Hydroxylation of lysine and glycosylation of hydroxylysine during collagen biosynthesis in isolated chick-embryo cartilage cells

Hydroxylation of lysine and glycosylation of hydroxylysine during collagen biosynthesis in isolated chick-embryo cartilage cells were studied by using continuous labelling and pulse-chase labelling experiments with [14C]lysine. Control experiments with [14C]proline indicated that in continuous labelling the hydroxylation of [14C]proline became linear with time after about 4 min and the secretion of collagen after about 35 min, as reported previously. In similar experiments with [14C]lysine the hydroxylation of [14C]lysine and the glycosylations of hydroxy[14C]lysine became linear at about 4 min, suggesting that these reactions were initiated while the polypeptide chains were growing on the ribosomes. Pulse-chase labelling experiments with [14C]lysine indicated that after a 5 min pulse-label the hydroxylation of [14C]lysine and the glycosylations of hydroxyl[14C]lysine continued during the chase period for about 20 min. The data suggest that these reactions are continued after the release of complete polypeptide chains into the cisternae of the endoplasmic reticulum, whereas the reactions are probably not continued after the formation of the triple helix and the movement of the molecules into the Golgi vacuoles.     

Local removal of phagocytic synovial lining cells by clodronate-liposomes decreases cartilage destruction during collagen type II arthritis

OBJECTIVE: To investigate whether local removal of phagocytic synovial lining cells (SLCs) from the knee joint before onset of collagen type II arthritis has an effect on development of cartilage destruction. METHODS: Phagocytic SLCs were selectively depleted by a single injection of clodronate laden liposomes in the knee joint seven days before induction of collagen type II arthritis (CIA). Clodronate laden liposomes were given in one knee joint either alone or in combination with a short-term oral treatment of dexamethasone. Cartilage damage including proteoglycan depletion and chondrocyte death was measured in total knee joints sections stained with safranin-o or haematoxylin. RESULTS: Local removal of phagocytic SLCs, seven days before arthritis onset, prevented cell influx for the larger part. Chondrocyte death was significantly decreased in the SLC depleted arthritic joint both at an early (6 days) and late (12 days) time point after CIA induction. However, depletion of proteoglycans from femoral and patellar cartilage layers was not prevented. If the mild acute inflammation caused by a single clodronate laden liposome injection in the left knee joint, was blocked by a short-term (on consecutive days 9, 8, 7, 6, 5 before CIA onset) oral treatment with dexamethasone, cell influx, but also proteoglycan depletion was almost completely blocked. In the contralateral control right knee joint prominent cell influx and severe cartilage damage was observed, indicating that there was no effect of dexamethasone anymore at the onset of CIA. CONCLUSIONS: This study shows that removal of phagocytic lining cells before CIA induction, particularly in the presence of a short-term treatment with dexamethasone, decreases cartilage destruction.     

Immunohistochemical findings type I and type II collagen in prenatal mouse mandibular condylar cartilage compared with the tibial anlage

In growing animals the mandibular condylar cartilage serves not only as an articular but also as a growth cartilage, yet, condylar cartilage has some characteristic features that are not found in growth cartilage. For example, some reports suggest that type I collagen, which is not seen in the growth plate cartilage of long bones, is present in the extracellular matrix of condylar cartilage postnatally. Here, the condylar and limb bud cartilage of fetal mice was examined. The distribution of type I and type II collagen in condylar cartilage was already different from that in the limb bud at the first appearance of the cartilage. Type I collagen was demonstrated in the extracellular matrix of the condylar cartilage that first appeared on day 15 of gestation. However, the reaction for type II collagen was much weaker than that for type I collagen. On day 18 of gestation, type I collagen was still found throughout the cell layers but became gradually weaker with depth. Type II collagen was limited exclusively to the deeper layers at this stage. These findings are different from those in the limb bud cartilage, indicating a characteristic feature of the cells in the condylar cartilage present from the prenatal period.     

Tenascin-R inhibits the growth of optic fibers in vitro but is rapidly eliminated during nerve regeneration in the salamander Pleurodeles waltl

Tenascin-R is a multidomain molecule of the extracellular matrix in the CNS with neurite outgrowth inhibitory functions. Despite the fact that in amphibians spontaneous axonal regeneration of the optic nerve occurs, we show here that the molecule appears concomitantly with myelination during metamorphosis and is present in the adult optic nerve of the salamander Pleurodeles waltl by immunoblots and immunohistochemistry. In vitro, adult retinal ganglion cell axons were not able to grow from retinal explants on a tenascin-R substrate or to cross a sharp substrate border of tenascin-R in the presence of laminin, indicating that tenascin-R inhibits regrowth of retinal ganglion cell axons. After an optic nerve crush, immunoreactivity for tenascin-R was reduced to undetectable levels within 8 d. Immunoreactivity for the myelin-associated glycoprotein (MAG) was also diminished by that time. Myelin was removed by phagocytosing cells at 8-14 d after the lesion, as demonstrated by electron microscopy. Tenascin-R immunoreactivity was again detectable at 6 months after the lesion, correlated with remyelination as indicated by MAG immunohistochemistry. Regenerating axons began to repopulate the distal lesioned nerve at 9 d after a crush and grew in close contact with putative astrocytic processes in the periphery of the nerve, close to the pia, as demonstrated by anterograde tracing. Thus, the onset of axonal regrowth over the lesion site was correlated with the removal of inhibitory molecules in the optic nerve, which may be necessary for successful axonal regeneration in the CNS of amphibians.     

Expression of hindlimb abnormalities under rearing temperature effects during the larval development of the salamander Pleurodeles waltl (urodele amphibian)

The Q-switched ruby laser (694 nm, 25-40 nsec) is an effective and safe therapeutic device for the treatment of tattoos and well-defined, benign, pigmented epidermal and dermal lesions. Because of its selective mode of action, dermal pigments of natural and artificial origin are destroyed photothermically and removed without scar. This method is exceptionally suited for the elimination of lay and professional tattoos, traumatic tattoos, and permanent makeup. Other frequent indications include benign pigmented lesions such as lentigines, freckles, café-au-lait spots, seborrheic keratosis, and Becker nevi. As a dermal pigmented lesion, the nevus of Ota is perfectly treatable. However, chloasma can no longer be considered an indication for ruby laser treatment due to unsatisfactory results. Melanocytic nevi and congenital nevi should be treated only in clinical studies. The effectiveness of the long-term epilation of dark hair with this laser device has to be verified in future investigations. Particularly attractive is the nonproblematic and straightforward removal of pigmented lesions in precarious anatomic regions like the lips, eyelids, and genitals (e.g., benign melanosis of the lips or of the penis, seborrheic keratosis of the lid angle).     

Evidence for the degradation of type XI collagen by bovine intervertebral disc- and articular cartilage extracts

Bovine intervertebral disc- and articular cartilage extracts contain a metalloproteinase system capable of degrading type XI collagen. The collagen-degrading activity is rather low in unmodified extracts but increases considerably on metalloproteinase activation. The similarity between intervertebral disc and articular cartilage in their patterns of (casein-degrading) metalloproteinases and type XI and type II collagen degradation is believed to suggest a similarity in the events underlying the degradative disorders of articular cartilage and intervertebral disc.     

Localization of carboxy-terminal type II procollagen peptide (pCOL-II-C) and type II collagen in the repair tissue of full-thickness articular cartilage defect

Apoptosis and aging share common mechanisms in oxidative stress and mitochondrial involvement. Treatment of cultured neuroblastoma cells with a radical initiator induced apoptosis; raise in hydrogen peroxide and release of cytochrome c from mitochondria preceded collapse of mitochondrial potential and cell death. In rat hepatocytes treated with adriamycin incubation with exogenous Coenzyme Q10 counteracted the drug-induced increase of hydrogen peroxide and the fall of the mitochondrial potential, thus demonstrating the quinone antioxidant effect. Complex I activity and its rotenone sensitivity decreased in brain cortex non-synaptic mitochondria from old rats; a 5 kb mitochondrial DNA deletion was found only in the old rats. A similar behavior was found in human platelets from old individuals. The postulated energy decline was confirmed by the inhibitor sensitivities of platelet aggregation and lactate production. The lack of the 5 kb deletion in platelets throws doubts on mitochondrial DNA lesions as the only causes of mitochondrial dysfunction in aging.     

Retinoic acid-induced type II collagen degradation does not correlate with matrix metalloproteinase activity in cartilage explant cultures

OBJECTIVE: To determine the role of matrix metalloproteinases (MMPs) in retinoic acid (RetA)-induced degradation of type II collagen in cartilage. METHODS: Bovine nasal cartilage explants were cultured with 1 microM RetA or in 3 nM interleukin-1alpha (IL-1alpha). Release of proteoglycan and type II collagen into the medium was measured by colorimetric assay and immunoassay, respectively. MMP activity in the medium was determined using a quenched fluorescent substrate assay, while specific collagenases were identified by Western immunoblotting. In some cases the effects of low molecular mass synthetic MMP inhibitors and serum on collagen degradation were studied. RESULTS: RetA promoted maximal breakdown of type II collagen after 4 or 5 weeks in culture, compared with 3 weeks in culture with IL-1alpha. In IL-1alpha-stimulated cultures, collagen degradation was coincident with a large increase in MMP activity in the culture medium, whereas in RetA-stimulated cultures, there was only a small increase. In Western immunoblots of culture media containing RetA, prointerstitial collagenase and active collagenase 3 were sometimes detected, but not in all experiments. In IL-1alpha cultures, active interstitial collagenase was always detected, and active collagenase 3 was detectable in some experiments. Neutrophil collagenase was not detected in any cultures. IL-1alpha-stimulated collagen degradation was effectively inhibited by a potent, broad-spectrum inhibitor of MMPs, whereas it was poorly inhibited by a weak MMP inhibitor. The same 2 compounds were both only weak inhibitors of RetA-induced collagen degradation. When fetal calf serum was included in cartilage cultures, MMP activity in the culture medium was reduced to low levels. This resulted in a marked inhibition of IL-1alpha-induced type II collagen degradation, whereas there was no inhibition of RetA-induced collagen degradation. CONCLUSION: Unlike IL-1alpha, RetA induces degradation of type II collagen in cartilage explants by a mechanism that is mainly independent of those MMPs that can be detected in the culture medium.     

Expression of basement membrane type IV collagen chains during postnatal development in the murine cochlea

An immunofluorescence study was performed to examine the temporal and spatial patterns of expression for the different type IV collagen chains during postnatal cochlear development. At birth, the classical chains (4A1 and 4A2) were widely expressed, while the novel chains (4A3, 4A4, and 4A5) were completely absent. Activation of the novel chains was observed at 4 days of age, with intense, widely distributed immunostaining suggesting that most of the cells in the cochlea express the novel chains at this developmental stage. From day 8 through day 14, developmental inactivation of the novel chains results in a reduction of generalized immunoreactivity with a concomitant elevation of specific staining in the membranous structures bounding the interdental cells of the spiral limbus, the inner sulcus, the basilar membrane, and in a fibrous bed of staining radiating from the spiral prominence into the region of the spiral ligament which corresponds to the location of the root cell processes. This pattern of intense immunostaining for the novel chains persists through adulthood. The classical chains are expressed in these same anatomical regions only transiently (from day 6 to day 10), after which a gradual developmental inactivation leads to the adult expression pattern where classical collagen chains are found primarily in the perineurium, in the membranes surrounding the spiral ganglion cell bodies, and in the vascular basement membranes of the spiral ligament and the stria vascularis. The complex developmental pattern of expression for the type IV collagen chains in the murine cochlea is similar to that observed in the murine kidney, which is the other major site for basement membrane pathology in Alport syndrome.     

Inhibition and axial deviation of limb regeneration in the newt by means of a digit implanted into the amputated limb

This research was designed to follow up the observation of Thornton and Kraemer ('51) that regressed, denervated limbs of Ambystoma larvae will not regenerate upon reinnervation if all digits on the limbs were not completely resorbed. The object of this experiment was to determine whether the presence of an apical structure, protruding past the amputation surface, would affect the regenerative process. Both forearms of adult newts were amputated midway between the elbow and the wrist. One limb served as a normal regeneration control, and in the other limb the third digit from the removed hand was implanted in place of the removed radius, so that the three distal phalangeal segments protruded past the plane of amputation. Blastema formation in the experimental limbs was delayed by several weeks as compared with control limbs. Approximately one third of the experimental limbs did not regenerate. The regenerates that did form were strongly deviated (45-90 degrees) radially from the longitudinal axis of the limb. Experimental analysis showed that the delay in regeneration is due largely to the projecting part of the digit. The radial deviation of the regenerates is not due to the digital implant, but rather to the removal of the radius. Trauma alone does not account for this phenomenon.     

Identification of distinct metabolic pools of aggrecan and their relationship to type VI collagen in the chondrons of mature bovine articular cartilage explants

The metabolism and distribution of newly synthesized aggrecan present in the extracellular matrix of intact explant cultures of mature articular cartilage was investigated with respect to type VI collagen-stained chondrons. Using biochemical, autoradiographical and novel confocal immunohistochemical techniques it was shown that aggrecan exists as a number of distinct pools that are located within the extracellular matrix of the tissue. The first was identified as a pool of high specific radioactivity, much of which appeared in the medium one day after incubation with radiolabeled sulfate. Of the radiolabeled aggrecan remaining within the extracellular matrix, three pools were differentiated on the basis of time and location within the extracellular matrix. One pool was resident within the pericellular microenvironment associated with the chondron, one migrated into the territorial matrix adjacent to the chondron and one was sequestered long term in the interterritorial matrix. Analysis of the kinetics of loss of radiolabeled aggrecan macromolecules present in the region of matrix defined by the chondron suggests that this pool rapidly turns over and is a precursor to the pools of aggrecan present in the territorial and interterritorial matrix. There were marked differences in the distribution of newly synthesized aggrecan present in these regions of the extracellular matrix in explant cultures maintained with or without fetal calf serum. In the absence of serum, more of the newly synthesized aggrecan moved into the territorial and interterritorial matrix indicating that the presence of serum in the culture medium influenced the tissue distribution of aggrecan. This work indicates that the pericellular microenvironment of the chondron plays an important role in the retention and maturation of aggrecan prior to the sequestration of aggrecan complexes into the functional load bearing matrices of adult articular cartilage.     

Molecular mechanisms in the control of limb regeneration: the role of homeobox genes

The Ca2+ sensitivity of cardiac myofibrillar force production can be decreased by acidosis or inorganic phosphate (P(i)) and increased by caffeine. To investigate whether the source of tissue influences the potency of these agents, we compared the actions of acidosis (change of pH from 7.0 to 6.2), P(i) and caffeine (both 20 mM) on force production of skinned cardiac muscles from adult ventricle, adult atrium and neonate ventricle of the rat. Maximum Ca(2+)-activated force was reduced by all three interventions and the responses of the different muscle types to a given intervention were similar. Acidosis reduced myofibrillar Ca2+ sensitivity by 1.09 and 1.04 pCa units in adult ventricle and atrium, respectively, and P(i) reduced it by 0.19 and 0.22 pCa units. However, each effect was only one-third as great in the neonate ventricle, which showed falls of 0.33 pCa units for acidosis and 0.06 for P(i). In contrast, caffeine raised the Ca2+ sensitivity by the same amount (approximately 0.4 pCa units) in all three muscle types. The differential effect between adult and neonate seen with both acidosis and P(i) suggests some similarity in the mechanisms by which these factors decrease Ca2+ sensitivity. In contrast, the equal effects of caffeine on neonate and adult suggests that caffeine acts by a completely different mechanism. The lower pH- and P(i)-sensitivity of the neonatal ventricle can help to explain why neonatal and adult myocardium exhibit differential force responses to ischaemia (or hypoxia alone).     

Transformation of skin from larval to adult types in normally metamorphosing and metamorphosis-arrested salamander, Hynobius retardatus

Transformation of skin from larval to adult types in a salamander, Hynobius retardatus, which had been reported to show neotenic reproduction in a specific environment, was examined morphologically in normally metamorphosing, precociously metamorphosing and metamorphosis-arrested larvae. Typical larval skin was composed of an epidermis constituted by three types of cells such as apical, Leydig, and basal cells. The Leydig cells were larval specific, and thus disappeared and were replaced by adult epidermal cells during the metamorphosis. Disappearance of the Leydig cells was accomplished by apoptosis as confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end-labeling method and electron microscopy during the normal metamorphosis and precocious metamorphosis induced by exogenously applied triiodothyronine. Typical adult skin was composed of epidermis constituted by stratified squamous cells and of dermis mainly occupied with two types of dermal glands, mucous and serous glands. When the metamorphosis was arrested by different procedures (thyroidectomy, hypophysectomy, goitrogen treatment, and rearing at low temperature), the larval-specific Leydig cells fully remained in the epidermis, suggesting that the disappearance of these depended on the thyroid activity. Contrary to this, dermal glands behaved differently from the Leydig cells, though they developed and differentiated from epidermal basal cells and constituted the same skin. Those in the metamorphosis-arrested (thyroidectomized, hypophysectomized, or goitrogen-treated) larvae, except in the larvae reared at 4 degrees C, appeared a little later than in the controls. Thus, the aged, metamorphosis-arrested larvae had skin which consisted of larval type epidermis (Leydig cells) and adult type dermis (mucous and serous glands).     

The role of type III collagen in spontaneous cervical arterial dissections

OBJECTIVE: To determine the efficacy of lymphadenectomy after nephroureterectomy in patients with transitional cell carcinoma (TCC) of the upper urinary tract. PATIENTS AND METHODS: Between January 1986 and December 1995, 72 patients (mean age 67 years, range 45-82) underwent nephroureterectomy for primary TCC of the upper urinary tract. In 35 patients, a lymphadenectomy was also performed. The clinicopathological data were analysed retrospectively, focusing on the significance of lymphadenectomy. RESULTS: Lymph vessel invasion was found in 28 patients and its incidence was closely correlated with both tumour grade and pathological stage. Of the 35 patients who underwent lymphadenectomy, lymph node metastases were found in 13 patients, all of whom had lymph vessel invasion. There was no significant difference in the survival rate between patients with and without lymphadenectomy; however, among the 44 patients with no lymph vessel invasion, the survival rate of those with lymphadenectomy was significantly higher than in those without (P<0.05). CONCLUSION: Lymphadenectomy may provide a therapeutic advantage in patients with upper urinary tract TCC and no lymph vessel invasion. However, patients with lymph vessel invasion seem to have systemic disease; therefore, aggressive systemic adjuvant therapies rather than regional lymphadenectomy should be applied in these patients.     

Posttranscriptional aspects of the biosynthesis of type 1 collagen pro-alpha chains: the effects of posttranslational modifications on synthesis pauses during elongation of the pro alpha 1 (I) chain

Early studies indicated that chain elongation pauses were prominent during the in vivo synthesis of type I procollagen chains, and it was postulated [Kirk et al., (1987): J Biol Chem 262:5540-5545.] that these might have a role in the coordination of procollagen I molecular assembly. To examine this postulate, polysomes isolated from [(14)C]-Pro-labeled 3T6 cells were subjected to SDS-PAGE. The resulting gels were Western blotted and screened with a monoclonal antibody (SP1 .D8) directed against the N-terminal region of the pro alpha 1 (I) chain. The blots were fluorographed, which also permitted analysis of the pro alpha 2 (I) chain. There was a prominent pro alpha1 synthesis pause near the completion of full-length chain elongation, not matched by a pro alpha 2 pause. The amount of labeled polysome-associated near-full length pro alpha 1 (I) chains increased in parallel with labeling time. After 24 h in culture -[(14)C-Pro], collagen synthesis ceased but unlabeled polysome-associated pro alpha1 chains were readily detected by SP1 .D8. Change to fresh culture medium +[(14)C-Pro] reinitiated synthesis and permitted tracing of the newly synthesized labeled pro a chains through the polysome and intracellular compartments. The secreted procollagen molecules had a 2:1 pro alpha 1 (1):pro alpha 2 (I) chain ratio but the polysome-bound peptides did not. Pulse-chase experiments showed that near-full length pro alpha 1 (I) chains remained bound to polysomes as long as 4 h after reinitiation of translation but there was no evidence for pro alpha 2 (I) chain accumulation. The hydroxylation inhibitor alpha, alpha'-dipyridyl, and triple-helix inhibitors cis-hydroxyproline and 3,4 dehydroproline had minimal effects on the buildup of polysome-associated pro al chains. The glycosylation inhibitor tunicamycin also failed to change the final pro alpha 1 chain pausing, but it did cause the appearance of several discrete lower molecular weight pro alpha 1-related polypeptides that could not be accounted for simply as the result of lack of N-linked glycosylation in the C-propeptide regions. Disulfide bond experiments showed that some of the paused nascent polysome-associated pro alpha 1 (I) chains were disulfide bonded. Thus, while synthesis of pro alpha 1 (I) and pro alpha 2 (I) chains proceeds in parallel within the same ER compartments, their elongation rates are not coordinated. Interactions leading to heterotrimer formation are a late event which may affect the rate of release of the completed pro alpha 1 (I) chain from the polysome. The release of completed nascent pro alpha 1 (I) chains from their polysomal complexes is regulated by a mechanism not operating in the synthesis of pro alpha 2 (I) chains. The pro alpha 1 (I) chain release process is not connected directly with hydroxylation, glycosylation or triple-helix formation.     

Upregulation of cathepsin D expression in the dedifferentiating salamander limb regenerates and enhancement of its expression by retinoic acid

The antiphospholipid antibodies (aPL) present in "antiphospholipid-protein syndrome and autoimmune disorders" are associated with thromboembolic episodes, such as venous and/or arterial thrombosis and fetal loss. Patients with antiphospholipid antibodies have, by definition, laboratory abnormalities in either coagulation assays or various solid phase immunoassays ELISA or radioimmunoassays (RIA). These assay systems were initially thought to detect antibodies against phospholipids. The problem was complicated when it was reported that phospholipid is not the sole antigen but only a part of it, the other contribution being due to b2-glycoprotein I (b2-GP I). More findings, demonstrate that the aPL are in fact anti-b2-GP I antibodies directed against a epitope which is expressed when b2-GP I is bound to anionic phospholipid or another suitable surface. Recent studies have demonstrated that antibodies related to lupus anticoagulant (LA) induce an anticoagulant activity in b2-GP I. Some of these LA require binding to phospholipid. However, not all LA require b2-GP I as a cofactor. Human prothrombin is an antigen for some LA IgG's. Finally, a subclassification of phospholipid-dependent coagulation test anticoagulants is described, there appear to be several subclasses of LA, and the clinical and laboratory criteria required to establish the diagnosis of antiphospholipid-protein syndrome is emphasised.