Biomedical subjects

Lixia Liu

Publications and source records attributed to Lixia Liu.

11 recordsLinked to original sources

PubMed2005

Development of a shuttle vector for Moraxella catarrhalis.

Efforts to perform genetic analysis in Moraxella catarrhalis have been hampered by the lack of a cloning vector. M. catarrhalis strain E22 was previously shown to contain plasmid pLQ510 which lacked a selectable antibiotic resistance marker. Several methods were used to eliminate unnecessary DNA from pLQ510. Then, a 1.2 kb spectinomycin resistance cartridge, a multiple cloning site, and the origin of replication from pACYC184 were cloned into this plasmid backbone to obtain the 7.2 kb plasmid pWW102B. This new plasmid could replicate in M. catarrhalis as well as in both Escherichia coli and Haemophilus influenzae. This shuttle vector was used to clone and express two different M. catarrhalis genes, respectively, encoding an adhesin and a protein involved in serum resistance. When these two plasmids were introduced into appropriate M. catarrhalis mutants, they complemented the phenotypic deficiency of each mutant. This is the first report of functional complementation in trans in this pathogen.

Adhesins, Bacterial↗

PubMed2005

Preparation and self-assembly of uniform TiO2/SiO2 composite submicrospheres.

Monodisperse spheres of silica were uniformly coated with titania through the hydrolysis of titanium alkoxide in order to increase the refractive index of complex submicrospheres and keep their monodispersity as well as their surface morphology. On the basis of the hydrolysis of tetrabutyl orthotitanate (TBOT), a continuous-feeding procedure was used to fabricate 10-nm titania coatings on monodisperse colloidal silica submicrospheres of diameter 200 nm. The TiO2/SiO2 composite spheres were assembled to achieve structures with three-dimensional order by gravity sedimentation and vertical deposition. The complex sphere composition, quality, and morphology were characterized by different techniques.

Journal Article↗

PubMed2005

SCAMP2 interacts with Arf6 and phospholipase D1 and links their function to exocytotic fusion pore formation in PC12 cells.

SNAP receptor (SNARE)-mediated fusion is regarded as a core event in exocytosis. Exocytosis is supported by other proteins that set up SNARE interactions between secretory vesicle and plasma membranes or facilitate fusion pore formation. Secretory carrier membrane proteins (SCAMPs) are candidate proteins for functioning in these events. In neuroendocrine PC12 cells, SCAMP2 colocalizes on the cell surface with three other proteins required for dense-core vesicle exocytosis: phospholipase D1 (PLD1), the small GTPase Arf6, and Arf6 guanine nucleotide exchange protein ARNO. Arf6 and PLD1 coimmunoprecipitate (coIP) with SCAMP2. These associations have been implicated in exocytosis by observing enhanced coIP of Arf6 with SCAMP2 after cell depolarization and in the presence of guanosine 5'-O-(3-thio)triphosphate and by inhibition of coIP by a SCAMP-derived peptide that inhibits exocytosis. The peptide also suppresses PLD activity associated with exocytosis. Using amperometry to analyze exocytosis, we show that expression of a point mutant of SCAMP2 that exhibits decreased association with Arf6 and of mutant Arf6 deficient in activating PLD1 have the same inhibitory effects on early events in membrane fusion. However, mutant SCAMP2 also uniquely inhibits fusion pore dilation. Thus, SCAMP2 couples Arf6-stimulated PLD activity to exocytosis and links this process to formation of fusion pores.

ADP-Ribosylation Factor 6↗

PubMed2005

Epigenetic alterations in neuroendocrine tumors: methylation of RAS-association domain family 1, isoform A and p16 genes are associated with metastasis.

Well-differentiated neuroendocrine tumors including pancreatic endocrine tumors and carcinoid tumors are uncommon neoplasms that have site-specific differences in clinicopathological features, clinical course and genetic alterations. The epigenetic alterations in these tumors are not well characterized. We therefore compared methylation of the RAS-association domain family 1, isoform A (RASSF1A), p14, p16 and O6-methyl-guanine methyltransferase genes in neuroendocrine tumors from 47 patients including 16 pancreatic, 15 nonileal and 16 ileal neuroendocrine tumors. Methylation of the RASSF1A gene was present in 57% of tumors, p14 in 49%, p16 in 26% and O6-methyl-guanine methyltransferase in 13% of tumors. Ileal neuroendocrine tumors lacked methylation of O6-methyl-guanine methyltransferase gene (P = 0.04). RASSF1A methylation was associated with histopathologic type of tumors (P = 0.03) and lymph node metastasis (P = 0.004), and p16 methylation with older patient age (P = 0.002) and liver metastasis (P = 0.04). Two or more genes were methylated in 53% of tumors, one gene was methylated in 30% of tumors, and all four genes were unmethylated in 17% of tumors. Methylation of one or more gene was associated with older age of patients (P = 0.01), and methylation of two or more genes was associated with liver metastasis (P = 0.044). Our study shows that in neuroendocrine tumors epigenetic alterations vary by tumor subsite and clinicopathologic features, including age of onset, histopathologic type and metastasis status.

Aged↗

PubMed2005

Significance of the depth of tumor invasion and lymph node metastasis in superficially invasive (T1) esophageal adenocarcinoma.

Superficially invasive esophageal adenocarcinomas are a heterogeneous group of tumors, including tumors invading into mucosa and submucosa. The prognostic significance of the depth of tumor invasion and lymph node status in this group of patients remain unclear. We evaluated 90 consecutive patients with resected T1 adenocarcinoma of esophagus or esophagogastric junction. The T1 tumors were classified into four groups based on the depth of invasion: T1a, invading into lamina propria; T1b, into muscularis mucosae; T1c, into superficial submucosa; and T1d, into deep submucosa. The depth of tumor invasion was compared with clinicopathologic features. The depth of tumor invasion was significantly associated with the presence of lymph node metastasis (36% in T1d, 8% in T1c, 12% in T1b, and 0% in T1a; P < 0.001) and with tumor size (76% > 1.2 cm in T1d, 75% in T1c, 35% in T1b, and 25% in T1a; P < 0.001). The 5-year recurrence-free and overall survivals were significantly better in patients with tumors confined to mucosa (100% and 91%, respectively) than invasive into submucosa (60% and 58%; P = 0.0005 and P = 0.02, respectively). Lymph node metastasis was associated with tumor recurrence (P = 0.01) but not overall survival. Lymphovascular invasion was associated with both tumor recurrence (P = 0.001) and overall survival (P < 0.001) and was an independent prognostic factor in multivariate analysis (P = 0.04). Our study indicated evaluation of depth of tumor invasion, status of lymph nodes, and lymphovascular invasion is important in resected superficially invasive esophageal adenocarcinoma and may provide supportive information for the decision about postoperative adjuvant therapy.

Adenocarcinoma↗

PubMed2003

Identification by subtractive hybridization of a novel insertion element specific for two widespread Burkholderia cepacia genomovar III strains.

Species of the Burkholderia cepacia complex cause chronic and life-threatening infections in persons with cystic fibrosis. Epidemic strains infect multiple patients, reside primarily in genomovar III, and have an apparent enhanced capacity for human infection and/or interpatient transmission. By using subtractive hybridization, a novel insertion element, designated IS1363, was identified in epidemic strain PHDC, known to infect many cystic fibrosis patients in the mid-Atlantic region of the United States. IS1363 was also found in most isolates of the ET12 lineage, responsible for infecting large numbers of patients in Ontario, Canada, and the United Kingdom. Southern blot analysis demonstrated that whereas multiple copies of IS1363 were present in strain PHDC, only one copy was present in ET12 isolates. IS1363 was used to probe a collection of 943 B. cepacia complex isolates, representing all nine genomovars, recovered from 761 cystic fibrosis patients or the natural environment. IS1363 was not found in other genomovar III strains and, with the exception of B. ambifaria, was absent from other B. cepacia complex species. Genotyping analyses of all IS1363-positive isolates demonstrated that strain PHDC was more widely distributed in the United States than previously appreciated; 212 cystic fibrosis patients in 24 states were identified as being infected with PHDC.

Bacterial Typing Techniques↗

PubMed2002

Perturbation of a very late step of regulated exocytosis by a secretory carrier membrane protein (SCAMP2)-derived peptide.

Secretory carrier membrane proteins (SCAMPs) are conserved four transmembrane-spanning proteins associated with recycling vesicular carriers. In mast cells, as in other cell types, SCAMPs 1 and 2 are present in secretory granule membranes and other intracellular membranes. We now demonstrate a population of these SCAMPs in plasma membranes. Although small, this population partially colocalizes with SNARE proteins SNAP-23 and syntaxin 4. A fraction of SCAMPs 1 and 2 also coimmunoprecipitates with SNAP-23. An oligopeptide, E peptide, within the cytoplasmic segment linking the second and third transmembrane spans, particularly of SCAMP2, potently inhibits exocytosis in streptolysin O-permeabilized mast cells. The E peptide is unique to SCAMPs and highly conserved among SCAMP isoforms, and minor changes in its sequence abrogate inhibition. It blocks fusion beyond the putative docking step where granules contact the cell surface and each other during compound exocytosis. Blockade is also beyond Ca(2+)/ATP-dependent relocation of SNAP-23, which regulates compound exocytosis, and beyond ATP-dependent priming of fusion. Kinetic ordering of exocytotic inhibitors has shown that E peptide acts later than other perturbants at a stage closely associated with membrane fusion. These findings identify a new reagent for analyzing the final stage of exocytosis and point to the likely action of SCAMP2 in this process.

Amino Acid Sequence↗

PubMed2002

Function of the t-SNARE SNAP-23 and secretory carrier membrane proteins (SCAMPs) in exocytosis in mast cells.

Mast cells acutely respond to allergens and other stimuli by releasing accumulated internal stores of inflammatory mediators and other secretory products by "compound" exocytosis involving massive granule-to-plasma membrane and granule-to-granule fusion. Our recent findings implicate the SNAP receptor (SNARE) protein SNAP-23 and secretory carrier membrane protein 2 (SCAMP2) as regulators of this process. We summarize evidence indicating that stimulus-induced relocation of SNAP-23 from foci in the plasma membrane to putative sites of membrane fusion both at the cell surface and intracellularly between granules is an essential link in coupling stimulation to exocytosis. We also review recent findings showing that the candidate exocytotic SNAREs SNAP-23 and syntaxin 4 colocalize with SCAMPs, especially SCAMP2, and that SCAMP2 is likely to be a partner in the compound exocytotic process.

Animals↗

PubMed2002

Role of secretory carrier membrane protein SCAMP2 in granule exocytosis.

In secretory carrier membrane proteins (SCAMPs), the most conserved structural segment is between transmembrane spans 2 and 3, facing the cytosol. A synthetic peptide, CWYRPIYKAFR (E peptide), from this segment of SCAMP2 potently inhibits exocytosis in permeabilized neuroendocrine (PC12) cells. E peptide blocked discharge of (35)S-labeled secretogranin with the same structural selectivity and potency as observed for hexosaminidase secretion in mast cells. SCAMPs 1 and 2 are concentrated primarily on intracellular membranes in PC12 cells. Both, however, are found on plasma membranes, but neither is present on large dense-core vesicles. Yet, large dense-core vesicles marked by secretogranin attach to plasma membranes at foci containing SCAMP2 along with syntaxin1 and complexin at putative cell-surface docking/fusion sites. Regulated overexpression of SCAMP2 with point mutations in its E peptide but not of normal SCAMP2 caused dose-dependent inhibition of depolarization-induced secretion. The SCAMP2 mutants also inhibited secretion stimulated by elevated calcium. Inhibition was largely overcome by adding lysophosphatidylcholine to the medium at concentrations that do not otherwise affect secretion. Although overexpression of normal or mutant SCAMP2 slightly inhibits endocytosis, this effect does not appear to be related to the specific effect of the mutant SCAMP on stimulated exocytosis. Thus, SCAMP2 not only colocalizes with fusion sites but also appears to have an essential function in granule exocytosis through actions mediated by its E peptide-containing domain.

Adaptor Proteins, Vesicular Transport↗

PubMed2002

Lack of cable pili expression by cblA-containing Burkholderia cepacia complex.

The Burkholderia cepacia complex consists of several closely related bacterial species (or genomovars) which although generally not pathogenic for healthy individuals, contribute significantly to morbidity and mortality among persons with cystic fibrosis (CF). Certain B. cepacia complex strains are more frequently recovered from CF sputum cultures than are others, and these typically reside in genomovar III. The ET12 clone is a genomovar III strain that predominates among CF patients in Canada and the United Kingdom and is characterized by distinctive cblA-encoded pili that have a cable-like morphology. In a previous survey of B. cepacia complex isolates recovered from 606 CF patients in the US, a single genomovar III ET12 isolate (isolate AU0007) was identified; several cblA-containing genomovar I isolates, however, were also detected. In the study reported here, analysis by PFGE revealed several distinct strain types among these genomovar I isolates, and sequence analysis of their cblA genes demonstrated 87.8-88.4% identity to the ET12 cblA sequence. Southern analysis indicated that the cblA variant from each genomovar I isolate resides on a 4 kbp EcoRI fragment, in contrast to ET12 isolates, in which cblA localizes to a 5 kbp EcoRI fragment. Western blot assay indicated expression of the 16 kDa major pilin subunit by ET12 isolates, including AU0007, but neither whole-cell nor surface-protein extracts of the genomovar I reacted. Electron microscopy revealed the complete absence of pili expression by the genomovar I isolates. In contrast to typical ET12 isolates, AU0007 appeared to be hyperpiliated with rigid pili that lacked the cable morphology and did not bind cytokeratin 13, which has been previously identified as the epithelial cell receptor for the ET12 cable-pili-associated adhesin.

Amino Acid Sequence↗

PubMed2002

Ribosomal DNA-directed PCR for identification of Achromobacter (Alcaligenes) xylosoxidans recovered from sputum samples from cystic fibrosis patients.

The opportunistic human pathogen Achromobacter (Alcaligenes) xylosoxidans has been recovered with increasing frequency from respiratory tract culture of persons with cystic fibrosis (CF). However, confusion of this species with other closely related respiratory pathogens has limited studies to better elucidate its epidemiology, natural history, and pathogenic role in CF. Misidentification of A. xylosoxidans as Burkholderia cepacia complex is especially problematic and presents a challenge to effective infection control in CF. To address the problem of accurate identification of A. xylosoxidans, we developed a PCR assay based on a 16S ribosomal DNA sequence. In an analysis of 149 isolates that included 47 A. xylosoxidans and several related glucose-nonfermenting species recovered from CF sputum, the sensitivity and specificity of this PCR assay were determined to be 100 and 97%, respectively. The availability of this assay will enhance identification of A. xylosoxidans, thereby facilitating study of the pathogenic role of this species and improving infection control efforts in CF.

Alcaligenes↗