Hyaluronate degradation in 3T3 and simian virus-transformed 3T3 cells

The cellular control of hyaluronate levels was examined in cultures of simian virus 40-transformed 3T3 (SV3T3) and 3T3 cells which are known to differ in their metabolism of hyaluronate. When [3H]hyaluronate was added to cultures of the two cell lines, four times more ligand was bound per mg of protein by the SV3T3 cells than by the 3T3 cells. Of the bound [3H] hyaluronate, 40% was degraded by the SV3T3 cells to oligosaccharides characteristic of the breakdown of hyaluronate, but only 2% was degraded by 3T3 cells. Hyaluronidase activity was found in the cell layer and medium of the SV3T3 cultures, but was not detectable in 3T3 cells. The SV3T3 enzyme was active only at acidic pH, but at neutral pH the secreted SV3T3 hyaluronidase was thermally more stable then the cell-associated enzyme. In contrast, both cell lines were found to contain similar amounts of beta-glucuronidase and beta-N-acetylglucosaminidase activity. We conclude that the elevated capacity of SV3T3 cells to degrade hyaluronate may be partially responsible for their lack of the hyaluronate-containing pericellular coat which is prominent around 3T3 cells.     

Cell density and amino acid transport in 3T3, SV3T3, and SV3T3 revertant cells

The transport of selected neutral and cationic amino acids has been studied in Balb/c 3T3, SV3T3, and SV3T3 revertant cell lines. After properly timed preincubations to control the size of internal amino acid pools, the activity of systems A, ASC, L, and Ly⁺ has been discriminated by measurements of amino acid uptake (initial entry rate) in the presence and absence of sodium and of transportspecific model substrates. L-Proline, 2-aminoisobutyric acid, and glycine were primarily taken up by system A; L-alanine and L-serine by system ASC; L-phenylalanine by system L; and L-lysine by system Ly⁺ in SV3T3 cells. L-Proline and L-serine were also preferential substrates of systems A and ASC, respectively, in 3T3 and SV3T3 revertant cells. Transport activity of the Na⁺-dependent systems A and ASC decreased markedly with the increase of cell density, whereas the activity of the Na⁺-independent systems L and Ly⁺remained substantially unchanged. The density-dependent change in activity of system A occurred through a mechanism affecting transport maximum (V_max) rather than substrate concentration for half-maximal velocity (K_m). Transport activity of systems A and ASC was severalfold higher in transformed SV3T3 cells than in 3T3 parental cells at all the culture densities that could be compared. In SV3T3 revertant cells, transport activity by these systems remained substantially similar to that observed in transformed SV3T3 cells. The results presented here add cell density as a regulatory factor of the activity of systems A and ASC, and show that this control mechanism of amino acid transport is maintained in SV40 virus-transformed 3T3 cells that have lost density-dependent inhibition of growth, as well as in SV3T3 revertant cells that have resumed it.     

Synthesis of 2'-O-deuteriomethyl ribonucleosides Gm-d3, Am-d3, Um-d3 and Cm-d3.

The synthesis of 2'-O-deuteriomethyl ribonucleosides by iodomethane-d3 (99.5 + atom % D) deuteriomethylation of 3',5'-O-tetra(isopropyldisiloxane)-diyl nucleosides, followed by deprotection, is described.     

Endogenous hyaluronate-cell surface interactions in 3T3 and simian virus-transformed 3T3 cells

3T3 cells have a large, pericellular coat which contains 30 times more hyaluronate than the amount of cell surface hyaluronate associated with simian virus 40-transformed 3T3 (SV-3T3) cells. On the other hand, SV-3T3 cells have high affinity binding sites for exogenously added hyaluronate, whereas 3T3 cells have much lower affinity sites. Removal of cell surface hyaluronate from SV-3T3 cells by treatment with hyaluronidase caused a reproducible increase in their maximum binding capacity for exogenous hyaluronate but no significant change in binding affinity or specificity. For 3T3 cells, however, the maximum amount of binding decreased and the affinity of binding increased after hyaluronidase treatment. When endogenous cell surface hyaluronate was labeled metabolically and then the cells incubated in the presence of exogenous unlabeled hyaluronate, the labeled cell surface hyaluronate was quantitatively displaced from the SV-3T3 cells but was not displaced from the 3T3 cells. Chondroitin sulfate and heparin did not displace cell surface hyaluronate from either cell type. Membranes isolated from SV-3T3 cells bound hyaluronate specifically and with high affinity, whereas membranes from 3T3 cells did not consistently bind a significant amount of hyaluronate. We conclude from these studies that the retention of endogenous hyaluronate on the surface of SV-3T3 cells is mediated by binding sites similar to those detected by the addition of exogenous hyaluronate, and the mechanism of retention of endogenous hyaluronate on the surface of 3T3 cells differs from SV-3T3 cells.     

Protein degradation in 3T3 cells and tumorigenic transformed 3T3 cells

To study the relation of overall rates of protein degradation in the control of cell growth, we determined if transformation of fibroblasts to tumorigenicity affected their rates of degradation of short- and long-lived proteins. Rates of protein degradation were measured in nontumorigenic mouse Balb/c 3T3 fibroblasts, and in tumorigenic 3T3 cells transformed by different agents. Growing 3T3 cells, and cells transformed with Moloney sarcoma virus (MA-3T3) or Rous sarcoma virus (RS-3T3), degraded short- and long-lived proteins at similar rates. Simian virus 40 (SV-3T3)- and benzo(a)pyrene (BP-3T3)-transformed cells had slightly lower rates of degradation of both short- and long-lived proteins. Reducing the serum concentration in the culture medium from 10% to 0.5%, immediately caused about a twofold increase in the rate of degradation of long-lived proteins in 3T3 cells. Transformed lines increased their rates of degradation of long-lived proteins only by different amounts upon serum deprivation, but none of them to the same extent as did 3T3. Greater differences in the degradation rates of proteins were seen among the transformed cells than between 3T3 cells and some transformed cells. Thus, there was no consistent change in any rate of protein degradation in 3T3 cells due to transformation to tumorigenicity.     

3-Bromo-3-deazaneplanocin and 3-bromo-3-deazaaristeromycin: Synthesis and antiviral activity

As an outgrowth of our program to explore 3-deazaadenine carbocyclic nucleosides, 3-bromo-3-deazaneplanocin (5) and 3-bromo-3-deazaaristeromycin (6) have been synthesized from a readily available cyclopentenol and cyclopentanone and either 4-amino- or 4-chloro-1H-imidazo[4,5-c]pyridine (6-amino- or 6-chloro-3-deazaadenine) in 5 steps and 7 steps, respectively. Antiviral analysis found 5 to display significant activity towards a number of (-)-ssRNA and a few dsDNA viruses. Compound 6 was less active than 5 against selected examples of those viruses affected by 5.     

Cyclic AMP-dependent protein kinases from Balb 3T3 cells and other 3T3 derived lines

Cyclic AMP-dependent protein kinase and 3H-cAMP-binding activities were determined in normal Balb 3T3 cells and compared with the same preparations from SV40, chemical, and spontaneous transformants of 3T3 cells. The cytosolic protein kinase activities and protein kinase activity ratios were similar in all cell lines, although when the normal 3T3 cytosol was prepared by homogenization it contained less 3H-cAMP binding activity than the transformed 3T3 cytosols. The Triton X-100 treated particulate fractions from the normal and transformed 3T3 cells contained similar protein kinase and binding activities. The isozymic profile of cAMP-dependent protein kinases was examined by DEAE-chromatography. The 3T3 cells contained only type II isozyme in either cytosolic or membrane fractions. All transformants of the 3T3 cells contained both type I and type II isozymes. Other cell cultures, including chicken embryo fibroblasts, rat kidney cells, and human or calf endothelial cells contained type I and type II isozymes. Binding of the photoaffinity analogue of cAMP, 8-N3 cAMP, to the regulatory subunits of protein kinases in sonicates obtained from Balb 3T3 and SV 3T3 cells followed by separation on SDS polyacrylamide electrophoresis showed that the amount of RII subunit was approximately equal in the two cell lines. RI in Balb 3T3 cells was detectable but in a much lower quantity than in SV 3T3 cells. The cyclic AMP dependent-protein kinases from Balb 3T3 cells appears to be different from SV 3T3 cells by three criteria: 3H-cAMP binding in homogenates, DEAE chromatographic separation of isozymes, and 8-N3 cAMP binding.     

Heparan sulfates from Swiss mouse 3T3 and SV3T3 cells: O-sulfate difference

A difference in the extent of sulfation between the heparan sulfate isolated from Swiss 3T3 mouse cells and that from Swiss 3T3 cells transformed by the DNA virus SV40 has been reported previously. This variance is manifested by different chromatographic and electrophoretic properties. Heparan sulfates from the two cell types were treated with nitrous acid under conditions that gave selective deaminative cleavage of glucosaminyl residues with sulfated amino groups in order to define the nature of the difference in sulfation further. The O-sulfate containing fragments from the heparan sulfates were compared by gel filtration and ion-exchange chromatography. The results showed that the 3T3 heparan sulfate contains 8% more O-sulfate than does the SV3T3 heparan sulfate. Analysis of uronic acids revealed that both types of heparan sulfates contain 45% L-iduronic acid and 55% D-glucuronic acid. These and other observations indicate that the primary difference in sulfation between the 3T3 and SV3T3 heparan sulfates lies in the extent of O-sulfation.     

Anaerobic degradation of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate

The anaerobic degradation of the polyesters poly-3-hydroxybutyrate (PHB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) was investigated with special regard to intermediate products, kinetics, and yields. During the degradation of PHBV acetate, propionate, n-butyrate, and n-valerate were detected. Additionally, 3-hydroxybutyrate and 3-hydroxyvalerate and four dimeric esters of these two molecules were identified by GC-MS measurements. Three different test systems for the anaerobic degradation of polyesters were studied. It was not possible to get reproducible results by means of the Anaerobic Sturm-test, a simple system based on carbon dioxide measurement. Secondly, a system based on the GC measurement of accumulated organic acids was investigated. A degradation of 90% in two days was calculated by a carbon balance. Best results were reached with the third test system based on the measurement of methane with a gas meter. A degradation of 99% was observed within 30 days.     

14-3-3 proteins--an update

14-3-3 is a highly conserved acidic protein family, composed of seven isoforms in mammals. 14-3-3 protein can interact with over 200 target proteins by phosphoserine-dependent and phosphoserine-independent manners. Little is known about the consequences of these interactions, and thus are the subjects of ongoing studies. 14-3-3 controls cell cycle, cell growth, differentiation, survival, apoptosis, migration and spreading. Recent studies have revealed new mechanisms and new functions of 14-3-3, giving us more insights on this fascinating and complex family of proteins. Of all the seven isoforms, 14-3-3σ seems to be directly involved in human cancer. 14-3-3σ itself is subject to regulation by p53 upon DNA damage and by epigenetic deregulation. Gene silencing of 14-3-3σ by CpG methylation has been found in many human cancer types. This suggests that therapy-targeting 14-3-3σ may be beneficial for future cancer treatment.     

The binding and processing of plasminogen by Balb/c 3T3 and SV3T3 cells

The binding and processing of plasminogen by Balb/c 3T3 and SV3T3 cells was studied using ¹²⁵I-labeled canine plasminogen. Throughout a 3-day period, ¹²⁵I-plasminogen in the incubation medium bound to the cells and was degraded, first to intermediate-sized macromolecules that were the same size as the large (74,600-dalton) and small (25,000-dalton) chains of active plasmin, and to smaller fragments including 3-iodo-L-tyrosine. Binding to SV3T3 cells was independent of the protease-dependent morphological change (PDMC)¹ characteristic of these and many other transformed cells. The SV3T3, and to a somewhat lesser extent, the 3T3 cells, both accumulated and released into the incubation medium 3-iodo-L-tyrosine, a terminal lysozymal digestion product. The results of a sublethal cell-surface trypsinization assay suggest that the cell-associated plasminogen was primarily bound to the surfaces of the 3T3 and SV3T3 cells while the macromolecular degradation products including active plasmin were inside the cells. The rate of ¹²⁵I plasminogen degradation exhibited by SV3T3 cells was approximately two times greater than that of 3T3 cells, which presumably reflects differences in endocytosis or lysosomal hydrolysis, or both. The rates were unaffected by addition of pancreatic or soybean trypsin inhibitor sufficient to inhibit PDMC. In the incubation medium, plasminogen was activated to plasmin by SV3T3, but not by 3T3 cells. However, 95–100% of plasmin covalently bound to a 47,000-dalton canine serum component, which could be dissociated from plasmin by hydroxylamine: 95–100% of the plasmin was inactive to reaction with DF³²P. Thus the serum component is a plasmin inhibitor. The plasmin-containing complex in the medium had an apparent molecular weight of 212,000. Under denaturing conditions, the complex dissociated into two covalently modified plasmin-containing species of 153,000 and 127,000 daltons. In addition to forming a complex with a serum component, the plasmin is cleaved into two small fragments (～10,000 and 12,000 daltons) by as-yet uncharacterized serum factors.     

Ether-linked lipids of Balb/c3T3, SV3T3 and concanavalin A-selected SV3T3 revertant cells

Ether-linked lipids were analyzed in Balb/c3T3, SV3T3 and Concanavalin A-selected SV3T3 revertant cells. The three cell lines were found to contain significant quantities of alk-1-enyl- and alkyl-linked phosphatidylethanolamine (PE) and phosphatidylcholine (PC) and small amounts of alkyldiacylglycerols. Compared to 3T3 cells, SV3T3 cells contain a higher amount of alk-1-enyl-linked PC, while in SV3T3 revertant cells the concentrations of the various ether lipids are similar to those of 3T3 cells. The major difference in the composition of ether groups of SV3T3 cells, compared to 3T3 cells, is an increase of 18:0 accompanied by a decrease of 18:1 in the alk-1-enyl-linked PE and PC. Alk-1-enyl-linked PC of SV3T3 revertant cells also shows an increase of 18:0, while the decrease of 18:1 was not statistically significant.     

Properties of 3-hydroxypropionaldehyde 3-phosphate.

Several modifications to the synthesis of the diethyl acetal of 3-hydroxypropionaldehyde-3-P (HPAP) are described. HPAP is liberated from its acetal by treatment with Dowex 50-H⁺ at 40 °C for 4 min, and longer time or higher temperature lower yields. Breakdown of the dianion of HPAP (pK 6.7) is self-catalyzed, with the phosphate acting as a general base to remove a proton from carbon 2 and allow elimination of phosphate to give acrolein. Monoanion breakdown is at least 400-fold slower. At 25 °C the dianion breaks down with k = 0.025 min^?1, and the activation energy for the process is 24 kcal/mol. Buffers have little effect on breakdown of HPAP, except for those containing primary or secondary amines. Thus morpholine enhances breakdown by forming a Schiff's base with a positively charged nitrogen, and Tris inhibits breakdown by forming one with an uncharged nitrogen. The aldehyde group of HPAP is 60% hydrated in water.     

Anaerobic degradation of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate

The anaerobic degradation of the polyesterspoly-3-hydroxybutyrate (PHB) andpoly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) wasinvestigated with special regard to intermediateproducts, kinetics, and yields. During the degradationof PHBV acetate, propionate, n-butyrate, andn-valerate were detected. Additionally,3-hydroxybutyrate and 3-hydroxyvalerate and fourdimeric esters of these two molecules were identifiedby GC-MS measurements. Three different test systemsfor the anaerobic degradation of polyesters werestudied. It was not possible to get reproducibleresults by means of the Anaerobic Sturm-test, a simplesystem based on carbon dioxide measurement. Secondly,a system based on the GC measurement of accumulatedorganic acids was investigated. A degradation of 90%in two days was calculated by a carbon balance. Bestresults were reached with the third test system basedon the measurement of methane with a gas meter. Adegradation of 99% was observed within 30 days.     

Synthesis of 3-(3-pyridyl)- and 3-(3-benzo[b]thienyl)-D-alanine

The DL-arylamino acid ethyl ester derivatives of beta-(3-pyridyl)-DL-alanine, and beta-(3-benzo[b]thienyl)-DL-alanine were synthesized by diethyl acetamidomalonate condensation with the respective arylmethyl halides followed by partial hydrolysis to the monoethyl ester and decarboxylation. Each derivative was enzymatically resolved to a separable mixture of the corresponding N-acetyl-L-amino acid and the unchanged D amino acid derivative. Acidic hydrolysis of the latter gave the corresponding D-amino acid, the optical purity of which was established by HPLC analysis of the 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC) derivative. The free D amino acids were converted to D-BOC derivatives by reaction with di-tert-butyldicarbonate in tert-butyl alcohol, water and sodium hydroxide.     

Phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase

Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase has been purified to apparent homogeneity by a process involving the following steps: solubilization from microsomes and chromatography on Affi-Gel Blue, phosphocellulose, Bio-Gel A 1.5m, and agarose-hexane-ATP. The apparent M_r of the purified enzyme as judged by gel-filtration chromatography is 205,000 and by sodium dodecyl sulfate-gel electrophoresis is 105,000. Immunoprecipitation of homogeneous reductase phosphorylated by reductase kinase and [γ-³²P]ATP produces a unique band containing ³²P bound to protein which migrates at the same R_f as the reductase subunit. Incubation of ³²P-labeled HMG-CoA reductase with reductase phosphatase results in a time-dependent loss of protein-bound ³²P radioactivity, as well as an increase in enzymic activity. Reductase kinase, when incubated with ATP, undergoes autophosphorylation, and a simultaneous increase in its enzymatic activity is observed. Tryptic treatment of immunoprecipitated, ³²P-labeled HMG-CoA reductase phosphorylated with reductase kinase produces only one ³²P-labeled phosphopeptide with the same R_f as one of the two tryptic phosphopeptides that have been reported in a previous paper. The possible existence of a second microsomal reductase kinase is discussed.     

Synchronization of mouse 3T3 and SV40 3T3 cells by way of centrifugal elutriation

Populations of G1 phase 3T3 and SV40 3T3 mouse fibroblasts have been isolated from exponentially growing cultures by the technique of centrifugal elutriation. Return of the G1 phase cells to growth conditions results in their synchronous passage through the cell cycle, as determined from monitoring of cell number, [³H]thymidine ([³H]TdR) incorporation and fraction of [³H]TdR labeled nuclei. The durations of G1, S and G2 phases are consistent with values obtained by previous investigators using conventional induction techniques for synchronization. The method for isolation of the G1 phase cells is rapid, the yield is high and the process does not appear to alter the temporal aspects of the cell cycle in either cell type.     

Export of Galectin-3 from Nuclei of Digitonin-Permeabilized Mouse 3T3 Fibroblasts

Galectin-3 is a galactose-/lactose-binding protein (M(r) approximately 30,000), identified as a required factor in the splicing of pre-mRNA. Immunofluorescence staining revealed that galectin-3 distributes differentially between the nucleus and the cytoplasm, depending on the proliferative state of the cells under analysis. Using digitonin-permeabilized mouse 3T3 fibroblasts, we provide evidence that galectin-3 is rapidly and selectively exported from the nucleus. Although both phosphorylated and nonphosphorylated isoforms of galectin-3 are found in the nuclear fraction, only phosphorylated galectin-3 is identified in the exported fraction, implying that phosphorylation is important for the nuclear export of the protein. The rate of galectin-3 export is temperature dependent and is decreased by the addition of wheat germ agglutinin. More strikingly, galectin-3 export can be inhibited by the addition of leptomycin B, a drug that disrupts the interaction between the leucine-rich nuclear export signal and its receptor, CRM1 (chromosome maintenance region 1). Indeed, a putative leucine-rich nuclear export signal can be found in residues 241-249 of the murine galectin-3 sequence. Finally, gel filtration of the exported material showed that galectin-3 can be found in at least two high molecular weight complexes (approximately 650 and approximately 60 kDa), both of which can be disrupted by lactose.     

Calcium transport and exchange in mouse 3T3 and SV40-3T3 cells

The kinetics of Ca++ uptake have been evaluated in 3T3 and SV40-3T3 mouse cells. The data reveal at least two exchangeable cellular compartments in the 3T3 and SV40-3T3 cell over a 50-min exposure to 45Ca++. A rapidly exchanging compartment may represent surface-membrane-localized Ca++ whereas a more slowly exchanging compartment is presumably intracellular. The transition of the 3T3 cell from exponential growth (at 3 day's incubation) to quiescence (at 7 days) is characterized by a 7.5-fold increase in the size of the fast component. Quiescence of the 3T3 cell is also characterized by a 3.2-fold increase in the unidirectional Ca++ influx into the slowly exchanging compartment and a 3.6-fold increase in its size. The increase in size of the slow compartment at quiescence may result from a redistribution of intracellular Ca++ to a more readily exchangeable compartment, possibly reflecting a release of previously bound Ca++. In contrast, no significant change in any of these parameters is observed in the proliferatively active SV40-3T3 cells after corresponding period of incubation, even though these cells attained higher growth densities and underwent postconfluence.