[Correlation between polymorphism of angiotensin-converting enzyme gene and the lower extremity atherosclerosis in type 2 diabetes mellitus patients].

Objective: To explore the correlation between polymorphism of the angiotensin-converting enzyme (ACE) gene and lower extremity atherosclerosis (LEA) in type 2 diabetes mellitus (T2DM) patients. Methods: A total of 380 patients diagnosed with T2DM in Department of Endocrinology from June 2015 to March 2016 were enrolled and divided into two groups: group A had no LEA (n=120) and group B had LEA(n=260). Color doppler ultrasound was used to detect the vascular lesions of the patients. For all patients in groups A and B, the polymerase chain reaction (PCR) was applied to determined the insertion/deletion polymorphism in intron 16 of the ACE gene of the patients. Then the blood pressure, blood lipid, glycated hemoglobin, and renal function were measured. Furthermore, the measured data was compared between the two groups. Multivariate logistic regression analysis was used to analyze the independent risk factors for LEA. Results: There was no significant statistical difference in age, sex, smoking and disease course between the two groups. The frequencies of DD genotype and D allele in the ACE gene of group B were much higher than those in group A. More specifically, DD genotype frequency was 18.8% in group B and 9.2% in group A, D allele frequency was 36.8% in group B and 29.2% in group A (all P<0.05). Multivariate logistic regression analysis showed that DD genotype in ACE gene (OR=2.744, 95% CI: 1.326-5.682), systolic blood pressure (OR=1.725, 95% CI: 1.072-2.778), total cholesterol (OR=3.785, 95% CI: 1.796-7.978), and glycated hemoglobin (OR=2.612, 95% CI: 1.602-4.258) were risk factors for LEA in T2DM patients. Conclusions: ACE gene insertion/deletion polymorphism was associated with the incidence of LEA in T2DM patients. DD genotype of the ACE gene may be a genetic risk factor for T2DM patients with concurrent atherosclerosis.

[Determination of Hair Shafts by InnoTyper® 21 Kit].

OBJECTIVES: To explore the application value of InnoTyper® 21 kit in forensic practice. METHODS: Samples of hair shafts and saliva were collected from 8 unrelated individuals. Template DNA was extracted by AutoMate Express™ forensic DNA automatic extraction system. DNA was amplified by InnoTyper® 21 kit and AmpFℓSTR™ Identifiler™ Plus kit, respectively, and then the results were compared. RESULTS: After the amplification by InnoTyper® 21 kit, complete specific genotyping could be detected from the saliva samples, and the peak value of genotyping profiles of hair shafts without sheath cells was 57-1 219 RFU. Allelic gene deletion could be found sometimes. When amplified by AmpFℓSTR™ Identifiler™ Plus kit, complete specific genotyping could be detected from the saliva samples, and the specific fragment was not detected in hair shafts without sheath cells. CONCLUSIONS: The InnoTyper® 21 kit has certain application value in the cases of hair shafts without sheath cells.

Association between the expression of HIF-1α and VEGF and prognostic implications in primary liver cancer.

The goal of this study was to investigate the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in primary liver cancer (PLC) and their association with prognosis. Tumor tissue, non-tumor tissue, and blood samples of 75 PLC patients were collected. Blood samples of 20 volunteers were also collected as healthy controls. Real-time quantitative reverse transcription-polymerase chain reaction was used to analyze the mRNA levels of HIF-1α and VEGF in the tissues. Protein expression of HIF-1α and VEGF was analyzed by immunohistochemistry. Enzyme-linked immunosorbent assay was used to detect the expression of HIF-1α and VEGF at the serum level. Univariate tests, multivariate Cox proportional hazards model, and the Student t-test were used to analyze the data. HIF-1α and VEGF showed higher expression in PLC tumor tissue both at the mRNA and protein levels. HIF-1α and VEGF expression was positive in 62.67 and 66.67% of PLC patients, respectively. HIF-1α and VEGF expression was significantly related to tumor stage and lymph nodes and lung metastases (P < 0.05). HIF-1α expression correlated with VEGF expression in PLC (r = 0.665, P < 0.05). Both HIF-1α and VEGF were significantly associated with overall survival (P < 0.05), while HIF-1α was identified as an independent prognostic factor. Both HIF-1α and VEGF, as the predictors of efficacy of TACE and metastasis of PLC, are biomarkers indicating PLC in advanced stage, and implied poor prognosis in patients with PLC. HIF-1α and VEGF could potentially be targets to improve outcomes in PLC.

Expression and clinical significance of CD4+ CD45+ peripheral blood T cells in patients with ulcerative colitis.

The objectives of this study were to explore the expression of peripheral blood CD4+CD45+ T cells in patients with ulcerative colitis (UC) and determine its clinical value. We selected 80 patients with UC from the First Affiliated Hospital of Liaoning Medical University from March 2012 to December 2013. Of these, 27 had mildly active, 28 moderately active, and 25 severely active UC. We also recruited 80 subjects to constitute the healthy control group. The percentages of CD4+CD45+ molecules on the peripheral blood T cell surfaces of patients were detected using flow cytometry and were compared between patients to determine the severity of illness. The percentage of peripheral blood CD4+CD45+T cells in the UC group was 52.93 ± 3.64% and in the controls it was 41.34 ± 2.94%; the UC group percentages were significantly higher (t = -22.159, P < 0.05). The average percentages in patients with mild, moderate, and severe activity were 50.99 ± 1.45, 52.66 ± 1.41, and 55.18 ± 2.18%, respectively; the moderate activity percentage was higher than that of mild activity, and the severely active stage percentage was overall the highest. Comparison between groups showed a statistically significant difference, F = 39.850, (P < 0.05). The expression levels of peripheral blood CD4+CD45+ T cells in the UC group were higher than those in the control group. Overall, our results showed that with the aggravation of disease the peripheral blood CD4+CD45+ T cell percentages were significantly increased, which might be useful as a marker for clinical diagnosis.

Genetic polymorphism of toxicant-metabolizing enzymes and prognosis of Chinese workers with chronic benzene poisoning.

Workers with chronic benzene poisoning (CBP) sometimes have a white blood cell count (WBC) below 4 x 10(9)/L even after cessation of workplace exposure to benzene for years. In order to explore this phenomenon, 120 workers with CBP were divided into two groups depending on the WBC, the mean diagnostic age of CBP, benzene exposure duration, and body mass index (BMI). The proportion of genotypes of cytochrome P450 2E1 (CYP2E1), glutathione-S-transferase mu-1 (GSTM1), glutathione-S-transferase theta-1 (GSTT1), myeloperoxidase (MPO), and NAD(P)H, quinone oxidoreductase 1 (NQO1) were compared between workers with WBC <4 x 10(9)/L and those with WBC > or =4 x 10(9)/L. With methods of logistic regression, a risk model was set up to predict the prognosis of CBP workers. The results indicated that the BMI of workers with WBC <4 x 10(9)/L was lower than that of workers with WBC of > or =4 x 10(9)/L (21.40 +/- 2.76 versus 23.09 +/- 3.36, P = 0.01), and the logistic regression model suggested there was a 4.5-fold increased risk among workers carrying GSTT1 null genotype (95% CI= 1.13- 17.54) compared with workers with GSTT1 non-null genotype. Our findings suggest that benzene exposure duration, BMI, and GSTT1 genotype may impact prognosis of the CBP workers.

GABA transporter 1 transcriptional starting site exhibiting tissue specific difference.

GABA transporter 1(GAT1) takes important roles in multiple physiological processes through the uptake and release of GABA, but the regulation of GAT1 gene expression in different tissues is rarely known. To address the question, first, 5' Rapid amplification of cDNA end (RACE) was used to determine GAT1 transcriptional starting sites in neonatal mouse cerebral cortex and intestine, adult mouse brain and adult rat testis. The products of 5'RACE were confirmed by DNA sequencing. We found that the transcript of GAT1 in neonatal mouse cerebral cortex and adult mouse brain starts at the same site (inside of exon 1), while in mouse intestine, GAT1 starts transcription in intron 1, and in rat testis, the transcript of GAT1 has an additional untranslation exon to the 5' direction.

Ultraviolet radiation and reactive oxygen generation as inducers of keratinocyte apoptosis: protective role of tea polyphenols.

Ultraviolet A (UVA) radiation produces serious damage to skin, especially to dermis, but its damage to epidermis and responsible mechanisms are not fully understood. Studies were thus undertaken to investigate the effects of UVA or reactive oxygen species (ROS) on lipid peroxidation, cell cycle, and apoptosis in primary cultured rat keratinocytes and to determine the possible protective effects of tea polyphenols (TPP). UVA or ROS increased the release of plasma enzyme lactate dehydrogenase (LDH), and increased lipid peroxidation production (malondialdehyde, MDA), but decreased the activity of glutathione peroxidase (GSH-Px), indicating that UVA or ROS were cytostatic and peroxidizing to keratinocytes. TPP stabilized and protected cell membranes from ROS or UVA by inhibiting the release of LDH, lowering MDA levels, and increasing GSH-Px activity. Flow cytometry (FCM) analysis revealed that UVA or ROS decreased the proliferative index (PI); hence the cell growth was blocked in the S/G2 phase, with an increase in the percentage of apoptosis in primary keratinocytes. TPP modified the UVA or ROS-induced changes in PI and apoptosis. TPP may be useful to protect keratinocytes from UVA irradiation. In summary, these data demonstrated that UVA damage to skin keratinocytes in vitro was similar to that for ROS and that TPP protects against UVA-induced cytotoxicity by inhibiting lipid peroxidation and apoptosis.

The effects on cell growth of tea polyphenols acting as a strong anti-peroxidatant and an inhibitor of apoptosis in primary cultured rat skin cells.

Studies during the past few years have indicated an inhibitory effect of green tea or tea polyphenols on tumorigenesis in animal and even in human. The purpose of this study was to observe the possible effects of tea polyphenols on skin cell growth and on apoptosis in rat primary cultured keratinocytes and fibroblasts. The release of a cell plasma enzyme (LDH), lipid peroxidation products (MDA production), and GSH-Px (glutathione peroxidase) into the medium in cultured cells was determined after treatment with tea polyphenols in a primary culture of skin cells. The percentage of cells in each cell cycle phase and in apoptosis were assayed by flow cytometry (FCM). Tea polyphenols may have a beneficial effect on skin cells at concentrations from 0.05% to 0.1%, showing a dose-dependent decrease in LDH, MDA (malondialdehyde) production, and a significant dose-dependent increase in GSH-Px and cell number. These effects were more obvious after exposure for 24 h than after 12 h. The results indicate that tea polyphenols may stabilize and protect the cell membrane against the release of cell plasma enzyme LDH, and its anti-peroxidation effect is also important for cell growth. FCM analysis revealed that treatment with 0.01% to 0.1% tea polyphenols decreased the percentage of cells in the G1/G0 (quiescent) phase from 81.32% to 74.38%, and increased the percentage of cells in S and G2/M phase from 9.87% to 15.26%, and from 6.51% to 10.36%, respectively. Tea polyphenols also increased the value of PI (proliferation index) from 18.17 to 25.62. At the same time it decreased the percentage of apoptosis from 27.10% to 17.97%, which indicates that green tea stimulates cell growth and inhibits the occurrence of apoptosis. Our results indicate that tea polyphenols are effective anti-oxidants and also inhibit apoptosis, which may improve the proliferative capacity of primary skin cells in vitro.

[Cloning and analyzing of members of excitatory amino acid transporter family from neonatal mouse brain].

Excitatory amino acid transporter family (EAAT) contains several structure-related membrane proteins. They are essential for the removal of glutamate released from pre-synaptic terminal to terminate its action of synaptic transduction and maintaining the normal concentration of neurotransmitters in nerve system. To study these proteins in single animal model, we cloned several members of EAAT family, named mGLAST-1, mGLT-1, mEAAC1 and mASCT1, from a neonatal mouse brain cDNA library. The cDNA sequence of mASCT1 was firstly reported in mouse, it is composed of 3787 bp which has an open reading frame (ORF) encoding a protein of 532 amino acid residues. The mASCT1 protein was expressed in Xenopus oocyte and the function was characterized by 3H-Ser uptaking. The homology between human ASCT1 and mouse ASCT1 is 89.3%. The DNA sequence data shows the variance in length and composition exists in the sequence of 5'UTR and 3'UTR of mRNA in the family members of EAAT. This phenomenon may indicate a post-transcription regulation mechanism might exist in the gene expression of mouse EAAT family members.

Xylene-induced effects on brain neurotransmitters, behavior and fos protein in rats.

Male Sprague-Dawley rats administered xylene intraperitoneally on alternate days at a dose of 125 or 250 mg/kg for 30 days exhibited no marked changes in locomotor activity, learning and memory capacity. However in rats given xylene on alternate day at a dose of 500 mg/kg for 30 days, a significant decrease in locomotor activity, deficits in learning ability and memory loss were detected. These xylene-induced behavioral changes were associated with a decrease in beta-endorphin and leuenkaphlin concentrations in the pons-medulla. On the contrary, xylene at a dose of 500 mg/kg increased the beta-endorphin level in caudate and c-fos expression in hippocampus. These data suggest that the xylene-induced behavioral alterations might be associated with the expression of Fos protein in the hippocampus.

Flow cytometric analysis of the toxicity of nitrofen in cultured keratinocytes.

Lactate dehydrogenase (LDH) release test, 3H-thymidine (3H-TdR) and 3H-leucine (3H-Leu) incorporation tests and flow cytometric analysis (FCM) of cell cycle were employed to elucidate cellular and molecular mechanism of nitrofen-induced toxicity in cultured keratinocytes. The results showed that cell morphologic damages were observed after exposure to 1.0 mmol/L and 10.0 mmol/L nitrofen. LDH release increased in a dose- and time-dependent manner. Depressions in 3H-TdR and 3H-Leu incorporation were found even at 0.01 mmol/L, and increased with the exposure dose. Cell cycle was analyzed from the DNA- histogram with propidium iodide stain. The results showed that there was no pronounced alteration in cell cycle after cells exposed to 0.01 and 0.1 mmol/L nitrofen. At dose of 1.0 mmol/L, S phase cells increased 2 times of that of control. With the increase of dose, G2/M phase cells became to increase about 5 times of that of the control. At 1.0 mmol/L, time course of cell cycle after exposure was observed. At the beginning of exposure, cells in S phase and G2/M phase were about 8.7% and 11%. Following 24 h incubation with nitrofen, cells in S phase increased to 18.0% with almost no change in G2/M. 72 h after exposure, G2/M phase cells increased to 63.3%. The above results demonstrated that S phase and G2/M phase blockage in cultured keratinocytes after exposed to nitrofen seems of importance in the mechanism of nitrofen-induced toxicity.

Preliminary validation of tumor cell attachment inhibition assay for developmental toxicants with mouse S180 cells.

This study was designed to explore the possibility of using ascitic mouse sarcoma cell line (S180) to validate the mouse tumor cell attachment assay for developmental toxicants, and to test the inhibitory effects of various developmental toxicants. The results showed that 2 of 3 developmental toxicants under consideration, sodium pentobarbital and ethanol, significantly inhibited S180 cells attachment to Concanavalin A-coated surfaces. Inhibition was dependent on concentration, and the IC50 (the concentration that reduced attachment by 50%), of these 2 chemicals was 1.2 x 10(-3) mol/L and 1.0 mol/L, respectively. Another developmental toxicant, hydrocortisone, did not show inhibitory activity. Two non-developmental toxicants, sodium chloride and glycine were also tested and these did not decrease attachment rates. The main results reported here were generally similar to those obtained with ascitic mouse ovarian tumor cells as a model. Therefore, this study added further evidence to the conclusion that cell specificity does not limit attachment inhibition to Con A-coated surfaces, so S180 cell may serve as an alternative cell model, especially when other cell lines are unavailable. Furthermore, after optimal validation, it can be suggested that an S180 cell attachment assay may be a candidate for a series of assays to detect developmental toxicants.

Mutagenicity of various organic fractions of diesel exhaust particles.

Treated with various organic fractions of Diesel Exhaust Particles (DEP), the Ames test withSalmonella typhimurium strains TA98 and TA100, and the mice micronucleus test were employed to study the mutagenic activity in the bacterial reverse mutation system, with and without a mammalian S(9) activation component, and the clastogenic activity in mice polychromatic erythrocyte (PCE) stem cells. Extracted ultrasonically with dichloromethane then using the acid and base separated reaction and column chromatography, DEP were divided into five organic fractions. They are the organic acid fraction (Fl), the organic base fraction (F2), the aliphatic hydrocarbon fraction (F3), the aromatic hydrocarbon fraction (F4) and the polar fraction (F5). Results showed that an increase in the counted numbers of histidine revertants on theSalmonella TA100 and TA98 was observed with or without (S(9) mix), but these activities were more pronounced in the TA98 strains especially in the absence of the S(9) mix. These results suggest that the organic fractions of DEP contain mainly compounds with direct frame-shift mutaganicity. Positive results were also obtained from mice micronucleus assay. The frequency of mice bone marrow micronucleated polychromatic erythrocytes (PCE) was increased using this assay and it showed a definite dose-response relationship. The results suggest that various organic fractions could affect spindle fiber function or formation in mammalian cells. Compared with the results of different organic fraction, the effects of the F2, F4 and F5 were found to be stronger than those of other fractions. Based on the findings obtaind in the Ames and micronucleus tests, DEPs have genotoxic effects in both of the test systems.

Ascertainment corrected prevalence rate (ACPR) of leukopenia in workers exposed to benzene in small-scale industries calculated with capture-recapture methods.

ACPRs of leukopenia in peripheral blood of workers exposed to benzene in small-scale industries are calculated using capture-recapture methods. The results from two figures with 6-month apart demonstrate that the ACPR in workers exposed to benzene is 36.81(29.14-44)%, significantly higher than that of control 12.71(7.20-18.22)% (P < 0.05), with a relative risk of 2.9. The prevalences of 4 cross-sectional investigations in exposure group calculated with routine method are 18.73%, 26.37%, 27.93%, and 36.76% respectively; in controls, 8.38%, 6.85%, 7.94%, and 15.00% respectively and all fall in the range of 95% CI of ACPR. It is suggested that the methods of calculating ACPR by capture-recapture methods is simple, feasible and efficient, with the results more precise than with traditional methods.

Influence of in vitro methods, receptor fluids on percutaneous absorption and validation of a novel in vitro method.

In vitro experiment using excised skin has been valuable for studying the mechanism of percutaneous absorption. Based on previously established static diffusion cell system in this laboratory, a novel model-perfused glass diffusion cell system is designed. The results of initial comparative study on percutaneous absorption between glass perfused diffusion cell and static diffusion cell, in vitro and in vivo permeation as well as factors affecting permeation with seven radiolabelled chemicals are presented. The results demonstrate that the perfused diffusion cell system, which used a perfusion fluid below the surface of skin to take up the materials which penetrated the skin, is more similar to physiologic condition, convenient and automatic than that of the static cell. It well predicts the in vivo percutaneous absorption if appropriate receptor fluid is chosen. The results also show that the selection of receptor fluid is critical for in vitro permeation of chemicals with different solubility.

[Alveolar air analysis for benzene as a biomonitoring index].

To establish a well validated biological monitoring procedure for benzene exposure, the concentration of benzene in alveolar air and phenol in urine was studied in conjunction with personal monitoring data both in workers and volunteers. There existed good correlation between benzene concentration in alveolar air, phenol concentration in urine by the end of workshift and the benzene concentration in workplace when the exposure level was above 5 mg/m3. When the exposure level was below 5 mg/m3, the urinary phenol was no longer a valid indicator of uptake/exposure in volunteers. The determination of benzene concentration in expired air collected after the workshift was a potentially useful index. The relationship between benzene concentration in alveolar air collected after shift and environmental air declined when the exposure level was very high (TWA greater than 200mg/m3). In general, the alveolar air analysis was valid as a biomonitoring index of benzene exposure under the exposure concentration ranged 3-120 mg/m3. The lest sampling time of alveolar air appears to be 30 minutes after the cessation of exposure and before the next shift.

Relationship between cell differentiation and binding of fluorescein isothiocyanate (FITC)-conjugated insulin of keratinocytes in culture.

Basal-type keratinocytes, isolated from newborn rat skin and separated on Percoll density gradients, proliferate in low (0.1 mM) calcium medium and, after raising the calcium level to normal (1.96 mM), stratify. Cells in the low calcium culture do not have extensive cell-cell connections, as seen with fluorescein isothiocyanate (FITC)-conjugated insulin. Fluorescein isothiocyanate-conjugated concanavalin A and Griffonia simplicifolia isolectin B4, but not peanut agglutinin (PNA), fluorescently label these cells. In 3-day-old low calcium cultures, within 2 h after raising the calcium of the medium to the normal level, intense binding of PNA to cells appears and neighboring cells are connected through bundles of filaments that are fluorescently labeled by FITC-insulin. After 2 days in normal calcium medium, the cultures exhibit relatively smooth, straightlined, cell boundaries that are labeled by FITC-insulin and cell boundaries and intracellular granules that are stained by hematoxylin. One day later, similar cell boundaries are present, but they are not significantly decorated by FITC-insulin and, under phase contrast microscopy, are dark. Free FITC gives labeling patterns similar to those given by FITC-insulin, but the FITC labeling is blocked by mercaptoethanol and dithiothreitol in contrast to FITC-insulin binding. The present results suggest the insulin moiety is involved in the labeling by FITC-insulin and the labeling is chronologically related to the stage of cell differentiation.

A neurophysiological study among Chinese CS2-exposed workers.

A neurophysiological study was carried out to examine peripheral neurotoxicity of extremely low levels of CS2, that is, less than 2 ppm (TWA-8hr) among Chinese viscose rayon workers. From the subjects who participated in a 1981 cross-sectional medical survey, 70 male workers exposed to CS2 and age-matched unexposed workers were randomly selected for the present neurophysiological examination. The conduction velocities of motor, sensory and slower motor fibres of the right ulnar nerve were measured using the same methods as those in the study by Seppäläinen (1974). Skin temperature was measured at the middle of the volar surface of the right forearm with a Thermistor thermometer. According to an earlier occupational hygiene survey in the plant, current personal exposures determined by a passive dosimeter method were very low; the average of daily exposure of 7 jobs studied was 1.45 ppm (range 0.2-5.0). Past and current area sampling data also suggested that occupational hygiene conditions regarding CS2 exposure in the plant had been extremely good for the previous 6 years. The present neurophysiological study clearly showed that significant reduction in the conduction velocities of motor and slower motor fibres of the ulnar nerve was detected as a consequence of chronic exposure to low levels of CS2. In the previous cross-sectional medical survey, no retinopathy was found among Chinese workers exposed to CS2 at this level. These results suggested that an effect of CS2 on the peripheral nerve would appear earlier than that on the retina.