Biomedical subjects

Lei Li

Publications and source records attributed to Lei Li.

At least 199 records · Page 11Linked to original sources

PubMed2003

The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development.

Jasmonic acid (JA) is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies in Arabidopsis have established that JA also performs a critical role in anther and pollen development but is not essential for other developmental aspects of the plant's life cycle. Here, we describe the phenotypic and molecular characterization of a sterile mutant of tomato (jasmonic acid-insensitive1 [jai1]) that is defective in JA signaling. Although the mutant exhibited reduced pollen viability, sterility was caused by a defect in the maternal control of seed maturation, which was associated with the loss of accumulation of JA-regulated proteinase inhibitor proteins in reproductive tissues. jai1 plants exhibited several defense-related phenotypes, including the inability to express JA-responsive genes, severely compromised resistance to two-spotted spider mites, and abnormal development of glandular trichomes. We demonstrate that these defects are caused by the loss of function of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. These findings indicate that the JA/COI1 signaling pathway regulates distinct developmental processes in different plants and suggest a role for JA in the promotion of glandular trichome-based defenses.

Amino Acid Sequence↗

PubMed2003

JNK-dependent phosphorylation of c-Jun on serine 63 mediates nitric oxide-induced apoptosis of neuroblastoma cells.

c-Jun NH2-terminal kinases (JNKs) potentiate transcriptional activity of c-Jun by phosphorylating serines 63 and 73. Moreover, JNK and c-Jun can modulate apoptosis. However, an involvement of nitric oxide (NO)-induced phosphorylation of c-Jun on Ser-63 and Ser-73 in apoptosis has not been explored. We report that in SH-Sy5y neuroblastoma cells, NO induced apoptosis following JNK activation and phosphorylation of c-Jun almost exclusively on Ser-63. Importantly, NO-induced apoptosis and caspase-3 activity were inhibited in cells stably transformed with dominant-negative c-Jun in which Ser-63 is mutated to alanine (S63A), but not in cells transformed with dominant-negative c-Jun (S73A). Ser-63 of c-Jun (but not Ser-73) was required for NO-induced, c-Jun-dependent transcriptional activity. NO-induced apoptosis, Ser-63 phosphorylation of c-Jun, and caspase-3 activity were all inhibited in SH-Sy5y cells transformed with dominant-negative jnk. A caspase-3 inhibitor prevented apoptosis but not c-Jun phosphorylation. In a different neuroblastoma cell line, NO-induced Ser-63 phosphorylation of c-Jun and apoptosis were blocked by a specific JNK inhibitor. We conclude that NO-inducible apoptosis is mediated by JNK-dependent Ser-63 phosphorylation of c-Jun upstream of caspase-3 activation in neuroblastoma cells.

Apoptosis↗

PubMed2003

Determination of bromine in flame-retardant plastics using pulsed glow discharge mass spectrometry.

A series of bromine-containing flame-retardant plastics was used to demonstrate the applicability of pulsed radio frequency glow discharge mass spectrometry to the determination of elements in commercial polymers. The direct analysis of bulk samples produced both atomic and molecular species, allowing elemental identification and molecular characterization. However, the low ion signal intensities hindered quantification. Atomization mechanisms for the plastic samples were studied in detail using scanning electron microscopy and mass spectrometry. Although some thermal desorption was observed, sputter atomization dominated when samples were not subjected to excessive discharge power. The sputter rates of various polymers, as evidenced by sputter weight loss measurements, showed a strong correlation with their ion production capabilities. Sputtering rates were related to the physical and chemical properties inherent to a polymer's composition. Analysis of samples compacted with a silver binder provided intense analyte signals allowing quantitative analysis. Signal stability, measurement accuracy, measurement precision, and detection limits were all assessed.

Journal Article↗

PubMed2003

Resonance light-scattering method for the determination of BSA and HSA with sodium dodecyl benzene sulfonate or sodium lauryl sulfate.

A new resonance light-scattering (RLS) assay of proteins such as bovine serum albumin (BSA) and human serum albumin (HSA) is presented. In the medium of phosphoric acid (pH=2.6), the weak RLS of sodium dodecyl benzene sulfonate (SDBS) or sodium lauryl sulfate (SLS) can be greatly enhanced by proteins, owing to interaction between the protein and the anionic surfactant and formation of an associate. The RLS intensity of the SDBS-protein system is stronger than that of the SLS-protein system under same experimental conditions. It is considered that the synergistic resonance caused by the absorption of both protein and SDBS could produce strong RLS, while absorption of protein only in the SLS system could cause relatively weak RLS. The enhanced intensity of RLS is proportional to the concentration of the protein. If SDBS is used as the probe the linear range is 7.5 x 10(-9)-1.5 x 10(-5) g mL(-1) for BSA and 1.0 x 10(-8)-1.0 x 10(-5) g mL(-1) for HSA. The detection limits are 1.8 and 2.8 ng mL(-1), respectively. When SLS is used as the probe the linear range is 2.0 x 10(-8)-1.0 x 10(-5) g mL(-1) and 2.5 x 10(-8)-1.0 x 10(-5) g mL(-1) for BSA and HSA, respectively, and the detection limits are 12.8 and 21.6 ng mL(-1), respectively. The biological mimics samples are synthetic concoctions of BSA and HSA with some interferents. In these samples, the concentration of interferents is higher than the concentration normally existing in organisms. The samples were determined satisfactorily.

Animals↗

PubMed2003

Synleurin, a novel leucine-rich repeat protein that increases the intensity of pleiotropic cytokine responses.

We have identified and characterized a novel single span transmembrane leucine-rich repeat protein, synleurin, that renders cells highly sensitive to the activation by cytokines and lipopolysaccharide (LPS). The major part of the extracellular domain consists of a leucine-rich repeats (LRR) cassette. The LRR central core has 12 analogous LRR repeating modules arranged in a seamless tandem array. The LRRs are most homologous to that of chondroadherin, insulin-like growth factor binding proteins, platelet glycoprotein V, slits, and toll-like receptors. Synleurin expression was detected at low levels in many tissues, including smooth muscle, brain, uterus, pancreas, cartilage, adipose, spleen, and testis. When synleurin is ecotopically expressed in transfected cells, the cells exhibit amplified responses to bFGF, EGF, PDGF-B, IGF-1, IGF-2, and LPS. Synleurin gene (slrn) maps to human chromosome at 5q12. The name synleurin reflects its synergistic effect on cytokine stimulation and its prominent leucine-rich repeats.

Amino Acid Sequence↗

PubMed2003

Identification and functional characterization of a novel R621C mutation in the synphilin-1 gene in Parkinson's disease.

Synphilin-1 is linked to the pathogenesis of Parkinson's disease (PD) based on its identification as an alpha-synuclein (PARK1) and parkin (PARK2) interacting protein. Moreover, synphilin-1 is a component of Lewy bodies (LB) in brains of sporadic PD patients. Therefore, we performed a detailed mutation analysis of the synphilin-1 gene in 328 German familial and sporadic PD patients. In two apparently sporadic PD patients we deciphered a novel C to T transition in position 1861 of the coding sequence leading to an amino acid substitution from arginine to cysteine in position 621 (R621C). This mutation was absent in a total of 702 chromosomes of healthy German controls. To define a possible role of mutant synphilin-1 in the pathogenesis of PD we performed functional analyses in SH-SY5Y cells. We found synphilin-1 capable of producing cytoplasmic inclusions in transfected cells. Moreover we observed a significantly reduced number of inclusions in cells expressing C621 synphilin-1 compared with cells expressing wild-type (wt) synphilin-1, when subjected to proteasomal inhibition. C621 synphilin-1 transfected cells were more susceptible to staurosporine-induced cell death than cells expressing wt synphilin-1. Our findings argue in favour of a causative role of the R621C mutation in the synphilin-1 gene in PD and suggest that the formation of intracellular inclusions may be beneficial to cells and that a mutation in synphilin-1 that reduces this ability may sensitize neurons to cellular stress.

Acetylcysteine↗

PubMed2003

Plasma membrane localization and function of the estrogen receptor alpha variant (ER46) in human endothelial cells.

Estrogen receptor (ER) alpha variants have been identified in an array of nonendothelial cells. We previously demonstrated that estrogen rapidly induces nitric oxide release via a phosphatidylinositol 3-kinase/Akt/endothelial nitric-oxide synthase (eNOS) pathway in EA.hy926 cells (immortalized human endothelial cells), which express a 46-kDa ER. We now confirm that, due to alternative splicing, the 46-kDa endothelial cell protein (ER46) is an amino-terminal truncated product of full-length ER alpha (ER66). ER46 is expressed in the plasma membrane, cytosol, and nucleus of resting, estrogen-deprived cells. Flow cytometric and immunofluorescence microscopic analyses demonstrated that the ER46 C but not N terminus is Ab-accessible in the plasma membrane. Inhibition of palmitoylation with tunicamycin and [(3)H]palmitic acid labeling demonstrated an estrogen-induced, palmitoylation-dependent plasma membrane ER46 recruitment, with reorganization into caveolae. In reconstituted, estrogen-stimulated COS-7 (ER-null) cells, membrane ER46 more efficiently triggered membrane eNOS phosphorylation than ER66. Conversely, ER66 more efficiently mediated estrogen response element reporter-gene transactivation than ER46. These results demonstrate that ER46 is localized and further dynamically targeted to the plasma membrane in a palmitoylation-dependent manner. ER46 more efficiently modulates membrane-initiated estrogen actions, including eNOS activation, than full-length ER66. These findings may have important implications in vascular-specific targeting of estrogen receptor agonists.

Alternative Splicing↗

PubMed2003

Genomic instability and endoreduplication triggered by RAD17 deletion.

Cell cycle checkpoints are critical for genomic stability. Rad17, a component of the checkpoint clamp loader complex (Rad17/Rfc2-5), is required for the response to DNA damage and replication stress. To explore the role of Rad17 in the maintenance of genomic integrity, we established somatic conditional alleles of RAD17 in human cells. We find that RAD17 is not only important for the Atr-mediated checkpoint but is also essential for cell viability. Cells lacking RAD17 exhibited acute chromosomal aberrations and underwent endoreduplication at a high rate. Therefore, RAD17 links the checkpoint to ploidy control and is essential for the maintenance of chromosomal stability.

Blotting, Southern↗

PubMed2003

Isolation and characterization of a novel human NM23-H1B gene, a different transcript of NM23-H1.

The NM23 gene is a conspicuous metastasis-suppressor gene. Eight human genes of the NM23/nucleoside diphosphate kinase family have been discovered. From our large cDNA cloning and sequencing project, we cloned a different transcript ( NM23-H1B) of human NM23-H1 from 18-week-old human fetal brain. The 987-bp cDNA encodes a protein of 177 amino acid residues. Compared with NM23H1, the cDNA contained an additional NH(2)-terminal region (25 amino acid residues). It was mapped to chromosome 17q21.3 using bioinformatics analysis, which shows that the second exon does not exist in NM23-H1. The expression pattern of NM23-H1B showed that it was ubiquitously expressed in normal tissues (15 tissues except colon) at different levels. Our data also indicated that the expression of the transcript in tumors related to tumor differentiation: in poorly differentiated breast carcinoma GI-101, pancreatic adenocarcinoma GI-103, and undifferentiated ovarian carcinoma GI-102, there was no expression. In poorly differentiated lung carcinoma LX-1, lung carcinoma GI-117, the expression level was very low. The transcript band in well-differentiated colon adenocarcinoma CX-1 was significantly higher than that in poorly differentiated colon adenocarcinoma GI-112. A high transcription level was also found in grade IV prostatic adenocarcinoma PC3.

Adenocarcinoma↗

PubMed2003

Gene fusion of molecular adjuvant C3d to hCGbeta enhances the anti-hCGbeta antibody response in DNA immunization.

OBJECTIVE: To express the hCGbeta-C3d3 fusion protein in a CHO cell continual expression system to investigate further the adjuvant effects of C3d on contraceptive vaccination. METHOD: We constructed a plasmid pcDNA3-hCGbeta-C3d3 which contains three copies of murine C3d cDNA and the hCGbeta gene by cloning the chimerical hCGbeta-C3d3 cDNA into the eukaryotic vector pcDNA3 downstream of the CMV promoter. The plasmid was transfected into a COS-7 cell transient expression system and a CHO cell continual expression system. RIA was used to detect hCGbeta in the culture supernatant. Western blot and Raji cell immunohistochemical assays were performed to evaluate the expressed protein. Then, 6-8-week-old female BALB/c mice were inoculated intramuscularly with pcDNA3-hCGbeta and pcDNA3-hCGbeta-C3d3, and ELISA was used to assess anti-hCGbeta IgG antibody in serum. RESULTS: In 72 h after COS-7 cells were transfected with the plasmid pcDNA3-hCGbeta-C3d3, 1.0x10(5) cells could secrete 152 ng of the recombinant protein (calculated by hCGbeta contained). The transfected CHO cells, which were then screened by G418, could continuously secrete the fusion protein at 660 ng/10(6) cells/48 h. The hCGbeta-C3d3 protein was purified by anti-hCGbeta immunoaffinity chromatography. Raji cell immunohistochemical assay demonstrated that both the hCGbeta and C3d3 were successfully fused. After DNA immunization intramuscularly, the anti-hCGbeta IgG antibody titer in the pcDNA3-hCGbeta-C3d3 immunized group was 243-fold higher than that of the pcDNA3-hCGbeta immunized group. CONCLUSION: We have expressed the hCGbeta-C3d3 protein successfully, both in a transient expression system (COS-7 cells) and in a stable expression system (CHO cells). The C3d3 molecular adjuvant can enhance significantly the immunogenecity of hCGbeta antigen in DNA immunization.

Animals↗

PubMed2003

ENU-induced late-onset night blindness associated with rod photoreceptor cell degeneration in zebrafish.

We describe a dominant mutation, night blindness d (nbd), that causes late-onset rod photoreceptor cell degeneration in zebrafish. The mutation was induced by treating male zebrafish with N-ethyl-N-nitrosourea (ENU). Visual sensitivity was tested using a behavioral assay based on a visually mediated escape response. At a young age, the heterozygous (nbd+/-) fish did not show any signs of night blindness or retinal degeneration. At 2 years, their behaviorally assessed visual sensitivity was decreased, albeit no alterations in the electroretinogram (ERG) were detected. Histology revealed that in the mutant retinas the rod photoreceptor cell outer segments (ROS) were thinned out. In homozygous larvae (nbd-/-), mass neural degeneration was detectable at about 2 days post fertilization (dpf). They died at an early age, usually no later than 8 dpf. In conclusion, nbd is a dominant mutation that causes late-onset night blindness with slow progression. However, nbd is not photoreceptor cell-specific, as becomes clear from the systemic dysfunctions of the homozygous larvae.

Animals↗

PubMed2003

The COP9 signalosome: an alternative lid for the 26S proteasome?

Two groups have recently reported ubiquitin-deconjugating activities associated with the COP9 signalosome (CSN). Together with a previously identified deneddylation activity, CSN now appears to possess biochemical activities towards the cullin-containing ubiquitin ligases (e.g. deneddylation) and ubiquitinated protein substrates in the proteasome-mediated degradation processes (e.g. deubiquitination). Here, we discuss how these findings support a central role for CSN in ubiquitination of substrate proteins and their proteolysis

Arabidopsis↗

PubMed2003

Spatio-temporal frequency characteristics of the optomotor response in zebrafish.

The optomotor response (OMR) is a simple experimental paradigm that is widely used in the study of visual system functions. In the current paper we investigated how spatial and temporal properties of repetitive stimuli determine the OMR in zebrafish. The experiments showed that the OMR has the temporal characteristic of a low-pass filter when the spatial frequencies are low and of a band-pass filter when the spatial frequencies are high. These findings are discussed on the basis of inherent sampling constraints of any motion detector. We found some indications that the strength and direction of the OMR vary with the spatio-temporal frequency of the stimulus pattern as has previously been described for other species.

Animals↗

PubMed2003

Slow-progressing photoreceptor cell degeneration in night blindness c mutant zebrafish.

We describe here the morphological and functional alterations of the retina of mutant zebrafish, night blindness c ( nbc ). The nbc mutant was isolated from the F1 generation of N -ethyl- N -nitrosourea mutagenized founders. Visual sensitivity of wildtype and heterozygous ( nbc (+/-)) mutant fish was determined using a behavioral assay based on visually mediated escape responses. Histology, immunocytochemistry, and electroretinography were used to study structural and functional changes of the outer retina. The behavioral visual response of nbc (+/-) mutants started to deteriorate at 12 months of age. Considerable variations existed between the extents of retinal degeneration of individual fish. In the most severe cases, both rod and cone outer segments were degenerated. In moderate cases, only rod outer segments were affected. Yet in other cases, no degeneration was detected. The retina of homozygous ( nbc (-/-)) larvae had a normal appearance. However, they develop abnormally and died before 9 days post fertilization. In conclusion, nbc causes late-onset and progressive dominant retinal degeneration of both rod and cone photoreceptor cells. However, nbc is not a retina-specific gene, as the homozygous fish displayed extra-retinal defects.

Aging↗

PubMed2003

Distribution of human rotaviruses, especially G9 strains, in Japan from 1996 to 2000.

A 4-year (1996-2000) survey of rotavirus infection involving 2,218 diarrheal fecal specimens of children collected from five regions of Japan was conducted. A total of 642 (28.9%) specimens were found to be rotavirus positive. A changed prevalence pattern of rotavirus G serotype was found with an increase of G9 and G2 and a decrease of G1, although G1 remained the prevailing serotype. Serotype G9 was unexpectedly determined to be the prevailing serotype in Sapporo (62.5%) and Tokyo (52.9%) in 1998-1999, and in Saga (78.4%) in 1999-2000. G9 strains isolated from 1998-1999 belonged to the P[8]-NSP4-Wa-group with long RNA pattern, while, G9 strains isolated from 1999-2000 belonged to three groups, the P[8]-NSP4-Wa-group with long RNA pattern, the P[4]-NSP4-KUN-group with short RNA pattern and a mixed-type group (P[4]/P[8]-NSP4-KUN/Wa-group with long RNA pattern). Both sequence and immunological analysis of VP7 revealed that the G9 strains from 1999-2000 were much more closely related to the G9 strains isolated worldwide in the 1990s, including G9 strains found in Thailand in 1997. However, the G9 strains from 1998-1999 were distinct from these and more closely related to the G9 prototype strains F45, AU32 and WI61 discovered in Japan and the US in the 1980s. Thus the G9 strains isolated in 1998-1999 had progenitors common to the G9 prototype strains, while the strains isolated in 1999-2000 did not directly evolve from them but were related to global G9 strains that have emerged in recent years. These data supported our previous report that G9 rotavirus might exist as two or more subtypes with diverse RNA patterns, P-genotype and NSP4 genogroup combinations (Y.M. Zhou et al., J. Med. Virol. 65: 619-628, 2001) and suggested that G9 rotavirus prevalent in Japan during two successive years belonged to different subtypes. The nucleotide sequences presented in this paper were submitted to DDBJ, EMBL and GenBank nucleotide sequence databases. The accession numbers are: 00-Ad2863VP7 (AB091746), 00-OS2986VP7 (AB091747), 00-SG2509VP7 (AB091748), 00-SG2518VP7 (AB091749), 00-SG2541 (AB091750), 00-SG2864 (AB091751), 00-SP2737VP7 (AB091752), 99-SP1542VP7 (AB091753), 99-SP1904VP7 (AB091754), 99-TK2082VP7 (AB091755) and 99-TK2091VP7 (AB091756).

Adolescent↗

PubMed2003

Nucleotide excision repair- and polymerase eta-mediated error-prone removal of mitomycin C interstrand cross-links.

Interstrand cross-links (ICLs) make up a unique class of DNA lesions in which both strands of the double helix are covalently joined, precluding strand opening during replication and transcription. The repair of DNA ICLs has become a focus of study since ICLs are recognized as the main cytotoxic lesion inflicted by an array of alkylating compounds used in cancer treatment. As is the case for double-strand breaks, a damage-free homologous copy is essential for the removal of ICLs in an error-free manner. However, recombination-independent mechanisms may exist to remove ICLs in an error-prone fashion. We have developed an in vivo reactivation assay that can be used to examine the removal of site-specific mitomycin C-mediated ICLs in mammalian cells. We found that the removal of the ICL from the reporter substrate could take place in the absence of undamaged homologous sequences in repair-proficient cells, suggesting a cross-link repair mechanism that is independent of homologous recombination. Systematic analysis of nucleotide excision repair mutants demonstrated the involvement of transcription-coupled nucleotide excision repair and a partial requirement for the lesion bypass DNA polymerase eta encoded by the human POLH gene. From these observations, we propose the existence of a recombination-independent and mutagenic repair pathway for the removal of ICLs in mammalian cells.

Animals↗

PubMed2003

Effect of fetal hemoglobin-stimulating medicines on the interaction of DNA and protein of important erythroid regulatory elements.

Beta-Thalassemia is the most common single gene disorder in the world, which is caused by the imbalance between alpha-globin chain and beta-globin chain synthesis. Several medicines, such as 5-azacytidine, hydroxyurea, cytarabine, vinblatine, butyrate, and myleran, have been shown to be able to reactivate gamma-globin chain synthesis during the adult stage, and some of them (5-azacytidine, hydroxyurea, myleran, and butyrate) have been used clinically to treat thalassemia and sickle cell disease. Much research efforts are focusing on the determination of the underlying mechanisms of medicine action. In this experiment, as an effort to probe the underlying mechanism of medicine action, we used ligation-mediated polymerase chain reaction and in vivo footprinting methods to study the DNA-protein interaction at critical erythroid regulatory elements after hydroxyurea or myleran administration to mice. Our results showed that the patterns of in vivo footprints at both the hypersensitive site 2 of the locus control region and the beta-globin gene promoter were changed after medicine treatment. We proposed based on these results that the medicines' administration might result in a change in the interaction between trans-acting factors and cis-acting elements at these regions. These changes might influence the assembly of the transcription complex and, lastly, influence the expression of the beta-globin gene.

Anemia, Sickle Cell↗

PubMed2003

Betacellulin improves glucose metabolism by promoting conversion of intraislet precursor cells to beta-cells in streptozotocin-treated mice.

Betacellulin (BTC) induces differentiation of pancreatic beta-cells and promotes regeneration of beta-cells in experimental diabetes. The present study was conducted to determine if BTC improved glucose metabolism in severe diabetes induced by a high dose of streptozotocin (STZ) in mice. Male ICR mice were injected with 200 microg/g ip STZ, and various doses of BTC were administered daily for 14 days. The plasma glucose concentration increased to a level of >500 mg/dl in STZ-injected mice. BTC (0.2 microg/g) significantly reduced the plasma glucose concentration, but a higher concentration was ineffective. The effect of BTC was marked by day 4 but became smaller on day 6 or later. The plasma insulin concentration and the insulin content were significantly higher in mice treated with 0.1 and 0.2 microg/g BTC. BTC treatment significantly increased the number of beta-cells in each islet as well as the number of insulin-positive islets. Within islets, the numbers of 5-bromo-2-deoxyuridine/somatostatin-positive cells and pancreatic duodenal homeobox-1/somatostatin-positive cells were significantly increased by BTC. These results indicate that BTC improved hyperglycemia induced by a high dose of STZ by promoting neoformation of beta-cells, mainly from somatostatin-positive islet cells.

Animals↗