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Biomedical subjects

C C Chang

Publications and source records attributed to C C Chang.

At least 343 records · Page 19Linked to original sources

Human dermal papilla cells and outer root sheath cells: no follicular differentiation in nude mice and chicken embryos.

Human scalp specimens were incubated in 5 U/ml dispase solution at 4 degrees C overnight before the isolation of dermal papillae and follicle epithelium. This pretreatment not only facilitated the attachment and cell outgrowth of dermal papillae but also made it easier to pluck out hairs with intact follicle epithelium. The outer root sheath cells were released from the follicle epithelium and grown on a feeder layer of mitomycin C-treated human dermal fibroblasts. The subcultured outer root sheath cells were grown in a serum-free medium. When the mixtures of early-passage dermal papilla cells and outer root sheath cells were injected into the subcutis of nude mice, an epidermal cyst surrounded by layers of fibrous tissue was found in three weeks. No hair follicles were found when the mixtures were implanted onto the chorioallantoic membrane of nine-day-old chicken embryos. A keratinized mass lying on the chorionic epithelium with or without smaller similar masses in the chorioallantoic membrane was found in eight days. No hair follicle-like structure could be found. Possible factors contributing to the failure to undergo follicular differentiation in this study are discussed.

Animals↗

Chemical modification of Lys-6 in Taiwan cobra phospholipase A2 with 4-chloro-3,5-dinitrobenzoate.

Phospholipase A2 (PLA2) from Naja naja atra snake venom was modified with 4-chloro-3,5-dinitrobenzoate, and one major carboxydinitrophenylated (CDNP) PLA2 was separated by high performance liquid chromato-graphy. CDNP-PLA2 contained only one CDNP group on Lys-6 and showed a 93% drop in enzymatic activity. However, carboxydinitrophenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca2+ binding and antigenicity of CDNP-PLA2 were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA2 to 35% of that of native enzyme. These results suggested that the positively charged side chain of Lys-6 played a role in the enzymatic mechanism of PLA2. However, the partial restoration in PLA2 activity reflects that a distortion of the active conformation arising from incorporation of a bulky CDNP group should occur.

Amino Acid Sequence↗

Improvement in the binding of cobrotoxin to microtiter plates by glutaraldehyde at neutral pH.

The coating of the wells of microtiter plates with cobrotoxin in Tris (pH 9.8) or PBS (pH 7.2) buffer was assessed by enzyme-linked immunoassay (ELISA). It was found that the poor binding in neutral buffer was improved by adding glutaraldehyde (GA), and the bound amount reached the same extent as that measured with alkali buffer. The optimal concentration of GA was approx. 0.02%. A decrease in optical density was observed with GA concentration higher than 0.02%. This may result from the modification of cobrotoxin by GA, which induced a decrease in the antigenicity of the cobrotoxin as revealed by competitive immunoassay. This study implies that the coating of physiological samples with GA for ELISA may be carried out with physiological buffers without the need to change the buffer to one of alkaline pH.

Antibodies↗

Biochemical regulation of the activity of gamma-glutamylcysteine synthetase from rat liver and kidney by glutathione.

Rat liver and kidney gamma-glutamylcysteine synthetase (gamma GCS) had similar catalytic properties and consisted of heavy and light subunits, but the molecular structure of the two enzymes was not the same as evidenced by the results of SDS-PAGE and disc gel electrophoresis. Unlike kidney enzyme, most of liver gamma GCS was in a reduced enzyme form which did not have disulfide linkage between heavy and light subunits. Although the oxidized form of the two enzymes which subunits were linked with disulfide bond(s) could be dissociated to a similar extent by GSH, liver gamma GCS was inhibited by GSH to a much greater extent. These results suggest that the relative sensitivity of the gamma GCS enzymes to inhibition by GSH might be related to the inherent dissociability of heavy and light subunit of gamma GCS.

Animals↗

Effect of intravenous calcitriol on secondary hyperparathyroidism in chronic hemodialysis patients.

BACKGROUND: It has become evident that calcitriol can suppress parathyroid hormone (PTH) secretion by direct genomic actions. Intravenous calcitriol that bypasses gastrointestinal degradation might cause less degree of hypercalcemia and greater suppressive effect on PTH secretion. We investigated this PTH-suppressive effect of intravenous calcitriol in hemodialysis patients with secondary hyperparathyroidism. METHODS: Calcitriol was administered at the end of each dialysis session three times a week in 20 uremic patients, for 12 weeks. RESULTS: The mean dosage of calcitriol was 2.72 +/- 0.21 microgram per dialysis session. Serum intact PTH and C-PTH decreased (P < 0.05) after 6 weeks of treatment, while serum alkaline phosphatase (Alk-P) decreased 3 weeks later than PTH did. The individual maximal reduction of intact PTH, C-PTH and Alk-P were 77.80%, 67.36% and 45.98%. This PTH-suppression was dose-dependent. Despite the significant reduction of intact PTH by 58.17% after 6 weeks of treatment, no significant increase of serum calcium was found. An increase in serum calcium is not essential for this PTH-suppressive effect of calcitriol. Our observations thus provide another evidence to support the direct inhibitory effect of calcitriol on PTH secretion. Serum calcium, phosphorus, magnesium, albumin and osteocalcin levels did not change significantly. No side effect was found during treatment. CONCLUSIONS: Intravenous calcitriol is effective and safe in treating hemodialysis patients with secondary hyperparathyroidism. This treatment is more important in patients who are intolerant to oral vitamin D supply and who are candidates for surgical parathyroidectomy.

Adult↗

Mechanism of expression and role in transcriptional control of the proto-oncogene NFKB-2/LYT-10.

The NFKB-2 gene (previously LYT-10, NF-kappa Bp100 or NF-kappa Bp97) codes for a NF-kappa B/rel related protein which is highly homologous to NFKB-1 (previously NF-kappa Bp105) within its rel, poly-glycine and ankyrin domains. The NFKB-2 gene is a candidate proto-oncogene since it is involved in lymphoma-associated chromosomal aberrations. In order to gain insight into the physiological function and role in tumorigenesis of NFKB-2, we have analysed its mechanism of expression and role in transcriptional regulation. We report that, contrary to previous studies, a single 3.2 kb mRNA species and its 100 kD (p100) primary translation product is detectable in all cell types tested. A second NFKB-2 protein, p52, corresponding to the amino-terminal half (rel domain) of NFKB-2 p100, is detectable in the same cell types and derives from the post-translational processing of p100. While p100 is constitutively localized in the cytoplasm, NF-kappa B induction by TPA treatment of Hela cells is associated with cytoplasmic/nuclear translocation of NFKB-2 p52 and its appearance within DNA-binding NF-kappa B complexes. NFKB-2 p52 differs from NFKB-1p50 in its differential affinity for kappa B sequences: by itself it binds H2/HLA-kappa B sites more efficiently than HIV/IgK-kappa B sites, while it can bind both sites efficiently when complexed with Rel-A(p65). Transient co-transfection of expression and reporter plasmids in cells devoid of endogenous NF-kappa B activity showed that p52 has no intrinsic transcriptional activation capabilities: it can stimulate Rel-A(p65)-driven transcription by formation of p65/p52 heterodimers, whereas, overexpressed, it down-regulates p65-dependent transcription by formation of inactive p52/p52 homodimers. These results indicate that the NFKB-2 gene codes for an inducible NF-kappa B transcription factor with the capability of differentially regulating NF-kappa B transcription depending on its abundance in the nucleus.

Amino Acid Sequence↗

Isolation and characterization of a toxic phospholipase A2 from the venom of the Taiwan habu (Trimeresurus mucrosquamatus).

A toxic phospholipase A2 (PLA2) was isolated from Taiwan-habu-snake (Trimeresurus mucrosquamatus) venom by sequential chromatographies on CM-52, Sephadex G-75 and S-Sepharose columns. This basic PLA2 has a single polypeptide with an estimated M(r) of 15,000. The PLA2 activity was Ca(2+)-dependent and inactivated by p-bromophenacyl bromide and its specific antibody. The toxic PLA2-induced myotoxic and direct haemolytic effects as well as respiratory distress in mice with an intraperitoneal LD50 of 1.17 micrograms/g body weight. The histological examination showed it caused haemorrhage and congestion in the viscera of mice. It was also cytotoxic to myeloma cells (NS-1), baby-hamster-kidney (BHK) cells and human umbilical endothelial cells. By neutralization experiments with a specific antibody against toxic PLA2, it was found that the enzymic activity of toxic PLA2 is essential for its myotoxicity, but it is not the only factor responsible for the lethal toxicity.

Acetophenones↗

Rearranged NFKB2 gene in the HUT78 T-lymphoma cell line codes for a constitutively nuclear factor lacking transcriptional repressor functions.

Rearrangements of the NFKB2 gene are associated with lymphoid malignancies, but the functional significance of these alterations is not known. Here we characterize structurally and functionally a rearranged NFKB2 gene identified at the T cell lymphoma line, HUT78. The rearrangement has truncated NFKB2 sequences within the 3' ankyrin domain, leading to the production of truncated mRNA species and proteins as detected by Northern blot and immunoprecipitation analysis, respectively. Cloning and sequencing of the corresponding cDNAs indicates that, via alternative splicing, the rearranged gene codes for two proteins of 84 and 85 kD (p84/85) which retain the DNA-binding rel domain and the first five ankyrin repeats, but have lost their carboxy-terminus including the seventh ankyrin repeat. Immunofluorescence and immunoprecipitation analysis of HUT78 cells indicate that p84/85 are abnormally located in the nucleus in an unprocessed form, suggesting that these proteins can escape the cytoplasmic retention typical of the normal NFKB2 p100 protein before it is processed into p52. Electrophoretic mobility shift assays performed on HUT78 nuclear extracts indicate that the abnormal NFKB2 proteins bind kappa B sites specifically and alter the composition of NF-kappa B complexes in HUT78 cells. Transient co-transfection assays involving NFKB2 expression vectors and kappa B-driven reporter plasmids indicate that NFKB2 p85 has lost the transcriptional repressor functions typical of normal NFKB2 p52. These data indicate that the NFKB2 gene rearrangement detected in HUT78 cells leads to the production of abnormal NFKB2 proteins capable of altering the function of the NF-kappa B transcription system. Since analogous rearrangements are found in lymphoid malignancies, these findings further support a role of NFKB2 alterations in tumorigenesis.

Cloning, Molecular↗

Brequinar sodium, mycophenolic acid, and cyclosporin A inhibit different stages of IL-4- or IL-13-induced human IgG4 and IgE production in vitro.

We investigated the effect of cyclosporin A (CsA), mycophenolic acid (MPA), and brequinar sodium (BQ) on human IgG4 and IgE synthesis induced by IL-4 or IL-13. BQ inhibited IL-4 and IL-13-induced IgG4 and IgE synthesis in cultures of peripheral blood mononuclear cells (PBMC) or highly purified B cells costimulated by anti-CD40 mAbs in a dose-dependent fashion. CsA and MPA had either suppressive or enhancing effects depending on the concentrations tested. Interestingly, BQ inhibited IgG4 and IgE synthesis at concentrations of 10(-6)-10(-8) M, which did not affect T or B cell proliferation, indicating that the inhibitory effects of BQ on Ig production were not directly related to inhibition of T or B cell proliferation. In contrast, the inhibitory effects of MPA on Ig production were directly associated with inhibitory effects on T and B cell proliferation. CsA blocked T and B cell proliferation at the same concentration (10(-7) M) which enhanced IgG4 and IgE synthesis, indicating that reduction in T or B cell proliferation correlated with enhanced IgE production. CsA also inhibited CD40 ligand expression and IL-2, IL-4, IL-5, IFN-gamma, and GM-CSF production by activated CD4+ T cell clones, whereas MPA and BQ were ineffective, indicating that these compounds do not inhibit early events in T cell activation. Collectively, our data indicate that BQ, MPA, and CsA block different stages of the IgG4 and IgE production process. In addition, we observed that CsA and MPA, in contrast to BQ, at lower concentrations can also have potentiating effects on the production of these Ig isotypes.

Antigens, CD↗

Identification of functional involvement of tryptophan residues in phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom.

Phospholipase A2 (PLA2) from Naja naja atra snake venom was subjected to Trp modification with 2-nitrophenylsulfenyl chloride (NPS-Cl), and six derivatives were separated by HPLC. The results of amino-acid analysis and sequence determination revealed that Trp-18, Trp-19 and Trp-61 were modified by NPS-Cl. The order of accessibilities of the three Trp residues for NPS-Cl was Trp-18 > Trp-19 > Trp-61. Sulfenylation of Trp-18 caused a 92% drop in enzymatic activity. Modification of Trp-19 and Trp-61 resulted in a decrease in enzymatic activity of PLA2 by 45.5% and 51%, respectively. The enzyme modified on both Trp-18 and Trp-19 or on both Trp-18 and Trp-61 retained little PLA2 activity. It is evident that Trp-18 plays a more crucial function in PLA2 than Trp-19 and Trp-61. Sulfenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca2+ binding and antigenicity of sulfenylated PLA2 was unaffected. These observations, together with the fact that Trp-18 is involved in the substrate binding of PLA2, suggest that incorporation of a bulky NPS group on Trp-18 might give rise to a direct distortion of the interaction between substrate and the enzyme molecule. Alternatively, modification of Trp-19 and Trp-61 might indirectly affect the interfacial binding of PLA2 with its substrate.

Amino Acid Sequence↗

Molecular cloning and functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells.

Accumulation of cholesterol esters as cytoplasmic lipid droplets within macrophages and smooth muscle cells is a characteristic feature of early lesions of atherosclerotic plaque. Intracellularly, an essential element in forming cholesterol ester from cholesterol is the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT). ACAT is a membrane protein located in the endoplasmic reticulum. The ACAT protein has never been purified to homogeneity, and no antibodies directed against ACAT have been reported. The gene(s) encoding this enzyme had not been isolated. This laboratory had previously reported the isolation of Chinese hamster ovary cells expressing human ACAT activity. From DNAs of these cells, we have cloned a 1.2-kb exonic human genomic DNA. This led to the eventual cloning of a 4-kb cDNA clone (K1) from a human macrophage cDNA library. Transfection of K1 in ACAT-deficient mutant Chinese hamster ovary cells complemented the mutant defect and resulted in the expression of human ACAT activity. K1 contained an open reading frame of 1650 bp encoding an integral membrane protein of 550 amino acids. Protein homology analysis showed that the predicted K1 protein shared homologous peptide sequences with other enzymes involved in the catalysis of acyl adenylate formation followed by acyl thioester formation and acyl transfer. These results indicate that K1 encodes a structural gene for ACAT. The cDNA reported here should facilitate future molecular studies on ACAT.

Amino Acid Sequence↗

O2 occlusion and cyanide induced immediate relaxation and contraction of murine skeletal muscle.

The acute changes of muscle tone and membrane current upon occlusion of oxygenation (O2 occlusion) were studied in vitro in mouse diaphragms. O2 occlusion immediately produced a contraction and a relaxation, respectively, in ryanodine- and high K(+)-contracted muscles while a biphasic change (an initial decrease then a late increase) of muscle tone was produced in muscles contracted with caffeine. The O2 occlusion effects were reversed after reoxygenation. CN- produced similar acute changes of muscle tone and abolished O2 occlusion effects. The O2 occlusion-induced relaxation in high K+ medium was converted into a contraction by 3,4-diaminopyridine and by low Cl- Tyrode's. O2 occlusion induced a small outward current and membrane hyperpolarization at a rate slower than the changes of muscle tone. Glybenclamide inhibited all of the changes induced by O2 occlusion. It is possible that the K+ and Cl- permeabilities of sarcoplasmic reticulum are highly sensitive to hypoxic challenge and related to the immediate changes of muscle tone after O2 occlusion.

Animals↗

Improvement by diazepam of neuromuscular transmission blocked by anticholinesterase agents in mouse diaphragms.

Effects of diazepam on the neuromuscular transmission blocked by neostigmine were studied in isolated mouse phrenic nerve-diaphragm preparations. Diazepam, in the absence of neostigmine, had no significant effect on the neuromuscular transmission at concentrations lower than 100 microM, except to enhance muscle contractility. Single and train pulses-evoked endplate potentials (e.p.p.s) and miniature e.p.p.s (m.e.p.p.s) were also unaffected. At concentrations of 175 microM or higher, diazepam caused an axonal conduction block. However, neostigmine-induced twitch potentiation, spontaneous fasciculation and tetanic fade were antagonized by diazepam at 3.5-35 microM. Diazepam did not decrease the amplitude of neostigmine-augmented e.p.p.s and m.e.p.p.s but slightly reduced their decay time. The incidence of regenerative depolarization of endplates induced by repetitive stimulation in the presence of neostigmine, was decreased from 92% to 35% junctions and the duration shortened from 650 ms to 230 ms. The amplitude of train e.p.p.s was increased. It is suggested that diazepam reverses the muscle paralysis induced by anticholinesterase agents by inhibiting the regenerative release of acetylcholine.

Animals↗

Comparative abilities of athymic nude mice and severe combined immune deficient (SCID) mice to accept transplants of induced rat mammary carcinomas: enhanced transplantation efficiency of those rat mammary carcinomas that have elevated expression of neu oncogene.

Grafts of primary carcinogen (DMBA)-induced mammary carcinomas from Sprague-Dawley rats have a poor transplantation efficiency in athymic nude mice. Further compromising these mice immunologically via whole-body irradiation and/or splenectomy, or the administration of hormonal growth factors (estrogen and progesterone) to these mice, did not significantly alter transplantation efficiency. Use of strains of mice that are more immune-impaired than the athymic nude mouse, i.e., the athymic nude-beige-XID mouse (T-cell and LAK-cell deficient) or mice with severe combined immune deficiency (SCID) (which lack functional T cells and B cells) also failed to improve transplantation efficiency. In contrast, transplantation efficiency was sharply increased when primary neu-induced rat mammary carcinomas from female Sprague-Dawley rats were used. These mammary carcinomas, unlike the DMBA-induced rat mammary carcinomas, have a very high level of expression of neu; transplantation of these tumors to either athymic nude mice or SCID mice was considerably more efficient. Thus, these data provide evidence that enhanced expression of neu confers heightened efficiency in the transplantation of primary rat mammary carcinomas to immune-deficient mice (athymic-nude or SCID). Increased neu expression was a greater determinant than more compromised immune states in the transplantation of these rat mammary carcinomas. This biological characteristic of neu expression in mammary carcinomas provides new, additional insight into the importance of this oncogene in mammary tumorigenic processes and may explain, at least in part, the reported inverse relationship between human breast carcinoma neu expression and patient prognosis.

9,10-Dimethyl-1,2-benzanthracene↗

Determination by heteronuclear NMR spectroscopy of the complete structure of the cell wall polysaccharide of Streptococcus sanguis strain K103.

Although complete structures of complex polysaccharides have traditionally been determined by chemical degradative methods, a number of recent developments in instrumentation have greatly facilitated this task. We illustrate the application of several of these methods in a determination of the complete covalent structure of the polysaccharide from Streptococcus sanguis K103, which is composed of an octasaccharide repeating subunit linked by phosphodiester bonds. Carbohydrate analysis by HPAE-PAD and by reverse-phase chromatography of benzoylated derivatives of the hydrolysis products of the polysaccharide gave glucose (3 mol), galactose (1 mol), rhamnose (2 mol), N-acetylglucosamine (1 mol), and galactose 6-phosphate (1 mol). Circular dichroism of the O-benzoylated monosaccharides showed the absolute configurations to be D for all residues except for rhamnose, which is L. The 1H NMR spectrum was completely assigned by two-dimensional homonuclear methods (DQF-COSY, NOESY, HOHAHA). The stereochemistry of pyranosides was assigned from 3JHH coupling constant values determined from these experiments. The 13C spectrum was assigned by 1H-detected heteronuclear multiple-quantum correlation (1H[13C] HMQC) and by the hybrid method of HMQC-COSY. The glycosidic linkage positions of the polymer were determined by 1H-detected multiple-bond correlation (1H[13C] HMBC) and by 2D-NOESY spectra. The position of the phosphodiester linkage was determined by splitting observed in the 13C resonances due to 31P couplings leading to the overall structure given in Chart I.

Acetylglucosamine↗

Sequence characterization of a novel alpha-neurotoxin from the king cobra (Ophiophagus hannah) venom.

Several postsynaptic neurotoxins (alpha-neurotoxins) with distinct pharmacological and biochemical properties were isolated and purified from the King cobra venom (Ophiophagus hannah) by employing sequentially preparative-scale cation-exchange chromatography on SP-Sephadex C-25 coupled with gel filtration and reversed-phase HPLC. The complete sequence of one neurotoxin was determined by N-terminal Edman degradation with the automatic pulsed-liquid phase sequencer on some peptide fragments generated from the endopeptidases, i.e. trypsin, S. aureus V8 protease and lysyl endopeptidase. This novel neurotoxin is a basic polypeptide of pI 9.05, consisting of 72 amino-acid residues with 10 cysteine residues. It is found to share about 60% sequence homology with Toxins a and b isolated from the same venom and the well established alpha-bungarotoxin, a major postsynaptic toxic ligand for acetylcholine receptor isolated from Bungarus multicinctus. The characterized alpha-neurotoxin molecules were also shown to bind specifically with nicotinic acetylcholine receptors of the electric eel, Torpedo californica.

Amino Acid Sequence↗

Restoration of gap-junctional intercellular communication in a communication-deficient rat liver cell mutant by transfection with connexin 43 cDNA.

To study the biochemical basis of gap-junctional intercellular communication (GJIC) and its role in tumorigenesis, a mammalian cell expression vector carrying both a rat connexin 43 (Cx43) cDNA and an amplifiable dihydrofolate reductase (DHFR) gene was transfected into the GJIC-deficient rat liver mutant cell line aB1. Two stable transfectants were selected for further amplification of the transfected Cx43 gene by increasing stepwise the concentration of methotrexate (MTX) in the culture medium. The results indicate that GJIC was restored in these two Cx43 cDNA transfectants after they became highly resistant to MTX but not in the control-vector transfectants, in which the DHFR gene was similarly amplified. The amount of Cx43 DNA revealed by Southern blot analysis and the expression of Cx43 gene revealed by northern and western blot analyses were concomitantly increased in the Cx43 cDNA transfectants resistant to high concentrations of MTX. Western blot analysis, using an antipeptide antibody that specifically recognizes Cx43 protein, further revealed that an approximately 46-kDa phosphorylated Cx43 protein that was prominent in the parental GJIC-competent cells was absent in the aB1 cells. This Cx43 protein, however, reappeared in the two Cx43 cDNA transfectants after amplification. After treatment of the membrane proteins with alkaline phosphatase in vitro, the approximately 46- and 44-kDa proteins disappeared, whereas the approximately 42-kDa proteins remained with increasing intensity, indicating that the higher molecular-weight proteins were the phosphorylated Cx43. These results indicate that a defect in posttranslational phosphorylation of Cx43 protein associated with low expression of the Cx43 gene might be responsible for the GJIC deficiency in aB1 cells and that increased expression of Cx43 by gene amplification might restore this phosphorylated Cx43 protein and so reestablish GJIC.

Animals↗

Silicon and silicone: theoretical and clinical implications of breast implants.

In the past 10 years, there have been multiple published reports associating silicone breast implants with scleroderma, morphea, SLE, rheumatoid arthritis, CREST syndrome and "human adjuvant disease." The alleged offending material, silicone, is a synthetic polymer containing a silicon-oxygen backbone. Beginning with the heating of SiO2 in the presence of carbon, elemental silicon is produced. Methylchloride is added and the resulting product is hydrolyzed to form low molecular weight prepolymers which are linked to form linear silicone polymers and cross-linked to yield silicone rubbers or elastomers. The polymeric and hydrophobic characteristics of silicone and the presence of electrostatic charges and organic sidegroups make silicone a potentially ideal immunogen, leading to cross-reactivity with autoantigens. Silicon is an essential constituent of proteoglycans which theoretically could result in immunological cross-reactions between silicone and connective tissues. Although the literature contains numerous examples of silicone-associated autoimmune disease, there is no consistent pattern of immunological abnormalities observed. There are, however, some intriguing and interesting observations. Further large-scale studies are needed to determine if a link between silicone exposure and autoimmunity exists. Also, since the inducing events of autoimmune diseases are unknown, studies on silicone could provide a model for autoimmune diseases associated with toxicological factors.

Adult↗