Degradation products of the mRNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean and transgenic petunia.

The degradation of a soybean ribulose-1,5-bisphosphate carboxylase small subunit RNA, SRS4, was investigated in soybean seedlings and in petunia plants transformed with an SRS4 gene construct. Polyacrylamide RNA gel blot, primer extension, and S1 nuclease analyses were used to identify and map fragments of the SRS4 mRNA generated in vivo. We showed that SRS4 mRNA is degraded to a characteristic set of fragments in soybean and transgenic petunia and that degradation is not dependent on position of insertion of the gene construct within the genome, on the expression level of the SRS4 mRNA, or on the rbcS promoter.

Laminin carbohydrates are implicated in cell signaling.

We have examined how laminin carbohydrates participate in cellular responses and have focused upon cell spreading and neurite outgrowth. Our earlier studies showed that unglycosylated laminin fully supported cell adhesion but did not promote subsequent spreading of mouse melanoma cells or neurite outgrowth of rat pheochromocytoma cells (Dean et al. (1990): J Biol Chem 265:12553-12562).

The major proteoglycan of adult rabbit skeletal muscle. Relationship to small proteoglycans of other tissues.

We have been interested in examining the putative biological role(s) of the major proteoglycan of adult skeletal muscle. The small proteoglycans of adult rabbit skeletal muscle and tendon were extracted and purified by sequential density-gradient ultracentrifugation, ion-exchange chromatography and gel filtration. They appeared to be homogeneous by the criterion of gel electrophoresis in SDS and to yield one major product, the core protein, after digestion with chondroitin ABC lyase, also observed after gel electrophoresis.

A biological role of the carbohydrate moieties of laminin.

The ways in which the carbohydrate moieties of laminin affect its cellular interactions have been examined by two different experimental approaches. In one approach, we used lectins in order to block specific carbohydrates on laminin which previously had been dried onto a plastic surface. We found that wheat germ agglutinin and Griffonia simplicifolia agglutinin I blocked the binding of the neuron-like rat pheochromocytoma cell line PC12.

Characterization of Chinese hamster ovary cells with impaired spreading properties on fibronectin.

The development of receptor-defective or -deficient mutants can be applied to the investigation of cell-matrix interactions including cell adherence and spreading. In the present study we developed a series of ethyl methyl sulfonate (EMS)-induced Chinese hamster ovary (CHO) cell mutants, which adhere to fibronectin but have impaired spreading characteristics. Using morphometric analysis, a significant suppression in the degree of cell spreading between the wild-type and the mutant cells (P less than 0.

The relationship of intercondylar notch size and content to notchplasty requirement in anterior cruciate ligament surgery.

A stenotic intercondylar notch is an indication for a notchplasty at the time of ACL reconstructive surgery. However, the absence of a stenotic notch does not ensure that impingement of the ligament upon the intercondylar notch will not occur. An excessively large ligament, even in the presence of a normal intercondylar notch, will result in abrasion of the ligament on the notch.

The natural history of flexion contracture in total knee arthroplasty. A prospective study.

A prospective study was carried out to document the natural history of flexion contractures of the knee after total knee arthroplasty (TKA). Thirty-five knees in 33 patients with TKA were followed for a mean duration of 55 weeks. In no case did the surgical procedure include excessive bony resection in order to correct a flexion contracture.

Partial structure of the gene for chicken cartilage proteoglycan core protein.

A genomic DNA fragment (gCORE-1), encoding a portion of the cartilage proteoglycan core protein, has been isolated from a phage library using cDNA as a probe. The genomic insert is about 17 kilobase pairs; two BamHI fragments of the insert (1.3 and 4.

Lectins inhibit cell binding and spreading on a laminin substrate.

This study examined the effects on cell binding and spreading of the exposure of laminin substrates to the lectins Wheat Germ Agglutinin or Concanavalin A. Exposure of laminin to Wheat Germ Agglutinin inhibited binding of mouse B16 F1 melanoma cells in a dose dependent manner. Exposure to Concanavalin A had no deleterious effects on binding but did inhibit cell spreading.

Temporal and spatial analysis of cartilage proteoglycan core protein gene expression during limb development by in situ hybridization.

As limb mesenchymal cells differentiate into chondrocytes they initiate the synthesis of a cartilage-specific sulfated proteoglycan, cartilage-characteristic type II collagen, and other cartilage-specific proteins. In the present study, in situ hybridization with a 32P-labeled cloned cDNA probe complementary to mRNA encoding the core protein of cartilage proteoglycan has been used to visualize and localize the accumulation of cartilage proteoglycan core protein mRNA sequences during development of the chick limb bud in vivo. When the probe was hybridized to sections through 7-day (stage 32) limbs, an intense hybridization signal was observed over the well-differentiated cartilage rudiments of the limb, while no signal above background was observed over nonchondrogenic tissues including muscle, loose connective tissue, and epidermis.

Separation and characterization of the subunits of the laminin of EHS sarcoma.

A rapid and sensitive method was developed for the preparative separation of laminin subunits. Laminin was extracted and purified from mouse EHS sarcoma. On SDS-PAGE, the reduced and carboxymethylated molecule separated into two components corresponding to molecular weights of about 400 KDa (subunit A) and 200 KDa (subunit B).

Isolation and characterization of a low molecular weight chondroitin sulfate proteoglycan from rabbit skeletal muscle.

Proteoglycans may be implicated in the process of aggregation of acetylcholine receptors in the basal lamina of skeletal muscle and possibly in the mechanism of reinnervation at the neuromuscular junction. In order to further deduce the role of such proteoglycans, we have sought to isolate them and define their molecular structures. In this study, proteoglycans were extracted from rabbit skeletal muscle by using 4 M guanidine hydrochloride and were purified by sequential cesium chloride density gradient ultracentrifugation, DEAE-cellulose ion-exchange chromatography, and Sepharose CL-6B and CL-2B gel filtration under dissociative conditions.

Cartilage proteoglycan core protein gene expression during limb cartilage differentiation.

Changes in the steady-state cytoplasmic levels of mRNA for the core protein of the major sulfated proteoglycan of cartilage were examined during the course of limb chondrogenesis in vitro using cloned cDNA probes. Cytoplasmic core protein mRNA begins to accumulate at the onset of overt chondrogenesis in micromass culture coincident with the crucial condensation phase of the process, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to depositing a cartilage matrix. The initiation of core protein mRNA accumulation coincides with a dramatic increase in the accumulation of mRNA for type II collagen, the other major constituent of hyaline cartilage matrix.

Fibronectin glycosylation modulates fibroblast adhesion and spreading.

The role of the carbohydrate residues of fibronectin concerning the specificities of that glycoprotein to interact with fibroblastic cell surfaces, gelatin, and heparin was examined. Tunicamycin was used to produce carbohydrate-depleted fibronectin; it was synthesized by cultured fibroblasts. Unglycosylated and glycosylated fibronectins were analyzed for their ability to bind gelatin and heparin, using affinity columns.

Structures of the asparagine-linked sugar chains of laminin.

This investigation describes the isolation and characterization of oligosaccharides of the basement membrane glycoprotein, laminin. Pronase-released glycopeptides of isolated laminin, from a mouse Engelbreth-Holm-Swarm tumor, were fractionated using a combination of gel permeation chromatography and Con A-Sepharose affinity chromatography. The glycopeptides were analyzed for sugar linkage patterns by methylation analysis.

Cloning and sequence analysis of a partial cDNA for chicken cartilage proteoglycan core protein.

A chicken embryo sternal cartilage cDNA library, created in the plasmid expression vector pUC9, was screened for sequences coding for immunologically detectable core protein of the large, major proteoglycan of cartilage. A 1229-base-pair cDNA clone was isolated that contained only one extended open reading frame, which had sequences coding for a polypeptide of 379 amino acid residues. These deduced sequences corresponded to those anticipated from current models of proteoglycan structure; a deduced sequence encompassing 21 amino acids was almost identical to a known sequence of bovine nasal cartilage proteoglycan.

Peptide repeats in a mussel glue protein: theme and variations.

The adhesive protein from Mytilus edulis contains 75-80 closely related, repeated peptide sequences in its primary structure. These peptides can be resolved following digestion with trypsin by reversed-phase high-pressure liquid chromatography. The most frequently repeated sequence is the decapeptide Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Dopa-Lys (peptide E).

Identification and quantitation of O-phosphoserine in human plasma fibronectin.

O-Phosphoserine was positively identified as the phosphorylated moiety in human plasma fibronectin by 31P-NMR of intact peptides. These data correlated completely with chemical analyses which demonstrated the presence of O-phosphoserine at a concentration of 2 residues/molecule. Neither O-phosphothreonine nor O-phosphotyrosine was detected in partial acid and partial alkaline hydrolysates, respectively.

Partial characteristics of an analog of pyridinoline isolated from human skin.

The most abundant amine in acid hydrolysates of human skin, eluting in the crosslink region of a reversed-phase HPLC chromatogram, has the same retention time as pyridinoline standard. This amine is not pyridinoline, since it is a weak fluorophore and its U/V spectrum does not agree with that of pyridinoline. The unknown amine was isolated and characterized by fast atom bombardment mass spectrometry and its structure is consistent with a deoxy-analogue of pyridinoline.

Reversibility of monensin inhibition of oligosaccharide processing of human fibronectin.

Monensin impairs oligosaccharide processing in fibronectin primarily by inhibiting the conversion of oligosaccharides from the high mannose type to the complex type. The separate effects of monensin and cations on alpha-mannosidase activity in fibroblasts were examined using an in vitro assay system. The results indicated that monensin did not directly inhibit alpha-mannosidase activity in vitro, although prior treatment of fibroblasts with monensin caused an irreversible suppression of enzyme activity.

High-performance liquid chromatographic separation of glycopeptides from Nereis cuticle collagen.

To facilitate the structural studies of invertebrate collagens, a sensitive and effective method was developed, using reverse-phase high-performance liquid chromatography for preparative isolation of the collagen subunits and their clostridial collagenase-derived peptides; the methods have been applied to Nereis cuticle collagen. The two subunits of denatured Nereis cuticle collagen, termed A and B, were initially separated by high-performance liquid chromatography. These polypeptides, with Mr of about 0.

Swainsonine inhibits macrophage receptor-mediated uptake and degradation of a mannosyl-oligosaccharide.

Rat pulmonary macrophages were incubated in the presence of a radiolabeled mannosyl-oligosaccharide obtained from ovalbumin. Receptor-mediated endocytosis and degradation of this ligand by the cells was followed in the presence or absence of swainsonine, an inhibitor of alpha-mannosidases. The results indicated that at higher concentrations (greater than 1 microgram/ml) of swainsonine, both the internalization and degradation of the radiolabeled ligand were inhibited.

Characterization of a large fragment from annelid cuticle collagen and its relationship to the intact molecule.

Digestion of the cuticle collagen from the annelid Nereis virens with Clostridium histolyticum collagenase yields a native, collagenase-resistant fragment (CCRF) of the molecule with an Mr of about 900,000 (Kimura and Tanzer, J. Biol. Chem.