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Biomedical subjects

L Li

Publications and source records attributed to L Li.

At least 865 records · Page 48Linked to original sources
PubMed1999

In vitro translation of type A Clostridium botulinum neurotoxin heavy chain and analysis of its binding to rat synaptosomes.

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. BoNTs consist of a toxifying light chain (LC; 50 kDa) and a binding/translocating heavy chain (HC; 100 kDa) linked through a disulfide bond. A DNA fragment encoding type A Clostridium botulinum heavy chain (BoNT/A HC) was amplified by polymerase chain reaction and cloned into an E. coli PET-15b vector. In vitro translated [35S]BoNT/A HC was identified by anti-BoNT/A polyclonal antibodies, and was used to investigate the binding of the toxin to rat synaptosomes. The binding of [35S]BoNT/A HC to synaptosomes was abolished by 500-fold excess of cold BoNT/A, and by incubation with trypsin. Treatment of BoNT/A HC with anti-BoNT/A or G(T1b) blocked its binding to synaptosomes. The radioactive BoNT/A HC recognized three proteins corresponding to a molecular mass of 150 (P150), 120 (P120), and 75 (P75) kDa in rat and bovine synaptosomal preparations. These results represent the first successful expression of functional full-length BoNT heavy chain.

Animals↗
PubMed1999

An ultra-metastatic model of human colon cancer in nude mice.

An ultra-high metastatic model of human colon cancer was developed in order to represent highly malignant patient disease for which there is no current model. Surgical orthotopic implantation (SOI) of a histologically intact liver metastasis fragment derived from a surgical specimen of a patient with metastatic colon cancer was initially implanted in the colon, liver and subcutaneously in nude mice. This tumor did not metastasize for the first 10 passages. At the eleventh passage, the tumor exhibited metastasis from the colon to the liver, spleen, and lymph nodes. At this time, two selective passages were carried out by transplanting resulting liver metastases in the nude mice to the colon of additional nude mice. After these two passages, the tumor became stably ultra-metastatic and was termed AC3488UM. One-hundred percent of mice transplanted with AC3488UM with SOI to the colon exhibited local growth, regional invasion, and spontaneous metastasis to the liver, lymph nodes, and spleen. While the maximum size of the primary tumor was 0.9 g, the metastatic liver was over 9 times the weight of the normal liver with the maximum weight of the metastatic liver over 12 g. Liver metastases were detected by the tenth day after transplantation in all animals. Half the animals died of metastatic tumor 25 days after transplantation. Histological characteristics of AC3488UM tumor were poorly differentiated adenocarcinoma of colon. Mutant p53 is expressed heterogeneously in the primary tumor and more homogeneously in the liver metastasis suggesting a possible role of p53 in the liver metastasis. The human origin of AC3488UM was confirmed by positive fluorescence staining for in situ hybridization of human DNA. The AC3488 human colon-tumor model with its ultra-high metastatic capability in each transplanted animal, short latency and a short median survival period is different from any known human colon cancer model and will be an important tool for the study of and development of new therapy for highly metastatic human colon cancer.

Animals↗
PubMed1999

Soluble TNF-alpha receptor and IL-1 receptor antagonist elevation in BAL in active pulmonary TB.

Accumulating evidence suggests that patients with active pulmonary tuberculosis (TB) have an alveolar inflammation resulting in the release of tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta in bronchoalveolar epithelial fluid. It was proposed that the levels of these cytokines would correlate with clinical status parameters (extent of pulmonary involvement, fever, and body weight loss) and that their naturally occurring inhibitors would be concomitantly released in the local inflammatory sites. To test this hypothesis lung epithelial lining fluid (ELF) obtained by bronchoalveolar lavage and serum were collected from 29 patients with active pulmonary TB and 15 healthy subjects to determine the levels of these variables using a sandwich enzyme-linked immunosorbent assay (ELISA). ELF levels of TNF-alpha, soluble (s)TNF receptor I (RI), sTNF-receptor II (RII) and interleukin-1 receptor antagonist (IL-1RA) but not IL-1beta, and their serum levels except for sTNF-RII and IL-1beta were significantly higher in TB patients. Nevertheless, only ELF levels of TNF-alpha and IL-1beta were significantly correlated with disease status. No correlation was found between TNF-alpha levels and those of sTNF-RI and sTNF-RII, nor between IL-1beta and IL-1RA in ELF and serum of TB patients, although there was a significant correlation between sTNF-RI and sTNF-RII levels both in ELF and serum. These findings suggest local release of tumour necrosis factor-alpha and interleukin-1beta and a correlation with disease status. Soluble tumour necrosis factor-alpha receptors and interleukin-1beta receptor antagonist, although increased in lung epithelial lining fluid and serum in tuberculosis patients, were not correlated with tumour necrosis factor-alpha and interleukin-1beta or with disease status.

Adult↗
PubMed1999

Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I.

Calcium plays a critical part in the regulation of cell growth, and growth factors stimulate calcium entry into cells through calcium-permeable channels. However, the molecular nature and regulation of calcium-permeable channels are still unclear at present. Here we report the molecular characterization of a calcium-permeable cation channel that is regulated by insulin-like growth factor-I (IGF-I). This channel, which we name growth-factor-regulated channel (GRC), belongs to the TRP-channel family and localizes mainly to intracellular pools under basal conditions. Upon stimulation of cells by IGF-I, GRC translocates to the plasma membrane. Thus, IGF-I augments calcium entry through GRC by regulating trafficking of the channel.

3T3 Cells↗
PubMed1999

Brain choline acetyltransferase activity in chronic, human users of cocaine, methamphetamine, and heroin.

Cognitive impairment has been reported in some chronic users of psychostimulants, raising the possibility that long-term drug exposure might damage brain neuronal systems, including the cholinergic system, which are responsible for normal cognition. We measured the activity of choline acetyltransferase (ChAT), the marker enzyme for cholinergic neurones, in autopsied brain of chronic users of cocaine, methamphetamine, and, for comparison, heroin. As compared with the controls, mean ChAT levels were normal in all cortical and subcortical brain areas examined. However, the two of 12 methamphetamine users, who had the highest brain/blood drug levels at autopsy, had a severe (up to 94%) depletion of ChAT activity in cerebral cortex, striatum, and thalamus. Based on the subjects examined in the present study, our neurochemical data suggest that brain cholinergic neurone damage is unlikely to be a typical feature of chronic use of cocaine, methamphetamine, or heroin, but that exposure to very high doses of methamphetamine could impair, at least acutely, cognitive function requiring a normal nucleus basalis cholinergic neuronal system. Reduced brain ChAT might be explained in part by a hyperthermia-related mechanism as low ChAT levels have also been observed in brain of some patients with neuroleptic drug-associated hyperthermia. Studies of cognitive and brain cholinergic status in high dose users of MA are warranted.

Adult↗
PubMed1999

Alcohol consumption and alcohol-related problems: Chinese experience from six area samples, 1994.

AIMS: To understand the drinking frequency, mean yearly volume of consumption and drinking-related problems in individuals living in six areas in China. DESIGN AND MEASUREMENTS: By two-stage sampling, 23,513 community household residents (15-65 years old) were assessed with data collected by trained psychiatrists using structured questionnaires and DSM-III-R criteria. FINDINGS: The male, female and total drinking rates were 84.1%, 29.3% and 59.5%, respectively. More than half of male drinkers and 90.0% of female drinkers used alcohol once a week or less. More than 16.1% of male drinkers and 2.5% of female drinkers drank once a day or more. The males reported a greater amount of alcohol consumed per session. The mean yearly volume of consumption of pure alcohol per year for all sample was 3.60 l; males consumed 18.6 times more alcohol than females. The rate of alcohol-related problems was higher in males than females. The male, female and total alcohol dependence point prevalence rates were 6.632%, 0.104% and 3.428%, respectively. Six-month incidence rates of acute intoxication were 5.162% 0.017% and 2.637%, respectively. CONCLUSION: Drinking problems in China will continue to be a public health problem in the next century.

Adolescent↗
PubMed1999

A PCR method for typing B-L beta II family (class II MHC) alleles in broiler chickens.

Certain haplotypes of the major histocompatibility (B) complex are strongly associated with resistance or susceptibility to several infectious diseases in Leghorn chickens. Identification of chicken haplotypes based on the nucleotide sequence of B complex loci could provide more precise identification of haplotypes than traditional serological methods. We report the development and application of polymerase chain reaction with sequence specific primers (PCR-SSP) to type broiler chicken B haplotypes based on the DNA sequence of B-L beta II family genes. Five well-defined standard B haplotypes from White Leghorns and 12 recently characterized B haplotypes from a broiler breeder line were used to develop the test system. The B-L beta II family loci were amplified from genomic DNA by B-L beta II family specific primers and then characterized by PCR-SSP. In total, ten pairs of primers, derived from the sequences of expressed B-L beta II family alleles, were used in the PCR typing test to discriminate the chicken B haplotypes identified previously by serological means. The PCR-SSP showed that each haplotype had a different amplification pattern, except those haplotypes known or suspected to have the same B-L beta alleles. Cloning and sequencing of the family specific PCR products indicated that two loci in the B-L beta II family, presumably B-L beta I and B-L beta II, were amplified. Finally, B-L beta PCR-SSP typing was used in combination with B-G RFLP analyses to characterize unusual (variant) B serotypes; the results indicate that some of these are natural recombinants within the B complex.

Alleles↗
PubMed1999

Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli.

Escherichia coli K-12 carries a gene for a protein denoted ClyA or SheA that can mediate a cytolytic phenotype. The ClyA protein is not expressed at detectable levels in most strains of E. coli, but overproduction suitable for purification was accomplished by cloning the structural gene in an hns mutant strain. Highly purified ClyA protein was cytotoxic to macrophage cells in culture and caused detachment and lysis of the mammalian cells. Results from osmotic protection assays were consistent with the suggestion that the protein formed pores with a diameter of up to 3 nm. Using Acholeplasma laidlawii cells and multilamellar liposomes, we studied the effect of ClyA on membranes with varying compositions and in the presence of different ions. ClyA induced cytolytic release of the fluorescent tracer from carboxyfluorescein-loaded liposomes, and the release was stimulated if cholesterol was present in the membranes whereas addition of calcium had no effect. Pretreatment of the ClyA protein with cholesterol inhibited the pore formation, suggesting that ClyA could bind to cholesterol. Efficient coprecipitation of ClyA with either cholesterol or 1,2,3-trioctadecanoylglycerol in aqueous solutions showed that ClyA directly interacted with the hydrophobic molecular aggregates. We tested the possible functional importance of selected ClyA protein regions by site-directed mutagenesis. Defined mutants of ClyA were obtained with alterations in postulated transmembrane structures in the central part and in a postulated membrane-targeting domain in the C-terminal part. Our results were consistent with the suggestion that particular amphiphilic segments are required for ClyA activity. We propose that these domains are necessary for ClyA to form pores.

Amino Acid Sequence↗
PubMed1999

Formation of N-pyroglutamyl peptides from N-Glu and N-Gln precursors in Aplysia neurons.

Matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry is used to examine the formation of N-pyroglutamate (pGlu) in single, identified neurons from Aplysia. Six pGlu peptides are identified in the R3-14 and the R15 neurons that result from in vivo processing of peptides containing either Glu or Gln at their respective N-termini. Moreover, we show that Glu-derived pGlu is not a sample collection or measurement artifact. The pGlu peptides are detected in isolated cell bodies, regenerated neurites in culture, interganglionic connective nerves, cell homogenates, and collected releasates. We also demonstrate that R3-14 cells readily convert a synthetic N-Glu peptide to its pGlu analogue, indicating the presence of novel enzymatic activity.

Amino Acid Sequence↗
PubMed1999

The amino terminus with a conserved glutamic acid of G protein-coupled receptor kinases is indispensable for their ability to phosphorylate photoactivated rhodopsin.

To investigate functions of the consensus amino terminus of G protein-coupled receptor kinases (GRKs), two amino terminus-truncated mutants (delta30 or delta15) and two single-amino-acid mutants of conserved acidic residues (D2A or E7A) of human GRK1 were constructed and expressed in human embryonic kidney 293 cells. It was shown that truncated mutations and one single-point mutation (E7A) greatly decreased GRK1's activity to phosphorylate photoactivated rhodopsin (Rho*), whereas the abilities of these mutants to phosphorylate a synthetic peptide substrate and to translocate from cytosol to rod outer segments on light activation were unaffected. Further experiments demonstrated that the same truncated mutations (delta30 or delta15) of GRK2, representative of another GRK subfamily, also abolished the kinase's activity toward Rho*. The similar single-point mutation (E5A) of GRK2 heavily impaired its phosphorylation of Rho* but did not alter its ability to phosphorylate the peptide, and the G329-rhodopsin-augmented peptide phosphorylation by GRK2 (E5A) remained unchanged. Our data, taken together, suggest that the amino terminus as well as a conserved glutamic acid in the region of GRKs appears essential for their ability to functionally interact with G protein-coupled receptors.

Acyltransferases↗
PubMed1999

Detection of ozone-induced DNA single strand breaks in murine bronchoalveolar lavage cells acutely exposed in vivo.

Single-strand breaks (SSBs) in DNA have been used a biomarker of oxidative damage. The comet assay, also known as single-cell gel electrophoresis, was used to investigate the ability of ozone (O(3)) to induce DNA SSBs in murine bronchoalveolar lavage (BAL) cells. The comet assay is more sensitive than other techniques currently utilized for detecting SSBs and requires fewer cells. In the present study, 3 mice were exposed for 3 h to 0.25 ppm of O(3), and 3 to 0.5 ppm of O(3) for 3 h. Two air-exposed mice served as negative controls. All mice were euthanized 3 h after exposure, at which time BAL cells were recovered from the lungs and stained with ethidium bromide. BAL cells recovered from an air-exposed mouse were exposed to various concentrations of H(2)O(2) in vitro for 1 h at 4 degrees C. Excluding cells from the H(2)O(2) group (n = 25), 50 randomly selected BAL cells were graded by comet tail length into 1 of 4 categories: no damage (0 mm), low damage (1-10 mm), medium damage (11-30 mm), and high damage (31 + mm). The nonparametric Wilcoxon rank-sum test was used for statistical analysis, and p values lower than .05 were considered significant. The H(2)O(2) and the 0.25 and 0.5 ppm O3 groups showed statistically significant increases in DNA SSBs as compared to air-exposed controls. The results of this study indicate that (1) O(3) induces DNA strand breaks in murine BAL cells at 0.25 and 0.5 ppm, as evidenced by statistically significant increases in the length of comet tails for O(3)-exposed groups, and (2) the comet assay can be used to assess O(3)-induced SSBs for in vivo exposures. Therefore, it has the potential as a biomarker for in vivo oxidant exposures.

Animals↗
PubMed1999

Effects of copper-aspirin complex on plasma 6-keto-prostaglandin F(1alpha) level and platelet cytosolic calcium in rabbits.

Effects of copper-aspirin complex on washed platelet aggregation, thromboxane B(2) formation and 6-ketoprostaglandin F(1alpha) level were monitored by Born's and Terashita's methods, respectively. The influence of copper-aspirin complex on cytosolic free calcium was examined using the fluorescent indicator, Fura 2-AM. Copper-aspirin complex significantly inhibited arachidonic acid-induced aggregation in washed platelets. The IC(50) value was 9.6 micromol L(-1). Copper-aspirin complex significantly decreased arachidonic acid-induced thromboxane B(2) formation by 87.1% in washed platelets. Ten mg kg(-1) of copper-aspirin complex given intragastrically markedly increased the plasma level of 6-keto-prostaglandin F(1alpha). Aspirin, however, reduced both thromboxane B(2) formation and 6-keto-prostaglandin F(1alpha) level. In the presence of CaCl2 1 mmol L(-1), copper-aspirin complex (20, 40 and 80 micromol L(-1)) markedly lowered arachidonic acid-induced increase in platelet calcium from the resting level (270+/-36 nmol L(-1)) to 213+/-14, 170+/-20 and 135+/-17 nmol L(-1), respectively. In the presence of ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid 1 mmol L(-1), copper-aspirin complex (20, 40 and 80 micromol l L(-1)) significantly suppressed the release of intracellular calcium induced by arachidonic acid from 127+/-23 nmol L-1 to 108+/-17, 93+/-12 and 70+/-13 nmol L(-1).

Journal Article↗
PubMed1999

Mucosal events in the pathogenesis of human immunodeficiency virus type 1 infection.

The interaction between human immunodeficiency virus type 1 (HIV-1) and primary mucosal cells isolated from normal human small intestine was investigated. Purified primary intestinal epithelial cells could transport cell-free HIV-1 to mononuclear cells, although the epithelial cells did not support viral replication. An unexpected finding was that primary intestinal macrophages were markedly less permissive to HIV-1 than were blood monocytes. The reduced permissiveness appeared to be due to the near absence of surface CCR5 on resident intestinal macrophages. Surface CCR5 could be up-regulated on the monocytes but not the intestinal macrophages by HIV-1 and gp120. Impaired permissiveness of intestinal macrophages to HIV-1 may play an important role in the low prevalence of HIV-1 mRNA-expressing macrophages in the lamina propria during HIV-1 infection in vivo. Characterization of the biologic properties of HIV-1 transport and infection in primary mucosal cells will be key to elucidating the pivotal role of mucosal surfaces in HIV-1 disease.

Disease Reservoirs↗
PubMed1999

Adeno-associated virus 2-mediated transduction and erythroid lineage-restricted expression from parvovirus B19p6 promoter in primary human hematopoietic progenitor cells.

Human parvovirus B19 gene expression from the viral p6 promoter (B19p6) is restricted to primary human hematopoietic cells undergoing erythroid differentiation. We have demonstrated that expression from this promoter does not occur in established human erythroid cell lines in the context of a recombinant parvovirus genome (Ponnazhagan et al. J Virol 69:8096-8101, 1995). However, abundant expression from this promoter can be readily detected in primary human bone marrow cells (Wang et al. Proc Natl Acad Sci USA 92:12416-12420, 1995; Ponnazhagan et al. J Gen Virol 77:1111-1122, 1996). In the present studies, we investigated the pattern of expression from the B19p6 promoter in primary human bone marrow-derived CD34+ HPC undergoing differentiation into myeloid and erythroid lineages. CD34+ cells were transduced with recombinant adeno-associated virus 2 (AAV) vectors containing the beta-galactosidase (lacZ) gene under the control of the following promoters/enhancers: the cytomegalovirus promoter (vCMVp-lacZ), B19p6 promoter (vB19p6-lacZ), B19p6 promoter with an upstream erythroid cell-specific enhancer element (HS-2) from the locus control region (LCR) from the human beta-globin gene cluster (vHS2-B19p6-lacZ), and the human beta-globin gene promoter with the HS-2 enhancer (vHS2-beta p-lacZ). Transgene expression was evaluated either 48 h after infection or following erythroid differentiation in vitro for 3 weeks. Whereas high-level expression from the CMV promoter 48 h after infection diminished with time, low-level expression from the B19p6 and the beta-globin promoters increased significantly following erythroid differentiation. Furthermore, in HPC assays, there was no significant difference in the level of expression from the CMV promoter in myeloid or erythroid cell-derived colonies. Expression from the B19p6 and the beta-globin promoters, on the other hand, was restricted to erythroid cell colonies. These data further corroborate that the B19p6 promoter is erythroid cell-specific and suggest that the recombinant AAV-B19 hybrid vectors may prove useful in gene therapy of human hemoglobinopathies in general and sickle cell anemia and beta-thalassemia in particular.

Anemia, Sickle Cell↗
PubMed1999

Polymorphisms of DNA repair gene XRCC1 in squamous cell carcinoma of the head and neck.

Because reduced DNA repair capacity (phenotype) has been suggested as a risk factor for squamous cell carcinoma of the head and neck (SCCHN), newly-identified DNA repair gene polymorphisms (genotype) may also be implicated in risk. To test this hypothesis, we conducted a case-control study of 203 SCCHN patients and 424 control subjects (matched for age, sex and ethnicity) to investigate the role of two XRCC1 polymorphisms (XRCC1 26304 T and XRCC1 28152 A, respectively) in SCCHN. Multivariate logistic regression analysis was performed to calculate the adjusted odds ratio (OR) and 95% confidence interval (CI). A total of 180 cases (88.7%) and 363 controls (85.6%) lacked the XRCC1 26304 T allele [adjusted OR = 1.34 (CI, 0.80-2.25)]. Lack of this polymorphism was a significant risk factor specifically for cancers of the oral cavity and pharynx [adjusted OR = 2.46 (CI, 1.22-4.97)]. Thirty-two cases (15.8%) and 46 controls (10.8%) were homozygous for the XRCC1 28152 A allele [adjusted OR = 1.59 (CI, 0.97-2.61) for all cases, and 1.41 (CI, 0. 80-2.48) for oral and pharyngeal cancer only]. Furthermore, when the two genotypes were combined into a three-level model of risk, a polymorphism-polymorphism interaction of increasing risk (trend test, P = 0.049) was evident: OR = 1.0 for those with neither risk genotype (referent group), adjusted OR = 1.51 (CI, 0.87-2.61) for those with either risk genotype, and 2.02 (CI, 1.00-4.05) for those with both risk genotypes. For oral and pharyngeal cancer, this trend was even more pronounced with the adjusted OR = 2.68 (CI, 1.28-5.61) for those with either risk genotype, and 3.22 (CI, 1.33-7.81) for those with both risk genotypes. The findings support the hypothesis that a polymorphic XRCC1 DNA repair gene contributes to risk of developing SCCHN.

Base Sequence↗
PubMed1999

Genome-wide screen for systemic lupus erythematosus susceptibility genes in multiplex families.

Systemic lupus erythematosus (SLE) is the prototype of human autoimmune diseases. Its genetic component has been suggested by familial aggregation (lambdas = 20) and twin studies. We have screened the human genome to localize genetic intervals that may contain lupus susceptibility loci in a sample of 188 lupus patients belonging to 80 lupus families with two or more affected relatives per family using the ABI Prism linkage mapping set which includes 350 polymorphic markers with an average spacing of 12 cM. Non-parametric multipoint linkage analysis suggests evidence for predisposing loci on chromosomes 1 and 18. However, no single locus with overwhelming evidence for linkage was found, suggesting that there are no 'major' susceptibility genes segregating in families with SLE, and that the genetic etiology is more likely to result from the action of several genes of moderate effect. Furthermore, the support for a gene in the 1q44 region as well as in the 1p36 region is clearly found only in the Mexican American families with SLE but not in families of Caucasian ethnicity, suggesting that consideration of each ethnic group separately is crucial.

Chromosomes, Human, Pair 1↗
PubMed1999

Embryo and yolk compositional relationships in broiler hatching eggs during incubation.

Developmental relationships between yolk, embryo body, and embryo liver compositions during incubation were determined in two trials. In Trial 1, embryo body moisture, fat, and CP contents and embryo liver moisture and fat contents were determined. In Trial 2, relative yolk weights, moisture, fat, and fatty acid contents, relative wet and dry embryo weights and moisture contents, and relative wet and dry liver weights and moisture contents were determined. In Trial 1, embryo moisture decreased sigmoidally between Days 6 and 21, whereas embryo fat increased between Days 12 and 21 of incubation; embryo CP displayed sequential fluctuations throughout incubation. However, an overall significant decrease in embryo CP occurred between Days 6 and 21. Liver fat content increased between Days 12 and 21, whereas liver moisture decreased through Day 18, with a subsequent increase by Day 21. In Trial 2, relative yolk weight and moisture content decreased, whereas percentage yolk lipid content increased between Days 6 and 15. Relative wet and dry embryo weights changed in a similar manner, with rapid increases between Days 12 and 18 of incubation. Embryo moisture and CP were negatively correlated to embryo fat content. Furthermore, relative embryo and liver DM were related to yolk palmitic acid concentration, whereas yolk oleic acid was correlated only with liver DM. In conclusion, embryos and their livers displayed differential accumulations of moisture and DM during incubation, and these differences exhibited distinctive associations with various yolk fatty acids.

Animals↗