Tony F. Cruz

Characterization of proteoglycan accumulation during formation of cartilagenous tissue in vitro

In order to study proteoglycan retention and accumulation, we optimized a chondrocyte cell culture system in which isolated bovine articular chondrocytes accumulate extracellular matrix to form a continuous layer of cartilagenous tissue. The tissue can attain a thickness of up to 110 microns by 35 days. The cells synthesize large keratan sulfate containing proteoglycans and type II collagen indicating that the chondrocytes maintain their phenotype in these culture conditions. Matrix accumulation is enhanced by increased cell density and the presence of serum and ascorbic acid. The amount of proteoglycans synthesized by the chondrocytes increases up to day 21 and then decreases to the same levels as are synthesized during the first week of culture. The percentage of newly synthesized proteoglycans retained in the matrix increases from 20% on day 6 to a maximum of 85% by day 35. The proteoglycan and collagen content in the tissue increases with time in culture. The changes in the percentage of proteoglycans retained parallels the increase in proteoglycan content. After day 35, there is no further increase in the amount of proteoglycans and collagen nor in the percentage of newly synthesized proteoglycans retained in the extracellular matrix. These studies demonstrate that the cultures are going through two phases: one of matrix accumulation and then one of maintaining the existing matrix. The period of matrix accumulation occurs between days 10-21 whereas matrix maintenance is observed after day 35. Using this culture system to study proteoglycan accumulation and maintenance during these culture periods may prove useful in identifying the mechanisms regulating these processes.

Development of Synaptic Glycoproteins: Effect of Postnatal Age on the Synthesis and Concentration of Synaptic Membrane and Synaptic Junctional Fucosyl and Sialyl Glycoproteins

Abstract: Synaptic plasma membranes (SPM) and synaptic junctions (SJ) were isolated from the cortices of rats varying in age between 5 and 28 days. Gel electrophoresis of SPM and SJ indicated a marked increase in the concentration of the “PSD protein” (M. W. 52,000) with development. The biosynthesis of glycoproteins was measured following the intracranial injection of [3H]fucose or [3H]N′‐acetylmannosamine. The incorporation of [3H]fucose into synaptic fractions decreased two‐ to threefold between 10 and 28 days whereas little change in the incorporation of [3H]N′‐acetylmannosamine occurred over the same period. Gel electrophoretic analyses of labeled synaptic membranes indicated major increases in the relative incorporation of radiolabeled precursors into glycoproteins with apparent molecular weights of 74,000, 65,000, 50,000, and 40,000 with increasing age. Identification of fucosyl and sialyl glycoproteins following reaction with 125I‐fucose‐binding protein or labeling of sialic acid with NaIO4NaB[3H4] demonstrated similar increases in the concentrations of these glycoproteins. Synaptic junctions contained three major glycoproteins with apparent molecular weights of 180,000, 130,000 and 110,000. The reaction of these glycoproteins with 125I‐fucose‐binding protein increased one‐ to twofold between 10 and 28 days but little variation in their relative distribution or synthesis occurred over this period. The reaction of synaptic junctional glycoproteins GP 180 and GP 110 with 125I‐wheat germ ag‐glutinin increased between 10 and 28 days. The results indicate that the molecular composition of the synapse continues to evolve after the initial synaptic contact has been formed.

In vitro and in vivo antineoplastic effects of ortrovanadate

In the present study we have demonstrated that orthovanadate at concentrations of 5–10 uM is cytotoxic to proliferating cells including primary cultures and tumour cell lines. However, concentrations of up to 50 uM did not affect the viability of non-proliferating cells. The cytotoxicity appears to be dependent on the vanadium concentration rather than on the oxidation state of vanadium or the vanadium compound. Furthermore, tumour cell lines with different proliferative rates were equally sensitive to orthovanadate cytotoxicity. Although the mechanisms responsible for the cytotoxicity are not known, addition of H2O2 potentiated orthovanadate cytotoxicity suggesting that hydroxyl or vanadium radicals may be involved.In vivo subcutaneous injections of orthovanadate into mice containing MDAY-D2 tumours resulted in the inhibition of tumour growth by 85–100%. These data indicated that orthovanadate at concentrations greater than 5 uM has antineoplastic properties and may be useful as a chemotherapeutic agent.

In vitro formation of mineralized cartilagenous tissue by articular chondrocytes.

SummaryStudy of the deep articular cartilage and adjacent calcified cartilage has been limited by the lack of an in vitro culture system which mimics this region of the cartilage. In this paper we describe a method to generate mineralized cartilagenous tissue in culture using chondrocytes obtained from the deep zone of bovine articular cartilage. The cells were plated on Millipore CMR filters. The chondrocytes in culture accumulated extracellular matrix and formed cartilagenous tissue which calcified when β-glycerophosphate was added to the culture medium. The cartilagenous tissue generated in vitro contains both type II and type X collagens, large sulfated proteoglycans, and alkaline phosphatase activity. Ultrastructurally, matrix vesicles were seen in the extracellular matrix. Selected area electron diffraction confirmed that the calcification was composed of hydroxyapatite crystals. The chondrocytes, as characterized thus far, appear to maintain their phenotype under these culture conditions which suggests that these cultures could be used as a model to examine the metabolism of cells from the deep zone of cartilage and mineralization of cartilagenous tissue in culture.

Interleukin 1 and phorbol 12-myristate 13-acetate induce collagenase and PGE2 production through a PKC-independent mechanism in chondrocytes

In the present study we demonstrate that interleukin 1 (IL 1) and phorbol 12-myristate 13-acetate (PMA) stimulate collagenase production by bovine chondrocytes in monolayer culture. Since it has been well established that PMA stimulates protein kinase C (PKC), we examined whether IL 1 and PMA also stimulate PKC in chondrocytes. In agreement with other studies, PMA induced the translocation of PKC, reflecting PKC activation by PMA. In contrast, IL 1 did not induce the translocation of PKC. Both IL 1 and PMA stimulated the release of [14C]arachidonic acid from chondrocyte phospholipids, suggesting that both agents stimulate phospholipase A2 (PLA2). Concomitantly, IL 1 and PMA also induced a pronounced increase in the production of PGE2. Pre-incubation of chondrocytes with staurosporine, a PKC inhibitor, did not affect the stimulation of collagenase production by IL 1 and only minimally that induced by PMA. Similarly, high concentrations of staurosporine did not inhibit prostaglandin E2 (PGE2) production induced by IL 1 or PMA. These data show that IL 1 and PMA stimulate the PLA2 pathway and collagenase production, however, these processes can occur in the absence of PKC activation.

FETAL BOVINE SERUM INHIBITS CHONDROCYTE COLLAGENASE PRODUCTION : INTERLEUKIN 1 REVERSES THIS EFFECT

Bovine articular chondrocytes incubated in medium which was serum free or contained low levels of fetal bovine serum (less than 5%) constitutively produced collagenase. Increasing the concentration of serum in the culture medium inhibited the production of collagenase. Addition of interleukin 1 and lipopolysaccharide reversed the inhibitory effect of serum. Phorbol esters only stimulated collagenase production when the serum concentration was at least 10%. These data suggest that there is a factor(s) in fetal bovine serum that inhibits collagenase production by chondrocytes and this can be reversed by agents such as interleukin 1.

The effects of development on activity, specificity and endogenous substrates of synaptic membrane sialidase.

Synaptic plasma membranes were prepared from cortices of rats varying in post-natal age between 4 and 30 days. Sialic acid associated with synaptic plasma membrane glycoproteins and gangliosides increased 75% and 50%, respectively, between 4 and 30 days. The amount of sialic acid released from these membrane constituents by intrinsic synaptic sialidase increased 2-4-fold over the same period. Incubation of synaptic plasma membranes with exogenous gangliosides or glycopeptides demonstrated a 2-3-fold increase in sialidase activity during development. The major gangliosides present in synaptic plasma membranes at all ages were GT1, GD1a, GD1b and GM1. Intrinsic sialidase hydrolyzed 50-70% of endogenous GT1 and GD1a gangliosides at all ages. Endogenous GD1b ganglioside was poorly hydrolyzed in young rats and its susceptibility to enzymic hydrolysis increased during development. When exogenous GD1a and GD1b were used as substrates a preferential increase in activity against GD1b occurred during development, the ratio of activity (GD1a/GD1b) decreasing from 3.6 to 1.6 between 7 and 30 days. 10- and 30-day-old synaptic plasma membranes contained complex mixtures of sialoglycoproteins, an increase in the relative concentrations of lower molecular weight sialoglycoproteins occurring during development. Intrinsic sialidase present in 10- and 30-day-old synaptic plasma membranes acted upon all molecular weight classes of sialoglycoproteins.

Identification of Intrinsic Sialidase and Sialoglycoprotein Substrates in Rat Brain Synaptic Junctions

Sialidase activity associated with rat brain synaptic junctions (SJ) and synaptic membranes (SM) was determined. Both fractions released sialic acid from exogenous glycopeptides and gangliosides. SJ accounted for 5–10% of the total sialidase activity recovered from SM following extraction with Triton X‐100, and the specific activity of SJ sialidase was 60% of that of the parent SM fraction. Intrinsic SJ sialidase hydrolysed 12–15% of the sialic acid associated with endogenous SJ glycoproteins. Sialic acid residues associated with SJ glycoproteins were labelled with sodium borotritide and SJ proteins fractionated by affinity chromatography on concanavalin A‐agarose. SJ glycoproteins that reacted with concanavalin A (con A+ glycoproteins) accounted for 25% of the total SJ [3H]sialic acid. Intrinsic SJ sialidase hydrolysed 20% of the [3H]sialic acid associated with these glycoproteins. Each molecular weight class of con A+ glycoprotein previously shown to be a specific component of the postsynaptic apparatus contained sialic acid and was acted on by intrinsic SJ sialidase.

Effects of development and aging on the concentration of a human brain antigen

The concentration of a glycoprotein reactive with monoclonal antibody 44D10 was studied during development and aging in 19 normal brains. Little change in the concentration of the antigen was found in white matter of brains ranging from the age of 1 to 54 years. However, a linear increase in the concentration of antigen was observed between the ages of 54 and 80 years. By the age of 80 years, the concentration of the glycoprotein had increased 2-3-fold. In contrast to white matter, gray matter did not contain detectable levels of antigen at ages from 1 to 54 years. Low levels of antigen were detected in brains of all older individuals examined. However, the relative concentration of this glycoprotein in gray matter was less than 5% of that in white matter. An examination of white matter homogenates from guinea pig and bovine brain showed that monoclonal antibody 44D10 was not reactive in these species.

An adaptation of the sodium periodate/sodium borotritide labeling procedure which allows the unambiguous identification of synaptic sialoglycoproteins

Abstract An adaptation of the sodium periodate/sodium borotritide procedure for the identification of membrane sialoglycoproteins is described which eliminates interference from nonspecifically incorporated tritium. Synaptic membranes were labeled using the NaIO 4 NaB 3 H 4 procedure and separated by polyacrylamide gel electrophoresis. Following electrophoresis the gels were fixed, sliced, and individual slices treated with neuraminidase. Treatment with neuraminidase selectively released [ 3 H]sialyl derivatives from the fixed glycoproteins allowing the unambiguous identification of sialoglycoproteins. The sialoglycoprotein composition of synaptic membranes and synaptic junctions was compared.