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P L Chang

Publications and source records attributed to P L Chang.

At least 19 recordsLinked to original sources
PubMed1992

Diagnosis of arylsulfatase A deficiency.

Metachromatic leukodystrophy (MLD) is a neurologically devastating autosomal recessive disorder in humans associated with deficient arylsulfatase A activity. However, clinically normal individuals described as being pseudo-arylsulfatase-A deficient also demonstrate the same deficiency. Genotypically, they may be homozygous for the pseudodeficiency mutation (associated with 2 A-->G transitions in the cDNA of arylsulfatase A) or heterozygous with one pseudodeficiency and one MLD allele. Using as examples 2 families in which the pseudo deficiency condition occurs either independently or together with MLD, we demonstrate the utility of a proposed diagnostic protocol to provide complete genotype identification of individuals suffering from arylsulfatase A deficiency. Patient fibroblasts are extracted for DNA and a cytoplasmic fraction, which is used for arylsulfatase A enzyme assay. This will identify an arylsulfatase A-deficient group, which is further analyzed electrophoretically. Cells from the clinically affected patients with MLD are completely deficient in arylsulfatase A activity, whereas those from the pseudodeficient individuals demonstrate a characteristic residual arylsulfatase A activity detectable only after electrophoresis. Within this pseudodeficient group, gene amplification of DNA specific for the A-->G mutations will distinguish between those who are homozygous for the pseudodeficiency allele and those who are compound heterozygous for the pseudodeficiency and MLD alleles. This protocol of complete genotype identification requires only about 10(6) fibroblasts (1 x 100 mm dish) and 2 days to complete. Such variant-specific genotype identification increases accuracy and prognostic value of the diagnosis. It will likely become the preferred choice for diagnosis of genetic disease in the future as more variant-specific mutations are identified at the molecular level.

Alleles↗
PubMed1992

Xanthogranulomatous pyelonephritis: experience in 36 cases.

A retrospective study of 36 patients with xanthogranulomatous pyelonephritis who underwent nephrectomy at our hospital was performed. The disease occurred most frequently in middle-aged women with a history of recurrent urinary tract disorder. There were 2 cases of focal xanthogranulomatous pyelonephritis, 2 associated with emphysematous pyelonephritis, 2 that manifested as fistula formation between the colon or skin, and 1 with deep sinus formation into the hip joint that presented as septic arthritis. Flank pain and fever were the most frequent complaints. Escherichia coli (67%) and Proteus mirabilis (26%) were the most common organisms isolated from the voided urine, kidney and blood stream. Cephalothin plus gentamicin or tobramycin were the drugs of choice before surgical intervention.

Adult↗
PubMed1992

Intrathymic implants of genetically modified fibroblasts.

Implantation of autologous rodent fibroblasts genetically altered to express human growth hormone has recently been shown to be a feasible approach to the delivery of new gene products in somatic gene therapy. However, the novel gene product elicited in its recipients an intense antibody response that would have curtailed the effectiveness of such therapy. The possibility of inducing tolerance to foreign gene product was explored by implanting allogeneic fibroblasts transfected with the human growth hormone gene into rat thymus, a site recently shown to be immunologically privileged and able to induce donor-specific tolerance to transplanted tissues. In the circulation of the implanted rats, human growth hormone was detected at 4-15 ng/ml serum within the first day and subsided to 0.6-9 ng/ml within the first wk in all animals implanted either thymically or intraperitoneally. Within 2-3 wk, high titers of anti-human growth hormone were detected in all animals regardless of the site of implantation. The failure of the thymus to offer immune protection for the foreign antigen was further confirmed when the animals were subsequently challenged with purified human growth hormone. An immediate twofold increase in titer within the first week of challenge was detected in animals previously implanted thymically. In contrast, animals implanted intraperitoneally but treated with short-term daily injection of cyclosporine A (28-41 days) did not mount any significant antibody response to human growth hormone throughout the experiment or even when challenged subsequently at weeks 8-10 with purified growth hormone. In conclusion, implantation of genetically modified fibroblasts in the thymus does not lead to tolerization toward soluble novel gene product secreted by these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Predictors of antibiotic prophylactic failure in post-cesarean endometritis.

Despite the use of prophylactic antibiotics, endometritis occurs in 1-31% of patients after cesarean delivery. We sought to identify predictors of prophylactic antibiotic failure. In a retrospective review of 1800 cesarean deliveries, 766 patients had either ampicillin or cefazolin prophylaxis; 240 (31.3%) developed endometritis. Stepwise logistic regression identified the number of vaginal examinations (P less than .001), nulliparity (P = .001), low gestational age (P = .033), and cefazolin use (P = .002) as predictors of endometritis. One in two women with six or more vaginal examinations before cesarean delivery will have prophylactic failure. In preterm gestations (N = 177), the number of vaginal examinations was the single predictor of prophylactic antibiotic failure (P = .002). The number of vaginal examinations can be used to design new strategies to prevent a high rate of prophylactic failure.

Ampicillin↗
PubMed1991

Characterization of arylsulfatase C isozymes from human liver and placenta.

Arylsulfatase C and steroid sulfatase were thought to be identical enzymes. However, recent evidence showed that human arylsulfatase C consists of two isozymes, s and f. In this study, the biochemical properties of the s form partially purified from human placenta were compared with those of the f form from human liver. Only the placental s form has steroid sulfatase activity and hydrolyses estrone sulfate, dehydroepiandrosterone sulfate and cholesterol sulfate. The liver f form has barely detectable activity towards these sterol sulfates. With the artificial substrate, 4-methylumbelliferyl sulfate, both forms demonstrated a similar KM but the liver enzyme has a pH optimum of 6.9 while the placental form displayed two optima at 7.3 and 5.5. The molecular weight of the native enzyme determined with gel filtration was 183,000 for the s form and 200,000 for the f form and their pI's were also similar at 6.5. However, the T50, temperature at which half of the enzyme activity was lost, was 49.5 degrees C for the f form and 56.8 degrees C for the s form. Polyclonal antibodies raised against the placental form reacted specifically against the s and not the f form. They immuno-precipitated concomitantly greater than 80% of the total placental arylsulfatase C and steroid sulfatase activities while less than 20% of the liver enzyme was immuno-precipitable. In conclusion, the two isozymes s and f of arylsulfatase C in humans purified from placenta and liver, respectively, have similar KM, pI' and native molecular weight. However, they are distinct proteins with different substrate specificity, pH optima, heat-lability and antigenic properties. Only the s form is confirmed to be steroid sulfatase.

Arylsulfatases↗
PubMed1991

1 alpha,25-dihydroxyvitamin D3 stimulates synthesis and secretion of nonphosphorylated osteopontin (secreted phosphoprotein 1) in mouse JB6 epidermal cells.

Murine JB6 epidermal cells can be irreversibly transformed into tumorigenic cells by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. One feature of this transformation is induction of the synthesis and secretion of the phosphoprotein osteopontin (also called secreted phosphoprotein 1 and previously referred to as transformation-related phosphoprotein, 2ar, bone sialoprotein 1, or Mr 44,000 bone phosphoprotein), an arginylglycylaspartic acid-containing cell adhesion glycoprotein the expression of which has been implicated in tumorigenesis and metastasis. Since 1 alpha,25-dihydroxyvitamin D3, calcitriol, also transforms JB6 cells and, in other cell types, regulates osteopontin synthesis, we hypothesized that calcitriol-mediated transformation of JB6 cells would also cause induction of osteopontin synthesis and secretion. Metabolic labeling with 32PO4 of near confluent JB6 cells (clone 41.5a) treated with calcitriol (0.1-100 ng/ml) for up to 48 h revealed only a minimal production of osteopontin, which is the major phosphoprotein secreted by 12-O-tetradecanoylphorbol-13-acetate-treated cells. Similar treatment followed by labeling with [35S]methionine showed a substantial dose-dependent increase in the synthesis and secretion of osteopontin. This induction was not associated with increased cell proliferation or with cell transformation, as assayed by anchorage-independent growth. Calcitriol-treated cells were morphologically indistinguishable from control cells, while 12-O-tetradecanoylphorbol-13-acetate-treated cells acquired a distinctive morphology. No induction of osteopontin was found with 25-hydroxyvitamin D3 or 24R,25-dihydroxyvitamin D3. These results show that calcitriol induces the synthesis and secretion of a nonphosphorylated form of osteopontin, in a cell type which normally makes little or none of this protein, and that the induction is not correlated with the tumorigenic transformation of these cells.

Animals↗
PubMed1991

Expression of a novel gene product by transplants of genetically modified primary fibroblasts in the central nervous system.

Primary fibroblasts initiated from skin biopsies of Wistar rats were transfected with a plasmid that encodes the human growth hormone and the neomycin resistance genes. Cell clones selected for G418 resistance and expressing high levels of human growth hormone were propagated in vitro and subsequently transplanted into the cerebral cortex of adult allogeneic rats. Grafts were examined by immunocytochemistry at weekly intervals up to 2 months. Fibroblasts in the transplants survived and expressed growth hormone clearly up to 4 weeks, but showed reduced expression at 6 to 8 weeks. Significant levels of human growth hormone were also detected by radioimmunoassay in the serum of the host rats up to 1 month. The experiments demonstrate that primary fibroblasts can be genetically modified to deliver a new gene product into the central nervous system, and the gene product can pass the blood-brain barrier to enter the systemic circulation. This model illustrates the potential to introduce desired products into the brain through the genetic alteration of autologous primary fibroblasts.

Animals↗
PubMed1991

Root reconstruction for bladder reinnervation: an experimental study in rats.

Nerve transfer (or nerve crossover) is a well established technique for achieving reinnervation of a valuable sensory or motor territory by reconnection using a functional nerve of lesser value. Patients with lower spinal cord lesions causing neurogenic bladder dysfunction could theoretically benefit from such an approach for return of useful micturition. Based on the known anatomical details of the spinal nerve, a new reconstructive method was created to provide intradural ventral root transfer for pure motor-to-motor reinnervation and extradural postganglionic spinal nerve transfer for pure sensory-to-sensory reinnervation. Experimental studies in rats were performed, demonstrating the feasibility of this approach. A modified method is further suggested that would use nerve grafts for extradural approaches to pure motor and sensory transfers, without the need for extensive laminectomy and dura opening. This proposed approach is anticipated to minimize the associated morbidity and mortality with such spinal nerve reconstruction.

Animals↗
PubMed1991

Growth inhibition by cyclosporine A in vivo and in vitro.

Cyclosporine A is a commonly used immunosuppressant in organ transplant. However, in addition to its nephro- and hepato-toxicity, we now report that it also exerts growth inhibiting effects in vivo and in vitro. Rats treated with daily Cyclosporine A I.P. injections of 10 mg or 20 mg/kg body weight gained weight at significantly lower rates than controls over a six week period. This growth retardation was completely reversible. By 6 weeks post treatment, the body weights of the treated animals were the same as controls. In contrast, rats treated with lower doses of cyclosporine A (5 mg/kg) did not suffer any growth retardation throughout. In vitro, rat primary cultured fibroblasts treated with greater than 1 microgram/ml Cyclosporine A suffered 25-60% cell loss in the post-log phase but the cytotoxic effect was not apparent during the log phase of growth. Fibroblasts treated with less than or equal to 1 microgram/ml of Cyclosporine A did not suffer any cell loss. In conclusion, Cyclosporine A exerts growth inhibitory effects in vivo when given at greater than 5 mg/kg body weight and cytotoxic effects in vitro at greater than 1 microgram/ml.

Animals↗
PubMed1990

Deficient glycosylation of arylsulfatase A in pseudo arylsulfatase-A deficiency.

Deficient arylsulfatase-A activity is diagnostic of a neurodegenerative human lysosomal storage disease, metachromatic leukodystrophy. Paradoxically, similar enzyme deficiency also occurs in normal individuals, who are known as being pseudo arylsulfatase-A deficient. We showed previously that this phenotype is associated with a structural gene mutation that produces an exceptionally labile enzyme. We now report on the nature and consequence of this mutation. When the mutant arylsulfatase-A is deglycosylated by endoglycosidase H, only one smaller molecular species was generated, instead of the two from the normal enzyme. This is consistent with the loss of one of the two N-linked oligosaccharide side chains known to be present on the wild-type enzyme. Quantitative analysis of mannose and leucine incorporation showed that the mutant enzyme incorporated two- to tenfold less mannose than the normal enzyme on a molar basis. This deficient glycosylation was specific to arylsulfatase-A. Another lysosomal enzyme not affected in this mutation, beta-hexosaminidase, was glycosylated normally in the mutant cells. The remaining single oligosaccharide side chain released from the mutant arylsulfatase-A by pronase digestion was normally processed to complex and high-mannose forms. However, the high-mannose side chains contained 30% fewer phosphorylated residues than those of the normal enzyme. Nevertheless, this reduced level of phosphorylation did not prevent targeting of the mutant enzyme to the lysosomes, a process normally mediated through phosphorylated mannose residues. In conclusion, pseudo arylsulfatase-A deficiency is a unique human mutation associated with reduced glycosylation and phosphorylation of a lysosomal enzyme with the loss of one of the two carbohydrate side chains. The mutation results in greatly reduced enzyme stability, thus indicating a role for oligosaccharides in maintaining enzyme stability within the degradative environment of the lysosomes. However, the residual catalytic activity or subcellular targeting of the mutant enzyme was not affected. These properties probably account for the benign clinical presentation of pseudo arylsulfatase-A deficiency.

Acetylglucosaminidase↗
PubMed1990

Energy dependence of DNA-mediated gene transfer and expression.

When cells are transfected with calcium phosphate-precipitated DNA, most of the DNA is internalized through active endocytosis but a small pool is postulated to be internalized by passive diffusion. The relative importance of these two DNA pools in gene transfer and expression has never been examined. We now show that mouse Ltk- fibroblasts internalize greater than 90% of radioactively labeled, calcium phosphate-precipitated DNA by a temperature- and energy-dependent process but a small pool of 2-3% of radioactively labeled DNA may be internalized even at 4 degrees C or under energy depletion conditions. The contribution of these two pools of DNA to gene expression was examined by transfecting Ltk- cells with DNA from a plasmid encoding marker for both transient expression (human growth hormone) and stable integration (neomycin resistance). When cells were depleted of ATP during transfection, transient expression of human growth hormone was reduced to only 2% of control values at day 4 posttransfection. Under the same condition, stable expression of neomycin resistance also was reduced to less than 1% of the control. Therefore, these data showed that only the pool of calcium phosphate-precipitated DNA internalized through an energy-dependent process is ultimately responsible for transient and stable expression of exogenous genes.

Adenosine Triphosphate↗
PubMed1990

Intracellular distribution of DNA internalized through calcium phosphate precipitation.

Although calcium phosphate precipitation is the most commonly used method for DNA-mediated gene transfer, the mechanism for its action is unknown. We showed recently that both transient and stable expression of exogenous genes in the transfected cells are entirely dependent on DNA internalized through active endocytosis. We now report on the subcellular distribution of the endocytosed DNA. After exposure to calcium phosphate-precipitated DNA, cultured fibroblasts internalized less than 10% of the DNA into the nuclei fraction. About 20% was recovered in each of the putative plasma membrane and vesicular organelle fractions. Although over 50% was recovered in the cytosolic fraction, it was completely degraded to oligonucleotides of smaller than 100 bp. In contrast, intact DNA molecules were recovered in all the other subcellular fractions. Similar patterns of DNA distribution were observed not only in the easily transformed mouse cells (Ltk-) but also in the transformation-resistant human primary fibroblasts. In conclusion, DNA-mediated gene transfer by calcium phosphate precipitation is an inefficient procedure because over 50% of the DNA is almost immediately degraded and released into the cytosol. Contrary to accepted views, DNA macromolecules do not seem to pass through the cytosol before reaching the nuclei. A novel transport pathway is proposed in which exogenous DNA molecules may be transferred directly by intermediary vesicles from the endocytic-lysosomal compartment to the nucleus.

Animals↗
PubMed1990

Hunter disease (mucopolysaccharidosis type II) in a karyotypically normal girl.

A female child of healthy, unrelated parents presented at 12 months of age with a history of moderately severe developmental delay, macrocephaly, dysmorphic facies, hypotonia, hepatosplenomegaly, mild generalized dysostosis multiplex, mucopolysacchariduria (dermatan and heparan sulfates), and Alder-Reilly bodies in peripheral blood leukocytes. Iduronate sulfatase activity in plasma was markedly depressed: 0.11 units/ml/h (normal, 1.75 +/- 0.56, N = 6). Analyses of arylsulfatases A, B, and C, heparan N-sulfatase, alpha-mannosidase, beta-mannosidase, beta-glucuronidase, beta-hexosaminidase, beta-galactosidase, and alpha-fucosidase activities in plasma, leukocytes, and/or cultured skin fibroblasts were all normal. Urinary sulfatide excretion was also within normal limits. Karyotypes of peripheral blood leukocytes and cultured skin fibroblasts were normal. Serum iduronate sulfatase activities in the parents were in the normal range (father, 1.63 units/ml/h; mother, 1.25 units/ml/h). The results of analyses of restriction fragment length polymorphisms (RFLP) of DNA from cultured skin fibroblasts with the use of probes for loci extending from Xpter to Xq28 showed X chromosome heterozygosity and confirmed the paternal origin of one of the X chromosomes. Studies on sulfur-35 uptake in mixed fibroblast cultures showed cross-correction of [35S]-glycosaminoglycan accumulation between cells from the patient and normal cells or cells from a patient with Hurler disease; however, there was no cross-correction between cells from the patient and those from boys affected with classical Hunter disease. This represents only the second confirmed case of Hunter disease reported in a karyotypically normal girl.

Arylsulfatases↗
PubMed1990

Comparison of arylsulfatase C and steroid sulfatase from human placenta and liver.

Human placental and hepatic arylsulfatase C (ASC) were purified to homogeneity and about 1,000-fold, respectively. Placental ASC hydrolyzed sterol sulfates at the same active site, whereas the major hepatic ASC did not. This major hepatic ASC isozyme was more thermolabile than placental ASC and steroid sulfatase from both placenta and liver. It was not precipitated by anti-bovine ASC IgG which quantitatively precipitated both placental ASC and steroid sulfatase activities from placenta and liver. A minor hepatic ASC isozyme with similar electrophoretic mobility to the placental enzyme copurified with the major hepatic ASC and is likely responsible for the steroid sulfatase activity in this organ. Hence, placental ASC and steroid sulfatase are biochemically and antigenically identical to hepatic steroid sulfatase. In contrast, the major hepatic ASC is a distinct protein whose catalytic and structural properties differ from all the above enzymes.

Arylsulfatases↗
PubMed1990

The human arylsulfatase-C isoenzymes: two distinct genes that escape from X inactivation.

Arylsulfatase C is a microsomal membrane-bound enzyme previously thought to be the same as steroid sulfatase, the only X-linked enzyme known to escape from X inactivation in man. We had shown that arylsulfatase C actually consists of two biochemically distinct isozymes, s and f. Only the s form has steroid sulfatase activity. The f and s forms were thought to be related through posttranslational or posttranscriptional modification of the same gene product. In part consistent with this hypothesis, we now report that in a panel of 28 rodent-human somatic cell hybrids, expression of both s and f was concordant only with the human X chromosome, thus showing that the f form is also X linked. In three separate somatic hybrids containing human X chromosomes in an inactive state, the f form was still expressed. Thus, similar to the s form, the f form also escapes from X inactivation. However, contrary to expectations, the s and f forms were not related by posttranslational modification of the same gene product. A full-length cDNA for the s form failed to hybridize to transcripts produced from an f-expressing cell line, showing that there is little sequence identity between the two. They are also not related by differential splicing of a common primary transcript, since fibroblasts from some patients with steroid sulfatase deficiency due to gene deletion of the s form continue to express the f form. Therefore, although the f and s isozymes of arylsulfatase C are X linked and escape from X inactivation, they are products from separate genes, thus providing a unique isoenzyme system to study possible gene duplication and regulation in the part of the human X chromosome that escapes inactivation.

Animals↗
PubMed1990

Anterograde axonal transport of horseradish peroxidase in the rat postganglionic sympathetic nerves.

Seven minutes after initiating the experiment, horseradish peroxidase (HRP)-labeled pinocytotic vesicles were observed on the plasma membranes of the postganglionic sympathetic neurons in the left superior cervical ganglia (SCG). HRP reaction product was also demonstrated in the vesicles of the smooth endoplasmic reticulum (sER) and multivesicular bodies in the neuronal perikarya. The HRP-positive vesicles of sER and multivesicular bodies were also detected in the adrenergic sympathetic nerve endings in the pineal gland 7 minutes after initiation of the experiment. This suggests that the HRP is rapidly transported anterogradely from the postganglionic sympathetic neurons in the SCG into the axon terminals in the pineal glands through the vesicles of the sER and multivesicular bodies. The present findings reveal that the rate of anterograde transport of HRP is 85 mm/h and that the majority of the adrenergic sympathetic nerves in either the left or right half of the pineal gland are derived from the postganglionic sympathetic neurons in both the left and right superior cervical ganglia.

Animals↗
PubMed1990

Autologous fibroblast implantation. Feasibility and potential problems in gene replacement therapy.

Autologous implants of genetically modified fibroblasts may be an ideal approach to gene replacement therapy. However, primary fibroblasts have limited life span and their use for gene therapy is questionable. We have developed a rat model to evaluate the feasibility of this approach. Primary fibroblasts initiated from rat skin biopsies were transfected with plasmid DNA. The transkaryotic fibroblasts were implanted into the original rat donors. A plasmid-encoded gene product, human growth hormone, was detected in the donor rats' circulation but less than 1% of the expected amount was recovered. Implantation at intraperitoneal, intramuscular or subcutaneous sites were effective in delivering the novel gene product. Fluctuating levels of human growth hormone were detected for over 6 months but their determination was rendered unreliable due to interference by extremely high titers of antibodies developed against the human growth hormone. In some cases, the antibody titer continued rising for more than 6 months, indicative of the continued presence of the antigen. One of the recipients developed two intra-abdominal fibrosarcomas that were shown to be derived from the implanted cells. In conclusion, this model demonstrates that autologous fibroblast implantation is a feasible approach to delivering novel gene products lasting for several months. Strong and prolonged immuno-reaction against the gene product will occur if the recipient's immune system is naive to the replacement product, as would occur in patients with CRM- mutations. The potential for neoplastic development from the implanted fibroblasts should prompt further evaluation of the long-term safety in gene replacement therapy.

Animals↗