An inhibitor of protein synthesis in cytoplasmic extracts of density inhibited cells

Cytoplasmic extracts of green monkey kidney-Vero M3 cells that have been grown to high cell density and have entered the stationary phase of growth lose the capacity to synthesize proteins after they have been frozen and thawed. The same loss of protein synthetic capacity is not observed when cytoplasmic extracts of low cell density Vero M3 or HeLa S-3 cells are frozen and thawed before assay, nor when high cell density Vero M3 cell extract is assayed without having been frozen. The loss of the protein synthesis capacity of the frozen and thawed extracts of stationary phase Vero M3 cells is accounted for by the appearance of an inhibitor. The inhibitor is precipitated in the 30 to 70% ammonium sulfate fraction of the 100,000 g supernatant. It is inactivated by heat and is non-dialyzable. It blocks the transfer of amino acids from tRNA to growing peptide chains. The amount of this inhibitor in the extract (measured by relative inhibition of in vitro protein synthesis) increases as a function of the density of the cells from which the extract was made, and the increase can be correlated with the progressive turnoff of protein synthesis in whole cells.     

Cryopreservation of human endothelial cells for vascular tissue engineering

To investigate the influence of cryopreservation on endothelial cell growth, morphology, and function human umbilical vein endothelial cells (HUVECs) were frozen following a standard protocol. Cell suspensions were exposed to 10% dimethyl sulfoxide in a high-potassium solution, cooled to -80 degrees C at 1 degrees C/min and stored in liquid nitrogen for 7-36 days. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution. The growth of cell suspensions was assayed by culturing 7300 cells/cm2 for 3-5 days in order to determine the cell multiplication factor. Fresh and cryopreserved/thawed cells were analyzed for their growth, and their anti-inflammatory and anti-coagulant function by using cellular ELISA. Cryopreservation resulted in a retrieval of 66 +/- 5% and a viability of 79 +/- 3%. Cryopreserved/thawed and fresh cells showed identical doubling times and identical cell counts in the confluent monolayers. However, the lag phase of thawed HUVECs was approximately 36 h longer, resulting in significant differences in the cell multiplication factor at 3 and 5 days after seeding. After expansion to a sufficient cell count the lag phases were identical. Fresh and cryopreserved/thawed cells showed comparable anti-inflammatory and anti-coagulant activity, as judged by the basal and TNF-induced VCAM-1, ICAM-1, E-selectin, and thrombomodulin expression. Cryopreserved/thawed and recultivated endothelial cells are suitable for endothelialization of autologous allograft veins. Such tissue-engineered grafts will offer the necessary clinical safety for those patients who lack autologous material.     

Multiple thymine dimer excising nuclease activities in extracts of human KB cells

Crude extracts of human KB cells grown in suspension culture contain enzyme activity that catalyzes the preferential excision of thymine-containing pyrimidine dimers from UV-irradiated E. coli DNA specifically incised adjacent to dimer sites. Fractionation of KB cell crude extracts reveals the presence of three such activities with distinct affinities for both DEAE-cellulose and phosphocellulose. One of the activities (activity B) is distinguished by its s 20,w (2.6) and isoelectric point (9.0) from the other two (activities A and C) which have similar s 20,w's (3.0-3.2) and isoelectric points (6.0). All three differ in their extent of stimulation by divalent cation and inhibition by NaCl or a sulfhydryl group inhibitor. These results indicate that multiple 5' leads to 3' dimer excision nuclease activities exist in human cells; however, there is as yet no direct evidence that these enzymes are functional in nucleotide excision repair in vivo.     

The c-myc DNA-unwinding element-binding protein modulates the assembly of DNA replication complexes in vitro

The presence of DNA-unwinding elements (DUEs) at eukaryotic replicators has raised the question of whether these elements contribute to origin activity by their intrinsic helical instability, as protein-binding sites, or both. We used the human c-myc DUE as bait in a yeast one-hybrid screen and identified a DUE-binding protein, designated DUE-B, with a predicted mass of 23.4 kDa. Based on homology to yeast proteins, DUE-B was previously classified as an aminoacyl-tRNA synthetase; however, the human protein is approximately 60 amino acids longer than its orthologs in yeast and worms and is primarily nuclear. In vivo, chromatin-bound DUE-B localized to the c-myc DUE region. DUE-B levels were constant during the cell cycle, although the protein was preferentially phosphorylated in cells arrested early in S phase. Inhibition of DUE-B protein expression slowed HeLa cell cycle progression from G1 to S phase and induced cell death. DUE-B extracted from HeLa cells or expressed from baculovirus migrated as a dimer during gel filtration and co-purified with ATPase activity. In contrast to endogenous DUE-B, baculovirus-expressed DUE-B efficiently formed high molecular mass complexes in Xenopus egg and HeLa extracts. In Xenopus extracts, baculovirus-expressed DUE-B inhibited chromatin replication and replication protein A loading in the presence of endogenous DUE-B, suggesting that differential covalent modification of these proteins can alter their effect on replication. Recombinant DUE-B expressed in HeLa cells restored replication activity to egg extracts immunodepleted with anti-DUE-B antibody, suggesting that DUE-B plays an important role in replication in vivo.     

Nuclear transfer of sand cat cells into enucleated domestic cat oocytes is affected by cryopreservation of donor cells

In the present study, we used the sand cat (Felis margarita) as a somatic cell donor to evaluate whether cryopreservation of donor cells alters viability and epigenetic events in donor cells and affects in vitro and in vivo developmental competence of derived embryos. In Experiment 1, flow cytometry analysis revealed that the percentage of necrosis and apoptosis in cells analyzed immediately after freezing/thawing (61 vs. 8.1%, respectively) was higher than that observed in frozen/thawed cells cultured for 18 h (6.9 vs. 3.3%, respectively) or 5 days (38 vs. 2.6%; respectively). The relative acetylation level of H3K9 was lower in frozen/thawed cells (5.4%) compared to that found in cultured cells (60.1%). In Experiment 2, embryos reconstructed with frozen/thawed cells had a lower cleavage rate (85%; day 2) than did embryos reconstructed with cultured cells (95%), while development to the blastocyst stage (day 8) was not affected by cell treatment (17.0% with frozen/thawed cells vs. 16.5% with cultured cells). In Experiment 3, pregnancy rates were similar between both cell treatments (32% with frozen/thawed cells vs. 30% with cultured cells), but the number of embryos that were implanted, and the number of fetuses that developed to term was lower for embryos reconstructed with frozen/thawed cells (1.2 and 0.3%, respectively) than those reconstructed with cultured cells (2.6 and 1.8%, respectively), while the number of fetuses reabsorbed by day 30 was higher (75%) for embryos reconstructed with frozen/thawed cells than those reconstructed with cultured cells (31%). A total of 11 kittens from cultured cells and three kittens from frozen/thawed cells were born between days 60 to 64 of gestation. Most kittens died within a few days after birth, although one kitten did survive for 2 months. In Experiment 4, POU5F1 mRNA expression was detected in 25% of blastocysts derived from frozen/thawed cells, whereas 88 and 87% of blastocysts derived from cultured cells and by in vitro fertilization, respectively, expressed POU5F1. We have shown that cell cryopreservation increased the incidence of necrosis and apoptosis and altered epigenetic events in donor cells. Consequently, the number of embryos that cleaved, implanted, and developed to term-gestation and POU5F1 expression in derived blastocysts indirectly was affected.     

ORF90, a Gene Required for Photoreactivation in Rhodobacter capsulatus SB1003 Encodes a Cyclobutane Pyrimidine Dimer Photolyase

Based on deduced amino-acid sequence similarities to class-I photolyases, the open reading frame ORF90 was identified from the genome sequence of Rhodobacter capsulatus SB1003. Photoreactivation activity is not detectable in an ORF90 deletion mutant of R. capsulatus SB1003. The phenotype of R. capsulatus wild-type cells was restored by plasmid borne ORF90 of R. capsulatus DeltaORF90. Furthermore, we detected an ORF90-related CPD-specific photoreactivation activity in R. capsulatus cell extracts. The results show that the gene product of ORF90 is involved in photoreactivation and encodes a class-I cyclobutane pyrimidine dimer photolyase.     

Regulation of phosphatidylinositol kinase activity in Saccharomyces cerevisiae.

The effects of growth phase and carbon source on membrane-associated phosphatidylinositol kinase in cell extracts of Saccharomyces cerevisiae were examined. Phosphatidylinositol kinase activity increased 2- and 2.5-fold in glucose- and glycerol-grown cells, respectively, in the stationary phase as compared with the exponential phase of growth. The increase in phosphatidylinositol kinase activity in the stationary phase of growth correlated with an increase in the relative amounts of phosphatidylinositol 4-phosphate, the product of the reaction. The increase in phosphatidylinositol kinase activity was not due to the presence of water-soluble effector molecules in cell extracts as indicated by mixing experiments. Phosphatidylinositol kinase activity decreased in cell extracts of exponential-phase cells preincubated under phosphorylation conditions which favor cyclic AMP-dependent protein kinase activity. Phosphatidylinositol kinase activity was not affected in cell extracts of stationary-phase cells preincubated under phosphorylation conditions.     

Protein degradation in extracts of exponential and stationary phase Vibrio cells

The degradation of the foreign protein [14C]methyl apohaemoglobin ([14C-me]globin) was stimulated by ATP in cell-free extracts from exponential phase and shaken and standing stationary phase Vibrio cells. A marked stimulation by ATP of the degradation of [14C-me]globin was observed with exponential phase cell extracts which were preincubated for 30 min at 30 degrees C. Maximum stimulation was obtained with 3 mM-ATP and optimum degradation was at pH 8.0-8.5. Preincubation of extracts from both types of stationary phase cells did not affect the degree of ATP stimulation. The amount of ATP stimulation of [14C-me]globin degradation by exponential phase extracts decreased markedly when the cells were starved in a growth limiting minimal medium before preparation of the cell extracts. In the exponential and both types of stationary phase extracts most of the activity was located in the cytoplasmic fractions. Although the periplasmic preparations contained a minor portion of the total activity, this activity showed a greater percentage stimulation by ATP. In the absence of ATP the specific proteolytic activities of the extracts from exponential and both types of stationary phase cells were similar. The proteolytic activities in all the cell extracts were inhibited to the same extent by phenylmethylsulphonyl fluoride, but the exponential and both types of stationary phase cell extracts were inhibited to different extents by EDTA and p-hydroxymercuribenzoate. The results suggest that the proteolytic systems responsible for the degradation of abnormal proteins are different in exponential and stationary phase Vibrio cells.     

Purification and properties of soluble hydrogenase from the cyanobacterium Anabaena cylindrica

Two soluble hydrogenase activities were separable from cell extracts of the cyanobacterium Anabaena cylindrica, one detectable by the tritium exchange assay, the other having a relatively low tritium exchange activity but catalyzing methyl viologen-dependent hydrogen formation. Their molecular weights, by gel filtration chromatography, were 42,000 and 100,000, respectively. The two hydrogenase activities were differentially inhibited. The methyl viologen-dependent activity has been purified to homogeneity from cells in which the enzyme was induced by gassing the growing cells with N2/H2/CO2 (95.7%/4%/0.3%, v/v/v). The procedure involved French pressure cell disruption of the cells, differential precipitation with ZnCl2, heat treatment (50 degrees C), and lyophilization of the heat-step supernatant. It was then subjected to DEAE-Sephacel chromatography, dye-ligand chromatography on Procion Red, and HPLC anion exchange on QMA-Accel. Polyacrylamide gel electrophoresis on both native and denaturing gels revealed two peptides with Mr's 42,000 and 50,000. The 42,000 protein alone catalyzed tritium exchange activity; both proteins appeared to be necessary for the methyl viologen activity. The native enzyme appears to be a readily dissociable dimer of two nonidentical subunits, one of which contains the hydrogen binding site and the other providing the ability to utilize electrons from a reductant for hydrogen formation.     

Repair of pyrimidine dimers in radiation-sensitive mutants rad3, rad4, rad6 and rad9 of Saccharomyces cerevisiae.

The ability to remove ultraviolet (UV)-induced pyrimidine dimers was examined in four radiation-sensitive mutants of Saccharomyces cerevisiae. The susceptibility of DNA from irradiated cells to nicking by either the T4 UV-endonuclease or an endonuclease activity found in crude extracts of Micrococcus luteus was used to measure the presence of dimers in DNA. The rad3 and rad4 mutants are shown to be defective in dimer excision whereas the rad6 and rad9 mutants are proficient in dimer excision.     

Light Regulation of delta-Aminolevulinic Acid Biosynthetic Enzymes and tRNA in Euglena gracilis

Chlorophyll synthesis in Euglena, as in higher plants, occurs only in the light. The key chlorophyll precursor, δ-aminolevulinic acid (ALA), is formed in Euglena, as in plants, from glutamate in a reaction sequence catalyzed by three enzymes and requiring tRNA^Glu. ALA formation from glutamate occurs in extracts of light-grown Euglena cells, but activity is very low in dark-grown cell extracts. Cells grown in either red (650-700 nanometers) or blue (400-480 nanometers) light yielded in vitro activity, but neither red nor blue light alone induced activity as high as that induced by white light or red and blue light together, at equal total fluence rates. Levels of the individual enzymes and the required tRNA were measured in cell extracts of light- and dark-grown cells. tRNA capable of being charged with glutamate was approximately equally abundant in extracts of light- and dark-grown cells. tRNA capable of supporting ALA synthesis was approximately three times more abundant in extracts of light-grown cells than in dark-grown cell extracts. Total glutamyl-tRNA synthetase activity was nearly twice as high in extracts of light-grown cells as in dark-grown cell extracts. However, extracts of both light- and dark-grown cells were able to charge tRNA^Glu isolated from light-grown cells to form glutamyl-tRNA that could function as substrate for ALA synthesis. Glutamyl-tRNA reductase, which catalyzes pyridine nucleotide-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde (GSA), was approximately fourfold greater in extracts of light-grown cells than in dark-grown cell extracts. GSA aminotransferase activity was detectable only in extracts of light-grown cells. These results indicate that both the tRNA and enzymes required for ALA synthesis from glutamate are regulated by light in Euglena. The results further suggest that ALA formation from glutamate in dark-grown Euglena cells may be limited by the absence of GSA aminotransferase activity.     

Assignment of a gene for tryptophanyl-transfer ribonucleic acid synthetase (E.C. 6.1.1.2) to human chromosome 14

We describe a simple method for locating tryptophanyl-tRNA synthetase (E.C. 6.1.1.2) on cellulose acetate gels (Cellogel) following electrophoresis. Employing electrophoretic conditions which result in the separation of mouse and human tryptophanyl-tRNA synthetases, we have analyzed extracts of a number of independently derived mouse-human somatic cell hybrids and subclones derived from these hybrids for the presence of human tryptophanyl-tRNA synthetase. Electrophoretic patterns of hybrid extracts which contain human tryptophanyl-tRNA synthetase exhibit three bands. This is consistent with published evidence that the enzyme from mammalian cells is a homologous dimer. The electrophoretic patterns derived from some hybrids are unusual in that the human and hybrid bands of activity are more intense than the mouse band from the same hybrid. An analysis of hybrid cells and extracts indicates that human tryptophanyl-tRNA synthetase segregates with human chromosome 14 and with the only enzyme marker which has previously been assigned to this chromosome, nucleoside phosphorylase.R. M. D. was supported by a postdoctoral fellowship from the Damon Runyon Fund for Cancer Research. The work described was supported in part by grants from Cancer Research Campaign, the Medical Research Council, and NATO.     

The mechanism of heparin stimulation of rat adipocyte lipoprotein lipase

Free fat cells and stromal-vascular cells were prepared from rat adipose tissue by incubation with collagenase. NH(4)OH-NH(4)Cl extracts of acetone-ether powders prepared from fat cells contained lipoprotein lipase activity but extracts of stromal-vascular cells did not. Intact fat cells released lipoprotein lipase activity into incubation medium, but intact stromal-vascular cells did not. The lipoprotein lipase activity of the medium was increased when fat cells were incubated with heparin, and this was accompanied by a corresponding decrease in the activity of subsequently prepared fat cell extracts. Heparin did not release lipoprotein lipase activity from stromal-vascular cells. The lipoprotein lipase activity of NH(4)OH-NH(4)Cl extracts of fat cell acetone powders is increased by the presence of heparin during the assay. This increase is not due to preservation of enzyme activity, but to increased binding of lipoprotein lipase to chylomicrons. Protamine sulfate and sodium chloride have little effect on the binding of lipoprotein lipase to chylomicrons, but they inhibit enzyme activity after binding to substrate has occurred. These inhibitors do, however, inhibit the stimulatory effect of heparin on enzyme-substrate binding.     

Anaerobic Aryl Reductive Dehalogenation of Halobenzoates by Cell Extracts of "Desulfomonile tiedjei"

We studied the transformation of halogenated benzoates by cell extracts of a dehalogenating anaerobe, "Desulfomonile tiedjei." We found that cell extracts possessed aryl reductive dehalogenation activity. The activity was heat labile and dependent on the addition of reduced methyl viologen, but not on that of reduced NAD, NADP, flavin mononucleotide, flavin adenine dinucleotide, desulfoviridin, cytochrome c(3), or benzyl viologen. Dehalogenation activity in extracts was stimulated by formate, CO, or H(2), but not by pyruvate plus coenzyme A or by dithionite. The pH and temperature optima for aryl dehalogenation were 8.2 and 35 degrees C, respectively. The rate of dehalogenation was proportional to the amount of protein in the assay mixture. The substrate specificity of aryl dehalogenation activity for various aromatic compounds in "D. tiedjei" cell extracts was identical to that of whole cells, except differences were observed in the relative rates of halobenzoate transformation. Dehalogenation was 10-fold greater in "D. tiedjei" extracts prepared from cells cultured in the presence of 3-chlorobenzoate, suggesting that the activity was inducible. Aryl reductive dehalogenation in extracts was inhibited by sulfite, sulfide, and thiosulfate, but not sulfate. Experiments with combinations of substrates suggested that cell extracts dehalogenated 3-iodobenzoate more readily than either 3,5-dichlorobenzoate or 3-chlorobenzoate. Dehalogenation activity was found to be membrane associated. This is the first report characterizing aryl dehalogenation activity in cell extracts of an obligate anaerobe.     

Effect of lipid mimetics of GM3 and lyso-GM3 dimer on EGF receptor tyrosine kinase and EGF-induced signal transduction

The tyrosine kinase activity associated with epidermal growth factor receptor (EGFR) has been a target in studies of pharmacological reagents to inhibit growth of cancer cells, which are mostly of epidermal origin. Lyso-GM3 dimer showed stronger inhibitory effect on the tyrosine kinase of EGFR than GM3, with minimal cytotoxicity [Y. Murozuka, et al. Lyso-GM3, its dimer, and multimer: their synthesis, and their effect on epidermal growth factor-induced receptor tyrosine kinase. Glycoconj. J. 24 (2007) 551-563]. Synthesis of lipids with sphingosine requires many steps, and the yield is low. A biocombinatory approach overcame this difficulty; however, products required a C(12) aliphatic chain, rather than the sphingosine head group [Y. Murozuka, et al. Efficient sialylation on azidododecyl lactosides by using B16 melanoma cells. Chemistry & Biodiversity 2 (2005) 1063-1078]. The present study was to clarify the effects of these lipid mimetics of GM3 and lyso-GM3 dimer on EGFR tyrosine kinase activity, and consequent changes of the A431 cell phenotype, as follows. (i) A lipid mimetic of lyso-GM3 dimer showed similar strong inhibitory effect on EGF-induced EGFR tyrosine kinase activity, and similar low cytotoxicity, as the authentic lyso-GM3 dimer. (ii) A lipid mimetic of lyso-GM3 dimer inhibited tyrosine phosphorylation of EGFR or its dimer to a level similar to that of the authentic lyso-GM3 dimer, but more strongly than GM3 or a lipid mimetic of GM3. (iii) Associated with the inhibitory effect of a lipid mimetic of lyso-GM3 dimer on EGF-induced EGFR kinase activity, only Akt kinase activity was significantly inhibited, but kinases associated with other signal transducers were not affected. (iv) The cell cycle of A431 cells, and the effects of GM3 and a lipid mimetic of lyso-GM3 dimer, were studied by flow cytometry, measuring the rate of DNA synthesis with propidium iodide. Fetal bovine serum greatly enhanced S phase and G(2)/M phase. Enhanced G(2)/M phase was selectively inhibited by pre-incubation of A431 cells with a lipid mimetic of lyso-GM3 dimer, whereas GM3 had only a minimal effect.     

Mixed spermatogenic germ cell nuclear extracts exhibit high base excision repair activity

Spermatogenic cells exhibit a lower spontaneous mutation frequency than somatic tissues in a lacI transgene and many base excision repair (BER) genes display the highest observed level of expression in the testis. In this study, uracil-DNA glycosylase-initiated BER activity was measured in nuclear extracts prepared from tissues obtained from each of three mouse strains. Extracts from mixed spermatogenic germ cells displayed the greatest activity followed by liver then brain for all three strains, and the activity for a given tissue was consistent among the three strains. Levels of various BER proteins were examined by western blot analyses and found to be consistent with activity levels. Nuclear extracts prepared from purified Sertoli cells, a somatic component of the seminiferous epithelium, exhibited significantly lower activity than mixed spermatogenic cell-type nuclear extracts, thereby suggesting that the high BER activity observed in mixed germ cell nuclear extracts was not a characteristic of all testicular cell types. Nuclear extracts from thymocytes and small intestines were assayed to assess activity in a mitotically active cell type and tissue. Overall, the order of tissues/cells exhibiting the greatest to lowest activity was mixed germ cells > Sertoli cells > thymocytes > small intestine > liver > brain.     

The oxidative DNA lesion 8,5'-(S)-cyclo-2'-deoxyadenosine is repaired by the nucleotide excision repair pathway and blocks gene expression in mammalian cells

Xeroderma pigmentosum (XP) patients with inherited defects in nucleotide excision repair (NER) are unable to excise from their DNA bulky photoproducts induced by UV radiation and therefore develop accelerated actinic damage, including cancer, on sun-exposed tissue. Some XP patients also develop a characteristic neurodegeneration believed to result from their inability to repair neuronal DNA damaged by endogenous metabolites since the harmful UV radiation in sunlight does not reach neurons. Free radicals, which are abundant in neurons, induce DNA lesions that, if unrepaired, might cause the XP neurodegeneration. Searching for such a lesion, we developed a synthesis for 8,5'-(S)-cyclo-2'-deoxyadenosine (cyclo-dA), a free radical-induced bulky lesion, and incorporated it into DNA to test its repair in mammalian cell extracts and living cells. Using extracts of normal and mutant Chinese hamster ovary (CHO) cells to test for NER and adult rat brain extracts to test for base excision repair, we found that cyclo-dA is repaired by NER and not by base excision repair. We measured host cell reactivation, which reflects a cell's capacity for NER, by transfecting CHO and XP cells with DNA constructs containing a single cyclo-dA or a cyclobutane thymine dimer at a specific site on the transcribed strand of a luciferase reporter gene. We found that, like the cyclobutane thymine dimer, cyclo-dA is a strong block to gene expression in CHO and human cells. Cyclo-dA was repaired extremely poorly in NER-deficient CHO cells and in cells from patients in XP complementation group A with neurodegeneration. Based on these findings, we propose that cyclo-dA is a candidate for an endogenous DNA lesion that might contribute to neurodegeneration in XP.     

Inhibition of in vitro SV40 DNA replication by ultraviolet light

Ultraviolet light-induced DNA damage was found to inhibit SV40 origin-dependent DNA synthesis carried out by soluble human cell extracts. Replication of SV40-based plasmids was reduced to approx. 35% of that in unirradiated controls after irradiation with 50-100 J/m2 germicidal ultraviolet light, where an average of 3-6 pyrimidine dimer photoproducts were formed per plasmid circle. Inhibition of the DNA helicase activity of T antigen (required for initiation of replication in the in vitro system) was also investigated, and was only significant after much higher fluences, 1000-5000 J/m2. The data indicate that DNA damage by ultraviolet light inhibits DNA synthesis in cell-free extracts principally by affecting components of the replication complex other than the DNA helicase activity of T antigen. The soluble system could be used to biochemically investigate the possible bypass or tolerance of DNA damage during replication.