Extracellular matrix metabolism by chondrocytes 7. Evidence that L-glutamine is an essential amino acid for chondrocytes and other connective tissue cells

THe incorporation of [³H]glycine into acid-insoluble protein and of [³H]acetate into glysoaminoglycans by cultured chick chondrocytes was stimulated by the addition of L-glutamine to the incubation medium. The effect of exogenous L-glutamine on protein synthesis was studied further by examining changes in the sedimentation patterns on sucrose gardients of ribosomes isolated from chondrocytes incubated in presence and absence of L-glutamine. It was found that the absence of L-glutamine caused a disaggregation of poly-ribosomes that was reversed by the addition of this amino acid to the culture medium. No detectable glutamine synthetase activity could be measured in avian articular cartilage. These results indicate that L-glutamine is an essential amino acid for cartilage in that an extracellular supply of this amino acid is required for the maintenance of protein and glycosaminoglycan synthesis. A dependence on L-glutamine was also demonstrated for other avain connective tissues.     

The Isolation and Characterization of Glycosylated Phosphoproteins from Herring Fish Bones

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.     

Catalysis by dienelactone hydrolase: A variation on the protease mechanism

Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.     

Immunofluorescent localization of collagen types I,III and IV,fibronectin, laminin,and basement membrane proteoglycan in developing mouse skin

Summary The distribution of various extracellular matrix components was studied in frozen sections of embryonic (14–18 days) and early postnatal (birth and 4 days post parturn) dorsal mouse skin using monospecific antibodies and indirect immunofluorescence. Basement membrane zone components — type IV collagen, laminin and heparan sulphate proteoglycan — were found to be uniformly and unchangingly distributed along the dermal-epidermal junction. In contrast, the distribution of interstitial matrix components — types I and III collagen, and fibronectin — was heterogeneous and varied with the stages of hair development. Collagens became sparse and were eventually completely removed from the prospective dermal papilla and from a one-cell-thick sheath of dermal cells around hair buds. They remained absent from the dermal papilla throughout hair organogenesis. Fibronectin was always present around dermal papilla cells and was particularly abundant along the dermal-epidermal junction of hair rudiments, as well as underneath hair buds. In contrast, in interfollicular skin, collagens accumulated in increasing density, while fibronectin became progressively sparser. It thus appears that interstitial collagens and fibronectin are distributed in a manner which is related to hair morphogenesis. In morphogenetically active regions, collagen density is low, while that of fibronectin is high. Conversely, in histologically stabilized zones, collagen is abundant and fibronectin is sparse. This microheterogeneous distribution of interstitial collagens and of fibronectin might thus constitute part of the morphogenetic message that the dermis is known to transmit to the epidermis during the development of skin and of cutaneous appendages.     

Formation of Metallogenium-like structures by a manganese-oxiding fungus

Structures resembling Metallogenium spp. were observed in agar and in liquid cultures of a Mn-oxidizing basidiomycetous fungus only when Mn²⁺ was oxidized. Fungal viability was necessary for formation of the structures; Mn²⁺ concentration and the presence or absence of agar in the medium were important factors determining their morphology. Slide cultures revealed no identifiable cells in any stage of development. Fluorescent dyes that stained nucleic acids and polysaccharides in the fungal hyphae did not stain the Metallogenium-like structures. Likewise, Rhodamine 123, a fluorescent probe for membrane potential, stained fungal mitochondria, but did not stain the structures. Thin sections through the structures showed no biological membranes or other cellular features. Only the characteristic ultrastructure of biological Mn oxides were observed in serial thin sections. In agar, unfixed structures disappeared permanently during reduction of Mn oxides with hydroxylamine. Glutaraldehyde fixation stabilized these structures. Fixed structures lost most of their original phase density during reduction with hydroxylamine, but continuous microscopic observations showed that their phase density could be restored by staining with Coomassie blue. Structures that formed in liquid medium did not require stabilization with glutaraldehyde during reduction of Mn oxides. They, too, lost their original phase density during reduction with hydroxylamine; phase density could be restored by staining with cationic colloidal iron or Coomassie blue. The results suggest that the Metallogenium-like structures were formed as a result of Mn oxidation associated with exopolymers produced by the fungus.Non-standard abbreviations HEPES (N-hydroxyethylpiperazine-N-2-ethane sulfonic acid) - DAPI (4,6-diamidino-2-phenylindole) - PIPES (piperazine-N,N-bis[2-ethane sulfonic acid])     

Extracellular matrix influences hormone and protein production by human chorionic villi

Summary Increasing evidence confirms that the extracellular matrix greatly influences cell behaviour and function. Collagen and fibrin are in contact with trophoblast throughout pregnancy. To investigate whether these two matrices influence hormon production by the trophoblast, explants from first-trimester chorionic villi were cultured for up to 30 days either a) in medium with agitation, b) embedded in type-I collagen (three-dimensional gels), or c) embedded in fibrin (three-dimensional gels). The supernatant culture medium was changed every 48 h and tested by radioimmunoassay for hCG, progesterone and pregnancy-associated plasma protein A. In addition, after 3, 7, 15, and 30 days of culture villi were fixed and studied by light and electron microscopy. Embedding in the extracellular matrix showed higher and longer-lasting production rates of all measured products and superior structural preservation as compared to cultures with agitation. Collagen matrix proved to be superior to fibrin. As established by several tests, this difference was neither due to thrombin used to polymerize fibrinogen, nor to differences in the diffusion rates through the two different matrices used. We conclude that extracellular matrix, particularly collagen, influences the synthesis of trophoblastic products. Embedding of the villous explants in three-dimensional gels constitutes a new method for long-term cultures of chorionic villi.This study was presented at the workshop Placental-and decidual-specific protein synthesis and secretion: regulation, role and interaction, Zemun, Belgrade, Yugoslavia, 19–20 May, 1988 (Bischof and Castellucci 1988; see also J. Aplin 1989), and at the 11th Rochester Trophoblast Conference, Rochester, N.Y. USA, 9–12 October 1988 (Castellucci et al. 1988)     

Immunocytochemical localization of lipophorins in the flight muscles of the migratory locust (Locusts migratoria) at rest and during flight

Summary Locust lipoproteins (lipophorins) were localized by indirect immunofluorescence- and immunogold labelling in cryosections of dorsolongitudinal flight muscles. Immunolabelling was performed with monoclonal antibodies against apolipoprotein epitopes that are exposed at the surfaces of the lipophorin particles. Both at rest and during flight, lipophorins were located only in the wider spaces of the extracellular matrix, in the basement membranes of the individual muscle fibers and in the extracellular spaces that surround interfibrillar tracheoles. No internalization of lipophorins by the flight muscle cells was observed. Our results indicate that the unloading of lipophorins at the flight muscles is an extracellular event. Similarities with the vertebrate system of chylomicron and very-low-density lipoprotein degradation are discussed.     

Extracellular enzyme localization during interspecific fungal interactions

Abstract Confronted colonies of Phlebia radiata, P. rufa, Coriolus versicolor, Stereum hirsutum, Phanerochaete velutina and Hypholoma fasciculare showed spatially and temporally heterogeneous patterns of loccase-α-naphthol and peroxidase activities. These activities were coincident in axenic cultures. but were not always so during interaction. Confrontation between species resulted in induction of phenoloxidase activities, even within coenocytic colony regions of Phlebia species which were normally void of such activities in axenic culture. These events resulted in restriction of C. versicolor growth during interaction with P. rufa.     

Lipolytic and peroxidative enzyme expressions in cultured potato tuber cells

Potato cells (cv. Norchip) were cultured from tuber parenchymal tissue and subcultured to dissociate and habituate the despecialized cells. After several subculturings on a minimal nutrient media, this line of cells demonstrated repeatable physical growth profiles for dry weight (DW), fresh weight (FW) and protein. Two enzymes of plant lipid metabolism were investigated, lipolytic acyl hydrolase (LAH) and lipoxygenase (LOX), which respectively liberate and peroxidize fatty acids from lipid in cellular membranes. LAH, measured as p-nitrophenyl palmitate hydrolase, was present in this line of cells in easily detectable amounts (317 units g^-1 DW) albeit much lower than that found in mother tuber (9878 units g^-1 DW). The presence of LAH in this line is significant because LAH isozymes are often described as storage proteins, yet activity per gram fresh weight in these unorganized cells is reasonably constant until culture growth exits the linear phase. However, LOX, the most active free fatty acid metabolizing enzyme in potato tubers (89,800 units g^-1 DW), was not detectable in this line of callus or suspension cultured cells. The absence of LOX activity in this line of cells was verified by a number of assay approaches and was confirmed by activity staining of extracted enzymes separated in polyacrylamide gels. The absence of LOX in these cultured cells is especially important in determining the functions of this lipid peroxidation system and how it may be genetically regulated.Mention of company or trade name does not imply endorsement by the United States Department of Agriculture over others not named.A laboratory cooperatively operated by the Midwest Area, Agricultural Research Service, U.S. Department of Agriculture, The Minnesota Agricultural Experiment Station, the North Dakota Agrcultural Experiment Station, and the Red River Valley Potato Grower's Association.     

Dihydrotetrabenazine Binding and Monoamine Uptake in Mouse Brain Regions

The objective of the present study was to estimate extracellular pH (pHe) and intracellular pH (pHi) during near-complete forebrain ischemia in the rat, and to evaluate the relative importance of lactic acidosis and rise in tissue Pco2 (Ptco2) in causing pHe and pHi to fall. The animals, which were ventilated, normoxic, normocapnic, and normothermic, were subjected to 15 min of ischemia, either without or with 30-60 min of recirculation. Ptco2 was measured with a tissue electrode, pHe with a double-barrel liquid ion-exchanger microelectrode, changes in extracellular fluid (ECF) volume by impedance measurements, tissue CO2 content by a microdiffusion technique, and labile tissue metabolites by enzymatic fluorometric methods. Ischemia caused Ptco2 to rise to between 95 and 190 mm Hg (mean 149 mm Hg), and pHe to fall by 0.45-1.05 units (mean 0.70 units). During recovery, Ptco2 normalized within 5 min and pHe after 15-30 min. During ischemia, high-energy phosphates were depleted and tissue lactate content increased to 15 mumol X g-1. The total CO2 content (Tco2) was minimally or moderately reduced (normal, 11.9 mumol X g-1; range of ischemic values, 7.9-12.1 mumol X g-1), this range probably reflecting variable amounts of remaining blood flow. Impedance measurements demonstrated that ECF volume during ischemia was reduced to 55% of control, with gradual normalization during the first 15-30 min of recirculation. From values for Ptco2, Tco2, [HCO3-]e, and ECF volume, [HCO3-]i and pHi could be calculated. These values pertain to an idealized homogeneous intracellular compartment, and the methods used cannot detect whether different intracellular compartments diverge in their acid-base responses.(ABSTRACT TRUNCATED AT 250 WORDS)