Glycopeptides prepared from mouse cerebrum inhibit protein synthesis and cell division in baby hamster kidney cells, but not in their polyoma virus-transformed analogs

Glycopeptides isolated from mouse cerebral cortex cell surfaces (BCSG) were shown to inhibit cell growth and protein synthesis in baby hamster kidney (BHK)-21 cells, whereas polyoma virus-transformed BHK-21 cells (pyBHK-21) were refractory to the inhibitory activity of the glycopeptides. Growth inhibition was shown to be reversible and non-lethal to BHK-21 cells. Despite that difference in sensitivity to the action of the glycopeptides, both cell lines could bind the inhibitor in a saturable fashion and in similar quantities. After trypsinization, BHK-21 cells appeared refractory to the inhibitor, whereas pyBHK-21 cells became sensitive. The data suggested the presence of a receptor for BCSG on the cell surface of both cell lines. Incubating BCSG with conditioned medium from pyBHK-21 cells resulted in loss of the glycopeptide's inhibitory activity. In contrast, medium conditioned by BHK-21 cells had no effect on the inhibitory activity of BCSG. We hypothesize that the refractoriness of pyBHK-21 cells to BCSG is related to their autonomous growth characteristics and failure to respond to topo-inhibitory growth control. BCSG may be a naturally occurring growth regulator whose function can be explored by use of the BHK-21/ pyBHK-21 model system.     

An inhibitor of protein synthesis prepared by protease treatment of mouse cerebral cortex cells

A substance was isolated from mouse brain cortical tissue that inhibits both cell division and protein synthesis by cells in culture. The inhibitor was released from cerebral cortex tissue by mild protease treatment. A single exposure of cells to as little as 1.25 μg of the isolated material was sufficient to inhibit BHK-21 cell protein synthesis by 20%. Higher concentrations and continual exposure resulted in 87% reduction in protein synthesis. The inhibition was shown to be independent of amino acid uptake and most effective against primary mouse embryo fibroblasts and neonatal mouse brain cell suspensions. Cells previously adapted to culture or transformed cells derived from the nervous system were less affected by, or refractory to, the inhibitor. The substance was shown to be nondialyzable, relatively resistant to thermal inactivation and the inhibitor activity was not removed by chloroform extraction. Two active fractions were identified by Bio-Gel P-100 chromatography and the protein synthetic inhibitor was removed by affinity chromatography with $\text{Ulex}$$\text{europus}$ agglutinin.     

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 $\text{Ulex}$$\text{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.     

The role of gangliosides in the interaction of a growth inhibitor with mouse LM cells

We have isolated and characterized glycopeptides, derived from mouse and bovine cerebral cortex cells, that inhibit protein synthesis and cell growth of normal but not transformed cells. The inhibitor binds to target cell surfaces, and gangliosides have previously been shown to influence cell sensitivity to the glycopeptides. Preincubation with 3.0 micrograms/ml ganglioside GM1 at 0 degrees C for 3 hr sensitized the mouse L-cell line to the inhibitor, as determined by protein synthesis assays. Preincubation of LM cells with ganglioside GM1 alone did not affect protein synthesis rates. In addition, the gangliosides GD1a and GM3 also sensitized the LM cells to the protein synthesis inhibitory effect of the glycopeptide inhibitor. Binding experiments were performed with 3T3 (sensitive) and LM (insensitive) cells to determine if sensitivity to the glycopeptide inhibitor was reflected in binding of the inhibitor to these cells. Binding of 125I-labeled inhibitor to 3T3 cells was maximal after 60 min at 0 degrees C and saturable at approximately 1 X 10(4) molecules/cell. Furthermore, binding of the inhibitor was dose-dependent, with half-maximal binding at 1.5-2.0 nM and saturation at 8.0-10.0 nM. Scatchard plot analysis indicated that the Kd was about 1 X 10(-9) M and that there are 1 X 10(4) receptors/cell. Binding of the inhibitor to LM cells was maximal after 30 min at 0 degrees C and saturation occurred at 5 X 10(3) molecules/cell. We then examined the possibility that gangliosides are the cellular receptor or co-receptor for the glycopeptide inhibitor. Binding of the inhibitor to ganglioside GM1 was first examined after the ganglioside had been preadsorbed to polystyrene tubes. These experiments indicated that the ganglioside did not bind the inhibitor. Ganglioside-containing liposomes from phosphatidylcholine or LM cell membrane components were also prepared; these artificial membranes did not bind appreciable amounts of the iodinated inhibitor. Competition experiments showed that the gangliosides GM1 and GD1a did not neutralize the protein synthesis inhibitory activity of the glycopeptides, indicating that gangliosides do not directly interact with the glycopeptide inhibitor. In addition, binding of the inhibitor to LM cells preincubated with ganglioside GM1 was studied. Although the binding of the inhibitor to LM cells was one-half that observed for 3T3 cells, incorporation of exogenous gangliosides into LM cells did not result in increased binding of the inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glycopeptides from brain inhibit rates of polypeptide chain elongation

In previous reports, we have identified cell-surface glycopeptides from mouse cerebrum (BCSG) that inhibited protein synthesis and mitosis in several cell types. When baby hamster kidney (BHK)-21 cells were infected with vesicular stomatitis virus (a negative strand RNA virus), BCSG extensively inhibited viral protein synthesis. This inhibition was effective against both protein and glycoprotein synthesis and was independent of amino acid uptake by infected cells, synthesis of viral RNA, and degradation of viral proteins. Analysis of polyribosome profiles in uninfected BHK-21 cells indicated that the degree of cellular protein synthesis inhibition could not be attributed to activation of RNase or solely to a disruption of chain initiation. When added directly to a cell-free protein-synthesizing system derived from BHK-21 cells, BCSG was ineffective, but if the inhibitory material was first allowed to react with cells, cell-free protein synthesis was substantially reduced. When BCSG were reacted with cells for 5 min at 0 degrees C, the cells tested, BHK-21 (a BCSG-sensitive line) and murine fibrosarcoma 2237 (a BCSG-insensitive line), both effectively adsorbed the inhibitor from the medium.     

A cell surface glycoprotein virus inhibitor that is not interferon

The possible relationship between a newly isolated glycoprotein virus inhibitor and interferon was assessed. Comparisons of the cell surface glycopeptide, obtained from mouse cerebral cortex, and interferon included antiviral activity, radioimmune assays, and the ability of antibodies raised against the brain cell surface glycoprotein (BCSG) and against mouse L cell interferon to precipitate the biological activity. BCSG was able to inhibit virus replication but only in a transient fashion. Although anti-BCSG precipitated a major portion of the radiolabelled inhibitor in a double antibody assay, anti-mouse interferon did not. Over 90% of the inhibitory activity was removed with anti-BCSG and $\text{Staphylococcus}$ protein A while anti-mouse interferon removed little, or none, of the activity under similar reaction conditions. Other properties of the BCSG that distinguish it from interferon are presented.     

Purification and partial-characterization of a protease inhibitor from Drosophila melanogaster

A protein from Drosophila melanogaster which inhibits bovine alpha-chymotrypsin activity was purified using an extensive extraction procedure. SP-Sephadex column chromatography and affinity column chromatography. The inhibitor has an estimated molecular weight of approx. 12 000 and is extremely pH and heat stable. It did not exhibit any inhibitory activity against trypsin from numerous sources nor mosquito larval chymotrypsin but did inhibit adult mosquito chymotrypsin. Chymotrypsin-like activity has not been found in Drosophila and therefore the function of the inhibitor is unknown. Preliminary work indicates that it effectively inhibits cathepsin D activity from a nematode parasite and rabbit liver.     

G2 cell cycle arrest induced by glycopeptides isolated from the bovine cerebral cortex

The ability of glycopeptides, isolated from bovine cerebral cortex, to alter cell division was studied by cell-cycle analyses. The results showed that glycopeptides arrested baby hamster kidney (BHK)-21 cells and Chinese hamster ovary (CHO) cells in the G2 phase of the cell cycle. Upon removal of the growth inhibition from arrested BHK-21 cells, the mitotic index in colchicine-treated cultures increased from 5 to 40% within 6 h and the increase in mitotic activity was accompanied by a complete doubling of all arrested cells within this 6- h time period. Determination of DNA content in growth-arrested BHK-21 cells showed that growth-arrested cells contained about twice the DNA of control cell cultures. Although CHO cells treated in a like manner with growth inhibitor could not be arrested for the same length of time as BHK-21 cells (18 h vs. 72 h before initiation of escape) and to the same degree (60% of the cell population vs. 99% of BHK-21 cells), the escape kinetics of CHO cells did indicate a G2 arrest. Approximately 3.5 h after escape began, CHO cell numbers in treated cultures attained the cell numbers found in control cultures. This rapid growth phase occurring in less than 4 h indicated that the growth inhibitor induced a G2 arrest-point in CHO cells that was not lethal since the entire arrested cell population divided.     

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.     

Growth inhibitory activity in extracts from human fibroblasts

Indirect evidence for the presence of a growth inhibitor in normal human fibroblasts has been obtained previously; the inhibitory activity has been found associated with crude cell extracts, but the molecule responsible for the growth inhibition has never been isolated. We have isolated a glycopeptide fraction from human fibroblast cultures, whose synthesis decreases when the cells are stimulated into the division cycle. It was separated by electric charge, lectin affinity, and molecular mass. When added to quiescent cells simultaneously with a growth stimulus, the glycopeptide reduces DNA synthesis activity. The relationship of the kinetics of the synthesis of the glycopeptide with the cell division cycle and its molecular weight are different from what has been described so far for other growth regulators. The decreased synthesis of this inhibitor, induced by growth factors, seems to be one of the requirements for the initiation of the division cycle by human fibroblasts. This response to growth factors was stable during the lifespan of the fibroblast population and became less pronounced only in cells at the end of their replicative potential. © 1993 Wiley-Liss, Inc.     

Purification and characterization of a cytostatic factor with anti-viral activity from the bitter melon

We have previously reported that a crude aqueous extract of the bitter melon (Momordica charantia) has both cytostatic and cytotoxic activities, and is a competitive inhibitor of guanylate cyclase activity. This crude preparation kills human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocytes at these same doses. In this report we describe the purification and characterization of one of these cytostatic factors which also exhibits anti-viral activity. The partially purified factor was both cytostatic to BHK-21 cells and inhibitory to VSV plaque formation in a dose-dependent manner. This preparation was inhibitory to both viral and host cell RNA and protein synthesis as early as 30 min after addition to these samples. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this purified factor is a single component with a molecular weight corresponding to 40,000 daltons. The factor is sensitive to boiling and to pre-treatments with trypsin, but not ribonuclease (RNAse), or deoxyribonuclease (DNAse). As determined by radioactive precursor uptake and incorporation studies, the purified factor inhibits both RNA and protein synthesis in intact tissue culture cells and inhibits protein synthesis in a cell-free wheat germ system. DNA synthesis was slightly stimulated. The purified factor is cytostatic for both BHK-21 and for the IM9 leukemic cell lines for at least 120 h. The cytostatic component had no effect on cellular cyclic GMP metabolism.     

Purification and Characterization of a Bovine Cerebral Cortex Cell Surface Sialoglycopeptide that Inhibits Cell Proliferation and Metabolism

A sialoglycopeptide from bovine cerebral cortex cells was purified to apparent homogeneity by a procedure that included chloroform/methanol extraction, diethylaminoethyl ion exchange chromatography, wheat germ agglutinin affinity chromatography, size-exclusion HPLC, and hydrophobic interaction chromatography. The cell surface inhibitor had a molecular weight of approximately 18,000, no subunit composition was detectable on reduction and polyacrylamide gel electrophoresis analysis, and the glycopeptide apparently contained sialic acid, as illustrated by its ability to bind to Limulus polyhemus lectin. Deglycosylation of the molecule, however, did not reduce its protein synthesis inhibitory activity. As little as 20 ng of the sialoglycopeptide was capable of inhibiting protein synthesis in a wide variety of fibroblast cell lines but not in transformed cells. Mice immunized with the sialoglycopeptide produced antibodies that, when bound to protein A-agarose gel, removed the inhibitory activity from solution. The antibodies were used to identify a single isoelectric focused band and to establish the pI of 3.0 for the molecule.     

Platelet adenylate cyclase and phospholipase C are affected differentially by ADP-ribosylation. Effects on thrombin-mediated responses.

During the isolation of the activator protein for glucosylceramide beta-glucosidase, we found that certain column fractions contained an inhibitor of the enzyme. After separation from the activator protein by a DEAE-Sephacel column, the inhibitor was purified further with a Spehadex G-75 column. The u.v. absorption spectrum of the purified material was similar to that of nucleic acids and the protein content of the purified material was negligible. Furthermore the purified inhibitor reacted with orcinol but not with diphenylamine, and its inhibitory activity was completely destroyed by treatment with RNAases. It seems likely that the purified inhibitor was tRNA. Authentic RNA, tRNA and DNA had similar inhibitory effects on beta-glucosidase (Ki 17 micrograms/ml for tRNA, noncompetitive toward the substrate). The inhibitory effect of nucleic acids was not fully overcome by an excess amount of the activator protein, but phosphatidylserine could restore the activity to normal. Tests with several other hydrolases revealed that the inhibitory effect of nucleic acids was fairly general.     

A synthetic operon containing 14 bovine pancreatic trypsin inhibitor genes is expressed in E. coli.

A synthetic gene encoding the protein sequence of mature bovine pancreatic trypsin inhibitor (BPTI) has been cloned into a novel E. coli expression vector. After in vitro gene amplification by successive DNA duplications, more than 600 000 mostly inactive inhibitor molecules may be recovered from a single cell. After purification the inhibitory activity can be reconstituted almost completely. The specificity of BPTI for trypsin is abolished by a single amino acid exchange from lysine to isoleucine at position 15. The altered protein is shown to be an efficient inhibitor of human leukocyte elastase.     

Inhibition of DNA synthesis and cell division by a cell surface sialoglycopeptide

We have isolated and purified a cell surface sialoglycopeptide (SGP) from bovine cerebral cortex cells that previously was shown to be a potent inhibitor of cellular protein synthesis. The following studies were carried out to characterize the potential ability of the SGP to inhibit DNA synthesis and to arrest cell division. Treatment of exponentially proliferating Swiss 3T3 cells with the SGP inhibitor resulted in a marked inhibition of thymidine incorporation within 24 h. When the SGP was removed from inhibited cultures, a sharp rise in 3H-thymidine incorporation followed within 3-4 h that peaked well above that measured in exponentially growing cultures, suggesting that the inhibitory action of the SGP was reversible and that a significant proportion of the arrested cells was synchronized in the mitotic cycle. In addition to DNA synthesis, the inhibitory action of the SGP was monitored by direct measurement of cell number. Consistent with the thymidine incorporation data, the SGP completely inhibited 3T3 cell division 20 h after its addition to exponentially growing cultures. Upon reversal there was a delay of 15 h before cell division resumed, when the arrested cells quickly doubled. Most, if not all, of the growth-arrested cells appeared to have been synchronized by the SGP. The SGP inhibited DNA synthesis in a surprisingly wide variety of target cells, and the relative degree of their sensitivity to the inhibitor was remarkably similar. Cells sensitive to the SGP ranged from vertebrate to invertebrate cells, fibroblast and epitheliallike cells, primary cells and established cell cultures, as well as a wide range of transformed cell lines.     

Glial fibrillary acidic protein from bovin and rat brain Degradation in tissues and homogenates

Compared with human material glial fibrillary acidic protein isolated from bovine, rat and mouse brain was remarkably homogeneous and migrated as a single band at 54 000 mol. wt. on sodium dodecyl sulfate gel electrophoresis. The protein was extremely susceptible to proteolysis and lower molecular weight components were invariably isolated together with the major species when the brain was not rapidly frozen. Further degradation of the 54 000 mol. wt. polypeptide in bovine tissues incubated at 24 °C resulted in preparations essentially identical to those previously isolated from human autopsy material and separating into a series of immunologically active polypeptides ranging in molecular weight from 54 000 to approximately 40 500. The gel band pattern obtained after progressively longer periods of autolysis suggested that small fragments were cleaved from the original polypeptide in successive steps of degradation. As in human brain, the lower molecular weight products in the 45 000–40 500 range were more resistant to proteolysis and still present after prolonged periods of tissue autolysis. The effect of the pH and of proteinase inhibitors on degradation was studied in homogenates of bovine brain stem incubated at 37 °C. At pH 8.0 proteolysis of the glial fibrillary acidic protein followed essentially the same pattern as in tissue. Cleavage of the major species was not prevented by the addition of proteinase inhibitors. At pH 6.0 and 6.5 a different type of degradation was observed, with rapid breakdown of the protein and loss of immunological activity. Increased solubility in buffer solutions was another effect of autolysis. Compared with cerebral cortex and brain stem, where most of the protein was water soluble, only a small fraction was extracted with buffer from bovine white matter. However, the solubility markedly increased following incubation and comparable amounts were extracted in buffer and in 6 M urea.     

Inhibition of pseudorabies virus replication by vesicular stomatitis virus. II Activity of defective interfering particles.

Purified defective interfering (DI) particles of vesicular stomatitis virus (VSV) inhibit the replication of a heterologous virus, pseudorabies virus (PSR), in hamster (BHK-21) and rabbit (RC-60) cell lines. In contrast to infectious B particles of VSV, UV irradiation of DI particles does not reduce their ability to inhibit PSR replication. However, UV irradiation progressively reduces the ability of DI particles to cause homologous interference with B particle replication. Pretreatment with interferon does not affect the ability of DI particles to inhibit PSR replication in a rabbit cell line (RC-60) in which RNA, but not DNA, viruses are sensitive to the action of interferon. Under similar conditions of interferon pretreatment, the inhibition of PSR by B particles is blocked. These data suggest that de novo VSV RNA or protein synthesis is not required for the inhibition of PSR replication by DI particles. DI particles that inhibit PSR replication also inhibit host RNA and protein synthesis in BHK-21 and RC-60 cells. Based on the results described and data in the literature, it is proposed that the same component of VSV B and DI particles is responsible for most, if not all, of the inhibitory activities of VSV, except homologous interference.     

Peptide analogs of the pseudosubstrate domain of smooth muscle myosin light chain kinase inhibit actomyosin ATPase activity at concentrations that do not inhibit superprecipitation.

The inhibitory effect of calmodulin antagonists, synthetic peptide analogs of the pseudosubstrate domain of smooth muscle MLC kinase, and an inhibitor based on the sequence of MLC were examined using bovine aortic actomyosin and isolated chicken gizzard MLC. Much lower concentrations of the peptides were necessary to inhibit actomyosin ATPase activity than to inhibit superprecipitation. In contrast, calmodulin antagonists inhibited both ATPase activity and superprecipitation at similar concentrations. The peptide analogs were competitive with isolated MLC, but not calmodulin, for inhibition of MLC kinase. These results suggest that in addition to the calmodulin dependence of MLC phosphorylation, a second calmodulin-like protein may be important in actin-myosin interactions. The data also suggest that the pseudosubstrate hypothesis may not completely account for regulation of MLC kinase activity.     

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.     

腺相关病毒载体介导的Endostatin及K1-5蛋白抗肿瘤血管生成协同作用

为研究腺相关病毒载体介导的内皮抑素(Endostatin)及K1-5蛋白抗肿瘤血管生成协同作用,实验中用含有Endostatin及K1-5基因的重组病毒rAAV-hE和rAAV-K5分别感染BHK-21细胞,并表达分泌出内皮抑素和K1-5蛋白,EIA法测定培养液上清中内皮抑素浓度达36.42ng/m l,免疫斑点印迹实验表明rAAV-K5可介导K1-5的体外表达。重组腺相关病毒(rAAV)介导所表达的内皮抑素和K1-5蛋白对血管内皮细胞增殖具有抑制作用。二者对人脐静脉内皮细胞(ECV-304)的抑制率分别为68.1%和63.1%,对牛毛细血管内皮细胞(BCE)的抑制率分别为41.6%和34.0%。同时二者还有协同效应,用rAAV-hE和rAAV-K5同时感染BHK-21细胞,表达产物对人脐静脉内皮细胞和牛毛细血管内皮细胞的抑制率分别为70.8%和43.8%。动物实验表明,重组病毒rAAV-hE和rAAV-K5转入荷有人肺肿瘤细胞的裸鼠,对肿瘤细胞生长具有抑制作用,同时二者具有协同抗肿瘤作用。