Resistin release by human adipose tissue explants in primary culture

Resistin, also known as Fizz3 or ADSF, is a protein found in murine adipose tissue and inflammatory lung exudates. The present studies found that resistin was released by explants of human adipose tissue but the release was quite variable ranging from 3 to 158 ng/g over 48 h. The release of resistin was 250% greater by explants of omental than by explants of human subcutaneous abdominal adipose tissue. Resistin release by adipocytes was negligible as compared to that by the non-fat cells of adipose tissue. Leptin formation by adipocytes was 8-fold greater than its formation by the non-fat cells, while the formation of PAI-1 by adipocytes was 38% of that by the non-fat cells. The conversion of glucose to lactate as well as the formation of PGE(2) and IL-8 was approximately 15% of that by the non-fat cells. In contrast the release of IL-6 and IL-1beta by adipocytes was 4-7% of that by the non-fat cells while the formation of resistin and IL-10 by adipocytes was 2% of that by non-fat cells. The release of adiponectin by explants ranged from 1000 to 5000 ng/g over 48 h but did not correlate with that of resistin. The present data suggest that resistin release by explants of human adipose tissue in primary culture is largely derived from the non-fat cells present in the explants.     

Thrombolytic properties of an inactive proenzyme form of human urokinase secreted from human kidney cells

The relative fibrin-binding, fibrinolytic and fibrinogenolytic properties of single-chain pro-urokinase, an inactive proenzyme form of human urokinase purified from cultured human kidney cells, and urokinase were compared. The affinity of single-chain pro-urokinase for fibrin was much higher than that of urokinase. In Vitro thrombolytic studies showed that single-chain pro-urokinase is approximately three times more potent in fibrinolysis than urokinase and that it does not degrade fibrinogen in the plasma at a concentration, at which complete plasma clot lysis takes place; whereas, urokinase extensively degrades the fibrinogen in the plasma. These specific, potent thrombolytic properties of single-chain pro-urokinase seem to be due to its high affinity for fibrin and to its conversion from the inactive single-chain form to the active two-chain form on the thrombus by the catalytic amount of plasmin generated during coagulation. This single-chain pro-urokinase obtained from human kidney cells by tissue culture should prove advantageous than urokinase in thrombolytic therapy.     

Synthesis of collagenase and collagenase inhibitors by osteoblast-like cells in culture

A rat osteosarcoma cell clone (ROS 17/2), and osteoblast-enriched populations from rat calvaria cultured in the presence of concanavalin A, have been shown to produce latent collagenase and collagenase inhibitors. The enzymes and inhibitor activities from the ROS 17/2 cells were concentrated by ammonium sulphate precipitation and separated by gel filtration on AcA 54 resin. The size of the latent collagenase (Mr approximately equal to 58000) was reduced on conversion to active enzyme (Mr approximately equal to 48000) by p-aminophenylmercuric acetate. Latent and active forms of gelatinase activity, similar in size to the corresponding forms of collagenase, were also resolved. The collagenase inhibitor activity, which was sensitive to organomercurials, was recovered in two peaks (Mr approximately equal to 68000 and 30000). The active collagenase cleaved interstitial collagens (type I = III greater than II) producing typical 3/4 and 1/4 fragments. This activity was inhibited by the metal ion chelators ethylenediaminetetraacetic acid and o-phenanthroline. Additional specific cleavages of native collagen were also observed which, from the susceptibility of this activity to phenylmethylsulphonyl fluoride, leupeptin and antipain, suggested the presence of a second collagenolytic enzyme. This synthesis of collagenolytic enzymes by these osteoblast-like cells suggests that individual osteoblasts, like fibroblasts, are capable of both synthesizing and degrading their respective organic matrices in vivo.     

The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen.

1. A latent neutral proteinase was found in culture media of mouse bone explants. Its accumulation during the cultures is closely parallel to that of procollagenase; both require the presence of heparin in the media. 2. Latent neutral proteinase was activated by several treatments of the media known to activate procollagenase, such as limited proteolysis by trypsin, chymotrypsin, plasmin or kallikrein, dialysis against 3 M-NaSCN at 4 degrees C and prolonged preincubation at 25 degrees C. Its activation often followed that of the procollagenase present in the same media. 3. Activation of neutral proteinase (as does that of procollagenase) by trypsin or plasmin involved two successive steps: the activation of a latent endogenous activator present in the media followed by the activation of neutral proteinase itself by that activator. 4. The proteinase degrades cartilage proteoglycans, denatured collagen (Azocoll) and casein at neutral pH; it is inhibited by EDTA, cysteine or serum. Collagenase is not inhibited by casein or Azocoll and is less resistant to heat or to trypsin than is the proteinase. Partial separation of the two enzymes was achieved by gel filtration of the media but not by fractional (NH4)2SO4 precipitation, by ion exchange or by affinity chromatography on Sepharose-collagen. These fractionations did not activate latent enzymes. 5. Trypsin activation decreases the molecular weight of both latent enzymes (60 000-70 000) by 20 000-30 000, as determined by gel filtration of media after removal of heparin. 6. The latency of both enzymes could be due either to a zymogen or to an enzyme-inhibitor complex. A thermostable inhibitor of both enzymes was found in some media. However, combinations of either enzyme with that inhibitor were not reactivated by trypsin, indicating that this inhibitor is unlikely to be the cause of the latency.     

A new synthetic inhibitor of mammalian tissue collagenase inhibits bone resorption in culture

A specific and potent synthetic inhibitor of mammalian tissue collagenase and related metallo-proteinases inhibits the collagen matrix resorption induced by parathyroid hormone (PTH) in cultured embryonic mouse calvaria. The inhibition is reversible, dose-dependent and virtually complete at 50 microM inhibitor concentration whereas that due to a less potent stereoisomer is much weaker. The PTH-enhanced secretion of calvarial lysosomal enzymes and the small spontaneous leakage of lactate dehydrogenase are not affected by the inhibitor. These results suggest that collagenase plays a critical role in bone resorption. Its role is discussed in relation to that of cysteine-proteinases that have also been implicated in this process.     

A complex of collagenase with low molecular weight inhibitors in the culture medium of embryonic chick skin explants.

Collagenase inhibitors with molecular weights of about 6,000 and 12,000 were isolated from latent chick skin collagenase treated with 3 M NaI and from the culture medium of embryonic skin explants. It is suggested that these inhibitors, which are possibly derived from connective tissue macromolecule metabolites, are candidates for regulating factors of collagenase activity in vivo.     

Further differentiation of early neural tube explants in organ culture as studied by electron microscopy

Summary Chick embryo spinal cord has been explanted at 2-day stages, when few or no cells have formed axons, and cultured organotypically with adjacent tissues or isolated from all other tissues.Relatively mature nerve cells and glial cells, neurites and synapses could be seen in electron micrographs of cultures maintained for three to four weeks. Histological organization and some aspects of cell differentiation differed in the two types of cultures. Ependymal cells and randomly arranged cells, possibly modified glia, were seen only in cultures of neural tube with adjacent tissue; neurons and macroglia seemed to be more numerous in cultures of isolated neural tube.The development of characteristic cells, with variations according to culture conditions, provides the opportunity for further study of factors controlling patterns of proliferation and differentiation in the central nervous system from very early to advanced stages.This work was supported by a grant from the United States Public Health Service (5 ROI NB 0637).The author wishes to thank Miss Geraldine McTiernan for her competent technical assistance.     

Chick bone collagenase inhibitor and latency of collagenase

Collagenase and collagenase inhibitor were isolated from the culture fluid of embryonic chick bone. The inhibitor, separated as a high molecular weight aggregate (160,000–200,000 daltons) during gel filtration in 1M NaCl, dissociated in 6M urea to species of approx 25,000 daltons. The inhibition of collagenase activity by the addition of inhibitor was not reversed by the addition of trypsin or p-aminophenylmercuric acetate. However, isolated inhibitor alone was inactivated by treatment with either trypsin or p-aminophenylmercuric acetate. The results suggest that the latent form of chick bone collagenase is a proenzyme which converts into an active form without a detectable change in molecular weight and that this occurs after the inactivation of collagenase inhibitor.     

Relationship between bone resorption and lysosomal enzyme release as demonstrated by the effect of 1α-hydroxy-vitamin D-3 in an organ culture system

The effect of 1α-hydroxy-vitamin D-3 on the release of calcium (⁴⁰Ca, ⁴⁵Ca), inorganic phosphate and lysosomal enzymes, on glucose consumption and lactate production was studied in a bone organ culture system using half calvaria from 6–7-day-old mice. 1α-Hydroxy-vitamin D-3 stimulated the mobilization of minerals and increased the release of β-glucuronidase, β-N-acetylglucosaminidase and acid phosphatase, while no effect on the release of lactate dehydrogenase was seen. 1α-Hydroxy-vitamin D-3 also caused a significant increase in the total activities of acid phosphatase in the bones after culture, indicating increased enzyme synthesis. The stimulatory effect of the release of P_i and β-glucuronidase was also obtained after a temporary exposure to 1α-hydroxy-vitamin D-3. The stimulation by 1α-hydroxy-vitamin D-3 on the release of Ca²⁺, P_i and β-glucuronidase was suppressed by a protein synthesis inhibitor cycloheximide. No effect by 1α-hydroxy-vitamin D-3 on glucose consumption and lactate production was registered, suggesting that increased mineral mobilization does not require increased lactate production. It is concluded that although the data in the present paper do not prove a cause-and-effect relationship between lysosomal enzyme release and bone resorption, they give further support to the concept that the processes are intimately associated.     

Apolipoprotein C-II synthesis as studied in explants of rat liver in culture

Adult male Sprague-Dawley rats (200-250 g) were used to study apolipoprotein C-II synthesis and secretion. Liver slices were prepared and incubated in RPMI 1629-medium (tissue amount and incubation time studies) and in Minimum Essential Medium (Eagle) with Earle's salts (hormone experiments). Incubation was performed in scintillation vials in a 95% O2-5% CO2 atmosphere, at 37 degrees C from 1 to 21 hr (2 and 4 hr with hormones). The hormones used and their amounts per millilitre medium were: oestradiol-17 beta 0.1 microgram, progesterone 3.0 micrograms and dexamethasone 1.5 micrograms. Apolipoprotein C-II was determined by specific double immunoprecipitation technique and TCA-insoluble protein fraction represented total protein. Optimal tissue amount was 100 mg/vial and the results show that liver slices quickly secrete the newly synthesized apo C-II (also total protein) into the surrounding medium. There were only minor differences between apo C-II values with the hormones used. The portion of apo C-II synthesis from total protein synthesis was 0.47-1.50%. After 4 hr incubation the [3H]leucine incorporation was almost equal for controls and hormone treated slices.     

Osteopetrotic (grey-lethal) bone produces collagenase and TIMP in organ culture: regulation by vitamin A

Evidence has recently accumulated suggesting that osteoblasts play a direct role in bone resorption by producing collagenase. In this paper we describe studies carried out with explants of bone from osteopetrotic grey lethal (gl/gl) mice and show that despite the lack of osteoclastic activity the production of both active and latent collagenase and its specific inhibitor TIMP (tissue inhibitor of metalloproteinases) is similar to that of normal bones. Synthesis of collagenase was stimulated by the bone resorptive agent vitamin A (retinol); concomitantly, TIMP levels fell to zero and active enzyme was detected in the culture medium. This work supports the view that bone collagenase is produced by cells other than osteoclasts, since the response of the osteoblastic population to resorptive signals appears normal.     

Synthesis of a collagenase inhibitor by gingival fibroblasts in culture

Human collagenase was inhibited by test solutions of human gingival fibroblast culture media. The fibroblast-derived collagenase inhibitor was only slightly affected by 10 micrograms trypsin but was inactivated with 100 micrograms trypsin. The chaotropic agent KSCN (3 M) completely inactivated the inhibitor, whereas the thiol-blocking reagent, p-aminophenylmercuric acetate, partially inactivated the inhibitor. Inhibitory activity was retained at 60 degrees C but was abolished at 100 degrees C. Following ammonium sulfate fractionation, the fibroblast inhibitor was recovered in the supernatant at concentrations of at least 70% saturation. It is suggested that collagenase latency in soft connective tissues may derive from a collagenase-inhibitor complex formed by interaction of collagenase and a fibroblast-derived inhibitor.     

Monensin inhibits collagenase production in osteoblastic cell cultures and also inhibits both collagenase release and bone resorption in mouse calvaria cultures

Monensin, a monovalent cation ionophore, inhibited collagenase production in mouse osteoblast-rich bone cell and clonal osteogenic cell cultures. Inhibition of parathyroid hormone-stimulated bone resorption by monensin was also studied in calvaria cultures. Collagenase activity levels in the medium decreased concomitantly with the inhibition of bone resorption by monensin, indicating that monensin inhibited bone resorption by blocking collagenase secretion from osteoblasts in bone explants.