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M L Chu

Publications and source records attributed to M L Chu.

At least 145 records · Page 8Linked to original sources
PubMed1993

Hyperglycemic glucose concentrations up-regulate the expression of type VI collagen in vitro. Relevance to alterations of peripheral nerves in diabetes mellitus.

Electron microscopy of peripheral nerves obtained from two diabetic patients revealed large deposits of microfibrils and the presence of Luse bodies in the vicinity of perineurial cells. Microfibrils were found to accumulate also in the sciatic nerves of diabetic BB rats; these microfibrillar deposits were shown to contain type VI collagen by immunoelectron microscopy. Connective tissue cells cultured from rat sciatic nerves were exposed to high glucose concentrations. High glucose concentrations up-regulated the mRNA steady-state levels of alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains of type VI collagen and caused accumulation of type VI collagen-containing fibrils in the cultures. Immunostaining and in situ hybridizations demonstrated that perineurial cells, Schwann cells, and fibroblasts expressed type VI collagen at the mRNA and protein levels. The results suggest that the turnover and supramolecular assembly of type VI collagen are perturbed in diabetic nerves and that glucose per se increases the expression of type VI collagen in vitro.

Adolescent↗
PubMed1992

Effect of transforming growth factor-beta on collagen type VI expression in human dermal fibroblasts.

Steady-state mRNA levels and protein synthesis of collagen type VI were determined after stimulation of human dermal fibroblasts with transforming growth factor-beta (TGF beta). While there was a 227% increase in the alpha 3(VI) subunit mRNA at maximal TGF-beta concentration, alpha 1(VI) and alpha 2(VI) subunit mRNA levels remained unchanged. Concomitantly collagen type VI immuno-reactive material increased up to 172% of controls in cell culture medium and cell layer extracts. Regulation of alpha 3(VI) gene expression is therefore critical for the control of collagen type VI synthesis and determines the deposition of collagen type VI heterotrimeric molecules.

Blotting, Northern↗
PubMed1992

Cloning and chromosomal location of human alpha 1(XVI) collagen.

We have characterized cDNA clones that encode a newly discovered collagenous polypeptide. A 4-kilobase (kb) cDNA clone was initially isolated by screening a human fibroblast cDNA library with a probe encoding the collagenous domain of the human alpha 3(VI) collagen. Subsequent screening of another fibroblast cDNA library yielded overlapping clones having a total length of 5.4 kb, which contained an open reading frame of 1603 amino acids including a 21-amino acid signal peptide. The predicted polypeptide consists of 10 collagenous domains 15-422 amino acids long, which were interspersed with 11 noncollagenous (NC) domains. Except for a large N-terminal NC11 domain of 312 residues, most of the NC domains were short (11-39 residues) and cysteine-rich. The overall structural arrangement differed significantly from other known collagen chains. Further analysis indicated that the deduced polypeptide exhibited several structural features characteristically seen in members of the fibril-associated collagen, types IX, XII, and XIV. In addition, the cysteine-rich motifs in the NC domains resembled those found in the cuticle collagen of Caenorhabditis elegans. Northern blot analyses showed hybridization of the cDNA to a 5.5-kb mRNA in human fibroblasts and keratinocytes. The gene was localized by in situ hybridization to band p34-35 of human chromosome 1. The data clearly support the conclusion that the cDNA encodes a collagen chain that has not been previously described. We suggest that the cDNA clones encode the alpha 1 chain of type XVI collagen.

Amino Acid Sequence↗
PubMed1992

Human alpha 2(VI) collagen gene. Heterogeneity at the 5'-untranslated region generated by an alternate exon.

Cosmid clones containing the 5' region of the human alpha 2(VI) collagen gene have been isolated and characterized. DNA sequencing indicates that the signal peptide and the amino-globular domain are encoded by four exons of 142, 596, 21, and 66 base pairs (bp). However, S1 nuclease and primer extension analyses show that the transcription start site is not present in the 142-bp exon. Two different 5' cDNA clones are generated by the anchored polymerase chain reaction. Using the 5' cDNA clones as probes, two untranslated exons (1, 1A) are found 12 kilobase pairs upstream of the first coding exon. These two exons are alternatively used in human fibroblasts, and most transcripts contain exon 1 sequence. Exon 1 shows, by primer extension and S1 nuclease protection assay, two major and several minor transcription start sites. The promoter region contains a canonical TATA box, seven GGGCGG sequences, two possible CAAT boxes, and two sequences resembling AP2 binding sites. Exon 1A contains three alternative splice donor sites and is located 650 bp downstream of exon 1. The most 3' splice donor site of exon 1A is found within an Alu repeat sequence. Exon 1A is preceded by five GGGCGG sequences and one resembling the AP2 binding site although neither TATA or CAAT boxes are found. Two additional GGGCGG sequences are located at the beginning of exon 1A. This study establishes that the human alpha 2(VI) collagen gene is 36 kilobase pairs long and contains 30 exons. The 5'-untranslated and promoter regions are significantly different from the corresponding segments of the chicken gene. The human gene produces by alternative processing multiple mRNAs differing in the 5'-untranslated region as well as the 3'-coding and noncoding sequences.

Amino Acid Sequence↗
PubMed1992

Stable ring chromosome 21: molecular and clinical definition of the lesion.

Ring chromosome 21 results in deletions of chromosome 21. We report on a cytogenetic and molecular analysis of a 4-generation family segregating a stable ring chromosome 21 in 4 relatives. To investigate the molecular structure of the ring chromosome, we have analyzed the DNAs of the transmitted ring in a mother and her daughter. The daughter presented at the age of 2 years with severe growth retardation and microcephaly, whereas her mother had microcephaly but normal intelligence. High resolution chromosome analysis of both cases showed the ring chromosome to be r(21)(p13q22) resulting in deletions of 21p and 21q22. The molecular content of the ring chromosome was determined using quantitative Southern blot analyses of 5 random DNA sequences and 4 expressed genes assigned to chromosome 21 and mapping in the region of q22.3. We have shown that collagen type VI, alpha 2 (COL6A2,) S100 protein, beta polypeptide (neural), (S100B), and D21S44 are present in only one copy in both ring carriers, while CRYA1, CBS, D21S43, D21S42, D21S41, and D21S39 are present in two copies. These data and the breakpoints defining the deletion in these patients show that deletion of COL6A2 and S100B is compatible with normal function and confirm the physical map of 21q22.3 by placing COL6A2, S100B, and D21S44 in very distal 21q22.3. Patients with such small deletions provide unique models for understanding the biological and clinical significance of aneuploidy for specific expressed genes.

Adult↗
PubMed1992

Headaches in children younger than 7 years of age.

Headache in young children is frequently a cause of concern to parents and physicians. We have reviewed our experience with 104 children with onset of headaches prior to 7 years of age seen by age 9 years. Headaches could be classified in more than 90% of cases. The most common headache type in this population referred to a child neurologist was migraine that constituted 75% of the cases. Seventy-two of 78 cases were common migraine. Posttraumatic headaches accounted for an additional 12%. Associated symptoms such as autonomic signs, nausea, and vomiting were common, particularly in the migraine group. Neuroimaging studies when performed did not reveal any significant abnormalities. Other laboratory tests were also generally unhelpful. No child has gone on to develop new neurologic abnormalities or evidence of an intracranial tumor. We conclude that even in young children headaches are generally benign. Even in this population, neuroimaging studies have a very low yield in the absence of other symptoms and findings and are not always indicated.

Child↗
PubMed1992

Structure of recombinant N-terminal globule of type VI collagen alpha 3 chain and its binding to heparin and hyaluronan.

A large portion of the N-terminal globule of human collagen VI was prepared from the culture medium of stably transfected human embryonic kidney cell clones. The recombinant product corresponds to sequence positions 1-1586 of the alpha 3 (VI) chain that consists of eight homologous approximately 200 residue motifs (N9 to N2) being similar to the A domain motif of von Willebrand factor. By ultracentrifugation fragment N9-N2 showed a molecular mass of 180 kDa and an asymmetric shape. Elongated structures that consist of eight small globes (diameter approximately 5 nm) were demonstrated by electron microscopy. The data indicate that each A domain motif represents a separate folding unit which are connected to each other by short protease-sensitive peptide segments. Circular dichroism studies demonstrated about 38% alpha helix, 14% beta sheets and 17% beta turns. Fragment N9-N2 showed binding to heparin which could be abolished by moderate salt concentrations. Heparin binding was assigned to domains N9, N6 and N3 which were obtained after partial proteolysis. Domains N7, N5 and N4 lacked affinity for heparin. In addition, N9-N2 showed strong binding to hyaluronan that required exposure to 6 M urea for full dissociation. Ligand binding studies indicated some affinity of N9-N2 for the triple helical region of collagen VI suggesting a role of the N-terminal globule in the self-assembly of microfibrils. No or only little binding was, however, observed to fibril-forming collagens I and III, several basement membrane proteins and other extracellular proteins. Fragment N9-N2 was also an inactive substrate for cell adhesion.

Amino Acid Sequence↗
PubMed1992

Effects of phenobarbital on leukocyte activation: membrane potential, actin polymerization, chemotaxis, respiratory burst, cytokine production, and lymphocyte proliferation.

Leukocyte activation is known to involve cell membrane potential changes. Phenobarbital, an anesthetic and anticonvulsant that can inhibit neuronal membrane depolarization, may also affect leukocyte activation. Measuring membrane potential, actin polymerization, chemotaxis, superoxide production, lymphocyte proliferation, intracellular calcium concentration, and cytokine production, we found that phenobarbital at a concentration of 15-30 micrograms/ml, which is considered a therapeutic serum level for controlling seizures, did not affect polymorphonuclear neutrophil (PMN) activation. At levels higher than 100 micrograms/ml, phenobarbital significantly suppressed formylmethionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis. Concentrations greater than 300 micrograms/ml also inhibited phorbol myristate acetate-stimulated membrane potential change. In contrast, 30 micrograms/ml phenobarbital significantly inhibited lymphocyte proliferation stimulated by phytohemagglutinin (PHA) and pokeweed mitogen. This concentration of phenobarbital also suppressed the increase of intracellular free calcium induced by PHA. However, only a higher concentration of phenobarbital (300 micrograms/ml) was able to inhibit PHA-induced interleukin-2 (IL-2) production and suppress the proliferation of PHA-induced IL-2 receptor-bearing lymphocytes. These results suggest that concentrations of phenobarbital associated with anticonvulsive levels do not affect PMN activation but suppress lymphocyte activation, possibly by affecting intracellular signal transduction.

Actins↗
PubMed1992

Epidemiology of penicillin-resistant Neisseria gonorrhoeae isolated in Taiwan, 1960-1990.

The epidemiology of penicillin-susceptible Neisseria gonorrhoeae isolated in Taiwan from 1960 to 1990 is summarized. The isolation of N. gonorrhoeae less sensitive to penicillin (i.e., with intrinsic resistance) was first reported in 1960. The rate at which organisms less sensitive to penicillin (MIC, greater than or equal to 0.5 microgram/mL) were isolated increased to 17%, 50%, 80.1%, and 88.8% in 1967, 1975, 1984, and 1990, respectively. Penicillinase-producing N. gonorrhoeae (PPNG) first appeared in Taiwan in late 1976, and the first six strains of PPNG isolated were from United States military servicemen who had relocated from Southeast Asia. The percentage of PPNG strains rose to 37.82% in 1982, and has remained high (50%-62%) since 1983. In the present study, resistance of N. gonorrhoeae to spectinomycin (MIC, greater than 32 micrograms/mL), third-generation cephalosporins (MIC, greater than 4 micrograms/mL), or quinolones (MIC, greater than 4 micrograms/mL) has not been found. Strains requiring arginine, hypoxanthine, and uracil for growth, which frequently cause disseminated gonococcal infections, were not isolated. PPNG strains tended to be of the prototrophic auxotype (55%); non-PPNG strains were mostly of the proline-requiring auxotype (48.8%). Two kinds of R plasmids were isolated in the PPNG strains: the 4.4-MD Asian type (82%-95%) and the 3.05-MD Toronto type (5%-18%). All of the PPNG strains possessing Toronto R plasmid were of the same auxotype/serotype (prototrophic/IB). Evidence suggests that the Asian-type R plasmid was imported into Taiwan in 1976, while the Toronto-type R plasmid may have first emerged in Taiwan in 1983.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Typing Techniques↗
PubMed1992

The large non-collagenous domain (NC-1) of type VII collagen is amino-terminal and chimeric. Homology to cartilage matrix protein, the type III domains of fibronectin and the A domains of von Willebrand factor.

Type VII collagen, the major component of anchoring fibrils, consists of a central collagenous triple-helical segment flanked by non-collagenous domains, NC-1 and NC-2. In this study, we examined the domain organization of human type VII collagen through analysis of deduced amino acid sequences derived from cloned complementary and genomic DNAs, as compared to peptide segments derived from amniotic membrane type VII collagen. The results revealed that the peptide segments derived from the NC-1 domain of type VII collagen could be assigned to the 5' portion of the composite cDNA, indicating that NC-1 resides at the amino terminal end of the molecule. Several sub-domains with homology to adhesive molecules were also identified within NC-1. These protein domains may confer adhesive properties to NC-1, thereby facilitating the binding of type VII collagen to the lamina densa in the cutaneous basement membrane and the anchoring plaques within the dermis.

Amino Acid Sequence↗
PubMed1992

[Survey of heavy metals in traditional Chinese medicinal preparations].

Traditional Chinese medicinal preparations are still widely used by natives in Taiwan. Some traditional Chinese medicine prepared with ancient formulas have been found to contain some heavy metals. There has been an occasional pediatric case admitted with heavy metal intoxication. In the last two years, we collected 11 ancient formula drug samples from the families of the patients. We detected the heavy metals with an inductively-coupled plasma atomic emission spectrometry and a graphite furnace atomic absorption spectrometry. The final result of our report reveals the problem of heavy metals in traditional Chinese medicinal preparations as being very serious.

Drugs, Chinese Herbal↗
PubMed1991

Genomic organization of collagenous domains and chromosomal assignment of human 180-kDa bullous pemphigoid antigen-2, a novel collagen of stratified squamous epithelium.

We have recently isolated a 1.0-kilobase (kb) cDNA encoding 180-kDa bullous pemphigoid antigen (BPAG2), an autoantigen in blistering skin disease, bullous pemphigoid (Giudice, G. J., Squiquera, H. L., Elias, P. M., and Diaz, L. (1991) J. Clin. Invest. 87, 734-738). The deduced amino acid sequence identified two collagenous domains characterized by Gly-X-Y repeats. In this study we have elucidated the genomic organization of the corresponding segment in the human BPAG2 gene. Screening of a genomic lambda-phage DNA library yielded six overlapping clones, and the sequences corresponding to the 1.0-kb cDNA spanned approximately 12 kb of genomic DNA. The coding segment consisted of 19 separate exons varying in size from 27 to 222 base pairs. The organization of these exons and the splice sites at the intron-exon junctions were clearly different from other fibrillar and nonfibrillar collagen genes described to date. The BPAG2 gene was mapped by chromosomal in situ hybridization to the long arm of human chromosome 10, at locus 10q24.3; this location is distinct from any previously mapped collagens, and it also distinguishes BPAG2 from BPAG1, a 230-kDa bullous pemphigoid antigen, which has been mapped to chromosome 6p (Sawamura, D., Nomura, K., Sugita, Y., Mattei, M.-G., Chu, M.-L., Knowlton, R.G., and Uitto, J. (1990) Genomics 8, 722-726). These data suggest that BPAG2 is a novel collagen present in stratified squamous epithelia.

Amino Acid Sequence↗
PubMed1991

Human bullous pemphigoid antigen (BPAG1). Amino acid sequences deduced from cloned cDNAs predict biologically important peptide segments and protein domains.

Bullous pemphigoid antigens are defined as the autoantigens in a blistering skin disease, bullous pemphigoid. One of them, a 230-kDa protein (BPAG1), is associated with hemidesmosomes, attachment complexes at the basal keratinocyte-lamina lucida interface within the dermal-epidermal basement membrane zone. The precise functions and cellular compartmentalization of BPAG1 are unknown. In this study, a human keratinocyte lambda gt11 cDNA library was screened for clones corresponding to BPAG1. The composite of overlapping cDNAs delineated 8,930 base pairs of nucleotide sequences that contained an open reading frame encoding 2,649 amino acids. Analysis of the deduced amino acid sequences predicted a putative signal peptide of 43 amino acids and the presence of a membrane-associated sequence of 17 amino acids. Several potential sites for N-glycosylation, as well as for protein kinase C or cAMP- and cGMP-dependent protein kinase-mediated phosphorylation were identified. Three peptide segments were predicted to be highly antigenic, potentially serving as epitopes for the formation of autoantibodies. Eight repeat segments of 38 residues each with a high degree of homology with sequences in desmoplakin I, a component of desmosomal cytoplasmic plaques, were detected in the carboxyl-terminal end of the molecule. In addition, the presence of three subdomains characterized by heptad repeats predicted an alpha-helical coiled coil dimer structure in the central portion of the protein. These data suggest that BPAG1 may be a membrane-associated protein that plays a role in the attachment of basal keratinocytes to the underlying basement membrane.

Amino Acid Sequence↗
PubMed1991

Human type VII collagen: cDNA cloning and chromosomal mapping of the gene.

A human keratinocyte cDNA expression library in bacteriophage lambda gt11 was screened with the purified IgG fraction of serum from a patient with epidermolysis bullosa acquisita, which had a high titer of anti-type VII collagen antibodies. Screening of approximately 3 x 10(5) plaques identified 8 positive clones, the largest one (K-131) being approximately 1.9 kilobases in size. Dideoxynucleotide sequencing of K-131 indicated that it consisted of 1875 base pairs and contained an open reading frame coding for a putative N-terminal noncollagenous domain of 439 amino acids and a collagenous C-terminal segment of 186 amino acids. The collagenous domain was characterized by repeating Gly-Xaa-Yaa sequences that were interrupted in several positions by insertions or deletions of 1-3 amino acids. The deduced amino acid sequence also revealed a peptide segment that had a high degree of identity with a published type VII collagen protein sequence. Northern hybridization of the K-131 cDNA with human epidermal keratinocyte and skin fibroblast RNA revealed an mRNA of approximately 8.5 kilobases. The fusion protein produced by the K-131 cDNA, when incubated with epidermolysis bullosa acquisita serum, bound to antibodies that reacted in Western blots with type VII collagen. The genomic location of the type VII collagen gene (COL7A1) was determined by chromosomal in situ hybridization with the K-131 cDNA. The results mapped the COL7A1 to the locus 3p21. The cDNA clones characterized in this study will be valuable for understanding the protein structure and gene expression of type VII collagen present in anchoring fibrils and its aberrations in the dystrophic forms of heritable epidermolysis bullosa.

Amino Acid Sequence↗
PubMed1991

Human alpha 3(VI) collagen gene. Characterization of exons coding for the amino-terminal globular domain and alternative splicing in normal and tumor cells.

We recently reported the isolation and sequencing of human cDNA clones corresponding to the alpha 3 chain of type VI collagen (Chu, M.-L., Zhang, R.-Z., Pan, T.-c., Stokes, D., Conway, D., Kuo, H.-J., Glanville, R., Mayer, U., Mann, K., Deutzmann, R., and Timpl, R. (1990) EMBO J. 9, 385-393). The study indicates that the amino-terminal globular domain of the alpha 3(VI) chain consists of nine repetitive subdomains of approximately 200 amino acid residues (N1-N9) and the gene appeared to undergo alternative splicing since some clones lacked regions encoding the N9 and part of the N3 subdomains. In the present study, we report the exon structure for the region encoding the amino-terminal globular domain of the human alpha 3(VI) chain. The nine repetitive subdomains are encoded by 10 exons spanning 26 kilobase pairs of genomic DNA. Eight of the repetitive subdomains (N2-N9) were found to be encoded by separate exons of approximately 600 base pairs each. The only exception is the N1 subdomain which is encoded by two exons of 417 and 146 base pairs. Characterization of the exon/intron structure showed that the cDNA variants were the result of splicing out of exon 9 (encoding the N9 subdomain) and part of exon 3 (encoding the N3 subdomain). Nuclease S1 analysis and the polymerase chain reaction demonstrated that exon 7 (N7 subdomain) was also subject to alternative splicing in normal skin fibroblasts. Examination of these splicing events by nuclease S1 analysis in normal fibroblasts, three different human tumor cell lines, and several human tissues showed that splicing out of exon 9 is much more efficient in normal as compared to tumor cells.

Adult↗
PubMed1991

The COL6A1 and COL6A2 genes exist as a gene cluster and detect highly informative DNA polymorphisms in the telomeric region of human chromosome 21q.

The genes that encode the alpha 1 (VI) and alpha 2 (VI) collagen chains, designated COL6A1 and COL6A2, map to human chromosomal band 21q22.3. Using pulsed-field gel electrophoresis and somatic cell hybrids, we found that COL6A1 and COL6A2 form a gene cluster on the most distal part of chromosome 21. Furthermore, we detected several DNA polymorphisms (both restriction site and VNTRs) associated with these loci. These polymorphisms make the COL6A1 and COL6A2 genes among the most informative markers on human chromosome 21.

Blotting, Southern↗
PubMed1991

Analysis of plasmid pattern in paediatric intensive care unit outbreaks of nosocomial infection due to Enterobacter cloacae.

Sequential outbreaks of nosocomial infection due to multiply-resistant Enterobacter cloacae occurred in September 1987, and between December 1988 and January 1989, in a paediatric intensive care unit. A total of eight neonates were affected and most had received ventilatory support. Initially, we were unable to determine whether the two outbreaks were caused by the same strain of E. cloacae. After applying plasmid profile analysis to identify epidemic strains, we established that the strain from the first outbreak was different from the second outbreak strain, as each had its own plasmid pattern. During the second outbreak, an environmental bacteriological survey was carried out. We found that the distilled water containers were contaminated with E. cloacae which had the same plasmid profile. After changing the distilled water containers and by reinforcement of aseptic techniques, the nosocomial outbreak was terminated.

Cross Infection↗