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Biomedical subjects

K S Cheah

Publications and source records attributed to K S Cheah.

At least 37 records · Page 2Linked to original sources
PubMed1995

Human COL2A1-directed SV40 T antigen expression in transgenic and chimeric mice results in abnormal skeletal development.

The ability of SV40 T antigen to cause abnormalities in cartilage development in transgenic mice and chimeras has been tested. The cis-regulatory elements of the COL2A1 gene were used to target expression of SV40 T antigen to differentiating chondrocytes in transgenic mice and chimeras derived from embryonal stem (ES) cells bearing the same transgene. The major phenotypic consequences of transgenic (pAL21) expression are malformed skeleton, disproportionate dwarfism, and perinatal/neonatal death. Expression of T antigen was tissue specific and in the main characteristic of the mouse alpha 1(II) collagen gene. Chondrocyte densities and levels of alpha 1(II) collagen mRNAs were reduced in the transgenic mice. Islands of cells which express cartilage characteristic genes such as type IIB procollagen, long form alpha 1(IX) collagen, alpha 2(XI) collagen, and aggrecan were found in the articular and growth cartilages of pAL21 chimeric fetuses and neonates. But these cells, which were expressing T antigen, were not properly organized into columns of proliferating chondrocytes. Levels of alpha 1(II) collagen mRNA were reduced in these chondrocytes. In addition, these cells did not express type X collagen, a marker for hypertrophic chondrocytes. The skeletal abnormality in pAL21 mice may therefore be due to a retardation of chondrocyte maturation or an impaired ability of chondrocytes to complete terminal differentiation and an associated paucity of some cartilage matrix components.

Animals↗
PubMed1994

Influence of digits, ectoderm, and retinoic acid on chondrogenesis by mouse interdigital mesoderm in culture.

We have cultured tissues isolated from the interdigital zones (IDZ) of the mouse footplate in the presence of the digits, ectoderm, and all-trans retinoic acid. The objective was to understand how these various factors influence the developmental fate of the interdigital tissues. Neutral red staining showed that these tissues normally differentiate by dying between day 12.5-14.5. However, if they were isolated from the footplate between day 12.5-13.5 (when cell death is not overtly obvious in the IDZ) and maintained in organ culture, these tissues would develop into cartilage and soft connective tissues. In culture, chondrogenesis is initiated very rapidly in the interdigital explants as revealed by in situ hybridization with riboprobes specific for type IIA and IIB procollagen mRNAs. The ability of interdigital tissues to form cartilage is not attributed to factors present in the serum of the culture medium as this phenomenon is also observed in serumless cultures. We have found that if all-trans retinoic acid, at concentrations of 10-50 ng/ml culture medium, were added to the explants it could inhibit chondrogenesis and promote cell death. Moreover, in some of the cultures, a single digit was left attached to the interdigital tissue. This also dramatically reduced the incidence of chondrogenesis. We have tried to determine whether the digits and ectoderm can produce a diffusible factor that can prevent cartilage from developing by culturing day 12.5 interdigital tissues in ectoderm and digit conditioned media. The ectoderm conditioned medium had no effects on interdigital growth or chondrogenesis. In contrast, the size of interdigital explants cultured in the presence of digit conditioned medium was shown to be significantly smaller than the control. These explants also produced a smaller quantity of cartilage as revealed by Alcian blue binding assay. In sum, our results showed that the fate of the interdigital tissues are not fully determined until after day 13.5. These tissues have the potentials to form cartilage and soft connective tissues. We tentatively propose that these interdigital tissues do not normally realize their histogenetic potentials because of the antichondrogenic influence of the digits and retinoic acid.

Animals↗
PubMed1994

Further evidence that the failure to cleave the aminopropeptide of type I procollagen is the cause of Ehlers-Danlos syndrome type VII.

Dermal fibroblasts from a Chinese Ehlers-Danlos syndrome type VII patient synthesized approximately equal amounts of normal pro-alpha 2(I) chains of type I procollagen and abnormal ones with electrophoretic mobility of pN alpha 2(I) chains, in which the amino-propeptide (N-propeptide) was retained. Reverse-transcriptase PCR analysis of the proband's RNA showed outsplicing of the 54 base exon 6 in half of the pro-alpha 2(I) mRNAs. Exon 6 encodes 18 amino acids of the N-telopeptide which contains the procollagen N-proteinase cleavage site and a cross-link precursor lysine. Loss of these sequences would result in failure to cleave the amino-propeptide of pro-alpha 2(I) and the accumulation of pN-alpha 2(I) chains. Nucleotide sequencing analyses of the proband's COL1A2 gene showed the presence of a T to C transition at position +2 of intron 6 in one allele and the proband is heterozygous for the defect. This mutation which destroyed the consensus GT dinucleotide at the 5' splice donor site of the intron is responsible for the loss of exon 6 by exon skipping. Electron microscopic analysis of the patient's dermis showed the presence of abnormal collagen I fibrils of irregular diameter and circularity. This mutation in COL1A2 in an EDS VII patient is the first reported case in the Chinese population and is identical to one reported for another EDS-VII (Libyan) patient. The occurrence of an identical mutation in two probands of different ethnic origin is direct evidence that the mutant genotype is the cause of the EDS VII phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

Intron-exon structure, alternative use of promoter and expression of the mouse collagen X gene, Col10a-1.

The entire mouse collagen X gene (Col10a-1) has been isolated. The gene is composed of three exons and two introns spanning 7.0 kb of the DNA sequence. Exons 2 and 3 together encode 15-bp of 5' untranslated sequence, a 2040-bp open reading frame and an 895-nucleotide 3' non-coding region. In the 5' flanking region of the gene, two consensus TATA-box sequences were found. Identification of the first exon by ribonuclease-protection assays and the determination of the 5' end of Col10a-1 mRNA transcripts by primer-extension analyses show that the more 3' TATA box is probably predominantly used and that there are at least three transcription start sites in the exon 1 sequence 3' to this, resulting in 5' untranslated regions of 78, 77 and 55 nucleotides. By means of rapid amplification of cDNA ends by polymerase chain reaction, an additional mRNA species was detected which overlapped the other Col10a-1 transcripts, including the 3' TATA box sequence, giving a 5' untranslated sequence of approximately 235 bases. This latter transcript starts approximately 20 bp 3' to the more 5' TATA box. The data suggest alternative use of promoters and transcription starts for the Col10a-1 gene. Comparison of the combined nucleotide and deduced amino acid sequences of exons 2 and 3 with chicken, bovine and human collagen X genes, showed a high degree of similarity indicating conservation of this gene throughout evolution. Mouse Col10a-1 mRNA was shown to be approximately 3.0 kb and the pepsinized protein, as detected by SDS/PAGE, was approximately 45 kDa. The mRNA and protein sizes correlate with that predicted by the open reading frame. Reverse-transcription polymerase chain reaction assays indicate that the mouse collagen X gene is first expressed at 13.5 days post coitum, temporally preceding the onset of endochondral ossification. In agreement with the generally accepted association of type-X collagen with endochondral ossification, in situ hybridization analyses indicate that Col10a-1 mRNA are restricted to the hypertrophic regions of growth cartilage.

Amino Acid Sequence↗
PubMed1993

A critical assessment of the RNAse protection assay as a means of determining exon sizes.

The RNAse protection assay is a highly sensitive assay which is commonly used to detect specific hybridization between complementary RNAs and to determine exon sizes in gene characterization studies. Unfortunately, each of the numerous steps involved in the assay could give artifacts depending on the probe used. In this study, common causes of artifacts have been identified using riboprobes which identify exons of known sizes. The RNAse concentration and duration of digestion used were found to be critical factors affecting exon size estimations. Five different riboprobes were tested to obtain a consensus optimum RNAse condition--10 micrograms/ml RNAse A, 0.5 microgram/ml RNAse T1--enabling the correct determination of exon sizes. This condition was further analyzed for its specificity when RNAse protection assays were performed between highly homologous RNA fragments from two different species. Results show that this concentration of RNAse would efficiently cleave a minimum of two nucleotide mismatches. Single nucleotide mismatches were frequently not cleaved by the same RNAse concentration making it possible to detect the correct exon size regardless of such sequence polymorphisms in gene sequences.

Animals↗
PubMed1993

Preferential expression of alternatively spliced mRNAs encoding type II procollagen with a cysteine-rich amino-propeptide in differentiating cartilage and nonchondrogenic tissues during early mouse development.

Type II procollagen mRNAs are alternatively spliced: type IIA mRNA contains an exon encoding a cysteine-rich domain in the amino-propeptide and type IIB mRNA lacks this exon. In mouse embryos between 9.5 and 13.5 days, type IIA mRNA was the major form of Col2a-1 transcript expressed in both prechondrogenic and nonchondrogenic tissues and type IIB mRNAs were present in small amounts. After 12.5 days, type IIB mRNA levels increased rapidly and finally exceeded type IIA mRNAs. Type IIB mRNAs became the major Col2a-1 transcript by 14.5 days, predominantly expressed in maturing chondrocytes. By 17.5 days type IIB mRNAs account for 80% of the Col2a-1 transcripts. Expression of type IIA mRNAs follows the change in the growth pattern of the cartilaginous model of the axial and appendicular skeleton and of the otic capsule and nasal septum. In nonchondrogenic tissues, type IIA mRNAs are more commonly expressed in epithelial structures of ectodermal and endodermal origin than in nonepithelial tissues. The switching of expression from type IIA to type IIB mRNA as major Col2a-1 transcript may be associated with the commitment of precursor cells to the chondrocyte lineage and sites of type IIA mRNA expression may mark regions of potential cartilage growth. The differential expression pattern of type IIA mRNAs therefore points to an association of type IIA procollagen with chondrocyte differentiation during cartilage growth and some function early in embryogenesis in the epithelial organization of nonchondrogenic tissues.

Alternative Splicing↗
PubMed1993

The alpha 2(XI) collagen gene lies within 8 kb of Pb in the proximal portion of the murine major histocompatibility complex.

A number of serious hereditary disorders are now known to be associated with defective expression of collagen genes, and these findings have underscored the important and varied roles that the collagen family of genes must play during normal mammalian development. Although the activities of genes encoding the quantitatively major types of collagen are fairly well characterized, functions of the many minor types of collagen remain a matter of speculation. As a first step toward a functional analysis of type XI collagen, a member of this class of poorly understand "minor" collagen proteins which is expressed primarily in hyaline cartilage, we have used human probes for the gene encoding the protein's alpha 2-subunit (COL11A2) to isolate and map homologous murine DNA sequences. Our results demonstrate that Col11a-2 is embedded within the major histocompatibility complex (MHC), within 8.4 kb of the class II pseudogene locus, Pb, and confirm that human and murine alpha 2(XI) collagen genes are located in very similar genomic environments. The conserved location of these genes raises the possibility that type XI collagen genes may contribute to one or more of the diverse hereditary disorders known to be linked to the MHC in mouse and human.

Amino Acid Sequence↗
PubMed1991

Genomic organization of the human procollagen alpha 1(II) collagen gene.

The nucleotide sequence of the human procollagen alpha 1(II) collagen gene extending from within the first intron through exon 15, and part of the 15th intron has been determined. This sequence analysis (7056 bases) identifies the intron/exon organization of the region of this gene encoding the N-propeptide and part of the triple-helical domain. Structural comparison of this with the genes of other human fibrillar collagens shows considerable diversity in terms of size and number of introns and exons that encodes the N-propeptide domain. Although the genomic structure of the human procollagen alpha 1(II) gene is quite different from the rat procollagen alpha 1(II) gene, the nucleotide coding sequences are 89% identical.

Base Sequence↗
PubMed1991

The mouse Col2a-1 gene is highly conserved and is linked to Int-1 on chromosome 15.

Type II collagen is the major extracellular matrix component of cartilage and correct expression of the alpha 1(II) collagen gene is important for vertebrate skeletal development. In order to provide the basis for studying the control of type II collagen gene expression in embryogenesis and in mouse models of human connective tissue disease, the complete mouse Col2-a1 gene has been isolated in a single cosmid clone, cosMco1.2, and partially characterized. The gene is approximately 30 kb and is highly conserved in exon/intron structure and nucleotide and amino acid sequence (greater than 80% homology) when compared with the human, rat, bovine and chicken equivalents. A high degree of conservation was also found in the 5' flanking region of the rat, human and mouse alpha 1(II) collagen genes, including the presence of several G + C and C + T rich, direct repeat motifs. The sites of transcription start, termination codon and polyadenylation have also been identified. Unlike chicken, bovine and human, where polyA attachment is at a single site, for the mouse Col2a-1 gene two polyadenylation sites are utilized. Col2a-1 has also been localized by interspecies backcross analysis to the central portion of mouse Chromosome (Chr) 15, approximately 8 centiMorgans (cM) proximal of Int-1 and 18 cM distal of Myc. Col2a-1 is therefore included in a linkage group which is conserved on human Chr 12q.

Amino Acid Sequence↗
PubMed1991

Expression of the mouse alpha 1(II) collagen gene is not restricted to cartilage during development.

The mouse alpha 1(II) collagen gene has been isolated and a 5' portion of the gene which has low homology to other collagen genes was used to study the pattern of expression during mouse embryogenesis. In situ hybridization studies show that in the mouse, like the chick, alpha 1(II) collagen is expressed in chondrogenic tissues in advance of chondrocyte differentiation. The gene is expressed early in embryogenesis at 9.5 days both in the cranial mesenchyme destined for the chondrocranium, and the sclerotome of the somites, and at 12.5 days in the primordia of the hyoid and the laryngeal cartilage. Type II collagen gene transcripts were found in all the chondrogenic tissues of the axial and appendicular skeleton until the onset of endochondral ossification. Expression of alpha 1(II) collagen mRNA was also observed in non-chondrogenic tissues such as the notochord which may be responsible for inducing chondrogenesis in somitic mesoderm, neural retina, the corneal and conjunctival epithelia and sclera of the developing eye. Expression in the tail tendon was late, at 16.5-18.5 days. Transient expression was also found in the heart at 9.5-12.5 days, the epidermis at 10.5-14.5 days, the calvarial mesenchyme at 12.5-16.5 days, the inner ear at 14.5 days and the fetal brain from 9.5-14.5 days. Within the neural tube, alpha 1(II) collagen mRNA was localized in the proliferative ventricular cells of the forebrain and midbrain of 9.5- to 10.5-day embryos. Subsequently, transcription of the alpha 1(II) collagen gene was confined to restricted areas of the rhombencephalic basal plate, the ventricular layer of the hindbrain and the cervical spinal cord. These examples of expression of the type II collagen gene in the developing nervous system seem to suggest that active transcription of this gene might be associated with early stages of neuroblast differentiation. Type II collagen may therefore have additional roles in development unrelated to chondrogenesis.

Amino Acid Sequence↗
PubMed1990

Calcium accumulation by sarcoplasmic reticulum in whole muscle homogenate preparations of malignant hyperthermia diagnostic patients and pigs.

Calcium accumulation by the sarcoplasmic reticulum in whole muscle homogenate preparations of malignant hyperthermia-susceptible (MH+) and non-susceptible (MH-) humans and pigs was investigated using a calcium electrode at 35 degrees C. Sarcoplasmic reticulum of MH+ humans and pigs showed normal Ca2+ accumulation, with no difference being observed in the rate and the time taken to achieve maximal accumulation. However, the capacity for Ca2+ accumulation by the sarcoplasmic reticulum in MH+ humans and pigs is considerably less stable than normal after prolonged ageing of the whole muscle homogenate preparations in ice. In MH+ patients, the capacity for Ca2+ accumulation by the sarcoplasmic reticulum showed a decline of 62% at 22 h ageing and 70% at 48 h ageing, as compared with a reduction of only 23% in MH- patients. In MH+ pigs, the sarcoplasmic reticulum showed a 96% deterioration in the capacity for Ca2+ accumulation as compared with a loss of only 40% in MH- pigs at 7 h ageing in ice. In both humans and pigs, the decline in Ca2+ accumulation was prevented by incubating the whole muscle homogenate preparations for 2 h at 35 degrees C prior to ageing the preparations. The diminished Ca2+ accumulating capacity of the sarcoplasmic reticulum in whole muscle homogenate preparations of MH-susceptible individuals in our experimental protocol provides a potential diagnostic test for malignant hyperthermia susceptibility.

Animals↗
PubMed1990

The gene for the alpha 2 chain of the human fibrillar collagen type XI (COL11A2) assigned to the short arm of chromosome 6.

A cosmid clone (CosHcol.11) containing the alpha 2(XI) collagen gene (COL11A2) has been isolated. The gene contains conserved DNA and amino-acid sequences characteristic of fibril forming collagen, which is in accordance with the classification of type XI collagen as a fibrillar collagen. The genomic clone containing the alpha 2(XI) gene has been used as probe in the Southern blot analysis of DNA from a panel of human/hamster somatic cell hybrids containing different numbers and combinations of human chromosomes. Synteny analysis revealed that only chromosome 6 showed complete concordant segregation with COL11A2. Furthermore, the gene was regionally mapped to the short arm of chromosome 6 by using a hybrid which contained only the long arm of the chromosome.

Blotting, Southern↗
PubMed1989

The human alpha 2(XI) collagen (COL11A2) chain. Molecular cloning of cDNA and genomic DNA reveals characteristics of a fibrillar collagen with differences in genomic organization.

We have isolated three overlapping cDNA clones encoding the pro alpha 2(XI) collagen chain from a human chondrocyte cDNA library. Together, the cDNAs code for 257 uninterrupted Gly-X-Y triplets (almost 80% of the triple helical domain) and about 200 amino acid residues of the carboxyl telopeptide and carboxyl propeptide. The identification of the clones as pro alpha 2(XI) cDNAs was based on the complete identity between the amino acid sequences of three tryptic peptides derived from human alpha 2(XI) collagen and the cDNA-derived sequence. We have also sequenced six exons within a human genomic alpha 2(XI) cosmid clone. This sequence shows that although type XI collagen belongs to the fibril-forming class of collagens, there are substantial differences in exon sizes at the 3' end of the gene when comparing the alpha 2(XI) gene with those of human types I, II, and III collagens. Finally, pro alpha 2(XI) cDNA has been used as a probe to determine the location of the gene by in situ hybridization of chromosome spreads. The results demonstrate that the gene is located close to the region p212 on chromosome 6. Northern blot analysis shows that the gene is expressed in cartilage but not in adult liver, skin, and tendon.

Amino Acid Sequence↗
PubMed1989

Skeletal muscle mitochondrial respiration of malignant hyperthermia-susceptible patients. Ca2+-induced uncoupling and free fatty acids.

1. Skeletal muscle mitochondria of malignant hyperthermia (MH)-susceptible patients showed normal oxidative phosphorylation but were more easily uncoupled than normal by exogenous Ca2+. 2. Fatty acids, in stimulating the mitochondrial ATPase activity, are responsible for the enhanced State 4 respiration in MH-susceptible patients. 3. These results imply that skeletal muscle mitochondria and free fatty acids are associated with the development of MH syndrome.

Calcium↗
PubMed1989

The human alpha 2(XI) collagen gene (COL11A2) maps to the centromeric border of the major histocompatibility complex on chromosome 6.

Type XI collagen, a minor structural component of cartilage fibrils, is composed of three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI). Using a cloned fragment of the human alpha 2(XI) collagen gene (COL11A2) as a molecular probe for in situ hybridization and somatic cell hybrid mapping, we have localized the gene to the short arm of chromosome 6, region 21.3. By exploiting the rich source of probes provided by the major histocompatibility complex (MHC) genes, which also map to this chromosomal band, we have constructed macrorestriction maps of the region by pulsed-field gel electrophoresis and have localized the alpha 2(XI) collagen gene to the centromeric extreme of the MHC. Finally, we have demonstrated, by the isolation of overlapping cosmid clones, that the gene is 45 kb centromeric to the HLA-DPB2 locus and oriented with the 3' end toward the MHC. The COL11A2 locus thus demarcates the proximal boundary of the MHC. This finding may have implications for the understanding of certain MHC-linked diseases.

Centromere↗