Biomedical subjects

D R Olsen

Publications and source records attributed to D R Olsen.

70 records · Page 4Linked to original sources

PubMed1991

Extracellular matrix gene expression by human endothelial and smooth muscle cells.

In this study, the expression of extracellular matrix genes by vascular cells from human iliac blood vessels was characterized on the mRNA steady-state level by slot blot and Northern transfer analyses, as well as by in situ hybridization. Endothelial cells were isolated from adult human iliac arteries and veins, as well as from umbilical veins; smooth muscle cells were isolated from adult human iliac arteries and inferior vena cava. The results show that confluent umbilical vein endothelial cells expressed the genes that encode types I, III, IV and VI collagens, as well as fibronectin and laminin. In contrast, the iliac endothelial cells expressed the genes for types IV and V collagens, fibronectin and laminin; mRNA transcripts for types I, III and VI collagens were not detectable. The smooth muscle cells from iliac arteries or inferior vena cava displayed gene expression for types I, III, IV, V and VI collagens, fibronectin and laminin. The results indicate major differences in gene expression for the various types of collagens by human iliac endothelial and smooth muscle cells. Furthermore, the fetal-derived umbilical endothelial cells displayed differential collagen gene expression from that of adult iliac endothelial cells.

Adolescent↗

PubMed1991

Magnetic resonance imaging (MRI) and model-free estimates of brain volume determined using the Cavalieri principle.

A complete set of parallel (coronal) slices through a fixed human forebrain was generated by magnetic resonance imaging (MRI) and the Cavalieri principle, combined with point counting, was used to estimate brain volume. Alternative sampling schemes for estimating volume were then assessed by taking systematic and simple random selections of slices. Later, the brain was weighed and its fixed volume determined by fluid displacement. For the complete set of n = 28 MRI slices, the volume (1025 ml) was estimated with a coefficient of error (CE) of less than 1%. Decreasing the number of slices by systematic sampling increased the CE but this was still only 5% when just 5-6 slices were analysed. Estimated volumes varied from 947 ml to 1098 ml. Simple random sampling was less efficient (estimated volumes for 5-6 slices were 644-1187 ml). The forebrain actually weighed 1090 g and displaced 1060 ml of fluid. A set of 14 other brains was physically sliced in order to assess sampling errors in the context of observed brain-to-brain variation. It was found that 5-6 slices per brain is enough to yield efficient estimates of mean brain volume. The findings demonstrate the practicability of using MRI to estimate brain volumes unbiasedly and efficiently. The methods have great potential for noninvasive, longitudinal studies on in vivo brains and other organs.

Brain↗

PubMed1990

Suppression of ornithine decarboxylase gene expression by retinoids in cultured human keratinocytes.

Modulation of ornithine decarboxylase (ODC) gene expression by retinoids was analyzed in human keratinocyte cultures maintained in serum-free medium containing 0.15 mM Ca++. Cells were incubated with all-trans-retinoic acid, 13-cis-retinoic acid or arotinoid Ro15-0778 (10(-10) to 10(-5) M), total RNA was isolated, and mRNA transcripts for ODC were analyzed by Northern and slot blot hybridizations with a human ODC cDNA. Treatment of cells for 24 h resulted in a dose-dependent decrease in ODC mRNA levels, with an estimated IC50 of approximately 1 X 10(-8) M for all-trans- and 13-cis-retinoic acid, while Ro15-0778 was somewhat less effective (IC50 approximately 1-5 X 10(-7) M). The suppression of ODC mRNA levels by retinoids was detectable at approximately 3 h of incubation, with essentially a maximal inhibition at 12 h. Reduced ODC mRNA levels noted after 24 h of incubation with 5 X 10(-7) M all-trans-retinoic acid were accompanied by a reduction in ODC enzyme activity. To determine if all-trans-retinoic acid was regulating ODC gene expression directly, or if protein synthesis was required, ODC expression was analyzed in cultures treated with protein synthesis inhibitors. In the presence of cycloheximide or puromycin, all-trans-retinoic acid did not suppress ODC mRNA levels. These findings suggest that suppression of ODC gene expression is not a direct effect of all-trans-retinoic acid, but depends on ongoing protein synthesis.

Cells, Cultured↗

PubMed1989

Expression of extracellular matrix genes by cultured human cells: localization of messenger RNAs and antigenic epitopes.

Simplified and expedient methodologies for examination of cellular gene expression at the mRNA and protein levels, utilizing in situ hybridization and peroxidase-anti-peroxidase immunodetection, were developed. These techniques were first optimized for the detection of extracellular matrix genes expressed by cultured human skin fibroblasts and keratinocytes, the two principal cell types of human skin. In situ hybridizations and Northern transfer analyses with human-sequence-specific cDNAs encoding collagenous and noncollagenous protein sequences demonstrated selective expression of different matrix genes by these two cell types, indicating different biosynthetic capacities of these cells and attesting to the specificity of the hybridizations. The utility of in situ hybridization was also demonstrated in mixed primary cell cultures established from cutaneous neurofibromas consisting of Schwann cells, perineurial cells, and fibroblasts. The methodologies developed here were further utilized for simultaneous detection of fibronectin mRNA and immunoreactive protein in fibroblast cultures. This procedure allowed detection of grains representative of radioactively labeled cDNA-mRNA hybrids and protein epitopes, as visualized by peroxidase-anti-peroxidase immunodetection on the same cells. This methodology, with appropriate modifications, may be applicable to other cell types as well as tissue specimens.

Cells, Cultured↗

PubMed1989

Human nidogen: complete amino acid sequence and structural domains deduced from cDNAs, and evidence for polymorphism of the gene.

Nidogen, a multifunctional glycoprotein, is an integral part of all basement membranes. In this study, human nidogen cDNAs were isolated and characterized from human placental and skin fibroblast cDNA libraries by hybridization with a mouse nidogen cDNA probe. Six overlapping clones covering 4.9 kb were characterized. The composite cDNA contained a 3,741-nucleotide open reading frame which coded for a 1,247-amino-acid peptide that included a hydrophobic signal sequence. The deduced amino acid sequence contains seven epidermal growth factor-like cysteine-rich repeats, one possible tyrosine O-sulfation site, and a possible N-glycosylation site. The tripeptide sequence -Arg-Gly-Asp- (RGD), a potential cell attachment site, was also present. Human and mouse nidogen sequences were 84% homologous at the nucleotide level and 85% homologous at the deduced amino acid level. Southern blotting of human leukocyte DNA from 23 individuals indicated that nidogen probably is a single-copy gene and shows multiple restriction fragment length polymorphisms when cleaved with Eco RI, Pvu II, Taq I, and Msp I. In particular, digestions with Pvu II revealed polymorphism in four discrete DNA fragments, which could be discriminated by hybridizations with nidogen subclones. One of the polymorphisms revealed an allelic frequency of 0.52/0.48. Thus, human nidogen gene displays RFLPs which provide analytical tools to establish genetic linkage between the nidogen gene and a clinical phenotype.

Amino Acid Sequence↗

PubMed1989

Differential expression of type IV procollagen and laminin genes by fetal vs adult skin fibroblasts in culture: determination of subunit mRNA steady-state levels.

Basement membrane zone gene expression by fibroblast cultures, established from individuals varying in age from 14 fetal weeks to 61 years, was examined by molecular hybridizations with human sequence specific cDNAs corresponding to type IV procollagen and laminin subunit polypeptides. Northern transfer analysis with poly(A)+RNA revealed the presence of specific mRNA transcripts for alpha 1(IV) and alpha 2(IV) chains of type IV procollagen as well as B1 and B2 chains of laminin. Laminin A chain mRNAs were not detected using the same RNA preparations. Quantitative estimates of the steady-state levels of type IV procollagen and laminin mRNAs indicated that they were of relatively low abundance, as compared with mRNA to type I procollagen. The expression of alpha 1(IV) and alpha 2(IV) collagen genes was high in fetal fibroblasts but was reduced to low, yet detectable, levels in cultures established from 3-d to 61-year-old individuals. In contrast, the laminin B1 and B2 chain mRNA levels showed little age-associated variation within the cultures examined. These results provide evidence for differential regulation of the expression of different basement membrane zone macromolecules during chronologic aging.

Aging↗

PubMed1989

Extracellular matrix of the skin: 50 years of progress.

The extracellular connective tissue matrix of the skin is a complex aggregate of distinct collagenous and non-collagenous components. Optimal quantities and delicate interactions of these components are necessary to maintain normal physiologic properties of skin. This overview summarizes the progress made in understanding the normal biology and biochemistry of the extracellular matrix, and will highlight cutaneous diseases with underlying molecular defects in the structure and expression of extracellular matrix components.

Animals↗

PubMed1989

Collagen gene expression by cultured human skin fibroblasts. Abundant steady-state levels of type VI procollagen messenger RNAs.

Previous studies have suggested that procollagen types I and III are the major collagenous gene products of cultured human skin fibroblasts. In this study the expression of 10 different genes, encoding the subunit polypeptides for collagen types I-VI, by human skin fibroblasts in culture was analyzed by molecular hybridizations. Northern transfer analysis demonstrated the presence of specific mRNA transcripts for collagen types I, III, IV, V, and VI, but not for type II collagen. Quantitation of the abundance of these mRNAs by slot blot hybridizations revealed that type I, III, and VI procollagens were the major collagenous gene products of skin fibroblasts in culture. The mRNAs for type IV and V collagens represented only a small percentage of the total collagenous mRNA transcripts. Further analysis by in situ hybridization demonstrated that the majority of the cultured cells coexpressed the genes for type I, III, and VI procollagen pro-alpha chains. Further in situ hybridization analyses revealed the expression of type VI collagen genes in normal human skin. These data demonstrate that human skin fibroblast cultures can be used to study the transcriptional regulation of at least nine genetically distinct procollagen genes. The data further suggest that type VI collagen, in addition to types I and III, may be a major collagenous component of human skin.

Cells, Cultured↗

PubMed1989

Human nidogen: cDNA cloning, cellular expression, and mapping of the gene to chromosome Iq43.

A human placental lambda gt11 expression cDNA library was screened for nidogen cDNAs by hybridizations with a heterologous mouse nidogen cDNA. A total of four positive overlapping clones were identified, and the sizes of the inserts were shown to vary from 0.8 to 2.8 kb. Nucleotide sequencing of the human cDNAs revealed that the largest clone, cHPN-16, contained both a 5' open reading frame encoding 582 amino acids and a 3' untranslated region of 1,063 nucleotides. Comparison of human cDNA sequences with mouse nidogen sequences revealed 84% identity on the nucleotide level and 88% identity with the deduced amino acid sequence. The deduced amino acid sequence of the human cDNAs revealed the presence of cysteine-rich epidermal growth factor-like repeats and the sequence Arg-Gly-Asp (RGD), a potential cell binding site, two features previously identified in mouse nidogen. The sequence Asn-Pro-Ser, a consensus sequence for N-linked glycosylation, was also noted. The newly isolated human cDNAs were utilized to analyze the expression of the nidogen gene by cultured human cells. Northern hybridizations revealed a single mRNA transcript of approximately 6.0 kb in human skin fibroblast and in HT 1080 fibrosarcoma cell cultures. However, the human choriocarcinoma cell line JEG-3, which expressed laminin genes, did not contain detectable levels of nidogen mRNAs. Quantitation of the relative nidogen mRNA abundance in HT 1080 fibrosarcoma cells indicated that nidogen mRNA levels were approximately the same as those for the laminin B2 chain. Finally, one of the nidogen cDNAs was used to map the nidogen gene onto locus q43 of chromosome 1.

Amino Acid Sequence↗

PubMed1989

Molecular mechanisms of cutaneous aging. Age-associated connective tissue alterations in the dermis.

Clinically detectable, age-associated cutaneous changes result from two independent processes: chronologic aging and actinic irradiation. Several lines of evidence suggest that these two processes have different biologic, biochemical, and molecular mechanisms. This review summarizes the current understanding of age-associated alterations in the biochemistry and molecular biology of the extracellular matrix.

Aged↗

PubMed1989

Skin aging: lessons from cutis laxa and elastoderma.

Several lines of evidence suggest that elastic fibers provide resilience to normal human skin, and that abnormalities in elastin may be the primary event leading to the clinical appearance of aged skin. Innately aged skin exhibits a paucity and fragmentation of elastic fibers, while actinically damaged skin consists of an abnormal accumulation of elastotic material within the dermis. Cutis laxa and elastoderma are two cutaneous diseases that manifest as selective alterations in elastic fibers. Clinical and histopathologic findings in these diseases are similar to those found in cutaneous aging. Thus, it appears that alterations in the quantity and/or quality of the elastic fibers may contribute to age-associated cutaneous changes.

Adolescent↗

PubMed1989

Sex vulnerability in the subtotal nephrectomy model of glomerulosclerosis in the rat.

Unilateral nephrectomy and contralateral ligation of two thirds of the renal arterial circulation were performed on male (N x M, n = 6) and female (N x F, n = 7) Sprague-Dawley rats. Sham nephrectomies were performed in 16 male and female control rats (SN x M, n = 8; SN x F, n = 8). Creatinine clearance corrected for body weight and systolic arterial blood pressure were equal in both nephrectomized groups after 1, 3, and 5 weeks. The appearance of proteinuria was delayed in N x F and was still significantly less than N x M after 5 weeks (79 +/- 22 vs 30 +/- 12 mg per 24 hours, p less than 0.05). Glomerular visceral epithelial cell protein reabsorption droplets and vacuolization were equal in N x M and N x F. N x F exhibited one fifteenth of the glomerulosclerosis seen in N x M (p less than 0.01). In addition, there was one fourth of the mesangial expansion, but this trend did not reach statistical significance (p greater than 0.05). Mean glomerular volume was similar in male and female sham-operated (p greater than 0.05) and nephrectomized rats (p greater than 0.05), and nephrectomy resulted in hypertrophy in both groups (p less than 0.01). Glomerular procollagen alpha 1(IV) mRNA levels were higher in N x M than in all other groups (p less than 0.05) and correlated with mesangial expansion and glomerular sclerosis (p less than 0.01) but not with systemic hypertension, proteinuria, or epithelial cell changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1988

Cutis laxa: reduced elastin gene expression in skin fibroblast cultures as determined by hybridizations with a homologous cDNA and an exon 1-specific oligonucleotide.

Fibroblast cultures were established from six patients with cutis laxa, and elastin gene expression was analyzed by RNA hybridizations with a 2.5-kilobase human elastin cDNA or an exon 1-specific 35-base oligomer. Northern analyses using either probe detected mRNA transcripts of approximately 3.5 kilobases, and no qualitative difference between the control and cutis laxa mRNAs was detected. However, quantitation of the elastin mRNA abundance by slot blot hybridizations revealed markedly reduced levels in all cutis laxa cell strains. Assuming equal translational activity of the control and cutis laxa mRNAs, the reduced mRNA levels could result in diminished elastin production, providing an explanation for the paucity of elastic fibers in the skin and other tissues in cutis laxa.

Base Sequence↗

PubMed1988

Cloning of full-length elastin cDNAs from a human skin fibroblast recombinant cDNA library: further elucidation of alternative splicing utilizing exon-specific oligonucleotides.

A human cDNA library was constructed utilizing RNA isolated from cultured skin fibroblasts. Recombinant clones containing elastin sequences were identified by plaque hybridizations with previously characterized human placental elastin cDNAs. Seven positive recombinant clones with inserts of approximately 3.2-2.2 kb were isolated. Characterization of the clones by restriction endonuclease analysis and dot-blot hybridizations with exon-specific synthetic oligonucleotides demonstrated considerable variability in the primary nucleotide sequence. Dideoxy nucleotide sequencing confirmed this finding. The variability is most likely a result of alternative splicing of exons from the primary elastin transcripts. The two largest clones contained approximately 1 kb of 3' untranslated sequence and approximately 2.2 kb of translated sequence encoding 730 amino acids. Six amino acids, encoded by exon 12A, have not been previously noted in human elastin cDNAs. In addition, these human skin fibroblast clones contained a 49 bp 5' untranslated sequence. These results demonstrate that there is considerable variability in the processed nucleotide sequence of the elastin mRNAs. These transcripts may code for isoforms of tropoelastin with different biologic properties.

Amino Acid Sequence↗

PubMed1988

Expression of basement membrane zone genes coding for type IV procollagen and laminin by human skin fibroblasts in vitro: elevated alpha 1 (IV) collagen mRNA levels in lipoid proteinosis.

Basement membrane zone gene expression by fibroblast cultures established from adult human skin was examined by molecular hybridizations with human sequence-specific cDNAs corresponding to pro-alpha 1 (IV) chain of type IV collagen and B2 chain of laminin. Northern transfer analysis of poly(A)+RNA isolated from fibroblast cultures clearly revealed the presence of specific mRNA transcripts, indicating the expression of the genes coding for pro-alpha 1 (IV) and laminin B2 polypeptides. Quantitative estimates of the relative levels of these mRNAs coding for basement membrane zone components indicated that they are of relatively low abundance in control fibroblasts, as compared with mRNAs coding for fibronectin or pro-alpha 1 (I) chain of type I procollagen. In fibroblast cultures established from the lesional skin of a 32-year-old patient with lipoid proteinosis, the levels of mRNA coding for pro-alpha 1 (IV) polypeptides were increased approximately 4.5-fold, as compared with age- and passage-matched control cultures. This increase was selective in that the levels of fibronectin, pro-alpha 1 (I), and beta-actin mRNAs were unaltered in the same cultures. The increase in pro-alpha 1 (IV) mRNA level was also uncoordinate with the expression of the laminin B2 chain gene, which was unaltered in lipoid proteinosis. The selective increase in pro-alpha 1 (IV) mRNA may have relevance to the accumulation of this basement membrane component in the skin in lipoid proteinosis.

Adult↗

PubMed1988

Isolation and characterization of human elastin cDNAs, and age-associated variation in elastin gene expression in cultured skin fibroblasts.

In attempts to isolate human elastin cDNAs, a human placental lambda gt11 cDNA library was screened with a 1.3 kilobase sheep genomic DNA subclone, corresponding to the 3'-end of the elastin mRNA. The four largest clones, the largest being approximately 3 kilobase, were characterized by Northern transfer analyses, restriction endonuclease digestions and dideoxy nucleotide sequencing. Northern transfer analyses of poly(A)+RNA revealed hybridization to mRNA transcripts in the region of 3.5 kilobase. Restriction endonuclease mapping and nucleotide sequencing demonstrated distinct domains characteristic of elastin, and identified areas of variability which apparently reflects alternative splicing of the primary elastin transcripts. To demonstrate the utilization of these cDNAs for studies on elastin gene expression in human cells, elastin mRNA was examined in fibroblast cultures established from the skin of several individuals of varying ages. Northern transfer analyses and slot blot hybridizations demonstrated that elastin gene expression is initiated early during fetal development, and continues at a relatively constant level through several decades. The lowest abundance of elastin mRNA was noted in the cell cultures established for the oldest individual studied (61-year-old female). Demonstration of elastin gene expression in cultured fibroblasts provides a system to study diseases affecting the elastic fibers.

Aging↗