West Nile virus strains differ in mouse neurovirulence and binding to mouse or human brain membrane receptor preparations.
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Animals↗
Biomedical subjects
L Li
Publications and source records attributed to L Li.
[Evaluation of immunochromatography based rapid detection kit of rotavirus and adenovirus].
We investigated the usefulness of Rapidtesta Rota-Adeno (Daiichi Pure Chemicals Co., Ltd., Japan) for rapid detection of group A rotavirus and adenovirus simultaneously using immunochromatography with clinical samples. In investigation of the reaction of the kit to 5 strains of group A rotavirus, 13 strains of adenovirus and other intestinal viruses, specific lines were formed in red to group A rotavirus and blue to adenovirus. No cross reaction was observed with other intestinal virus. In measurement of detection limit, group A rotavirus (SA11) was detected at 10(4.4)TCID50/ml, serotype 3 adenovirus at 10(4.45)TCID50/ml. The detection limit of the kit was similar to other immunochromatographic assay or enzyme immnoassay kits and approximately 10 times higher than that of kits using latex agglutination test. In comparison with other testing kits in clinical samples, the concordance with other immunochromatographic assay was 99.2% (121/122) in group A rotavirus and 98.6% (138/140) in adenovirus. The rate of concordance with latex agglutination test kit was 94.5% (69/73) in group A rotavirus and 92.3% (84/91) in adenovirus. The kit had high rates of concordance with other immunochromatographic assay kits and higher virus detection rates than those of latex agglutination test kits. Rapidtesta Rota-Adeno is able to detect group A rotavirus and adenovirus simply and rapidly. In addition, the two kinds of virus can be easily differentiated by color difference in reaction lines, suggesting that the kit is useful in clinical diagnosis.
Adenoviridae↗
False-positive reduction in CAD mass detection using a competitive classification strategy.
High false-positive (FP) rate remains to be one of the major problems to be solved in CAD study because too many false-positively cued signals will potentially degrade the performance of detecting true-positive regions and increase the call-back rate in CAD environment. In this paper, we proposed a novel classification method for FP reduction, where the conventional "hard" decision classifier is cascaded with a "soft" decision classification with the objective to reduce false-positives in the cases with multiple FPs retained after the "hard" decision classification. The "soft" classification takes a competitive classification strategy in which only the "best" ones are selected from the pre-classified suspicious regions as the true mass in each case. A neural network structure is designed to implement the proposed competitive classification. Comparative studies of FP reduction on a database of 79 images by a "hard" decision classification and a combined "hard"-"soft" classification method demonstrated the efficiency of the proposed classification strategy. For example, for the high FP sub-database which has only 31.7% of total images but accounts for 63.5% of whole FPs generated in single "hard" classification, the FPs can be reduced for 56% (from 8.36 to 3.72 per image) by using the proposed method at the cost of 1% TP loss (from 69% to 68%) in whole database, while it can only be reduced for 27% (from 8.36 to 6.08 per image) by simply increasing the threshold of "hard" classifier with a cost of TP loss as high as 14% (from 69% to 55%). On the average in whole database, the FP reduction by hybrid "hard"-"soft" classification is 1.58 per image as compared to 1.11 by "hard" classification at the TP costs described above. Because the cases with high dense tissue are of higher risk of cancer incidence and false-negative detection in mammogram screening, and usually generate more FPs in CAD detection, the method proposed in this paper will be very helpful in improving the performance of early detection of breast cancer with CAD.
Biophysical Phenomena↗
Mercapturic acids (N-acetylcysteine S-conjugates) as endogenous substrates for the renal organic anion transporter-1.
Mercapturic acids are N-acetyl-L-cysteine S-conjugates that are formed from a range of endogenous and exogenous chemicals. Although the kidney is a major site for elimination of mercapturic acids, the transport mechanisms involved have not been identified. The present study examined whether mercapturic acids are substrates for the renal basolateral organic anion transporter-1 (Oat1) from rat kidney. This carrier mediates uptake of organic anions from the bloodstream in exchange for intracellular alpha-ketoglutarate. Uptake of [(3)H]p-aminohippuric acid (PAH) in Oat1-expressing Xenopus laevis oocytes was strongly inhibited by S-(2,4-dinitrophenyl)-N-acetyl-L-cysteine (DNP-NAC) and by all other mercapturic acids tested, including the endogenous mercapturic acid N-acetyl-leukotriene E(4). Inhibition by the mercapturic acids was competitive, which is consistent with the hypothesis that these compounds are substrates for Oat1. This conclusion was supported by the direct demonstration of saturable [(35)S]DNP-NAC uptake in Oat1-expressing oocytes. [(35)S]DNP-NAC uptake was inhibited by PAH and other mercapturic acids and was stimulated in oocytes preloaded with glutarate. The apparent K(m) value for DNP-NAC uptake was only 2 microM, indicating that this mercapturic acid is a high affinity substrate for Oat1. Together, these data indicate that clearance of endogenous mercapturic acids is an important function of the renal organic anion transporter.
Acetylcysteine↗
Activation of interleukin-1 receptor-associated kinase by gram-negative flagellin.
Flagellin from various species of gram-negative bacteria activates monocytes to produce proinflammatory cytokines. We have analyzed the pathway by which Salmonella enteritidis flagellin (FliC) activates murine and human monocyte/macrophage-like cell lines. Since lipopolysaccharide (LPS), the principal immune stimulatory component of gram-negative bacteria, is known to signal through Toll-like receptor 4 (TLR4), we tested the possibility that FliC also signals via TLR4. When murine HeNC2 cells were stimulated with LPS in the presence of a neutralizing anti-TLR4 monoclonal antibody, tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) production were markedly reduced. In contrast, FliC-mediated TNF-alpha and NO production were minimally affected by the anti-TLR4 antibody. Furthermore, FliC, unlike LPS, stimulated TNF-alpha production in the TLR4 mutant cell line, GG2EE, indicating that TLR4 is not essential for FliC-mediated signaling. To test the possibility that FliC signals via another TLR, we measured FliC-mediated activation of interleukin-1 (IL-1) receptor-associated kinase (IRAK), a central component in IL-1R/TLR signaling. FliC induced IRAK activation in HeNC2 and GG2EE cells as well as in the human promonocytic cell line THP-1. IRAK activation was rapid in HeNC2 cells, with maximal activity observed after 5 min of treatment with FliC. In addition, FliC-mediated IRAK activation exhibited the same concentration dependence as was demonstrated for the induction of TNF-alpha. These results represent the first demonstration of IRAK activation by a purified bacterial protein and strongly suggest that a TLR distinct from TLR4 is involved in the macrophage inflammatory response to FliC.
Animals↗
In vitro culture, ultrastructure, antigenic, and molecular characterization of Encephalitozoon cuniculi isolated from urine and sputum samples from a Spanish patient with AIDS.
In this report we describe the cultivation of two isolates of microsporidia, one from urine and the other from sputum samples from a Spanish AIDS patient. We identified them as Encephalitozoon cuniculi, type strain III (the dog genotype), based on ultrastructure, antigenic characteristics, PCR, and the sequence of the ribosomal DNA internal transcribed spacer region.
AIDS-Related Opportunistic Infections↗
Genotyping Encephalitozoon hellem isolates by analysis of the polar tube protein gene.
To develop an alternative genotyping tool, the genetic diversity of Encephalitozoon hellem was examined at the polar tube protein (PTP) locus. Nucleotide sequence analysis of the PTP gene divided 24 E. hellem isolates into four genotypes, compared to two genotypes identified by analysis of the internal transcribed spacer of the rRNA gene. The four PTP genotypes differed from each other by the copy number of the 60-bp central repeat as well as by point mutations. A simple PCR test was developed to differentiate E. hellem genotypes based on the difference in the size of PTP PCR products, which should facilitate the genotyping of E. hellem in clinical samples.
Animals↗
Genotyping Encephalitozoon cuniculi by multilocus analyses of genes with repetitive sequences.
Encephalitozoon cuniculi infects a wide range of mammalian hosts. Three genotypes based on the number of GTTT repeats in the internal transcribed spacer (ITS) of the rRNA have been described, of which genotypes I and III have been identified in humans. In this study, the genetic diversity of E. cuniculi was examined at the polar tube protein (PTP) and spore wall protein I (SWP-1) loci. Nucleotide sequence analysis of the PTP gene divided 11 E. cuniculi isolates into three genotypes in congruence with the result of analysis of the ITS of the rRNA gene. The three PTP genotypes differed from one another by the copy number of the 78-bp central repeat as well as point mutations. These E. cuniculi isolates also differed from one another in the number of 15- and 36-bp repeats in the SWP-1 gene. In addition, some E. cuniculi isolates had heterogeneous copies of the SWP-1 gene with various numbers of repeats. Intragenotypic variation was also observed at the SWP-1 locus. Based on the length polymorphism and sequence diversities of the PTP and SWP-1 genes, two simple PCR tests were developed to differentiate E. cuniculi in clinical samples.
Animals↗
Phylogenetic and evolutionary relationships among yellow fever virus isolates in Africa.
Previous studies with a limited number of strains have indicated that there are two genotypes of yellow fever (YF) virus in Africa, one in west Africa and the other in east and central Africa. We have examined the prM/M and a portion of the E protein for a panel of 38 wild strains of YF virus from Africa representing different countries and times of isolation. Examination of the strains revealed a more complex genetic relationship than previously reported. Overall, nucleotide substitutions varied from 0 to 25.8% and amino acid substitutions varied from 0 to 9.1%. Phylogenetic analysis using parsimony and neighbor-joining algorithms identified five distinct genotypes: central/east Africa, east Africa, Angola, west Africa I, and west Africa II. Extensive variation within genotypes was observed. Members of west African genotype II and central/east African genotype differed by 2.8% or less, while west Africa genotype I varied up to 6.8% at the nucleotide level. We speculate that the former two genotypes exist in enzootic transmission cycles, while the latter is genetically more heterogeneous due to regular human epidemics. The nucleotide sequence of the Angola genotype diverged from the others by 15.7 to 23.0% but only 0.4 to 5.6% at the amino acid level, suggesting that this genotype most likely diverged from a progenitor YF virus in east/central Africa many years ago, prior to the separation of the other east/central African strains analyzed in this study, and has evolved independently. These data demonstrate that there are multiple genotypes of YF virus in Africa and suggest independent evolution of YF virus in different areas of Africa.
Africa↗
Role of CD8(+) lymphocytes in control of simian immunodeficiency virus infection and resistance to rechallenge after transient early antiretroviral treatment.
Transient antiretroviral treatment with tenofovir, (R)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than 1 year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8(+) lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8(+) cells and returned to predepletion levels (as low as <100 copy Eq/ml) as circulating CD8(+) cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.
Adenine↗
Structural analysis of the -1 ribosomal frameshift elements in giardiavirus mRNA.
The RNA polymerase of giardiavirus (GLV) is synthesized as a fusion protein through a -1 ribosomal frameshift in a region where gag and pol open reading frames (ORFs) overlap. A heptamer, CCCUUUA, and a potential pseudoknot found in the overlap were predicted to be required for the frameshift. A 68-nucleotide (nt) cDNA fragment containing these elements was inserted between the GLV 5' 631-nt cDNA and the out-of-frame luciferase gene that required a -1 frameshift within the 68-nt fragment for expression. Giardia lamblia trophozoites transfected with the transcript of this construct showed a frameshift frequency at 1.7%, coinciding with the polymerase-to-capsid protein ratio in GLV. The heptamer is required for the frameshift but can be replaced with other sequences of the same motif. Mutations placing stop codons in the 0 or -1 frame, located directly before or after the heptamer, implicated the latter as the site for the -1 frameshift. Shortening or destroying the putative stem decreased the frameshift efficiency threefold; the efficiency was fully recovered by mutations to restore the stem. Deleting 18 nt from the 3' end of the 68-nt fragment, which formed the second stem in the putative pseudoknot, had no effect on the frequency of the frameshift. Chemical probing of the RNA secondary structure in the frameshift region showed that bases resistant to chemical modification were clustered in the putative stem structures, thus confirming the presence of the postulated stem-loop, while all the bases in the loop were chemically modified, thus ruling out their capability of forming a pseudoknot. These results confirmed the conclusion based on data from the mutation study that there is but a simple stem-loop downstream from the heptamer. Together, they constitute the structural elements for a -1 ribosomal frameshift in the GLV transcript.
Giardiavirus↗
Involvement of nucleotide excision repair in a recombination-independent and error-prone pathway of DNA interstrand cross-link repair.
DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion. Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner. However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways. To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells. A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids. By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombination-independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue. Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.
Animals↗
Evaluation of the Latrobe Valley Better Health Injury Prevention Program.
OBJECTIVE: To evaluate the Latrobe Valley Better Health Injury Prevention Program, a regional community based intervention in south east Victoria, Australia. METHOD: The evaluation design was quasiexperimental including pre-intervention and post-intervention observations in a predominately town dwelling population of approximately 76,000. There was no comparison community. Process measures included key informant interviews. Impact evaluation utilised self reported changes in injury risk and protective factors, gathered by a random household telephone survey. Outcome evaluation was based on five years of emergency department injury surveillance data for the Latrobe Valley. RESULTS: The program built strategic partnerships, increasing the emphasis on local safety. Activities were implemented in the targeted areas of home, sport, and playground injuries. Some 47,000 educational contacts were made with the community and at least 6,000 resource items distributed. There were significant increases in home safety knowledge. Some changes in the areas of playground and sport safety were achieved after partnership development with relevant agencies. Poisson regression models showed significant decreases in the presentation rate for all home injury and for the more severe home injuries. CONCLUSION: This study clearly demonstrates the difficulty of conducting robust evaluation in the absence of readily available and reliable data and adequate budgets. The Latrobe Valley Better Health Program activities contributed to structural, environmental, and organisational changes that have the potential to reduce injury. The extent of this contribution beyond that made by the statewide injury prevention strategy is not able to be determined.
Accident Prevention↗
Injuries among teens employed in the homebuilding industry in North Carolina.
OBJECTIVE: To describe injuries of teens employed in the residential construction industry and to assess whether their injury experiences are significantly different from those of adults in this high risk industry. METHODS AND SETTING: North Carolina homebuilders workers' compensation data for a 41 month period were analyzed. Injuries of teens were identified and described by body part injured, nature, and cause of injury. Proportionate injury ratios were used to summarize and compare the injury experience of teens with those of adult construction workers. RESULTS: Teens had proportionately more injuries to the eye and foot and fewer injuries to the back than adults. They had more cuts and scratches and fewer sprains and strains. They also had proportionately fewer injuries from falls from elevations and overexertion, injuries that account for a significant cost burden in construction. Consistent with these findings, teens had significantly fewer injuries resulting in medical costs or lost time costs of $1,000 or more. CONCLUSIONS: The analyses indicate that injuries of teens are less serious than those of adults. This finding may indicate that their work exposures are less dangerous than those of adults in comparable broad categories of construction. However, the data also provide documentation of injuries to teens resulting from work at heights, use of power tools, and motor vehicles with the majority of more expensive claims involving one of these exposures. Construction is dangerous work and these results add to the documentation of the need for additional measures to prevent work related injuries among all workers-teens and adults-in this industry.
Accidents, Occupational↗
Ca2+-RhoA signaling pathway required for polyamine-dependent intestinal epithelial cell migration.
Expression of voltage-gated K(+) (Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated ([Ca2+](cyt)) following activation of K(+) channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced whole cell K+ currents [I(K(v))] through Kv channels and caused membrane depolarization, which was associated with decreases in ([Ca2+](cyt)), RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO on I(K(v)), E(m), ([Ca2+](cyt)), and RhoA protein and restored cell migration to normal. Elevation of ([Ca2+](cyt)) induced by the Ca2+ ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca2+ decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C(3) transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca2+-activated RhoA activity.
Animals↗
Polyamine depletion stabilizes p53 resulting in inhibition of normal intestinal epithelial cell proliferation.
The p53 nuclear phosphoprotein plays a critical role in transcriptional regulation of target genes involved in growth arrest and apoptosis. The natural polyamines, including spermidine, spermine, and their precursor putrescine, are required for cell proliferation, and decreasing cellular polyamines inhibits growth of the small intestinal mucosa. In the current study, we investigated the mechanisms of regulation of p53 gene expression by cellular polyamines and further determined the role of the gene product in the process of growth inhibition after polyamine depletion. Studies were conducted both in vivo and in vitro using rats and the IEC-6 cell line, derived from rat small intestinal crypt cells. Levels for p53 mRNA and protein, transcription and posttranscription of the p53 gene, and cell growth were examined. Depletion of cellular polyamines by treatment with alpha-difluoromethylornithine (DFMO) increased p53 gene expression and caused growth inhibition in the intact small intestinal mucosa and the cultured cells. Polyamine depletion dramatically increased the stability of p53 mRNA as measured by the mRNA half-life but had no effect on p53 gene transcription in IEC-6 cells. Induction of p53 mRNA levels in DFMO-treated cells was paralleled by an increase in the rate of newly synthesized p53 protein. The stability of p53 protein was also increased after polyamine depletion, which was associated with a decrease in Mdm2 expression. When polyamine-deficient cells were exposed to exogenous spermidine, a decrease in p53 gene expression preceded an increase in cellular DNA synthesis. Inhibition of the p53 gene expression by using p53 antisense oligodeoxyribonucleotides significantly promoted cell growth in the presence of DFMO. These findings indicate that polyamines downregulate p53 gene expression posttranscriptionally and that growth inhibition of small intestinal mucosa after polyamine depletion is mediated, at least partially, through the activation of p53 gene.
Animals↗
Insulin-like growth factor I and insulin-like growth factor binding protein 5 in Crohn's disease.
Insulin-like growth factor (IGF)-I and its binding protein IGF binding protein 5 (IGFBP-5) were highly expressed in inflamed and fibrotic intestine in experimental Crohn's disease. IGF-I induced proliferation and increased collagen synthesis by smooth muscle cells and fibroblasts/myofibroblasts in vitro. Here we studied IGF-I and IGFBP-5 in Crohn's disease tissue. Tissue was collected from patients undergoing intestinal resection for Crohn's disease. IGF-I and IGFBP-5 mRNAs were quantitated by RNase protection assay and Northern blot analysis, respectively. In situ hybridization was performed to localize mRNA expression, and Western immunoblot was performed to quantitate protein expression. IGF-I and IGFBP-5 mRNAs were increased in inflamed/fibrotic intestine compared with normal-appearing intestine. IGF-I mRNA was expressed in multiple cell types in the lamina propria and fibroblast-like cells of the submucosa and muscularis externa. IGFBP-5 mRNA was highly expressed in smooth muscle of the muscularis mucosae and muscularis externa as well as fibroblast-like cells throughout the bowel wall. Tissue IGFBP-5 protein correlated with collagen type I (r = 0.82). These findings are consistent with a mechanism whereby IGF-I acts on smooth muscle and fibroblasts/myofibroblasts to increase collagen synthesis and cellular proliferation; its effects may be modulated by locally expressed IGFBP-5.
Blotting, Northern↗
NF-kappaB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells.
The maintenance of intestinal mucosal integrity depends on a balance between cell renewal and cell death, including apoptosis. The natural polyamines, putrescine, spermidine, and spermine, are essential for mucosal growth, and decreasing polyamine levels cause G(1) phase growth arrest in intestinal epithelial (IEC-6) cells. The present study was done to determine changes in susceptibility of IEC-6 cells to apoptosis after depletion of cellular polyamines and to further elucidate the role of nuclear factor-kappaB (NF-kappaB) in this process. Although depletion of polyamines by alpha-difluoromethylornithine (DFMO) did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced apoptosis increased significantly as measured by changes in morphological features and internucleosomal DNA fragmentation. In contrast, polyamine depletion by DFMO promoted resistance to apoptotic cell death induced by the combination of tumor necrosis factor-alpha (TNF-alpha) and cycloheximide. Depletion of cellular polyamines also increased the basal level of NF-kappaB proteins, induced NF-kappaB nuclear translocation, and activated the sequence-specific DNA binding activity. Inhibition of NF-kappaB binding activity by sulfasalazine or MG-132 not only prevented the increased susceptibility to STS-induced apoptosis but also blocked the resistance to cell death induced by TNF-alpha in combination with cycloheximide in polyamine-deficient cells. These results indicate that 1) polyamine depletion sensitizes intestinal epithelial cells to STS-induced apoptosis but promotes the resistance to TNF-alpha-induced cell death, 2) polyamine depletion induces NF-kappaB activation, and 3) disruption of NF-kappaB function is associated with altered susceptibility to apoptosis induced by STS or TNF-alpha. These findings suggest that increased NF-kappaB activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the death stimulus.
Animals↗