[Correlation of damage-associated molecular pattern molecules with age and body mass index].

Objective: To explore the correlation of damage-associated molecular pattern molecules(DAMPs) serum S100, C-reactive protein (CRP), serum amyloid A (SAA) and uric acid (UA) with age and body mass index (BMI) to provide direction for further study of metabolic inflammation and inflammaging. Methods: The observational study method was used,and three hundred and sixty-six healthy people (131 males and 235 females) were selected from the physical examination center of the Second People's Hospital of Hunan Province from May to October 2020. They were divided into three age groups according to the age interval of 20 years, including 156 (53 males and 103 females) aged 20-40 years, 110 (36 males and 74 females) aged 41-60 years, and 100 (42 males and 58 females) aged 61-80 years. Kruskal Wallis H test was used to compare the differences of serum S100, CRP, SAA and UA levels among different age groups. According to the Health Industry Standards of the People's Republic of China-Weight Determination for Adults, the boundary is BMI =24 kg/m2. The healthy people were divided into non overweight (BMI<24 kg/m2) and overweight (BMI ≥ 24 kg/m2) two groups. The 1∶1 propensity score was used to match the age and gender. There were 96 non overweight subjects [43 males, 53 females, age 52 (35, 66) years], 96 overweight subjects [44 males, 52 females, age 52 (36, 64) years]. The serum levels of S100, CRP, SAA and UA in different BMI groups were compared by Mann-Whitney U test. Results: The median serum UA concentrations in males and females were 356 and 277 µmol/L, and the levels of serum UA of male was significantly higher than that of female (Z=-10.428, P<0.001); the median serum SAA concentrations in males and females were 3.1 mg/L and 4.4 mg/L, while the serum SAA level of female was significantly higher than that of male (Z=3.652, P<0.001); for 20-40, 41-60, and 61-80 years old group, the median concentration of serum S100 was 0.058, 0.057, 0.070 µg/L, and the median concentration of serum CRP was 0.32, 0.58, 0.93 mg/L; the median serum SAA concentrations were 3.2, 4.0, 5.2 mg/L; serum uric acid concentrations were (301.8±61.5), (298.6±69.8), (329.0±77.8) µmol/L. The levels of serum S100, CRP, SAA, UA in 61-80 years group were significantly higher than those of 20-40 years group (H=-2.749, H=-6.731, H=-5.033, H=-2.521, P=0.018, P<0.001, P<0.001, P=0.035) and 41-60 years old group (H=-2.719, H=-2.539, H=-2.540, H=-2.486, P=0.020, P=0.033, P=0.033, P=0.039).The levels of serum CRP of 41-60 years group was significantly higher than that of 20-40 years group (H=-4.108,P<0.001). There was no significant difference in levels of serum S100, SAA and UA between 20-40 years group and 41-60 years group (H=0.189, H=-2.360, H=-0.165, P=1.000, P=0.055, P=1.000); the levels of serum CRP and SAA were positively correlated with age (rs =0.342, rs =0.301, P<0.001, P<0.001); for overweight, non-overweight group, the median concentrations of serum S100 were 0.065 µg/L, 0.059 µg/L, the median concentrations of serum CRP were 0.92 mg/L, 0.47 mg/L, the median concentrations of serum SAA were 5.0 mg/L, 4.1 mg/L, the median concentrations of serum UA were 339.5 µmol/L, 301.5 µmol/L, the levels of CRP, SAA and UA in the overweight group were higher than those in the non-overweight group (Z=4.278, Z=2.025, Z=3.787, P<0.001, P=0.043, P<0.001); the levels of S100 in the overweight group was higher than those in the non-overweight group, but there was no significant difference in S100 between the two groups (Z=0.862, P=0.388); the levels of Serum CRP and UA were positively correlated with BMI (rs =0.348, rs =0.264, P<0.001, P=0.009). Conclusions: With the increase of age, the serum S100, CRP, SAA and UA levels of healthy people may be on the rise, especially the serum CRP and SAA levels are positively correlated with age; the serum S100, CRP, SAA and UA levels of overweight people may be higher than those of non-overweight people, especially the serum CRP, UA levels are positively correlated with BMI.

[Effects of miR-141-3p on proliferation, migration and NF-κB signaling pathways in gastric cancer cells].

Objective: To investigate the effects of miR-141-3p on proliferation and migration of gastric cancer cells and nuclear factor-κB (NF-κB) signaling pathway. Methods: Human gastric cancer cell line BGC-823 was cultured, and miR-141-3p mimetic (miR-141-3p mimics) was transfected into BGC-823 cells by lipofection. The miR-141-3p overexpressed BGC was constructed. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the transfection effect. The proliferation of BGC-823 cells was determined by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Transwell assay was used to detect the effect of miR-141-3p on BGC-823 cell migration. The expressions of NF-κB p65, p-IKK-α and p-IKB-α protein in NF-κB signaling pathway were detected by western blot. Results: Compared with the control group and the negative control group, the expression level of miR-141-3p in BGC-823 cells of the miR-141-3p group was (2.39±0.27), which was higher than (1.00±0.09) of the control group and (1.01±0.10) of the negative control group (P<0.05). The number of migrating cells in the miR-141-3p group was (47.64±5.65), which was lower than (106.22±12.14) in the control group and (110.40±12.26) in the negative control group (P<0.05). The expression levels of NF-κB p65, p-IKK-α and p-IKB-α protein in BGC-823 cells were down-regulated (P<0.05). Conclusion: MiR-141-3p can inhibit the proliferation and migration of human gastric cancer BGC-823 cells, which may be related to the inhibition of NF-κB signaling pathway activation.

[Impacts of early metoprolol intervention on connexin 43 and phosphorylated connexin 43 expression in rabbits with experimental myocardial infarction].

Objective: To investigate the early intervention effects of metoprolol on connexin 43(Cx43) and phosphorylated Cx43 (p-Cx43) expression in rabbits with post myocardial infarction. Methods: A total of 24 adult male New Zealand white rabbits were divided into sham group (n=6), early treatment group(n=6), routine treatment group(n=6), and myocardial infarction group(n=6) with a randomized block design blocked by weight. Myocardial infarction was induced by left anterior descending coronary artery (LAD) ligation. Rabbits in sham group received similar surgical procedure without LAD ligation. Metoprolol (12.5 mg/kg dissolved in 2 ml distilled water) was applied to rabbits in early treatment group and routine treatment group per gavage immediately after recovery from anesthesia and at 24 hours after myocardial infarction, respectively, then treated daily for 40 days. Rabbits in sham group and myocardial infarction group received 2 ml distilled water per gavage daily for 40 days. Plasma lactate dehydrogenase (LDH) and creatine kinase (CK) level were detected by automatic biochemistry analyzer after 6 hours in all rabbits. Ventricular fibrillation threshold (VFT) was measured in vivo by bipolar pacing electrodes at 40 days. Cx43 and p-Cx43 distribution in ventricular tissue was detected by immunofluorescence analyses. Cx43 and p-Cx43 protein level in ventricular tissue was determined by Western blot. Results: (1) Plasma LDH ((851.7±85.9)U/L vs. (332.3±39.6)U/L, P<0.01) and CK ((1 192.7±105.3)U/L vs. (462.3±65.6)U/L, P<0.01) were significantly higher in myocardial infarction group than in sham group (both P<0.01). (2) VFT was significantly lower in myocardial infarction group than that in sham group ((470.0±91.0) beats per minute vs. (683.3±60.9) beats per minute, P<0.05), and VFT was significantly higher in early treatment group ((633.3±43.2) beats per minute) and routine treatment group ((645.0±30.8) beats per minute) than in the myocardial infarction group (both P<0.05). (3) Immunofluorescence analyses showed that Cx43 was mainly localized in the intercalated disk, which was perpendicular to the cell long axis with linear arrangement, and less lateral distribution in sham group, early treatment group and routine treatment group, which was significantly different as the case in the myocardial infarction group. The expression of p-Cx43 in myocardial infarction group was less than in sham group, which was significantly upregulated in in early treatment group and routine treatment group when compared with myocardial infarction group, and expression of p-Cx43 was significantly higher in early treatment group than in routine treatment group. (4)The p-Cx43/Cx43 ratio of protein was significantly lower in myocardial infarction group than in sham group (0.165±0.011 vs. 0.363±0.046, P<0.05), and significantly higher in early treatment group (0.720±0.063) and routine treatment group (0.364±0.030) than in myocardial infarction group (both P<0.05), and this ratio was significantly higher in early treatment group than in routine treatment group (P<0.05). Conclusion: Metoprolol treatment, especially the early metoprolol treatment (within 24 hours after LAD ligation), could significantly improve VFT by ameliorating the distribution and dephosphorylation of myocardial Cx43 in rabbits with experimental myocardial infarction.

Impact of citrate pretreatment on ventricular arrhythmia and myocardial capase-3 expression in ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury often triggers ventricular arrhythmia. Citrate binds calcium ions, forming a soluble calcium citrate complex that may reduce I/R injury by affecting calcium ion concentration. We tested the effects of citrate pretreatment on ventricular heart rate and related factors in a rat I/R model. Fifty male Sprague Dawley rats weighing 350-400 g were randomly divided into equally sized control (A), model (B), and 0.1 M (C), 0.05 M (D), and 0.025 M (E) citrate groups. An I/R model was established by ligating the left anterior descending coronary artery. Serum calcium ion concentration was measured before and after citrate treatment. Triphenyltetrazolium chloride staining and spectrophotometry were used to determine infarction area and caspase-3 protein levels in myocardial tissue, respectively. Polymerase chain reaction was performed to test myocardial calmodulin (CAM) expression. The frequency of ventricular arrhythmia in group B was significantly higher than in the sham surgery group (P < 0.05). Citrate pretreatment resulted in lower and higher frequencies than those observed in the model and control groups, respectively, in a dose-independent manner. The most obvious reduction in ventricular arrhythmia was seen in Group D. Serum calcium ion concentration decreased markedly after citrate treatment (P < 0.05), with a specific pattern emerging over time. Infarction area and caspase-3 and CAM levels were significantly lower in the citrate groups compared with the model group (P < 0.05). Citrate can reduce myocardial cell apoptosis, alleviating ventricular arrhythmia and protecting the myocardium by reducing serum calcium ion concentration and downregulating caspase-3 and CAM expression.

First Report of a Soft Rot of Philodendron 'Con-go' in China Caused by Dickeya dieffenbachiae.

Philodendron is a popular foliage plant cultivated in interiorscapes of homes, offices, and malls throughout China. A severe outbreak of a soft rot of Philodendron 'Con-go' occurred in Guangzhou, China from 2010 to 2011. The disease was characterized by leaf infections starting as pinpoint spots that are water soaked and yellow to pale brown. The lesions are sometimes surrounded by a diffuse yellow halo. When the humidity is high and temperatures are warm to hot, the spots expand rapidly, becoming slimy, irregular, and sunken with light tan centers, darker brown borders, and diffused yellow margins and may involve the entire leaf in a few days. An invasion of the midrib and larger veins by the causal bacterium often results in advancement into the petiole and stem. A survey of three areas of production of Philodendron 'Con-go' (5 ha) in Guangzhou revealed that 91% of the fields were affected at an incidence ranging from 15 to 30%. Of 41 bacterial isolates obtained from lesions, three were selected randomly for further characterization. All strains were gram negative, negative for oxidase and positive for catalase and tryptophanase (indole production), and utilized citrate, tartrate, malonate, glucose, sucrose, fructose, and maltose but not glucopyranoside, trehalose, or palatinose. Biolog analysis (version 4.20.05, Hayward, CA) identified the isolates as Pectobacterium chrysanthemi (SIM 0.804 to 0.914). According to Samson et al. (1), it was renamed as a Dickeya sp. PCR was performed on the 16S rDNA gene with primers 27f and 1495r (3) and 1,423 bp of the 16S rDNA gene (GenBank No. JN709491) showed 99% identity to P. chrysanthemi (GenBank No. AF373202), and 98% to Dickeya dieffenbachiae (GenBank No. JF311644). Additionally, the gyrB gene was amplified with primers gyrB-f1 (5'-atgtcgaattcttatgactcctc-3') and gyrB-r1 (5'-tcaratatcratattcgcygctttc-3') designed based on all the submitted gyrB gene sequences of Dickeya spp. The dnaX gene was amplified with primers dnaXf and dnaXr (2). The products were sequenced and phylogeny analyses were performed by means of MEGA 5.05. Results showed that the gyrB and the dnaX genes of the strains were 98% homologous to those of D. dieffenbachiae (GenBank Nos. JF311652 and GQ904757). Therefore, on the basis of phylogenetic trees of the 16S rDNA, gyrB, and dnaX gene sequences, the bacterial isolate named PC1 is related to D. dieffenbachiae (100% bootstrap values). Pathogenicity of each of the three strains on Philodendron 'Con-go' was confirmed by injecting 60 50-day-old seedlings each with 0.1 ml of the isolate suspension (108 CFU/ml) into the leaves. Another 60 were injected with sterile water to serve as the control treatment. Plants were enclosed in plastic bags and returned to the greenhouse under 50% shade at 32°C day and 28°C night temperatures with high humidity. After 72 h, all the injected plants started to show symptoms similar to those observed on field plants, but no symptoms appeared on the control plants. The reisolates were identical to the inoculated strains in biochemical characteristics. Bacteria characteristic of the inoculated strains were not reisolated from the control plants. To our knowledge, this is the first report of D. dieffenbachiae causing soft rot of Philodendron 'Con-go' in China. References: (1) R. Samson et al. Evol. Microbiol. 55:1415, 2005. (2) M. Slawiak et al. Eur. J. Plant Pathol. 125:245, 2009. (3) W. G. Weisbury et al. J. Bacteriol. 173:697, 1991.

First Report of Bacterial Soft Rot of Potato Caused by Pectobacterium carotovorum subsp. carotovorum in Guangdong Province of China.

Potato (Solanum tuberosum L.) is a major crop in China, with 80.0 million tons being produced in 2010 on 3.3 million ha. Pectobacterium carotovorum subsp. carotovorum Jones 1901; Hauben et al. 1999 causes soft rot worldwide on a wide range of hosts including potato, carrot, and cabbage. During spring 2010, a soft rot with a foul smell was noted in stored potato tubers of different cultivars in the Guangdong Province. Symptoms on tubers appeared as tan, water-soaked areas with watery ooze. The rotted tissues were white to cream colored. Stems of infected plants with typical inky black symptoms could also be found in the fields prior to harvest. Three different potato fields were surveyed, and 13% of the plants had the symptoms. Twenty-seven samples (three symptomatic tubers per sample) were collected. Bacteria were successfully isolated from all diseased tissues on nutrient agar media supplemented with 5% sucrose and incubated at 26 ± 1°C for 36 h. After purification on tripticase soy agar media, four typical strains (7-3-1, 7-3-2, 8-3-1, and 8-3-2) were identified using the following deterministic tests: gram-negative rods, oxidase negative, facultatively anaerobic, able to degrade pectate, sensitive to erythromycin, negative for phosphatase, unable to produce acid from α-methyl-glucoside, and produced acid from trehalose. Biolog analysis (Ver 4.20.05, Hayward, CA) identified the strains as P. carotovorum subsp. carotovorum (SIM 0.808, 0.774, 0.782, and 0.786, respectively). The identity of strains 7-3-1 (GenBank Accession No. JX258132), 7-3-2 (JX258133), and 8-3-1 (JX196705) was confirmed by 16S rRNA gene sequencing (4), since they had 99% sequence identity with other P. carotovorum subsp. carotovorum strains (GenBank Accession Nos. JF926744 and JF926758) using BLASTn. Further genetic analysis of strain 8-3-1 was performed targeting informative housekeeping genes, i.e., acnA (GenBank Accession No. JX196704), gabA (JX196706), icdA (JX196707), mdh (JX196708), mtlD (JX196709), pgi (JX196710), and proA (JX196711) (2). These sequences from strain 8-3-1 were 99 to 100%, homologous to sequences of multiple strains of P. carotovorum subsp. carotovorum. Therefore, strain 8-3-1 grouped with P. carotovorum subsp. carotovorum on the phylogenetic trees (neighbor-joining method, 1,000 bootstrap values) of seven concatenated housekeeping genes when compared with 60 other strains, including Pectobacterium spp. and Dickeya spp. (3). Pathogenicity of four strains (7-3-1, 7-3-2, 8-3-1, and 8-3-2) was evaluated by depositing a bacterial suspension (106 CFU/ml) on the potato slices of cultivar 'Favorita' and incubating at 30 ± 1°C. Slices inoculated with just water served as non-inoculated checks. The strains caused soft rot within 72 h and the checks had no rot. Bacteria were reisolated from the slices and were shown to be identical to the original strains based on morphological, cultural, and biochemical tests. Although this pathogen has already been reported in northern China (1), to our knowledge, this is the first report of P. carotovorum subsp. carotovorum causing bacterial soft rot of potato in Guangdong Province of China. References: (1) Y. X. Fei et al. J. Hexi Univ. 26:51, 2010.(2) B. Ma et al. Phytobacteriology 97:1150, 2007. (3) S. Nabhan et al. Plant Pathol. 61:498, 2012. (4) W. G. Weisbury et al. J. Bacteriol. 173:697, 1991.

First Report of a Soft Rot of Phalaenopsis aphrodita Caused by Dickeya dieffenbachiae in China.

Phalaenopsis orchids, originally from tropical Asia, are mainly planted in Thailand, Singapore, Malaysia, the Philippines, and Taiwan and have gained popularity from consumers all over the world. The cultivation area of Phalaenopsis orchids has been rising and large-scale bases have been established in mainland China, especially South China because of suitable environmental conditions. In September 2011, a soft rot of Phalaenopsis aphrodita was found in a Phalaenopsis planting base in Guangzhou with an incidence of ~15%. Infected plants initially showed water-soaked, pale-to-dark brown pinpoint spots on leaves that were sometimes surrounded by a yellow halo. Spots expanded rapidly with rising humidity and temperatures, and in a few days, severely extended over the blade with a light tan color and darker brown border. Lesions decayed with odorous fumes and tissues collapsed with inclusions exuding. The bacterium advanced to the stem and pedicle. Finally, leaves became papery dry and the pedicles lodged. Six diseased samples were collected, and bacteria were isolated from the edge of symptomatic tissues after sterilization in 0.3% NaOCl for 10 min, rinsing in sterile water three times, and placing on nutrient agar for culture. Twelve representative isolates were selected for further characterization. All strains were gram negative, grew at 37°C, were positive for indole production, and utilized malonate, glucose, and sucrose but not glucopyranoside, trehalose, or palatinose. Biolog identification (version 4.20.05, Hayward, CA) was performed and Pectobacterium chrysanthemi (SIM 0.868) was confirmed for the tested isolates (transfer to genus Dickeya). PCR was used to amplify the 16S rDNAgene with primers 27f and 1492r, dnaX gene with primers dnaXf and dnaXr (3), and gyrB gene with primers gyrBf (5'-GAAGGYAAAVTKCATCGTCAGG-3') and gyrB-r1 (5'-TCARATATCRATATTCGCYGCTTTC-3') designed on the basis of the published gyrB gene sequences of genus Dickeya. BLASTn was performed online, and phylogeny trees (100% bootstrap values) were created by means of MEGA 5.05 for these gene sequences, respectively. Results commonly showed that the representative tested strain, PA1, was most homologous to Dickeya dieffenbachiae with 98% identity for 16S rDNA(JN940859), 97% for dnaX (JN989971), and 96% for gyrB (JN971031). Thus, we recommend calling this isolate D. dieffenbachiae PA1. Pathogenicity tests were conducted by injecting 10 P. aphrodita seedlings with 100 µl of the bacterial suspension (1 × 108 CFU/ml) and another 10 were injected with 100 µl of sterile water as controls. Plants were inoculated in a greenhouse at 28 to 32°C and 90% relative humidity. Soft rot symptoms were observed after 2 days on the inoculated plants, but not on the control ones. The bacterium was isolated from the lesions and demonstrated identity to the inoculated plant by the 16S rDNA sequence comparison. Previously, similar diseases of P. amabilis were reported in Tangshan, Jiangsu, Zhejiang, and Wuhan and causal agents were identified as Erwinia spp. (2), Pseudomonas grimontii (1), E. chrysanthemi, and E. carotovora subsp. carovora (4). To our knowledge, this is the first report of D. dieffenbachiae causing soft rot disease on P. aphrodita in China. References: (1) X. L. Chu and B. Yang. Acta Phytopathol. Sin. 40:90, 2010. (2) Y. M. Li et al. J. Beijing Agric. Coll. 19:41, 2004. (3) M. Slawiak et al. Eur. J. Plant Pathol. 125:245, 2009. (4) Z. Y. Wu et al. J. Zhejiang For. Coll. 27:635, 2010.

[Application of internal carotid artery stent in skull base surgery].

Objective: To report the experience of the application of internal carotid artery stent in skull base surgery, and to clarify the important role of internal carotid artery stent in skull base surgery. Methods: A retrospective study of 22 cases with skull base neoplasms implanted with internal carotid artery stents in the Department of ENT Head and Neck Surgery at the Sixth People's Hospital affiliated with Shanghai Jiao Tong University between July 2019 and January 2021 was conducted. Among them, 17 were male and 5 were female, aged between 33 and 75 years. There were 5 cases on the left, 16 cases on the right, and 1 case on both sides. Of these, there were 4 cases of jugular paraganglioma, 1 case of chondrosarcoma in the jugular foramen, 1 case of carotid body paraganglioma, and 16 cases of nasopharyngeal carcinoma after radiotherapy. Results: The degree of internal carotid artery erosion was assessed by computed tomography angiography (CTA), magnetic resonance imaging and digital subtraction angiography (DSA) images in 22 patients before surgery. It was found that the internal carotid artery was involved to varying degrees in all patients, so internal carotid artery stents were implanted before surgery. Tumor tissue was found to surround the internal carotid artery to varying degrees. Total or subtotal tumor resection was performed in all patients, and no intraoperative and postoperative complications occurred. The postoperative follow-up was 5 months to 2 years, and all patients had no complications such as spontaneous bleeding and pseudo aneurysm. There were no signs of stenosis or occlusion of the internal carotid artery stent segment in all cases. Conclusions: For patients with skull base tumors, preoperative imaging indicates the limited involvement of the internal carotid artery, and internal carotid artery stent implantation before surgery is a safe and effective treatment.

Cyclophilin A up-regulates MMP-9 expression and adhesion of monocytes/macrophages via CD147 signalling pathway in rheumatoid arthritis.

OBJECTIVES: To investigate whether cyclophilin A (CypA) can up-regulate the expression of MMP-2 and MMP-9 in monocytes/macrophages and whether CD147 facilitates this regulation in RA. Methods. Peripheral blood monocytes were isolated from RA patients and differentiated into macrophages by M-CSF (15 ng/ml). Under CypA stimulation (200 ng/ml), the protein release and activation of MMPs were detected by gelatin zymography and invasion assay. Human monocyte cell line THP-1 cells were selected for the advanced searching for potential interaction between CypA and CD147 in production of MMPs and cell adhesion to extracellular matrix (ECM). RESULTS: CypA significantly increased production and activation of MMP-9, not MMP-2, in the monocytes/macrophages derived from RA SF. CSA and HAb18G/CD147 antagonistic peptide AP-9 against CD147, respectively, dramatically decreased MMP-2 and MMP-9 expression, both in the absence or presence of CypA. Similar effects of CypA on MMP-9 production and cell invasion were observed in THP-1 cells. CypA-induced nuclear factor kappaB (NF-kappaB) activity for MMP-9 transcription were strongly blocked by extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) inhibitors (U0126 and SP600125, respectively), but not by p38 mitogen-activated protein kinase (MAPK) inhibitors (SB203580). CypA also induced calcium mobilization and increased the adhesion of THP-1 cells to ECM. CONCLUSIONS: These findings suggest that in RA, the abundant CypA, by its direct binding to CD147, up-regulates MMP-9 expression and adhesion of monocytes/macrophages to ECM, and the cyclophilin-CD147 interactions might contribute to the destruction of cartilage and bone.

High efficiency reconstitution of a human-mouse chimeric Fab of CAb-1 antibody specific to human colon cancer.

Monoclonal antibody (mAb) has been widely applied in the treatment of human diseases, especially in malignant tumours. However, most antibodies produced in mouse by hybridoma technology might induce severe human anti-mouse reactions. We had reported a murine mAb CAb-1 of therapeutic interest for its specifically binding to a cell surface glycoprotein of human colon cancer. Here, we attempted to generate a reconstituted human-mouse chimeric Fab (cFab) of CAb-1 in vitro to reduce its antigenicity and increase its capacity of penetration. First, the genes of heavy and light chain variable region (VH, VL) of CAb-1 were cloned. Then, the chimeric light chain (cL) and Fd (cFd) were constructed and expressed in Escherichia coli. Finally, the reconstituted cFab was obtained by gradient dialysis of the mixture of cFd and cL. SDS-PAGE and western blot analysis showed the reconstituted cFab with a recovery rate of 70.2% when the initial total concentration of cL and cFd proteins to be 100 microg/ml. The reconstituted cFab maintained the affinity and specificity to colon cancer cells compared with its parental antibody as determined by immunostaining analysis, FACS and ELISA. Our results established a foundation for further application of the cFab in diagnosis and treatment of colon cancer.

[The correlation analysis of the cochlear hydrops degree found by MRI angiography with Meiniere's disease classification].

Objective:To investigate the correlation between the levels of cochlear hydrops with the clinic classification of Meniere's disease(MD).Method:3D-FlAIR MRI was performed 24 hours after intratympanic injection of 8-fold diluted Gadopentetate Dimeglumine in 31 patients with unilateral MD. We evaluated the levels of cochlear hydrops and further analyzed the correlation between the levels of cochlear hydrops and thresholds of pure tone audiometry and clinic classification of MD. Result:MRI image clearly distinguished perilymph from endolymph in the labyrinth. The images showed different levels of enhancement of perilymphatic fluid spaces. In the 31 patients, obvious signs of endolymphatic hydrops were found, including 4 cases of level 0, 6 cases of level 1, 11 cases of level 2 and 10 cases of level 3. Their average hearing threshold was(54.37±3.88)dB HL. The levels of cochlear hydrops were significantly correlated with pure tone audiometry thresholds （r=0.636，P<0.01） and MD classification(r=0.516,P<0.01). None of the patients after intratympanic injection complained about discomfort or happened with any complications such as eardrum perforation, infection, and so on. Conclusion:The degree of endolymphatic hydrops based on MRI in MD patient has significant correlation with the pure tone audiometry and classification of disease. The examination can act as an objective index for MD diagnosis.

Regulation of a TGF-ß1-CD147 self-sustaining network in the differentiation plasticity of hepatocellular carcinoma cells.

Cellular plasticity has an important role in the progression of hepatocellular carcinoma (HCC). In this study, the involvement of a TGF-ß1-CD147 self-sustaining network in the regulation of the dedifferentiation progress was fully explored in HCC cell lines, hepatocyte-specific basigin/CD147-knockout mice and human HCC tissues. We demonstrated that TGF-ß1 stimulation upregulated CD147 expression and mediated the dedifferentiation of HCC cells, whereas all-trans-retinoic acid induced the downregulation of CD147 and promoted differentiation in HCC cells. Overexpression of CD147 induced the dedifferentiation and enhanced the malignancy of HCC cells, and increased the transcriptional expression of TGF-ß1 by activating ß-catenin. CD147-induced matrix metalloproteinase (MMP) production activated pro-TGF-ß1. The activated TGF-ß1 signaling subsequently repressed the HNF4α expression via Smad-Snail1 signaling and enhanced the dedifferentiation progress. Hepatocyte-specific basigin/CD147-knockout mice decreased the susceptibility to N-nitrosodiethylamine-induced tumorigenesis by suppressing TGF-ß1-CD147 signaling and inhibiting dedifferentiation in hepatocytes during tumor progression. CD147 was positively correlated with TGF-ß1 and negatively correlated with HNF4α in human HCC tissues. Positive CD147 staining and lower HNF4α levels in tumor tissues were significantly associated with poor survival of patients with HCC. The overexpression of HNF4α and Smad7 and the deletion of CD147 by lentiviral vectors jointly reprogrammed the expression profile of hepatocyte markers and attenuated malignant properties including proliferation, cell survival and tumor growth of HCC cells. Our results highlight the important role of the TGF-ß1-CD147 self-sustaining network in driving HCC development by regulating differentiation plasticity, which provides a strong basis for further investigations of the differentiation therapy of HCC targeting TGF-ß1 and CD147.

Synthesis, characterization, and antiinflammatory activity of naproxen complexes with rare earth (III).

RE(III) complexes of Naproxen(HNap) have been synthesized and characterized by elemental analyses, conductance measurements, solubilities, thermal analysis, infrared, proton magnetic resonance, and electronic spectral data. The elemental analyses reveal the presence of 1:3 (metal:ligand) stoichiometry and the IR spectra suggest the carboxylate group of HNap functions, as a bridging ligand to coordinate to RE(III) ions. The electronic spectra recorded in solid exhibit only slight shifts in visible regions, on which beta, delta and b1/2 of covalent parameters have been calculated. Formalin-induced rat paw edema and croton oil-induced rat ear edema inflammatory models were chosen to examine the antiinflammatory activity of Nd(III) complex, which ascertained enhanced antiinflammatory activity relative to the ligand.

Expression and analysis of McAbs antigen against human hepatocellular carcinoma.

AIM: To analyze the cause of tumor antigen heterogeneity and solve the problem of targeting diagnosis and therapy. METHODS: Using flow cytometry, the expression of McAbs antigen against human hepatocellular carcinoma (HAb18, E5, F11 and HAb27) was investigated. Analyses of the antigen sites were made quantitatively on the human hepatoma cell lines (SMMC-7721; QGY-7701; BEL-7402; HHCC and 9204). In particular, expression of human hepatoma, its association with antigen HAg18, and its relation s with cell cycle in four human hepatoma cell lines using the methods of indirect immunofluorescence and dual-parameter DNA dyeing were studied. RESULTS: The corresponding antigen of McAbs HAb18, HAb27, E5 were expressed on the five hepatoma cell lines and F1 was expressed only on the 7721 and 7701 hepatoma cell lines, but their mean fluorescence intensity showed different values on each cell line. HAb18 and HAb27 showed a relatively high level of expression, while E5 and F11 showed a lower level of expression. The value of AI (additivity index) was 136% for HAb18 and E5, 108% for HAb18 and HAb27, and 118.6% for E5 and H27As AI < 30% shows that both antibodies sites are the same, AI > 40% shows that both anti bodies sites are different, so the HAb18, HAb27 or E5 McAbs were combined in pairs, showing that their antigen sites were different. Furthermore, HAg18 antigen was expressed very highly and the positive rate of HAg18 was 100% in all the four human hepatoma cell lines. The was a mean intensity fluorescence was 8.237 ± 1.168 for SMMC-7721; 5.627 ± 1.678 for QGY-7701; 4.378 ± 1.525 for BEL-7402 is 4.378 ± 1.525 and 7.38 ± 1.919 for HHCC. However, in the normal human liver cell (QZG), HAg18 antigen showed low expression (0.534 ± 0.018) and its positive rate was only 9%. The relationship between human hepatoma associated antigen HAb18 and the cell cycle was expressed at the lowest level in G0-G1 stages, a higher level in S stage and the highest level in G2-M stage. CONCLUSION: Analysis of the anti-hepatoma McAbs corresponding to antigen may provide the basis for targeted diagnosis and therapy. The expression heterogeneity of human hepatoma-associated antigen HAg18 is related to the stage of cell cycle in the same cell lines, but not related to the stage of cell cycle in different cell lines.

Preparation and activity of conjugate of monoclonal antibody HAb18 against hepatoma F(ab')(2) fragment and staphylococcal enterotoxin A.

AIM: To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')(2) fragment of mAb HAb18 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC. METHODS: MAb HAb18 was extracted from the abdominal dropsy of Balb/c mice, and was purified through chromatography column SP 40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')(2) fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')(2) fragment and SEA was prepared with chemical conjugating reagent N succinimidyl 3 (2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12 with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F(ab')(2) against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay. RESULTS: The IgG mAb HAb18 was extracted, and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab')(2) SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F(ab') (2) SEA conjugate and HAb18 F(ab') (2), i.e.the cytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F(ab') (2) SEA conjugate was 0.182 +/- 0.012, that of negative control was 0.033 +/- 0.009, and there was significant difference between them (P < 0.05). CONCLUSION: SPDP is a good protein conjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18 F(ab') (2) fragment and SEA protein was prepared successfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti HCC mAbs or their fragments.

[Significance and application of anti-malignant hepatoma MAb HAb18 in radioimmunal diagnosis of human hepatocellular carcinoma].

In this study, 125I or 131I labelled intact IgG and its F(ab')2 fragments of an antihuman hepatoma monoclonal antibody, HAb18, was used for in vivo radioimmunoimaging of malignant hepatomas. Clear imaging of the tumor was obtained in 168 hours for intact IgG or in 72 hours for F(ab')2 fragments after iv injection or via selective catheterization of the arteriae hepatica communis. In addition, 99mTc-PHY (500 microCi) was simultaneously injected for static scanning of the liver, with which the tumor appeared as defects. All the 20 patients in this study were operated and the lesions were pathologically examined to verify the imaging results. The tumor: liver isotopic ratios were 2.15 +/- 0.15 and 2.63 +/- 0.21 for intact IgG and F(ab')2 fragments, respectively. In other vital organs, such as heart, brain, spleen, lungs and kidneys, as well as non-hepatoma neoplasms, including 3 cases of liver cavernous hemangioma and 2 cases of gastric cancer metastasized to the liver, no radioisotopic concentration was observed. Both the labelled IgG and F(ab')2 fragments had the same targeting potential, but a better contrast was obtained with F(ab')2 fragments. Furthermore, its clearance rate was faster than intact IgG. The smallest tumor diagnosed with this antibody was 0.5 cm in diameter and the positive rate for imaging primary hepatoma was 86.7% (13/15). The results obtained in this study promisingly indicate that HAb18 antibody may become the first choice for the early radioimmunodetection of human hepatoma.

[Studies on triterpenoids of Populus yunnanensis Dode].

Three triterpenoids (I, II and III) were isolated from the roots of Populus yunnanensis Dode, which has been used as a folk remedy for the treatment of urinary tract infection and calculus in Yunnan Province. Compound I and III were identified as friedelin and 21 alpha-hydroxyfriedelan-3-one and compound II as 21 alpha-acetoxyfriedelan-3-one which is a new triterpenoid. 13C NMR data of II showed that the presence of an alpha-acetoxy group at C-21 does not affect the boat conformation of ring E similar to the results in the study of some other D:A-friedo-oleananes.

[Studies on some derivatives of crotepoxide--an antitumor constituent of Piper futokadzura].

Through reactions of deacetylation and partial deacetylation with selected opening of epoxide ring, seven derivatives of crotepoxide (futoxide), a known antitumor constituent from Piper futokudzura, were prepared. Preliminary pharmacological tests showed that individual derivative possess significant inhibitory activity at 2 x 10(-4) mol/L to the PAF-induced platelet aggregation, while crotepoxide has no effect even at fivefold of this concentration.

[Determination of adenosine and thymidine in Fritillaria bulbs by dual wavelength ultraviolet spectrophotometry].

A convenient method for the quantitative analysis of adenosine and thymidine in Fritillaria bulbs was developed by means of dual wavelength ultraviolet spectrophotometry. This method has good linear relationship and the interrelated coefficient of standard curve for adenosine and thymidine were all found to be 0.9999. The methanol extracts of four species of Fritillaria have been analyzed with this method. The results show that the the bulbs of four species contain different quantities of adenosine and thymidine, which indicates that the nucleosides may be responsible for the anti-coagulation activity of Fritillaria.