Biomedical subjects
B C Starcher
Publications and source records attributed to B C Starcher.
Biochemical and ultrastructural demonstration of elastin accumulation in the skin lesions of the Buschke-Ollendorff syndrome.
The Buschke-Ollendorff syndrome is an association of cutaneous lesions, dermatofibrosis lenticularis disseminata, with osteopoikilosis. This condition is inherited in an autosomal dominant pattern. In order to clarify the biochemical nature of the skin lesions, we have examined 12 patients with the Buschke-Ollendorf syndrome, representing 2 unrelated kindreds. Histologically, the lesions were characterized by excessive amounts of unusually broad, interlacing elastic fibers in the dermis. Digestion of skin secretions with pancreatic elastase completely removed these fibers. Electron microscopy of the dermis further revealed markedly branched elastic fibers without fragmentation. The accumulation of elastin in the skin was also demonstrated by measurements of desmosine employing a radioimmunoassay. The desmosine content of the skin lesions increased 3- to 7-fold when compared to the skin either from healthy controls or from uninvolved skin adjacent to a lesion. The results indicate that the skin lesions of the Buschke-Ollendorff syndrome are connective tissue nevi of the elastin type. Cell cultures from these patients may provide a convenient model to study the control mechanisms involved in elastin metabolism.
Desmosine radioimmunoassay as a means of studying elastogenesis in cell culture.
Elastin is synthesized by fibroblasts and chondroblasts in cell culture shortly before the cells become confluent. Fibroblasts secrete elastin into the medium as soluble tropoelastin molecules, which form desmosine crosslinks and become constituents of the cell layer only after three weeks in culture. Even then only a small fraction of the available tropoelastin molecules from crosslinks. Conversely, the chondrocytes secrete an elastin which never reaches the media as soluble elastin in significant quantities. Crosslinking occurs immediately in the chondroblast cell layer forming stable, insoluble elastic fibers. Both cells in culture produce lysyl oxidase at approximately the same levels. The reason for the marked differences between these cells in the mode of conversion of soluble elastin to insoluble elastin is not known. The suggestion of Mecham that the extracellular matrix may play a major role in the development of elastogenesis may provide an answer.
Bleomycin-induced synthesis of type I procollagen by human lung and skin fibroblasts in culture.
Bleomycin is a chemotherapeutic agent sometimes associated with pulmonary fibrosis and skin lesions in patients undergoing treatment. We examined the mechanisms of increased collagen deposition on bleomycin-induced fibrosis by incubating human lung and skin fibroblast cultures with [14C]proline; the synthesis of [14C]hydroxyproline relative to DNA or cell protein was taken as an index of procollagen formation. Procollagen synthesis by lung cells in the presence of 0.1 and 1.0 microgram/ml bleomycin was significantly increased and similar results were obtained with skin fibroblasts. The relative synthesis of genetically distinct types of collagen was measured by isolating the newly synthesized type I and type III procollagens by DEAE-cellulose chromatography. The proportion of type III procollagen of total newly synthesized procollagen in control lung fibroblast cultures was 17.4 +/0 0.6% (mean +/- S.E.) while the corresponding value in cells incubated in 1 microgram/ml bleomycin was 12.5 +/- 0.6% (n = 6, P < 0.01). Similar results were obtained when the ratios of newly synthesized type I and type III collagens were estimated by interrupted polyacrylamide disc gel electrophoresis in sodium dodecyl sulfate after a limited proteolytic digestion with pepsin. The results indicate that the increased procollagen synthesis induced by bleomycin in fibroblast cultures is predominantly directed towards the synthesis of type I procollagen.
The measurement of elastin turnover by the radioimmunoassay of urinary desmosine excretion.
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Effect of zinc deficiency on bone collagenase and collagen turnover.
The effect of zinc deficiency on bone collagenase activity and collagen turnover was studied in the chick. Zinc deficiency symptoms, evident after 8 days on the low zinc diet, included tibia deformities and decreased alkaline phosphatase. Bone collagen metabolism was markedly altered, with a significant reduction in collagen synthesis and turnover. Half-turnover time for tibia collagen was 13 days in the control and 35 days in the zinc-deficient chicks. Tibia collagenase activity was reduced by 40-80% in the zinc-deficient as compared to the control chicks. Heparin markedly increased collagenase activity in the zinc-deficient tibias elevating activity to control levels. But commercially available heparin was found high in zinc content which may explain this effect entirely. These data show that zinc deficiency decreases bone collagen turnover and probably accounts for the leg deformities seen in zinc-deficient chicks.
Zinc absorption and its relationship to intestinal metallothionein.
This study examined the relationship between zinc absorption and metallothionein. Mice injected intraperitoneally with ZnCl2 (2 mmoles) were found within 18 hours to have increased levels of intestinal metallothionein but an apparent decrease in 65Zn absorption. Induction of metallothionein with lower levels of ZnCl2 (0.2 or 0.5 mumoles) resulted in an apparent increase in 65Zn absorption. Isotope dilution experiments showed that intraperitoneal injections of 2 mumoles of ZnCl2 had resulted in a 500-fold dilution of the available 65Zn pool. Mild stress of the animals was shown to increase both 65Zn absorption and intestinal metallothionein. Actinomycin D administered 4 hours prior to ZnCl2 or stress, prevented the induction of metallothionein and obliterated the increase in 65Zn absorption. These results indicate that zinc absorption is directly proportional to intestinal metallothionein levels and imply a significant role for metallothionein in zinc absorption.
The effect of lathyrogens on the evolution of elastase-induced emphysema.
When pancreastic elastase is introduced into the lungs of hamsters to produce emphysema, there is an initial rapid destruction of elastin followed by a subsequent resynthesis. In order to investigate its pathologic significance, we attempted to interfere with this resynthesis by feeding inhibitors of elastin cross-linking. The feeding of the lathyrogens beta-aminoproprionitrile (beta APN) or penicillamine resulted in a marked worsening of the elastase-induced emphysema, as measured by the average distance between alveolar walls and the internal alveolar surface area, when compared with the effect of elastase in animals fed a normal diet. The lathyrogens produced no effect on lung morphology without elastase injections. In elastase-injected animals, the principlal biochemical effect of beta APN was a decrease in the aldehyde content of the elastin without a measurable decrease in desmosine cross-links. These results indicated that the formation of normal connective tissue proteins during the replair of elastolytic injury helps to limit the degree of anatomic deformity that is produced.
The presence of desmosine and isodesmosine in eggshell membrane protein.
Investigation of eggshell membrane by amino acid analysis and radioimmunoassay established the presence of the crosslinking amino acids desmosine and isodesmosine at levels of 28 microgram/100 mg dry weight. Specific enzymatic and chemical hydrolysis indicates that these crosslinks are present as constituents of eggshell membrane protein and do not reflect the presence of small amounts of elastin. The eggshell membrane protein has approximately 10 times the level of lysine-derived aldehyde found in elastin.
Radioimmunoassay for desmosine.
A radioimmunoassay was developed for the determination of desmosine. Desmosine conjugated to albumin was injected into rabbits which developed useful titers of serum antibodies after six months. A radioactive probe was prepared with desmosine using [125I]-Bolton-Hunter reagent. Bound desmosine was separated from free desmosine by cellulose acetate filter binding. The sensitivity of the assay is 1-50 picomoles of desmosine. The antibody is highly selective for desmosine, reacting less than 1% with other known crosslinks. Some prepurification may be necessary with complex samples which contain trace amounts of elastin peptides.
Collagen biosynthesis in bleomycin-induced pulmonary fibrosis in hamsters.
Bleomycin results in pulmonary interstitial fibrosis characterized by accumulation of collagen. We studied the mechanisms of this accumulation in hamsters 4 to 24 days after intratracheal injection of 1 U of bleomycin. Lung collagen was significantly increased within 11 days. Collagen synthesis was increased 10-fold in explant cultures obtained 8 days after injection. Prolyl hydroxylase activity and the degradation of newly synthesized collagen were also increased proportionally to collagen synthesis. Total protein synthesis also increased but not as much as collagen synthesis. We conclude that the accumulation of collagen in the lung following proceeds from an increased rate of collagen synthesis that is maximal early after injection and persists for at least 24 days after exposure.
Fibrogenesis and biosynthesis of elastin in cartilage.
This study presents direct evidence that dissociated chondroblasts from rabbit ear cartilage grown in vitro are capable of synthesizing insoluble elastin. Ultrastructural examination indicated that at an early stage of tissue development, elastogenesis is initiated producing a form of primary fibrils which later condense into an electron dense amorphous material which, unlike other elastin-containing tissues, is heavily stained by metal cations and lacks peripheral microfibrils. Native elastic fibrils and the mature elastic fiber bundles are both susceptible to elastase digestion. Transmission electronmicroscopy demonstrated the presence of many intracellular filaments all showing a substructural organization and localized in close proximity to the nucleus. Their possible contractile nature is discussed. Amino acid analysis of cartilage elastin and of the elastin synthesized in vitro revealed a close chemical similarity between the two molecules. Ultrastructural analysis of the in vitro elastin demonstrated a substructural organization quite similar to that of the elastin observed in an in vivo system.
Studies on the absorption of desmosine and isodesmosine.
Intestinal absorption of desmosine and isodesmosine was investigated in several animal species. Hamsters given desmosine injections intraperitoneally excreted 70% of the injected dose in the urine. When desmosines or elastin were given via gastric intubation, desmosines failed to appear in the urine, indicating a lack of desmosine absorption. Additional studies with everted hamster intestine in vitro showed that norleucine was actively transported from the mucosal to the serosal side but that desmosines and lysinonorleucine were not transported. Intestinal slice experiments with hamster, rabbit, and dog intestine indicated desmosines only in the extracellular space and not absorbed by the mucosal cells. These studies strongly suggest that desmosines are not absorbed via the gastrointestinal tract. The importance of this study rests with the conclusion that desmosines recovered in the urine must represent the degradation products of elastin destruction in the tissue. Newly synthesized elastin cannot interfre, since elastin precursor molecules do not contain desmosines. Thus quantitation of urinary desmosines may be of clinical value as a method of evaluating elastin catabolism.
Increased elastin and collagen content in the lungs of hamsters receiving an intratracheal injection of bleomycin.
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Urinary excretion of elastin peptides containing desmosin after intratracheal injection of elastase in hamsters.
The intratracheal injection of pancreatic elastase results in an acute loss of elastin from the lungs of hamsters and the development of emphysema. We used measurements of the unique covalent cross linking amino acids of elastin, desmosine and isodesmosine, to quantitate elastin. Direct measurements on the lungs estimated an average loss of elastin of 57% after elastase injection. Elastin breakdown products were also quantitated in the urine and feces after injection. An average of 8.8 nmol of desmosines was recovered from the urine of each hamster. This amount represented the desmosines from 61% of the elastin lost from the lungs. Desmosine and isodesmosine existed in the urine in peptide fractions that ranged from 9 to 27,000 daltons with an average of 13,000. Only trace quantities of desmosines could be detected in feces. Desmosines injected intraperitoneally were completely recovered in the urine, and radioactive tracer studies failed to reveal in vivo catabolism of injected desmosines. These results suggest that measurement of urinary desmosines holds promise for the study of elastin turnover.
Elastin content of the scleral spur, trabecular mesh, and sclera.
The scleral spur and trabecular mesh of the human eye contain approximately 5% elastic tissue. Elastic tissue forms less than 2% of the sclera.
Lysyl oxidase deficiency in lung and fibroblasts from mice with hereditary emphysema.
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Determination of the elastin content of tissues by measuring desmosine and isodesmosine.
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