Hydrophobic component in noncovalent binding of fusion peptides to lipids as observed by electrospray mass spectrometry.
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Buffers↗
Biomedical subjects
Yan Li
Publications and source records attributed to Yan Li.
Establishment of green fluorescent protein-expressing hepatocellular carcinoma cell lines with different metastatic potential: relevant models for in vivo monitoring of metastasis and angiogenesis.
PURPOSE: To establish stable green fluorescent protein (GFP)-expressing metastatic human hepatocellular carcinoma (HCC) cell lines with different metastatic potential for long-term in vivo studies of metastasis and angiogenesis. METHODS: The pIRES2-EGFP vector, which contains an enhanced GFP gene, was transfected into MHCC97-H and MHCC97-L, HCC cell lines with different metastatic potential. The stability of GFP expression, basic biological characteristics, invasion abilities in vitro, and spontaneous metastasis in vivo of the new cell lines (MHCC97-HG and MHCC97-LG) were studied. Microvessel density (MVD) of orthotopic implanted tumors was compared by anti-CD31 immunohistochemical staining, and real-time angiogenesis and metastasis of GFP-transfected tumors were detected by intravital fluorescent microscope. RESULTS: The GFP-transfected cell lines stably expressed green fluorescence in the absence of G418 over a 36-day period. Compared with the parental cell lines, they exhibited no distinct differences in biological characteristics. MHCC97-HG showed more aggressive invasion and spontaneous metastatic behavior than MHCC97-LG, and even its parental cell line, MHCC97-H (P<0.01). MVD levels induced by MHCC97-HG orthotopic implanted tumors were significantly higher than MHCC97-LG (P<0.01). Real-time angiogenesis and sequential steps of metastasis could be detected clearly under intravital fluorescent microscope. CONCLUSIONS: These two stable GFP-expressing HCC cell lines with the same genetic background and different metastatic potential were established, which could be useful models for monitoring metastasis and angiogenesis of HCC.
Animals↗
Indoor allergens, asthma, and asthma-related symptoms among adolescents in Wuhan, China.
PURPOSE: Information on indoor allergen exposures among non-Western populations, which have lower prevalence of atopic illness, is scant. We examined whether exposures to common indoor allergens were associated with doctor-diagnosed asthma and asthma-related symptoms among Chinese adolescents. METHODS: A cross-sectional study of 4,185 ninth grade students was conducted at 22 randomly selected schools in Wuhan, China. Information on respiratory health and exposures to indoor allergens was obtained by a self-administered questionnaire completed in class. RESULTS: Having animals currently was associated with persistent cough [prevalence odds ratio (POR)=1.54, 95% confidence interval (CI ): 1.21-2.11] and wheeze (POR=1.41, 95% CI: 1.03-1.94). Early-life exposure to animals was also associated with doctor-diagnosed asthma (POR=1.95, 95% CI: 1.35-2.82). Associations with respiratory symptoms strengthened with higher levels of exposure and for exposure in both early childhood and in adolescence. Exposure to cockroaches and having mold/water damage in the home contributed especially to wheezing (POR=2.03, 95% CI: 1.41-2.90 for cockroaches; POR=2.49, 95% CI: 1.82-3.40 for mold/water damage). CONCLUSIONS: Indoor allergen exposures were positively associated with asthma diagnosis and persistent respiratory symptoms among Chinese adolescents. Neither early-life nor current exposure to animals was protective for asthma or asthma-related symptoms.
Adolescent↗
Effects of four chlorobenzenes on serum sex steroids and hepatic microsome enzyme activities in crucian carp, Carassius auratus.
Four chlorobenzenes (chlorobenzene, 1,3-dichlorobenzene, 1,4-dichlorobenzene, p-chloro-methylbenzene) were administrated to the crucian carps (Carassius auratus) by peritoneal injections in the laboratory for 30 days. Serum testosterone and 17 beta-estradiol concentrations were detected using radioimmunology assay (RIA), and the activities of two hepatic microsome enzymes, glutathione s-transferase (GST) and UDP-glucuronosyltransferase (UDPGT), were measured using the modified methods as described by Habig and Owens. Results showed that the four chlorobenzenes caused significant increases in serum testosterone concentration in the crucian carps (P < 0.05) compared to the controls, but they caused no significant effect on 17 beta-estradiol level. All test chemicals caused a change in hepatic GST activity in crucian carps, with significant increases in enzyme activity (P < 0.05). Chlorobenzene, 1,3-dichlorobenzene and p-chloro-methylbenzene resulted in a marked inhibition to UDPGT activity in crucian carp (P < 0.05) except 1,4-dichlorobenzene. The changes in hepatic microsome enzyme activities may have resulted in the alterations of serum sex steroids levels in the crucian carps. The results indicated that these four chlorobenzenes may result in the changes of endocrine functions and may affect the reproductive success of this and other species.
Animals↗
Detection of antibodies against SARS-CoV in serum from SARS-infected donors with ELISA and Western blot.
Recombinant fragments of S proteins from the Severe Acute Respiratory Syndrome (SARS) coronavirus (SARA-CoV) were generated and used in a Western blot (WB) assay that was compared to a commercial SARS ELISA method. In 85% of confirmed SARS cases (n = 20), the S2 recombinant fragment based WB was positive and this was comparable to the commercial ELISA using heat killed SARS-CoV. WB using the other four recombinant fragments in confirmed SARS cases generated lower rates of detection (S1--75%, S1-N--25%, S1-C--55%). Evaluation of sera from healthy controls (n = 60) resulted in two weakly positive ELISA results with the remainder being negative while the S2 protein WB demonstrated three positive results from the 20 controls with a history of SARS contact and no positive results in 40 noncontact controls. A discrepancy between the ELISA and S2 WB arose when evaluating per-2003 sera from individuals (n = 10) with SARS-like symptoms (ELISA--100% positive, S2 WB--30% positive). These data suggest that the S2 WB assay may be particularly useful in ELISA-negative SARS cases and in some ELISA-positive non-SARS cases.
Adult↗
Purification and refolding of a novel cancer/testis antigen BJ-HCC-2 expressed in the inclusion bodies of Escherichia coli.
BJ-HCC-2 is one of the cancer/testis antigens that may be the most promising targets for tumor immunotherapy. To investigate the expression of BJ-HCC-2 protein in tumor cells and its capacity to elicit CTL response, the recombinant protein of BJ-HCC-2 was expressed in the inclusion bodies in Escherichia coli. The inclusion bodies were solubilized effectively with 0.3% N-lauroyl sarcosine in alkaline buffer. Under this denatured form, the BJ-HCC-2 protein carrying 6x histidine tag was purified with Ni-NTA affinity chromatography in a single step with a purity of over 97%. The yield of the purified protein was about 78%. The purified recombinant protein was refolded in a simple way. The correct refolding of the recombinant protein was verified in the recovery of its secondary and tertiary structures as assessed by circular dichroism and fluorescence emission spectra. The recovery rate of refolded protein was 92.1%. The renatured protein displayed its immunoreactivity with the antibodies to BJ-HCC-2 protein by Western blotting. This method of protein purification and refolding is easy to manipulate and may be applicable to the hydrophobic proteins that are unable to be purified by other methods.
Antigens, Neoplasm↗
The design of antagonist peptide of hIL-6 based on the binding epitope of hIL-6 by computer-aided molecular modeling.
The interaction between human interleukin-6 (hIL-6) and human interleukin-6 receptor (hIL-6R) is the initial and most specific step in the hIL-6 signaling pathway. Understanding its binding core and interaction mechanism at amino acid level is the basis for designing small IL-6 inhibiting molecules, such as peptides or lead compounds. With Docking method, the complex structure composed of hIL-6 and its alpha-subunit receptor (hIL-6R) was analyzed theoretically. By using structure-based analysis and phage display methods, the loop AB (from Lys67 to Glu81) of hIL-6 was found to be the important binding epitope of hIL-6R. By means of computer-aided design, the mimic antagonist peptide (14 residues) was designed and synthesized. Using multiple myeloma cell line (XG7), IL-6 dependent cell line, as test model, the influence of antagonist peptides on the proliferation of XG7 cells was investigated. The results showed that the synthetic peptide could be competitive to bind to hIL-6R with hIL-6, and the effect was concentration dependent. The theoretical design approach is a powerful alternative to phage peptide library for protein mimics. Such mini-peptide is more amenable to synthetic chemistry and thus may be useful starting points for the design of small organic mimics.
Binding Sites↗
In utero exposure to tobacco and alcohol modifies neurobehavioral development in mice offspring: consideration a role of oxidative stress.
OBJECTIVE: To determine whether in utero tobacco and alcohol exposure induces long-term neurobehavioral alterations and whether oxidative stress/damage is a possible causal factor. METHODS: Gravid mice were subjected to tobacco smoking and alcohol consumption. Their offspring were subsequently evaluated in developmental and behavioral tests. Antioxidative enzymes and erythrocyte membrane fluidity of adult offspring were measured. RESULTS: The intrauterine tobacco and alcohol exposure has resulted in significant reduced postnatal body and organ weights accompanied by reduced gestational body weight gain in their mothers. Such exposure also induced remarkable developmental delay in neonatal reflexes and notable behavioral deficit in adulthood, namely reduced motive coordination and locomotor activity as well as impaired learning and memory abilities. Furthermore, the formation of malondialdehyde (MDA) increased significantly whereas the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (Cat) and glutathione S-transferases (GST) decreased in the cerebral cortex and liver of prenatal intoxicated offspring. The embryonic intoxication also markedly reduced erythrocyte membrane fluidity in offspring. CONCLUSION: Our study shows the long-term neurotoxicity associated with prenatal tobacco and alcohol exposure, and suggests that the deleterious outcome may be in relation to increased free radicals formation and oxidative stress.
Alcohol Drinking↗
Characterization of protein-protein interactions between the nucleocapsid protein and membrane protein of the SARS coronavirus.
The human coronavirus, associated with severe acute respiratory syndrome (SARS-CoV), was identified and molecularly characterized in 2003. Sequence analysis of the virus indicates that there is only 20% amino acid (aa) identity with known coronaviruses. Previous studies indicate that protein-protein interactions amongst various coronavirus proteins are critical for viral assembly. Yet, little sequence homology between the newly identified SARS-CoV and those previously studied coronaviruses suggests that determination of protein-protein interaction and identification of amino acid sequences, responsible for such interaction in SARS-CoV, are necessary for the elucidation of the molecular mechanism of SARS-CoV replication and rationalization of anti-SARS therapeutic intervention. In this study, we employed mammalian two-hybrid system to investigate possible interactions between SARS-CoV nucleocapsid (N) and the membrane (M) proteins. We found that interaction of the N and M proteins takes place in vivo and identified that a stretch of amino acids (168-208) in the N protein may be critical for such protein-protein interactions. Importantly, the same region has been found to be required for multimerization of the N protein (He et al., 2004) suggesting this region may be crucial in maintaining correct conformation of the N protein for self-interaction and interaction with the M protein.
Binding Sites↗
Limited lentiviral transgene expression with increasing copy number in an MGMT selection model: lack of copy number selection by drug treatment.
Retroviral vector integration into the human genome carries increased risk of oncogenesis with increasing integrations. To boost transgene expression for gene therapy, multiple integrations are often sought. We studied the relationship between the number of vector integrations and transgene expression and the effect that drug selection in an MGMT-selection model would have on vector copy number. K562 cells were transduced using a lentiviral vector and a library of clones was generated. Median proviral copy number was 4 and a positive correlation with transgene expression was observed. Transgene expression increased at a linear rate between 1 and 4 vector copies/cell, but was unpredictable at >4 integrations/cell. When lentivirus MGMT(P140K)-transduced K562 cells were treated with O(6)-benzylguanine (BG)/BCNU, there was no selection for increased median copy number in colony-forming units, despite strong selection pressure and an increase in transgene expression and activity. These data show a direct and linear correlation between MGMT(P140K) transgene expression and vector copy number. Strong BG/BCNU selective pressure does not result in preferential survival of high-copy-number clones but does select for strong transgene expression. Thus drug selection would not be expected to increase the risk of oncogenesis due to exaggerated selection in favor of high-copy-number vector integration.
Alkyl and Aryl Transferases↗
Optical coherence tomography of the anterior segment of the eye.
Optical coherence tomography is a versatile tool for visualization and measurement of corneal and anterior segment anatomy. Keratorefractive surgery, anterior chamber biometry, and angle assessment are some of the applications that should benefit from this technology.
Anterior Eye Segment↗
Phelligridins C-F: cytotoxic pyrano[4,3-c][2]benzopyran-1,6-dione and furo[3,2-c]pyran-4-one derivatives from the fungus Phellinus igniarius.
Three unique pyrano[4,3-c][2]benzopyran-1,6-dione derivatives and a new furo[3,2-c]pyran-4-one, named phelligridins C-F (2-5), together with hispolon (8), (E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one (9), 4-hydroxybenzaldehyde, protocatechualdehyde, syringic acid, protocatechuic acid, caffeic acid, isoergosterone, and octadecyl ferulate were isolated and identified from the ethanolic extract of Phellinus igniarius. Their structures were determined by spectroscopic methods including IR, MS, and 1D and 2D NMR experiments. The structures of the new compounds were characterized as 3-(4-hydroxystyryl)-8,9-dihydroxypyrano[4,3-c]isochromene-4-one (2), 3-(3,4-hydroxystyryl)-8,9-dihydroxypyrano[4,3-c]isochromene-4-one (3), 8,9-dihydroxy-3-[5',6'-dihydroxy-5' '-methyl-3' '-oxo-spiro[fural-2' '(3' 'H),1'-indene]-2'-yl]-1H,6H-pyrano[4,3-c][2]benzopyran-1,6-dione (4), and (3Z)-3-(3,4-dihydroxybenzylidene)-6-(3,4-dihydroxystyryl)-2,3-dihydro-2-methoxy-2-(2-oxo-propyl)furo[3,2-c]pyran-4-one (5), respectively. Some compounds including 2 and 3 showed in vitro selective cytotoxicity against a human lung cancer cell line (A549) and a liver cancer cell line (Bel7402). Possible biogenetic sequences to the formation of 1-9 are postulated.
Antineoplastic Agents↗
Equine catechol estrogen 4-hydroxyequilenin is a more potent inhibitor of the variant form of catechol-O-methyltransferase.
Catechol-O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. It has been shown that COMT is genetically polymorphic with a wild-type and variant form where a valine has been substituted with a methionine. Several, but not all, epidemiological studies have shown that women, homozygous with the variant form, have an increased risk of developing breast cancer. Previously, we showed that 4-hydroxyequilenin (4-OHEN), a cytotoxic/genotoxic equine catechol estrogen metabolite, is both a substrate of COMT and an irreversible inhibitor of the methylation activity of COMT in vitro. To further understand the mechanism(s) of the association between the breast cancer risk and the COMT polymorphism, it was of interest to study the effect of the Val/Met polymorphism on COMT-catalyzed catechol estrogen methylation and 4-OHEN-mediated inhibition. In the present study, Michaelis-Menten analysis showed no difference between the relative ability of each form to methylate 4-OHEN. However, we found that the COMT variant form was more susceptible to 4-OHEN-mediated irreversible inactivation. Electrospray ionization mass spectrometry and SDS-gel analysis of COMT modified by 4-OHEN revealed that inhibition mechanisms include alkylation and/or oxidation of certain amino acids. In addition, site-directed mutagenesis experiments showed that Cys33 played a more important role in the variant form of COMT demonstrated by the fact that the C33A mutant of the variant form of COMT decreased its catalytic capability more dramatically as compared with that of wild type. Furthermore, thermotropic studies indicated that the variant form was more thermolabile, which suggested that the valine to methionine substitution may have changed the secondary/tertiary structure of the variant form of COMT, making it more susceptible to 4-OHEN and heat inactivation. These data suggest that 4-OHEN-mediated inhibition of the variant form of COMT in vivo might affect the detoxification efficiency of endogenous and/or exogenous catechol estrogens and play a role in the association between breast cancer risk and COMT polymorphism.
Breast Neoplasms↗
The thymic stromal cell line MTSC4 induced thymocyte apoptosis in a non-MHC-restricted manner.
Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4+8+ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class I and class II molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC-restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis.
Animals↗
Preparation and characterization of monoclonal antibody against protein tyrosine phosphatase PRL-3.
PRL-3 is a newly identified nonclassical protein tyrosine phosphatase associated with colorectal cancer metastasis. To prepare specific monoclonal antibody (MAb) against PRL-3, cDNAs of PRL-1, -2, and -3 were cloned by RT-PCR and inserted into prokaryotic expression vector pGEX-4T1, respectively. The fusion proteins were expressed in Escherichia coli. Monoclonal antibody against PRL-3 was obtained with hybridoma technique and specific ELISA screening. Western blotting and immunohistochemistry assays showed that MAb 3B6 had specific binding ability with PRL-3 protein in eukaryotic cells. This MAb will be a helpful tool for the detection of PRL-3 protein in the tissues and serum of colorectal cancer and other cancer patients.
Animals↗
Structure-based preliminary analysis of immunity and virulence of SARS coronavirus.
The research on SARS-associated coronavirus (SARS-CoV) has not stopped since its discovery, but the pathogenesis of SARS is still unclear. To explore the possible molecular mechanisms of the invasion and virulence of SARS-CoV, we investigated the structural basis of the viral proteins using computational biology. Forty-five motifs relating to superantigens, toxins and other bioactive molecules were detected in the proteins of SARS-CoV. The results showed that the distribution of the motifs varied in different proteins. Enzyme-like motifs were located in the R protein, while ICAM- 1-like and toxin-like molecules were located in the spike, envelop, nucleocapsid, PUP1, PUP 2 and PUP 4 proteins. Comparison of SARS-CoV with other viruses (OC43, PEDV, HRSV, HHerpV and HAdenoV) showed that each group of motifs was different for each type of virus. Data suggest that the proteins of SARS-CoV with toxic motifs might play crucial roles in targeting host cells and interfering with the immune system. This study provides new information for drug and vaccine design, as well as therapeutic strategies against SARS.
Amino Acid Motifs↗
Effect of three resistant soybean genotypes on the fecundity, mortality, and maturation of soybean aphid (Homoptera: Aphididae).
The fecundity, longevity, mortality, and maturation of the soybean aphid, Aphis glycines Matsumura (Homoptera: Aphididae), were characterized using three resistant soybean, Glycine max (L.) Merrill, genotypes ('Dowling', 'Jackson', and PI200538 'Sugao Zarai') and two susceptible genotypes ('Pana' and 'Loda'). Antibiosis in the resistant genotypes was demonstrated by a significant decrease in fecundity and longevity and increased mortality of A. glycines. Aphid fecundity, measured as number of offspring produced in the first 10 d by each viviparous aptera, was higher on Pana than on the resistant genotypes. Aphid longevity, the mean number of days a 1-d-old adult lived, was 7 d longer on Pana than on Dowling and Jackson. The mortality of both viviparous apterae and nymphs on resistant genotypes was significantly higher than on susceptible genotypes. A greater number of first instars survived to maturation stage (date of first reproduction) on susceptible plants than on resistant plants. None of the first instars placed on Dowling and PI200538 leaves survived to maturation. Observations of aphid behavior on leaves indicated that aphids departed from the leaves of resistant plants 8-24 h after being placed on them, whereas they remained indefinitely on leaves of susceptible cultivars and developed colonies. Reduced feeding due to ingestion of potentially toxic compounds in soybean may explain the possible mechanism of resistance to the soybean aphid.
Animals↗
Family-based associations between the angiotensin- converting enzyme insertion/deletion polymorphism and multiple cardiovascular risk factors in Chinese.
OBJECTIVE: We investigated, in a sample of Chinese families, the associations of body mass index (BMI), blood pressure (BP), serum lipids, fasting plasma glucose, serum creatinine and uric acid with the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene. METHODS: We genotyped 902 subjects from 186 nuclear families recruited in Shanghai, China, via a specialized hypertension clinic. We performed family-based association analyses for continuous and dichotomous phenotypic measurements using the quantitative and sib- transmission/disequilibrium tests (QTDT and Sib-TDT), respectively. RESULTS: The study sample included 121 parents and 781 offspring from 25 two-parent families, 71 one-parent families, and 90 families without parental information. The median number of offspring was four (range from 2 to 10). Of the 654 (85.4%) hypertensive offspring, 458 took antihypertensive drugs. The 354 male offspring were slightly younger than the 427 female siblings (48.1 versus 49.2 years, P = 0.03), but they had similar BMI (25.1 kg/m). In 482 informative offspring, QTDT analyses demonstrated a significant association between BMI and the transmission of the ACE D allele (regression coefficient 0.563, chi 2 = 4.02, P = 0.04). In 106 families with at least one hypertensive offspring and at least one normotensive sibling, Sib-TDT analyses showed that the ACE D allele was slightly over-transmitted from heterozygous parents to hypertensive offspring (P = 0.08). CONCLUSIONS: Our family-based study suggests that in Chinese, the ACE I/D polymorphism might play a role in the development of obesity and hypertension, which are closely linked cardiovascular risk factors.
Adult↗