Search PubMed⌕ Search

Biomedical subjects

D Liu

Publications and source records attributed to D Liu.

At least 595 records · Page 33Linked to original sources
PubMed1995

Rapid identification of Acremonium lolii and Acremonium coenophialum endophytes through arbitrarily primed PCR.

Using a random decamer 5'-CCGAGGTGAC-3' in an arbitrarily primed PCR, similar band patterns were observed between Acremonium lolii and A. coenophialum DNA, which were somewhat different from those formed by other fungal DNA. Despite sharing bands of around 0.7, 0.9 and 2.1 kb, A. lolii can be distinguished from A. coenophialum by the presence of an additional band at around 0.5 kb in the arbitrarily primed PCR.

Acremonium↗
PubMed1995

Evidence that pH-titratable groups control the activity of a large epithelial chloride channel.

The effects of pH changes were examined on the properties of a large, voltage dependent, epithelial Cl- channel from bovine tracheal cells. Alkaline solutions in the range pH = 7.4-9.2 had no detectable effects on channel conductance or gating. However, acid solutions significantly reduced channel open probability, raising the voltage required to open the channel. Analysis of channel activity in the acidic pH range suggested that at least one charged group on the channel with an apparent pK = 6.09, is responsible for its voltage dependence. Neutralization of this charge does not eliminate the voltage dependence, but changes the energy difference between the closed and open states. The absence of any change in channel conductance over this wide pH range suggests that the protonation site is far removed from the channel permeation pathway.

Animals↗
PubMed1995

Discovery of an epidermal stearoyl-acyl carrier protein thioesterase. Its potential role in wax biosynthesis.

Plant epicuticular, or surface, waxes are synthesized primarily, if not exclusively, by epidermal cells. The epicuticular wax constitutes almost 20% of the chloroform-extractable lipids in developing leek leaf and is derived predominantly from saturated fatty acids. The significant requirement for saturated fatty acids in epidermal tissues led us to investigate whether or not epidermal extracts have thioesterase activities that prefer saturated acyl-acyl carrier protein (ACP) substrates, rather than the 18:1-ACP more commonly hydrolyzed by total leaf extracts. Epidermal extracts from Brassica, pea, and leek exhibited higher activities toward saturated acyl-ACPs relative to 18:1-ACP when compared to total leaf or leaf parenchymal extracts. We identified and purified a stearoyl-ACP (18:0-ACP)-specific thioesterase from leek epidermal extracts which could be separated from 18:1-ACP thioesterase using hydroxyapatite chromatography. The stearoyl-ACP thioesterase exhibited a high preference for 18:0-ACP, having less than 10% of the 18:0-ACP hydrolyzing activity when presented with 18:1-ACP, 16:0-ACP, or 18:0-CoA substrates. The stearoyl-ACP thioesterase was predominantly, if not exclusively, expressed in epidermis and may play a role in generating the saturated fatty acid pool required for wax production.

Cross Reactions↗
PubMed1995

Crystal structure of the zeta isoform of the 14-3-3 protein.

The 14-3-3 family of proteins have recently been identified as regulatory elements in intracellular signalling pathways: 14-3-3 proteins bind to oncogene and proto-oncogene products, including c-Raf-1 (refs 2-5), c-Bcr (ref. 6) and polyomavirus middle-T antigen; overexpression of 14-3-3 activates Raf kinase in yeast and induces meiotic maturation in Xenopus oocytes. Here we report the crystal structure of the major isoform of mammalian 14-3-3 proteins at 2.9 A resolution. Each subunit of the dimeric protein consists of a bundle of nine antiparallel helices that form a palisade around an amphipathic groove. The groove is large enough to accommodate a tenth helix, and we propose that binding to an amphipathic helix represents a general mechanism for the interaction of 14-3-3 with diverse cellular proteins. The residues in the dimer interface and the putative ligand-binding surface are invariant among vertebrates, yeast and plants, suggesting a conservation of structure and function throughout the 14-3-3 family.

14-3-3 Proteins↗
PubMed1995

Recognition and clearance of liposomes containing phosphatidylserine are mediated by serum opsonin.

Liver uptake of liposomes containing phosphatidylserine was studied in a single pass liver perfusion system and found to be serum dependent. The effectiveness of serum in mediating liposome uptake by the liver depends on liposomes size. Large liposomes appeared to be opsonized more efficiently and, therefore, taken up more by the liver than the smaller ones. The effects of liposomes size on liver uptake did not occur in the absence of serum. Treatment of serum at 56 degrees C for 30 min abolished the serum activity, suggesting the involvement of complement components. Inhibition of the hemolytic activity of complement through the alternative pathway by PS-containing liposomes suggests that components in this pathway are responsible for liposome opsonization. Liposomes containing phosphatidic acid, phosphatidylglycerol, and dicetyl phosphate compete in different degrees for serum components which mediate the liver uptake of PS-containing liposomes. These results suggest that the opsonization of liposomes by serum opsonins are the determining factors for the recognition and clearance of liposomes by the RES. Complement components are most likely involved in this process. The results presented here are relevant to the use of liposomes as drug delivery vehicle in vivo and to the PS-mediated clearance of red blood cells from the blood circulation.

Animals↗
PubMed1995

Assay of pancreatic lipase with the surface acoustic wave sensor system.

A new type of surface acoustic wave sensor system for assaying the activity of pancreatic lipase has been proposed. The assay of this enzyme is based on the change in conductance of the solution caused by the release of a fatty acid, using triolein as a substrate. A linear relationship between frequency response and enzyme concentration is obtained. Kinetic parameters of pancreatic lipase, i.e., the Michaelis constant for triolein and the corresponding maximum initial rate, are estimated to be 5.46 mM and 7551.6 Hz/min, respectively. The effects of temperature and pH value are also investigated.

Acoustics↗
PubMed1995

Organisation and gel contraction by human colonic carcinoma (HCA-7) sublines grown in 3-dimensional collagen gel.

The role of cell-matrix interactions in controlling phenotypic heterogeneity in human colonic carcinoma sublines has been investigated. Four cell lines (colony 1, colony 3, colony 6 and colony 30) previously isolated from a single human colonic carcinoma cell line, HCA-7, were grown in 3-dimensional collagen gels. In collagen, the growth of the 4 sublines ranged from well-organised glandular structures (colony 30) to elongated branching structures (colony 3). The capability of cells to organise into glandular structures in collagen correlated with the degree of differentiation observed in their xenografts. Certain sublines, most notably colony 3, were able to contract the collagen gel. Gel contraction could be partially inhibited by a function-blocking antibody directed to the alpha 2 integrin chain but not by an antibody directed to the alpha 3 integrin chain demonstrating a role for alpha 2 integrin in the contraction process. In addition, colony 3 cultures treated with the function-blocking alpha 2 antibody formed more compact structures with limited outgrowth, suggesting a role for alpha 2 integrin in cell migration. Gel contraction and cell migration in collagen gel was largely restricted to 1 subline, colony 3. The subsequent demonstration that alpha 2 integrin is involved in both of these processes suggests that integrin expression and function has a role in generating the phenotypic heterogeneity exhibited by these cell lines.

Collagen↗
PubMed1995

Loligomers: design of de novo peptide-based intracellular vehicles.

Defined branched peptides (loligomers) incorporating cytoplasmic translocation signals, nuclear localization sequences, and fluorescent probes were designed and synthesized to demonstrate the feasibility and simplicity of creating novel classes of intracellular vehicles. Loligomers containing all the above signals were rapidly internalized by Chinese hamster ovary (CHO) cells and accumulated in their nucleus. At 4 degrees C, the interaction of peptide constructs with CHO cells was limited to membrane association. Loligomers entered cells at higher temperatures by adsorptive endocytosis. Inhibitors of ATP synthesis affected cytoplasmic import only weakly but abolished nuclear uptake. The peptide signals guided both cytoplasmic and nuclear localization events. The properties exhibited by loligomers suggest a strategy for the facile design of "guided" classes of intracellular agents.

Amino Acid Sequence↗
PubMed1995

High-affinity monoclonal antibodies against P-glycoprotein.

Cancer patients treated with one anticancer agent often develop resistance to a broad spectrum of chemotherapeutic agents. This type of multiple drug resistance (MDR) is often accompanied by a decrease in drug accumulation and an increase in expression of a 170,000-Da plasma membrane glycoprotein (P-170) that can effectively pump various anticancer agents out of cytoplasm. A panel of 12 IgG1, IgG2a, or IgG2b monoclonal antibodies was generated against the extracellular portion of P-glycoprotein by immunizing mice with a human MDR1 gene-transfected BA3T3 fibroblast line. We have characterized two of the anti-P-glycoprotein monoclonal antibodies, 15D3 and 17F9, in some detail. Both antibodies immunoprecipitate a 170- to 180-kDa protein from MDR cells, but do not block binding of the known anti-P-glycoprotein antibody MRK16, suggesting that 15D3 and 17F9 bind to a different epitope on the extracellular domain of P-glycoprotein than MRK16. Scatchard analysis revealed that 15D3 and 17F9 had association constants of 1.3 and 1.1 x 10(8) M-1, respectively. 15D3 and 17F9 had little effect on MDR cell growth except for a minor inhibition of KB-V1 cells when the cells were incubated in the presence of vinblastine. Neither antibody inhibited the efflux of P-glycoprotein substrates from MDR cells. Because of their strong binding activity, these antibodies may be useful for diagnostic detection of MDR in patients undergoing chemotherapy or as targeting components of immunotherapeutic agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Fine structure and function of the osmotin gene promoter.

The gene encoding osmotin, a tobacco pathogenesis-related protein, has been shown to be regulated by an array of hormonal and environmental signals. The osmotin promoter fragment -248 to -108 upstream of the transcription start site (fragment A), was sufficient to direct reporter gene expression when fused to a minimal CaMV 35S promoter in transient assays using microprojectile bombardment. This was consistent with previous 5'-deletion analyses of the osmotin promoter which showed that the promoter sequence from -248 to -108 is absolutely required for reporter gene activity. Nuclear protein factors from salt-adapted tobacco cells, ABA-treated unadapted cells, and young cultured tobacco leaves were shown to interact with fragment A by gel mobility-shift assays. DNase I footprinting revealed that three conserved promoter elements in fragment A interact specifically with nuclear factors. These elements are: (1) a cluster of G-box-like sequences (G sequence); (2) an AT-1 box-like sequence, 5'-AATTATTTTATG-3' (AT sequence); (3) a sequence highly conserved in ethylene-induced PR gene promoters, 5'-TAAGA/CGCCGCC-3' (PR sequence). Transient expression assays performed with fragment A deletions fused to GUS indicated that osmotin promoter activity correlated with the presence of these elements. UV cross-linking analysis showed that the protein complex bound to fragment A consisted of at least four individual proteins with approximate molecular masses of 28, 29, 40 and 42 kDa. One component of this protein complex, which was associated with the G sequence, was a 14-3-3 like protein.

Base Sequence↗
PubMed1995

Changes in E-cadherin immunoreactivity in the adenoma-carcinoma sequence of the large bowel.

We have used an avidin-biotin immunoperoxidase technique to localise epithelial cadherin (E-cadherin), a calcium-dependent cell-cell adhesion molecule, in 107 paraffin-embedded sections from 93 patients consisting of 24 with colorectal adenoma, 55 with rectal carcinoma and 14 with liver metastases. The corresponding primary colorectal tumours were also studied in these cases. E-cadherin was expressed by normal colorectal epithelial cells with typical membranous staining at the intercellular junctions. Loss of normal membranous E-cadherin expression and presence of cytoplasmic staining were found frequently in adenomas larger than 1 cm (P < 0.01), with high grade dysplasia and villous histology (P < 0.01). In primary rectal cancers, loss of membranous expression correlated with high tumour grade. No correlation was seen with Dukes and Jass stage, local extramural spread and 5-year recurrence rate. Complete loss of membranous E-cadherin immunoreactivity was seen in 7/14 (50%) liver metastases in which 6/7 (86%) showed intense membranous E-cadherin immunoreactivity in the corresponding primary tumour. Our data indicate that changes in E-cadherin immunoreactivity and cellular localisation correlate with size, severe dysplasia in adenomas and tumour grade in carcinomas. However, there seems to be no correlation between loss of membranous E-cadherin immunoreactivity and the invasive and metastatic potential of the carcinomas.

Adenoma↗
PubMed1995

Relationship between clinical manifestations of footrot and specific DNA products of Dichelobacter nodosus amplified through PCR.

A total of 141 Dichelobacter nodosus isolates from 46 merino sheep farms with various clinical forms of footrot was examined by the gelatin gel test and the polymerase chain reaction (PCR) using virulent (Vf2 and Vr2) and benign (Bf and Br) specific primers. Isolates from sheep with virulent and high intermediate footrot usually produced relatively thermostable proteases, but a decreasing proportion of the isolates from sheep with medium and low intermediate or benign footrot had thermostable proteases, as determined by the gelatin gel test. The amplification by PCR of a major band of 857 bp by Vf2 and Vr2 was often associated with isolates from the more virulent forms of footrot whereas the presence of a major band of 1300 bp by Vf2 and Vr2 and/or a band of 609 bp by Bf and Br was associated with isolates from less virulent forms of footrot. Nevertheless, the virulent and benign gene regions represented by Vf2 and Vr2 and Bf and Br are only two of the many factors involved in determining the virulence of D nodosus. As a result the relationship observed between the clinical manifestations of footrot and specific DNA products amplified by PCR was not complete.

Animals↗
PubMed1995

A polymerase chain reaction assay for improved determination of virulence of Dichelobacter nodosus, the specific causative pathogen for ovine footrot.

Three sets of oligonucleotide primers (Vf1 and Vr1, Vf2 and Vr2, Bf and Br) were derived from a D. nodosus virulent-specific clone pV470-13 (2146 bp) and a benign-specific clone pB645-335 (737 bp), respectively. Using the virulent-specific primers Vf1 and Vr1 in a polymerase chain reaction (PCR) enabled amplification of a DNA fragment of 460 bp in 25/27 virulent, 9/25 high intermediate, 9/24 low intermediate and 2/20 benign isolates of D. nodosus. On the other hand, using the second set of the virulent-specific primers Vf2 and Vr2 resulted in the production of either a fragment of 857 bp or 1300 bp in virulent, intermediate and 3/20 benign isolates. There appeared to be some correlation between the amplification of the smaller fragment (857 bp) and higher elastase activity and between the amplification of the larger fragment (1300 bp) and lower elastase activity of D. nodosus. The use of the benign-specific primers Bf and Br in PCR enabled amplification of a fragment of 609 bp in all 20 benign, 23/24 low intermediate, 20/25 high intermediate and 0/27 virulent isolates. The combination of the virulent-specific primers Vf2 and Vr2 and benign-specific primers Bf and Br in PCR would provide a rapid, specific and sensitive differentiation of strains of D. nodosus causing virulent, intermediate or benign footrot.

Animals↗
PubMed1995

Prostaglandin F2 alpha rises in response to hydroxyl radical generated in vivo.

Free radicals and some free fatty acids, such as arachidonic acid metabolites, have been hypothesized to be contributors to secondary damage to the spinal cord upon injury. These two types of species may form a feedback loop in which generation of one type leads to formation of the other. In this study, to determine whether hydroxyl radical causes generation of arachidonic acid metabolites in vivo, we generated hydroxyl radical, a most reactive oxygen radical, in the rat spinal cord and measured resulting changes in levels of prostaglandin F2 alpha, an arachidonic acid metabolite that rises following traumatic injury. The hydroxyl radical was generated in the rat spinal cord by administering H2O2 through one microdialysis fiber and FeCl2/EDTA through a parallel fiber. The prostaglandin F2 alpha in the collected microdialysates was measured by HPLC as its 3-bromomethyl-6,7-dimethoxy-1-methyl-2-(1H)-quinoxalinone derivative. Prostaglandin F2 alpha dramatically increased in response to hydroxyl radical generation, but declined substantially after 3 h of exposure. Prostaglandin F2 alpha was undetectable when either H2O2 or FeCl2/EDTA was administered alone in control experiments, demonstrating that its formation was caused by generated hydroxyl radical.

Animals↗