Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.     

The bacterial metabolism of 2,4-xylenol

1. Measurements of the rates of oxidation of various compounds by a fluorescent Pseudomonas indicated that metabolism of 2,4-xylenol was initiated by oxidation of the methyl group para to the hydroxyl group. 2. 4-Hydroxy-3-methylbenzoic acid was isolated as the product of oxidation of 2,4-xylenol by cells inhibited with alphaalpha'-bipyridyl. 3. 4-Hydroxyisophthalic acid accumulated at low oxygen concentrations when either 2,4-xylenol or 4-hydroxy-3-methylbenzoic acid was oxidized by cells grown with 2,4-xylenol. 4. When supplemented with NADH, but not with NADPH, cell extracts oxidized 4-hydroxy-3-methylbenzoic acid readily. 2-Hydroxy-5-methylbenzoic acid was not oxidized. 5. Both 4-hydroxyisophthalic acid and p-hydroxybenzoic acid were oxidized to beta-oxoadipic acid by cell extracts supplemented with either NADH or NADPH. 4,5-Dihydroxyisophthalic acid was not oxidized. 6. From measurements of oxygen consumed and carbon dioxide evolved it was concluded that protocatechuic acid is an intermediate in the conversion of 4-hydroxyisophthalic acid into beta-oxoadipic acid.     

Co-correction of the ERCC1, ERCC4 and xeroderma pigmentosum group F DNA repair defects in vitro.

The mammalian ERCC1-encoded polypeptide is required for nucleotide excision repair of damaged DNA and is homologous to Saccharomyces cerevisiae RAD10, which functions in repair and mitotic intrachromosomal recombination. Rodent cells representing repair complementation group 1 have nonfunctional ERCC1. We report that repair of UV-irradiated DNA can be reconstituted by combining rodent group 1 cell extracts with correcting protein from HeLa cells. Background repair was minimized by employing fractionated rodent cell extracts supplemented with human replication proteins RPA and PCNA. Group 1-correcting activity has a native molecular mass of 100 kDa and contains the 33 kDa ERCC1 polypeptide, as well as complementing activities for extracts from rodent group 4 and xeroderma pigmentosum group F (XP-F) cells. Extracts of group 1, group 4 or XP-F cells do not complement one another in vitro, although they complement extracts from other groups. The amount of ERCC1 detectable by immunoblotting is reduced in group 1, group 4 and XP-F extracts. Recombinant ERCC1 from Escherichia coli only weakly corrected the group 1 defect. The data suggest that ERCC1 is part of a functional protein complex with group 4 and XP-F correcting activities. The latter two may be equivalent to one another and analogous to S. cerevisiae RAD1.     

Escherichia coli produces linoleic acid during late stationary phase.

Escherichia coli produces linoleic acid in the late stationary phase. This was the case whether the cultures were grown aerobically or anaerobically on a supplemented glucose-salts medium. The linoleic acid was detected by thin-layer chromatography and was measured as the methyl ester by gas chromatography. The linoleic acid methyl ester was identified by its mass spectrum. Lipids extracted from late-stationary-phase cells generated thiobarbituric acid-reactive carbonyl products when incubated with a free radical initiator. In contrast, extracts from log-phase or early-stationary-phase cells failed to do so, in accordance with the presence of polyunsaturated fatty acid only in the stationary-phase cells.     

Trypanosoma cruzi, Leishmania donovani, and L. mexicana: extract factor that lyses mammalian cells.

Cell-free extracts of Trypanosoma cruzi, Leishmania donovani, and L. mexicana, cultivated in a medium supplemented with 5% fetal calf serum, contain a factor that induces lysis of mammalian red blood cells and Vero cells. All the lytic activity was found in the insoluble fraction of parasite extracts obtained after centrifugation at 100,000g for 2 hr. The lytic agent is pronase, trypsin, and temperature resistant. The optimum pH of the lytic effect is pH 6.5. Normal red blood cells of several mammalian species had different sensitivities to the lytic agent. The lipid phase of T. cruzi extract contains the total lytic activity. Albumins of different animal species at 1 mg/ml, completely inhibit the lytic activity of parasite extracts.     

Purification and characterization of actinogelin, a calcium-sensitive actin-accessory protein, from rat liver

Although cell-free extracts prepared from several types of free-living cells, including Ehrlich tumor cells, macrophages and sea-urchin eggs, readily form gels under low Ca2+ conditions, no such ability to induce actin-related gel has been detected in tissue-cell extracts. Ca2+ -insensitive gelation activity was discovered, however, in several tissue-cell extracts, including liver and brain, provided that the extracts were supplemented with skeletal muscle actin. Based on sodium dodecylsulfate/polyacrylamide gel electrophoretic analysis of the gel, these extracts seem to contain both a Ca2+ -insensitive gelation factor and Ca2+ -sensitive one, actinogelin. A procedure for purification of actinogelin from rat liver was developed, and the properties of actinogelin thus purified were compared with those of Ehrlich tumor cell actinogelin. No appreciable difference was found in these two proteins, and Ca2+ sensitivity (50% inhibition of gelation at 1 microM) was very similar. Some of the molecular characteristics are described, and the importance of the presence of actinogelin in tissue cells is discussed.     

A practical method for uniform isotopic labeling of recombinant proteins in mammalian cells.

A method to obtain uniformly isotopically labeled (15N and 15N/13C) protein from mammalian cells is described. The method involves preparation of isotopically labeled media consisting of amino acids isolated from bacterial and algal extracts supplemented with cysteine and enzymatically synthesized glutamine. The approach is demonstrated by producing 15N-labeled and 15N/13C-labeled urokinase from Sp2/0 cells and successfully growing Chinese hamster ovary (CHO) cells on the labeled media. Thus, using the procedures described, isotopically labeled proteins that have been expressed in mammalian cells can be prepared, allowing them to be studied by heteronuclear multidimensional NMR techniques.     

Control of Lysine Biosynthesis in Yeast by a Feedback Mechanism

Homocitric acid (β-hydroxy-β-carboxyadipic acid; HC) is accumulated by a lysine-requiring yeast mutant when grown in a chemically defined medium, supplemented with limited amounts of lysine. A study of the formation of HC in relation to the depletion of lysine from the growth medium indicates that HC accumulated only when the concentration of lysine was low. The enzymatic formation of HC from α-ketoglutarate plus acetyl-coenzyme A in cell-free extracts of the same organism was also inhibited by lysine. The inhibitory effect of lysine on the formation of HC in both whole cells and cell-free extracts is indicative of the functional existence of a feedback control mechanism in the pathway for lysine biosynthesis in yeast.     

Green tea extracts can counteract the modification of fatty acid composition induced by doxorubicin in cultured cardiomyocytes

Doxorubicin cardiotoxicity is associated with the generation of free radicals, and involves not only lipid peroxidation but also a decreased biosynthesis of highly unsaturated fatty acids, leading to significant modification in cardiomyocyte fatty acid composition. We have evaluated whether naturally occurring antioxidants could counteract this side-effect. Green tea is an excellent source of catechins; we supplemented cultured rat cardiomyocytes with different green tea extracts to relate their catechin content and composition to their ability in protecting cells against doxorubicin-induced damage. The determination of total lipid fatty acid composition, of conjugated diene production (indicator of lipid peroxidation), and of lactate dehydrogenase release revealed that supplementation with tea extracts could counteract significant modifications in the fatty acyl pattern due to doxorubicin exposure, although to different extents. These differences could be ascribed to the different total catechin content and to qualitative differences among the tea extracts, determined by HPLC analysis.     

Immunocytochemical studies of the evolution of islet hormones.

By using both immunofluorescence and peroxidase-anti-peroxidase procedures to detect cells producing the four islet hormones, supplemented by biochemical, biological, and radioimmunological assays of tissue extracts, it has been shown that insulin seems to be the most original hormone, apparently occurring already in invertebrates in cells of open type in the alimentary tract mucosa. Insulin cells also predominate in the first islet organ, namely that of the cyclostomes. The order of appearance in the endocrine pancreas during the subsequent evolution is: somatostatin; glucagon; and the pancreatic polypeptide. Even in lower vertebrates pancreatic polypeptide cells occur in those parts of the pancreas situated in close proximity to the gut.     

Defective 30S ribosomal particles in a polyamine auxotroph of Escherichia coli

Polypeptide synthesis directed by poly(U) or MS 2 phage RNA is several fold more active in cell-free systems prepared from polyamine supplemented bacteria than in extracts of polyamine depleted cells. This effect depends on the presence of defective 30S ribosomal subunits in the starved bacteria. It is concluded that polyamines play a role in the normal biosynthesis, maturation and/or assembly of the small ribosomal subparticles.     

Evaluation of a short term in vitro test for granulocyte chalone activity.

A short term in vitro test for granulocyte chalone activity eas examined for its specificity and reliability. The test used the inhibition, by granulocyte extracts, of 3H-thymidine (3H-Tdr) uptake in to the acid-insoluble material by rat bone marrow cells in vitro to measure possible chalone activity. Among the many possible 3H-Tdr artifacts pool size dilution by Tdr contained in the extracts was excluded using an E. coli mutant requiring thymine. Several amino acids and biogenic amines do not affect the test. However, continuous and pulse labelling of bone marrow cells with 3H-Tdr, viability tests and micro flow fluorometric measurements of the cell cycle distribution following colcemid treatment strongly suggests that the cells do not proliferate in vitro during short term incubation, since practically no cells enter the S-phase, cells in the S-phase die and few if any cells proceed through G2 and mitosis. Moreover, the test cannot exclude cytotoxicity. Thus, the in vitro test may only sceem for an unspecific S-phase inhibitor and must hence be supplemented by another assay to prove the chalone nature of an extract or fraction. The test per se fails to meet most of the requirements of a valid granulocyte chalone assay.     

Conventional laboratory agar media provide an iron-limited environment for bacterial growth

Abstract The outer membrane proteins of Escherichia coli and Pseudomonas aeruginosa grown in a number of conventional laboratory media were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) High-molecular-weight proteins similar to those produced by these strains in an iron-limited chemically defined medium were detected in cells grown on the surface of various agar media. In contrast, these proteins were not produced or were only poorly expressed by the corresponding broth cultures or by cells grown an agar supplemented with iron. A catecholic substance could be detected in DST agar extracts subsequent to bacterial growth which was produced to a lesser extent in IST agar and in broth cultures.     

Acquisition of thymidylate by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

The pathway for the acquisition of thymidylate in the obligate bacterial parasite Rickettsia prowazekii was determined. R. prowazekii growing in host cells with or without thymidine kinase failed to incorporate into its DNA the [3H]thymidine added to the culture. In the thymidine kinase-negative host cells, the label available to the rickettsiae in the host cell cytoplasm would have been thymidine, and in the thymidine kinase-positive host cells, it would have been both thymidine and TMP. Further support for the inability to utilize thymidine was the lack of thymidine kinase activity in extracts of R. prowazekii. However, [3H]uridine incorporation into the DNA of R. prowazekii was demonstrable (973 +/- 57 dpm/3 x 10(8) rickettsiae). This labeling of rickettsial DNA suggests the transport of uracil, uridine, uridine phosphates (UXP), or 2'-deoxyuridine phosphates, the conversion of the labeled precursor to thymidylate, and subsequent incorporation into DNA. This is supported by the demonstration of thymidylate synthase activity in extracts of R. prowazekii. The enzyme was determined to have a specific activity of 310 +/- 40 pmol/min/mg of protein and was inhibited greater than or equal to 70% by 5-fluoro-dUMP. The inability of R. prowazekii to utilize uracil was suggested by undetectable uracil phosphoribosyltransferase activity and by its inability to grow (less than 10% of control) in a uridine-starved mutant cell line (Urd-A) supplemented with 50 microM to 1 mM uracil. In contrast, the rickettsiae were able to grow in Urd-A cells that were uridine starved and supplemented with 20 microM uridine (117% of control). However, no measurable uridine kinase activity could be measured in extracts of R. prowazekii. Normal rickettsial growth (92% of control) was observed when the host cell was blocked with thymidine so that the host cell's dUXP pool was depressed to a level inadequate for growth and DNA synthesis in the host cell. Taken together, these data strongly suggest that rickettsiae transport UXP from the host cell's cytoplasm and that they synthesize TTP from UXP.     

Alternative fermentation conditions for improved Escherichia coli-based cell-free protein synthesis for proteins requiring supplemental components for proper synthesis

Escherichia coli-based cell-free protein synthesis is a powerful emerging tool for protein engineering due to the open, accessible nature of the reaction and its straightforward, economical potential for many diverse applications. One critical limitation of this system is the inability to express some complex, eukaryotic, and/or unnatural proteins at high expression yields. A potential solution is a synthetic-biology-like approach where cell-free reactions are supplemented by expressing the required supplemental components in the E. coli cells during the fermentation, which cells are used to prepare the extract for cell-free protein synthesis. Here we report adjustments to the fermentation conditions that increase yields of complex proteins upwards of 150% over standard conditions. We consider extracts containing GroEL/ES protein folding chaperones and extracts containing orthogonal tRNA/tRNA synthetase pairs for noncanonical amino acid incorporation. In contrast to standard cell-free synthesis, delaying the harvest of supplemented fermentations lead to increased and more consistent yields of proteins that required supplemental components. Protein yields enhanced by buffering the fermentation media pH lead to an average 52% decrease in yield cost, while costs for cases unchanged or negatively affected by buffering increased an average 14%. An apparent balance is required between the supplemental components and general extract protein profile.     

Binding of a 50-kD Protein to a U-Rich Sequence in an mRNA Encoding a Proline-Rich Protein That Is Destabilized by Fungal Elicitor

The mRNA encoding the bean proline-rich protein PvPRP1 has been shown previously to be destabilized in elicitor-treated cells. In this study, we identified a 50-kD protein in cellular extracts that binds specifically to the PvPRP1 mRNA by UV cross-linking assays. Using 32P-labeled RNAs transcribed in vitro from a series of 5[prime] deleted PvPRP1 cDNA clones, we demonstrated that the PvPRP1 mRNA binding protein (PRP-BP) binds to a 27-nucleotide U-rich (~60%) domain in the 3[prime] untranslated region. Poly(U) and, to a lesser extent, poly(A-U) competed for the PRP-BP binding activity. PRP-BP activity is redox regulated in vitro, as shown by the effects of sulfhydryl-modifying reagents on the RNA binding activity. Treatment of cellular extracts with the reducing agents DTT and [beta]-mercaptoethanol increased binding activity, whereas treatment with the oxidizing agent diamide and the alkylating agent N-ethylmaleimide inhibited binding. In extracts from elicitor-treated cells, PRP-BP activity increased approximately fivefold prior to rapid PvPRP1 mRNA degradation. The increase in PRP-BP activity was apparently due to post-translational regulation because control and elicitor-treated cell extracts supplemented with DTT showed high comparable levels of RNA binding activity. The kinetics of PRP-BP activation after elicitor treatment and its capacity for redox regulation in vitro suggested that PRP-BP may function in the elicitor-induced destabilization of PvPRP1 mRNA.     

Enhancement of insulin sensitivity in adipocytes by ginger

Antidiabetic and hypoglycemic drugs have been reported to enhance adipocyte differentiation of 3T3-L1 preadipocytes. We previously reported that ginseng (active constituents: ginsenosides) enhanced the differentiation [1]. In this experiment, effect of some ginger group food extracts on the adipocyte differentiation was investigated using cultured mouse 3T3-L1 preadipocytes. 3T3-L1 cells were grown as monolayer cultures at 37 degrees C in DMEM supplemented by 10% FBS under the atmosphere of 5% CO(2)-95% air. Ginger extracts were found to enhance the adipocyte differentiation. Active constituent was purified and identified as gingerol. In the gingerol-treated cells, insulin-sensitive glucose uptake was increased. It is expected that ginger enhance the insulin-sensitivity, and improve chronic disease, such as diabetes.     

Regulation of purine biosynthesis in cultured Drosophila melanogaster cells: I.--Conditional activity of hypoxanthine-guanine-phosphoribosyltransferase and 5-nucleotidase.

In cultured cells established from Drosophila melanogaster embryos, and grown in usual medium, no hypoxanthine-guanine-phosphoribosyl transferase (HG-PRT) could be measured, and only traces of 5'-nucleotidase activity were detectable. On the contrary, it was observed that if the same medium is supplemented with purine bases, nucleosides, orotate, glutamine, azaserine or antifolates, de novo purine biosynthesis is inhibited, and HGPRT is detectable, along with an important 5'-nucleotidase activity. Moreover, dialysis or treatment of extracts from cells untreated by purines, with activated charcoal restored HGPRT and 5'-nucleotidase activities. These activities were abolished completely by inosinic acid (IMP) and guanosine 5'-monophosphoric acid (GMP). Similar results were obtained with fly extracts. These results suggest that de novo purine biosynthesis masks HGPRT activity, the endogenous synthesis leading to the accumulation of purine nucleotides which are inhibitors of the HGPRT activity.     

Cell-free translation of frog virus 3 messenger RNAs. Initiation factors from infected cells discriminate between early and late viral mRNAs

Cell-free protein-synthesizing extracts prepared from rabbit reticulocytes, wheat germ, or cultured baby hamster kidney cells efficiently translated frog virus 3 early mRNAs; in contrast, late mRNAs were translated poorly under similar conditions. However, the translational efficiency of the late viral mRNAs was markedly enhanced in cell-free extracts prepared from frog virus 3 (FV 3)-infected baby hamster kidney cells and in nuclease-treated rabbit reticulocyte extracts by the addition of a 0.5 M KCl wash from FV 3-infected cell ribosomes; the 0.5 M KCl wash (initiation factors) from uninfected cells had no such effect. Total cytoplasmic RNA from infected cells was fractionated according to size on sucrose gradients and fractions containing different concentrations, and relative proportions of early and late mRNAs were translated in either native or initiation factor-supplemented extracts. Under these conditions, the translation efficiency of early mRNAs was unchanged, while the translation of late mRNAs increased 2-7-fold. Thus, the in vitro discriminatory activity of the 0.5 M wash was not dependent on the complexity of the mRNAs present in the translation mixture. We show also that in native extracts, under conditions of blocked polypeptide chain elongation, early mRNAs are initiated preferentially. However, late as well as early mRNAs are initiated equally well in reticulocyte extracts under similar experimental conditions when supplemented with crude initiation factors from infected cells. These data support the conclusion that the translational enhancement of FV 3 mRNAs in vitro is mediated by a virus-specified or virus-modified initiation factor(s) and likely represents a regulatory mechanism of protein synthesis operative in vivo (Willis, D. B., Goorha, R., Miles, M., and Granoff, A. (1977) J. Virol. 24, 326-342).     

Activation of pyruvate dehydrogenase in adipose tissue by insulin. Evidence for an effect of insulin on pyruvate dehydrogenase phosphate phosphatase.

1. The mechanism by which insulin activates pyruvate dehydrogenase in rat epididymal adipose tissue was further investigated. 2. When crude extracts, prepared from tissue segments previously exposed to insulin (2m-i.u/ml) for 2min, were supplemented with Mg-2+, Ca-2+, glucose and hexokinase and incubated at 30 degrees C, they displayed an enhanced rate of increase in pyruvate dehydrogenase activity compared with control extracts. 3. When similar extracts were instead supplemented with fluoride, ADP, creatine phosphate and creatine kinase, the rate of decrease in pyruvate dehydrogenase activity observed during incubation at 30 degrees C was unaffected by insulin treatment. 4. It is suggested that insulin increases the fraction of pyruvate dehydrogenase present in the tissue in the active dephospho form by increasing the activity of pyruvate dehydrogenase phosphate phosphatase.