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Biomedical subjects

X Gu

Publications and source records attributed to X Gu.

At least 199 records · Page 11Linked to original sources
PubMed1997

Two novel mutations in phenylalanine hydroxylase gene and in vitro expression analysis on mutation Arg252Gln.

We report novel mutations in exon 7 of human phenylalanine hydroxylase (PAH) gene of phenylketonuria (PKU) in southern Chinese, analysed by using PCR-DGGE (denaturing gradient gel electrophoresis), solid phase DNA sequencing and in vitro expression. One of the 2 novel mutations, IVS6nt-1, is an intron-exon junctional mutation which results a splicing defect in mRNA. Arg252Gln is another novel mutation with residual PAH activity only 24% compared to wild type in in vitro mutagenesis and expression in Cos-1 cell. Other 3 known mutations and polymorphism including Arg241Cys, Arg243Gln and Val245Val (GTG to GTA) together with these novel mutations composed the mutational profile of exon 7 in the PAH gene of PKUs in this populations.

Exons↗
PubMed1997

[In vitro effects of hematopoietic growth factors on CD34+ cell from patients with myelodysplastic syndromes].

OBJECTIVE: To investigate the role of recombinant human hematopoietic growth factors (HGFs) in the treatment of myelodysplastic syndromes (MDS) patients. METHODS: CD34+ cells from 16 MDS patients were purified up to 96% approximately 99% by means of a immunomagnetic beads sorting system. The effects of individual and combination of HGFs on the proliferation and differentiation of the CD34+ cells were studied in semi -solid cultures. RESULTS: CD34+ cells were impaired and none of the individual HGF (GM-CSF, IL-3, SCF) could effectively support their proliferation and differentiation. rh-SCF combined with GM-CSF, IL-3 or Epo could partly increase the colony forming capacity of MDS hematopoietic progenitor, however, an increase of undifferentiated blast cells colonies occurred in some high-risk MDS patients at the same time. CONCLUSION: Proper combination of HGFs was essential for improving hematopoiesis in MDS patients.

Animals↗
PubMed1997

[Expression of human betaIVS- II -nt 654 C --> T allele in HeLa cell].

OBJECTIVE: To establish a cell model expressing human betaIVS- II -nt 654C --> T allele (beta654 mutant). METHODS: DNA fragment of entire beta-globin gene encompassing the codon region and poly(A) signal of the allele was amplified by long PCR from the genomic DNA of a homozygote with beta65.4 mutation. The amplified fragments were cloned into the Hind III and Xba I sites of the pcDNA3.1 vector. After reidentification of the clone with beta654 mutant by DNA sequencing, the recombinant plasmid was transfected into HeLa cell using liposome method. The expression of the beta654 mutant gene in the transfected cells was detected by RT-PCR. RESULTS: Both the normally processed beta globin mRNA (183bp) and aberrant processed beta globin mRNA (256bp) were identified in the transfected HeLa cells, while no RT-PCR product was detected in the controls. CONCLUSION: The transfected HeLa cells can express human beta654 allele.

Alleles↗
PubMed1997

Quantification of infectious duck hepatitis B virus by radioimmunofocus assay.

A simple method is described for the precise quantification of infectious duck hepatitis B virus (DHBV) in cell culture, using a radioimmunofocus assay (RIFA). Primary duck hepatocyte cell cultures were infected with serial dilutions of viral samples as for a plaque assay, but then maintained with liquid overlay medium. After incubation for up to 14 days, cell monolayers were fixed with acetone, then stained with a monoclonal antibody to DHBV L protein followed by secondary antibody labelled with 125I. Foci of infection (representing individual infectious particles in the inoculum) were detected by autoradiography. The number of foci recovered was increased by addition of dimethyl sulphoxide to culture medium, but was not appreciably altered by the use of semi-solid medium. The titre of virus suspensions determined by RIFA correlated well with titration in ducklings. The RIFA is a useful method for titration of DHBV, as it has a wide dynamic range and is well suited to parallel titration of large numbers of samples. This assay will have wide use for the analysis of DHBV growth kinetics, antiviral efficacy, and virus inactivation procedures.

Animals↗
PubMed1997

Impact mass chemotherapy with praziquantel on schistosomiasis control in Fanhu village, People's Republic of China.

The paper describes the endemic situation of schistosomiasis japonica in Fanhu village, Poyang Lake region, China and the effect of the strategy of combining annual mass chemotherapy with health education on schistosomiasis control in the community. The results showed that the prevalence of infection with schistosome reduced form 26.0% in 1992 to 10.7% in 1994, the intensity of infection in residents decreased from 1.92 in 1992 to 0.55 in 1994 and the condition of hepatomegaly, splenomegaly and liver fibrosis also improved after chemotherapy in the individuals in the case prospective study. Moreover, the future strategies of schistosomiasis control in this area have been suggested according to the transmission of schistosomiasis in the lake region and the effect of anti-schistosomiasis control indifferent populations.

Adolescent↗
PubMed1997

Transmission factors of schistosomiasis japonica in the mountainous regions with type of plateau canyon and plateau basin.

OBJECTIVE: To provide the scientific basis for formulating the control strategies of schistosomiasis japonica in mountainous regions in China Schistosomiasis Control Programme. METHODS: We selected Chuanxing Village in Sichuan Province and Zhonghe Village in Yunnan Province as the two pilot areas for an epidemiological investigation according to their geographical and topographical characteristics, i.e. plateau canyon and plateau basin, respectively. Concentrated-egg hatching method in nylon tissue bag and Kato-Katz technique were used for the survey on prevalence rate and intensity of infection of man and domestic animals. Questionnaires were applied to investigating the infection sources, community behavior and the socioeconomic status. Snail survey and detection of cercaria-infested water bodies were conducted using routine techniques. RESULTS: The results showed that in the endemic areas with plateau canyon, the key infection source was cattle. The infection rate in women was higher than that in men. The pattern of water-pollution was wild feces and the pollution source was cattle feces. Snails and infectious snails were distributed over the rice fields and the resident was infected through production activities. In plateau basin, the key infection source was human beings. The infection rate in men was higher than that in women. The pattern of water pollution was fertilization. The pollution source was human feces. Snails and infectious snails were distributed over the canals and ditches and the resident was infected through production and daily activities. CONCLUSIONS: Therefore, it was suggested that the mountainous endemic areas should be divided into two sub-types, i.e., plateau basins and plateau canyons. The different control strategies should be formulated in accordance with the environmental situations and socioeconomic factors in the two kinds of endemic areas.

Adolescent↗
PubMed1997

A preliminary study on interruption of HBV transmission in uterus.

OBJECTIVE: The infection of Hepatitis B virus (HBV) in the uterus may occur in neonates born to HBV carrier mothers. The rates of intrauterine transmission in these neonates were 10%-16%, Recent studies on the methods of combined passive and active immunizations indicate an efficacy approaching 70%-90%, but intrauterine infection of HBV is the major cause of failure of vaccination to combat hepatitis B in neonates born to HBV carrier mothers. We studied the interruptive effect of HBV specific immunoglobulin (HBIG) before delivery in the prevention of intrauterine transmission of HBV. METHODS: Of 3632 pregnant women, two hundred and four were HBV carriers; they were randomly divided into an HBIG group and a control group. Each subject in the HBIG group received 200 IU of HBIG intramuscularly at 3, 2 and 1 month before delivery. The subjects in the control group did not receive any specific treatment. Blood tests were conducted for all the subjects and their neonates after birth. Blood specimens were tested for HBsAg and HBeAg by enzyme immunoassay (Abbott Kits). RESULTS: The results showed that the rates of intrauterine transmission in the two groups were 5.7% and 14.7% respectively (X2 = 4.58 P < 0.05). No significant differences were observed for the positive rates of HBsAg and HBeAg between the two groups after delivery, but titer of HBsAg was reduced significantly in the HBIG group (t = 4.82, P < 0.01). CONCLUSIONS: Our study suggests that HBV infection in the uterus may be interrupted using multiple HBIG intramuscularly before delivery without any side effects.

Adult↗
PubMed1997

[Repair of the splicing defect of thalassemic allele beta IVS-2-654 C-->T in cultured human erythroid cells by antisense RNA].

OBJECTIVE: To investigate the repair of the splicing defect of thalassemic allele beta IVS-2-654 C-->T (beta 654) in cultured human adult erythroid cells (hAE) by antisense RNA A2. METHODS: Vectors expressing antisense RNA fragments A2 which targeted against the aberrant splice sites of beta 654 pre-mRNA were introduced into hAE by lipid-mediated DNA-transfection method. Quantitation of beta-globin mRNA by RT-PCR and micro-biosynthesis of globin chain assay were used to measure the expression of globin genes in hAE from day 0 to day 8 after transfection. Meanwhile, hAE transfected with random antisense sequence was used as control. RESULTS: Antisense RNA A2 treatment restored the correct splicing of beta 654 pre-mRNA. The proportions of correctly spliced beta-globin mRNA (beta/beta + beta*) increased from 0.024 (before transfection) to 0.380 (8th day after transfection) in hAE obtained from individual with beta 654/ beta 41-42 mutations and, from 0.396 to 0.883 in hAE obtained from individual with beta 654/beta A genotype. Correspondently, microglobin biosynthesis of chain revealed that the ratio of beta/alpha increased from 0.052 to 0.359 in the former and, from 0.624 to 0.820 in the later case, respectively. There was no difference with or without antisense RNA A2 treatment on hAE obtained from normal controls. CONCLUSIONS: Antisense RNA A2 treatment could restore efficiently the correct splicing of beta 654 pre-mRNA and, consequently improve the imbalance of globin chains in thalassemic cells. This method is of potential clinical interest in gene therapy of the disease.

Adult↗
PubMed1997

[The relationship between expression of basement membrane in squamous cell carcinoma of oral cavity and cervical lymph node metastasis].

Fifty seven cases of oral squamous cell carcinoma (SCC) were studied by immunohistochemical ABC method using type IV collagen and laminin antibody to investigate the relationship between expression and distribution of basement membrane in oral SCC and clinicopathologic characteristics and cervical lymph node metastasis. The distribution of basement membrane of oral SCC was discontinuous and in some cases the membranes disappeared. There was highly significant correlation between the staining patterns together with histologic differentiation degrees and cervical lymph node metastasis (P < 0.05). These indicate that the expression of baasement membrane in oral SCC may be a useful parameter for evaluation of tumor histologic differentiation and tumor invasion and metastasis.

Basement Membrane↗
PubMed1996

3' Processing and termination of mouse histone transcripts synthesized in vitro by RNA polymerase II.

The highly expressed mouse histone H2a-614 gene is located 800 nt 5' of the histone H3-614 gene. There is a 140 nt sequence located 500 nt from the end of the H2-614 mRNA which has been defined as a transcription termination site for RNA polymerase II. We established an in vitro transcription system in which both 3' end processing and transcription termination occur. A template containing the adenovirus major late promoter, a portion of the histone H2a-614 coding region, its 3' processing signal, followed by the transcription termination site was transcribed in a nuclear extract prepared from mouse myeloma cells. Some of the transcripts synthesized in the extract were cleaved at the histone processing site in a reaction which was dependent both on the hairpin binding factor and the U7 snRNP. The efficiency of histone 3' end formation was similar both on synthetic transcripts and transcripts synthesized by RNA polymerase II. Defined transcripts, which were not processed and which mapped to the transcription termination site, were released from the template, suggesting that they were formed by transcription termination. Termination in vitro was dependent on a functional histone processing signal.

Animals↗
PubMed1996

Recognition of the T-arm of tRNA by tRNA (m5U54)-methyltransferase is not sequence specific.

tRNA (m5U54)-methyltransferase (RUMT) catalyzes the methylation of U54 of tRNAs. In contrast to enzymes which recognize a particular tRNA, RUMT recognizes features common to all tRNAs. We have shown that these features reside in the T-arm of tRNA and constructed a minimal consensus sequence for RUMT recognition and catalysis (Gu et al., 1991b). Here, we have mutated each conserved T-loop residue and conserved T-stem base pair to bases or base pairs which are not observed in Escherichia coli tRNA. The substrate specificity of RUMT for 30 in vitro synthesized T-arm mutants of tRNAPhe and 37 mutants of the 17-mer analog of the T-arm derived from tRNA1Val was investigated. A 2-5 base pair stem was essential for recognition of the T-arm by RUMT, but the base composition of the stem was unimportant. The 7-base size of the T-loop maintained by the stem was essential for RUMT recognition. For tRNA, most base substitutions in the 7-base loop did not eliminate RUMT activity, except for any mutation of the methyl acceptor U54 and the C56G mutation. The effect of base and base pair mutations on Kcat or the rate of methylation by RUMT was more striking than the effect on the Kd for binding to RUMT. In comparison with mutations in the T-loop of intact tRNA, base mutation in the T-loop of the 17-mer T-arm had a more deleterious effect on binding and methylation. Surprisingly, recognition of tRNA by RUMT appears to reside in the three-dimensional structure of the seven-member T-loop rather than in its primary structure.

Base Sequence↗
PubMed1996

Modulation of honey bee thermoregulation by adrenergic compounds.

The effects of adrenergic compounds on the thermoregulation of resting honey bees were investigated using a non-traumatizing approach. Bees fed ad lib were treated orally and kept at an ambient temperature of 22 degrees C; the surface thoracic temperature was monitored by infrared thermography. Bees were treated with the adrenergic agonists epinephrine and ephedrine, and with the beta-blocking agent alprenolol. Low doses of adrenergic agonists had no significant effect on thermogenesis, but high doses caused a hypothermia. Alprenolol triggered a dose-dependent hypothermia that was reversed by low doses of epinephrine and ephedrine. Results are discussed with reference to the metabolic and neural adrenergic pathways that may be involved in bee thermoregulation.

Alprenolol↗
PubMed1996

A general additive distance with time-reversibility and rate variation among nucleotide sites.

As additivity is a very useful property for a distance measure, a general additive distance is proposed under the stationary time-reversible (SR) model of nucleotide substitution or, more generally, under the stationary, time-reversible, and rate variable (SRV) model, which allows rate variation among nucleotide sites. A method for estimating the mean distance and the sampling variance is developed. In addition, a method is developed for estimating the variance-covariance matrix of distances, which is useful for the statistical test of phylogenies and molecular clocks. Computer simulation shows (i) if the sequences are longer than, say, 1000 bp, the SR method is preferable to simpler methods; (ii) the SR method is robust against deviations from time-reversibility; (iii) when the rate varies among sites, the SRV method is much better than the SR method because the distance is seriously underestimated by the SR method; and (iv) our method for estimating the sampling variance is accurate for sequences longer than 500 bp. Finally, a test is constructed for testing whether DNA evolution follows a general Markovian model.

Analysis of Variance↗
PubMed1996

Rapid monitoring of site-specific glycosylation microheterogeneity of recombinant human interferon-gamma.

An analytical system is presented for rapid assessment of site-specific microheterogeneity of the two potential N-linked glycosylation sites of recombinant human interferon-gamma (IFN-gamma) derived from Chinese hamster ovary cell culture. The target protein is first purified from culture supernatant by immunoaffinity chromatography, and the acidic eluent is neutralized via an in-line mixing tee. On-line proteolysis is rapidly performed by an immobilized trypsin cartridge, and reversed-phase chromatography isolates the two pools of glycopeptides representing the potential glycosylation sites. Following off-line analysis by matrix-assisted laser-desorption ionization/time-of-flight (MALDI/TOF) mass spectrometry, observed mass shifts of glycopeptides relative to the known masses of their amino acid portions are correlated to site-specific oligosaccharide structures. Desialylation of glycopeptides by sialidase treatment on the MALDI sample plate allows for quantitative estimations of asialoglycan structures by MALDI/TOF. This methodology permits glycoprotein microheterogeneity to be evaluated in a time frame of approximately 2 h, utilizing as little as 0.5 microgram (25 pmol) of product. Results of monitoring a batch culture are presented as well as analysis of a culture containing deoxymannojirimycin, an inhibitor of glycoprotein processing.

1-Deoxynojirimycin↗
PubMed1996

Interaction of tRNA (uracil-5-)-methyltransferase with NO2Ura-tRNA.

tRNA in which uracil is completely replaced by 5-nitro-uracil was prepared by substituting 5-nitro-UTP for UTP in an in vitro transcription reaction. The rationale was that the 5-nitro substituent activates the 6-carbon of the Ura heterocycle towards nucleophiles, and hence could provide mechanism-based inhibitors of enzymes which utilize this feature in their catalytic mechanism. When assayed shortly after mixing, the tRNA analog, NO2Ura-tRNA, is a potent competitive inhibitor of tRNA-Ura methyl transferase (RUMT). Upon incubation, the analog causes a time-dependent inactivation of RUMT which could be reversed by dilution into a large excess of tRNA substrate. Covalent RUMT-NO2Ura-tRNA complexes could be isolated on nitrocellulose filters or by SDS-PAGE. The interaction of RUMT and NO2Ura-tRNA was deduced to involve formation of a reversible complex, followed by formation of a reversible covalent complex in which Cys 324 of RUMT is linked to the 6-position of NO2Ura 54 in NO2Ura-tRNA.

Binding Sites↗
PubMed1996

Phragmoplastin, a dynamin-like protein associated with cell plate formation in plants.

Cytokinesis in a plant cell is accomplished by the formation of a cell plate in the center of the phragmoplast. Little is known of the molecular events associated with this process. In this study, we report the identification of a dynamin-like protein from soybean and demonstrate that this protein is associated with the formation of the cell plate. Plant dynamin-like (PDL) protein contains 610 amino acids showing high homology with other members of the dynamin protein family. Western blot experiments demonstrated that it is associated with the non-ionic detergent-resistant fraction of membranes. Indirect immunofluorescence microscopy localized PDL to the cell plate in dividing soybean root tip cells. Double labeling experiments demonstrated that, unlike phragmoplast microtubules which are concentrated on the periphery of the forming plate, PDL is located across the whole width of the newly formed cell plate. Based on the temporal and spatial organization of PDL in the phragmoplast, we termed this protein 'phragmoplastin'. The data suggest that phragmoplastin may be associated with exocytic vesicles that are depositing cell plate material during cytokinesis in the plant cell.

Amino Acid Sequence↗
PubMed1996

The crystal structure of a high oxygen affinity species of haemoglobin (bar-headed goose haemoglobin in the oxy form).

We have determined the crystal structure of bar-headed goose haemoglobin in the oxy form to a resolution of 2.0 A. The R-factor of the model is 19.8%. The structure is similar to human HbA, but contacts between the subunits show slightly altered packing of the tetramer. Bar-headed goose blood shows a greatly elevated oxygen affinity compared to closely related species of geese. This is apparently due to a single proline to alanine mutation at the alpha 1 beta 1 interface which destabilises the T state of the protein. The beta chain N and C termini are well-localized, and together with other neighbouring basic groups they form a strongly positively charged groove at the entrance to the central cavity around the molecular dyad. The well-ordered conformation and the three-dimensional distribution of positive charges clearly indicate this area to be the inositol pentaphosphate binding site of bird haemoglobins.

2,3-Diphosphoglycerate↗