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Biomedical subjects

M L Chu

Publications and source records attributed to M L Chu.

At least 109 records · Page 6Linked to original sources
PubMed1996

Conversion of the Kunitz-type module of collagen VI into a highly active trypsin inhibitor by site-directed mutagenesis.

The recombinant Kunitz protease inhibitor module (domain C5) of human collagen alpha 3(VI) chain was previously shown to lack inhibitory activity for proteases with trypsin-like specificity and some other proteases. We have now prepared mutants in the binding loop region including the P1' site (D2889-->A), the P2' site (F2890-->R) and the P3 site (T2886-->P) and in a more remote region (W2907-->V) either as individual substitutions or combinations of them. These mutants were analyzed for their kinetics of binding to trypsin by surface plasmon resonance and for their capacity to inhibit various proteases. Single substitutions (D-->A, T-->P, W-->V) showed an effect only for D->A which bound to trypsin with Kd = 0.25 microM. A 25-100-fold increase in affinity was observed for the double mutants T-->P/D-->A and F-->R/D-->A and approached the affinity of aprotinin (Kd approximately 0.01 nM) in two different triple mutants. These affinities correlated well with the inhibitory capacities of the mutants for trypsin in the cleavage of a large protein and a small peptide substrate. A similar but not completely identical improvement in inhibitory capacity was also observed for leucocyte elastase but not for thrombin. These data could be interpreted in terms of steric interferences or lack of hydrogen bonding of a few critical residues based on three-dimensional structures available for the C5 domain.

Aprotinin↗
PubMed1996

Structure and multiple conformations of the kunitz-type domain from human type VI collagen alpha3(VI) chain in solution.

BACKGROUND: The Kunitz-type inhibitor motif is found at the C terminus of the human collagen alpha3(VI) chain. This 76-residue module (domain C5) was prepared in recombinant form and showed high stability against proteases; however, it lacked any inhibitory activity against trypsin, thrombin, kallikrein and several other proteases. We have undertaken the determination of the three-dimensional (3D) structure of domain C5 in solution, by nuclear magnetic resonance (NMR), in order to establish the structural basis for the properties of this protein. RESULTS: The 7 N-terminal and 12 C-terminal residues of domain C5 are disordered in the solution structure. The 55-residue core, which shows high homology to bovine pancreatic trypsin inhibitor, retains the characteristic fold of all members of the Kunitz-type inhibitor family. 24 residues of this main structural body show more than one resonance, symptomatic of multiple conformations slowly exchanging on the NMR time scale. In addition, significant proton chemical exchange line broadening is observed for residues in the vicinity of the disulfide bridge between residues 20 and 44: this indicates interconversion, on the micro- to millisecond time scale, between multiple conformations. CONCLUSION: The NMR study demonstrates that domain C5 is a highly dynamic molecule at temperatures studied (between 10 and 30 degrees C). Indeed, some 44% of the main body structure of C5 showed multiple conformations. The existence of multiple conformations was not necessarily expected in view of the conformational constraints imposed by the 3D structure of proteins as rigid as C5; it should therefore be considered in the interpretation of its structural and dynamical properties. The accessibility of the inhibitory binding loop (Gly18 [P4] to Leu25 [P4']) should be relatively unaffected by this conformational exchange and thus would not explain the unusual specificity of C5. Most serine proteinase inhibitors that, like C5, have an arginine at the P1 position inhibit trypsin; the lack of trypsin inhibition of C5 must therefore arise from the amino-acid side-chain composition of the adjoining positions in the binding loop.

Amino Acid Sequence↗
PubMed1996

Differential regulation of fibulin, tenascin-C, and nidogen expression during wound healing of normal and glucocorticoid-treated mice.

Expression and distribution of fibulins, nidogen, and tenascin-C were analyzed in healing skin wounds of normal and dexamethasone-treated mice. In normal mice both tenascin-C and fibulin-2 showed a marked increase in mRNA expression, which declined to normal levels after completion of skin repair. These two matrix proteins are found throughout the granulation tissue and persisted there after mRNA expression had ceased. Fibulin-1 is present in normal skin and in wounds but is not distinctly upregulated during the healing process. Nidogen, however, is expressed uniformly throughout the granulation tissue early in wound healing, has a peak expression around Day 7, and selectively localizes to basement membranes after healing is accomplished. Dexamethasone treatment led to a decreased expression of tenascin-C in healing wounds but had no effect on fibulin-2 expression. In vitro experiments revealed that growth factors like TGF-beta 1 can partly counteract glucocorticoid action. These data therefore provide some molecular interpretations for the well-known glucocorticoid suppression of wound healing. They also indicate that repair involves complex regulatory processes which are obviously different for each of the four proteins studied.

3T3 Cells↗
PubMed1996

Fibulin-1 and fibulin-2 expression during organogenesis in the developing mouse embryo.

Fibulin-1 and fibulin-2 are extracellular matrix proteins with unique structural features. We used in situ hybridization and immunofluorescence staining to examine the expression of fibulin-1 and fibulin-2 during mouse embryogenesis. Both fibulins have previously been shown to be deposited at sites where polarized cells convert into mesenchyme, during early stages of development of endocardial cushion tissue and in neural crest cells. By Northern blots we confirm that expression of fibulin-2 is particularly high in the developing and newborn heart. We also show that fibulin-2 mRNA and protein remained highly expressed during organogenesis in tissues derived from neural crest mesenchyme. In addition, a locally restricted expression pattern of fibulin-1 and fibulin-2 mRNA and protein at sites of epithelial-mesenchymal interactions was detected in two tissues, the developing tooth and hair follicles. In other tissues where epithelial-mesenchymal interactions occur, fibulin-1 mRNA and its corresponding protein were detected rather uniformly around mesenchymal cells, and no expression of fibulin-2 was noted. Fibulin-1 protein was located in some embryonic epithelial basement membranes. Fibulin-1 mRNA was also expressed in the epidermal layer of brain and in the mesenchyme of choroid plexus and the meninges which surround the spinal cord. Overall, fibulin-2 expression was much more limited than fibulin-1 expression. A very prominent expression of fibulin-2 was seen during early stages of chondrogenesis in all cartilages analyzed. These studies show that the differential expression of the fibulin family contributes to the formation of molecularly distinct extracellular matrices already during early developmental stages of a large number of tissues.

Animals↗
PubMed1996

The extracellular matrix proteins fibulin-1 and fibulin-2 in the early human embryo.

Fibulin-1 and fibulin-2, two recently identified extracellular matrix proteins with a homologous domain structure, are known to bind various extracellular ligands and calcium. In this study, they have been localized at the light microscopical level in human embryos of gestational weeks 4-10, using polyclonal antibodies. Identical localization patterns were observed for the two fibulins in most of the tissues. In the heart, the endocardial cushion tissue and endocardium, but not the myocardium, were stained, as were the basement membrane zones and adventitia of blood vessels. Staining was also observed in the perichondrium and calcifying regions of developing bones. Moreover, reactions occurred with the gut subepithelium and epithelial basement membranes of the skin. Differences in staining patterns, however, were observed in various neural structures. Fibulin-1 was prominent in the matrix of the leptomeningeal anlage, in basement membranes of the neuroepithelium and the perineurium of peripheral nerves. Fibulin-2 was detected primarily within the neuropithelium, spinal ganglia and peripheral nerves. The early embryonic expression of both fibulins indicates specific roles during organ development and, in particular, involvements in the differentiation of heart, skeletal and neuronal structures.

Animals↗
PubMed1996

Tissue distribution and developmental expression of type XVI collagen in the mouse.

The expression of a recently identified collagen, alpha 1 (XVI), in adult mouse tissue and developing mouse embryo was examined by immunohistochemistry and in situ hybridization. A polyclonal antiserum was raised against a recombinant fusion protein, which contained a segment of 161 amino acids in the N-terminal noncollagenous domain of the human alpha 1 (XVI) collagen. Immunoprecipitation of metabolically labelled human or mouse fibroblast cell lysates with this antibody revealed a major, bacterial collagenase sensitive polypeptide of approximately 210 kDa. The size agrees with the prediction from the full-length cDNA. Immunofluorescence examination of adult mouse tissues using the affinity purified antibody revealed a rather broad distribution of the protein. The heart, kidney, intestine, ovary, testis, eye, arterial walls and smooth muscles all exhibited significant levels of expression, while the skeletal muscle, lung and brain showed very restricted and low signals. During development, no significant expression of the mRNA or protein was observed in embryo of day 8 of gestation, but strong signals was detected in placental trophoblasts. Expression in embryos was detectable first after day 11 of gestation with weak positive signals appearing in the heart. In later stages of development, stronger RNA hybridizations were observed in a variety of tissues, particularly in atrial and ventricular walls of the developing heart, spinal root neural fibers and skin. These data demonstrate that type XVI collagen represents another collagenous component widely distributed in the extracellular matrix and may contribute to the structural integrity of various tissues.

Aging↗
PubMed1996

Fibulin-2: genetic mapping and exclusion as a candidate gene in Marfan syndrome type 2.

Fibulin-2 (FBLN2) is a new extracellular matrix protein that has been considered a candidate gene for Marfan syndrome type 2 (locus MFS2) based on chromosomal colocation at 3p24.2-p25 and disease phenotype. In the absence of polymorphic markers reported for FBLN2, direct sequencing of the gene was performed and two intragenic polymorphisms were identified. Linkage was excluded between FBLN2 and the MFS2 gene. Furthermore, two-point lod scores were generated between these markers and anonymous markers arrayed on the genetic map of 3p and closely linked to MFS2. These analyses placed FBLN2 at marker D3S1585.

Calcium-Binding Proteins↗
PubMed1996

Expression of fibulin-2 by fibroblasts and deposition with fibronectin into a fibrillar matrix.

The extracellular matrix protein fibulin-2 was shown to be a typical product of cultured human and mouse fibroblasts by several immunological assays. It is secreted and deposited in cells and tissues as a disulfide-bonded oligomer identical in size to the previously described recombinant fibulin-2. Most of the fibroblast fibulin-2 is deposited into a dense fibrillar meshwork which requires treatment with EDTA and/or 6 M urea for solubilization. Fibulin-2 and fibronectin are synthesized at equivalent levels and both colocalize in the fibrils as shown by immunofluorescence. Metabolic labelling and pulse-chase studies demonstrated fibulin-2 oligomers in detergent extracts of cells and their rapid translocation to extracellular EDTA-sensitive assembly forms. Unlike for fibronectin and fibulin-1 only a little fibulin-2 was found in the cell culture medium. Immunogold staining of confluent human fibroblasts showed localization of fibulin-2 to a fine meshwork or bundles of amorphous microfibrils in the matrix. This also demonstrated a distinct colocalization of fibulin-2 and fibronectin at the electron microscope level, indicating that the interaction between these two protein shown in in vitro assays may also exist in situ. No distinct colocalization of both proteins could, however, be observed with cross-striated fibrils of collagen I and collagen VI microfibrils.

Animals↗
PubMed1996

Clinical isolation of vancomycin-resistant Enterococcus faecalis in Taiwan.

We report the isolation of a vancomycin-resistant strain of Enterococcus faecalis, designated AH803, from a 76-year-old Taiwanese woman with pneumonia and bacteremia. This is the first documented clinical isolation of a vancomycin-resistant enterococcus in Taiwan. AH803 was repeatedly isolated from sputum specimens of the patient. AH803 had a high level of vancomycin (minimal inhibitory concentration, MIC = 512 micrograms/mL) and gentamicin (MIC > 2,000 micrograms/mL) resistance, but was susceptible to teicoplanin (MIC = 8 micrograms/mL) and ampicillin (MIC = 2 micrograms/mL). AH803 was shown by polymerase chain reaction to have the vanA gene, but not the vanB gene. Despite treatment efforts, the patient's condition continued to deteriorate. She requested to be discharged, against medical advice. The patient died at home the following day after discharge.

Aged↗
PubMed1995

Binding of mouse and human fibulin-2 to extracellular matrix ligands.

Recombinant mouse and human fibulin-2 were obtained as disulfide-bonded trimers from transfected kidney cell clones and used in solid phase, biosensor and radioligand binding assays. Strong binding occurred with fibronectin and required calcium. A distinct interaction was also observed with nidogen but this was only partially blocked by EDTA. Distinctly weaker affinities were detected for collagen IV, perlecan and the N-terminal globule of collagen VI alpha 3 chain, while no or only little binding activity could be detected for several other collagen types, laminin-1, BM-40, fibulin-1 and vitronectin. This weaker binding reactions were also dependent on calcium. Surface plasmon resonance assays demonstrated for fibulin-2 binding to nidogen and fibronectin high equilibrium dissociation constants (0.5 to 1 microM) due to a rapid initial dissociation of the complexes. This is apparently followed by a slower stabilizing reaction. The fibulin-2 binding site of nidogen could be localized to its C-terminal globular domain G3, which also possesses a high-affinity binding site for laminin-1. Several tests demonstrated competition between the two ligands, probably due to steric hindrance. Binding of nidogen to immobilized fibulin-2 allowed the formation of ternary complexes with collagen IV, perlecan and fibulin-1, which, as shown previously, bind independently of the G3 domain. This indicated multifunctional binding properties for fibulin-2 and several alternative routes for its integration into basement membranes and other extracellular structures.

Amino Acid Sequence↗
PubMed1995

Characterization of the human alpha 1(VI) collagen promoter and its comparison with human alpha 2(VI) promoters.

From a human cosmid library, we isolated a clone (5B) with an insert of 32 kb, encoding the amino-terminal and the 5'-end flanking region of the alpha 1(VI) collagen gene. Exon 1 was found to be 194 bp and contain the 5' untranslated region plus 97 bp coding sequence. Exon 2 consists of 130 bp, a size that is conserved across the chicken and mouse species. S1-nuclease-protection assays and primer-extension analysis, using mRNA from human dermal fibroblasts, show the presence of multiple transcription start sites located in a region of approximately 20 nucleotides. Canonical TATA and CAAT boxes, as found in the chicken and mouse alpha 1 promoters, were absent in the human alpha 1(VI) promoter. The promoter region from positions -1 to -190, is a polypyrimidine/polypurine-rich region containing 12 CCCTCCCC (CT element consensus) sequences and has multiple potential binding sites for the Sp1, and AP2 transcription factors. These regulatory proteins bind to the alpha 2(VI) promoters [Saitta, B. & Chu, M.-L. (1994) Eur. J. Biochem. 223, 675-682]. To test the transcriptional activity of the alpha 1 promoter, transient transfection experiments of the DNA constructs were performed in human dermal fibroblasts and in human fibrosarcoma (HT1080) cell lines. The DNA constructs drive the expression of the chloramphenicol acetyl transferase (CAT) gene. The results show strong CAT activity for the constructs at positions -1700, -298 and -257, while low activity was found for the constructs at positions -4400, -142 and -5 when transfected in fibroblasts. The experiments also identified positive and negative regulatory regions in the alpha 1(VI) promoter CAT constructs when transfected in fibroblasts, but did not identify them in the fibrosarcoma cells.

Animals↗
PubMed1995

Head to tail organization of the human COL6A1 and COL6A2 genes by fiber-FISH.

Two type VI collagen genes, COL6A1 and COL6A2, both map to 21q22.3, but the order, distance, and organization of these two genes relative to one another were not known. Recently developed high-resolution fluorescence in situ hybridization (FISH) techniques have great potential to facilitate the construction of fine-resolution maps of telomeric regions where gene density is high. Here we have determined the distance separating the COL6A1 and COL6A2 genes (150 kb), the size of the COL6A1 gene (29 kb); and the 5'-3' orientation of these genes (5' COL6A1 3'-5' COL6A2 3') using fiber-FISH.

Chromosome Mapping↗
PubMed1995

Collagen type VI in the human bone marrow microenvironment: a strong cytoadhesive component.

Collagen type VI, which forms characteristic microfibrillar structures, is assembled from three individual alpha(VI) chains that form a short triple helix and two adjacent globular domains. Expression of all three alpha (VI) collagen chains in the human bone marrow (BM) microenvironment could be detected by chain-specific antibodies in tissue sections and in the adherent stromal layer of long-term BM cultures. In functional studies, collagen type VI was shown to be a strong adhesive substrate for various hematopoietic cell lines and light-density BM mononuclear cells. The adhesive site within the molecule seems to be restricted to the triple helical domain of all three alpha (VI) chains, because individual alpha (VI) chains were not active in the attachment assays. Adhesion of the hematopoietic cell lines to collagen VI was dose-dependent and could be inhibited by heparin. Although the triple helix contains several RGD sequences, adhesion of the hematopoietic cell types to collagen VI could be blocked neither by RGD-containing peptides nor by a neutralizing antibody to the beta 1 integrin subunit. In combination with an antiadhesive substrate, the binding properties of collagen VI could be downregulated. These data suggest that this collagen type may play an important role in the adhesion of hematopoietic cells within the BM microenvironment.

Bone Marrow↗
PubMed1995

Recombinant analysis of human alpha 1 (XVI) collagen. Evidence for processing of the N-terminal globular domain.

The N-terminal non-collagenous domain NC11 of the human collagen alpha 1 (XVI) chain was obtained as a recombinant 35-kDa protein from stably transfected kidney cell clones. This form had undergone proteolytic trimming at a basic cleavage motif indicating a similar release in vivo. Domain NC11 showed a globular shape after rotary shadowing and was resistant to neutral proteases. Specific antibodies could be raised against recombinant NC11 and were used for the analysis of other cell clones transfected with the full-length alpha 1 (XVI) chain. Immunoprecipitation of detergent extracts of metabolically labelled cells demonstrated the presence of disulfide-bonded 200-kDa polypeptides possessing NC11 epitopes. This material was partially resistant to pepsin, indicating the formation of alpha 1 (XVI) chain homotrimers with a triple-helical conformation. Yet a substantial proportion of these homotrimers was degraded to fragments of variable size (35-150 kDa) when secreted into the culture medium. Several of these fragments could be obtained on a semi-preparative scale from cells grown in hollow fiber cassettes and showed substantial hydroxylation of proline, consistent with triple-helix formation. Edman degradation demonstrated the origin of some from the N-terminal and of one from a more C-terminal position of collagen XVI. This extensive degradation may be explained by the release of NC11 and by further cleavages within some of the nine interruptions of the triple-helical domain of the alpha 1(XVI) chain. Whether this process also occurs in situ remains to be shown.

Amino Acid Sequence↗
PubMed1995

Structural characterization of two variants of fibulin-1 that differ in nidogen affinity.

Two C-terminal variants C and D of mouse fibulin-1 were purified from the culture medium of stably transfected human kidney cell clones. They showed, after rotary shadowing, a dumbbell-like structure of about 33 nm in length. Pepsin digestion demonstrated stability of the disulfide-bonded domains 1 (anaphylatoxin-like) and II (multiple EGF-like motifs) but not for domain III which is different in the variants. A close similarity of the variants was observed in immunochemical assays indicating that domain III epitopes are not very antigenic. Binding analysis in solid phase assays demonstrated for variant C a 100-fold stronger binding to the basement membrane protein nidogen than for variant D. Both interactions were sensitive to EDTA. Surface plasmon resonance assays confirmed this difference and showed KD = 60 nM for variant C and KD > 1 microM for variant D. Lower binding activities and smaller differences between both variants were observed for the calcium-dependent binding to fibronectin, laminin-1 and collagen IV. Self aggregation into nest-like oligomers was observed at high concentrations of fibulin-1 which was not sensitive to EDTA.

Amino Acid Sequence↗
PubMed1995

Extracellular matrix protein fibulin-2 is expressed in the embryonic endocardial cushion tissue and is a prominent component of valves in adult heart.

Fibulins-1 and -2 are two recently characterized extracellular matrix proteins of a novel protein family. They are found in many tissues in the adult stage, but several differences in their expression patterns were noted in cultured cells and adult tissues. Fibulin-1 is particularly well expressed in the endocardial cushion tissue (ECT) during embryogenesis and we therefore compared the expression patterns of fibulin-1 and -2 in the developing mouse heart. Antibodies against mouse fibulin-1 and -2 were used in immunofluorescence, and the expression of mRNAs were studied with synthetic oligonucleotides by in situ hybridization. A striking codistribution of fibulin-1 and -2 was seen at all stages of the development of the endocardial cushion tissue. Fibulin-2 was found to be a more specific marker for the embryonic ECT than fibulin-1, which was also transiently expressed in cardiac muscle. Immunofluorescence showed that the fibulins remained expressed in valves of the heart in the adult stage. The results suggest a role of the two fibulins in the development of the ECT and in the maintenance of the tensile strength of the cardiac valves.

Animals↗
PubMed1995

Developmental expression and molecular cloning of REMP, a novel retinal epithelial membrane protein.

The retinal pigment epithelium (RPE), like other transport epithelia, has a polarized distribution of membrane and cytoskeletal proteins. The establishment of a polarized phenotype is an essential step in the differentiation of the RPE and the development and maintenance of visual function. Using a monoclonal antibody (MAb 3C4) we have identified a novel membrane protein that is uniquely expressed in chick RPE. We have referred to this protein as REMP for retinal epithelial membrane protein. In these studies we characterized the expression and distribution of this protein during embryonic development and determined its primary structure by cDNA cloning. The developmental expression of REMP was examined by immunocytochemical localization. REMP was first detected in the chick RPE at Embryonic Day 5 (E5) in both apical and basolateral membranes. By E14 the distribution of REMP was restricted to the basolateral surface of the RPE cells. Biochemical fractionation and surface labeling of RPE cells suggested that REMP was an integral protein. The gene encoding REMP was isolated from an E15 chick RPE cDNA library, cloned into lambda gt11, and screened with MAb 3C4. The cDNA was sequenced and found to contain one 1350-bp open reading frame encoding for a 450-amino-acid protein. The deduced amino-acid sequence of REMP shares 32.9% identity with MCT1, a monocarboxylate transporter (Garcia, Goldstein, Pathak, Anderson, and Brown, Cell, 76, 865-873, 1994). By Northern blot analysis, REMP mRNA was detected only in RPE cells. There was an increase in the expression REMP transcript during development but when RPE cells were grown in primary culture the expression of REMP was turned off. The unique expression of REMP in the RPE in vivo would suggest a role for this protein in development and maintenance of normal retinal function.

Amino Acid Sequence↗