Identification of the lysine and histidine transporter family in Camellia sinensis and the characterizations in nitrogen utilization

In plants,the lysine and histidine transporter(LHT)family represent a class of proteins that mediate the uptake,translocation,and utilization of amino acids.The tea plant(Camellia sinensis)is a perennial evergreen with a relatively high level of amino acids.However,systematic identification and molecular characterization of the LHT gene family has rarely been reported in tea plants.In this study,22 CsLHTs were identified from the‘Shuchazao’genome and classified into two groups.The modeled three-dimensional structure and the conserved domains presented a high similarity among the LHTs proteins.Moreover,it was predicted that a few genes were conserved through the analysis of the physiochemical characters,structures and cis-elements in promoters.The expression patterns in tea plants revealed that CsLHT7 was mainly expressed in the roots,and CsLHT4 and CsLHT11 exhibited relatively high expression in both the roots and leaves.Moreover,the expression of all three genes could be induced by organic nitrogen.Additionally,heterogeneous expression of CsLHT4,CsLHT7 and CsLHT11 in Arabidopsis thaliana decreased the aerial parts biomass compared with that in WT plants while significantly increased the rosette biomass only for CsLHT11transgenic plants versus WT plants.Overall,our results provide fundamental information about CsLHTs and potential genes in N utilization for further analysis in tea plants.

Effect of fragile histidine triad gene transduction on proliferation and apoptosis of human hepatocellular carcinoma cells

AIM:To evaluate the inhibitory effects of human fragile histidine triad(FHIT) gene on cell proliferation and apoptosis in human hepatocellular carcinoma line Hep3B in vitro. METHODS:A recombinant pcDNA3.1(+) /FHIT including the functional region of FHIT gene was constructed and transferred into human hepatocellular carcinoma cells in vitro. mRNA and protein expression of the FHIT gene in the transfected cells was detected by RT-PCR and Western blot,respectively. The effect of FHIT on proliferation was detected by MTT assay. Changes in cell cycle and apoptosis were assayed by flow cytometry. Five mice received subcutaneous transplantation of Hep3B-FHIT;5 mice received subcutaneous transplantation of normal Hep3B and Hep3B-C as controls. The body weight of nude mice and tumor growth were measured. RESULTS:RT-PCR and Western blot analysis showed that the expression level of FHIT-mRNA and FHIT protein was higher in Hep3B cells after infection withpcDNA3.1(+) /FHIT. The growth of Hep3B cells treated with pcDNA3.1(+) /FHIT was significantly inhibited. The pcDNA3.1(+) /FHIT-transfected Hep3B cells showed a significantly higher cell rate at G0-G1 phase and increased apoptosis in comparison with controls(P < 0.05) . The growth of transplanted tumor was inhibited markedly by FHIT. Tumors arising from the Hep3B-FHIT cells occurred much later than those arising from the Hep3B and Hep3B-C cells. The growth of Hep3B-FHIT cells was slow and the tumor volume was low. CONCLUSION:Transduction of FHIT gene inhibits the growth of human hepatocellular carcinoma cells and induces cell apoptosis in vivo and in vitro.

Fragile histidine triad gene alterations are not essential for hepatocellular carcinoma development in South Korea

AIM: To establish the role of FHIT in the pathogenesis hepatocellular carcinoma (HCC). METHODS: We examined genomic alterations, as well as, mRNA and protein expression patterns from the FHIT gene, in 48 surgically resected hepatocellular carcinoma (HCC) tissues. Additionally, p53 mutations were analyzed. RESULTS: Aberrant FHIT transcripts were detected in 11 of 48 surrounding non-tumor liver tissues and 27 of 48 HCC samples (22.9% vs 56.3%, P = 0.002). No point mutations were identified within the open reading frame region of FHIT. Loss of heterozygosity (LOH) of the FHIT locus was detected in 4 of 42 informative cases for D3S1300, and 3 of 29 informative cases for D3S1313. Reduced expression of FHIT protein (Fhit) was observed in 8 (16.7%) of 48 HCC samples, with complete loss of Fhit in only 1 case. There were no associations with abnormal transcripts, LOH, and Fhit expression. p53 mutations were identified in 9 of the 48 HCC cases. However, none of the cases displayed a G to T transversion at p53 codon 249. CONCLUSION: Aberrant FHIT transcripts were more common in HCC tissues as compared to non-cancerous liver tissues. However, Fhit expression was lost or reduced in a minor fraction of HCC tissues, while it was strongly expressed in non-cancerous liver tissues.Therefore, our study suggests that FHIT plays a role in relatively few HCC cases in South Korea.

Compared efficacy of University of Wisconsin and histidine-tryptophan-ketoglutarate solutions in ex-situ liver resection and autotransplantation for end-stage hepatic alveolar echinococcosis patients

Background: The University of Wisconsin (UW) and histidine-tryptophan-ketoglutarate (HTK) solutions are the two most frequently used liver graft preservation fluids. The present study aimed to compare their efficacy in end-stage hepatic alveolar echinococcosis patients who underwent ex-situ liver resection and autotransplantation (ELRA). Methods: A total of 81 patients received ELRA from August 2010 to March 2018. They were allocated into UW ( n = 48) and HTK groups ( n = 33) based on the type of solutions used. Demographic and operational data were retrospectively analyzed. Primary outcomes included 90-day mortality, incidence of early graft loss, primary dysfunction, and postoperative complications. Results: Demographic and operational characteristics were similarly distributed in the two groups. No statistically significant differences were observed with regard to 90-day mortality (12.77% vs. 12.12%) and early graft loss rate (8.51% vs. 9.09%) between the two groups. Patients in the UW and HTK groups showed a primary dysfunction rate of 27.66% and 27.27%, respectively. The UW group exhibited a higher incidence tendency of biliary complications, albeit with no statistical significance. Conclusions: This is the largest cohort study comparing the efficacy of the UW and HTK organ-preserving solutions in end-stage hepatic alveolar echinococcosis patients in ELRA settings. UW and HTK solutions presented similar efficacy and safety. A randomized clinical trial with larger scale is needed for further investigation in future clinical applications.

Flow Injection Analysis of Histidine with Enhanced Electrogenerated Chemiluminescence of Luminol

A simple and sensitive flow injection method is presented for the determination of histidine based on its enhancement of electrogenerated chemiluminescence (ECL) of luminol. After optimization of the experimental parameters, the working range for histidine was in 1.0 x 10-6 to 1.0 x 10 -3 mol/L with a detection limit (S/N = 3) of 0.56 mmol/L. The relative standard deviation was 1.6% for 11 measurements of 5 x 10 5 mol/L histidine solution. The proposed method has been successfully applied to the determination of histidine in real pharmaceutical preparation.

RNA Cleavage by N-Phosphoryl Serine in Histidine Buffer

In the presence of histidine, N-(O,O-diisopropyl)phosphoryl serine could catalyze the cleavage of RNA in aqueous solution at neutral pH. The results were detected by sub-marine agarose gel electrophoresis and capillary zone electrophoresis. It was found that at high concentration histidine could cleave the RNA slightly. While the participation of DIPP-Ser could significantly accelerate the cleavage reaction. N-phosphodipeptide---N-(O,O-diisopropyl) Ser-His are proposed to be involved in the mechanism.

[(99m)~Tc(CO)_3]^+ labeled histidine derivative containing 4-nitroimidazole:Synthesis,biodistribution as a tumor hypoxia imaging agent

A novel histidine derivative containing 4-nitroimidazole,(S)-2-(4-((4-nitro-1H-imidazol-1-yl) methyl) benzamido)-3-(1H-imidazol-4-yl)propanoic acid (His-NI),was synthesized and labeled with [99mTc(CO)3(H2O)3]+.The tricarbonyl technetium complex,the 99mTc(CO)3-His-NI,showed a 99% yield under mild conditions at a low His-NI ligand concentration of 10-4 molL-1,and its biodistribution in mice bearing S180 tumor had a selective accumulation in tumor (2.01±0.40%ID/g at 1 h postinjection) and a slow clearance.The tumor/muscle ratio was 1.64 at 1 h,3.10 at 4 h,and 3.88 at 24 h,indicating that the 99mTc(CO)3-His-NI has a potential to image tumor hypoxia.

猪伪狂犬病病毒gE蛋白单克隆抗体的制备与鉴定

为了开发检测猪伪狂犬病病毒(PRV)的免疫诊断试剂,使用HEK293F细胞表达的gE重组蛋白免疫BALB/c小鼠。将免疫小鼠的脾细胞与SP2/0细胞融合,应用酶联免疫吸附试验(ELISA)和免疫过氧化物酶单层细胞试验(IPMA)方法筛选阳性杂交瘤细胞株,以期获得对PRV反应性良好的抗gE重组蛋白单克隆抗体。结果显示,获得2株(2B5和8F7)稳定分泌抗PRV gE蛋白单克隆抗体的杂交瘤细胞株,其腹水单克隆抗体的ELISA效价均为1∶1 000000;单克隆抗体亚型鉴定结果表明,2株单克隆抗体的重链均为IgG1亚型,轻链均为Kappa链。单克隆抗体特异性测定结果显示,2株单克隆抗体仅与PRV反应,不与其他常见病毒发生反应;SDS-PAGE结果显示,纯化后的单克隆抗体均在约50 ku和25 ku处有纯度较高的特异性条带;间接免疫荧光试验(IFA)结果表明,制备的2株单克隆抗体与转染gE质粒的293T细胞发生特异性反应。根据Western blot结果,筛选获得的2株单克隆抗体均在120 ku附近出现特异条带,表明这2株单克隆抗体均能特异性识别PRV。综上,成功制备了2株特异性强、效价高的抗gE蛋白单克隆抗体,为后期PRV诊断试剂盒的制备提供重要的生物材料。

平胃胶囊对恶变后GES-1细胞氧化应激的抑制作用及机制研究

目的:观察平胃胶囊对亚硝酸胺类化合物N-甲基-N’-硝基-N-亚硝基胍(N-methyl-N’-nitro-N-nitrosoguanidine,MNNG)诱导的胃癌前病变(precancerous lesions of gastric cancer,PLGC)细胞模型的影响,并初步探讨其作用机制。方法:制备空白血清和平胃胶囊含药血清备用;MNNG诱导人胃黏膜上皮细胞系GES-1制备PLGC细胞模型,采用倒置显微镜观察细胞形态,免疫荧光法检测增殖细胞相关抗原Ki67的表达水平,进行模型评价。CCK-8法筛选含药血清最佳干预浓度及时间;采用荧光探针DCFH-DA检测细胞内活性氧(reactive oxygen species,ROS)含量;ELISA检测丙二醛(malondialdehyde,MDA)含量;采用相关试剂盒检测超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性;采用新型荧光探针JC-10检测细胞线粒体膜电位的变化;采用实时荧光定量PCR检测Ki67和黑色素瘤分化相关基因7(melanoma differentiation-associated gene-7,MDA-7)的mRNA表达水平;采用Western blot法测定Ki67、白细胞介素6(interleukin-6,IL-6)和MDA-7的蛋白表达水平。结果:与正常组相比,模型组和空白血清组ROS和MDA含量显著升高(P<0.01),SOD和GSH-Px的活性显著降低(P<0.01),线粒体膜电位显著下降(P<0.01),Ki67和IL-6蛋白表达显著升高(P<0.01),MDA-7蛋白表达显著下降(P<0.01);与空白血清组相比,模型组ROS和MDA含量,SOD和GSH-Px活性,Ki67和MDA-7 mRNA表达水平,Ki67、IL-6和MDA-7蛋白表达水平,以及线粒体膜电位均无显著差异(P>0.05);与空白血清组相比,平胃胶囊含药血清组中ROS和MDA含量显著降低(P<0.01),SOD和GSH-Px活性显著上升(P<0.05),线粒体膜电位显著升高(P<0.01),Ki67和IL-6蛋白表达水平显著下降(P<0.01),MDA-7的mRNA及蛋白表达水平显著升高(P<0.01)。结论:平胃胶囊可显著减轻MNNG诱导的胃黏膜上皮细胞氧化应激损伤和炎症反应,调控促癌基因和抑癌基因的表达,从而发挥防治PLGC的作用。

Expression of fragile histidine triad in primary hepatocellular carcinoma and its relation with cell proliferation and apoptosis

AIM: To evaluate the expression of fragile histidine triad (FHIT) gene protein, product of a candidate tumor suppressor, and to investigate the relationship between FHIT, cell apoptosis and proliferation, and pathological features of primary hepatocellular carcinoma (HCC). METHODS: Forty-seven HCC and ten normal liver specimens were collected during surgical operation between 2001 and 2003. FHIT and proliferating cell nuclear antigen (PCNA) expression were detected by immunohistochemistry, and apoptotic level was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay on the tissue sections. RESULTS: All normal liver tissues showed a strong expression of FHIT, whereas 28 of 47 (59.6%) carcinomas showed a significant loss or absence of FHIT expression (P= 0.001). The proportion of reduced FHIT expression in those carcinomas at stages Ⅲ-Ⅳ (70.6%) and in those with extrahepatic metastasis (86.7%) showed an increasing trend compared with those at stages HI (30.8%, P= 0.013) and those without metastasis (46.9%, P = 0.010) respectively. Apoptotic incidence in advanced TNM stage carcinoma and those with positive FHIT expression was higher than that in early stage carcinoma (P=0.030) and in those with negative FHIT expression (P=0.044) respectively. The proliferating potential of hepatocellular carcinoma was associated with FHIT expression (P= 0.016) and the aggressive feature (P = 0.019). Kaplan-Meier analysis demonstrated that the survival time of these 47 patients correlated with TNM stage, FHIT expression and metastasis. CONCLUSION: There is marked loss or absence of FHIT expression, as well as abnormal apoptosis-prdiferation balance in HCC. FHIT may play an important role in carcinogenesis and development of HCC.

Bile acid increases expression of the histamine-producing enzyme,histidine decarboxylase,in gastric cells

AIM: To investigate the effect of bile acid on the expression of histidine decarboxylase (HDC), which is a major enzyme involved in histamine production, and gene expression of gastric transcription factors upon cooperative activation.

Decreased fragile histidine triad expression in colorectal cancer and its association with apoptosis inhibition

AIM： To detect the expression of fragile histidine triad （FHIT） in normal colorectal tissue, colorectal adenoma and colorectal cancer （CRC） tissue, and to analyze its relationship with the clinicopathological features of CRC, and apoptosis-associatecl proteins （Bcl-2, Bax, survivin） and apoptosis in colorectal cancer. METHODS： FHIT mRNA analysis was performed by nested reverse transcription-polymerase chain reaction （RT-PCR） assay. Tissue microarray （TMA） was established to detect the expression of FHIT, Bcl-2, Bax and survivin genes in 80 CRC tissue specimens, 16 colorectal adenoma tissue specimens and 16 hemorrhoid （PPH） tissue specimens during the same period of time as the control. Citrate-microwave-SP was used as immunohistochemical method. The relationship between clinicopathological factors, such as differentiation grades and 5-year survival rate was observed. TUNEL assay was used to detect the apoptosis index in 80 CRC tissue specimens. RESULTS： Ten out of 26 （38.5%） CRC tissue specimens expressed aberrant FHIT transcripts, none of the aberrant FHIT transcripts was observed in the matchednormal tissue and colorectal adenoma tissue by nested RT-PCR assay. The positive rate of FHIT gene expression in normal colorectal tissue, colorectal adenoma and carcinoma tissue was 93.75%, 68.75% and 46.25%, respectively. Clinicopathological analysis of patients showed that the decreased FHIT gene expression was not associated with age, sex, serum CEA levels, tumor site and size, histological classification. However, the expression of FHIT was correlated with differentiation grades, pathological stages, lymph node metastases and 5-year survival rate after operation. The positive rate of apoptosis-associated proteins （Bax, Bcl-2 and survivin） in CRC tissue was 72.50%, 51.25% and 77.50%, respectively. The expression of these apoptosisassociated proteins in CRC tissue was correlated with the expression of FHIT. The mean apoptosis index in FHIT negative tumors was significantly lower than that in FHIT- positive tumors （5.41 ± 0.23 vs 0.56 ± 0.10, P 〈 0.01）. CONCLUSION： The FHIT gene plays an important role in the regulation of apoptosis and decreased FHIT expression plays a key role in the initiation and progression of colorectal carcinoma.

Thermokinetics of Liquid-Liquid Reaction of Dy(NO_3)_3 with Histidine

The thermokinetics of liquid liquid reaction of dysprosium nitrate with histidine were studied using a microcalorimeter. On the basis of experimental and calculated results, three thermodynamic parameters (the activation enthalpy, the activation entropy and the activation free energy), the rate constant, three kinetic parameters (the activation energy, the pre exponential constant and the reaction order) were obtained. On the basis of thermodynamics and kinetics, the formation reaction of the complex was discussed.

Aberrant crypt focus and fragile histidine triad protein in sporadic colorectal carcinoma

AIM:To characterize aberrant crypt focus (ACF) in adjoining mucosa in sporadic colorectal carcinoma and to evaluate fragile histidine triad (Fhit) protein and Ki67. METHODS:ACF was identified grossly and classified histologically in 75 resected specimens. ACF was typed into hyperplastic ACF (HACF) and dysplastic ACF (DACF). Sections of ACF, carcinoma and normal colonic mucosa as control were studied for Fhit and Ki67 expressions by immunohistochemistry and were grouped according to staining intensity and the number of positive stained cells observed in different histological groups. Comparison was done between the different groups by Pearson's χ 2 test and γ test for the ordinal data. P value < 0.05 was considered as significant.RESULTS:Age range was 40 to 86 years in males (mean = 43.36) and 45 to 70 years in females (mean = 56). HACF was identified in all cases studied in the non-tumorous colonic mucosa; ACF was observed as non-contiguous scattered foci, which supports the hypothesis of acquisition of single focus monoclonality by colonic epithelial cells in tumor generation. Twenty-four (32%) had DACF and were observed as closure to carcinoma foci. Intensity of Fhit expression:(1) HACF 40% exhibited strong intensity, similar to normal, moderate in 36% and weak in 24%; (2) DACF strong in 25%, moderate in 37.5% and weak in 37.5%; and (3) carcinoma negative in 16%, strong in 43% and moderate and weak in 28.5% each. Significant difference was observed in intensity of the Fhit protein expressions by HACF and DACF (P < 0.05). Tumor in older patients showed a stronger Fhit intensity compared to younger patients (P = 0.036). Vegetarian diet intake and nonsmokers showed stronger Fhit intensities. Advanced stage tumor, non-vegetarian diet and younger age was associated with loss of Fhit protein. Ki67 positivity was an extended crypt pattern in HACF and DACF showed extension up to the neck region of the crypts and surface epithelium. Carcinomas showed a marked increase in Ki67 expression (P < 0.05). Fhit protein had an inverse association with Ki67 expression. CONCLUSION:Weaker Fhit intensity was associated with smoking, non-vegetarian diet intake and increasing Ki67 expression. Loss of Fhit protein expression is possibly influenced by environmental factors like smoking and non-vegetarian diet intake.

山橿中生物碱类成分Laetanine、Launobine对乙酸致GES-1细胞损伤的保护作用及机制

目的利用乙酸建立胃黏膜上皮细胞(GES-1细胞)损伤模型,研究山橿中生物碱类成分Laetanine、Launobine对GES-1细胞损伤的保护作用及机制。方法通过MTT细胞增殖/毒性实验确定Laetanine、Launobine的最佳给药浓度。分别以浓度为0.01%~0.2%的乙酸培养液作用于GES-1细胞,作用时间分别为3、4、5h,筛选最佳造模条件。利用乙酸建立GES-1细胞损伤模型,测定Laetanine、Launobine含药培养基处理后的细胞存活率;Griess法测定细胞上清液中NO的浓度;ELISA法测定各组细胞上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和前列腺素E2(PGE2)水平;WST-1法检测各实验组超氧化物歧化酶(SOD)抑制率,计算SOD活力值。结果0.1%乙酸溶液处理3h为GES-1细胞损伤的最佳造模条件;与模型组比较,Laetanine、Launobine给药组均能显著升高GES-1细胞存活率(P<0.01);经Laetanine、Launobine处理后,细胞上清液中的NO、TNF-α、IL-6和PGE2水平显著降低,SOD活力显著升高(P<0.01或P<0.05)。结论山橿中生物碱类成分Laetanine和Launobine均能保护乙酸损伤的GES-1细胞,减轻GES-1细胞的受损程度,其作用机制可能与其降低NO、TNF-α、IL-6和PGE2水平,升高SOD水平有关,表明Laetanine和Launobine可能为山橿发挥抗胃溃疡作用的有效成分。

Examination of Correlation between Histidine and Cadmium Absorption by Eleagnus angustifolia L.,Vitisvinifera L.and Nerium oleander L.Using HPLC-MS and ICP-MS

In this study,HPLC-MS and ICP-MS methods wereused for the determination of histidine and cadmiumin Eleagnusangustifolia L.,Vitisvinifera L.and Nerium oleander L.leaves taken from industrial area including Gaziantep and Bursa cities.To histidine determination by HPLC-MS,flow rate of mobile phase,fragmentor potential,injection volume and column temperature were optimized as 0.2mL·min^(-1),70V,15μL and 20℃,respectively.For extraction of histidine from plants,distilled water was used by applying on 90℃and 30min.The concentrations(as mg·kg^(-1))of histidine were found to be in range of 8~22for Eleagnusangustifolia L.,10~33for Vitisvinifera L.and 6~11for Nerium oleander L.The concentrations of cadmium were found to be in ranges of 6~21μg·kg^(-1) for Vitisvinifera L.15~110μg·kg^(-1) for Eleagnusangustifolia L.and 63~218μg·kg^(-1) for Nerium oleander L.

Enthalpies of Solution of Complexes of Rare Earth Nitrate with L-α-Histidine in Water

The enthalpies of solution in water of complexes of RE(NO 3) 3 (RE=La～Nd, Sm～Lu, Y) with L α Histidine (His) were measured at 298.15 K. The standard enthalpies of formation of RE(His) 3+ (aq) were calculated. The 'tetrad effect' regularity was observed from the curve, which is the enthalpies of solution plotted against the atomic numbers of the elements in lanthanide series.

Urinary Concentrations of Hydroxyproline and 3-Methyl Histidine in Postpartum Cow

The urinary concentrations of hydroxyproline (HYPRO)and 3 -methyl histidine (3 - MEHIS) were de- termined in 16 Chinese-Holstein cows. The objectives of the experiment were to find out thhe relationship between collagen and myosin ddegradation and uterine involution in the postpartum cow. The results in the experiment showed that the mean concentrations of HYPRO and 3-MEHIS were 138.32±22.99 and 37.09 ±3.90 nmol·mL-1,respectively,for the cows during the days between 60-90 postpartum,and for the cows immediately after calving the concentrations of HYPRO and 3 -MEHIS reduced from 284.30 and 65.48 nmol ·mL-1 on the day one after calving to the normal level of 109.18 and 33.51 nmol·mL^-1 on the day 50 post- partum,respectively. There was a good correlation between the urinary concentrations of both HYPRO and 3 -MEHIS and the diameters of the involuting uterus (r = 0. 79).

Peripheral Concentration of Hydroxyproline and 3-methyl-histidine in Postpartum Cow

A total of 16 Friesian cows were used in the study to determine the peripheral concentration of hydroxyproline(HYPRO) and 3 methyl histidine(3 MEHIS).The objectives of the experiment were to find out the relationship between collagen and myosin degradation and uterine involution in the postpartum cow.The results showed tht the mean concentrations of HYPRO and 3 MEHIS were 13.47±2.38 and 18.35±2.77 n mole/mL,respectively,for the cows during the days 60～90 postpartum.And for the cows immediately after calving the concentrations of HYPRO and 3 HEHIS reduced from 34.29 and 28.06 nmole/mL on the day one after calving to the normal lever of 13.23 and 17.97 n mole on the day 50 postpartum,respectively.There was a good correlation between the peripheral concentrations of both HYPRO and 3 MEHIS and the diameters of the involuting uterus(r=0.8128).

Crystal Structure of 2,6-Dihistidine Dimethyl Ester Pyridine Acylamine Monohydrate

The crystal structure of the title compound,C21H25N7O7,has been determined in the orthorhombic space group C222（1） with a=8.993（10）,b=12.149（14）,c=22.20（2）A and Z=4.There exist intramolecular C-H…O and N-H…N hydrogen bonds in the title crystal structure.The intermolecular N-H…N and C-H…O hydrogen bonds together with π-π stacking interactions（face-to-face） link the molecules into an infinite three-dimensional supramolecular network.