed elevated risk under the four genetic models employed, as did those using population-based controls, except under heterozygote comparison. The TNRC9 rs3803662 C>T polymorphism is greatly related to increased risk of BC, in both Asian and Caucasian populations.

Identifying the trend of reactivity for sp2 materials: an electron delocalization model from first principles calculations.

The reactivity of sp(2) carbon materials is studied using the adsorption and dissociation of O2 on graphene and graphene oxide as model systems. The reactions on the basal plane, zigzag and armchair edges of graphene and graphene oxide with different oxygen-containing groups are calculated using first principles calculations. Two Brønsted-Evans-Polanyi relationships are identified and an electron delocalization model is suggested to understand the general trend of reactivity for sp(2) carbon materials.

Fusarium solani and Fusarium oxysporum Associated with Root Rot of Glycyrrhiza uralensis in China.

Liquorice (root of Glycyrrhiza uralensis Fisch.) is an important Chinese traditional medicine for many ailments (4). From 2002, severe outbreaks of root rot occurring on cultivated G. uralensis plants in Ningxia, China, have affected the yield and quality of liquorice and been considered as a major threat to commercial production of liquorice. Approximately 30% of the plants die from this disease in Ningxia every year. The disease, mainly affecting 2- to 4-year-old G. uralensis seedlings, begins with brown rot of root tips or lateral roots followed by internal decay of taproots during June to August every year. The infected plants are wilted with chlorotic foliage and easily pulled up from the soil. Root rot is clearly visible as a severe brown discoloration of vascular tissue along taproots. In severe cases, white mycelia are clearly visible on the surface of diseased roots and roots are decomposed. Isolations from diseased roots were made on potato dextrose agar (PDA) amended with streptomycin sulfate. Isolates (n = 78) were recovered from symptomatic roots (n = 105) and pure cultures were established by the single spore method. The two most frequently isolated fungi were transferred to potato sucrose agar and identified as Fusarium solani (61.5%) and F. oxysporum (30.8%) (1). The monophialides bearing microconidia of F. solani are long when compared to those of F. oxysporum. Genomic DNA of strains F. solani G013 and F. oxysporum FLR were extracted from mycelia with the cetyltrimethylammonium bromide (CTAB) method. Primers EF1-728F and EF1-986R were used to amplify the translation elongation factor-1α (TEF-1α) gene (2). The TEF-1α sequences of F. solani G013 (GenBank Accession No. AB777258) and F. oxysporum FLR (AB777257) shared 99 and 100% similarity with F. solani isolate NRRL52790 and F. oxysporum isolate NRRL 38328, respectively. Pathogenicity tests with one representative isolate of each species were conducted in the greenhouse on 1-month-old potted G. uralensis seedlings (12 plants per treatment). Isolates of the tested fungi were transferred to PDA and incubated in darkness at 24 ± 1°C for 7 days. Plant taproots about 5 cm below the soil surface were wounded with a sterile needle and five 5-mm-diameter fungal disks on the margin of colony were taken and firmly placed on the wounded location of each taproot with tinfoil; wounded taproots of seedlings inoculated with sterile PDA disks were used as controls (3). Root rot was assessed 2 months after inoculation. F. solani G013 and F. oxysporum FLR produced root rot symptoms on inoculated plants that were the same as those observed in field plants, and the fungi were reisolated from roots with typical symptoms. Control plants inoculated with sterile PDA disks remained asymptomatic, and no pathogen was isolated from them. To our knowledge, this is the first report of root rot caused by F. solani and F. oxysporum on G. uralensis in China. Effective control strategies are needed to minimize losses. References: (1) C. Booth. The Genus Fusarium. Commonwealth Mycological Institute, Farnham Royal, 1971. (2) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999. (3) M. Guo et al. Plant Dis. 96:909, 2012. (4) T. Wu et al. Am. Assoc. Pharm. Sci. J. 13:1, 2011.

Morphology of the developing muscularis externa in the mouse esophagus.

Muscularis externa of mouse esophagus is composed of two skeletal muscle layers in the adult. But less attention is paid to the histogenesis of the muscularis externa of the esophagus, and controversies still exist about the developmental process and the spatio-temporal expression characteristics of muscle-specific proteins during the development of esophageal muscularis externa. To further probe into the developmental pattern of muscularis externa of the mouse esophagus and the expression characteristics of different muscle-specific proteins, immunohistochemical and terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick-end labeling apoptotic staining methods are used to investigate the expression patterns of different muscle-specific proteins and to elucidate the relationship of these protein expressions with the development of muscularis externa of the mouse esophagus. Thus, an understanding of the developing esophageal muscularis externa may be important for developing therapeutic strategies for the treatment of human esophagus diseases. Serial sections of mouse embryos from embryonic day (ED) 12 to ED18, and full-length esophagi from postnatal first to 5th day were stained with monoclonal antibodies against α-smooth muscle actin (α-SMA), α-sarcomerical actin (α-SCA), desmin, and monoclonal anti-skeletal myosin (MHC), while apoptosis was determined using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling assay. The expression of α-SMA was started at ED12. During the development of ED14-ED15, α-SMA positive cells were seen extending from the walls of left three, four, and six arch arteries toward the dorsal wall of esophagus. Stronger expression of α-SCA and desmin could be detected at ED14 and ED15, expression intensity in caudal segment and inner layer was stained stronger than that of cranial segment and outer layer, but after ED16, strong expression of α-SCA and desmin was found in the outer layer of muscularis externa. Expression of MHC was first detected in the outer layer of cranial segment of muscularis externa at ED17. At ED18, MHC had extended to the level of thyroid gland, staining intensity in the outer layer and cranial segment was stronger than that of inner layer and caudal segment. One to five days after birth, the thickness of the esophageal muscle layer was obviously increased. Most of the muscle cells in the cranial segment of esophagus showed strong expression of α-SCA and clear cross striations at higher magnification. With progression toward the caudal segment, expression intensity of α-SCA became weaker, but the expression intensity of desmin was the same at different levels of esophagus. The muscle fibers were arranged densely with high expression of MHC in the cranial segment. During the development of esophageal muscularis externa, few apoptotic cells were observed. α-SMA, α-SCA, desmin, and MHC show different expression patterns. The differentiation of outer layer of esophageal muscularis externa is quicker than that of inner layer, and the caudal segment is quicker than that of the cranial segment. Besides, apoptosis may not participate in the development of esophageal muscularis externa. The smooth muscle cells from arch arteries may participate in the development of esophageal muscularis externa.

Hepatoma-derived growth factor and its role in keloid pathogenesis.

Hepatoma-derived growth factor (HDGF) is a novel mitogenic growth factor that has been implicated in many different carcinomas. Its role in keloid biology has not yet been investigated. The present study is aimed at examining the role of HDGF in keloid pathogenesis. Immunohistochemical staining and Western blot analyses were used to examine in vivo localization and expression of HDGF in keloid and normal skin tissue. This was followed by the detection of HDGF expression in fibroblasts cultured in vitro and fibroblasts exposed to serum. To investigate the effect of epithelial-mesenchymal interactions, a two-chamber system was employed in which keratinocytes on membrane inserts were co-cultured with the fibroblasts. HDGF expression levels in all cell extracts and conditioned media were assayed through Western blot analysis. In another set of experiments, the effect of exogenous recombinant HDGF on keloid fibroblasts (KF) and normal fibroblasts (NF) was examined. Cell proliferation was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by quantifying proliferating cell nuclear antigen (PCNA) expression. Downstream targets of HDGF were identified by detecting their expression through Western blot analysis. Our results indicate that there was an increase in HDGF expression in the dermis of keloid compared with normal skin tissue. The application of serum and epithelial-mesenchymal interactions did not seem to have any effect on intracellular HDGF expression levels. However, co-culturing keloid keratinocytes with KFs resulted in increased HDGF secretion when compared with monoculture or normal controls. Furthermore, treatment with exogenous recombinant HDGF was found to increase the proliferation of KFs, activate the extracellular signal-regulated kinase (ERK) pathway and up-regulate the secretion of vascular endothelial growth factor (VEGF).

MTMR2 promotes the progression of NK/T cell lymphoma by targeting JAK1.

OBJECTIVE: The aim of this study was to investigate the expression characteristics of MTMR2 in NK/T cell lymphoma (NKTCL), and to further study its relationship with clinical parameters and the prognosis of patients with NKTCL. In addition, the potential mechanisms of MTMR2 promoting the progression of NKTCL was further explored. MATERIALS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine MTMR2 level in peripheral blood of 45 patients with NK/T-cell lymphoma and 45 healthy volunteers. The interplay between MTMR2 expression and clinical indicators, as well as the prognosis of patients with NK/T-cell lymphoma was analyzed. Meanwhile, MTMR2 expression in NKTCL cell lines was verified by qRT-PCR. Subsequently, MTMR2 knockdown and the overexpression models were constructed using lentivirus in NKTCL cell lines, including SNK-6 and KHYG-1. Transwell invasion and cell wound healing assays were applied to analyze the effect of MTMR2 on the biological function of NKTCL cells. Finally, an in-depth study of the relationship between MTMR2 and JAK1 was conducted to explore the underlying mechanism. RESULTS: QRT-PCR results showed that the expression level of MTMR2 in the serum of patients with NKTCL was remarkably higher than that of healthy volunteers, and the difference was statistically significant (p<0.05). Compared with patients with low expression of MTMR2, patients with high expression of MTMR2 exhibited significantly higher incidence of distant metastasis and lower overall survival rate (p<0.05). The metastasis ability of NKTCL SNK-6 cells was remarkably attenuated in MTMR2 knockdown group when compared with the negative control sh-NC group (p<0.05). Meanwhile, the metastatic ability of NKTCL KHYG-1 cells in MTMR2 overexpressing group was remarkably enhanced when compared with the control NC group (p<0.05). The Luciferase reporter gene assay confirmed that MTMR2 could target JAK1, thereby jointly regulating the malignant progression of NKTCL. In addition, cell recovery experiment verified that JAK1 could partially reverse the enhanced metastatic ability of NKTCL cells induced by the overexpression of MTMR2. CONCLUSIONS: MTMR2 was highly expressed in NKTCL serum samples and cell lines, leading to high risk of distant metastasis and poor prognosis. In addition, MTMR2 might promote the malignant progression of NKTCL by regulating JAK1.

Methylation of an immediate-early inducible gene as a mechanism for B cell tolerance induction.

Stage-specific gene regulation is important in determining cell function during development. Immature B cells expressing membrane-bound immunoglobulin M (mIgM) are sensitive to antigen-induced tolerance, whereas mature B cells are activated by antigen. Previous studies have established an association between Egr-1 gene induction and antigen receptor (mIgM)-mediated activation of mature B cells. Here it is shown that the immature B cell line WEHI-231 and tolerance-sensitive bone marrow-derived B cells do not express Egr-1. It is further shown that lack of inducible expression in these cells is due to specific methylation of the Egr-1 gene. Thus, covalent inactivation of an activation-associated gene may explain tolerance sensitivity at specific stages of B cell development.

[Clinical analysis of 234 esophageal foreign bodies].

Objective: To explore the clinical characteristics and treatment methods of esophageal foreign body. Method: The clinical data of 234 patients with esophageal foreign bodies admitted to our department from January 2015 to August 2018 were retrospectively analyzed, including course time, foreign body types, surgical methods, imaging manifestations and treatment related complications. Result: The diagnosis of esophageal foreign bodies was confirmed by esophageal CT or esophageal barium meal X-ray examination before operation in 234 patients. Course time varied from 3 hours to 7 days, and the jujube nucleus was the most common food-borne foreign body.223 patients underwent esophagoscopic exploration and foreign body removal under general intravenous anesthesia, 11 of them had no definite esophageal foreign body, 22 had esophageal perforation and periesophagitis. After removal of foreign body, the nasogastric feeding tube was inserted. About 10 days later, the nasogastric feeding tube was removed when they got healthy. Nine cases underwent cervical abscess incision and drainage under general anesthesia. The average postoperative hospital day was 11 days. Conclusion: The rigid esophagoscopy is a safe and effective method for the esophageal foreign bodies. And neck abscess incision must be necessary,when they suffered from esophageal perforation with neck abscess and other serious complications.

Transcriptional activation and repression by Fos are independent functions: the C terminus represses immediate-early gene expression via CArG elements.

The Fos-Jun complex has been shown to activate transcription through the regulatory element known as the AP-1 binding site. We show that Fos down regulates several immediate-early genes (c-fos, Egr-1, and Egr-2) after mitogenic stimulation. Specifically, we demonstrate that the target for this repression is a sequence of the form CC(A/T)6GG, also known as a CArG box. Whereas Fos bound to the AP-1 site through a domain rich in basic amino acids and associated with Jun via a leucine zipper interaction, mutant Fos proteins lacking these structures were still capable of causing repression. Furthermore, Jun neither enhanced nor inhibited down regulation by Fos. Critical residues required for repression are located within the C-terminal 27 amino acids of c-Fos, since v-Fos and C-terminal truncations of c-Fos did not down regulate. In addition, transfer of 180 c-Fos C-terminal amino acids to Jun conferred upon it the ability to repress. Finally, Fra-1, a Fos-related protein which has striking similarity to Fos in its C-terminal 40 amino acids, also down regulated Egr-1 expression. Thus, Fos is a transcriptional regulator that can activate or repress gene expression by way of two separate functional domains that act on distinct regulatory elements.

Identification and characterization of the Egr-1 gene product, a DNA-binding zinc finger protein induced by differentiation and growth signals.

Egr-1 is an immediate-early response gene induced by diverse signals that initiate growth and differentiation. Its cDNA sequence predicts a protein with zinc fingers. We have generated an antiserum to the Egr-1 gene product and identified it as an 80-kilodalton short-lived protein in serum-stimulated mouse fibroblasts. The rat Egr-1 product has also been identified in nerve growth factor-induced PC12 cells. In addition, we show by cell fractionation and immunocytochemistry that the Egr-1 protein is located in the nucleus. We also demonstrate that it is phosphorylated. In vitro-generated Egr-1 protein binds with high affinity to the sequence CGCCCCCGC in a zinc-dependent manner.

Investigation on the regulatory effect of PGE2 on ESCC cells through the trans-activation of EGFR by EP2 and the relevant mechanism.

OBJECTIVE: To determine whether the combination of prostaglandin E2 (PGE2) and EP2, the subtype receptor of PGE2, could trans-activate the epidermal growth factor receptor (EGFR). PATIENTS AND METHODS: In this experiment, we selected epithelial cells from normal esophageal mucosa as the negative control group, and the ESCC EC109 and TE-1 cell strain as the observation group. Real-time PCR and Western-blotting were used to detect the expression of EP2, EGFR and phosphorylated EGFR (p-EGFR). The pre-treatment of ESCC cell strains was carried out using Butaprost (special agonist of PGE2 and EP2) and RNAi of EP2, and we observed the expression of EP2, EGFR, and p-EGFR. WST-8 (CCK-8) was applied for the detection of the cell proliferation rate. The transwell invasion experiment was conducted for the detection of the invasion capability of cells. The expression of MMP-9 (matrix metalloproteinase-9), VEGF (vascular endothelial growth factor), pro-inflammatory factors (IL-6 and TNF-α) in the cell supernatant were detected using ELISA. RESULTS: The high mRNA and protein expression of EP2, EGFR, and p-EGFR were found in the EC109 and TE-1 cell strains in the observation group, which were higher than those in the control group (p < 0.05). After the intervention of PGE2, EP2 expression was decreased and the p-EGFR expression was increased (p < 0.05). There was no variation found in the expression of EGFR (p > 0.05). After cells were intervened using Butaprost, the expressions of EP2 and p-EGFR were increased (p < 0.05), and there were no changes identified in the expression of EGFR (p > 0.05). After the intervention of RNAi, the expression of EP2 and p-EGFR was decreased (p < .05), and no changes were identified in the expression of EGFR (p > 0.05). After the intervention of PGE2 and Butaprost, great increases were seen in the cell proliferation rate, invasion capability, and the expression of MMP-9, VEGF, IL-6, and TNF-α in EC109 and TE-1 cell strains (p < 0.05), however, the intervention of RNAi could reduce above indexes (p < 0.05). CONCLUSIONS: Through cell experiments, we verified that the combination of prostaglandin E2 (PGE2) and EP2, the subtype receptor of PGE2, could trans-activate the epidermal growth factor receptor (EGFR) to regulate the proliferation and invasion capability of esophageal squamous cell carcinoma (ESCC) cells, and secrete and express multiple cytokines, thus discovering the pathological mechanism of inflammation to carcinoma transition in the occurrence of ESCC, and providing the experimental evidence for the search of new target in the treatment of ESCC. ESCC cells can highly express the receptor subtype EP2 of PGE2 that can transactivate the EGFR, through which PGE2 is involved in the transition mechanism from inflammation to cancer.

Effects of protein and phosphate buffer concentrations on thermal denaturation of lysozyme analyzed by isoconversional method.

Thermal denaturation of lysozymes was studied as a function of protein concentration, phosphate buffer concentration, and scan rate using differential scanning calorimetry (DSC), which was then analyzed by the isoconversional method. The results showed that lysozyme thermal denaturation was only slightly affected by the protein concentration and scan rate. When the protein concentration and scan rate increased, the denaturation temperature (Tm) also increased accordingly. On the contrary, the Tm decreased with the increase of phosphate buffer concentration. The denaturation process of lysozymes was accelatated and the thermal stability was reduced with the increase of phosphate concentration. One part of degeneration process was not reversible where the aggregation occurred. The other part was reversible. The apparent activation energy (Ea) was computed by the isoconversional method. It decreased with the increase of the conversion ratio (α). The observed denaturation process could not be described by a simple reaction mechanism. It was not a process involving 2 standard reversible states, but a multi-step process. The new opportunities for investigating the kinetics process of protein denaturation can be supplied by this novel isoconversional method.

Ligation of highly modified bacteriophage DNA.

After digestion by TaqI or nicking by DNAase I, five highly modified bacteriophage DNAs were tested as substrates for T4 DNA ligase. The DNAs used were from phages T4, XP12, PBS1, SP82, and SP15, which contain as a major base either glucosylated 5-hydroxymethylcytosine, 5-methylcytosine, uracil, 5-hydroxymethyluracil, or phosphoglucuronated, glucosylated 5-(4',5'-dihydroxypentyl)uracil, respectively. The relative ability of cohesive-ended TaqI fragments of these DNAs and of normal, lambda DNA to be ligated was as follows: lambda DNA = XP12 DNA greater than SP82 DNA approximately equal to nonglucosylated T4 DNA greater than T4 DNA = PBSI1 DNA much greater than SP15 DNA. Taq I-T4 DNA fragments were also inefficiently ligated by Escherichia coli DNA ligase. However, annealing-independent ligation of DNAase I-nicked T4, PBS1, and lambda DNAs was equally efficient. We conclude that the poor ligation of Taq I fragments of T4 and PBS1 DNAs was due to the hydroxymethylation (and glucosylation) of cytosine residues at T4's cohesive ends and the substitution of uracil residues for thymine residues adjacent to PBS1's cohesive ends destabilizing the annealing of the restriction fragments. Only SP15 DNA with its negatively charged, modified base was unable to serve as a substrate for T4 DNA ligase in an annealing-independent reaction; therefore, its modification directly interfered with enzyme binding or catalysis.

[Determination of caffeine metabolite for the evaluation of N-acetyltransferase, CYP1A2 and xanthine oxidase activities].

Caffeine was used as a metabolic probe to measure, in 120 healthy volunteers, the activities of three enzymes, deduced to be N-acetyltransferase(NAT2), CYP1A2 and xanthine oxidase (XO). The caffeine metabolites of 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 1-methylxanthine(1X), 1-methyluric acid(1U), 1, 7-dimethylxanthine(17X), and 1, 7-dimethyluric acid(17U) in urine were determined with HPLC after 4-5 hours of caffeine drink. The ratios of AFMU/1X or AFMU/(AFMU + 1X + 1U), (AFMU + 1X + 1U)/17X or (AFMU + 1X + 1U)/17U, and 1U/1X or 1U/(1X + 1U) were used as the index of NAT2, CYP1A2, and XO activities respectively. Frequency distribution analysis of the metabolic ratios of NAT2 indicated two distinct group with 20 slow acetylators and 100 rapid acetylators. Similar CYP1A2 activity was found in Chinese compared with European volunteers. Frequency analysis of CYP1A2 indicated the log normal distribution in 120 Chinese. The CYP1A2 index was much higher in smokers than that in nonsmokers. But no obvious difference was observed between young and old volunteers. The XO index also showed log normal distribution and has the similar value compared with European volunteers. The concentration variations of 1X and 1U in young volunteers were much lower than that in old volunteers.

[HPLC determination of five caffeine metabolites].

An HPLC method for the determination of caffeine metabolites in urine was established. Shim Pack CLC-ODS column (5 microns) was eluted with the mobile phase of methanol--acetonitrile--0.05% acetic acid = 12:1:87 (v/v) at a flow rate of 1.2 ml.min-1, and the ultraviolet absorbance was monitored at 280 nm. The 13 caffeine metabolites and caffeine were well separated and the concentrations of the five metabolites, AFMU, 1U, 1X, 17U, and 17X, were determined. The recoveries of the five metabolites were above 87%, the inter- and intra-day variations were less than 3%. The concentrations of the five metabolites in 120 volunteers were determined. The ratios of the metabolites were employed for the assessment of CYP1A2, NAT, and XO enzymes successfully.

[Effect of angiotensin converting enzyme inhibitor on arterial wall thickening in coarctation hypertension].

In order to clarify the role of renin-angiotensin systems on arterial wall thickening in hypertension, the effect of angiotensin converting enzyme inhibitor, captopril, in rats with aortic coarctation was studied. Captopril was given by intragastric instillation, 3 mg/kg B, W. twice a day, since the 2nd day after the operation, and the animals were examined 4 to 6 weeks later. Blood pressures from carotid and femoral arteries were recorded directly; the heart and standardized segments of the aorta were weighted; aorta, coronary and renal arterioles were studied morphometrically; renin activity, angiotensin II and aldosterone concentrations were assayed. The results showed that captopril reduced significantly thickening of the aortic and arteriolar wall in both the hypertensive forequarters and the normotensive hindquarters. This suggests that renin-angiotensin system plays a role in the development of arterial wall thickening in hypertension.