Preservation of elastic system fibers and of collagen molecular arrangement and stainability in an Egyptian mummy

The present findings show that both elastic system fibers and collagen markedly resisted change in tissues more than 2000 years old. The distribution of elastic fibers and elastic-related fibers (namely, oxytalan and elaunin fibers) in mummified tissues coincided with the observations made on the modern human tissues used as controls. The collagenous structures present in tissue sections obtained from the Egyptian mummy studied took on a deeply red colour when stained in the Picrosirius solution indicating that, as well as in the fresh controls, the basic groups in the collagen molecules were available for reacting with the strongly acidic dye Sirius Red. When viewed with polarized light, the collagen in the same tissue sections displayed an increased birefringence, which shows that the collagen molecules in mummified tissues maintain the oriented disposition which is typical of the modern human tissues used as controls. The methods employed have proved to be useful for the delineation of the elastic system fibers and of the collagenous scaffolding, which may be used as valuable landmarks in the study of the histoarchitecture of organs that have undergone considerable distortion.     

Immunohistochemical distribution of collagens types IV, V, and VI and of pro-collagens types I and III in human alveolar bone and dentine

The aim of the present study was to characterize the composition of the organic matrix in alveolar jaw bone and dentine using antibodies against pro-collagens Types I and III and collagens Types IV, V, and VI. After demineralization of oral hard tissues in 0.2 N HCl, antigenicity was well preserved and the distribution of the pro-collagens and collagens could be demonstrated. Staining for pro-collagen Type I was prominent around osteoblasts and in pre-dentine, indicating active de novo synthesis of Type I pro-collagen. Pro-collagen Type I was ubiquitous but was less abundant in bone and dentine, whereas pro-collagen Type III was seen only in areas of bone remodeling, in peritubular spaces, and in pre-dentine. Type IV collagen was limited to the basement membranes of vessels in osteons and bone marrow. Type V collagen was detected neither in pre-dentine nor in bone. In contrast, Type VI collagen was found in dentine and bone, showing a faint but homogeneous staining which, similarly to pro-collagen Type III, was pronounced around osteoblasts and in pre-dentine, areas of active bone and dentine formation. This study showed that the organic matrix of dentine and bone contains Type VI as well as Type I collagen. Pro-collagen Type III (and to a lesser extent collagen Type VI) is transiently produced during new formation and remodeling of oral hard tissues, and disappears once the matrix calcifies. Type I pro-collagen qualifies as a general marker protein for increased osteoblastic activity. We conclude that immunostaining for the different collagen/pro-collagen types can be used to assess normal or abnormal stages of bone/dentine formation.     

Preservation of elastic system fibers and of collagen molecular arrangement and stainability in an Egyptian mummy

Summary The present findings show that both elastic system fibers and collagen markedly resisted change in tissues more than 2000 years old.The distribution of elastic fibers and elastic-related fibers (namely, oxytalan and elaunin fibers) in mummified tissues coincided with the observations made on the modern human tissues used as controls.The collagenous structures present in tissue sections obtained from the Egyptian mummy studied took on a deeply red colour when stained in the Picrosirius solution indicating that, as well as in the fresh controls, the basic groups in the collagen molecules were available for reacting with the strongly acidic dye Sirius Red. When viewed with polarized light, the collagen in the same tissue sections displayed an increased birefringence, which shows that the collagen molecules in mummified tissues maintain the oriented disposition which is typical of the modern human tissues used as controls.The methods employed have proved to be useful for the delineation of the elastic system fibers and of the collagenous scaffolding, which may be used as valuable landmarks in the study of the histoarchitecture of organs that have undergone considerable distortion.Supported in part by Grant no. 43.83.0610/00 from Financiadora de Estudos e Projetos (FINEP-FNDCT). G.S. Montes is Career Investigator of the Brazilian National Research Council (CNPq)     

Nanostructure and mechanics of mummified type I collagen from the 5300-year-old Tyrolean Iceman

Skin protects the body from pathogens and degradation. Mummified skin in particular is extremely resistant to decomposition. External influences or the action of micro-organisms, however, can degrade the connective tissue and lay the subjacent tissue open. To determine the degree of tissue preservation in mummified human skin and, in particular, the reason for its durability, we investigated the structural integrity of its main protein, type I collagen. We extracted samples from the Neolithic glacier mummy known as ‘the Iceman’. Atomic force microscopy (AFM) revealed collagen fibrils that had characteristic banding patterns of 69 ± 5 nm periodicity. Both the microstructure and the ultrastructure of dermal collagen bundles and fibrils were largely unaltered and extremely well preserved by the natural conservation process. Raman spectra of the ancient collagen indicated that there were no significant modifications in the molecular structure. However, AFM nanoindentation measurements showed slight changes in the mechanical behaviour of the fibrils. Young''s modulus of single mummified fibrils was 4.1 ± 1.1 GPa, whereas the elasticity of recent collagen averages 3.2 ± 1.0 GPa. The excellent preservation of the collagen indicates that dehydration owing to freeze-drying of the collagen is the main process in mummification and that the influence of the degradation processes can be addressed, even after 5300 years.     

Involvement of lysine 1047 in type I collagen-mediated activation of polymorphonuclear neutrophils

Oxidative functions of polymorphonuclear neutrophils (PMNs), which play a deciding role in the phagocytosis process, are stimulated by extracellular matrix proteins such as type I collagen. Previous studies have demonstrated the involvement of a DGGRYY sequence located within the alpha(1) chain C-terminal telopeptide in type I collagen-induced PMN activation, but so far the mechanism has not been completely elucidated. We have recently demonstrated that collagen carbamylation (i.e. post-translational binding of cyanate to lysine epsilon-NH(2) groups) impairs PMN oxidative functions, suggesting the potential involvement of lysine residues in this process. The present study was devoted to the identification of lysine residues involved in the collagen-induced activation of PMNs. The inhibition of PMN activation by collagen in the presence of 6-amino-hexanoic acid, a structural analogue of lysine residues, confirmed the involvement of specific lysine residues. Modification of lysine residues by carbamylation demonstrated that only one residue, located within the alpha(1)CB6 collagen peptide, was involved in this mechanism. A recombinant alpha(1)CB6 peptide, designed for the substitution of lysine 1047 by glycine, exhibited decreased activity, demonstrating that the lysine residue at position 1047 within the collagen molecule played a significant role in the mechanism of activation. These results help to understand in more detail the collagen-mediated PMN activation mechanism and confirm the prominent involvement of lysine residues in interactions between extracellular matrix proteins and inflammatory cells.     

Collagen defects in lethal perinatal osteogenesis imperfecta.

Quantitative and qualitative abnormalities of collagen were observed in tissues and fibroblast cultures from 17 consecutive cases of lethal perinatal osteogenesis imperfecta (OI). The content of type I collagen was reduced in OI dermis and bone and the content of type III collagen was also reduced in the dermis. Normal bone contained 99.3% type I and 0.7% type V collagen whereas OI bone contained a lower proportion of type I, a greater proportion of type V and a significant amount of type III collagen. The type III and V collagens appeared to be structurally normal. In contrast, abnormal type I collagen chains, which migrated slowly on electrophoresis, were observed in all babies with OI. Cultured fibroblasts from five babies produced a mixture of normal and abnormal type I collagens; the abnormal collagen was not secreted in two cases and was slowly secreted in the others. Fibroblasts from 12 babies produced only abnormal type I collagens and they were also secreted slowly. The slower electrophoretic migration of the abnormal chains was due to enzymic overmodification of the lysine residues. The distribution of the cyanogen bromide peptides containing the overmodified residues was used to localize the underlying structural abnormalities to three regions of the type I procollagen chains. These regions included the carboxy-propeptide of the pro alpha 1(I)-chain, the helical alpha 1(I) CB7 peptide and the helical alpha 1(I) CB8 and CB3 peptides. In one baby a basic charge mutation was observed in the alpha 1(I) CB7 peptide and in another baby a basic charge mutation was observed in the alpha 1(I) CB8 peptide. The primary defects in lethal perinatal OI appear to reside in the type I collagen chains. Type III and V collagens did not appear to compensate for the deficiency of type I collagen in the tissues.     

Immunocytochemical localization of collagen types I,III, IV,and fibronectin in the human dermis

Summary The distribution of collagen types I, III, IV, and of fibronectin has been studied in the human dermis by light and electron-microscopic immunocytochemistry, using affinity purified primary antibodies and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies. Type I collagen was present in all collagen fibers of both papillary and reticular dermis, but collagen fibrils, which could be resolved as discrete entities, were labeled with different intensity. Type III collagen codistributed with type I in the collagen fibers, besides being concentrated around blood vessels and skin appendages. Coexistence of type I and type III collagens in the collagen fibrils of the whole dermis was confirmed by ultrastructural double-labelling experiments using colloidal immunogold as a probe. Type IV collagen was detected in all basement membranes. Fibronectin was distributed in patches among collagen fibers and was associated with all basement membranes, while a weaker positive reaction was observed in collagen fibers. Ageing caused the thinning of collagen fibers, chiefly in the recticular dermis. The labeling pattern of both type I and III collagens did not change in skin samples from patients of up to 79 years of age, but immunoreactivity for type III collagen increased in comparison to younger skins. A loss of fibronectin, likely related to the decreased morphogenetic activity of tissues, was observed with age.     

Hounsfield Units ranges in CT-scans of bog bodies and mummies

Mummification processes, either artificial or natural, preserve the tissues from postmortem decay, but change them from their original state. In this study we provided the first comprehensive set of Hounsfield Unit (HU) ranges specific for tissues mummified under different environmental conditions (peat bog, cold-dry and hot-dry environment). We also analyzed the impact of different museal preservation techniques on the HU ranges, as e.g. in the Tollund Man and Grauballe Man, two bog bodies from Denmark. The HU results for mummies were compared with HU results from forensic cases, cremated and inhumated ancient human skeletal remains, and fossil animal bones. Knowledge of the typical HU range for the different tissues in mummies may help to avoid misinterpretation of increased or reduced radiodensity as evidence of paleopathological conditions. Finally, we demonstrate the practical benefit of using our re-defined HU ranges by showing the improved results of 3D visualization from automatic segmentation in an Inca mummy from Mount Llullaillaco.     

Distribution of laminin and collagens during avian neural crest development

The distribution of type I, III and IV collagens and laminin during neural crest development was studied by immunofluorescence labelling of early avian embryos. These components, except type III collagen, were present prior to both cephalic and trunk neural crest appearance. Type I collagen was widely distributed throughout the embryo in the basement membranes of epithelia as well as in the extracellular spaces associated with mesenchymes. Type IV collagen and laminin shared a common distribution primarily in the basal surfaces of epithelia and in close association with developing nerves and muscle. In striking contrast with the other collagens and laminin, type III collagen appeared secondarily during embryogenesis in a restricted pattern in connective tissues. The distribution and fate of laminin and type I and IV collagens could be correlated spatially and temporally with morphogenetic events during neural crest development. Type IV collagen and lamin disappeared from the basal surface of the neural tube at sites where neural crest cells were emerging. During the course of neural crest cell migration, type I collagen was particularly abundant along migratory pathways whereas type IV collagen and laminin were distributed in the basal surfaces of the epithelia lining these pathways but were rarely seen in large amounts among neural crest cells. In contrast, termination of neural crest cell migration and aggregation into ganglia were correlated in many cases with the loss of type I collagen and with the appearance of type IV collagen and laminin among the neural crest population. Type III collagen was not observed associated with neural crest cells during their development. These observations suggest that laminin and both type I and IV collagens may be involved with different functional specificities during neural crest ontogeny. (i) Type I collagen associated with fibronectins is a major component of the extracellular spaces of the young embryo. Together with other components, it may contribute to the three-dimensional organization and functions of the matrix during neural crest cell migration. (ii) Type III collagen is apparently not required for tissue remodelling and cell migration during early embryogenesis. (iii) Type IV collagen and laminin are important components of the basal surface of epithelia and their distribution is consistent with tissue remodelling that occurs during neural crest cell emigration and aggregation into ganglia.     

Fluorimetry of bovine myotendon junction by fibre-optics and microscopy of intact and sectioned tissues

Summary The autofluorescence of tendon, epimysium and endomysium at the myotendon junction of the deep digital flexor in the bovine forelimb was measured with a fluorescence microscope and with a bifurcated light guide composed of quartz optical fibres. Data were adjusted for spectral variation in the radiance of the halogen illuminator used to standardize the photometer. Samples of myotendon junction were examined intact, in slices several millimetres thick and after being frozen in liquid nitrogen and sectioned at 20 µm. Sections were examined with and without a mounting medium and with and without immersion oil objectives. Type I collagen fibres were identified by their scarcity of branching, relatively large size and yellow staining with silver. Type III collagen fibres were identified by their extensive branching, small size and black staining with silver. Purified Types I and III collagen were also examined. Type I collagen fibres had a strong fluorescence emission peak between 410 and 450 nm and a shoulder at 510 nm. For the strong peak, results obtained by fibre-optics were positively biased relative to those obtained by microscopy. Type III collagen reticular fibres lacked a strong emission peak at 410 to 450 nm. Although their overall fluorescence was weaker than that of Type I collagen fibres, Type III collagen fibres had similar or slightly stronger emissions around 510 nm. The Type I emission spectrum of collagen fibres was converted to a spectrum similar to the Type III spectrum by conditions that caused the fading of fluorescence (storage as dry or mounted sections and exposure of sections to UV light). It is suggested that, with fibre-optic fluorimetry of intact tissues, Type I collagen fibres may emit a pre-fading spectrum while Type III collagen fibres may emit a post-fading spectrum, and that the preservation of Type I and the fading of Type III collagen is a consequence of the surface to volume ratio of their fibres.     

Aortal collagen polymorphism in monkey and man

Aortal collagen typing in monkey and man showed the presence of types I, HI and V in human aorta and types I and III in monkey aorta. Type III collagen was found to be a predominate type in both species. The molecular weight of type III collagen was similar in these species while type I collagen was different. Both monkey and human collagen types I and III were found to be immunogenic. Type I collagen was significantly increased while type III was decreased in human atherosclerotic plaque. Collagen typing in fatty streak remained unaltered.     

Paleopathologic diagnosis based on experimental mummification

The difficulties of diagnosis of pathologic conditions are immensely magnified when the subject of a postmortem examination has been postmortem for several hundreds to thousands of years. Artefacts of decomposition and bacterial and fungal invasion are compounded upon those of rehydration when mummified tissue is examined. As an approach to these problems, a study of the changes seen in experimentally mummified and rehydrated tissues was undertaken. Normal and pathologic tissues were studied in comparison to sections prepared from the fresh tissue. The experimentally mummified tissues were generally similar to, but somewhat better preserved than, actual human mummies. There was organ and tissue specific variability in preservation, and different classes of pathology likewise showed differential preservation. Inflammatory reactions were not very well-preserved although infecting microorganisms were easily identified. Degenerative processes such as atherosclerosis and others characterized by the accumulation of abnormal products, were well preserved, while necrosis, as in acute myocardial infarction, was not. Malignancies were particularly well preserved. The implications of these findings for previous and future mummy studies is discussed in terms of our understanding of the evolution of disease processes.     

Tensile forces attenuate estrogen-stimulated collagen synthesis in the ACL

The purpose of this study was to examine whether mechanical tensile forces affect estrogen regulation of collagen synthesis of anterior cruciate ligament fibroblasts at the mRNA level. Estrogen was studied at three physiologic levels, 10(-11), 10(-10), and 10(-9)M. The results revealed that estrogen alone stimulated Type I and III collagen synthesis at the mRNA level, and application of mechanical force decreased the expression of collagen Type I and III genes at all tested estrogen levels. These findings suggest that estrogen may directly regulate ligament structure and function by alteration of Type I and III collagen synthesis. This regulation is dependent on mechanical loading.     

Natural taxonomy of collagen based on amino acid composition

Hierarchical cluster analysis and principal components have been used to provide a more detailed separation of the collagens into natural taxonomic groupings than previously obtained. These groups strongly reflect the evolutionary development of collagen. The first component separates land- from sea-based animals, primarily based on the hydroxylation of lysine and proline, indicating that control of hydroxylation, a post-translational event, has exerted a dominant influence during evolutionary adaptation. The power of the technique is illustrated by the ability to partially separate the evolutionarily closely related main homothermic species. Furthermore, the genetically different fibrous collagens, Types I and III, are well separated from basement membrane Type IV collagen and the filamentous collagens. The technique could, therefore, in addition to providing a taxonomic grouping, classify any new collagen and provide clues to its evolutionary development.     

Investigation of relationships between collagens, elastin and proteoglycans in bovine thoracic aorta by immunofluorescence techniques

Types I, III and V collagens and proteoglycan were localized in the aorta by indirect immunofluorescence techniques. Type I collagen was more prominent in media and adventitia than in intima while type III collagen predominated in intima and media but appeared less significant in adventitia. Type V collagen was observed in intima and media only and was seen surrounding smooth muscle cells. Type I collagen was located between elastic fibres but type III collagen appeared to envelop the fibres, suggesting an interaction between elastic fibres and type III collagen. Pretreatment of sections with testicular hyaluronidase caused no changes in staining for type I collagen, but adventitial areas showed increased staining for type III collagen. After digestion with chondroitinase ABC, intimal and medial areas showed increased staining for type III collagen. Therefore, type III collagen forms stronger interactions with proteoglycans and hyaluronic acid than does type I collagen and type III collagen in adventitia is largely masked by hyaluronic acid, while type III collagen in intima and media is associated with proteoglycan. Thus, type III collagen is a more significant component of adventitia than previously recognized. Proteoglycan was also partly localized along elastic fibres. It is, therefore, suggested that elastic fibres are coated with type III collagen, which itself is coated with proteoglycan.     

Immunofluorescent localization of collagen types I, II, and III in the embryonic chick eye.

The appearance and distribution of type I, II, and III collagens in the developing chick eye were studied by specific antibodies and indirect immunofluorescence. At stage 19, only type I collagen was detected in the primary corneal stroma, in the vitreous body, and along the lens surface. At later stages, type I collagen was located in the primary and secondary corneal stroma and in the fibrous sclera, but not around the lens. Type II collagen was first observed at stage 20 in the primary corneal stroma, neural retina, and vitreous body. It was particularly prominent at the interface of the neural retina and vitreous body and, from stage 30 on, in the cartilaginous sclera. The primary corneal stroma consisted of a mixture of type I and II collagens between stages 20 and 27. Invasion of the primary corneal stroma by mesenchyme and subsequent deposition of fibroblast-derived collagen corresponded with a pronounced increase of type I collagen, throughout the entire stroma, and of type II collagen, in the subepithelial region. Type II collagen was also found in Bowman's and Descemet's membranes. A transient appearance of type III collagen was observed in the corneal epithelial cells, but not in the stroma (stages 20–30). The fully developed cornea contained both type I and II collagens, but no type III collagen. Type III collagen was prominent in the fibrous sclera, iris, nictitating membrane, and eyelids.     

Untersuchungen über den Vitamin B2-Bedarf der Legehenne

Because of the well established function of carnitine possible effects of carnitine were studied in poultry. In trial I it was investigated if carnitine and its precursors (lysine, methionine) reduce the formation of abdominal fat in broilers. Chickens (10 groups of 10 chickens each) were fed different diets (control, lysine and methionine in excess and deficient, respectively, with or without 5% fat supplement, L‐carnitine and DL‐carnitine supplement, respectively).Performance (body weight gain, feed conversion), amount of abdominal fat and carnitine concentration in blood, muscles (M. sartorius, M.pectoralis superficialis, cardiac), liver and kidney were determined. Performance and abdominal fat were influenced by dietary fat, lysine and methionine as expected and were not altered by carnitine. Excess and deficiency of lysine and methionine did not influence, fat supplement reduced and carnitine supplementation significantly increased tissue concentration of carnitine.

In trial II it was studied if supplementation of a commercial layers’ ration with either 500 mg L‐carnitine or 500 mg nicotinic acid or both per kg reduces the cholesterol concentration in yolk. Influence on body weight, feed intake, laying performance, serum and yolk cholesterol concentration could not be observed, but yolk concentration of carnitine was significantly increased in supplemented groups.

Trial III should clarify if the L‐carnitine content in broiler parentstock ration influences hatchability. Four groups of 1350 hens each were fed a commercial all‐mash supplemented with 0, 20, 50 and 100 mg L‐carnitine, respectively. Hatching rate was increased from 83% to 87% and from 82.4% to 85.3% in groups supplemented with 50 and 100 mg L‐carnitine, respectively, and in randomly sampled eggs of these groups carnitine concentration in yolk was higher.     

Differential expression of type I and type III collagen genes during tooth development

Collagen gene expression during mouse molar tooth development was studied by quantitative in situ hybridization techniques. Different expression patterns of type I and type III collagen mRNAs were observed in the various mesenchymal tissues that constitute the tooth germ. High concentration for pro-alpha 1(I) and pro-alpha 2(I) collagen mRNAs were found within the osteoblasts. We found that the cellular content of type I collagen mRNAs in the odontoblasts varies throughout the tooth formation: whereas mRNA concentration for pro-alpha 1(I) collagen decreases and that of pro-alpha 2(I) increases, during postnatal development. Moreover, different amounts of pro-alpha 1(I) and pro-alpha 2(I) collagen mRNAs were observed in crown and root odontoblasts, respectively. Type III collagen mRNAs were detected in most of the mesenchymal cells, codistributed with type I collagen mRNAs, except in odontoblasts and osteoblasts. Finally, this study reports differential accumulation of collagen mRNAs during mouse tooth development and points out that type I collagen gene expression is regulated by distinct mechanisms during odontoblast differentiation process. These results support the independent expression of the collagen genes under developmental tissue-specific control.     

Changes in the proportion of type I and type III collagen in the developing and retained bovine placentome

The proportions of Type I and Type III collagen were evaluated from gestational, postpartum-retained, and released bovine placental membranes. Placentomes were excised at 90, 150, 210, and 270 days of gestation (n = 32) and from postpartum-retained (2 and 12 h, n = 8) and released (2 h, n = 4) membranes. Placentome components were processed for collagen, hydroxyproline, protein, and dry weight determination. Collagen extracts were separated by SDS-PAGE. Densitometry was used to establish the proportions of collagen alpha chains (Type I = 2 alpha 1 + 1 alpha 2; Type III = 3 alpha 1). No difference in the proportion of maternal caruncular Type I and Type III collagen was found. The proportion of Type I fetal cotyledonary collagen was lowest (p less than 0.05) at Day 90 of gestation but did not differ between Days 150, 210, 270, or between retained and released fetal membranes. The proportion of Type III fetal cotyledonary collagen was greatest (p less than 0.05) at Day 90. Retained fetal cotyledons had a greater (p less than 0.05) proportion of Type III collagen than did released fetal cotyledons. Therefore, although hydroxyproline content was not different between retained and released fetal membranes, the retained bovine fetal cotyledon was characterized by disproportionate amounts of Type III collagen as compared to the fetal cotyledon that was not retained.     

Carbamylation differentially alters type I collagen sensitivity to various collagenases.

Carbamylation is a post-translational modification due to nonenzymatic binding of cyanate, a by-product of urea, on free amino groups of proteins. Post-translational modifications are known to induce alterations in structural and functional properties of proteins, thus disturbing protein-protein or cell-protein interactions. We report the impact of carbamylation on type I collagen sensitivity to enzymatic proteolysis. Type I collagen was extracted from rat tail tendons and carbamylated by incubation with 0.1 M potassium cyanate at 37 degrees C for 2, 6 or 24 h. Degradation assays revealed that carbamylated collagen exhibited a greater resistance to collagenases (i.e. bacterial collagenase, matrix metalloproteinase(MMP)-1, MMP-8 and MMP-13), together with an increased sensitivity to MMP-2. Evaluation of collagen triple helix conformation by polarimetry indicated that local destabilizations of triple helix structure related to carbamylation could be responsible for the observed differences in sensitivity. These results confirm the crucial role of triple helix integrity in the degradation of type I collagen by MMPs, and support the deleterious impact of post-translational modifications in vivo by altering the balanced remodeling of collagen within connective tissue.