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Biomedical subjects

C H Wu

Publications and source records attributed to C H Wu.

At least 19 recordsLinked to original sources
PubMed1992

Specific inhibition of hepatitis B viral gene expression in vitro by targeted antisense oligonucleotides.

A 21-mer oligodeoxynucleotide complementary to the polyadenylation signal for human hepatitis B virus (HBV) was complexed to a soluble DNA-carrier system that is targetable to hepatocytes via asialoglycoprotein receptors present on those cells. A cell line, HepG2 (2.2.15) that possesses asialoglycoprotein receptors and is permanently transfected with hepatitis B virus (ayw subtype) was exposed to complexed antisense DNA or controls. In the presence of complexed antisense DNA, the concentration of hepatitis B surface antigen in medium was 80% lower than controls after 24 h. Furthermore, during the next 6 days, there was no significant increase in surface antigen concentration in the presence of complexed antisense DNA. The inhibition could be effectively blocked by competition with an excess of free asialoglycoprotein. Total protein synthesis remained unchanged by exposure to complexed antisense sequences under identical conditions. In addition, HBV DNA in the medium and cell layers after 24-h exposure to complexed antisense sequences was 80% lower than in controls. The data indicate that antisense oligonucleotides complexed by a soluble DNA-carrier system can be targeted to cells via asialoglycoprotein receptors resulting in specific inhibition of hepatitis B viral gene expression and replication.

Asialoglycoprotein Receptor↗
PubMed1992

A novel mechanism for achieving transgene persistence in vivo after somatic gene transfer into hepatocytes.

Infusion of hepatocyte-specific DNA-protein complexes into rats leads to transient recombinant gene expression in liver. The eventual deterioration of gene expression is due in part to instability of the targeted DNA. In a previous report, we noted retention of transgene sequences in liver and persistent recombinant gene expression when the animals were subjected to partial hepatectomy following in vivo gene transfer. In an attempt to define the mechanism(s) responsible for persistent gene expression following partial hepatectomy, we characterized the molecular state of the retained, liver-associated transgenes. Southern blot analysis of DNA from liver tissues harvested various times after in vivo gene transfer and partial hepatectomy (10 min to 11 weeks) demonstrated high levels of transgene DNA (100-10,000 copies/cell). The predominant form of this DNA appeared to be episomal based on analyses of uncut DNA or DNA restricted by an endonuclease with one site in the plasmid. Livers from several animals contained a small proportion of transgene sequences of unknown structure. The existence of episomal DNA in liver was confirmed in experiments in which intact plasmid was rescued from total hepatocyte DNA by transformation of bacteria. Both strands of DNA in the liver-associated plasmid retained a bacterial pattern of methylation suggesting that the plasmid had not replicated in the eukaryotic cell. These results are consistent with the hypothesis that the majority of transgene sequences are retained as stabilized plasmids. The specific form of DNA which is transcriptionally active was not identified in these studies. This represents a new mechanism for retaining foreign DNA in eukaryotic cells in vivo and has implications both for the development of somatic gene therapies and the pathogenesis of viral diseases.

Animals↗
PubMed1992

Hepatocyte-directed gene transfer in vivo leads to transient improvement of hypercholesterolemia in low density lipoprotein receptor-deficient rabbits.

Familial hypercholesterolemia is an inherited disease in humans, caused by a deficiency of low density lipoprotein (LDL) receptors, that we have used as a model for developing liver-directed gene therapies. Our strategy is to reconstitute hepatic LDL receptor expression in vivo by administering a DNA-protein complex that is capable of targeting the delivery of functional LDL receptor genes to hepatocytes. Infusion of this DNA-protein complex into the peripheral circulation of a rabbit animal model for familial hypercholesterolemia resulted in hepatocyte-specific gene transfer and a temporary amelioration of hypercholesterolemia. This noninvasive approach to gene therapy should have applications in the treatment of a wide spectrum of human diseases.

Animals↗
PubMed1992

Evidence that an iron chelator regulates collagen synthesis by decreasing the stability of procollagen mRNA.

Iron chelation has been shown previously to decrease collagen synthesis at a posttranslational level by inhibiting prolyl 4-hydroxylase, one of the key enzymes in collagen metabolism. On the other hand, recent in vivo studies of iron overload in rats suggest that iron could specifically activate collagen gene expression in liver tissues. These findings led us to investigate whether iron chelation might also affect collagen gene expression and posttranslational modification. Our data indicate that alpha,alpha'-dipyridyl, an iron chelator, at a concentration of 1 mmol/L, decreased steady-state levels of type I procollagen messenger RNA by 42% (p less than 0.001) without affecting beta-actin messenger RNA levels. Nuclear runoff studies demonstrated that transcription of the type I procollagen gene was unchanged by alpha,alpha'-dipyridyl. However, the turnover rate of type I procollagen messenger RNA was increased by 30%. This pretranslational inhibition of collagen synthesis was not due to decreased lipid peroxidation, because thiobarbituric acid-reactive substances were unchanged by alpha,alpha'-dipyridyl. However, cycloheximide totally abolished the effect, indicating that de novo protein synthesis was required.

2,2'-Dipyridyl↗
PubMed1992

The primary application of three-dimensional ultrasonography in obstetrics.

To date, no report of three-dimensional ultrasonography in obstetrics has been available in the literature. In this study we describe the primary experience with three-dimensional ultrasonography for the prenatal evaluation of nine fetuses between 21 and 33 weeks of gestation. We found that it provides more information for clinical diagnosis than the traditional two-dimensional ultrasonography. The following advantages are notable: (1) the anatomic area of interest can be located first with the conventional real-time two-dimensional mode, then switched to reveal three-dimensional images, instantly; (2) three images are exhibited concurrently; (3) each of the three images can be arrayed separately and scrolled to search for the area of interest within the scanned volume; (4) the three-dimensional ultrasonography can be equipped with Doppler color flow mapping for the study of the fetal cardiovascular system. In conclusion, we believe that three-dimensional ultrasonography can greatly strengthen diagnostic potential, which deserves obstetricians' attention.

Cardiovascular System↗
PubMed1992

Chlamydia antibody by enzyme-linked immunosorbent assay and associated severity of tubal factor infertility.

We have evaluated the clinical utility of the serum chlamydial antibody value by ELISA in predicting the severity of tubal factor infertility quantified by the use of a previously reported pelvic scoring system. Significant differences in salpingitis scores, tubal occlusion scores, adhesion scores, and pelvic stages were found with increasing chlamydia antibody grades. The level of ELISA value strongly correlated with salpingitis, tubal occlusion, and total pelvic scores and pelvic stages. The serum chlamydia antibody level by ELISA now appears to be a useful prognostic tool for predicting the severity of tubal factor infertility in addition to its value in screening for its presence.

Antibodies, Bacterial↗
PubMed1992

Detection of Mycobacterium tuberculosis antigen in synovial fluid of patients with rheumatoid arthritis.

By use of antimycobacterial saline extract antibodies, Mycobacterium tuberculosis (MT) antigens have been detected in synovial fluid (SF) of patients with rheumatoid arthritis (RA) by a highly specific and sensitive double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Absorbance for 15 gout, 14 osteoarthritis (OA) patients, 12 patients with spondylarthropathies (SA) and eight patients with other inflammatory disorders (OID) ranged from 0.001 to 0.025 with the mean values of 0.0086 +/- 0.0078, 0.0077 +/- 0.0051, 0.0069 +/- 0.0059 and 0.0113 +/- 0.0059, respectively. Among 65 SF of patients with RA examined, 34 were found to be negative for MT antigen with absorbance ranging from 0.002 to 0.024 and a mean value of 0.0114 +/- 0.0070. For 31 (47.7%) MT antigen-positive specimens of RA, optical density ranged from 0.052 to 2.446 with a mean value of 0.5564 +/- 0.7354. Significant statistical difference (P less than 0.05) was found when MT antigen positives were compared with MT antigen negatives, gout, OA, SA and OID groups. Our results indicate that MT may be relevant to the pathogenesis of RA.

Analysis of Variance↗
PubMed1992

Enhanced osteoinduction by intramuscular grafting of BMP-beta-TCP compound pellets into murine models.

The osteoinductive effects of bone morphogenetic protein (BMP, derived from murine osteosarcoma) were studied with regard to its use combined with beta-tricalcium phosphate (beta-TCP). BMP and beta-TCP were molded into pellets by the "pressure method", originated by us and transplanted to ddY mice. Control mice received interdorsal muscular implantations of either the BMP or beta-TCP pellets. The animals were sacrificed 1, 2 and 3 weeks after grafting, for radiological, histochemical, and ultrastructural observations. The BMP-beta-TCP compound pellets induced faster cartilage and bone formation, whereas these activities were slower when pellets made solely of BMP were used. The beta-TCP pellets demonstrated no osteoinductive properties. Observations revealed two types of beta-TCP resorbing multinuclear giant cells. One was osteoclastic, expressing calcitonin receptors, having numerous mitochondria and ruffled border-like structures; the other was not osteoclastic in nature. In animals grafted with the compound pellets, a great number of osteoclastic cells gathered on the pellets, much earlier than those grafted with the pellets made of BMP alone. Then, osteoblastic bone formation over the cement lines followed an osteoclastic resorption of both beta-TCP and newly formed bone. In contrast, BMP induced few osteoclastic cells, resulting in slower bone coupling. Furthermore, the faster bone formation induced by the compound pellets seemed to be associated with the presence of beta-TCP. Porous by nature, beta-TCP would entrap BMP within its micropores, and thus, the intrinsically diffusible BMP is retained and its action consequently prolonged. In addition, the compound pellet offered increased surface contact between BMP and mesenchymal cells. Therefore, BMP-beta-TCP compound pellets induce cartilage and bone formation more rapidly than does BMP alone.

Animals↗
PubMed1992

A quantitative and morphometric study of the transformation of amoeboid microglia into ramified microglia in the developing corpus callosum in rats.

The morphometric and quantitative changes associated with the differentiation of amoeboid microglia into ramified microglial cells in the corpus callosum of rats between 21 d postconception (E21) and 15 d postnatally are described. Using lectin labelling, 5 morphological types of labelled cells (R, SP, KLP, TLP, AP) based on cell body shape, the configuration of their cytoplasmic processes and their staining intensity, were recognised. Round cells (R) and cells with stout processes (SP) were aggregated in the central part of the developing corpus callosum whereas the highly branched labelled cells were distributed at its periphery. When the morphometric data and labelling intensities of labelled cells were analysed with the aid of an image analysis system, the values for cell length, area and perimeter increased as the complexity of branching increased, whereas the lectin-labelling intensity became reduced. Quantitative study showed that the proportion of the different morphological types of lectin-labelled cells peaked at different ages. The sequential peaking of R, SP and highly branched cells with advancing age suggests a similar chronological order of differentiation of R into branched cells. The quantitative study also showed a rapid increase in the density of lectin-labelled cells in the postnatal period between P4 and P8, attributed primarily to the active proliferation of the cell type. The consequent reduction of cell density (after P13) was probably due to cell death, a feature which appeared to increase with development.

Animals↗
PubMed1992

The effectiveness of the air-powder abrasive device for root planing during periodontal surgery.

The efficacy of the air-powder abrasive device (APAD) in root planing during flap surgery was assessed in vitro and in vivo. Two teeth on which full-thickness flaps were raised underwent root planing by hand-scaling alone and hand-scaling combined with APAD. One tooth extracted prior to surgery underwent unrestricted hand-scaling under a binocular microscope (control). The two teeth root planed in situ and the tooth root planed in vitro were observed by scanning electron microscopy (SEM). Then, 10 patients with periodontitis requiring flap surgery were selected for a clinical study. After raising full-thickness flaps, 29 sites from five patients underwent root planing using hand-scaling combined with APAD, whereas 22 sites from six patients underwent root planing by hand scalers only. SEM revealed that root surfaces planed in situ by hand-scaling combined with APAD were smoother than those root-planed using hand scalers alone. The control tooth surface was as smooth as those submitted to combined root planing. The clinical study showed that sites submitted to combined root planing displayed enhanced attachment level gain and pocket reduction. These results, associated with the shortened instrumentation time, suggest APAD as a useful instrument for root planing during flap surgery.

Air↗
PubMed1992

Electroejaculation in spinal cord injured males.

Electroejaculation is a newly developed method to retrieve sperm in anejaculatory spinal cord injured (SCI) males. We studied 25 completely traumatic SCI males from August 1990 to May 1991. The patients' ages ranged from 18.7 to 43.3 years, and the interval since injury ranged from four months to 14.1 years. The level of injury varied from C5 to T12. Bi-directional emission was found in 12 patients, antegrade in nine, retrograde in one and failure in three. Electroejaculatory stimulation parameters were 434 +/- 54 mA for mean maximum current, 21.7 +/- 2.7 volts for mean maximum voltage and 35.9 +/- 3.1 degrees C for mean maximum probe temperature. The antegrade semen obtained showed wide variations in sperm quality and quantity between subjects. The total sperm count was 478 +/- 809 x 10(6) in the antegrade portion, and the sperm motility was below 5% in most cases. The retrograde portion was usually worse. There was no correlation between sperm quality and quantity with patient age, injury level or injury period. Bladder management had no effect on the results of electrical stimulation. Epididymitis had a negative impact on the success of retrieval. Low-level injury victims needed analgesia or anesthesia to complete the stimulation. The major side effects were minimal autonomic dysreflexia and mild rectal mucosal change. Repeated stimulation may improve sperm counts, but semen quality deteriorates if the procedure is performed once a week. As a whole, electroejaculation is a safe, effective and simple procedure to retrieve sperm in anejaculatory persons, especially SCI patients.

Adolescent↗
PubMed1991

Receptor-mediated gene delivery in vivo. Partial correction of genetic analbuminemia in Nagase rats.

A plasmid (palb3) was constructed containing the structural gene for human serum albumin driven by mouse albumin enhancer-rat albumin promoter elements. Using an asialoglycoprotein-polycation conjugate consisting of asialoorosomucoid coupled to poly-L-lysine, a soluble DNA complex was formed that was capable of targeting specifically to hepatocytes via asialoglycoprotein receptors present on these cells. Groups of Nagase analbuminemic rats were injected with complexed DNA or controls, followed by two-thirds partial hepatectomy to stimulate hepatocyte replication. Using a cDNA probe for the human albumin structural gene, hybridizable sequences were detected in analbuminemic rats treated with complex as determined by Southern blot analysis. Two weeks post-injection, the targeted DNA was found to exist primarily in plasmid form with an average copy number of 1000/diploid cell. Human albumin mRNA was detected by dot-blot hybridization with a specific oligonucleotide cDNA probe and confirmed by RNase protection assay using a vector-specific probe. Circulating human albumin was detected in the serum of palb3-treated Nagase analbuminemic rats by Western blots using an antibody specific for human serum albumin. A time course demonstrated that circulating human albumin was not detectable 24 h after injection, but became measurable at a level of 0.05 micrograms/ml within 48 h and increased in concentration to a maximum of 34 micrograms/ml by 2 weeks post-injection. This level of expression remained stable through 4 weeks after injection and partial hepatectomy.

Animals↗
PubMed1991

Chemical modification of an ecotropic murine leukemia virus results in redirection of its target cell specificity.

An ecotropic virus was chemically modified in order to determine whether its target cell specificity could be altered. We hypothesized that chemical coupling of galactose residues to a virus might permit specific infection of hepatocytes mediated by asialoglycoprotein receptors unique to these cells. To test this hypothesis, we took advantage of the fact that: 1) artificial asialoglycoproteins can be created by chemical coupling of lactose to proteins; and 2) viruses that are ecotropic have a narrow species specificity. An ecotropic, rodent-specific, replication-defective murine leukemia virus containing the gene for beta-galactosidase was chemically modified with lactose to contain 5.9 mumol of lactose per mg of viral RNA. Modified and unmodified viruses were incubated for 5 days with HepG2, a human hepatoma line that possesses asialoglycoprotein receptors, and SK Hep1, a human cell line that does not. As expected from the ecotropism, unmodified virus did not produce beta-galactosidase activity in either cell type. Modified virus did not produce beta-galactosidase activity in SK Hep1 cells. However, modified virus did produce beta-galactosidase activity, 71.2 units/mg of cell protein, in the human receptor (+) HepG2 cells. Interestingly, modification of the virus also resulted in decreased enzyme activity in previously susceptible host rodent cells. Competition with modified virus by an excess of an asialoglycoprotein completely prevented development of enzymatic activity in HepG2 cells. Histochemical treatment of cells with 5-bromo-4-chloro-3-indoyl beta-D-galactoside to detect in situ beta-galactosidase activity demonstrated that only HepG2 cells treated with modified virus were positive and that 36% of these cells were stained after 5 days. These data indicate that chemical modification of a virus can result in a redirection of the infectivity of the virus toward hepatocyte-derived cells mediated by the presence of asialoglycoprotein receptors.

Animals↗
PubMed1991

Reiterated gene amplifications at specific short homology sequences in phage T4 produce Hp17 mutants.

Bacteriophage T4 gene 17 amplification mutants (Hp17) selected by growth of gene 17 amber mutants on ochre suppressor strains of Escherichia coli carry two to more than sixfold tandem head-to-tail repeats of the gene 17-18 region (Wu & Black, 1987). We characterized the structures of Hp17 isolates by restriction enzyme mapping and Southern blot analysis. The left and right boundaries of the amplified sequences were mapped within genes 16 and gene 18 or 19, respectively. The TaqI-restriction fragments containing the novel junctions arising from fusion of the amplified gene were then cloned and sequenced. Three Hp17 mutants arose from rearrangement in one five base-pair (bp) block within a G + C-rich region of partial homology (24 bp with 4 mismatches) between genes 16 and 19. Moreover, an oligonucleotide probe showed that 190/191 mutants isolated had recombined within the 5 bp block, and other rearrangements within this 24 bp region were not detected. Only one anomalous Hp mutant rearranged elsewhere between genes 16 and 18 in a 14 bp homology region with one mismatch. Elimination of gene alt of phage T4 is required for isolation of Hp17 mutants, apparently because more DNA can be packaged into alt- heads. Requirements for the dispensable replication and recombination genes of T4 were probed; T4 topoisomerase (39, 52, 60), primase (58/61), and uvsX are required, whereas the host recA gene and T4 denV gene do not appear to be required for isolation of the Hp17 mutants. The evidence suggests an initiating sequence-specific rearrangement leads to the T4 Hp17 amplification mutants.

Base Sequence↗
PubMed1991

Propeptide-mediated regulation of procollagen synthesis in IMR-90 human lung fibroblast cell cultures. Evidence for transcriptional control.

We have demonstrated previously that the carboxyl- and amino-terminal propeptides of type I procollagen can inhibit procollagen synthesis by specifically decreasing procollagen mRNA levels. The objective of the present experiments was to determine the mechanism by which propeptides cause these pretranslational effects. IMR-90 fibroblasts were exposed to medium containing carboxyl-terminal propeptide of type I procollagen, and nuclear run-off assays were performed by hybridization to a specific alpha 1 chain type I procollagen cDNA probe. Specific type I procollagen transcription rates were found to be decreased by 50% in the presence of 75 nM carboxyl-terminal propeptide compared with control (untreated) cells. Total cellular transcription rates as well as beta-actin mRNA rates were not affected significantly by any concentration of carboxyl-terminal propeptide. Propeptide radiolabeled with 125I was found to be taken up by cultured cells. Furthermore, exogenous carboxyl-terminal propeptide levels increased in the cytosolic compartment and eventually reached a steady-state level of 18 +/- 2 pmol/g cell protein by 30 min. Of particular interest was the finding that levels of radiolabeled carboxyl-terminal propeptide were also detected in the nuclear fraction and increased with time, reaching a plateau after 60 min of incubation. Incubation of nuclei from IMR-90 cells in medium containing varying concentrations of carboxyl-terminal propeptide resulted in nuclear transcription rates that were decreased by 40% compared with untreated controls. beta-Actin nuclear message levels remained unchanged under identical conditions. We conclude that carboxyl-terminal propeptide of type I procollagen can be internalized and become associated with the nuclear compartment. This suggests a feedback regulatory role on procollagen synthesis by a direct effect on procollagen gene transcription.

Animals↗