Norrie disease protein (norrin) forms disulfide-linked oligomers associated with the extracellular matrix

COS-7 cells transfected with a DNA construct encoding the 133 amino acids in norrin plus six histidine residues at its carboxyl terminus were pulse-labeled with [35S]cysteine, and the labeled norrin was examined in cell lysates, the medium, and the extracellular matrix. SDS-gel electrophoresis under reducing conditions showed that the norrin expressed had an apparent Mr = 14,000 and was present only in cell lysates and the extracellular matrix. Under nonreducing conditions, most of the norrin in the extracellular matrix was oligomers that contained up to approximately 20 monomers. One of the major extracellular species of norrin under reducing conditions after cross-linking of norrin oligomers with bis(sulfosuccinimidyl)suberate had an apparent Mr = 28,000, consistent with covalent cross-linked dimers. Thus the covalently cross-linked dimers are key structural components of norrin oligomers. By site-directed mutagenesis, the codon for half-cystine 95 in norrin was changed to one encoding alanine. The norrin C95A found in the extracellular matrix of cells transfected with this mutant was the size of dimers, indicating that half-cystine 95 is involved in oligomer formation. The corresponding half-cystine residue in human prepro-von Willebrand factor is also involved in interchain disulfide bond formation, which is consistent with the sequence identity of the half-cystine residues in norrin and part of the half-cystine residues in a disulfide-rich domain of von Willebrand factor. Replacement of valine at residue 60 in norrin by glutamic acid, a mutation found in humans with a severe type of Norrie disease, results in a considerable reduction (50%) in the amount of norrin in the extracellular matrix of transfected COS-7 cells. Replacement of arginine at residue 121 by glutamine, which is associated with a less severe type of Norrie disease, results in a reduction in the amount of norrin R121Q in the extracellular matrix (26%). These studies suggest that norrin is a secreted protein that forms disulfide-bonded oligomers that are associated with the extracellular matrix upon secretion from cells. Moreover, the disulfide-rich motif of norrin and prepro-von Willebrand factor promotes interchain disulfide bond formation among polypeptides in which it is found.     

Primary structure of matrilin-3, a new member of a family of extracellular matrix proteins related to cartilage matrix protein (matrilin-1) and von Willebrand factor

A mouse cDNA encoding for matrilin-3, the third member of the novel matrilin family of extracellular matrix proteins, was cloned. The protein precursor of 481 amino acids consists of a putative signal peptide, a short positively charged sequence, a single vWFA-like domain followed by four epidermal growth factor-like modules and a potential coiled-coil alpha-helical oligomerization domain at the C-terminus. It is the smallest member of the matrilin family with a predicted Mr of the mature protein of 48 902. The primary structure of a C-terminal portion of 310 amino acids of the human matrilin-3 was determined and showed a sequence identity to the mouse matrilin-3 of 84.8%. Northern blot hybridization of mouse matrilin-3 mRNA showed a 2.9 kb mRNA expressed in sternum, femur and trachea and indicates a cartilage-specific expression.     

Distribution and subcellular immunolocalization of 1,25-dihydroxyvitamin D3 receptors in rat epiphyseal cartilage

The distribution and subcellular localization of the 1,25-dihydroxyvitamin D3 receptor (VDR) in the epiphyseal cartilage of normal weaning rats were examined immunocytochemically at the light and electron microscope level using a monoclonal anti-VDR antibody (9A7 gamma). VDR immunoreactivity was detected in the nuclei of chondrocytes in all zones of the epiphyseal plate cartilage from the resting to calcifying chondrocytes, and at much lower concentrations, in the cytoplasms. Perichondrial mesenchymal cells contained no VDR immunoreactivity. VDR immunoreactivity developed in the nuclei of cells in the lateral margin area as they acquired the chondroblast phenotype. VDR immunoreactivity was also found over the nucleoli of chondrocytes in all cells zones of the epiphyseal plate and appeared in the nucleoli of the cells in the lateral margin area before immunostaining of the nuclei, as the mesenchymal cells differentiated into chondroblasts. Electron microscopy showed that the immunoreactivity for 1,25(OH)2D3 receptor, indicated by gold particles, was associated with scattered clumps of compact chromatin and small clumps of dispersed chromatin. But the nuclei immunostaining patterns before and after mitosis were different in proliferative chondrocytes. The heterochromatin along the nuclear envelope was immunonegative in interphase chondrocytes, but there was VDR immunostaining over the rim of the perinuclear chromatin just after mitosis. In the nucleoli, the dense fibrillar component was immunostained, but the fibrillar centers and the perinuclear chromatin were not. This distribution of VDR immunoreactivity suggests that the hormone is directly involved in differentiation, proliferation and maturation of cartilage cells, and also with extracellular calcification in epiphyseal cartilage. The presence of immunoreactive VDR receptors in nucleoli of chondrocytes, particularly the fibrillar component, suggests that 1,25(OH)2D3 may be involved in regulation of ribosomal genes.     

Reaction of hypochlorous acid with bovine nasal cartilage comparison to pig articular cartilage

The action of sodium hypochlorite (NaOCl) on bovine nasal cartilage was studied by proton nuclear magnetic resonance (1H-NMR) spectroscopy in order to model degradation processes of cartilage caused by neutrophil-derived hypochlorous acid. Nasal cartilage was chosen as a mean of comparison because it differs from articular cartilage in its composition. It contains some more proteoglycans, i.e. polymeric carbohydrates and less collagen than articular cartilage. This is important for studying the influence of hypochlorous acid on cartilage components (collagen and polysaccharides). Cartilage samples were incubated at 37 degrees C with phosphate buffer in the presence or absence of NaOCl. Supernatants were collected and assayed by NMR-spectroscopy. In the presence of pure phosphate buffer, the supernatants of bovine nasal cartilage were less rich in low molecular mass metabolites (e.g. amino acids, lactate) than articular cartilage. However, intense signals for highly mobile N acetyl groups of cartilage polysaccharides were detectable in nasal cartilage. NaOCl caused an increase in signals for acetate and formiate. Signals for N-acetyl groups rose only during the first 25 minutes of incubation with NaOCl. Then, their concentration decreased markedly. These changes were related to an enhanced release of chondroitinsulfate from nasal cartilage.     

25-hydroxyvitamin D: autoradiographic evidence of sites of action in epiphyseal cartilage and bone

Tritiated 25-hydroxyvitamin D3 was administered to growing rats to morphologically documents its sites of action. Highly selective incorporation occurred in epiphyseal hypertrophic cells, epiphyseal matrix, osteoid, osteoblasts, and osteocytes of metaphyseal bone spicules. The labeled metabolite appeared in chondrocytic lacunar matrix coincident with hypertrophic cell death as evidenced by histological examination. The tritiated 25-hydroxyvitamin D3 became localized only in areas of active mineralization.     

Calnexin associates with monomeric and oligomeric (disulfide-linked) CD3delta proteins in murine T lymphocytes

The antigen-binding receptor expressed on most T lymphocytes consists of disulfide-linked clonotypic alphabeta heterodimers noncovalently associated with monomeric CD3gamma,delta,epsilon proteins and disulfide-linked zeta zeta homodimers, collectively referred to as the T cell antigen receptor (TCR) complex. Here, we examined and compared the disulfide linkage status of newly synthesized TCR proteins in murine CD4(+)CD8(+) thymocytes and splenic T cells. These studies demonstrate that CD3delta proteins exist as both monomeric and oligomeric (disulfide-linked) species that differentially assemble with CD3epsilon subunits in CD4(+)CD8(+) thymocytes and splenic T cells. Interestingly, unlike previous results on glucose trimming and TCR assembly of CD3delta proteins in splenic T cells (Van Leeuwen, J. E. M., and K. P. Kearse (1996) J. Biol. Chem. 271, 9660-9665), we found that glucose residues were not invariably removed from CD3delta glycoproteins prior to their assembly with CD3epsilon subunits in CD4(+)CD8(+) thymocytes. Finally, these studies show that calnexin associates with both monomeric and disulfide-linked CD3delta proteins in murine T cells. The data in the current report demonstrate that CD3delta proteins exist as both monomeric and disulfide-linked molecules in murine T cells that differentially associate with partner TCR chains in CD4(+)CD8(+) thymocytes and splenic T cells. These results are consistent with the concept that folding and assembly of CD3delta proteins is a function of their oxidation state.     

Multiple forms of endocytosis in bovine adrenal chromaffin cells

BACKGROUND: Halothane is a potent dilator of cerebral arteries. The predominant site of cerebrovascular resistance is thought to be intracerebral arterioles, and the effects of halothane on these vessels were not previously examined. This study compared the effects of halothane with those of the vasodilator and nitric oxide donor, sodium nitroprusside, on intraparenchymal microvessel responsiveness in a brain slice preparation. METHODS: Anesthetized Sprague-Dawley rats underwent thoracotomy and intracardiac perfusion and then were decapitated. Hippocampal brain slices were prepared and placed in a perfusion/recording chamber and superfused with artificial cerebrospinal fluid. An arteriole was located within the brain parenchyma and its diameter was monitored with videomicroscopy before, during, and after various concentrations of halothane or sodium nitroprusside were equilibrated in the perfusate. All vessels were preconstricted with prostaglandin F2 alpha before halothane or sodium nitroprusside treatment. An observer blinded to treatment analyzed vessel diameter changes with a computerized videomicrometer. RESULTS: Baseline microvessel diameter was 18 +/- 2 microns in the halothane group (n = 14) and 15 +/- 1 microns in the sodium nitroprusside group (n = 15). Prostaglandin F2 alpha (0.5 micron) preconstricted vessels by approximately 15% from resting diameter in both groups. Halothane significantly and dose dependently dilated intracerebral microvessels by 54% +/- 6%, 74% +/- 8%, 108% +/- 13%, and 132% +/- 7% (normalized to the preconstricted diameter) at 0.5%, 1.0%, and 2.5% halothane, respectively. This dilatation corresponds to a decrease in a calculated index of cerebrovascular resistance index of up to 117% +/- 2% at 2.5% halothane. Sodium nitroprusside, in concentrations ranging from 10(-8) to 10(-3)M, also dose dependently dilated these intraparenchymal vessels by 129% +/- 7% at the highest concentration. These alterations in microvessel diameter corresponded to a decrease in the cerebrovascular resistance index of up to 116 +/- 4% for the largest dose. CONCLUSIONS: Halothane produces dose-dependent vasodilatation of intraparenchymal cerebral microvessels, thus predicting marked decreases in cerebrovascular resistance in this in vitro brain slice preparation. The effects of halothane on these cerebral microvessels are similar to those of the potent vasodilator sodium nitroprusside. These findings suggest that direct effects of halathane on cerebral microvessels diameter contribute substantially to alterations in cerebrovascular resistance and flow produced by this agent.     

Vibrational spectra of glycine, alanine and proline oligomers in relation to their polymeric forms

The cutaneous recording of the gastric electrical activity according to the modified method of Sobakin was utilized. In fasting individuals changes in potential occured between 2 and 5 per min., in most of the cases after standard breakfast the frequency increased while lower amplitudes appeared. The frequency of electrical waves from the duodenum increased remarkably. It could be demonstrated that the peristalsis recorded by gastroscopy was synchronous with the electrogastrographic potentials. The applicability of the method for clinical diagnostic purposes is limited at the present time, because a "normal" electrogastrogram has not yet been established. Specific changes of the electrogastrogram have not been observed in various gastric diseases, which may be verified by routine diagnostic procedures.     

Detection of bovine herpesvirus 1 (BHV-1) genomic sequences in bovine semen inoculated with BHV-1 by polymerase chain reaction

Polymerase chain reaction (PCR) technique was applied to detect BHV-1 in bovine semen inoculated with BHV-1. The technique was found to be 10(6) times more sensitive than a non-isotopic dot-blot hybridization method in detecting viral genomic DNA. Of the three primer pairs used, the one chosen from glycoprotein gC appeared to be most sensitive as it could detect up to 0.01 TCID50 of BHV-1 in the semen. The technique could be useful in screening breeding bulls or samples of frozen semen prior to use in artificial insemination.     

Identification of alpha-carboxamidated and carboxy-terminal glycine forms of peptides in bovine hypothalamus, bovine pituitary and porcine heart extracts

Two chemical assays have been developed for identifying and quantifying peptides which either could be biologically active by virtue of their alpha-carboxamidation or could be substrates for peptidylglycine alpha-amidating mono-oxygenase. The first assay is specific for the alpha-carboxamide of peptides. Using bis[trifluoroacetoxy]iodobenzene, the alpha-carboxamide was converted via a Hoffman reaction into a primary amine, which was then quantified by ninhydrin. The second assay is specific for glycine at the carboxy-terminus of a peptide. Glycine at the carboxy-terminus was derivatized to form 2-thiohydantoin, which was then separated and quantified by reverse phase HPLC. These assays were used to detect peptides in HPLC-separated extracts of bovine hypothalamus, bovine anterior lobe pituitary and porcine heart which may be of biological interest.     

Characterization of the protein and glycan moieties in different forms of bovine lactoferrin

The BrCN cleavage of lactoferrin-a or -b (LF-a or LF-b) led to the observation of four fragments by SDS-PAGE, whose molecular masses were 77, 58, 52, and 30 kDas, or 74, 54, 47, and 30 kDas, respectively. N-Terminal amino acid sequence analyses show that the sequences of 58, 52, and 30 kDa fragments (residues 64-471, 130-471, and 472-689) of LF-a coincide with those of the 54, 47, and 30 kDa fragments of LF-b. respectively. All these fragments, which were positive by PAS staining, were not stained after being treated with glycopeptidase F. This treatment changed the 58 and 52 kDa fragments of LF-a to the 54 and 47 kDa fragments, respectively, whose molecular masses were the same as those of the treated fragments of LF-b. The 58 and 52 kDa fragments of LF-a bound to the lectin, Ricinus communis agglutinin, while the 54 and 47 kDa fragments of LF-b hardly bound to it.     

Interactions of lactone, carboxylate and self-aggregated forms of camptothecin with human and bovine serum albumins

Highly degenerate primers to conserved regions of the eukaryotic phosphoinositol-specific phospholipase C (PLC) were used to amplify fragments of plant PLCs from Arabidopsis thaliana genomic DNA. Eight completely different fragment sequences that showed high homology to PLCs of both animals and plants were isolated. The variation between these putative PLCs was high and suggests that, like animals, plants have multiple isoforms of PLC. Using one of the PCR clones, we isolated a corresponding full-length Arabidopsis PLC gene (ATHATPLC1G), and sequence analysis indicated that it was most like a delta-type PLC. This gene is 2.5 kb and contains seven introns, all but one of which has intron/exon border sequences that conform to the Arabidopsis consensus. The structural complexity of the gene is relatively simple compared to mammalian beta-type PLCs that can be 15 kb long with up to 30 introns. The plant gene is a single copy and was mapped to four Arabidopsis YACs, one located on chromosome 2. The promoter region contained two TATA-like elements at -43 and -185 and other putative regulatory elements that suggest that this PLC is hormonally regulated. This is the first plant PLC gene and the first delta type-PLC gene from a higher organism to be sequenced.     

Local disappearance of epiphyseal growth plates in rats with hypervitaminosis A

Epiphyseal growth plates of proximal tibiae in rats with high doses of vitamin A (V-A) were observed. Of 4 groups, each consisting of 5 rats, three groups were given V-A at doses, IU/100 g by body weight/day, of 50,000, 100,000, and 150,000, respectively. The other group rats were given no V-A (control). Rats were administered V-A for the 5 days from 4 weeks after birth and sacrificed at 12 weeks after birth. Three rats of the 150,000 IU group died during the period of observation. The decalcified sections were stained with hematoxylin-eosin or toluidine blue. In the ground sections, microradiography, backscattered electron imaging, and energy-dispersive X-ray microanalysis were performed. These observations suggest that the local disappearance of epiphyseal growth plates under high doses of V-A goes in the order of the increased doses through the process of (1) calcified cartilage areae appearing in the resting cell zone, (2) some of the calcified areae extending in the growth plate towards the diaphysial side, (3) bone tissue replacing the calcified areae, and (4) the local disappearing of the growth plate. Such a local disappearance may be formed in the stressed proximal regions of tibiae.     

Identification of chondromodulin I as a novel endothelial cell growth inhibitor. Purification and its localization in the avascular zone of epiphyseal cartilage

Cartilage is unique among tissues of mesenchymal origin in that it is resistant to vascular invasion due to an intrinsic angiogenic inhibitor. During endochondral bone formation, however, calcified cartilage formed in the center of the cartilaginous bone rudiment allows vascular invasion, which initiates the replacement of cartilage by bone. The transition of cartilage from the angioresistant to the angiogenic status thus plays a key role in bone formation. However, the molecular basis of this phenotypic transition of cartilage has been obscure. We report here purification of an endothelial cell growth inhibitor from a guanidine extract of bovine epiphyseal cartilage. The N-terminal amino acid sequence indicated that the inhibitor was identical to chondromodulin I (ChM-I), a cartilage-specific growth-modulating factor. Purified ChM-I inhibited DNA synthesis and proliferation of vascular endothelial cells as well as tube morphogenesis in vitro. Expression of ChM-I cDNA in COS7 cells indicated that mature ChM-I molecules were secreted from the cells after post-translational modifications and cleavage from the transmembrane precursor at the predicted processing signal. Recombinant ChM-I stimulated DNA synthesis and proteoglycan synthesis of cultured growth plate chondrocytes, but inhibited tube morphogenesis of endothelial cells. In situ hybridization and immunohistochemical studies indicated that ChM-I is specifically expressed in the avascular zone of cartilage in developing bone, but not present in calcifying cartilage. These results suggest a regulatory role of ChM-I in vascular invasion during endochondral bone formation.     

Ultrastructural localization of the C-propeptide released from type II procollagen in fetal bovine growth plate cartilage

We used immunochemical and immunoelectron gold techniques to determine whether the C-propeptide previously identified in the matrix of endochondral cartilage (CPII) was still a part of the Type 11 procollagen molecule or had been released from it. Guanidinium hydrochloride extraction, followed by SDS-PAGE and Western blotting techniques and immunoelectron localization, revealed that predominantly only the released form (hereafter referred to as released CPII) was detected. The ultrastructural distribution of this CPII was examined with affinity-purified antibodies and with immunogold or immunoperoxidase localization techniques in the presence or absence of embedding resins. These methods yielded similar results. Although no significant amount of this CPII was retained in the matrix after guanidinium hydrochloride extraction, it was present in two recognizable sites under normal conditions, i.e., locally concentrated in a random association with collagen fibrils in the nonmineralized matrix and mainly concentrated in interfibrillar mineralizing sites in the mineralized matrix. These results suggest that the C-propeptide that has been released from Type II procollagen associates with collagen fibrils and then preferentially associates with mineralizing sites when these form in the endochondral cartilage. The significance of this preference for mineral is not known but may have something to do with its high affinity for hydroxyapatite.     

Abortion in heifers inoculated with a thymidine kinase-negative recombinant of bovine herpesvirus 1

The Copper isolate of bovine herpesvirus 1 (BHV-1) was used to produce a thymidine kinase-negative (TK-) recombinant by insertion of a beta-galactosidase (bgal) expression cassette into the TK coding region. The recombinant virus (TK- bgal+) was tested for abortifacient activity in cattle by inoculation of 5 pregnant heifers at 25 to 29 weeks gestation. Five additional heifers were inoculated with the Cooper TK-positive (TK+) virus to serve as controls. After inoculation, both groups of heifers developed similar febrile responses and neutralizing antibody titers. Virus was isolated from blood of all heifers during the first postinoculation (PI) week, and isolation frequencies were similar for both groups. In contrast, whereas virus was isolated from many of the nasal and vaginal swab specimens of heifers inoculated with TK+ virus, only rare virus isolations were made from the heifers given TK- bgal+ virus. All heifers inoculated with TK+ virus aborted between PI days 19 and 35. The finding of characteristic microscopic lesions and viral antigen in fetal tissues indicated that the abortions were caused by BHV-1 infection. Virus was isolated from 3 fetuses, and all isolates were TK+ virus. Two heifers inoculated with TK- bgal+ virus aborted at PI days 25 and 39. Fetal tissues had typical BHV-1 microscopic lesions and viral antigen. Virus was isolated from blood of both fetuses, and the isolates were TK- bgal+. Results of this study indicate that inactivation of the TK gene reduces, but does not eliminate, the abortifacient activity of BHV-1.     

Modulation of IL-1-induced cartilage injury by NO synthase inhibitors: a comparative study with rat chondrocytes and cartilage entities

Nitric oxide (NO) is produced in diseased joints and may be a key mediator of IL-1 effects on cartilage. Therefore, we compared the potency of new [aminoguanidine (AG), S-methylisothiourea (SMT), S-aminoethylisothiourea (AETU)] and classical [Nomega-monomethyl-L-arginine (L-NMMA), Nomega-nitro-L-arginine methyl ester (L-NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin-1beta (rhIL-1beta) on rat cartilage anabolism. Three different culture systems were used: (1) isolated chondrocytes encapsulated in alginate beads; (2) patellae and (3) femoral head caps. Chondrocyte beads and cartilage entities were incubated in vitro for 48 h in the presence of rhIL-1beta with a daily change of incubation medium to obtain optimal responses on proteoglycan synthesis and NO production. Proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate [Na2(35)SO4] and NO production by cumulated nitrite release during the period of study. Chondrocytes and patellae, as well as femoral head caps, responded concentration-dependently to IL-1beta challenge (0 to 250 U ml(-1) and 0 to 15 U ml(-1) respectively) by a large increase in nitrite level and a marked suppression of proteoglycan synthesis. Above these concentrations of IL-1beta (2500 U ml(-1) and 30 U ml(-1) respectively), proteoglycan synthesis plateaued whereas nitrite release still increased thus suggesting different concentration-response curves. When studying the effect of NOS inhibitors (1 to 1000 microM) on NO production by cartilage cells stimulated with IL-1beta (25 U ml(-1) or 5 U ml(-1)), we observed that: (i) their ability to reduce nitrite level decreased from chondrocytes to cartilage samples, except for L-NMMA and AETU; (ii) they could be roughly classified in the following rank order of potency: AETU > L-NMMA > or = SMT > or = AG > or = L-NAME and (iii) AETU was cytotoxic when used in the millimolar range. When studying the effect of NOS inhibitors on proteoglycan synthesis by cartilage cells treated with IL-1beta, we observed that: (i) they had more marked effects on proteoglycan synthesis in chondrocytes than in cartilage samples; (ii) they could be roughly classified in the following rank order of potency: L-NAME > or = L-NMMA > > AG > SMT > > AETU and (iii) potentiation of the IL-1 effect by AETU was consistent with cytotoxicity in the millimolar range. D-isomers of L-arginine analog inhibitors (1000 microM) were unable to correct nitrite levels or proteoglycan synthesis in IL-1beta treated cells. L-arginine (5000 microM) tended to reverse the correcting effect of L-NMMA (1000 microM) on proteoglycan synthesis, thus suggesting a NO-related chondroprotective effect. However, data with L-NAME and SMT argued against a general inverse relationship between nitrite level and proteoglycan synthesis. Dexamethasone (0.1 to 100 microM) (i) failed to inhibit NO production in femoral head caps and chondrocytes beads whilst reducing it in patellae (50%) and (ii) did not affect or worsened the inhibitory effect of IL-1beta on proteoglycan synthesis. Such results suggested a corticosteroid-resistance of rat chondrocyte iNOS. Data from patellae supported a possible contribution of subchondral bone in NO production. In conclusion, our results suggest that (i) NO may account only partially for the suppressive effects of IL-1beta on proteoglycan synthesis, particularly in cartilage samples; (ii) the chondroprotective potency of NOS inhibitors can not be extrapolated from their effects on NO production by joint-derived cells and (iii) L-arginine analog inhibitors are more promising than S-substituted isothioureas for putative therapeutical uses.