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Biomedical subjects

J Yan

Publications and source records attributed to J Yan.

At least 73 records · Page 4Linked to original sources
PubMed2001

The cross-sectional study of the health effects of occupational exposure to carbon disulfide in a Chinese viscose plant.

This article presents results of carbon disulfide exposure assessment in a Chinese viscose rayon factory and represents the cross-sectional study beginning phase of the occupational epidemiological project. The authors measured external exposure levels to carbon disulfide at a Chinese viscose rayon factory and monitored results as internal exposure indexes by testing urine samples of workers exposed to carbon disulfide levels. The rate at which carbon disulfide is metabolized by exposed subjects was demonstrated by 2-thio-thiazolidine-4-carboxylic acid (TTCA) concentrations in urine of exposed subjects, which was studied to determine best time points for internal exposure sampling. Carbon disulfide concentrations in the air of the workplace were analyzed with gas chromatography with flame photometric detector (GC-FPD); presence of TTCA in urine samples of subjects was determined using a modified high-performance liquid chromatography method. Kinetics of TTCA excretion was studied by analysis at different time points during and after exposure of workers to carbon disulfide. A total of 155 personal samples were obtained. Carbon disulfide concentration at staple viscose hall was found to be 13.72 +/- 1.12 mg/m3 in terms of geometric mean and geometric standard deviation. Carbon disulfide concentration at filament spinning hall was found to be 20.05 +/- 1.33 mg/m3. TTCA values of subjects at staple spinning hall were 1.18 +/- 0.43 and 1.07 +/- 0.38 mg/g creatinine for subjects working at filament spinning hall. The best time for TTCA sampling is at the end of the exposure-working shift.

Adult↗
PubMed2001

The mechanical study of vascular endothelial growth factor on the prevention of restenosis after angioplasty.

The mechanism of vascular endothelial growth factor (VEGF) on the prevention of restenosis after angioplasty was investigated. The cultured vascular endothelial cells (VEC) were incubated with the conditioned medium (CM) from vascular smooth muscle cells (VSMC) infected with recombinant adenoviruses containing the hVEGF165 gene. To observe the effects of VEGF on proliferation and NO, ET, 6-keto-PGF1 alpha secretion of VEC, WST-1 method, Griess method and radioimmunoassay were used respectively. The PDGF-B mRNA transcription in VECs was detected by RT-PCR. It was showed that NO, 6-keto-PGF1 alpha and OD value were markedly increased in a dose-dependent manner in the VEGF-treated groups as compared with those in the control group, while ET and PDGF-B mRNA were significantly decreased in the VEGF-treated groups (P < 0.05 or P < 0.01). Adenovirus vector mediated hVEGF165 gene could promote the proliferation of VECs and improve NO, PGI2 secretion, inhibit ET secretion and PDGF-B mRNA transcription in the VECs. The above results offered further theoretical evidence for VEGF on the prevention of restenosis after angioplasty.

6-Ketoprostaglandin F1 alpha↗
PubMed2001

Cloning and expression of the binary toxin gene from Bacillus sphaericus IAB872 in a crystal-minus Bacillus thuringiensis subsp. israelensis.

Bacillus sphaericus IAB872 has high toxicity against susceptible Culex spp. and medium larvicidal activity against binary toxin-resistant Culex spp. Sequence analysis revealed that the sequence of the binary toxin gene from IAB872 was totally identical to that of the reference strain 2362. The recombinant plasmids were introduced into an acrystalliferous B. thuringiensis strain 4Q7, and the resulting transformants produced parasporal inclusion composed of 51 kDa and 42 kDa proteins during sporulation. SDS-PAGE and Western blot further confirmed that B. thuringiensis transformants were able to express the binary toxin in a high level. Toxicity bioassays showed that they performed high toxicity to susceptible Culex spp. larvae, but no toxicity to resistant Culex larvae. It was, therefore, suggested that other unknown toxins perhaps existed in the wild strain IAB872 except the binary toxin. Their modes of action might be different from that of binary toxin, and they were possibly responsible for the activity of the wild strain IAB872 against resistant mosquito larvae.

Animals↗
PubMed2001

Creating a new color by omission of 3 end blocking step for simultaneous detection of different chromosomes in multi-PRINS technique.

In the multiple color primed in situ labeling (multi-PRINS) technique, the blocking step using ddNTPs, incorporated by a DNA polymerase, is an important procedure that blocks the free 3' end generated in the previous PRINS reaction, thus avoiding the next PRINS reaction using it as a primer to perform spurious elongation at non-desired sites. However, we found that omission of the blocking step never affected the correct identification of two chromosomes because the signals from the second PRINS reaction site always showed the pure original color. Nevertheless, taking advantage of the color mixing, we successfully used a multi-PRINS technique to create a third color using the two most common forms of labeled dUTP (biotin- and digoxigenin-labeled dUTP) and two fluorochromes (fluorescein and rhodamine) in order simultaneously to detect three chromosomes in the same cell. By arranging the labeling either in bio-dig-bio or in dig-bio-dig order in the sequential PRINS reaction, then detecting with a mixture of avidin-fluorescein/anti-dig-rhodamine or a mixture of anti-dig-fluorescein/avidin-rhodamine, the signals at the centromeres of three different chromosomes displayed perfect yellow, red and green colors, respectively. The entire procedure could be completed in less than 90 min because the blocking step was omitted. We showed that this is a practical and efficient way to carry out multiPRINS so that even more than three chromosome targets could be detected in the same cell.

Base Sequence↗
PubMed2001

Tetrasomy 8 is associated with a major cellular proliferative advantage and a poor prognosis. two cases of myeloid hematologic disorders and review of the literature.

We report two cases of acute myeloid leukemia (AML) with tetrasomy 8 detected in patients' bone marrow samples using chromosome GTG-banding, fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) techniques. Case 1 was a myelodysplastic syndrome (MDS) in transition to AML-M4 and case 2 was an AML-M2. In case 1, the tetrasomy 8 was found in 40% of metaphase cells and constituted the only chromosome abnormality. In case 2, it was accompanied by a double Ph, trisomy 18 and disomy Y and was found in 68% of metaphase cells. However, FISH and PRINS techniques revealed the coexistence of tetrasomy 8 and trisomy 8 in interphase nuclei of both cases. When the proportion of cells with tetrasomy 8 was compared between metaphases and interphase nuclei, it showed a much higher percentage of cells with tetrasomy 8 in metaphases than in interphase nuclei. Moreover, in case 2, although multi-PRINS and FISH-PRINS techniques showed other populations of interphase nuclei with different combinations of chromosome anomalies with respect to the copy numbers for chromosomes 8, 18, Y and Ph, only cells that contained either a single Ph or tetrasomy 8 plus trisomy 18, disomy Y, and double Ph could be seen in metaphases. This strongly suggests that tetrasomy 8 confers a higher proliferative advantage to cells. Our cases also show that the tetrasomy 8 is associated with a poor prognosis.

Aged↗
PubMed2001

Trisomy 8 and monosomy 7 detected in bone marrow using primed in situ labeling, fluorescence in situ hybridization, and conventional cytogenetic analyses. A study of 54 cases with hematological disorders.

Trisomy 8 and monosomy 7 are the two most frequent aneuploidies found in hematological disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, primed in situ labeling (PRINS), fluorescence in situ hybridization (FISH) and conventional cytogenetic approaches were used to investigate 54 cases of hematopoietic disorders. Of these cases, there were 22 cases of trisomy 8, 2 cases of tetrasomy 8, 14 cases of monosomy 7, and 16 cases with two copies of both chromosomes 7 and 8. PRINS was carried out in interphase nuclei of bone marrow samples using primers that can specifically detect alpha-satellite DNA sequences of chromosomes 7 and 8. In parallel, using the alpha-satellite probes for chromosomes 7 and 8, FISH was performed for all the cases. PRINS and FISH techniques showed similar specificity and sensitivity. In comparison to FISH, PRINS is more advantageous since it is a faster, easier, and more cost-effective technique. Additionally, for most of the cases, a higher proportion of aneuploidy was detected in metaphases using conventional cytogenetics, as compared to the one found in interphase nuclei identified with PRINS and FISH techniques. In other words, for these cases, the cells with trisomy 8 or monosomy 7, had a distinct proliferative advantage compared to the disomic cell population. Therefore, to better quantify the proportion of aneuploid cells in bone marrow, we recommend to investigate the frequency of aneuploidy in nuclei using PRINS, rather than studying only the dividing cells as detected by conventional cytogenetic techniques.

Adult↗
PubMed2001

Assay of ochratoxin A in wine and beer by high-pressure liquid chromatography photodiode array and gas chromatography mass selective detection.

To routinely assay the concentrations of ochratoxin A (OTA) in wines and beers, two new methods were developed and evaluated. The first utilized solid-phase extraction on a C(18) cartridge to achieve a 100-fold sample concentration followed by high-performance liquid chromatography on a C(18) column with gradient elution and quantitation at 333 nm by means of a photodiode array detector. Positive confirmation can be carried out by purity and match-factor analysis as well as peak shift following esterification with BF(3). Total run time is 28 min. The limits of detection (LOD) and quantitation (LOQ) are 0.05 and 0.10 microg/L, respectively. Recovery and imprecision ranged from 83 to 94% and from 4.0 to 8.9%, respectively. With a throughput of 35 assays per working day, this method is ideal for routine OTA analysis. It was used to survey the concentrations of OTA in 942 wines (2 of which gave values between 0.1 and 0.2 microg/L) and 107 beers (2 of which gave values between 0.05 and 0.1 microg/L). OTA was detected more frequently in red than white wines, with the highest incidence in red wines from Spain and Argentina. There was no association between OTA and country of origin or beverage type among the beers analyzed. The second method utilized gas chromatography with mass selective detection monitoring eight specific ions, preceded by extraction in dichloromethane and derivatization with bis[trimethylsilyl]trifluoroacetamide. LOD and LOQ were 0.1 and 2 microg/L, respectively; recovery and imprecision were 69-75 and 9.0-11.1%, respectively. The method is not suitable for routine quantitation but is potentially useful as a confirmatory tool for samples with OTA > or =0.1 microg/L.

Beer↗
PubMed2001

Development of a (32)P-postlabeling/HPLC method for detection of dehydroretronecine-derived DNA adducts in vivo and in vitro.

Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. Metabolism of pyrrolizidine alkaloids in vivo and in vitro generates dehydroretronecine (DHR) as a common reactive metabolite. In this study, we report the development of a (32)P-postlabeling/HPLC method for detection of (i) two DHR-3'-dGMP and four DHR-3'-dAMP adducts and (ii) a set of eight DHR-derived DNA adducts in vitro and in vivo. The approach involves (1) synthesis of DHR-3'-dGMP, DHR-3'-dAMP, and DHR-3',5'-dG-bisphosphate standards and characterization of their structures by mass and (1)H NMR spectral analyses, (2) development of optimal conditions for enzymatic DNA digestion, adduct enrichment, and (32)P-postlabeling, and (3) development of optimal HPLC conditions. Using this methodology, we have detected eight DHR-derived DNA adducts, including the two epimeric DHR-3',5'-dG-bisphosphate adducts both in vitro and in vivo.

Animals↗
PubMed2001

Metabolic activation of the tumorigenic pyrrolizidine alkaloid, riddelliine, leading to DNA adduct formation in vivo.

Riddelliine is a representative naturally occurring genotoxic pyrrolizidine alkaloid. We have studied the mechanism by which riddelliine induces hepatocellular tumors in vivo. Metabolism of riddelliine by liver microsomes of F344 female rats generated riddelliine N-oxide and dehydroretronecine (DHR) as major metabolites. Metabolism was enhanced when liver microsomes from phenobarbital-treated rats were used. Metabolism in the presence of calf thymus DNA resulted in eight DNA adducts that were identical to those obtained from the reaction of DHR with calf thymus DNA. Two of these adducts were identified as DHR-modified 7-deoxyguanosin-N(2)-yl epimers (DHR-3'-dGMP); the other six were DHR-derived DNA adducts, but their structures were not characterized. A similar DNA adduct profile was detected in the livers of female F344 rats fed riddelliine, and a dose-response relationship was obtained for the level of the total (eight) DHR-derived DNA adducts and the level of the DHR-3'-dGMP adducts. These results suggest that riddelliine induces liver tumors in rats through a genotoxic mechanism and the eight DHR-derived DNA adducts are likely to contribute to liver tumor development.

Animals↗
PubMed2001

Effect of p58GTA on beta-1,4-galactosyltransferase 1 activity and cell-cycle in human hepatocarcinoma cells.

Beta-1,4-galactosyltransferase 1 (beta1,4-GT 1) is the key enzyme transferring galactose to the terminal N-acetylglucosamine (GlcNAc) forming Galbeta3-->4GlcNAc structure in the Golgi apparatus. In addition, it also serves as a cell adhesion molecule by recognizing and binding to terminal GlcNAc of glycoconjugates on the adjacent cell surface and matrix through a subpopulation of the enzyme distributed on the cell surface. Transient expression of the p58GTA protein kinase, which belongs to the p34cdc2-related supergene family, could enhance beta1,4-GT 1 total activity in COS cells. In this study, the p58GTA interaction with beta1,4-GT 1 was confirmed using an in vitro assay with the TNT Coupled Reticulocyte Lysate System. An expression vector containing p58GTA was stably transfected into 7721 cells, a human hepatocarcinoma cell line, expression was confirmed by Northern and Western blot analyses. The cells transfected with p58GTA (p58GTA/7721) contained 1.9 times higher total beta1,4-GT 1 activity and 2.6 times higher cell-surface beta1,4-GT 1 activity than the mock transfected cells (pcDNA3/7721). However, Ricinus communis agglutinin-I lectin blot analysis revealed that the enhanced beta1,4-GT1 activity did not increase the Galbetal-->4GlcNAc groups on most of the membrane proteins in p58GTA/7721 cells. By flow cytometry analysis, it was found that the p58GTA/7721 cells were G2/M phase arrested, compared with the pcDNA3/7721 cells. These results suggest that the p58GTA stable transfection into human hepatocarcinoma cells could enhance the two beta1,4-GT1 subcellular pool activities independently and change its cell-cycle without modifying the beta-1,4-linked galactose residues on most membrane proteins.

Animals↗
PubMed2001

Quantification of all fetal nucleated cells in maternal blood between the 18th and 22nd weeks of pregnancy using molecular cytogenetic techniques.

Different types of nucleated fetal cells (trophoblasts, erythroblasts, lymphocytes, and granulocytes) have been recovered in maternal peripheral blood. In spite of many attempts to estimate the number of fetal cells in maternal circulation, there is still much controversy concerning this aspect. The numbers obtained vary widely, ranging from 1 nucleated cell per 104 to 1 per 109 nucleated maternal cells. The purpose of our project was to determine the absolute number of all different types of male fetal nucleated cells per unit volume of peripheral maternal blood. Peripheral blood samples were obtained from 12 normal pregnant women known to carry a male fetus between 18 and 22 weeks of pregnancy. Three milliliters (3 ml) of maternal blood has been processed without any enrichment procedures. Fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) were performed, and fetal XY cells were identified (among maternal XX cells) and scored by fluorescent microscopy screening. The total number of male fetal nucleated cells per milliliter of maternal blood was consistent in each woman studied and varied from 2 to 6 cells per milliliter within the group of normal pregnancies. The number of fetal cells in maternal blood, at a given period, is reproducible and can therefore be assessed by cytogenetic methods. This confirms the possibility of developing a non-invasive prenatal diagnosis test for aneuploidies. Furthermore, we demonstrate that it is possible to repeatedly identify an extremely small number of fetal cells among millions of maternal cells.

Adult↗
PubMed2001

GTP-dependent segregation of H-ras from lipid rafts is required for biological activity.

Different sites of plasma membrane attachment may underlie functional differences between isoforms of Ras. Here we show that palmitoylation and farnesylation targets H-ras to lipid rafts and caveolae, but that the interaction of H-ras with these membrane subdomains is dynamic. GTP-loading redistributes H-ras from rafts into bulk plasma membrane by a mechanism that requires the adjacent hypervariable region of H-ras. Release of H-ras-GTP from rafts is necessary for efficient activation of Raf. By contrast, K-ras is located outside rafts irrespective of bound nucleotide. Our studies identify a novel protein determinant that is required for H-ras function, and show that the GTP/GDP state of H-ras determines its lateral segregation on the plasma membrane.

Animals↗
PubMed2001

The gene encoding alsin, a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS) are neurodegenerative conditions that affect large motor neurons of the central nervous system. We have identified a familial juvenile PLS (JPLS) locus overlapping the previously identified ALS2 locus on chromosome 2q33. We report two deletion mutations in a new gene that are found both in individuals with ALS2 and those with JPLS, indicating that these conditions have a common genetic origin. The predicted sequence of the protein (alsin) may indicate a mechanism for motor-neuron degeneration, as it may include several cell-signaling motifs with known functions, including three associated with guanine-nucleotide exchange factors for GTPases (GEFs).

Amino Acid Sequence↗
PubMed2001

Molecular characterization of two serine proteases expressed in gut tissue of the African trypanosome vector, Glossina morsitans morsitans.

Serine proteases are major insect gut enzymes involved in digestion of dietary proteins, and in addition they have been implicated in the process of pathogen establishment in several vector insects. The medically important vector, tsetse fly (Diptera:Glossinidiae), is involved in the transmission of African trypanosomes, which cause devastating diseases in animals and humans. Both the male and female tsetse can transmit trypanosomes and both are strict bloodfeeders throughout all stages of their development. Here, we describe the characterization of two putative serine protease-encoding genes, Glossina serine protease-1 (Gsp1) and Glossina serine protease-2 (Gsp2) from gut tissue. Both putative cDNA products represent prepro peptides with hydrophobic signal peptide sequences associated with their 5'-end terminus. The Gsp1 cDNA encodes a putative mature protein of 245 amino acids with a molecular mass of 26 428 Da, while the predicted size of the 228 amino acid mature peptide encoded by Gsp2 cDNA is 24 573 Da. Both deduced peptides contain the Asp/His/Ser catalytic triad and the conserved residues surrounding it which are characteristic of serine proteases. In addition, both proteins have the six-conserved cysteine residues to form the three-cysteine bonds typically present in invertebrate serine proteases. Based on the presence of substrate specific residues, the Gsp1 gene encodes a chymotrypsin-like protease while Gsp2 gene encodes for a protein with trypsin-like activity. Both proteins are encoded by few loci in tsetse genome, being present in one or two copies only. The mRNA expression levels for the genes do not vary extensively throughout the digestive cycle, and high levels of mRNAs can be readily detected in the gut tissue of newly emerged flies. The levels of trypsin and chymotrypsin activities in the gut lumen increase following blood feeding and change significantly in the gut cells throughout the digestion cycle. Hence, the regulation of expression for trypsin and chymotrypsin occurs at the post-transcriptional level in tsetse. Both the coding sequences and patterns of expression of Gsp1 and Gsp2 genes are similar to the serine proteases that have been reported from the bloodfeeding insect Stomoxys calcitrans.

Amino Acid Sequence↗
PubMed2001

Ascorbic acid increases the yield of dopaminergic neurons derived from basic fibroblast growth factor expanded mesencephalic precursors.

CNS precursors derived from E12 rat mesencephalon proliferate in the presence of basic fibroblast growth factor and differentiate in vitro into functional dopaminergic neurons, which upon transplantation alleviate behavioral symptoms in a rat model of Parkinson's disease. Here we show that the efficiency of dopaminergic differentiation decreases in the mesencephalic precursors that were proliferated or passaged for extended periods in vitro. Ascorbic acid treatment restored dopaminergic differentiation in these precursors and led to a greater than 10-fold increase in dopamine neuron yield compared with untreated cultures. The effect of ascorbic acid was stereospecific and could not be mimicked by any other antioxidants. The expression of sodium-dependent vitamin C transporter, a recently identified stereospecific ascorbic acid transporter, was maintained in mesencephalic precursors for extended in vitro periods. Pre-treatment of in vitro expanded mesencephalic precursors with ascorbic acid might facilitate the large-scale generation of dopaminergic neurons for clinical transplantation.

Animals↗
PubMed2001

A dinucleotide deletion in amyloid precursor protein (APP) mRNA associated with sporadic Alzheimer's disease results in efficient secretion of truncated APP isoforms from neuroblastoma cell cultures.

Recently, two dinucleotide deletions were detected in the mRNA of the amyloid precursor protein (APP) from cerebral cortex neurons of patients with sporadic Alzheimer's disease (AD) or Down's syndrome. These deletions resulted in truncation of APP, producing an APP isoform with a 38-kDa N-terminus and a novel carboxyl terminus (APP+1). We investigated the subcellular localization and the processing of APP+1 in the neuroblastoma cell line B103. cDNA constructs were generated encoding fusion proteins of APP+1 or full-length APP with the enhanced green fluorescent protein (eGFP). In transient transfection experiments using B103 cells, the APP+1-eGFP fusion protein showed a reticular localization with intense staining in the Golgi complex. Unlike full-length APP fused to eGFP, the APP+1-eGFP fusion protein did not localize to the perinuclear area or to the plasma membrane. Western blot analysis of cell extracts confirmed the translation of the expected fusion proteins. Analysis of the supernatant by western blot indicated that the APP+1-eGFP fusion protein was efficiently secreted from B103 cells, whereas the secreted form of full-length APP fusion protein (APPs) was hardly detectable. Thus, both dinucleotide deletions in the APP mRNA result in truncated APP+1 that is not membrane associated and is readily secreted from neurons.

Alzheimer Disease↗
PubMed2001

Production of gamma-linolenic acid by Mortierella isabellina grown on hexadecanol.

AIMS: To optimize the production of linolenic acid by Mortierella isabellina grown on hexadecanol. METHODS AND RESULTS: Effects of culture conditions such as culture time, pH of medium, hexadecanol concentration, incubation temperature and ageing of mycelia on production of linolenic acid were studied. The production of gamma-linolenic acid reached 2.44 mg ml-1 (271 mg g-1 dry cells) when Mortierella isabellina was cultivated in a medium consisting of 2% hexadecanol and 1% yeast extract at 23 degrees C for 120 h and then the mycelia, after removal of medium by suction filtration, were allowed to stand for a further 15 d at 5 degrees C. CONCLUSION: Ageing of mycelia and incubation temperature showed predominant effects on the increased linolenic acid production. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights effective conditions for increasing linolenic acid production by Mortierella isabellina grown on hexadecanol.

Culture Media↗