PubMed Health⌕ Search

Biomedical subjects

A J Sinskey

Publications and source records attributed to A J Sinskey.

At least 19 recordsLinked to original sources
PubMed1999

Apoptosis in batch cultures of Chinese hamster ovary cells.

One of the main problems in the culture of Chinese Hamster Ovary (CHO) cells continues to be the inability to maintain the viability of the cultures over an extended period of time. The rapid decline in viability at the end of the culture is exacerbated by the absence of serum. In trying to reduce the extent of death in these cultures, we first tried to determine the mode of death. We found that more than 80% of the cells in a standard serum-free batch culture of CHO cells in suspension died via apoptosis--as evidenced by condensed chromatin and the appearance of a characteristic DNA ladder. Furthermore, when protein synthesis was inhibited using cycloheximide, the cells underwent rapid apoptosis indicating that death proteins were present in greater abundance than survival proteins in our CHO cells. Cell lysate from CHO cells showed evidence of cysteine protease (caspase) activity. Caspases of the Interleukin-1-beta-Converting Enzyme (ICE) family, e.g., CPP32, Mch-1, etc., have been implicated in the apoptotic process. Surprisingly, a caspase peptide inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoro-methyl-ketone (z-VAD.fmk), was unable to substantially extend the life of a serum-free batch culture of CHO cells. In addition, z-VAD.fmk was only marginally able to extend viability in response to withdrawal of growth and survival factors, insulin and transferrin. In both these instances, z-VAD.fmk was able to prevent cleavage of caspase substrates, but not protect cells from death. However, we found that bcl-2 expression was able to significantly extend viabilities in CHO batch culture. Bcl-2 expression also substantially extended the viability of cultures in response to insulin and transferrin withdrawal. These results provide interesting insights into the pathways of death in a CHO cell.

Amino Acid Chloromethyl Ketones↗
PubMed1999

Metabolite and isotopomer balancing in the analysis of metabolic cycles: II. Applications.

In a previous paper (Klapa et al., 1999), we presented a model for the analysis of isotopomer distributions of the TCA cycle intermediates resulting from 13C (or 14C) labeling experiments. Results allow the rigorous determination of the degree of enrichment at specific carbon atoms of metabolites, of the molecular weight distribution of metabolite isotopomers, as well as of the fine structure of NMR spectra in terms of a small number of metabolic fluxes. In this paper we validate the model by comparing model predictions with experimental data and then apply it to the analysis of metabolic networks that have been investigated in previous studies. The results have allowed us to conclude that: (1) there is no evidence of propionyl-CoA carboxylase pathway in Escherichia coli; and (2) the possibility that acetone utilization in mammals occurs solely via the "lactate/methylglyoxal" pathway is consistent with available labeling data. The presented modeling framework provides additional constraints that must be satisfied by experimental data in a biochemical network structure and therefore enhances the power of labeling methods for resolving in vivo metabolic fluxes.

Acetic Acid↗
PubMed1999

PHA synthase from chromatium vinosum: cysteine 149 is involved in covalent catalysis.

Polyhydroxyalkanoate synthase (PHA) from Chromatium vinosum catalyzes the conversion of 3-hydroxybutyryl-CoA (HB-CoA) to polyhydroxybutyrate (PHB) and CoA. The synthase is composed of a approximately 1:1 mixture of two subunits, PhaC and PhaE. Size-exclusion chromatography indicates that in solution PhaC and PhaE exist as large molecular weight aggregates. The holo-enzyme, PhaEC, has a specific activity of 150 units/mg. Each subunit was cloned, expressed, and purified as a (His)6-tagged construct. The PhaC-(His)6 protein catalyzed polymerization with a specific activity of 0.9 unit/mg; the PhaE-(His)6 protein was inactive (specific activity <0.001 unit/mg). Addition of PhaE-(His)6 to PhaC-(His)6 increased the activity several 100-fold. To investigate the priming step of the polymerization process, the PhaEC was incubated with a trimer of HB-CoA in which the terminal hydroxyl was replaced with tritium ([3H]-sT-CoA). After Sephadex G50 chromatography, the synthase contained approximately 0.25 equiv of the labile label per PhaC. Incubation of [3H]-sT-synthase with HB-CoA resulted in production of [3H]-polymer. Digestion of [3H]-sT-synthase with trypsin and HPLC analysis resulted in isolation of three labeled peptides. Sequencing by ion trap mass spectrometry showed that they were identical and that they each contained an altered cysteine (C149). One peptide contained the [3H]-sT while the other two contained, in addition to the [3H]-sT, one and two additional monomeric HBs, respectively. Mutation of C149 to alanine gave inactive synthase. The remaining two cysteines of PhaC, 292 and 130, were also mutated to alanine. The former had wild-type (wt) activity, while the latter had 0.004 wt % activity and was capable of making polymer. A mechanism is proposed in which PhaC contains all the elements essential for catalysis and the polymerization proceeds by covalent catalysis using C149 and potentially C130.

Acyltransferases↗
PubMed1999

Flux through the tetrahydrodipicolinate succinylase pathway is dispensable for L-lysine production in Corynebacterium glutamicum.

The N-succinyl-LL-diaminopimelate desuccinylase gene (dapE) in the four-step succinylase branch of the L-lysine biosynthetic pathway of Corynebacterium glutamicum was disrupted via marker-exchange mutagenesis to create a mutant strain that uses only the one-step meso-diaminopimelate dehydrogenase branch to overproduce lysine. This mutant strain grew and utilized glucose from minimal medium at the same rate as the parental strain. In addition, the dapE- strain produced lysine at the same rate as its parent strain. Transformation of the parental and dapE- strains with the amplified meso-diaminopimelate dehydrogenase gene (ddh) on a plasmid did not affect lysine production in either strain, despite an eightfold amplification of the activity of the enzyme. These results indicate that the four-step succinylase pathway is dispensable for lysine overproduction in shake-flask culture. In addition, the one-step meso-diaminopimelate dehydrogenase pathway does not limit lysine flux in Corynebacterium under these conditions.

Acyltransferases↗
PubMed1999

Isolation and characterization of indene bioconversion genes from Rhodococcus strain I24.

Rhodococcus strain 124 is able to convert indene into indandiol via the actions of at least two dioxygenase systems and a putative monooxygenase system. We have identified a cosmid clone from 124 genomic DNA that is able to confer the ability to convert indene to indandiol upon Rhodococcus erythropolis SQ1, a strain that normally can not convert or metabolize indene. HPLC analysis reveals that the transformed SQ1 strain produces cis-(1R,2S)-indandiol, suggesting that the cosmid clone encodes a naphthalenetype dioxygenase. DNA sequence analysis of a portion of this clone confirmed the presence of genes for the dioxygenase as well as genes encoding a dehydrogenase and putative aldolase. These genes will be useful for manipulating indene bioconversion in Rhodococcus strain 124.

Amino Acid Sequence↗
PubMed1999

Expression of the Escherichia coli catabolic threonine dehydratase in Corynebacterium glutamicum and its effect on isoleucine production.

The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1. 16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to alpha-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by the ilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and a tdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.

Biomass↗
PubMed1998

Growth factor and bcl-2 mediated survival during abortive proliferation of hybridoma cell line.

Cultures of the CRL-1606 hybridoma (ATCC) have been reported to undergo continuous proliferation with simultaneous death during nutrient limited fed-batch fermentations. The bcl-2 proto-oncogene has been shown to prevent cell death under a variety of otherwise death inducing conditions. We were interested in elucidating the nature of the massive death observed in cultures of CRL-1606, specifically with respect to the possible environmental causes, and the ability of overexpressed human bcl-2 (hbcl-2) to mitigate cell death. Abortive proliferation, or continuous proliferation in the presence of continuous death, could be induced in serum free cultures of CRL-1606 through the withdrawal of insulin provided the culture was competent for cell proliferation. Culture competency for proliferation was found to be solely determined by the presence of cell culture nutrients. Abortive proliferation was defective in cultures transfected with hbcl-2 and the enhanced viability observed resulted from an increased viable cell population and at the expense of the nonviable cell population normally found in untransfected cultures. Abortive proliferation was also observed in serum containing cultures upon serum shiftdowns. Like the insulin-supplemented serum free culture system, hbcl-2 transfected cultures exhibited defects in the abortive proliferation process. These results suggest that the massive death observed during nutrient-limited fed-batch fermentation originate, in part, from growth or survival factor limitations. Hence, approaches to design cell culture media that account for the cell's proliferation requirements without accounting for the cell's survival requirements may represent a cell death sentence. Given the transformed nature of the hybridomas, we conclude that the abortive proliferation of CRL-1606 is a consequence of inappropriate cell cycle entry in a survival factor limited environment.

Animals↗
PubMed1998

Sequence of the Corynebacterium glutamicum pyruvate carboxylase gene.

Pyruvate carboxylase is an important anaplerotic enzyme replenishing oxaloacetate consumed for biosynthesis during growth, or lysine and glutamic acid production in industrial fermentations. We used regions of homology from pyruvate carboxylase sequences of 12 different species (corresponding to the ATP- and pyruvate-binding sites), to design polymerase chain reaction (PCR) primers for amplifying a fragment of the pyruvate carboxylase (pc) gene from C. glutamicum genomic DNA. This 850-base-pair fragment was used to probe a C. glutamicum cosmid library and four candidate pc cosmids were identified. The fragment was sequenced and the sequence of the complete gene was obtained by several rounds of primer synthesis, PCR on one of the positive cosmids, and sequencing. The C. glutamicum pc sequence shows 64% homology with the pc gene of Mycobacterium tuberculosis and 44% homology with the human pc gene. Regions of ATP, pyruvate and biotin binding have also been identified.

Amino Acid Sequence↗
PubMed1997

PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo.

A synthetic operon for polyhydroxyalkanoate (PHA) biosynthesis designed to yield high levels of PHA synthase activity in vivo was constructed by positioning a genetic fragment encoding beta-ketothiolase and acetoacetyl-CoA reductase behind a modified synthase gene containing an Escherichia coli promoter and ribosome binding site. Plasmids containing the synthetic operon and the native Alcaligenes eutrophus PHA operon were transformed into E. coli DH5 alpha and analyzed for polyhydroxybutyrate production. The molecular weight of polymer isolated from recombinant E. coli containing the modified synthase construct, determined by multiangle light scattering, was lower than that of the polymer from E. coli containing the native A. eutrophus operon. A further decrease in polyester molecular weight was observed with increased induction of the PHA biosynthetic genes in the synthetic operon. Comparison of the enzyme activity levels of PHA biosynthetic enzymes in a strain encoding the native operon with a strain possessing the synthetic operon indicates that the amount of polyhydroxyalkanoate synthase in a host organism plays a key role in controlling the molecular weight and the polydispersity of polymer.

Acyltransferases↗
PubMed1995

Intracellular flux analysis applied to the effect of dissolved oxygen on hybridomas.

Quantitative estimates of intracellular fluxes and measurements of intracellular concentrations were used to evaluate the effect of dissolved oxygen (DO) concentration on CRL 1606 hybridoma cells in batch culture. The estimates of intracellular fluxes were generated by combining material balances with measurements of extracellular metabolite rates of change. Experiments were performed at DO levels of 60% and 1% air saturation, as well as under oxygen-limited conditions. Cell extracts were analyzed to evaluate the effect of DO on the intracellular concentrations of the glutamate dehydrogenase reactants, as well as the redox state of the pyridine nucleotides in the cytosol and mitochondria. The relationship between cell density and pyridine nucleotide redox state was also investigated. Dissolved oxygen concentration had a significant effect on nitrogen metabolism and the flux through glutamate dehydrogenase was found to reverse at low DO, favoring glutamate formation. The NAD in the cytosol and mitochondria was more reduced under low DO conditions while the cytosolic NAD was more oxidized at low DO. Cytosolic NAD was reduced at higher cell densities while the redox states of cytosolic NADP and mitochondrial NAD did not exhibit significant variation with cell density. These results point to the fundamental role of the intracellular oxidation/reduction state in cell physiology and the possibility of controlling physiological processes through modulation of the dissolved oxygen level or the oxidation/reduction potential of the culture.

Adenosine Triphosphate↗
PubMed1995

Effect of different levels of aspartokinase on the lysine production by Corynebacterium lactofermentum.

A 2.9-kb SacI fragment containing the ask-asd operon, encoding aspartokinase and aspartatesemialdehyde dehydrogenase, was cloned from an aminoethylcysteine-resistant, lysine-producing Corynebacterium lactofermentum strain. Enzymatic analysis showed that the aspartokinase (ASK) activity was completely resistant to inhibition by mixtures of lysine and threonine. Comparison of the deduced amino acid sequence of the beta submit of the ask gene showed three amino acid residue changes with ask gene encoding wild-type, feedback-sensitive enzymes. Three C. lactofermentum strains, one being aspartokinase-negative, one carrying two ask genes on the chromosome and one having a sixfold higher specific ASK activity than the parental strain, were constructed by transconjugation and electroporation, and used to analyse the role of ASK in the lysine production by C. lactofermentum. The results indicate that, in this study, feed-back-resistant ASK is necessary for high-level lysine production, but dispensable for lysine and diaminopimelate synthesis required for cell growth.

Amino Acid Sequence↗
PubMed1995

Production of isoleucine by overexpression of ilvA in a Corynebacterium lactofermentum threonine producer.

Overproduction of isoleucine, an essential amino acid, was achieved by amplification of the gene encoding threonine dehydratase, the first enzyme in the threonine to isoleucine pathway, in a Corynebacterium lactofermentum threonine producer. Threonine overproduction was previously achieved with C. lactofermentum ATCC 21799, a lysine-hyperproducing strain, by introduction of plasmid pGC42 containing the Corynebacterium homdr and thrB genes (encoding homoserine dehydrogenase and homoserine kinase respectively) under separate promoters. The pGC42 derivative, pGC77, also contains ilvA, which encodes threonine dehydratase. In a shake-flask fermentation, strain 21799(pGC77) produced 15 g/l isoleucine, along with small amounts of lysine and glycine. A molar carbon balance indicates that most of the carbon previously converted to threonine, lysine, glycine and isolecine was incorporated into isoleucine by the new strain. Thus, in our system, simple overexpression of wild-type ilvA sufficed to overcome the effects of feedback inhibition of threonine dehydratase by the end-product, isoleucine.

Acetolactate Synthase↗
PubMed1995

Purification and properties of oxaloacetate decarboxylase from Corynebacterium glutamicum.

Oxaloacetate (OAA) decarboxylase (E.C. 4.1.1.3) was isolated from Corynebacterium glutamicum. In five steps the enzyme was purified 300-fold to apparent homogeneity. The molecular mass estimated by gel filtration was 118 +/- 6 kDa. SDS-PAGE showed a single subunit of 31.7 KDa, indicating an alpha 4 subunit structure for the native enzyme. The enzyme catalyzed the decarboxylation of OAA to pyruvate and CO2, but no other alpha-ketoacids were used as substrate. The cation Mn2+ was required for full activity, but could be substituted by Mg2+, CO2+, Ni2+ and Ca2+. Monovalent ions like Na+, K+ or NH4+ were not required for activity. The enzyme was inhibited by Cu2+, Zn2+, ADP, coenzyme A and succinate. Avidin did not inhibit the enzyme activity, indicating that biotin is not involved in decarboxylation of OAA. Analysis of the kinetic properties revealed a Km for OAA of 2.1 mM and a Km of 1.2 mM for Mn2+. The Vmax was 158 mumol of OAA converted per min per mg of protein, which corresponds to an apparent kcat of 311 s-1.

Carboxy-Lyases↗
PubMed1995

Regulated secretion of prolactin by the mouse insulinoma cell line beta TC-3.

Our aim is to use cultured cells capable of regulated protein secretion for the production of recombinant proteins that require particular types of post-translational modifications. Here we have generated a stable transfected beta TC-3 cell line, beta TC-IPR9, that secretes high levels of recombinant prolactin. Transfected cells synthesize both the 27 kDa glycosylated and a 23 kDa nonglycosylated prolactin; the 23 kDa nonglycosylated species was secreted preferentially when cells were placed in secretion medium containing isobutylmethylxanthine (IBMX) and high concentrations of glucose, K+, and Ca2+. When the cells were cultured in medium containing low concentrations of glucose, K+, and Ca2+, most of the prolactin and insulin were not secreted; much of the prolactin was proteolytically converted to a 16 kDa form. Within the first 30 minutes after transferring the cells to medium containing secretagogues there was a 20-fold increase in the rate of secretion of prolactin; all of the 16 kDa species was secreted. The recombinant cells could be cycled several times between medium in which prolactin was biosynthesized and medium in which it was secreted. Preferential secretion of proteolytically processed prolactin in a medium without contaminating proteins offers an example of the advantage of this technology for production of other recombinant proteins.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Effect of inducible thrB expression on amino acid production in Corynebacterium lactofermentum ATCC 21799.

Amplification of the operon homdr-thrB encoding a feedback-insensitive homoserine dehydrogenase and a wild-type homoserine kinase in a Corynebacterium lactofermentum lysine-producing strain resulted in both homoserine and threonine accumulation, with some residual lysine production. A plasmid enabling separate transcriptional control of each gene was constructed to determine the effect of various enzyme activity ratios on metabolite accumulation. By increasing the activity of homoserine kinase relative to homoserine dehydrogenase activity, homoserine accumulation in the medium was essentially eliminated and the final threonine titer was increased by about 120%. Furthermore, a fortuitous result of the cloning strategy was an unexplained increase in homoserine dehydrogenase activity. This resulted in a further decrease in lysine production along with a concomitant increase in threonine accumulation.

Corynebacterium↗
PubMed1995

Recent advances in the physiology and genetics of amino acid-producing bacteria.

Corynebacterium glutamicum and its close relatives, C. flavum and C. lactofermentum, have been used for over 3 decades in the industrial production of amino acids by fermentation. Since 1984, several research groups have started programs to develop metabolic engineering principles for amino acid-producing Corynebacterium strains. Initially, the programs concentrated on the isolation of genes encoding (deregulated) biosynthetic enzymes and the development of general molecular biology tools such as cloning vectors and DNA transfer methods. With most of the genes and tools now available, recombinant DNA technology can be applied in strain improvement. To accomplish these improvements, it is critical and advantageous to understand the mechanisms of gene expression and regulation as well as the biochemistry and physiology of the species being engineered. This review explores the advances made in the understanding and application of amino acid-producing bacteria in the early 1990s.

Amino Acids↗
PubMed1994

Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a required posttranslational modification for catalytic activity.

Polyhydroxyalkanoate (PHA) synthase has been expressed in Escherichia coli by reengineering the 5'-end of the wild-type (wt) gene and subsequent transformation of this gene into protease-deficient E. coli UT5600 (ompT-). Induction with IPTG results in soluble PHA synthase, which is approximately 5% of the total protein. The soluble synthase has been purified to > 90% homogeneity using FPLC chromatography on hydroxylapatite and Q-Sepharose and has a specific activity of 5 mumol min-1 mg-1. The molecular weight of the PHA product is approximately 10(6) Da based on PlGel chromatography and calibration using polystyrene molecular weight markers. The synthase in the absence of substrate appears to exist in both monomeric and dimeric forms. Incubation of the synthase with an excess of substrate converts it into a form that is now extractable into CHCl3 and sediments on sucrose density ultracentrifugation with PHA. Studies in which the ratio of substrate, 3-D-hydroxybutyrylCoA, to synthase is varied suggest that during polymerization the elongation process occurs at a rate much faster than during the initiation process. A mechanistic model has been proposed for the polymerization process [Griebel, R., Smith, Z., & Merrick, J. (1968) Biochemistry 7, 3676-3681] in which two cysteines are required for catalysis. This model is based on the well-characterized enzymes involved in fatty acid biosynthesis. To test this model, several site-directed mutants of synthase, selected based on sequence conservation among synthases, have been prepared. The C459S mutant has activity approximately 90% that of the wt protein, while the C319S and C319A synthases possess < 0.01% the activity of the wt protein. CD and antibody studies suggest that the mutant proteins are properly folded. The detection of only a single essential cysteine by mutagenesis and the requirement for posttranslational modification by phosphopantetheine to provide a second thiol in many enzymes utilizing coenzyme A thiol ester substrates made us consider the possibility that posttranslational modification was required for synthase activity as well. This hypothesis was confirmed when the plasmid containing PHA synthase (pKAS4) was transformed into E. coli SJ16, requiring beta-alanine for growth. Growth of SJ16/pKAS4 on [3H]-beta-alanine followed by Coomassie staining of the protein and autoradiography revealed that PHA synthase is overexpressed and that beta-alanine is incorporated into the protein. These results suggest PHA synthase is posttranslationally modified by phosphopantetheine.(ABSTRACT TRUNCATED AT 400 WORDS)

Acyltransferases↗