Morphogenesis and origin of fibrous long-spacing collagen fibers in collagenase-treated mouse skin tissues

Morphogenesis and origin of fibrous long-spacing collagen (FLS) fibers in newborn mouse skin tissues treated with collagenase were examined using ultrastructural observation, morphometry, histochemical methods, and immunoelectron microscopy. The enzyme caused both the partial destruction of basal laminae and the formation of abundant FLS fibers in the dermal matrix. The fibers were usually distributed in the vicinity of basal laminae in the capillaries or basal layer cells. The fibers were characterized by the cross-striated dark bands with about 91 nm periodicity and longitudinally aligned filaments with a diameter of about 6.5 nm. The dark bands of FLS fibers were often continuous with the basal laminae. Histochemical results showed that the dark bands contained the similar mucopolysaccharides which were involved in the basal laminae. Immunoelectron microscopic results showed that laminin was present in the dark bands as well as in the basal laminae, and that type VI collagen was located in the filaments of FLS fibers. These results suggest that the dark bands are formed by products similar to basal laminae and that the products were precipitated on type VI collagen-contained filaments with periodic intervals of about 91 nm. Morphometric examination revealed that there was no differences in ultrastructure between FLS fibers of a collagenase-treated mouse and those of a human neural tumor.     

The collagen fibril: the almost crystalline structure

Blood vessels from aged animals and humans have impaired relaxation and cAMP production to beta-adrenergic stimulation. Direct activators of adenylyl cyclase (AC) such as forskolin are not affected. We hypothesized that analogous findings would occur in membrane preparations. Aortic media membrane preparations from Fischer 344 rats of four age groups (6 weeks to 24 months) were studied. Basal AC activity increased significantly with age. Forskolin-stimulated activity compared to basal tended to be greater in the 6-week and 6-month preparations compared to the 12- and 24-month preparations. AC activity was assessed in the presence of the G protein activators (GTP, GppNHp, NaF). There was no age-related decrease in responsiveness. The receptor agonists isoproterenol (beta-adrenergic), and PGE-1 (prostaglandin), were studied. There was no significant age-related change in responsiveness over basal activity to either of these agonists. There was a slight, but significant increase in the isoproterenol responsiveness over GTP responsiveness in the 6-week-old animals which also approached significance in the 6-month-old animals, but was not seen in the 12- and 24-month-old animals. These data suggest that using a membrane system to assess age-related changes in beta-adrenergic responsiveness in vascular smooth muscle does not retain the robust differences seen in whole vessels.     

Increased diameters of collagen fibrils precipitated in vitro in the presence of decorin from various connective tissues

Proteoglycans were isolated from bovine skin, sclera, deep flexor tendon and the periphery of the temporomandibular joint disc with urea. Decorin was purified from each of these extracts by ion-exchange, hydrophobic-interaction and gel-filtration chromatography. Purities were assessed by amino acid analysis and by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the protein cores released by digestion with chondroitin-ABC-lyase. In these respects the decorins were indistinguishable. However the glycosaminoglycan chains released by digesting the proteoglycans with papain varied widely in mobility on SDS-PAGE: that from skin decorin migrating fastest and that from tendon decorin slowest. The effects of each of the decorins on collagen fibrillogenesis in vitro were similar, all reducing the rate of fibril growth (by 55 to 71%, depending on the source of the proteoglycan) and increasing the diameters of the fibrils formed (by 27 to 66%). Core protein alone, isolated from skin decorin, reduced the rate of fibril growth as effectively as intact decorin, but had no effect on the diameter of fibrils formed. The dermatan sulphate chain and the protein thus appear to play different roles in the interaction of intact decorin with collagen. These data suggest that decorin found in fibrous connective tissues may increase Type I collagen fibril diameters, resulting in tissues that are better able to withstand tensile forces.     

Metabolism of rat skin collagen: the effect of arrested development and subsequent catch-up growth on the stability of the connective tissue fibers

The growth of 33 day-old rats was temporarily arrested by feeding a low-protein diet for 6 weeks. The skin collagen hydroxyproline was labelled previously at 31 days by giving a single intraperitoneally injection of L-[5-3H] proline. This label was rapidly incorporated during the subsequent 2 days of normal growth and then remained at a steady level in the skin collagen when growth was halted. Control animals continued to incorporate label for 2 weeks and then the level of [3H] hydroxyproline declined as this label was subsequently lost from the skin collagen. During arrested development, the skin collagen became increasingly insoluble and the analysis of reducible crosslinks revealed a pattern similar to the collagen of an older animal. In addition the isometric tension developed per unit mass of collagen during thermal denaturation was three-fold greater than that from the age-matched control animals. Refeeding the normal diet gave an immediate burst of rapid growth. The pre-labelled collagen did not turnover during this period since there was no significant change in the level of labelled hydroxyproline in the skin. The newly synthesized collagen, which diluted out these older fibers, appeared to be immature, since it was initially rich in type III, it was very poorly crosslinked, and it caused a fall in the isometric tension value. It was concluded that there need not be limited degradation of the older collagen fibers in skin as an a priori requirement for growth.     

Symmetry and phase-selected NMR spectra of liquid crystalline samples

It is demonstrated that the NMR spectra of liquid crystalline samples can be simplified by using multiple quantum filtering. In a system of N spin-12 nuclei, the N or (N-1)-multiple quantum filtered spectra (NQF or (N-1)QF) contain lines which originate only from transitions among the eigenstates belonging to the highest symmetry class of the spin permutation group. In addition the NQF spectra are divided further into two sets of lines which differ in phase by 180 degrees. A method for simulating and analysing multiple quantum filtered spectra is described, with examples from molecules with up to eight interacting spins.     

The role of oncostatin M in animal and human connective tissue collagen turnover and its localization within the rheumatoid joint

OBJECTIVE: To study the interaction of interleukin-1alpha (IL-1alpha) and oncostatin M (OSM) in promoting cartilage collagen destruction. METHODS: Bovine, porcine, and human cartilage and human chondrocytes were studied in culture. The levels of collagenase (matrix metalloproteinase 1 [MMP-1]) and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by bioassay and enzyme-linked immunosorbent assay (ELISA). The levels of OSM in rheumatoid synovial fluid were measured by ELISA. RESULTS: When combined with OSM, IL-1alpha, IL-1beta, and tumor necrosis factor alpha released proteoglycan and collagen from cartilage. OSM was the only member of the IL-6 family to have this effect. Human tendon also responded to IL-1alpha and OSM. OSM increased the production of MMP-1 and TIMP-1 but when combined with IL-1alpha, synergistically promoted MMP-1 production in human chondrocytes and synovial fibroblasts. High levels of OSM were found in human rheumatoid synovial fluids, and confocal microscopy showed that OSM was produced by macrophages in rheumatoid synovial tissue. CONCLUSION: These results highlight an important new mechanism by which there is irreversible loss of collagen from cartilage.     

Details of the orbital connective tissue system in the adult

The study of serial thick (60 mu) histological sections reveals a highly complicated but orderly constructed connective tissue system within the orbit. Between this system adipose tissue compartments are present built up in a comparable orderly fashion. In this intraorbital continuum, to which Tenon's capsule, the so-called check ligaments, the fascial sheaths of the muscles and the periorbital membrane belong, other different areas of characteristic connective tissue septa originating from the fascial sheaths of the eye muscles are recognizable. These different areas are linked to the presence of the different eye muscles and can be regarded as belonging to these eye muscles. That is why, in our opinion, this total of orbital connective tissue, together with the adipose tissue compartments lying in between, must be regarded as an additional mechanism playing an important role when eye movements are performed. The highly regular pattern and intra- and interindividual uniformity within this system will be clarified in the following paper, using models which specially highlight spatial architecture.     

Chemically-defined medium for growth and differentiation of mixed epithelial and connective tissues in organ culture

The effect on tissue differentiation and growth in vitro of certain of the factors implicated in collagen synthesis (ascorbic acid, alpha-ketoglutarate and oxygen) and the influence of hydrocortisone was studied using organ cultures of fetal mouse mandible as a mixed epithelial and connective tissue system. Using serum-free Waymouth's MB 752/1 chemically-defined medium, addition of high levels of ascorbic acid (300mug per ml), hydrocortisone (1mug per ml) and oxygen (95%) enhanced differentiation in a number of tissues, in particular skin and appendages, tooth germs and bone, while osteoid and dentine production were noticeable promoted. It is suggested that an essential aspect of media design for organ culture involves the incorporaation of collagen-promoting factors to the in vitro enviornment particularly with regard to the controlling role implicated for collagen in a variety of biological processess.     

Heart and autonomic nervous system in connective tissue disorders

Heart rate variability (HRV) is a suitable diagnostic tool in identifying patients with autonomic nervous system (ANS) disorders even in pre-clinical stage. We have enrolled in this study all patients with large variety of connective tissue disorders, given the possibility of an involvement of ANS in these diseases. The study population consisted in eighty-five patients (68 females and 17 males), 35 of whom affected by systemic lupus erythematosus, 16 by rheumatoid arthritis, 14 by Sj?gren syndrome, 12 by progressive systemic sclerosis, 3 by Beh?et syndrome and 5 by antiphospholipid antibodies syndrome. The mean age ranged between 33.7 of patients with lupus erythematosus and 51.8 of those with Sj?gren syndrome. As control, we enrolled healthy subjects of different age, divided into two groups, to rule out the aging as potential source of considered parameters alteration. The autonomic function has been evaluated by 24 hours ambulatory monitoring, using a Zymed 1210 Scanner with Zymed 3.74-PC 1990 software. We have considered: in the time domain, the standard deviation of the RR intervals average (SDNN) and the percentage of RR adjacent intervals differing each other more than 50 msec (pNN50); in the frequency domain, the low (LF) and high (HF) frequencies, the LF/HF ratio, and the total power (RT). The HRV parameters resulted abnormal in every type of the connective tissue diseases considered: particularly SDNN, pNN50, LF, HF and RT (p < or = 0.01). In conclusion: the results of our study suggest that autonomic neuropathy may be present in any kind of connective tissue disorders even in preclinical stage.     

Swelling of collagen fibers in neutral salt solutions (author's transl)

When applying various concentrations of NaC104 (0.15-7 M) to tendon fibres from rat tails, we found denaturation beginning at 2M, and with increasing concentration a linear increase of the maximal contraction tension and an expoential shortening of the contraction time down to a minimal value of 7.5 min. In a second group of experiments we investigated the weight changes of tendons of old (26-30 months) and young (8 months) rats treated with 5M NaC104 and compared the weight curves and the tension curves. We found an increase in weight up to 900% as the tension decreased. This indicates a connection between the swelling and the relaxation of collagen fibres. Other solutions of neutral salts (5M NaC103, NaC1; 0.15 M NaC104, NaC103, NaC1) tested on young and old tendons did not produce any tension nor other visible changes. The weight curves show an immediate increase up to about 200% which remains constant and which is low compaired with the effect of 5M NaC104. The reason for the contraction of tendon fivres seems to be the denaturation caused by the increased lipophilia of the aqueous salt solutions, while the relaxation is merely the result of the subsequent swelling of the collagen fibre.