A simple method for site-directed mutagenesis with double-stranded plasmid DNA.
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DNA↗
Biomedical subjects
X Zhu
Publications and source records attributed to X Zhu.
A Knowledge-Based Systems Approach to Design of Spatial Decision Support Systems for Environmental Management
/ This paper describes a framework for designing spatial decision support systems for environmental management using a knowledge-based systems approach. An architecture for knowledge-based spatial decision supportsystems (KBSDSS) is presented that integrates knowledge-based systems with geographical information systems (GIS) and other problem-solving techniques. A method based on spatial influence diagrams is developed for representation of environmental problems. The spatial influence diagram provides an interface through which knowledge-based systems techniques can be applied to build capabilities for problem formulation, automated design, and execution of a solution process. In addition to the flexibility and developmental advantages of knowledge-based systems, the KBSDSS incorporates expert knowledge to provide assistance for structuring spatial influence diagrams and executing a solution process that automatically integrates the GIS, data base, knowledge base, and different types of models. The framework is illustrated with a system, known as the Islay Land Use Decision Support System (ILUDSS), designed to assist planners in strategic planning of land use for the development of the island of Islay, off the west coast of Scotland.KEY WORDS: Geographical information systems; Spatial decision support systems; Knowledge-based systems; Spatial influence diagrams; Environmental management
Journal Article↗
Differences in the 5.8S rDNA sequences among ascarid nematodes.
Sequences of the 5.8S rDNA were obtained for 14 species of nematode from different superfamilies and families within the order Ascaridida. All sequences were 157 bp in length. Sequence differences among species ranged from 0 to 18 bp (0-11.5%). A phenetic analysis of the sequence data groups the 14 taxa into their respective superfamilies and families, but does not discriminate fully at the subfamily level. A phylogenetic analysis of the sequence data failed to resolve the evolutionary relationships at the superfamily level. The 5.8S gene may be useful for phylogenetic studies of the phylum Nematoda at the ordinal level.
Animals↗
Dideoxy fingerprinting: application to the genotyping of Echinococcus.
Dideoxy fingerprinting is an efficient method for the detection of sequence variation in PCR-amplified DNA segments. It is a hybrid between single-strand conformation polymorphism and dideoxy sequencing, employing only one dideoxynucleotide in the sequencing reaction. Herein, we report the application of dideoxy fingerprinting to genetically type cestodes of the genus Echinococcus, utilising the mitochondrial cytochrome c oxidase subunit I as the gene sequence for analysis. All of the seven genotypes (G1, G4, G6, G8, O, V and M2) examined could be readily differentiated from one another by their characteristic and reproducible dideoxy fingerprinting profiles. Only subtle variation in profiles was detected among some of the eight isolates representing genotype G1, and no variation was detected between two samples of genotype G4 and of genotype M2. The capacity of dideoxy fingerprinting to detect all nucleotide variations over 150-250bp fragments indicates that it should be possible to distinguish among all of the genotypes of Echinococcus thus far described. Although employed herein to display sequence variation in the cytochrome c oxidase subunit I of Echinococcus, dideoxy fingerprinting could be used for the high-resolution analysis of nucleotide variations in other parasite genes, without the need for DNA sequencing. This has important implications for studying the genetic structure of parasite populations.
Animals↗
Characterisation of anisakid nematodes with zoonotic potential by nuclear ribosomal DNA sequences.
Larvae of three species of anisakid nematode from fish, Anisakis simplex, Hysterothylacium aduncum and Contracaecum osculatum, were characterised genetically using a molecular approach. The nuclear ribosomal DNA region spanning the first internal transcribed spacer, the 5.8S gene and the second internal transcribed spacer was amplified and sequenced. The lengths of the first and second internal transcribed spacer sequences of the three species ranged from 392 to 449 bp and 262 to 347 bp, respectively, whereas the 5.8S sequence was 157 bp. For the three species, the G+C contents for the three regions of ribosomal DNA ranged from 42.4 to 52.2%. While no intraspecific variation was detected in the second internal transcribed spacer or 5.8S sequence of any species examined, one polymorphic nucleotide position was detected in the first internal transcribed spacer sequence for A. simplex and H. aduncum. The extent of sequence differences in the first (approximately 34-45%) and second (approximately 50-53%) internal transcribed spacers among the species was greater than in the 5.8S gene (approximately 3-5%). Based on the sequence differences, PCR-based restriction fragment length polymorphism and single-strand conformation polymorphism methods were established for the unequivocal delineation of the three species. These methods should provide valuable tools for studying the life-cycle, transmission pattern(s) and population structure of each of the three anisakid nematodes examined herein, and for the diagnosis of anisakiasis in humans and animals.
Animals↗
CD95L confers immune priviledge to liver grafts which are spontaneously accepted.
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Animals↗
Spontaneous liver graft acceptance is mediated by intragraft FAS-ligand expression and viable passenger leukocytes.
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Animals↗
Primary associates of mortality among dialysis patients: trends and reassessment of Kt/V and urea reduction ratio as outcome-based measures of dialysis dose.
Information from a large clinical database was used to construct time trends for the leading associates of mortality among dialysis patients. The changing strengths of association of those measures with mortal risk were also evaluated. Strength did not change in meaningful ways for serum albumin, creatinine, or anion gap concentrations. It declined for the urea reduction ratio (URR), however, as prevalent values of the URR increased. Irrational patterns of association between the URR and other measures suggested reevaluation of the urea kinetic method for prescribing and judging dialysis dose. Two premises on which the urea kinetic equations rest are not valid if the context for their use is clinical outcome instead of predicting blood urea nitrogen (BUN) concentration. Rigorous use of the Kt/V criterion for dialysis dose could lead to clinical judgment errors, particularly underdialysis for small or malnourished persons. Changes for prescribing dose and judging therapy are recommended.
Acid-Base Equilibrium↗
Gland atrophy following retrograde injection of methyl violet as a treatment in chronic obstructive parotitis.
OBJECTIVE: The purpose of this study was to evaluate the clinical effectiveness of retrograde injection of 1% methyl violet as a treatment for chronic obstructive parotitis. STUDY DESIGN: Sixteen patients with chronic obstructive parotitis were treated with retrograde injection of 1% methyl violet. Pretreatment evaluation and assessment of the treatment effect were performed by clinical and imaging methods, including sialography and sonography of the treated glands. RESULTS: Clinical symptoms following treatment included initial acute swelling and then a decrease in parotid swellings; the final outcome was a clinical cure of the affected glands characterized by disappearance of all symptoms, absence of secretion, and complete obliteration of the main duct orifice. Posttreatment imaging showed total atrophy of the diseased glands. CONCLUSION: Retrograde injection of 1% methyl violet caused total atrophy of the diseased glands and brought about complete relief in all 16 patients, with no detectable side effects.
Adolescent↗
Eucalyptus has a functional equivalent of the Arabidopsis floral meristem identity gene LEAFY.
Two genes cloned from Eucalyptus globulus, Eucalyptus LeaFy (ELF1 and ELF2), have sequence homology to the floral meristem identity genes LEAFY from Arabidopsis and FLORICAULA from Antirrhinum. ELF1 is expressed in the developing eucalypt floral organs in a pattern similar to LEAFY while ELF2 appears to be a pseudo gene. ELF1 is expressed strongly in the early floral primordium and then successively in the primordia of sepals, petals, stamens and carpels. It is also expressed in the leaf primordia and young leaves and adult and juvenile trees. The ELF1 promoter coupled to a GUS reporter gene directs expression in transgenic Arabidopsis in a temporal and tissue-specific pattern similar to an equivalent Arabidopsis LEAFY promoter construct. Strong expression is seen in young flower buds and then later in sepals and petals. No expression was seen in rosette leaves or roots of flowering plants or in any non-flowering plants grown under long days. Furthermore, ectopic expression of the ELF1 gene in transgenic Arabidopsis causes the premature conversion of shoots into flowers, as does an equivalent 35S-LFY construct. These data suggest that ELF1 plays a similar role to LFY in flower development and that the basic mechanisms involved in flower initiation and development in Eucalyptus are similar to those in Arabidopsis.
Amino Acid Sequence↗
38 000 MW antigen-specific major histocompatibility complex class I restricted interferon-gamma-secreting CD8+ T cells in healthy contacts of tuberculosis.
CD8+ T lymphocytes are required to protect mice against Mycobacterium tuberculosis, although in early infection the mechanism appears not to be via perforin or granzyme-mediated lysis of the infected target, and may be via interferon-gamma (IFN-gamma) production. We therefore investigated whether CD8+ T cells specific for the immunoprotective 38 000 MW antigen of M. tuberculosis could be detected in infected humans. Using a recombinant vaccinia virus expressing the 38 000 MW antigen of M. tuberculosis (rV38) and a control vaccinia virus (rVras) we demonstrated that both viruses stimulated IFN-gamma production from freshly isolated peripheral blood mononuclear cells (PBMC) in a 36-hr enzyme-linked immunospot assay. Cell depletion and antibody blockade established that the bulk of the 38 000 MW antigen-specific IFN-gamma response was mediated by CD8+, major histocompatibility complex class I-restricted T cells, whereas the anti-vaccinia virus response was predominantly mediated by CD4+ T cells. In further evaluations PBMC from all seven healthy tuberculosis-exposed contacts had a 38 000 MW antigen-specific IFN-gamma response, whereas seven patients with untreated sputum-positive pulmonary tuberculosis had very low levels of 38 000 antigen-specific IFN-gamma-producing cells. These preliminary observations demonstrate the utility of recombinant vaccinia viruses in restimulating freshly isolated CD4+ and CD8+ T cells. The bias towards a higher frequency of IFN-gamma-producing CD8+ T cells in contacts rather than patients may indicate a protective role for CD8+ cells in human tuberculosis.
Adult↗
Recombinant fertilization antigen-1 causes a contraceptive effect in actively immunized mice.
Recombinant (r) fertilization antigen (FA)-1 was investigated for its immunocontraceptive effect using the mouse as a model. Active immunization with the murine rFA-1 antigen raised high antibody titers in all the immunized mice (n = 16 in two trials); these titers were long lasting and reached preimmunization levels by the 255th day. There was a significant (p < 0.0001) effect (64% reduction in trial I and 70% reduction in trial II) in fertility of immunized animals compared to PBS-control animals (n = 22 in two trials). The effect on fertility was reversible. When the antibody titers declined to control levels, all the animals conceived and delivered healthy babies without a significant (p > 0.05) effect on the litter size compared to that of controls. There was a significant (p = 0.025) correlation (r = 0.76) between the reduction in fertility and the circulating rFA-1 antibody titers. Anti-rFA-1 antibodies from immunized mice, and not the immunoglobulins from the PBS-control mice, significantly (p < 0.001) blocked murine sperm binding to zona pellucida and in vitro fertilization of murine oocytes. In a Western blot procedure, the anti-rFA-1 antibodies specifically recognized the protein band of approximately 47 kDa (dimeric form of cognate FA-1 antigen) only in the protein extract of testes and not in the extracts of somatic tissues tested, namely kidney, liver, intestine, spleen, muscle, heart, lung, brain, and ovary. In conclusion, our data indicate that active immunization with rFA-1 antigen induces a strong and sperm/testis-specific antibody response that causes a reversible inhibition of fertility by affecting sperm-zona binding and the fertilization process. These findings suggest that rFA-1 antigen is an exciting candidate for the development of a contraceptive vaccine.
Animals↗
Protein kinase C inhibition enhances platelet-activating factor-induced eicosanoid production in human eosinophils.
Previous investigations have suggested that protein kinase C (PKC) may regulate guinea pig eosinophil responses through a suppressive "negative feedback" mechanism. Using the selective PKC inhibitors bisindolylmaleimide I (Bis I, GF 109203X) and calphostin C, we examined the role of PKC in platelet-activating factor (PAF)-induced respiratory burst and generation of arachidonic acid metabolites in human peripheral blood eosinophils. Bis I inhibited PAF-induced generation of superoxide anion with substantially lower potency (geometric mean IC50 = 1.41 microM, 95% CI 0.94-2.11 microM) than it exhibited against responses to the phorbol esters 4-beta-phorbol 12-myristate 13-acetate (PMA; IC50 = 0.25 microM, 0.09-0.72 microM; P < 0.01) and 4-beta-phorbol 12,13-dibutyrate (IC50 = 0.48 microM, 0.20-1.14 microM; P < 0.05). The production of thromboxane (measured as TxB2) induced by 1 microM PAF was increased significantly by Bis I at concentrations of 1 microM (162 +/- 7.5% of control PAF response; P < 0.01) and 10 microM (194 +/- 17%; P < 0.001); TxB2 release induced by PMA was unaffected by concentrations of Bis I up to 1 microM and inhibited by 10 microM Bis I (48 +/- 11%; P < 0.05). Bis I (1 microM) significantly increased both thromboxane and leukotriene C4 (LTC4) production induced by 2 microM (P < 0.01 and P < 0.05, respectively) or 20 microM PAF (both P < 0.001). The actions of Bis I on PAF-stimulated thromboxane and leukotriene production were mimicked by a second PKC inhibitor, calphostin C, whereas the non-PKC-inhibitory analog, bisindolylmaleimide V, caused no enhancement of TxB2 or LTC4 production. The increase in intracellular free calcium induced by 1 microM PAF was heightened and prolonged in cells pre-treated with 1 microM Bis I or 1 microM calphostin C (peak increase, P < 0.05 for both drugs; level 60 s after addition of PAF, P < 0.001 and P < 0.05 for Bis I and calphostin C, respectively; time to return to 50% of peak, P < 0.05 for Bis I). We conclude that PKC inhibition causes augmentation of thromboxane and LTC4 production in PAF-stimulated human eosinophils despite suppressing respiratory burst activity, indicating that different signaling pathways predominate in these two responses and that PKC mediates a suppression of an early stage in an alternative pathway of activation.
Arachidonic Acid↗
Purification and characterization of a novel cysteine synthase isozyme from spinach hydrated seeds.
A novel type of cysteine synthase (CSase, EC 4.2.99.8) isozyme, designated as CSase 1', was purified to homogeneity from hydrated spinach seeds. The enzyme had a molecular weight of 68,000 and consisted of two identical subunits of M(r), 34,000. The apparent K(m) for O-acetyl-L-serine was 8.33 mM and that for sulfide was 0.66 mM. The activity of CSase 1' was maintained when it was treated at 60 degrees C for 1 min. This novel enzyme was similar to CSases 1, 2, and 3 already purified from spinach leaves, in results of double immunodiffusion, molecular weight, subunit composition, K(m) values for O-acetyl-L-serine and sulfide, and heat stability. On the other hand, N-terminal amino acid sequence, effects of immunotitration, pH optimum, and effects of hydroxylamine on purified CSase 1' were different from those of the other CSases. Furthermore, it was found that CSases 2S and 3S isolated from hydrated spinach seeds were identical with the CSases 2 and 3 reported previously. It was also disclosed that CSases 1, 2, and 3 were localized in chloroplasts, cytosol, and mitochondria, respectively.
Amino Acid Sequence↗
Complexes of serine acetyltransferase and isozymes of cysteine synthase in spinach leaves.
Polyclonal antibodies against cysteine synthase (CSase; EC 4.2.99.8) isozymes 1, 2, and 3 were used for the detection of complexes of these isozymes with serine acetyltransferase (SATase; EC 2.3.1.30). SATase was partially purified and found to complex with these isozymes by western blotting and immunotitration. When the complexes were treated with a high concentration of O-acetyl-L-serine, they did not dissociate. However, some complexes with CSase 1 or 3 dissociated when left for 24 h at 4 degrees C. Results of western blotting on SDS-PAGE showed that CSase 2 complexed with SATase. CSases 1, 2, and 3 all could complex with SATase, but the tightness of the bond differed.
Acetyltransferases↗
[Effects of plendil on serum oxygen free radicals and insulin resistance in patients with essential hypertension].
To evaluate the effects of plendia on oxygen free radicals and insulin resistance in patients with essential hypertension, thirty non-obesive moderate hypertensive patients were treated with plendil for 30 days. Antihypertensive effect and effects on serum lipid peroxide (LPO), superoxide dismutase (SOD), insulin sensitivity index (ISI) were studied. Results showed: 1. SOD activity and ISI decreased but contents of LPO and insulin increased in the treatment group. After a course of plendil therapy the above mentioned parameters fell to normal ranges. 2. Plendil had obvious antihypertensive effect but with no significant changes in heart rate. 3. Plendil had no significant effect on fasting blood glucose and lipids. Plendia was an effective and safe drug for treatment of essential hypertension. It also had the effects of antioxidation which improved the sensitivity to insulin.
Calcium Channel Blockers↗
The pathogenetic role of endogenous angiotensin II in stress ulcer in obstructive jaundice rats.
OBJECTIVE: To investigate the pathogenetic role of endogenous angiotensin II (Ang II) in the mechanism of stress ulcer in obstructive jaundice rats and to detect the effect of angiotensin converting enzyme inhibitor (ACEI) on stress ulcer in obstructive jaundice rats. METHODS: After common bile duct ligation (CBDL) in Wistar rats, the content of plasma and gastric mucosal Ang II, gastric mucosal blood flow (GMBF) and gastric mucosal damage were measured, and the relationship among them was analyzed. RESULTS: The plasma Ang II contents increased much more significantly at 1, 3, 7 and 14 days following CBDL than those in non-CBDL rats (P < 0.05, < 0.01, < 0.01 and < 0.01, respectively). Within 120 minutes following cold-restraint stress, plasma and gastric mucosal Ang II contents were elevated, GMBF decreased, and ulcer index and gastric mucosal damage increased more significantly than those in non-cold-restraint stress rats (P < 0.05, < 0.05, < 0.01, < 0.01 and < 0.05, respectively). Administration of an ACEI, enalaprili, to CBDL rats (5 mg.kg-1.day-1, orally for two days) before stress reduced both the plasma and gastric mucosal Ang II levels, inhibited the decrease of GMBF and decreased ulcer index and gastric mucosal damage (P < 0.001, < 0.01, < 0.01, < 0.01 and < 0.05, respectively). CONCLUSION: The endogenous Ang II plays a significant pathogenetic role in the development of stress ulcer in obstructive jaundice rats, and ACEI may prevent stress ulcer.
Angiotensin II↗
[Experimental study of proliferation effects of platelet derived wound healing factor on tissue cells of wound].
This experiment was designed to observe the proliferative effects of platelet derived wound healing factor (PDWHF) of different concentrations on fibroblasts from rat wounds and on epithelial from human wounds. Cultured fibroblasts from rat wound and epithelia from human wound were randomly divided into three groups. (1) In blank control, the cells were treated with basic medium (BM, contains 1640/0.5% FCS); (2) the positive control, the cells were treated with 1640/10% FCS and (3) in the PDWHF group, the cells were treated respectively with BM/1% PDWHF, BM/3% PDWHF, BM/5% PDWHF, BM/7% PDWHF, BM/10% PDWHF, BM/12% PDWHF, respectively. The Cells were collected after 48 hours culturing with BM or PDWHF, and the cell proliferation was measured by MTT method according to the OD values. The result showed that the PDWHF could remarkably enhance the proliferation of fibroblasts and epithelial cells when its concentration was between 1% and 7%, which was obviously higher than that of the blank control (P < 0.01). When the concentration of PDWHF reached 10%, its proliferative effect was not remarkable when compared with the blank control, When the concentration of PDWHF reached 12%, it showed inhibitory effect on fibroblasts and manifested no obvious inhibitory effect on epithelial cells. It was concluded that the PDWHF was a combination of a variety of growth factors. In a certain range of concentration, the PDWHF might effectively promote the proliferation of fibroblasts and epithelial cells. Howerve, when its concentration reached to relatively higher level, its effect was not remarkable any more, or even showed inhibitory effect on cell proliferation.
Animals↗