Biomedical subjects

Lei Li

Publications and source records attributed to Lei Li.

At least 127 records · Page 7Linked to original sources

PubMed2005

Mapping neutral microclimate pH in PLGA microspheres.

A quantitative ratiometric method based on laser scanning confocal microscopic imaging was developed to create a pixel-by-pixel neutral microclimate pH (5.8-8.0) map inside poly(lactic-co-glycolic acid) (PLGA) microspheres. The method was accurate to within +/-0.2 pH units. PLGA microspheres with encapsulated bovine serum albumin and the pH-sensitive dye, SNARF-1(R) dextran (MW=10 kDa), were prepared by a water-in-oil-in-water solvent evaporation method. The imaging method was used to monitor the pH changes in PLGA microspheres with or without co-encapsulation of acid-neutralizing MgCO3, and for three different microsphere sizes (2.0+/-0.3, 40+/-10 and 137+/-32 microm) during incubation under physiological conditions. The pH of microspheres was significantly higher with co-encapsulated base compared to base-free microspheres, whose microclimate pH was predominantly below the dye detection limit of pH=5.8. The small microspheres displayed a more neutral pH during incubation probably due to their much shorter diffusion path for polymer-dissolved acids compared with medium- and large-size microspheres. The pH mapping technique discussed here may be useful in routine formulation development and for elucidation of mechanisms of microclimate pH development and polymer erosion.

Drug Delivery Systems↗

PubMed2005

Recovery of cDNAs encoding ribosomal proteins S9 and L26 from Aedes albopictus mosquito cells and identification of their homologs in the malaria vector, Anopheles gambiae.

We used PCR-based approaches to obtain the full-length cDNA sequences encoding ribosomal protein (Rp) S9 and L26 from a mosquito (Aedes albopictus) C7-10 cell line. The deduced mosquito RpS9 protein has a mass of 22,826 Da and a pI of 11.41, while RpL26 had a mass of 17,442 Da and a pI of 11.52. Both cDNAs initiated with the 5'-polypyrimidine motif characteristic of ribosomal protein transcripts. Using the Aedes protein and nucleic acid sequences, we identified rpS9 and rpL26 as single copy genes in the Anopheles gambiae genome. In An. gambiae, the RpS9 coding region was distributed over 3 exons, spanning 2.6 kb, but the Anopheles rpL26 protein coding region lacked introns. The Aedes and Anopheles RpS9 and RpL26 proteins shared 96 and 92% identity, respectively. Despite low numbers of parsimony-informative amino acid substitutions, phylogenies based on the ribosomal protein sequences accurately group the Aedes and Anopheles proteins with high bootstrap values.

Aedes↗

PubMed2005

Transgenic zebrafish that express tyrosine hydroxylase promoter in inner retinal cells.

We have generated a transgenic zebrafish line [Tg(Th:GFP)] that expresses green fluorescence proteins (GFP) driven by rat tyrosine hydroxylase (TH) promoter. In zebrafish, the transgene was expressed as early as 16 hr postfertilization (hpf). The first transgene expression was detected in the midbrain. Within a few hours of development, the expression spread to the forebrain and hindbrain. In the retina, the first transgene expression was detected at approximately 40 hpf, at which time a single GFP-positive cell was seen in the ventral-nasal patch of the retina. In late development, GFP spread across the inner retina. GFP was found in retinal cells that expressed TH or phenylethanolamine N-methyl-transferase (PNMT), the first and last enzymes for synthesis of catecholamine, respectively. This suggests that the transgene is expressed in catecholaminergic neurons. Of interest, GFP was also detected in some retinal cells that release gamma-aminobutyric acid. These latter data suggest that the transgene may also be expressed in noncatecholaminergic cells.

Animals↗

PubMed2005

A pilot study of transcription unit analysis in rice using oligonucleotide tiling-path microarray.

As the international efforts to sequence the rice genome are completed, an immediate challenge and opportunity is to comprehensively and accurately define all transcription units in the rice genome. Here we describe a strategy of using high-density oligonucleotide tiling-path microarrays to map transcription of the japonica rice genome. In a pilot experiment to test this approach, one array representing the reverse strand of the last 11.2 Mb sequence of chromosome 10 was analyzed in detail based on a mathematical model developed in this study. Analysis of the array data detected 77% of the reference gene models in a mixture of four RNA populations. Moreover, significant transcriptional activities were found in many of the previously annotated intergenic regions. These preliminary results demonstrate the utility of genome tiling microarrays in evaluating annotated rice gene models and in identifying novel transcription units that will facilitate rice genome annotation.

Carbocyanines↗

PubMed2005

In vitro differentiation of mouse bone marrow mononuclear cells into hepatocyte-like cells.

AIM: : It is imperative to explore some ways to gain the functional hepatocytes for hepatocyte transplantation. Bone marrow stem cells can differentiate into hepatocytes in vivo and in vitro. We select fibroblast growth factor-4 (FGF-4), oncostatin M (OSM), hepatocyte growth factor (HGF) and epithermal growth factor (EGF) as differentiation factors, and design the appropriate directed differentiation medium in order to gain hepatocytes through directed differentiation of bone marrow stem cells. METHODS: : Bone marrow mononuclear cells (BMMCs) were cultured in the directed differentiation media including FGF-4, OSM, HGF and EGF. In the course of cell differentiation, cell morphology was observed, and the expression patterns of some genes of the hepatocyte were validated and confirmed by RT-PCR. The ALB-, and CK18-expressed cells were gone further step to be confirmed by Western blot analysis, immunofluorescence and flow cytometric analysis. Hepatocyte functional activity, including glycogen synthesis and urea production, were confirmed by periodic acid-Shiff (PAS) staining and urea assay. RESULTS: : Some epithelial-like cells or polygonal cells appeared in the directed differentiation medium within 12 days, and the number and sizes of colonies of epithelial-like cells or polygonal cells increased in the course of the cell directed differentiation. AFP, HNF-3ss, ALB and CK18 mRNA expressions first appeared within day 7, and lasted throughout the later directed differentiation. TTR, G-6-P and TAT mRNA expressions could be detected within day 14, and their expressions lasted in the course of the later directed differentiation. ALB and CK18 were confirmed to exist in the differentiated BMMCs by Western blot analysis. ALB was found in the cytoplasm and cell membrane, while CK18 scattered in the cytoplasm by immunofluorescent staining. On day 21,the ratio of ALB-positive cells was 69.45%, and the ratio of CK18-positive cells was 67.36%. The accumulation of glucogen was detected in the cytoplasm of the differentiated cells. The directed differentiated BMMCs produced urea 3 days later, and they produced urea in a time-dependent manner. CONCLUSIONS: : BMMCs could differentiate into hepatocytes or hepatocyte-like cells in the differentiation media including HGF, FGF-4, EGF, and OSM. These hepatocyte-like cells were identified at the gene level and protein level. Furthermore, these hepatocyte-like cells had some hepatocellular synthesis and metabolism functions.

Journal Article↗

PubMed2005

Analysis of gene expression profile in yeast aging chronologically.

The use of simple model systems such as Saccharomyces cerevisiae and Caenorhabditis elegans has played a primary role in the identification of proteins and pathways that regulate the aging process in eukaryotes. Recent findings have shown that analogous pathways regulate aging in higher eukaryotes and suggest a conserved origin for the molecular mechanisms that regulate stress-resistance and longevity. Genomics approaches that allow the simultaneous monitoring of the expression of thousands of genes are beginning to reveal the complexity of the molecular changes required to extend life span. Here we describe how analysis of the gene expression profiles of wild-type and long-lived yeast aging chronologically can be used to identify proteins that increase stress-resistance and longevity. We also discuss a novel genomics method for the identification of chronologically long-lived yeast mutants.

Gene Expression Regulation, Fungal↗

PubMed2005

Behavioral screening for nightblindness mutants in zebrafish reveals three new loci that cause dominant photoreceptor cell degeneration.

Here we report three dominant nightblindness mutations in zebrafish: nightblindness e (nbe), nightblindness f (nbf) and nightblindness g (nbg). The mutants were isolated in the F1 generation of N-ethyl-N-nitrosourea (ENU) mutagenized zebrafish using a behavioral assay based on visually mediated escape responses. Subsequently, electroretinographic (ERG) recordings were made, and histological sections were screened for degenerative processes. For each mutant line, correlation analysis between behavioral, ERG and histological parameters was performed, and their relationships were determined by either calculating the Pearson correlation coefficient or by ANOVA. nbe is characterized by severe rod outer segments (ROS) degeneration. The degeneration correlates weakly with behavioral threshold and ERG b-wave amplitude, however, behavioral threshold correlates strongly with ERG b-wave. nbf is characterized by a dual histological pathology: patchy ROS-degeneration and 'gaps' homogeneously distributed over the outer nuclei layer (ONL) and between cone outer segments (COS). The correlations between histological pathology and behavioral threshold, and between behavioral threshold and ERG b-wave amplitude are obvious, but the correlation between histology and b-wave amplitude is less prominent. nbg is characterized by moderate ROS degeneration and moderate correlation between histology and behavioral threshold. Interestingly, behavioral threshold correlated inversely with ERG b-wave amplitude and threshold. Thus, contrary to what is normally seen in other nightblindness mutants, in nbg, the fish with the lowest behavioral threshold had the smallest b-waves amplitudes and the highest b-wave threshold. In our interpretation, the major impairment in nbe is photoreceptor-specific. In nbf, both photoreceptor degeneration and altered post-photoreceptor signaling are responsible for the behavioral deficit. In nbg, we find hypersensitivity at a post-photoreceptoral level concurrently with behavioral impairment.

Animals↗

PubMed2005

High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris.

Penaeidins, members of a new family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp, display antimicrobial activity against bacteria, and fungi. Here, a DNA sequence encoding the mature Ch-penaeidin peptide was cloned into the pPIC9K vector and transformed into Pichia pastoris. The transformed cells were screened for multi-copy plasmids using increasing concentrations of G418. Positive colonies carrying chromosomal integrations of the Chp gene were identified by phenotype and PCR. When transformed cells were induced with methanol, SDS-PAGE and Western blotting revealed the production of a approximately 6100 Da recombinant CHP (rCHP) expression product. Large scale expression revealed that rCHP was produced at 108 mg/L under optimal conditions in the highest Chp-producing P. pastoris clone. The antimicrobial activities of rCHP were studied by liquid phase analysis, which revealed that rCHP exhibited activities against some Gram-negative and Gram-positive bacteria, but had a relatively low activity against some fungi. Purification of rCHP by cation exchange chromatography and subsequent automated amino acid sequencing revealed the presence of four additional amino acids (YVEF) at the N-terminus that belonged to the cleaved fusion signal peptide; these residues may account for the observed decrease in antifungal activity. Together, these observations indicate that rCHP is an effective antimicrobial peptide that can be successfully produced at high levels in the yeast, and therefore may be a potential antimicrobial candidate for practical use.

Amino Acid Sequence↗

PubMed2005

Prokaryotic expression and polyclonal antibody preparation of novel ZLG10 protein involved in infection of RSV on SPC-A1 cells.

Differentially expressed genes between normal SPC-A1 cells and SPC-A1 cells infected by RSV were investigated using differential display. The novel zlg10 gene codes for a novel protein, ZLG10, which has previously been reported to be up-regulated in RSV-infected SPC-A1 cells. Its putative open reading frame was also identified. To better understand the structure, function, and possible role of ZLG10 as a potential candidate for diagnosis and vaccine studies, the intact region encoding ZLG10 was obtained by PCR and expressed in Escherichia coli as a GST-fusion protein. After purification, GST-ZLG10 fusion protein was used to immunize the adult rabbits following standard protocols. Consequently, we found that the produced antiserum of the novel fusion protein significantly suppressed the infection by RSV on SPC-A1 cells by using neutral red uptake assay and quantitative measurement. Together, our data demonstrate that ZLG10, a novel protein expressed and purified in this report, might be a potential effective therapeutic candidate for treating RSV infections.

Amino Acid Sequence↗

PubMed2005

It runs in the family: regulation of brassinosteroid signaling by the BZR1-BES1 class of transcription factors.

Research of the molecular events underlying brassinosteroid (BR) signaling is emerging as a new paradigm for studying plant hormone signal transduction pathways. Two recent reports have shown that the BZR1-BES1 family of proteins is a class of novel plant-specific transcription factors that directly bind to and regulate BR-responsive genes, thus filling in key details of the link between hormonal signal transmission in the cytoplasm and transcriptional status change in the nucleus.

Arabidopsis↗

PubMed2005

Treatment of praziquantel wastewater using the integrated process of coagulation and gas membrane absorption.

Praziquantel is an anti-schistosoma drug, its wastewater contains numerous cyanide and other colloid pollutant. A novel integrated treatment process was proposed in this study, i.e., using coagulation to eliminate colloid pollutant and using gas membrane absorption (GMA) to remove cyanide. The optimization of coagulation condition, determination of cyanide removal rate, elimination of membrane fouling, and overall evaluation of the integrated process for the treatment of praziquantel wastewater were investigated in this study. Good results were achieved: turbidity reduced from 700 NTU to 10-40 NTU, 92% COD was removed, cyanide concentration dropped from 3500 mg/L to below 0.5 mg/L, recovery rate of cyanide reached to 98%, and operation cost could be entirely compensated from the gain of recovered cyanide. The results demonstrate that the novel integrated process offers a number of advantages over alkaline chlorination method in the treatment of praziquantel wastewater: cyanide can be reused rather than destroyed, no secondary pollutants are produced, the operation cost is low and the equipment etching is avoided.

Absorption↗

PubMed2005

Evolutionary expansion, gene structure, and expression of the rice wall-associated kinase gene family.

The wall-associated kinase (WAK) gene family, one of the receptor-like kinase (RLK) gene families in plants, plays important roles in cell expansion, pathogen resistance, and heavy-metal stress tolerance in Arabidopsis (Arabidopsis thaliana). Through a reiterative database search and manual reannotation, we identified 125 OsWAK gene family members from rice (Oryza sativa) japonica cv Nipponbare; 37 (approximately 30%) OsWAKs were corrected/reannotated from earlier automated annotations. Of the 125 OsWAKs, 67 are receptor-like kinases, 28 receptor-like cytoplasmic kinases, 13 receptor-like proteins, 12 short genes, and five pseudogenes. The two-intron gene structure of the Arabidopsis WAK/WAK-Likes is generally conserved in OsWAKs; however, extra/missed introns were observed in some OsWAKs either in extracellular regions or in protein kinase domains. In addition to the 38 OsWAKs with full-length cDNA sequences and the 11 with rice expressed sequence tag sequences, gene expression analyses, using tiling-microarray analysis of the 20 OsWAKs on chromosome 10 and reverse transcription-PCR analysis for five OsWAKs, indicate that the majority of identified OsWAKs are likely expressed in rice. Phylogenetic analyses of OsWAKs, Arabidopsis WAK/WAK-Likes, and barley (Hordeum vulgare) HvWAKs show that the OsWAK gene family expanded in the rice genome due to lineage-specific expansion of the family in monocots. Localized gene duplications appear to be the primary genetic event in OsWAK gene family expansion and the 125 OsWAKs, present on all 12 chromosomes, are mostly clustered.

Arabidopsis↗

PubMed2005

Molecular epidemiology of adenovirus infection among pediatric population with diarrhea in Asia.

A total of 3,577 fecal specimens from infants and children with acute gastroenteritis in Japan, Korea and Vietnam during 1998 and 2001 were tested for adenovirus by the ELISA method. Of these, adenovirus was detected in 158 (4.4%). The detection rate of adenovirus was highest in Korea (8.7%, 20/231) followed by 5.0% (100/1,991) in Japan and 2.8% (38/1,355) in Vietnam. All adenoviruses were further serotyped by PCR-RFLP. The diversity of adenovirus serotypes including Ad2, 3, 5, 8, 31, 40 and 41 was demonstrated. Worth of note was a decrease in the rate of isolation of Ad40 (7.6%, 12/158) and a concomitant increase of Ad41 (63.9%, 101/158) to become the predominant serotype. Another interesting feature of the study was the presence of Ad3 (4.0% and 30%) and Ad8 (8.0% and 20%) in Japan and Korea, respectively, which is mainly associated with keratoconjunctivitis worldwide. Our result underscored the importance of adenovirus in association with acute gastroenteritis in Asian countries.

Adenoviridae↗

PubMed2005

Preoperative diagnosis of gastric cancer using 2-D magnetic resonance imaging with 3-D reconstruction techniques.

OBJECTIVE: To investigate the clinical value of 2-D magnetic resonance imaging (MRI) with 3-D reconstruction techniques for the preoperative diagnosis and TNM-staging of gastric cancer. METHODS: Using a Philips Gyroscan NT 1.0T superconductive unit, MRI using the water-filling method was performed in 15 patients with suspected gastric cancers. The 2-D MRI sequences included TSE-T1WI, TSE-T2WI and fat suppression (SPIR). The source images of magnetic resonance hydrography (heavily TSE-T2WI sequence) were reconstructed using the Philips EasyVision viewing workstation. Four 3-D postprocessing algorithms, including maximum intensity projection, surface shaded viewing, volume rendering and virtual endoscopy, were performed and compared with the results of a barium study and endoscopy. All 15 patients with 16 gastric cancers had their diagnosis confirmed by postoperative pathological findings. RESULTS: 2-D MRI and 3-D reconstruction images were successfully obtained for all 15 patients. The maximum intensity projection, surface shaded viewing, and volume rendering images corresponded to the upper gastrointestinal series findings, and the virtual endoscopy images corresponded to the gastroscopic views. In 16 gastric lesions, MRI correctly diagnosed 14 (87.5%) advanced gastric cancers, and the tumor location, size and classification were also accurately identified. The accuracy of MRI for determining the preoperative TNM stage was 64.3% (9/14), and there was significant correlation between these results and those from the histopathological studies (P < 0.01). Based on T, N and M factors, the staging accuracy of MRI was 71.4% (10/14), 57.1% (8/14) and 85.7% (12/14), respectively. CONCLUSIONS: 2-D MRI with 3-D reconstruction is an effective method for the preoperative diagnosis and TNM staging of gastric cancer. However, the detection of early cancers or benign lesions and N-staging should be further studied.

Aged↗

PubMed2005

Transfection of promyelocytic leukemia in retrovirus vector inhibits growth of human bladder cancer cells.

AIM: To construct a recombinant retrovirus vector carrying human promyelocytic leukemia (PML) cDNA and identify its expression and biology role in bladder cancer UM-UC-2 cells for future gene therapy. METHODS: PML full-length cDNA was inserted into the EcoR I and BamH I site of pLXSN vector containing the long terminal repeat (LTR) promoter. The vector was identified by restriction enzyme digestion and then transfected into PA317 packaging cell line by calcium phosphate coprecipitation. PML cDNA was detected by polymerase chain reaction (PCR) and the protein was identified by laser confocal microscopy and Western blot in bladder cancer cells, respectively. The morphology was observed by inverted phase contrast microscope, and MTT assay determined growth curve of the bladder cancer cells. RESULTS: Restriction enzyme digestion proved that a 2.1 kb PML cDNA was inserted into the pLXSN vector. PCR assay demonstrated that 304 bp fragments were found in UM-UC-2/pLPMLSN transfects. Laser confocal microscopy showed speck dots fluorescence in the UM-UC-2/pLPMLSN nucleus. A 90 kD specific brand was found by Western blot. MTT assay demonstrated the UM-UC-2/pLPMLSN bladder cancer growth inhibition. CONCLUSION: The retrovirus pLPMLSN vector was successfully constructed and could generate high effective expression of human PML in bladder cancer cell UM-UC-2, suggesting that PML recombinant retrovirus have potential utility in the gene therapy for bladder cancer.

Cell Line, Tumor↗

PubMed2005

Hepatocyte-like cells from directed differentiation of mouse bone marrow cells in vitro.

AIM: To design the effective directed differentiation medium to differentiate bone marrow cells into hepatocyte-like cells. METHODS: Bone marrow cells were cultured in the directed differentiation media including fibroblast growth factor-4 (FGF-4) and oncostatin M (OSM). Hepatocyte-like cells from directed differentiation of bone marrow cells were identified through cell morphology, RNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR), protein expressions by Western blot, and hepatocellular synthesis and metabolism functions by albumin ELISA, Periodic acid-Shiff staining and urea assay. RESULTS: Some epithelial-like cells or polygonal cells appeared and increased in the course of the cell directed differentiation. Hepatocyte nucleur factor-3beta (HNF-3beta, albumin (ALB), cytokeratin 18 (CK18), transthyretin (TTR), glucose-6-phosphate (G-6-Pase), and tyrosine aminotransferase (TAT) mRNA were expressed in the course of the directed differentiation. The directed differentiated cells on d 21 expressed HNF-3? ALB, and CK18 proteins. The directed differentiated cells produced albumin and synthesized urea in a time-dependent manner. They could also synthesize glycogen. CONCLUSION: Our differentiation media, including FGF-4 and OSM, are effective to differentiate bone marrow cells into hepatocyte-like cells, which could be used for hepatocyte resources for bioartificial liver or hepatocyte transplantation.

Albumins↗

PubMed2005

Gene cluster responsible for validamycin biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008.

A gene cluster responsible for the biosynthesis of validamycin, an aminocyclitol antibiotic widely used as a control agent for sheath blight disease of rice plants, was identified from Streptomyces hygroscopicus subsp. jinggangensis 5008 using heterologous probe acbC, a gene involved in the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone of the acarbose biosynthetic gene cluster originated from Actinoplanes sp. strain SE50/110. Deletion of a 30-kb DNA fragment from this cluster in the chromosome resulted in loss of validamycin production, confirming a direct involvement of the gene cluster in the biosynthesis of this important plant protectant. A sequenced 6-kb fragment contained valA (an acbC homologue encoding a putative cyclase) as well as two additional complete open reading frames (valB and valC, encoding a putative adenyltransferase and a kinase, respectively), which are organized as an operon. The function of ValA was genetically demonstrated to be essential for validamycin production and biochemically shown to be responsible specifically for the cyclization of D-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone in vitro using the ValA protein heterologously overexpressed in E. coli. The information obtained should pave the way for further detailed analysis of the complete biosynthetic pathway, which would lead to a complete understanding of validamycin biosynthesis.

Amino Acid Sequence↗

PubMed2005

Identification in the ancient protist Giardia lamblia of two eukaryotic translation initiation factor 4E homologues with distinctive functions.

Eukaryotic translation initiation factor 4E (eIF4E) binds to the m(7)GTP of capped mRNAs and is an essential component of the translational machinery that recruits the 40S small ribosomal subunit. We describe here the identification and characterization of two eIF4E homologues in an ancient protist, Giardia lamblia. Using m(7)GTP-Sepharose affinity column chromatography, a specific binding protein was isolated and identified as Giardia eIF4E2. The other homologue, Giardia eIF4E1, bound only to the m(2,2,7)GpppN structure. Although neither homologue can rescue the function of yeast eIF4E, a knockdown of eIF4E2 mRNA in Giardia by a virus-based antisense ribozyme decreased translation, which was shown to use m(7)GpppN-capped mRNA as a template. Thus, eIF4E2 is likely the cap-binding protein in a translation initiation complex. The same knockdown approach indicated that eIF4E1 is not required for translation in Giardia. Immunofluorescence assays showed wide distribution of both homologues in the cytoplasm. But eIF4E1 was also found concentrated and colocalized with the m(2,2,7)GpppN cap, 16S-like rRNA, and fibrillarin in the nucleolus-like structure in the nucleus. eIF4E1 depletion from Giardia did not affect mRNA splicing, but the protein was bound to Giardia small nuclear RNAs D and H known to have an m(2,2,7)GpppN cap, thus suggesting a novel function not yet observed among other eIF4Es in eukaryotes.

Amino Acid Sequence↗