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Biomedical subjects

B Fang

Publications and source records attributed to B Fang.

At least 91 records · Page 5Linked to original sources
PubMed1995

Calmodulin-dependent cyclic AMP phosphodiesterase in liver plasma membranes: stimulated by insulin.

In vivo insulin consistently stimulates the plasma membrane, high-affinity (low Km) cyclic AMP phosphodiesterase (PDE) from diabetic rat liver or adipose tissue. In vitro stimulation of membrane PDE by insulin has been reported to be inconsistent. Also, the involvement of calmodulin (CaM) in insulin stimulation of PDE has been controversial. In this report, conditions for the isolation of rat liver plasma membranes containing PDE that is sensitive to in vitro insulin stimulation and the involvement of CaM in insulin stimulation of PDE were investigated. In vitro insulin raised the Vmax of the enzyme without altering its apparent Km and was dose dependent. Insulin stimulation was lost after freezing, sonication, solubilization with detergents, or storage of the membranes at 4 degrees C for 4 h after isolation. Insulin stimulation was completely blocked by the CaM antagonist compound 48/80, EGTA, or trifluoperazine. Two isoforms of membrane-bound PDE were separated by ion-exchange chromatography following solubilization of the plasma membranes. The activities of both isoforms were stimulated by exogenous CaM. Plasma membrane PDE eluted after the application of exogenous CaM plus Ca2+. The data support the concept of a critical involvement of CaM in insulin activation of liver membrane PDE.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Gene therapy for hemophilia B: host immunosuppression prolongs the therapeutic effect of adenovirus-mediated factor IX expression.

Hemophilia B is caused by a deficiency of blood clotting factor IX (FIX). Previous studies have shown that the delivery of a recombinant adenoviral vector expressing canine FIX (cFIX) resulted in a complete correction of hemophilia B in FIX-deficient dogs, but that cFIX expression decreased to only about 1-2% of normal levels 3 weeks after treatment. In the present study, therapeutic levels of cFIX expression capable of producing a partial correction of hemophilia B were maintained for at least 6 months after the coadministration of the cFIX-expressing adenovirus and the immunosuppressive agent cyclosporin A (CsA). These findings support a recent report (Yang et al., 1994) that host T-cell-mediated immunity against virally transduced cells is a major contributing factor to the transient nature of adenovirus-mediated gene expression in immunocompetent animals. Although a second administration of the cFIX-expressing adenovirus 6 months after the first infusion had only a minimal effect on plasma FIX levels in a dog that had been continuously treated with CsA, the prolonged expression of the transgene indicates that immunosuppression may be applicable in attaining long-term treatment of clinically relevant disorders.

Adenoviridae↗
PubMed1995

PET and SPECT imaging for stereotactic localization.

Stereotactic localization using PET and SPECT has been used together with the Leksell stereotactic frame and the other imaging tools routinely provided to Gamma Knife radiosurgery sites. The accuracy of the measurements has been confirmed with CT and MR using a Radiation Analog Dosimetry phantom. The activity of the radionuclide solution placed in the tubes of Elekta Radiosurgery's MR imaging box is somewhat critical because the window levels chosen for the scan will affect the apparent size of the lesion. A technique is presented to resolve areas of activity more than about 12-14 mm apart, and it may prove useful in targeting epileptogenic sites in patients with medically intractable epilepsy.

Humans↗
PubMed1995

In vivo gene therapy for hyperlipidemia: phenotypic correction in Watanabe rabbits by hepatic delivery of the rabbit LDL receptor gene.

Elevations of plasma total or LDL cholesterol are major risk factors for cardiovascular disease. Efforts directed at preventing and treating cardiovascular disease have often focused on reducing the levels of these substances in the blood. The Watanabe Heritable Hyperlipidemic Rabbit, which has exceedingly high plasma cholesterol levels resulting from an LDL receptor deficiency, provides an excellent animal model for testing new treatments. A recombinant adenoviral vector containing the rabbit LDL receptor cDNA was administered to Watanabe rabbits. Plasma total cholesterol levels in the treated animals were reduced from 825.5 +/- 69.8 (mean +/- SD) to 247.3 +/- 61.5 mg/dl 6 d after infusion. These animals also demonstrated a 300-400% increase in plasma levels of HDL cholesterol and apo AI 10 d after treatment. As a result, the LDL:HDL ratio exhibited a dramatic decrease. Because only the rabbit LDL receptor gene was used for treatment, the results strongly suggest that the elevations of plasma HDL cholesterol and apo AI were secondary to a reduction in plasma total cholesterol in the treated animals. These results suggest an inverse relationship between plasma LDL and HDL cholesterol levels and imply that reduction of LDL cholesterol levels may have a beneficial effect on plasma HDL cholesterol.

Animals↗
PubMed1995

[Analysis of short tandem repeats polymorphism in the phenylalanine hydroxylase gene and its application to prenatal gene diagnosis of phenylketonuria].

There is a polymorphic tetranucleotide short tandem repeats (STR) sequence in the intron 3 of the phenylalanine hydroxylase (PAH) gene. The polymorphism of this STR sequence in Chinese was analysed on 36 normal individuals and 16 PKU families. After PCR amplification, the DNA fragments were separated on polyacrylamide sequencing gel and visualized by silver-staining. Seven alleles were identified with frequencies of 0.028, 0.042, 0.014, 0.138, 0.542, 0.208 and 0.028 in normal populations, and 0.016, 0.078, 0.047, 0.312, 0.375, 0.156 and 0.016 in PKU families, and PICs of 0.585 and 0.693, respectively. This polymorphic marker is very useful in quick prenatal gene diagnosis in PKU families, and prenatal gene diagnosis was performed by linkage analysis in two pregnancies at risk.

Alleles↗
PubMed1995

[Prediction of retinoblastoma: linkage analysis of families with hereditary retinoblastoma by using polymorphic sites within the Rb gene].

Retinoblastoma (Rb) is one of the most common intraocular tumors in childhood. 40% of the cases are hereditary in nature. 10% are due to the transmission of a germline mutation from an affected parent, and 30% are due to new germ cell mutation. In the hereditary form, the mutation is inherited via a germ cell; therefore, the mutant gene is present in all somatic cells. The segregations of four polymorphic sites within the Rb gene were analyzed in six families with hereditary retinoblastoma. Two families were informed with probe p123M1.8, three were informed with probe p68RS2.0 and one was informed with PCR amplification of XbaI site; all six families were informed with PCR amplification of intragenic VNTR site RB1.20. Our results showed that linkage analysis of families with hereditary retinoblastoma by using intrageneic polymorphic sites could be applied to detect carriers and for prenatal diagnosis in families with hereditary retinoblastoma.

Base Sequence↗
PubMed1994

Overexpression in Escherichia coli, purification and characterization of the molecular chaperone HSC70.

The 70-kDa heat-shock cognate protein (HSC70), a constitutively expressed protein in mammalian cells, plays a major role in several cellular processes such as protein folding and assembly, uncoating of clathrin-coated vesicles and transport of protein through membranes. HSC70 has been overexpressed in Escherichia coli in a soluble form using a designed two-cistron expression vector, and purified to homogeneity in a two-step procedure involving ion-exchange and affinity chromatography. Up to 20 mg of pure protein could be obtained from 11 of cell culture. Amino-terminal sequencing of the recombinant protein gives the expected sequence, and non-denaturing gel electrophoresis as well as gel filtration analysis reveal the presence of self-associating species that could be dissociated by ATP. Crosslinking studies confirm the presence of multiple species and the dissociating effect of ATP. Temperatures above 42 degrees C induce the aggregation of HSC70; ATP shifts this effect to higher temperatures. The recombinant protein displays a low intrinsic ATPase activity that can be stimulated about threefold by binding to apocytochrome c, a permanently unfolded protein, while native cytochrome c has no effect on the ATPase activity indicating that recombinant HSC70 binds specifically unfolded protein but not their native counterpart. Thus, efficient production of recombinant HSC70 having structural and functional properties comparable to those of the natural protein could be achieved, thereby allowing the molecular basis of the chaperone function and its regulation through ATP hydrolysis to be probed.

Adenosine Triphosphatases↗
PubMed1994

In vivo hepatic gene therapy: complete albeit transient correction of factor IX deficiency in hemophilia B dogs.

Hemophilia B is a bleeding disorder caused by mutations in the factor IX gene. The disorder is X-linked recessive with a prevalence of about 1 in 30,000 Caucasian males. Factor IX is naturally synthesized in the liver and secreted into blood. Here we report the construction of recombinant adenoviral vectors containing the canine factor IX cDNA that are capable of transducing hepatocytes in mice at high efficiencies in vivo without partial hepatectomy. The recombinant viral vector was used to treat hemophilia B dogs by direct vector infusion into the portal vasculature of deficient animals. Plasma factor IX concentrations in the treated hemophilia B dogs increased from 0 to 300% of the level present in normal dogs, resulting in complete amelioration of the disease as demonstrated by normal blood coagulation and hemostatic measurements. Although plasma factor IX concentration started to decline after a few days, therapeutic levels of factor IX persisted for 1-2 months in the treated animals. The results validate the principle of in vivo hepatic gene delivery to reconstitute the genetic deficiency in a large animal model and suggest that gene therapy is achievable when long-acting vectors are developed.

Adenoviridae↗
PubMed1994

Gene therapy for phenylketonuria: phenotypic correction in a genetically deficient mouse model by adenovirus-mediated hepatic gene transfer.

Classical phenylketonuria (PKU), which predisposes affected individuals to severe mental retardation, is caused by a deficiency of hepatic phenylalanine hydroxylase (PAH). A recombinant adenoviral vector containing the human PAH cDNA was constructed and administered to PAH-deficient mice (strain PAHenu2). The hyperphenylalaninemic phenotype of these animals was completely normalized within 1 week of treatment. Although this therapeutic effect did not persist, analysis of the relationship between hepatic PAH activity and serum phenylalanine levels indicated that only 10-20% of normal enzymatic activity in the mouse liver is sufficient to restore normal serum phenylalanine levels. These results demonstrate that PKU and other metabolic disorders secondary to hepatic deficiencies can be completely corrected by gene therapy when more persistent vector systems are developed.

Adenoviridae↗
PubMed1994

Application of a generalized logistic equation to simulate a fed-batch process.

The whole of fed-batch with two constant flow is successfully described by a generalized logistic equation. The research results show that (1) the adequacy of the simulation of the process depends to a considerable degree on the selection of simulated accuracy; (2) there is a quantitative relation between the specific growth rate of cells mu and the specific substrate consumption rate qs and content of RNA in microbial cells.

Data Interpretation, Statistical↗
PubMed1992

Distribution of ADH2 and ALDH2 genotypes in different populations.

The distribution of the human liver alcohol dehydrogenase, ADH2, and aldehyde dehydrogenase, ALDH2, genotypes in 21 different populations comprising Mongoloids, Caucasoids, and Negroids was determined by hybridization of the amplified genomic DNA with allele-specific oligonucleotide probes. Whereas the frequency of the ADH1(2) allele was found to be relatively high in the Caucasoids, Mexican Mestizos, Brazilian Indios, Swedish Lapps, Papua New Guineans and Negroids, the frequency of the ADH2(2) gene was considerably higher in the Mongoloids and Australian Aborigines. The atypical ALDH2 gene (ALDH2(2)) was found to be extremely rare in Caucasoids, Negroids, Papua New Guineans, Australian Aborigines and Aurocanians (South Chile). In contrast, this mutant gene was found to be widely prevalent among the Mongoloids. Individuals possessing the abnormal ALDH2 gene show alcohol-related sensitivity responses (e.g. facial flushing), have the tendency not to be habitual drinkers, and apparently suffer less from alcoholism and alcohol-related liver disease.

Alcohol Dehydrogenase↗
PubMed1992

Estimation for model parameters of batch fermentation kinetics.

Based on the widely accepted mathematical model of fermentation kinetics, an analytical solution is deduced in this paper. To describe the feature of batch fermentation, the parameters of the fermentation kinetics in the analytical solution (i.e., mumax, Ks, beta, YG, YP, and m) are estimated at one strike with POWELL optimization algorithm coded in FORTRAN-77. The experimental data in the example is quoted from a batch lysine fermentation process using Corynebacterium glutamicum. The result shows that: 1) the calculated values of the mathematical model agree very well with the experimental data; 2) the synthesis rate of lysine depends on both the growth rate and the concentration of the biomass.

Algorithms↗
PubMed1992

Detection of point mutations of the phenylalanine hydroxylase gene and prenatal diagnosis of phenylketonuria.

The known mutant alleles of the human phenylalanine hydroxylase (PAN) gene were analyzed in 25 phenylketonuria (PKU) families from North China by using polymerase chain reaction and allele-specific oligonucleotide dot blot hybridization techniques. The results showed that the six mutations analyzed accounted for 62% of all PKU genes. The three most frequent mutations were R243Q, R413P and Y204C. Seven prenatal gene diagnoses were carried out in 6 PKU families and were confirmed after birth or by examination of aborted materials.

Base Sequence↗
PubMed1992

[Analysis of RFLP haplotypes and point mutations at the phenylalanine hydroxylase (PAH) locus in PKU families from north China].

A study of DNA polymorphisms at the phenylalanine hydroxylase (PAH) locus was performed using 28 classical phenylketonuria (PKU) families from North China. In the families analyzed, haplotype 4 accounted for 77% of normal chromosomes and 79% of PKU gene bearing chromosomes. Two new haplotypes, haplotypes 49 and 50, were found. On the basis of haplotype analysis, only 37%-40% of PKU carriers in North China were heterozygous and therefore informative for linkage studies. Exon 3 (Arg111----stop) and exon 6 (Tyr204----Cys204) mutations of the PAH gene were studied using the polymerase chain reaction (PCR) and allele specific oligonucleotide probe hybridization in 42 PKU families from North China. These accounted for 3.6% and 9.5% of PKU mutations in North China, respectively.

Base Sequence↗
PubMed1992

[Polymorphisms of the D13S26 locus in Chinese and its application to linkage analysis of Wilson disease].

The D13S26 locus has been mapped to 13q21.1-q21.2 and is linked with Wilson disease gene at a distance of 3.8 centimorgans. The polymorphic alleles detected by HphI, EcoRI and BclI at the D13S26 locus are the same in Chinese as in Caucasians, but the allele frequencies are quite different. As calculated from 30 unrelated Chinese individuals, the allele frequencies were as follows: HphI 2.8 kb(0.47)/2.0kb(0.53); EcoRI 9.0 kb (0.02)/8.0 kb (0.98); BclI 6.3 kb (0.02)/5.6 kb (0.98). Cosegregation analysis of the D13S26 locus and Wilson disease locus was carried out in 3 families with the disease. In one of these families, the proband and his younger sister (7-years-old and phenotypically normal) were both heterozygous for this site. We predict with 85.6% confidence that the younger sister is in the presymptomatic stage of Wilson disease.

Alleles↗