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Biomedical subjects

W S Chang

Publications and source records attributed to W S Chang.

At least 19 recordsLinked to original sources
PubMed2002

Isolation and identification of a novel satellite DNA family highly conserved in several Cervidae species.

In an attempt to amplify cervid satellite II DNA from the genomes of Indian muntjac and Chinese muntjac, a pair of primers derived from the white tailed deer satellite II DNA clone (OvDII) yielded a prominent approximately 1 kb polymerase chain reaction (PCR) product (in addition to the expected 0.7 kb satellite II DNA fragments) in both species. The approximately 1 kb products were cloned, sequenced, and analyzed by Southern blotting and fluorescence in situ hybridization (FISH). This revealed that the approximately 1 kb cloned sequences indeed represent a previously unknown cervid satellite DNA family, which is now designated as cervid satellite IV DNA. Approximately 1 kb PCR clones were also obtained from the genomes of the black tailed deer and Canadian woodland caribou with similar primer pairs. Extremely high sequence conservation (over 90% homology) was observed among the clones generated from all four deer species and PCR-Southern hybridization experiments further verified the co-amplification of two kinds of satellite DNA sequences with the same pair of primers. This satellite DNA was found to co-localize with centromeric proteins at the kinetochore by a simultaneous FISH and immunofluorescence study. Due to its high sequence conservation and close association with kinetochores, the newly identified satellite DNA may have a functional centromeric role.

Animals↗
PubMed2000

Simultaneous detection of gfp-marked Moraxella sp. G21r and lux-marked Ralstonia eutrophas H850Lr using most-probable-number method.

The green fluorescent protein encoded by gfp gene and the luminescent protein encoded by luxAB genes were used as markers to detect p-nitrophenol (PNP)-degrading Moraxella sp. G21r and polychlorinated biphenyl (PCB)-degrading Ralstonia eutrophas H850Lr cells, respectively, in mixed liquid cultures and in soil samples using a most-probable-number (MPN) assay. Population estimates for both gfp-marked G21r and lux-marked H850Lr by using MPN assays were similar to viable colony counts. The MPN assay with microtiter plates permitted the simultaneous detection of fluorescent and luminescent bacteria in soil samples faster than conventional plate counting.

Bacteriological Techniques↗
PubMed2000

Inactive conformation of the serpin alpha(1)-antichymotrypsin indicates two-stage insertion of the reactive loop: implications for inhibitory function and conformational disease.

The serpins are a family of proteinase inhibitors that play a central role in the control of proteolytic cascades. Their inhibitory mechanism depends on the intramolecular insertion of the reactive loop into beta-sheet A after cleavage by the target proteinase. Point mutations within the protein can allow aberrant conformational transitions characterized by beta-strand exchange between the reactive loop of one molecule and beta-sheet A of another. These loop-sheet polymers result in diseases as varied as cirrhosis, emphysema, angio-oedema, and thrombosis, and we recently have shown that they underlie an early-onset dementia. We report here the biochemical characteristics and crystal structure of a naturally occurring variant (Leu-55-Pro) of the plasma serpin alpha(1)-antichymotrypsin trapped as an inactive intermediate. The structure demonstrates a serpin configuration with partial insertion of the reactive loop into beta-sheet A. The lower part of the sheet is filled by the last turn of F-helix and the loop that links it to s3A. This conformation matches that of proposed intermediates on the pathway to complex and polymer formation in the serpins. In particular, this intermediate, along with the latent and polymerized conformations, explains the loss of activity of plasma alpha(1)-antichymotrypsin associated with chronic obstructive pulmonary disease in patients with the Leu-55-Pro mutation.

Chromatography, Affinity↗
PubMed2000

Alveolar mononuclear cells can develop into multinucleated osteoclasts: an in vitro cell culture model.

Previous studies have shown that osteoclasts are derived from mononuclear cells of hemopoietic bone marrow and peripheral blood. The purpose of this study was to demonstrate the presence of multinucleated osteoclasts after adding alveolar mononuclear cells to new-born rat calvaria osteoblasts in vitro. To utilize osteoclast-free bone, fetal calvariae were obtained from newborn Wistar-rats and cultured in DMEM medium for 14 days. On the day of osteoblast culture, alveolar mononuclear cells were isolated from newborn Wistar rats with a serial washing method and then co-cultured with the calvarial osteoblasts. Bone resorption characteristics were observed both with light and scanning electron microscopy. When alveolar mononuclear cells were cultured for 14 days on the calvarial osteoblasts in response to 1 alpha, 25-dihydroxyvitamin D3, they formed tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multinucleated cells. Resorption pits were seen in the 7-14 days long-term cultures. These results indicate that osteoclasts can be derived from alveolar mononuclear cells in vitro when a suitable microenvironment is provided by calvarial osteoblasts and vitamin D(3).

Animals↗
PubMed2000

Tissue-specific cancer-related serpin gene cluster at human chromosome band 3q26.

Approximately one quarter of the identified human serpin genes are cancer-related and clustered mainly at two distinct loci: 6p25 and 18q21. We have studied a novel serpin gene cluster at 3q26 containing at least two recently identified members: the pancreas-specific protease inhibitor, pancpin (PI14), and the brain-associated protease inhibitor, neuroserpin (PI12). In this, unlike a previous study, both PI14 and PI12 at 3q26 were found to consist of 9 exons and 8 introns and to share a perfectly conserved gene organization whose pattern is very different from that of the ov-serpin family. This distinct pattern appears identical in the genomic structures of human plasminogen activator inhibitor-1 (PAI1) at 7q21 and protease nexin 1 (PI7) at 2q33-35, confirming that these four genes in three different chromosomes form a discrete subset within the serpin superfamily. As in the other three members whose gene expression is altered during tumorigenesis, PI12 expression was found to be down-regulated in tumor brain tissues and in two brain cancer cell lines: U-87 MG and H4. By screening genomic libraries, we isolated two overlapping clones showing that the marker SGC32223 (centromere) is located within intron F of PI12 and the marker WI-10077 (telomere) is located downstream of the 3'-flanking region of PI14. This finding indicates that the distance between human PI14 and PI12 is approximately 100 kb, and hence we speculate that other tissue-specific cancer-related serpin genes are likely to reside within this 3q26.1 cluster region.

Amino Acid Sequence↗
PubMed1999

Heteropolymerization of S, I, and Z alpha1-antitrypsin and liver cirrhosis.

The association between Z alpha1-antitrypsin deficiency and juvenile cirrhosis is well-recognized, and there is now convincing evidence that the hepatic inclusions are the result of entangled polymers of mutant Z alpha1-antitrypsin. Four percent of the northern European Caucasian population are heterozygotes for the Z variant, but even more common is S alpha1-antitrypsin, which is found in up to 28% of southern Europeans. The S variant is known to have an increased susceptibility to polymerization, although this is marginal compared with the more conformationally unstable Z variant. There has been speculation that the two may interact to produce cirrhosis, but this has never been demonstrated experimentally. This hypothesis was raised again by the observation reported here of a mixed heterozygote for Z alpha1-antitrypsin and another conformationally unstable variant (I alpha1-antitrypsin; 39Arg-->Cys) identified in a 34-year-old man with cirrhosis related to alpha1-antitrypsin deficiency. The conformational stability of the I variant has been characterized, and we have used fluorescence resonance energy transfer to demonstrate the formation of heteropolymers between S and Z alpha1-antitrypsin. Taken together, these results indicate that not only may mixed variants form heteropolymers, but that this can causally lead to the development of cirrhosis.

Adult↗
PubMed1998

Latent alpha1-antichymotrypsin. A molecular explanation for the inactivation of alpha1-antichymotrypsin in chronic bronchitis and emphysema.

alpha1-Antichymotrypsin is an acute phase protein that protects the tissues from damage by proteolytic enzymes, but previous studies have shown that alpha1-antichymotrypsin within the lungs of patients with chronic bronchitis and emphysema is intact but inactive as an inhibitor. Ammonium sulfate fractionation followed by blue Sepharose and DNA-Sepharose chromatography was used to isolate small amounts of intact, monomeric but inactive alpha1-antichymotrypsin from the plasma of 30 healthy blood donors. This species had a higher DNA binding affinity with more anodal electrophoretic mobility than native alpha1-antichymotrypsin and was conformationally stable against thermal denaturation, 8 M urea, and 7 M guanidinium chloride. The protein was unable to accept synthetic reactive loop peptides, and the reactive loop was resistant to proteolytic cleavage at the P5-P4 bond but could be cleaved between P1' and P3'. These data suggest that this new alpha1-antichymotrypsin species was in a conformation similar to those of the crystallographically determined latent serpins, plasminogen activator inhibitor-1 and antithrombin. alpha1-Antichymotrypsin from lung lavage migrated with the same electrophoretic mobility as the putative latent alpha1-antichymotrypsin, suggesting that this is the inactive conformation described previously in the lungs of patients with chronic bronchitis and emphysema. This conformational transition of alpha1-antichymotrypsin, from an active to an inactive state, within the lung may play an important role in the pathogenesis of chronic lung disease.

Bronchitis↗
PubMed1998

Implications for function and therapy of a 2.9 A structure of binary-complexed antithrombin.

The crystal structure of a binary complex of human antithrombin with a peptide of the same sequence as its reactive loop (P14-P3) has been determined at 2.9 A. The peptide binds as the middle strand s4A in the A beta-sheet, homologously to that of the reactive loop in the latent and cleaved forms of antithrombin. Peptide binding results in the complete expulsion of the hinge region of the loop from the A beta-sheet although the conformation differs from that of heparin-activated antithrombin. The 36-fold increase in the rate of reaction of the binary complex with factor Xa indicates that full loop expulsion alone is not sufficient for complete heparin activation of antithrombin but that this is also dependent on the overall conformation of the molecule. Previous studies have demonstrated that reactive loop peptides can block or reverse the polymerisation of serpins associated with cirrhosis and thrombosis. The antithrombin binary complex structure defines the precise localisation of the blocking peptide in a serpin and provides the basis for rational drug design for mimetics that will prevent polymerisation in vivo and so ameliorate the associated disease.

Amino Acid Sequence↗
PubMed1998

Sequential genesis and determination of cone and rod photoreceptors in Xenopus.

In this study, we addressed the temporal sequence of photoreceptor fate determination in Xenopus laevis by examining a number of key events during early cone and rod development. We compared the relative timing and spatial pattern of cone and rod specification using a number of cell type-specific markers, including probes to a long wavelength-sensitive opsin which is expressed by the major cone subtype. Our results show that cones are initially more numerous, and can arise in less mature regions of the retina than rods, although both types of photoreceptors begin to express their respective opsins at about the same time. We applied these markers to an assay of cellular determination to identify the stages of embryonic development at which the earliest photoreceptor fates are induced in vivo. The relative birth order of the major cone and rod subtypes was revealed by simultaneous labeling with markers of cell proliferation and terminal differentiation. Although there is much temporal overlap between the periods of cone and rod genesis and determination in Xenopus, we could discern that the earliest cones are both born and determined before the first rods. Thus, even in the rapidly developing retina of Xenopus, photoreceptors achieve their identities in a sequential fashion, suggesting that the inductive cues which determine specific photoreceptor fates may also arise sequentially during development.

Amino Acid Sequence↗
PubMed1997

The effects of reactive centre loop length upon serpin polymerisation.

The clinical effects of serpin polymerisation include thromboembolism, emphysema, and liver disease. A through understanding of serpin polymerisation mechanisms and the structures involved will permit the rational design of therapeutic polymerisation inhibitors. Here we show that serpin polymerisation can be delayed by extending the length of the serpin reactive centre loop. The heat stability of three chimeric serpins was examined. One of them, an active alpha 1-antitrypsin variant with a reactive centre loop C-terminal extension of four amino acid residues, was shown to have increased resistance to inactivation by polymerisation. This variant could also form serpin/peptide binary complexes with a reactive centre loop peptide, which indicates that the increase in thermostability was not due to the A-beta-sheet being unable to accept reactive centre loop residues, an essential requirement for polymerisation. Rather, we conclude that the additional residues within the reactive centre loop delay the release of strand 1C from the C-sheet, a process essential for polymer formation.

Amino Acid Sequence↗
PubMed1997

Preparative induction and characterization of L-antithrombin: a structural homologue of latent plasminogen activator inhibitor-1.

The inhibitory mechanism of the serpin family of serine protease inhibitors is characterized by a remarkable degree of conformational flexibility. Various conformational states have been elucidated by X-ray crystallography and indicate that the inhibitory loop, the central A-beta-sheet, and the outside edge of the C-beta-sheet are particularly mobile. However, no crystal structure of a serpin-enzyme complex is yet available, and the likely nature of the protease-complexed serpin remains for biochemical and biophysical researchers to examine. Here, we show that the biochemical induction of the latent state of antithrombin is slow relative to polymer formation, and infer that this may reflect structural features that are important for the regulation of the initial docking and subsequent locking of serpins with cognate proteases. L-Antithrombin was induced by incubation of native antithrombin at 60 degrees C for 10 h in the presence of citrate to prevent polymerization. L-Antithrombin was more stable to denaturation by both heat and urea than native antithrombin. Whereas native antithrombin formed binary complexes with synthetic peptide homologues of the inhibitory loop, biochemically induced L-antithrombin did not, indicating that the inhibitory loop of L-antithrombin is probably fully inserted into the A-beta-sheet as in the crystal structure. This was confirmed by limited proteolysis studies which demonstrated that the inhibitory loop of L-antithrombin could not be cleaved by five proteases which do cleave the loop of native antithrombin. The limited proteolysis studies also indicated that the "gate" region (residues 236-248) of the biochemically induced L-antithrombin was in a conformation substantially different from that of the native antithrombin. This again is similar to L-antithrombin in the crystal structure in which the gate has "opened" away from the body of the molecule by a rotation of 24 degrees to facilitate the relocation of strand 1C from its ordered position in the C-beta-sheet to a disordered surface loop. At 60 degrees C in the absence of citrate, antithrombin (and other serpins) rapidly polymerizes. In the presence of citrate, the formation of L-antithrombin is slow and increases with time, indicating that the inhibition of polymer formation by citrate allows the time necessary for the much slower formation of the L form. We therefore suggest that L-antithrombin formation is a two-step process: an initial rapid conformational change, probably including partial incorporation of the reactive loop into the A-sheet (as in the active molecule in the crystal structure) and displacement of s1C from the C-beta-sheet which supports polymer formation, and a much slower transition to complete loop insertion within the A-beta-sheet. It is likely that both the first rapid transitional step and the structural features that impose resistance to the second more extensive conformational change reflect the optimization of the unique inhibitory function in the serpins.

Amino Acid Sequence↗
PubMed1997

Inhibitory mechanism of serpins. Mobility of the C-terminal region of the reactive-site loop.

The reactive-site loops of serpins are characterized by a defined mobility where the loop adopts a new secondary structure as an essential part of the inhibitory process. While the importance of mobility in the N-terminal region of the reactive-site loop has been well studied, the role of mobility in the C-terminal portion has not been investigated. The requirements for mobility of the C-terminal portion of the reactive-site loop of alpha1-antitrypsin were investigated by creating a disulfide bridge between the P'3 residue and residue 283 near the top of strand 2C; this disulfide would restrict the mobility of the C-terminal portion of the reactive-site loop by locking together strands 1 and 2 of the C beta-sheet. The engineered disulfide bond had no effect on the inhibitory activity of alpha1-antitrypsin, indicating that there is no requirement for mobility in this region of the molecule. Moreover, these results, coupled with those from molecular modeling, indicate that insertion into the A beta-sheet of the intact reactive-loop beyond P12 is not rate-limiting for the formation of the stable complex. The engineered disulfide bond should also prove useful in the creation of more stable serpin variants; for example, such a bond in plasminogen activator inhibitor-1 would prevent it from becoming latent by locking strand 1C onto the C beta-sheet.

Binding Sites↗
PubMed1997

Regulation of neuronal diversity in the Xenopus retina by Delta signalling.

To generate the variety of mature neurons and glia found in the developing retina, the competence of pluripotent progenitor cells to respond to extracellular signals must be controlled. Delta, a ligand of the Notch receptor, is a candidate for regulating progenitor competence on the grounds that activation of the pathway involving Notch and Delta can inhibit cellular differentiation. Here we test this possibility in the developing Xenopus retina by misexpression of Delta messenger RNA. We find that Delta-misexpressing cells with wild-type neighbours adopt earlier fates, primarily becoming ganglion cells and cone photoreceptors. Progenitors transfected with Delta later in development also produce rod photoreceptors, but not the latest-generated cell types, demonstrating the importance of timing in Delta function. We conclude that Delta signalling in the vertebrate retina is a basic regulatory mechanism that can be used to generate neuronal diversity.

Animals↗
PubMed1997

Importance of the release of strand 1C to the polymerization mechanism of inhibitory serpins.

Serpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12-m34 peptide (homologous to P14-P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6-mer peptide (homologous to P14-P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5-mer peptide with s1C sequence of antithrombin or by an unrelated peptide representing residues 26-31 of cholecystokinin. The s1C or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 361-->Cys, Ser 283-->Cys, was engineered for the purpose of forming a disulfide bond between s1C and s2C to prevent movement of s1C. This mutant was resistant to polymerization if the disulfide bridge was intact, but, under reducing conditions, it regained the potential to polymerize. We have also modeled long-chain serpin polymers with acceptable stereochemistry using two previously proposed loop-A-sheet and loop-C-sheet polymerization mechanisms and have shown both to be sterically feasible, as are "mixed" linear polymers. We therefore conclude that the release of strand 1C must be an element of the mechanism of serpin polymerization.

Amino Acid Sequence↗
PubMed1997

Mechanisms of antithrombin polymerisation and heparin activation probed by the insertion of synthetic reactive loop peptides.

Incubation of antithrombin with a series of synthetic reactive loop peptides showed that 6-mer and 7-mer peptides, P14-P9 and P14-P8 of antithrombin respectively, induced loop-sheet polymerisation and binary complex formation. These peptides are likely to anneal to the upper part of the dominant A-sheet, favouring sheet opening and allowing insertion of a second reactive loop in the lower part of the A-sheet to form polymers. The insertion of longer peptides filled the A-sheet beyond the P7 position and prevented polymerisation. Heparinised antithrombin was more resistant to polymerisation and peptide insertion, indicating that heparin induces a conformational change that closes the A-sheet and expels the reactive loop.

Antithrombin III↗
PubMed1997

Commercial antithrombin concentrate contains inactive L-forms of antithrombin.

The preparation of antithrombin concentrate for clinical use requires a viral inactivation step. In most commercial preparations this is achieved by heat pasteurisation. This process would be expected to alter the conformation of antithrombin from the active native species to an inactive latent (L-form) state (1, 2). To determine if this occurs during commercial preparation and to identify the proportion of the product in the inactive state, we examined the various antithrombin conformations within a therapeutic concentrate. The antithrombin concentrate was separated into five fractions by heparin-Sepharose chromatography. The fraction with the highest heparin affinity retained full activity, whereas the four fractions with reduced heparin affinity (approximately 40% of the total antithrombin) had lost their inhibitory function. These inactive antithrombins were intact, monomeric, thermostable and resistant to unfolding in 8 M urea. Moreover, the protein patterns on isoelectric focusing and non-denaturing-PAGE showed that there were at least two different L-forms with isoelectric points separate from the native active species. Our findings demonstrate that approximately 40% of the antithrombin preparation examined exists as inactive L-forms. The clinical significance of administering this altered material is uncertain.

Antithrombin III↗