A Randomized Clinical Trial Comparing Posterior Cruciate-Stabilizing vs Posterior Cruciate-Retaining Prostheses in Primary Total Knee Arthroplasty: 10-Year Follow-Up.

BACKGROUND: This 10-year follow-up compares health-related quality of life (HRQL) and reoperations in 100 subjects who were randomized to receive posterior cruciate ligament substituting (PS) or posterior cruciate ligament retaining (CR) total knee arthroplasty. We previously reported 2-year results. METHODS: Subjects were enrolled preoperatively and randomized at surgery. Subjects completed HRQL questionnaires at all evaluation points. Subjects were re-evaluated at 2 and 10 years with reoperations determined through regional medical record review and patient report. RESULTS: Over 10 years, 25 (25%) subjects died, 2 subjects were revised and withdrew, and 11 (11%) subjects were lost to follow-up. Of survivors, 62 of 75 (83%) were evaluated at 10 years. Twenty-eight (37%) subjects provided HRQL, radiographic, and reoperation status, 28 (37%) subjects completed HRQL evaluations and reoperation status only, and 6 (8%) subjects provided radiographic and reoperation follow-up. Both groups retained good HRQL between 2 and 10 years with no group differences noted (P > .35). One revision (CR subject), secondary to deep joint infection, occurred within 2 years with 1 further revision (PS subject) occurring at 3 years postoperatively. One subject (PS subject) required manipulation under anesthesia within 3 months of surgery. Four subjects required late patellar resurfacing (1 CR subject, 3 PS subjects) but were retained in the 10-year evaluation. Overall, reoperations were not significantly different between groups (P = .26). CONCLUSION: Over 10 years postoperatively, both the PS and CR total knee arthroplasty performed well with subjects reporting acceptable levels of HRQL up to 10 years postoperatively; low levels of revision or reoperation were reported in both groups.

Abundant expression of parathyroid hormone-related protein in primary rat aortic smooth muscle cells accompanies serum-induced proliferation.

Parathyroid hormone-related protein (PTHrP), which is responsible for producing hypercalcemia in patients with humoral hypercalcemia of malignancy, has recently been identified in several normal tissues. Because PTHrP, like parathyroid hormone (PTH), is known to exhibit vasodilatory properties, we investigated the expression and regulation of PTHrP mRNA in cultured rat aortic smooth muscle cells (SMC). We report here that PTHrP mRNA is expressed in SMC and is markedly induced by serum in a time- and concentration-dependent fashion. Addition of 10% fetal calf serum to serum-deprived, confluent cells, resulted in a marked induction of PTHrP mRNA by 2 h with a peak at 4-6 h. PTHrP was detected in SMC by immunocytochemistry and radioimmunoassay of conditioned medium, and was shown to be up-regulated within 24 h after the addition of serum. The serum induction of PTHrP mRNA was blocked by actinomycin D and by cycloheximide indicating the need for protein synthesis to evoke the serum effect on PTHrP gene transcription. In addition, treatment with dexamethasone, which has been previously shown to reduce the constitutive expression of PTHrP in human cancer cells, also blunted the serum induction of PTHrP mRNA in SMC. Treatment of quiescent cells with the serum mitogens platelet-derived growth factor or insulin-like growth factor-I had no effect on PTHrP, whereas the vasoactive peptides endothelin, norepinephrine and thrombin stimulated PTHrP expression. Exogenous addition of recombinant PTHrP-(1-141) had no significant effect on SMC DNA synthesis as measured by [3H]thymidine incorporation. In summary, the abundance of PTHrP mRNA and the characteristics of its regulation in SMC suggest a major role for PTHrP as a local modulator in vascular smooth muscle.

Oxidized low-density lipoprotein regulates matrix metalloproteinase-9 and its tissue inhibitor in human monocyte-derived macrophages.

BACKGROUND: Macrophages in human atherosclerotic plaques produce a family of matrix metalloproteinases (MMPs), which may influence vascular remodeling and plaque disruption. Because oxidized LDL (ox-LDL) is implicated in many proatherogenic events, we hypothesized that ox-LDL would regulate expression of MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in monocyte-derived macrophages. MWRHOSA AND RESULTS: Mononuclear cells were isolated from normal human subjects with Ficoll-Paque density gradient centrifugation, and adherent cells were allowed to differentiate into macrophages during 7 days of culture in plastic dishes. On day 7, by use of serum-free medium, the macrophages were incubated with various concentrations of native LDL (n-LDL) and copper-oxidized LDL. Exposure to ox-LDL (10 to 50 microg/mL) increased MMP-9 mRNA expression as analyzed by Northern blot, protein expression as measured by ELISA and Western blot, and gelatinolytic activity as determined by zymography. The increase in MMP-9 expression was associated with increased nuclear binding of transcription factor NF-kappaB and AP-1 complex on electromobility shift assay. In contrast, ox-LDL (10 to 50 microg/mL) decreased TIMP-1 expression. Ox-LDL-induced increase in MMP-9 expression was abrogated by HDL (100 microg/mL). n-LDL had no significant effect on MMP-9 or TIMP-1 expression. CONCLUSIONS: These data demonstrate that unlike n-LDL, ox-LDL upregulates MMP-9 expression while reducing TIMP-1 expression in monocyte-derived macrophages. Furthermore, HDL abrogates ox-LDL-induced MMP-9 expression. Thus, ox-LDL may contribute to macrophage-mediated matrix breakdown in the atherosclerotic plaques, thereby predisposing them to plaque disruption and/or vascular remodeling.

Membrane type 1 matrix metalloproteinase expression in human atherosclerotic plaques: evidence for activation by proinflammatory mediators.

BACKGROUND: Matrix metalloproteinases (MMPs) are expressed in atherosclerotic plaques, where in their active form, they may contribute to vascular remodeling and plaque disruption. In this study, we tested the hypothesis that membrane type 1 MMP (MT1-MMP), a novel transmembrane MMP that activates pro-MMP-2 (gelatinase A), is expressed in human atherosclerotic plaques and that its expression is regulated by proinflammatory molecules. METHODS AND RESULTS: MT1-MMP expression was examined in normal and atherosclerotic human arteries by immunocytochemistry with specific antibodies. MT1-MMP expression in human saphenous vein-derived smooth muscle cells (SMCs) maintained in tissue culture was determined under basal conditions and in response to proinflammatory molecules (interleukin [IL]-1alpha, tumor necrosis factor [TNF]-alpha, and oxidized LDL [ox-LDL]) by use of Northern blot and ribonuclease protection assays for mRNA, Western blot and immunoprecipitation for protein, and gelatin zymography for catalytic activity. Medial SMCs of normal vessel wall expressed MT1-MMP. In atherosclerotic arteries, MT1-MMP expression was noted within the complex atheroma colocalizing with SMCs and macrophages (Mphi). Cultured SMCs constitutively expressed MT1-MMP mRNA and protein, which increased 2- to 4-fold over control in a time-dependent manner within 4 to 8 hours of exposure to IL-1alpha, TNF-alpha, and ox-LDL (thiobarbituric acid-reactive substances, 13.4 nmol/mg LDL protein), whereas native LDL had no effect. Flow cytometry revealed MT1-MMP expression by human monocyte-derived Mphi, which increased 3.8-fold over baseline within 6 hours after exposure to 10 ng/mL TNF-alpha. CONCLUSIONS: This study demonstrates that MT1-MMP, an activator of pro-MMP-2, is expressed by SMCs and Mphi in human atherosclerotic plaques. Furthermore, proinflammatory molecules upregulate MT1-MMP expression in vascular SMCs and Mphi. Thus, activation of SMCs and Mphi by proinflammatory molecules may influence extracellular matrix remodeling in atherosclerosis by regulating MT1-MMP expression.

Arterialization of human vein grafts is associated with tenascin-C expression.

OBJECTIVES: This study was performed to test the hypothesis that tenascin-C (TN-C), an extracellular matrix (ECM) protein with counteradhesive chemotactic and vascular growth-promoting effects, is expressed in "arterialized" human saphenous vein grafts (SVGs). BACKGROUND: Tenascin-C is expressed in the vessel wall after vascular injury in the experimental model, where it has been implicated in the formation of neointima. Overexpression of TN-C has also been implicated in the development and progression of pulmonary hypertension. Saphenous vein grafts are exposed to hemodynamic stress when interposed in the arterial circulation and mechanical stress upregulates expression of TN-C, whereas stress-relaxation suppresses its synthesis. We hypothesized that the hemodynamic stress of increased arterial pressure could also induce TN-C expression in SVG. METHODS: We examined the expression of TN-C protein and mRNA in normal vein and "arterialized" human SVG using immunohistochemistry and in situ hybridization, respectively. RESULTS: TN-C protein was not detected in control human saphenous veins; however, it was uniformly and strongly expressed in the adventitia and media of patent human vein grafts, with minimal or no expression in the neointima (n = 27, 100%). In situ hybridization showed that TN-C mRNA was not detected in the neointima, but was strongly upregulated in the adventitia and media, corroborating immunostaining data (n = 10, 100%). Unlike patent SVG, TN-C was not expressed in the adventitia of occluded grafts, except for a low level of expression around the newly formed vessels in neointima (n = 5, 100%). Smooth muscle cell-specific staining demonstrated that the lack of expression of TN-C in occluded vein grafts is not due to the lack of presence of smooth muscle cells in the graft. CONCLUSIONS: These findings suggest that placement of a venous graft in the arterial system leads to expression of TN-C, which may in turn facilitate graft remodeling. Conversely, loss of flow and intravascular pressure, associated with vein graft occlusion, is accompanied by disappearance of TN-C expression.

Adventitial remodeling after angioplasty is associated with expression of tenascin mRNA by adventitial myofibroblasts.

OBJECTIVES: The purpose of this study was to determine the temporospatial expression of tenascin-C (TnC) in balloon-injured rat and porcine arteries. BACKGROUND: Recent studies suggest that cell migration, in addition to cell proliferation, is a critical component of neointima formation after vascular injury. We have previously shown that adventitial myofibroblasts synthesize growth factors that contribute to the formation of neointima after arterial injury. We have also shown that the extracellular matrix protein, TnC, regulates cell migration. Consequently, we investigated the temporospatial expression of TnC by myofibroblasts after vascular injury. METHODS: In situ hybridization and immunohistochemistry were used to investigate the temporospatial expression of TnC in injured arteries. Northern and Western blots were used to determine the in vitro expression of TnC. RESULTS: In situ hybridization revealed that the major site of TnC expression early after vascular injury was the adventitial myofibroblasts. Immunohistochemical staining demonstrated that TnC expression began in adventitial myofibroblasts three days after injury. Tenascin-C expression, however, did not persist in this region. Rather, it moved progressively across the vascular wall toward the luminal surface. By one week, TnC expression reached the developing neointima. In vitro, myofibroblasts did not express TnC mRNA under basal conditions. In contrast, angiotensin II and PDGF-BB, factors that have been implicated in remodeling of balloon-injured arteries, markedly upregulated TnC mRNA. CONCLUSIONS: Tenascin-C is expressed in response to balloon injury. Tenascin-C expression begins with adventitial myofibroblasts. Over a period of 7 to 14 days, expression moves progressively across the vessel wall to the neointima. We hypothesize that adventitial myofibroblasts are actively involved in the formation of neointima and that TnC facilitates migration of these cells during adventitial remodeling.

A variant of mirror hand. A case report.

We describe a rare variant of mirror hand in a 20-year-old man who presented with multiple fingers. Radiographs revealed two ulnae (one vestigial) and a radius. There was duplication of the humeral head. The unique features of this case are the age of patient before the start of treatment and extension of the duplication proximal to the elbow.

Sympathetic nerve sprouting, electrical remodeling and the mechanisms of sudden cardiac death.

The purpose of this article is to review the nerve sprouting hypothesis of sudden cardiac death. It is known that sympathetic stimulation is important in the generation of sudden cardiac death. For example, there is a diurnal variation of sudden death rate in patients with myocardial infarction. Beta blockers, or drugs with beta blocking effects, are known to prevent sudden cardiac death. It was unclear if the cardiac nerves in the heart play only a passive role in the mechanisms of sudden death. To determine if nerve sprouting and neural remodeling occur after myocardial infarction, we performed immunocytochemical studies of cardiac nerves in explanted native hearts of transplant recipients. We found that there was a positive correlation between nerve density and a clinical history of ventricular arrhythmia. Encouraged by these results, we performed a study in dogs to determine whether or not nerve growth factor (NGF) infusion to the left stellate ganglion can facilitate the development of ventricular tachycardia (VT), ventricular fibrillation (VF), and sudden cardiac death (SCD). The results showed that augmented myocardial sympathetic nerve sprouting through NGF infusion plus atrioventricular (AV) block and MI result in a 44% incidence (four of nine dogs) of SCD and a high incidence of VT in the chronic phase of MI. In contrast, none of the six dogs (with AV block and MI) without NGF infusion died suddenly or had frequent VT episodes. Based on these findings, we propose the nerve sprouting hypothesis of ventricular arrhythmia and SCD. The hypothesis states that MI results in nerve injury, followed by sympathetic nerve sprouting and regional (heterogeneous) myocardial hyperinnervation. The coupling between augmented sympathetic nerve sprouting with electrically remodeled myocardium results in VT, VF and SCD. Modification of nerve sprouting after MI may provide a novel opportunity for arrhythmia control.

Overexpression of insulin-like growth factor-binding protein-4 (IGFBP-4) in smooth muscle cells of transgenic mice through a smooth muscle alpha-actin-IGFBP-4 fusion gene induces smooth muscle hypoplasia.

Insulin-like growth factor I (IGF-I) has been postulated to function as a smooth muscle cell (SMC) mitogen and to play a role in the pathogenesis of bladder hypertrophy, estrogen-induced uterine growth, and restenosis after arterial angioplasty. IGF-binding protein-4 (IGFBP-4) inhibits IGF-I action in vitro and is the most abundant IGFBP in the rodent arterial wall. To explore the function of this binding protein in vivo, transgenic mouse lines were developed harboring fusion genes consisting of a rat IGFBP-4 complementary DNA cloned downstream of either a -724 bp fragment of the mouse smooth muscle alpha-actin 5'-flanking region (SMP2-BP-4) or -1074 bp, 63 bp of 5'-untranslated region, and 2.5 kb of intron 1 of smooth muscle alpha-actin (SMP8-BP-4). SMP2-BP-4 mice expressed low levels of the exogenous IGFBP-4 messenger RNA (mRNA), which was not specifically targeted to SMC-rich tissue environments, and were therefore not analyzed further. Six SMP8-BP-4 transgenic lines derived from separate founders were characterized. Mating of hemizygous SMP8-BP-4 mice with controls produced about 50% transgenic offspring, with equal sex distribution. Expression of IGFBP-4 mRNA in nontransgenic littermates was maximal in liver and kidney. By contrast, transgenic IGFBP-4 mRNA expression, distinguished because of a smaller transcript size, was confined to SMC-containing tissues, with the following hierarchy: bladder > aorta > stomach = uterus. There was no transgene expression in skeletal muscle, brain, or cardiac myocytes. The abundance of IGFBP-4 measured by Western ligand blotting or by immunoblotting, was 8- to 10-fold higher in aorta and bladder of SMP8-BP-4 mice than in their nontransgenic littermates, with no change in plasma IGFBP-4 levels. Transgenic mice exhibited a significant reduction in wet weight of SMC-rich tissues, including bladder, intestine, aorta, uterus, and stomach, with no change in total body or carcass weight. In situ hybridization showed that transgene expression was targeted exclusively to the muscular layers of the arteries, veins, bladder, ureter, stomach, intestine, and uterus. Overexpression of IGFBP-4 was associated with SMC hypoplasia, a reciprocal phenotype to that of transgenic mice overexpressing IGF-I under control of the same promoter (SMP8-IGF-I). Double transgenic mice derived from mating SMP8-BP-4 with SMP8-IGF-I animals showed a modest decrease in wet weight at selected SMC tissues. Although we cannot exclude that the effects of IGFBP-4 may be IGF independent, these data suggest that IGFBP-4 is a functional antagonist of IGF-I action on SMC in vivo.

Growth factor production by human thyroid carcinoma cells: abundant expression of a platelet-derived growth factor-B-like protein by a human papillary carcinoma cell line.

As papillary thyroid carcinoma cells grow surrounding finger-like structures of stromal tissue, we postulated they may secrete a growth factor(s) for mesenchymal cells and that these would be distinct from any mitogenic factors elaborated by follicular carcinomas. Conditioned medium from both the human papillary carcinoma cell line NPA and the follicular carcinoma cell line WRO evoked a 20- to 30-fold increase in [3H]thymidine incorporation into NIH3T3 cell DNA. NPA cell growth factor activity largely eluted with 0.5 mol/L NaCl from a heparin-Sepharose column. NPA-conditioned medium competed in a platelet-derived growth factor-B (PDGF-B) RRA, and the mitogenic activity was partially blocked by an anti-PDGF-BB antibody. An immunoprecipitated PDGF-B-like protein from NPA cells was about 17 kilodaltons in a reducing gel, but, in contrast to wild-type PDGF-BB, did not change its electrophoretic mobility in an unreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NPA cells expressed an abundant 1.4-kilobase RNA that hybridized to probes for the 5'-untranslated and amino-terminal domains of PDGF-B and was distinct from the 4.2-kilobase wild-type PDGF-B chain transcript. There were no structural changes in the PDGF-B gene, as determined by cytogenetic analysis and restriction mapping. However, the PDGF-B gene in the NPA cells was hypomethylated compared to that in normal thyroid tissue or WRO cells. In contrast, the mitogenic activity of WRO cells bound to heparin with high affinity and was blocked by a basic fibroblast growth factor (bFGF) antibody. WRO cells contained abundant bFGF mRNA. Both cell lines abundantly expressed transforming growth factor-beta mRNA. Thus, NPA and WRO cells express powerful, yet distinct, mesenchymal cell growth factors. Whereas WRO cells express abundant bFGF, NPA cells produce a novel PDGF-B-like protein, which may correspond to a mutated form of PDGF-B-chain.

Stimulation of rat vascular smooth muscle cell glycosaminoglycan production by angiotensin II.

Matrix production by smooth muscle cells (SMC) appears to play a major role in the intimal thickening process. Proteoglycans (PG) are the predominant extracellular matrix component of early restenotic lesions. As angiotensin II (A II) has been proposed as a mediator of restenotic process, we hypothesized that A II may directly affect PG synthesis by SMC. SMC were cultured in the presence of [35S]sulfate and angiotensin II, and both the secreted and membrane-bound proteoglycans were analyzed. A II (1 to 100 nM) evoked a dose- and time-dependent increase in both cell- and media-associated PG production, an effect abrogated by the A II receptor antagonist, saralasin. SMC constitutively synthesize small amounts of PG with a molecular mass of 170-250 kDa. After treatment with A II, the abundance of PG is increased, as well as its molecular mass (230-300 kDa). Selective degradation by chondroitinases and heparinase identified chondroitin and dermatan sulfate PG as the predominant form being induced. These results demonstrate that the effect of A II is not general and is specific to certain classes of PGs. In order to further examine the specificity of the A II effect, we compared the synthesis of PG induced by A II with that induced by platelet-derived growth factors AA and BB (PDGF-AA and -BB), insulin-like growth factor I (IGF-I), and tumor necrosis factor alpha (TNF alpha). This comparison demonstrated that the profile of PG induced by A II is different from the other factors examined. Taken together, these data indicate that A II may not only function as a hypertrophic factor for SMC, but in addition may also be a potent modulator of specific PG synthesis by these same cells, which could significantly contribute to the formation of atherosclerotic and restenotic lesions.

Growth factors in pathogenesis of coronary arterial restenosis.

Restenosis occurs in 25% to 55% of patients within 6 months of successful angioplasty. The major histologic component of the restenotic lesion is intimal hyperplasia, which is almost certainly driven by growth factors. After vascular injury, smooth muscle cells proliferate, reaching a maximum rate at day 2. Smooth muscle cell proliferation diminishes as the vessel surface is re-endothelialized at about day 7, and by week 4 the smooth muscle cell mitotic rate returns to baseline of less than 1% per day. The events of the histologic evolution of arterial injury can be used to create a hypothetical paradigm for the role of growth factors in restenosis. Restenosis might logically be prevented by an inhibitory intervention at any of the various steps in the healing process.

A unique sialoglycopeptide growth regulator that inhibits mitogenic activity of a phorbol ester tumor promoter.

The ability of a naturally occurring cell surface sialoglycopeptide growth inhibitor to antagonize the induction of DNA synthesis by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied with mouse 3T3 cells. The bovine sialoglycopeptide was shown to be a potent antagonist of TPA-induced DNA synthesis in confluent 3T3 cell cultures. Kinetic studies demonstrated that inhibition of TPA-induced DNA synthesis required the addition of the sialoglycopeptide within 15 min of TPA treatment. Addition of the sialoglycopeptide 30 min or longer after the cells were exposed to TPA did not block stimulation of DNA synthesis by TPA. The inhibition of TPA action was shown not to be restricted to DNA synthesis in 3T3 cultured cells since the sialoglycopeptide also inhibited TPA-induced ornithine decarboxylase (ODC, L-ornithine carboxylase, EC 4.1.1.17) activation in suspensions of mouse epidermal and 3T3 cells.

Purification of a cell growth inhibitor from bovine cerebral cortex cells.

A glycopeptide, isolated from bovine cerebral cortex cells and added in only nanogram levels to cells in culture, has been shown to inhibit both cell protein synthesis and cell division. When purified by gel filtration and Ulex europaeus lectin affinity chromatography, the radioiodinated preparation was subjected to high resolution isoelectric focusing and shown to contain three species of macromolecules. The glycopeptide focusing at pH 8.1 comprised over 75% of the radioiodinated material and possessed inhibitory activity against both cell protein synthesis and cell division. A second species that focused at pH 8.3 was also found to be inhibitory to cell metabolism and may have represented a variant of the major glycopeptide.

Cell agglutination by a novel cell surface sialoglycopeptide inhibitor and the relationship between its protease and biological activities.

A bovine sialoglycopeptide, purified to homogeneity and capable of inhibiting cellular protein synthesis and proliferation, was shown to agglutinate a wide variety of nontransformed and transformed cells. The cell agglutination activity was shown to be independent of the biological inhibitory action and most likely related to a protease activity that could not be physically separated during purification of the sialoglycopeptide. Samples that were completely biologically inactivated retained full protease activity and their ability to agglutinate target cells. Balb/c 3T3 cells were not agglutinated by the sialoglycopeptide and they elicited a protein that interfered with the agglutination reaction and even redispursed cells that already had been aggregated by the inhibitor.

Affinity labeling of the sialoglycopeptide antimitogen receptor.

An 18-kDa 125I-sialoglycopeptide growth inhibitor was covalently cross-linked to its binding site on intact cultured Swiss 3T3 cells by three bifunctional cross-linkers with short (dimethyl adipimate), medium (disuccinimidyl suberate), and long (bis(2-succinimidooxycarbonyloxyethyl)sulfone) chain lengths. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated a band of Mr approximately 168,000 regardless of which cross-linker was used. The labeling of this band was specific in that it was prevented by excess unlabeled inhibitor and the apparent molecular weight of the cross-linked receptor-ligand complex was unchanged by treatment with reducing agent. The efficiency of the cross-linking was increased by increasing pH, and the extent of covalent cross-linking was dependent on the concentration of the bifunctional reagent. Octyl glucoside and sodium dodecyl sulfate were effective in solubilizing the receptor while Triton X-100 did not extract the receptor from the plasma membrane. These observations suggest that the 168-kDa binding species represents the 125I-sialoglycopeptide cross-linked to a specific plasma membrane receptor and that the receptor does not appear to contain interchain disulfide bonds.

Abrogation of the mitogenic activity of bombesin by a cell surface sialoglycopeptide growth inhibitor.

The ability of a cell surface sialoglycopeptide (SGP) growth inhibitor to abrogate the mitogenic activity of bombesin, in serum-free medium, was investigated. The SGP was shown to be a potent antagonist of bombesin-induced DNA synthesis as 2.7 nM of the inhibitor completely blocked the mitogenic activity of 5.0 nM of bombesin. Exposure of 3T3 cells to bombesin for only 10 hr was sufficient to initiate the induction of DNA synthesis 10 hr later. Kinetic studies demonstrated that the SGP was able to block bombesin-induced DNA synthesis only when added within 2 hr after exposure of the cells to the mitogen. The SGP inhibitor blocked bombesin mitogenesis at a post-receptor, intracellular, early event in the signal cascade.

The effects of a calcium and a sodium ionophore on protein synthesis inhibition by a bovine cell surface sialoglycopeptide.

The ability of the calcium ionophore A23187 and the sodium ionophore Monensin to antagonize the inhibition of 3T3 cell protein synthesis by a bovine cell surface sialoglycopeptide was measured. A23187, when added before and shortly after the sialoglycopeptide, significantly reduced the biological activity of the inhibitory glycopeptide. In contrast, Monensin had little, if any, influence on protein synthesis inhibition by the sialoglycopeptide. The ability of A23187 to circumvent the inhibitory action of the bovine glycopeptide was shown to be independent of the time the ionophore was incubated with the cells and the binding of the sialoglycopeptide to the 3T3 target cells. Neither the total amount of sialoglycopeptide bound to the cells, nor its affinity to the cell surface receptor, was influenced by the presence of A23187.

Receptor occupancy by a bovine sialoglycopeptide inhibitor correlates with inhibition of protein synthesis.

We have isolated from bovine cerebral cortex cells and purified to homogeneity an 18,000 dalton, pl 3.0 sialoglycopeptide that inhibits protein synthesis and DNA synthesis of nontransformed but not transformed cells without affecting uptake of radiolabeled precursors. In this paper, we examine the relationship between the binding of the sialoglycopeptide inhibitor to 3T3 cells and inhibition of protein synthesis. Binding of the sialoglycopeptide to 3T3 cells was rapid at 37 degrees C and reached a maximum at 30 min; the binding at 37 degrees C was shown to be saturable and specific. Scatchard analysis of the binding indicated that 3T3 cells contained about 2 X 10(4) receptors/cell with a dissociation constant of 1.0-1.5 nM. Several lines of evidence indicated that receptor occupancy on 3T3 cells correlated with the protein synthesis inhibitory activity of the sialoglycopeptide. A comparison of the kinetics of inhibitor binding with the kinetics of protein synthesis inhibition demonstrated that binding directly correlated with the inhibition of protein synthesis, concentration-dependent inhibition of protein synthesis directly correlated with concentration-dependent receptor occupancy, and a direct correlation was also observed between the kinetics of inhibitor dissociation from its specific cell surface receptor and the kinetics of recovery from protein synthesis inhibition.

Cell surface interaction is sufficient for the biological activity of a bovine sialoglycopeptide inhibitor.

A sialoglycopeptide inhibitor, isolated from bovine cerebral cortex cells, that reversibly inhibits protein and DNA synthesis, was coupled to either Sepharose or polyacrylamide beads. Whereas over 1 ng of the inhibitor was released from Sepharose beads after 30 min at 37 degrees C, less than 0.2 ng of the sialoglycopeptide was released from the polyacrylamide beads. When added to 3T3 cells, the immobilized sialoglycopeptide efficiently inhibited protein synthesis. No detectable sialoglycopeptide inhibitor was released into the assay medium in the presence or absence of 3T3 cells. Addition of [125I]sialoglycopeptide, coupled to acrylamide P100 beads, to 3T3 cells also demonstrated that the sialoglycopeptide was not internalized by the cells. Thus we conclude that an interaction of the sialoglycopeptide at the cell surface is sufficient for biological inhibitory activity.