Biomedical subjects

L Wang

Publications and source records attributed to L Wang.

At least 811 records · Page 45Linked to original sources

PubMed2000

Mitochondrial cytochrome b gene analysis of Aspergillus fumigatus and related species.

Nucleotide sequences of 426 bp from the mitochondrial (mt) cytochrome b genes of six anamorph species and two species of Neosartorya teleomophs of Aspergillus section Fumigati were determined. These sequences were used to build nucleotide- and amino acid-based trees for phylogenetic analysis. Thirteen strains of A. fumigatus including 10 clinical isolates of A. fumigatus, 1 type culture of A. fumigatus var. fumigatus, 1 type culture of A. fumigatus var. ellipticus, and 1 strain of A. fumigatus var. albus, had the same nucleotide sequences. One strain of A. fumisynnematus, two strains labeled A. neoellipticus, two strains of A. viridinutans, and one strain of A. duricaulis had distinct nucleotide and amino acid sequences. Two strains of A. brevipes were divided into two types. One produced a 1,500-bp fragment that included an intron. The nucleotide sequences of its two exons were similar to those of the A. fumigatus, and the derived amino acid sequence was the same as that for A. fumigatus. The other produced a 426-bp fragment and had the same nucleotide and amino acid sequences as A. unilateralis. Neosartorya fischeri var. fischeri and N. stramenia had nucleotide sequences that differed from that of A. fumigatus. These species possessed their own characteristic nucleotide sequences that differed from each other. In comparisons of homologous sequences from four other pathogenic species of Aspergillus, regions specific to section Fumigati were found. The mt cytochrome b gene analysis was valuable for the identification, classification, and phylogenetic analysis of isolates of section Fumigati.

Amino Acid Sequence↗

PubMed2000

Sequence diversity of the Escherichia coli H7 fliC genes: implication for a DNA-based typing scheme for E. coli O157:H7.

Flagellar (H) antigens are mostly encoded by genes at the fliC locus in E. coli. We have sequenced 11 H7 fliC genes from Escherichia coli strains that belong to seven O serotypes. These sequences, together with those of nine other H7 fliC genes (from strains of three different O serotypes) sequenced recently (S. D. Reid, R. K. Selander, and T. S. Whittam, J. Bacteriol. 181:153-160, 1999), include 10 different sequences. The differences between these 10 sequences range from 0.06 to 3.12%. By comparison with other E. coli flagellin genes, we have identified primer length sequences specific for H7 genes in general and others specific for H7 genes of O157 and O55 strains: the specificity was confirmed by PCR testing the type strains for all 53 E. coli H types. We have previously identified genes specific for the E. coli O157 antigen, and use of the combination of O157- and H7-specific primers allows the sensitive and rapid detection of O157:H7 E. coli strains, which cause the majority of hemorrhagic colitis cases.

Base Sequence↗

PubMed2000

Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome.

A competitive PCR (cPCR) assay was developed for monitoring porcine circovirus (PCV) DNA in serum samples from piglets. The cPCR was based on competitive coamplification of a 502- or 506-bp region of the PCV type 1 (PCV1) or PCV2 ORF2, respectively, with a known concentration of competitor DNA, which produced a 761- or 765-bp fragment, respectively. The cPCR was validated by quantification of a known amount of PCV wild-type plasmids. We also used this technique to determine PCV genome copy numbers in infected cells. Furthermore, we measured PCV DNA loads in clinical samples. More than 50% of clinically healthy piglets could harbor both types of PCV. While PCV1 was detected in only 3 of 16 pigs with postweaning multisystemic wasting syndrome (PMWS), all the sick piglets contained PCV2. A comparison of the PCV2 DNA loads of healthy and sick animals revealed a significant difference, indicating that the development of PMWS may require a certain amount of PCV2.

Animals↗

PubMed2000

Loss of G(1)/S checkpoint in human immunodeficiency virus type 1-infected cells is associated with a lack of cyclin-dependent kinase inhibitor p21/Waf1.

Productive high-titer infection by human immunodeficiency virus type 1 (HIV-1) requires the activation of target cells. Infection of quiescent peripheral CD4 lymphocytes by HIV-1 results in incomplete, labile reverse transcripts and lack of viral progeny formation. An interplay between Tat and p53 has previously been reported, where Tat inhibited the transcription of the p53 gene, which may aid in the development of AIDS-related malignancies, and p53 expression inhibited HIV-1 long terminal repeat transcription. Here, by using a well-defined and -characterized stress signal, gamma irradiation, we find that upon gamma irradiation, HIV-1-infected cells lose their G(1)/S checkpoints, enter the S phase inappropriately, and eventually apoptose. The loss of the G(1)/S checkpoint is associated with a loss of p21/Waf1 protein and increased activity of a major G(1)/S kinase, namely, cyclin E/cdk2. The p21/Waf1 protein, a known cyclin-dependent kinase inhibitor, interacts with the cdk2/cyclin E complex and inhibits progression of cells into S phase. We find that loss of the G(1)/S checkpoint in HIV-1-infected cells may in part be due to Tat's ability to bind p53 (a known activator of the p21/Waf1 promoter) and sequester its transactivation activity, as seen in both in vivo and in vitro transcription assays. The loss of p21/Waf1 in HIV-1-infected cells was specific to p21/Waf1 and did not occur with other KIP family members, such as p27 (KIP1) and p57 (KIP2). Finally, the advantage of a loss of the G(1)/S checkpoint for HIV-1 per se may be that it pushes the host cell into the S phase, which may then allow subsequent virus-associated processes, such as RNA splicing, transport, translation, and packaging of virion-specific genes, to occur.

CDC2-CDC28 Kinases↗

PubMed2000

Herpes simplex virus type 1 glycoprotein E domains involved in virus spread and disease.

Herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) functions as an immunoglobulin G (IgG) Fc binding protein and is involved in virus spread. Previously we studied a gE mutant virus that was impaired for IgG Fc binding but intact for spread and another that was normal for both activities. To further evaluate the role of gE in spread, two additional mutant viruses were constructed by introducing linker insertion mutations either outside the IgG Fc binding domain at gE position 210 or within the IgG Fc binding domain at position 380. Both mutant viruses were impaired for spread in epidermal cells in vitro; however, the 380 mutant virus was significantly more impaired and was as defective as gE null virus. gE mutant viruses were inoculated into the murine flank to measure epidermal disease at the inoculation site, travel of virus to dorsal root ganglia, and spread of virus from ganglia back to skin to produce zosteriform lesions. Disease at the inoculation and zosteriform sites was reduced for both mutant viruses, but more so for the 380 mutant virus. Moreover, the 380 mutant virus was highly impaired in its ability to reach the ganglia, as demonstrated by virus culture and real-time quantitative PCR. The results indicate that the domain surrounding amino acid 380 is important for both spread and IgG Fc binding and suggest that this domain is a potential target for antiviral therapy or vaccines.

Animals↗

PubMed2000

TEL, a putative tumor suppressor, modulates cell growth and cell morphology of ras-transformed cells while repressing the transcription of stromelysin-1.

TEL is a member of the ETS family of transcription factors that interacts with the mSin3 and SMRT corepressors to regulate transcription. TEL is biallelically disrupted in acute leukemia, and loss of heterozygosity at the TEL locus has been observed in various cancers. Here we show that expression of TEL in Ras-transformed NIH 3T3 cells inhibits cell growth in soft agar and in normal cultures. Unexpectedly, cells expressing both Ras and TEL grew as aggregates. To begin to explain the morphology of Ras-plus TEL-expressing cells, we demonstrated that the endogenous matrix metalloproteinase stromelysin-1 was repressed by TEL. TEL bound sequences in the stromelysin-1 promoter and repressed the promoter in transient-expression assays, suggesting that it is a direct target for TEL-mediated regulation. Mutants of TEL that removed a binding site for the mSin3A corepressor but retained the ETS domain failed to repress stromelysin-1. When BB-94, a matrix metalloproteinase inhibitor, was added to the culture medium of Ras-expressing cells, it caused a cell aggregation phenotype similar to that caused by TEL expression. In addition, TEL inhibited the invasiveness of Ras-transformed cells in vitro and in vivo. Our results suggest that TEL acts as a tumor suppressor, in part, by transcriptional repression of stromelysin-1.

3T3 Cells↗

PubMed2000

Molecular analyses of a repetitive DNA sequence in wheat (Triticum aestivum L.).

A repetitive sequence designated WE35 was isolated from wheat genomic DNA. This sequence consists of a 320-bp repeat unit and represents approximately 0.002% of the total wheat DNA. It is unidirectionally distributed either continuously or discretely in the genome. Ladder-like banding patterns were observed in Southern blots when the wheat genomic DNA was restricted with endonuclease enzymes EcoRI, HincII, NciI, and NdeI, which is characteristic for tandemly organized sequences. Two DNA fragments in p451 were frequently associated with the WE35 repetitive unit in a majority of lambda wheat genomic clones. A 475-bp fragment homologous to the 5'-end long terminal repeat (LTR) of cereal retroelements was also found in some lambda wheat genomic clones containing the repetitive unit. Physical mapping by fluorescence in situ hybridization (FISH) indicated that one pair of wheat chromosomes could be specifically detected with the WE35 positive probe p551. WE35 can be considered a chromosome-specific repetitive sequence. This repetitive unit could be used as a molecular marker for genetic, phylogenetic, and evolutionary studies in the tribe Triticeae.

Amino Acid Sequence↗

PubMed2000

Mesial temporal sclerosis and temporal lobe epilepsy: MR imaging deformation-based segmentation of the hippocampus in five patients.

In five patients with mesial temporal sclerosis, the authors verified the precision and reproducibility of hippocampal segmentations with deformation-based magnetic resonance (MR) imaging. The overall percentage overlap between automated segmentations was 92.8% (SD, 3.5%), between manual segmentations was 73.1% (SD, 9.5%), and between automated and manual segmentations was 74.8% (SD, 10.3%). Deformation-based hippocampal segmentations provided a precise method of hippocampal volume measurement in this patient population.

Adult↗

PubMed2000

Age-dependent response of the electrocardiogram to K(+) channel blockers in mice.

Developmental changes in electrocardiogram (ECG) and response to selective K(+) channel blockers were assessed in conscious, unsedated neonatal (days 1, 7, 14) and adult male mice (>60 days of age). Mean sinus R-R interval decreased from 120 +/- 3 ms in day 1 to 110 +/- 3 ms in day 7, 97 +/- 3 ms in day 14, and 81 +/- 1 ms in adult mice (P < 0.001 by ANOVA; all 3 groups different from day 1). In parallel, the mean P-R interval progressively decreased during development. Similarly, the mean Q-T interval decreased from 62 +/- 2 ms in day 1 to 50 +/- 2 ms in day 7, 47 +/- 8 ms in day 14 neonatal mice, and 46 +/- 2 ms in adult mice (P < 0.001 by ANOVA; all 3 groups are significantly different from day 1). Q-T(c) was calculated as Q- interval. Q-T(c) significantly shortened from 179 +/- 4 ms in day 1 to 149 +/- 5 ms in day 7 mice (P < 0.001). In addition, the J junction-S-T segment elevation observed in day 1 neonatal mice resolved by day 14. Dofetilide (0.5 mg/kg), the selective blocker of the rapid component of the delayed rectifier (I(Kr)) abolished S-T segment elevation and prolonged Q-T and Q-T(c) intervals in day 1 neonates but not in adult mice. In contrast, 4-aminopyridine (4-AP, 2.5 mg/kg) had no effect on day 1 neonates but in adults prolonged Q-T and Q-T(c) intervals and specifically decreased the amplitude of a transiently repolarizing wave, which appears as an r' wave at the end of the apparent QRS in adult mice. In conclusion, ECG intervals and configuration change during normal postnatal development in the mouse. K(+) channel blockers affect the mouse ECG differently depending on age. These data are consistent with the previous findings that the dofetilide-sensitive I(Kr) is dominant in day 1 mice, whereas 4-AP-sensitive currents, the transiently repolarizing K(+) current, and the rapidly activating, slowly inactivating K(+) current are the dominant K(+) currents in adult mice. This study provides background information useful for assessing abnormal development in transgenic mice.

4-Aminopyridine↗

PubMed2000

Slowing of intestinal transit by fat depends on naloxone-blockable efferent, opioid pathway.

Slowing of transit through the proximal small intestine by fat in the distal gut is termed the ileal brake. Intravenous naloxone, an opioid receptor antagonist, abolished the fat-induced ileal brake, suggesting that an endogenous opioid pathway may be involved in this response. To test the hypothesis that slowing of intestinal transit by fat in the distal half of the gut depends on an opioid pathway located on the efferent limb of this response, we compared intestinal transit in dogs equipped with duodenal and midgut fistulas while naloxone was either compartmentalized with oleate to the distal half of the gut or with buffer to the proximal half of the gut. We found that intestinal transit depended on the perfusion conditions (P<0.00001). Specifically, compared with ileal brake (marker recovery of 35.7+/-7.4%), intestinal transit was accelerated when naloxone was delivered into the proximal half of the gut (76.2+/-5.2%) (P<0.005) but not the distal half of the gut (29.4+/-5.4%). We conclude that slowing of intestinal transit by fat in the distal half of the gut depends on an opioid pathway located on the efferent limb of the ileal brake.

Animals↗

PubMed2000

Activation of mRNA translation in rat cardiac myocytes by insulin involves multiple rapamycin-sensitive steps.

Insulin acutely activates protein synthesis in ventricular cardiomyocytes from adult rats. In this study, we have established the methodology for studying the regulation of the signaling pathways and translation factors that may be involved in this response and have examined the effects of acute insulin treatment on them. Insulin rapidly activated the 70-kDa ribosomal S6 kinase (p70 S6k), and this effect was inhibited both by rapamycin and by inhibitors of phosphatidylinositol 3-kinase. The activation of p70 S6k is mediated by a signaling pathway involving the mammalian target of rapamycin (mTOR), which also modulates other translation factors. These include the eukaryotic initiation factor (eIF) 4E binding proteins (4E-BPs) and eukaryotic elongation factor 2 (eEF2). Insulin caused phosphorylation of 4E-BP1 and induced its dissociation from eIF4E, and these effects were also blocked by rapamycin. Concomitant with this, insulin increased the binding of eIF4E to eIF4G. Insulin also activated protein kinase B (PKB), which may lie upstream of p70 S6k and 4E-BP1, with the activation of the different isoforms being in the order alpha>beta>gamma. Insulin also caused inhibition of glycogen synthase kinase 3, which lies downstream of PKB, and of eEF2 kinase. The phosphorylation of eEF2 itself was also decreased by insulin, and this effect and the inactivation of eEF2 kinase were attenuated by rapamycin. The activation of overall protein synthesis by insulin in cardiomyocytes was substantially inhibited by rapamycin (but not by inhibitors of other specific signaling pathways, e.g., mitogen-activated protein kinase), showing that signaling events linked to mTOR play a major role in the control of translation by insulin in this cell type.

Age Factors↗

PubMed2000

beta(1)-integrin and PI 3-kinase regulate RhoA-dependent activation of skeletal alpha-actin promoter in myoblasts.

RhoA GTPase, a regulator of actin cytoskeleton, is also involved in regulating c-fos gene expression through its effect on serum response factor (SRF) transcriptional activity. We have also shown that RhoA plays a critical role in myogenesis and regulates expression of SRF-dependent muscle genes, including skeletal alpha-actin. In the present study, we examined whether the RhoA signaling pathway cross talks with other myogenic signaling pathways to modulate skeletal alpha-actin promoter activity in myoblasts. We found that extracellular matrix proteins and the beta(1)-integrin stimulated RhoA-dependent activation of the alpha-actin promoter. The muscle-specific isoform beta(1D) selectively activated the alpha-actin promoter in concert with RhoA but inhibited the c-fos promoter. In addition, focal adhesion kinase (FAK) and phosphatidylinositol (PI) 3-kinase were required for full activation of the alpha-actin promoter by RhoA. Expression of a dominant negative mutant of FAK, application of wortmannin to cultured myoblasts, or expression of a dominant negative mutant of PI 3-kinase inhibited alpha-actin promoter activity induced by RhoA. These results suggest that RhoA, beta(1)-integrin, FAK, and PI 3-kinase serve together as an important signaling network in regulating muscle gene expression.

Actins↗

PubMed2000

Free radical-mediated transgene inactivation of macrophages by endotoxin.

Endotoxin, the lipopolysaccharide component of gram-negative bacteria, is a common contaminant of plasmid DNA preparations. The present study investigated the effect of endotoxin on gene transfection efficiency and the role of reactive oxygen species (ROS) in this process. Gene transfection studies were performed in various cell types with cytomegalovirus-luciferase as a reporter plasmid and cationic liposome as a transfecting agent. The presence of endotoxin in plasmid DNA preparations severely limited transgene expression in macrophages but had little or no effect in other cell types tested. This decreased transfection was dependent on ROS-mediated cellular toxicity induced by endotoxin. Neutralizing the endotoxin by the addition of polymyxin B effectively increased transfection efficiency and reduced toxicity. Electron spin resonance studies confirmed the formation of ROS in endotoxin-treated cells and their inhibition by free radical scavengers. The ROS scavenger N-t-butyl-alpha-phenylnitrone, the H(2)O(2) scavenger catalase, and the.OH scavenger sodium formate effectively inhibited endotoxin-induced effects, whereas the O(2)(-) scavenger superoxide dismutase had lesser effects. These results indicate that multiple oxidative species are involved in the transfection inactivation process and that.OH formed by H(2)O(2)-dependent, metal-catalyzed Fenton reaction play a major role in this process.

Animals↗

PubMed2000

Inhibition of type I and III IP(3)Rs by TGF-beta is associated with impaired calcium release in mesangial cells.

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) mediate cytosolic free calcium concentration ([Ca(2+)](c)) signals in response to a variety of agonists that stimulate mesangial cell contraction and proliferation. In the present study, we demonstrate that mesangial cells express both type I and III IP(3)Rs and that these receptors occupy different cellular locations. Chronic treatment with transforming growth factor-beta1 (TGF-beta1; 10 ng/ml, 24 h) leads to downregulation of both type I and III IP(3)Rs as measured by immunoblot and confocal analysis. TGF-beta1 treatment does not affect IP(3) levels, and downregulation of type I IP(3)R is not due to enhanced degradation of the protein, as the half-life of type I IP(3)R is unchanged in the presence or absence of TGF-beta1. Functional effects of TGF-beta1-induced downregulation of the IP(3)Rs were evaluated by measuring [Ca(2+)](c) changes in response to epidermal growth factor (EGF) in intact cells and sensitivity of [Ca(2+)](c) release to IP(3) in permeabilized cells. TGF-beta1 pretreatment led to a significant decrease of [Ca(2+)](c) release induced by EGF in intact cells and by submaximal IP(3) (400 nm) in permeabilized cells. Total IP(3)-sensitive [Ca(2+)](c) stores were not changed, as assessed by stimulation with maximal doses of IP(3) (10.5 microm) and thapsigargin-mediated calcium release in permeabilized cells. We conclude that prolonged exposure to TGF-beta1 leads to downregulation of both type I and III IP(3)Rs in mesangial cells and this is associated with impaired sensitivity to IP(3).

Animals↗

PubMed2000

Airway narrowing and internal structural constraints.

A computer model has been developed to simulate the movement restriction in the lamina propria-submucosa (L-S) layer (sandwiched by the basement membrane and the muscle layer) in a cartilage-free airway due to constriction of the smooth muscle layer. It is assumed that the basement membrane is inextensible; therefore, in the two-dimensional simulation, the perimeter outlining the membrane is a constant whether the airway is constricted or dilated. The cross-sectional area of the L-S layer is also assumed to be constant during the simulated airway narrowing. Folding of the mucosal membrane in constricted airways is assumed to be a consequence of the L-S area conservation and also due to tethering between the basement membrane and the muscle layer. The number of tethers determines the number of folds. The simulation indicates that the pressure in the L-S layer resulting from movement restriction can be a major force opposing muscle contraction and that the maximum shortening of the muscle layer is inversely proportional to the number of tethers (or folds) and the L-S layer thickness.

Animals↗

PubMed2000

Mechanical properties of the tracheal mucosal membrane in the rabbit. I. steady-state stiffness as a function of age.

Airway responsiveness is exaggerated in infancy and declines with maturation. These age-related differences (R.S. Tepper, T. Du, A. Styhler, M. Ludwig, and J.G. Martin. Am. J. Respir. Crit. Care Med. 151: 836-840, 1995; R.S. Tepper, S.J. Gunst, C.M. Doerschuk, Y. Shen, and W. Bray. J. Appl. Physiol. 78: 505-512, 1995; R.S. Tepper, J. Stevens, and H. Eigen. Am. J. Respir. Crit. Care Med. 149: 678-681, 1994) could be due to changes in the smooth muscle, the lung, and/or the airway wall. Folding of the mucosal membrane can provide an elastic load (R.K. Lambert, J. Appl. Physiol. 71: 666-673, 1991), which impedes smooth muscle shortening. We hypothesized that increased stiffness of the mucosal membrane occurs during aging, causing an increased mechanical load on airway smooth muscle and a decrease in airway responsiveness. Forty female New Zealand White rabbits between 0.75 and 35 mo of age were studied. Rectangular mucosal membrane strips oriented both longitudinally and circumferentially to the long axis of the trachea were dissected, and the stress-strain relationships of each strip were tested. The results showed that the membrane was stiffer in the longitudinal than in the circumferential direction of the airway. However, there was no significant change with age in either orientation. We conclude that the mechanical properties of the airway mucosal membrane did not change during maturation and were not likely to influence age-related changes in airway responsiveness.

Aging↗

PubMed2000

Mechanical properties of the tracheal mucosal membrane in the rabbit. II. Morphometric analysis.

Folding of the airway mucosal membrane provides a mechanical load that impedes airway smooth muscle contraction. Mechanical testing of rabbit tracheal mucosal membrane showed that the membrane is stiffer in the longitudinal than in the circumferential direction of the airway. To explain this difference in the mechanical properties, we studied the morphological structure of the rabbit tracheal mucosal membrane in both longitudinal and circumferential directions. The collagen fibers were found to form a random meshwork, which would not account for differences in stiffness in the longitudinal and circumferential directions. The volume fraction of the elastic fibers was measured using a point-counting technique. The orientation of the elastic fibers in the tissue samples was measured using a new method based on simple geometry and probability. The results showed that the volume fraction of the elastic fibers in the rabbit tracheal mucosal membrane was approximately 5% and that the elastic fibers were mainly oriented in the longitudinal direction. Age had no statistically significant effect on either the volume fraction or the orientation of the elastic fibers. Linear correlations were found between the steady-state stiffness and the quantity of the elastic fibers oriented in the direction of testing.

Animals↗

PubMed2000

Effects of length oscillation on the subsequent force development in swine tracheal smooth muscle.

It has been shown that deep inspiration (DI) taken before application of bronchoconstricting stimuli causes a reduction in the subsequent bronchoconstriction; a fast DI has a greater inhibitory effect than a slow DI. We hypothesize that periodic length changes imposed on a relaxed airway smooth muscle (ASM) would attenuate subsequent bronchoconstriction by disrupting the organization of the contractile apparatus, and this could be an important mechanism for the observed bronchoprotective effect of DI and tidal breathing. Length oscillations of different amplitude, frequency, and duration were applied to a relaxed muscle. The effects of such perturbations on force development were then assessed. Results show that oscillations reduce the subsequent force generation and that the magnitude of force reduction is proportional to amplitude and duration of the length oscillation. After the oscillation, isometric force recovered to the preoscillation level in a series of isometric contractions, and the rate of recovery was facilitated by frequent stimulation. The in vitro behavior of ASM found in this study could account for the observed temporary reduction in bronchoconstriction subsequent to a DI.

Animals↗