Biomedical subjects

A J Sinskey

Publications and source records attributed to A J Sinskey.

At least 19 recordsLinked to original sources

PubMed2006

Glioblastoma cell death induced by asiatic acid.

Asiatic acid (AA), a triterpene, is known to be cytotoxic to several tumor cell lines. AA induces dose- and time-dependent cell death in U-87 MG human glioblastoma. This cell death occurs via both apoptosis and necrosis. The effect of AA may be cell type-specific as AA-induced cell death was mainly apoptotic in colon cancer RKO cells. AA-induced glioblastoma cell death is associated with decreased mitochondrial membrane potential, activation of caspase-9 and -3, and increased intracellular free Ca2+. Although treatment of glioblastoma cells with the caspase inhibitor zVAD-fmk completely abolished AA-induced caspase activation, it did not significantly block AA-induced cell death. AA-induced cell death was significantly prevented by an intracellular Ca2+ inhibitor, BAPTA/AM. Taken together, these results indicate that AA induces cell death by both apoptosis and necrosis, with Ca2+-mediated necrotic cell death predominating.

Amino Acid Chloromethyl Ketones↗

PubMed2005

Characterization of Streptomyces MITKK-103, a newly isolated actinomycin X2-producer.

A new actinomycete strain designated MITKK-103 was isolated from the soil of a flowerpot using a humic acid agar medium. The newly isolated strain was able to produce a large amount of actinomycin X2 even under nonoptimized growing conditions and serves as a promising source of this antibiotic. Actinomycin X2 has higher cytotoxicity toward cultured human leukemia (HL-60) cells than does actinomycin D, and it induces cell death via apoptosis. A nearly complete 16S ribosomal DNA (rDNA) sequence from the isolate was determined and found to have high identity (98.5-100%) with Streptomyces galbus, Streptomyces griseofuscus, and Streptomyces padanus, indicating that MITKK-103 belongs to the genus Streptomyces. The isolate clustered with species belonging to the S. padanus clade in a 16S-rDNA-based phylogenetic tree and showed 75% overall homology to S. padanus ATCC 25646 in DNA-DNA relatedness analysis. Although the growth of the isolate was somewhat different from the three species mentioned, the strain MITKK-103 most closely resembles S. padanus on the basis of the morphological and phenotypic characteristics, phylogenetic analysis, and genotypic data. As such, this is the first report of a strain of S. padanus capable of producing actinomycins.

Bacterial Typing Techniques↗

PubMed2002

Disparity between changes in mRNA abundance and enzyme activity in Corynebacterium glutamicum: implications for DNA microarray analysis.

The relationship between changes in mRNA abundance and enzyme activity was determined for three genes over a span of nearly 3 h during amino acid production in Corynebacterium glutamicum. Gene expression changes during C. glutamicum fermentations were examined by complementary DNA (cDNA) microarrays and by a second method for quantitating RNA levels, competitive reverse transcriptase-PCR (RT-PCR). The results obtained independently by both methods were compared and found to be in agreement, thus validating the quantitative potential of DNA microarrays for gene expression profiling. Evidence of a disparity between mRNA abundance and enzyme activity is presented and supports our belief that it is difficult to generally predict protein activity from quantitative transcriptome data. Homoserine dehydrogenase, threonine dehydratase, and homoserine kinase are enzymes involved in the biosynthesis of l-isoleucine and other aspartate-derived amino acids in C. glutamicum. Our data suggest that different underlying regulatory mechanisms may be connected with the expression of the genes encoding each of these three enzymes. Indeed, whereas in one case the increases in enzyme activity exceeded those in the corresponding mRNA abundance, in another case large increases in the levels of gene expression were not congruent with changes in enzyme activity.

Aspartic Acid↗

PubMed2002

Engineering an indene bioconversion process for the production of cis-aminoindanol: a model system for the production of chiral synthons.

Cis-aminoindanol, a key chiral precursor to the HIV protease inhibitor CRIXIVAN, can be derived from indene oxidation products of (2R) stereochemistry. A number of different microorganisms, notably strains of the genera Pseudomonas and Rhodococcus, have been isolated that catalyze the oxygenation of indene to indandiol with greater stereospecificity than is achievable through traditional chemical synthesis. The yield and ultimate optical purity of indandiol produced in such biocatalytic processes is influenced by the intrinsic stereospecificity of the oxygenase(s), enantioselective dehydrogenation, and the loss of substrate to alternate, undesirable metabolites. Metabolic engineering of any indene bioconversion system for the commercial-scale production of cis-aminoindanol must account for these influences, as well as pathway fluxes and enzyme regulation, to optimize the formation of oxygenated precursors with useful stereochemistry. As such, the process by which bacterial systems carry out the bioconversion of indene to indandiol serves as a model for biological production of industrially relevant chiral synthons.

Biotechnology↗

PubMed2001

Class I and III polyhydroxyalkanoate synthases from Ralstonia eutropha and Allochromatium vinosum: characterization and substrate specificity studies.

Class I and III polyhydroxyalkanoate (PHA) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to polyhydroxybutyrate. The Class I PHA synthase from Ralstonia eutropha has been purified by numerous labs with reported specific activities that vary between 1 and 160 U/mg. An N-terminal (His)6-PHA synthase was constructed and purified with specific activity of 40 U/mg. The variable activity is shown to be related to the protein's propensity to aggregate and not to incomplete post-translational modification by coenzyme A and a phosphopantetheinyl transferase. The substrate specificities of this enzyme and the Class III PHA synthase from Allochromatium vinosum have been determined with nine analogs of varied chain length and branching, OH group position within the chain, and thioesters. The results suggest that in vitro, both PHA synthases are very specific and provide further support for their active site structural similarities. In vitro results differ from studies in vivo.

Acyltransferases↗

PubMed2001

Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: class I and III synthases share a similar catalytic mechanism.

The Class I and III polyhydroxybutyrate (PHB) synthases from Ralstonia eutropha and Chromatium vinosum, respectively, catalyze the polymerization of beta-hydroxybutyryl-coenzyme A (HBCoA) to generate PHB. These synthases have different molecular weights, subunit composition, and kinetic properties. Recent studies with the C. vinosum synthase suggested that it is structurally homologous to bacterial lipases and allowed identification of active site residues important for catalysis [Jia, Y., Kappock, T. J., Frick, T., Sinskey, A. J., and Stubbe, J. (2000) Biochemistry 39, 3927-3936]. Sequence alignments between the Class I and III synthases revealed similar residues in the R. eutropha synthase. Site-directed mutants of these residues were prepared and examined using HBCoA and a terminally saturated trimer of HBCoA (sT-CoA) as probes. These studies reveal that the R. eutropha synthase possesses an essential catalytic dyad (C319-H508) in which the C319 is involved in covalent catalysis. A conserved Asp, D480, was shown not to be required for acylation of C319 by sT-CoA and is proposed to function as a general base catalyst to activate the hydroxyl of HBCoA for ester formation. Studies of the [(3)H]sT-CoA with wild-type and mutant synthases reveal that 0.5 equiv of radiolabel is covalently bound per monomer of synthase, suggesting that a dimeric form of the enzyme is involved in elongation. These studies, in conjunction with search algorithms for secondary structure, suggest that the Class I and III synthases are mechanistically similar and structurally homologous, despite their physical and kinetic differences.

Acyl Coenzyme A↗

PubMed2001

Metabolic redirection of carbon flow toward isoleucine by expressing a catabolic threonine dehydratase in a threonine-overproducing Corynebacterium glutamicum.

Carbon destined for lysine synthesis in Corynebacterium glutamicum ATCC 21799 can be diverted toward threonine by overexpression of genes encoding a feedback-insensitive homoserine dehydrogenase (hom(dr)) and homoserine kinase (thrB). We studied the effects of introducing two different threonine dehydratase genes into this threonine-producing system to gauge their effects on isoleucine production. Co-expression of hom(dr), thrB, and ilvA, which encodes a native threonine dehydratase, caused isoleucine to accumulate to a final concentration of 2.2+/-0.2 g l(-1), five-fold more than accumulates in the wild-type strain, and approximately twice as much as accumulates in the strain expressing only hom(dr) and thrB. Comparing these data with previous results, we found that overexpression of the three genes, hom(dr), thrB, and ilvA, in C. glutamicum ATCC 21799 is no better in terms of isoleucine production than the expression of a single gene, tdcB, encoding a catabolic threonine dehydratase from Escherichia coli. Co-expression of hom(dr), thrB, and tdcB, however, caused the concentration of isoleucine to increase 20-fold compared to the wild-type strain, about four times more than the corresponding ilvA-expressing strain. In this system, the apparent yield of isoleucine production was multiplied by a factor of two [2.1 mmol (g dry cell weight)(-1)]. While the balance of excreted metabolites showed that the carbon flow in this strain was completely redirected from the lysine pathway into the isoleucine pathway, it also showed that more pyruvate was diverted into amino acid synthesis.

Base Sequence↗

PubMed2001

Application of radiolabeled tracers to biocatalytic flux analysis.

Radiolabeled tracers can provide valuable information about the structure of and flux distributions in biocatalytic reaction networks. This method derives from prior studies of glucose metabolism in mammalian systems and is implemented by pulsing a culture with a radiolabeled metabolite that can be transported into the cells and subsequently measuring the radioactivity of all network metabolites following separation by liquid chromatography. Intracellular fluxes can be directly determined from the transient radioactivity count data by tracking the depletion of the radiolabeled metabolite and/or the accompanying accumulation of any products formed. This technique differs from previous methods in that it is applied within a systems approach to the problem of flux determination. It has been used for the investigation of the indene bioconversion network expressed in Rhodococcus sp. KY1. Flux estimates obtained by radioactive tracers were confirmed by macroscopic metabolite balancing and showed that indene oxidation in steady state chemostat cultures proceeds primarily through a monooxygenase activity forming (1S,2R)-indan oxide, with no dehydrogenation of trans-(1R,2R)-indandiol. These results confirmed the significance of indan oxide formation and identified the hydrolysis of indan oxide as a key step in maximizing the production of (2R)-indandiol, a chiral precursor of the HIV protease inhibitor, Crixivan.

Carbon Radioisotopes↗

PubMed2001

Development and validation of corynebacterium DNA microarrays.

We have developed DNA microarray techniques for studying Corynebacterium glutamicum. A set of 52 C. glutamicum genes encoding enzymes from primary metabolism was amplified by PCR and printed in triplicate onto glass slides. Total RNA was extracted from cells harvested during the exponential-growth and lysine production phases of a C. glutamicum fermentation. Fluorescently labeled cDNAs were prepared by reverse transcription using random hexamer primers and hybridized to the microarrays. To establish a set of benchmark metrics for this technique, we compared the variability between replicate spots on the same slide, between slides hybridized with cDNAs from the same labeling reaction, and between slides hybridized with cDNAs prepared in separate labeling reactions. We found that the results were both robust and statistically reproducible. Spot-to-spot variability was 3.8% between replicate spots on a given slide, 5.0% between spots on separate slides (though hybridized with identical, labeled cDNA), and 8.1% between spots from separate slides hybridized with samples from separate reverse transcription reactions yielding an average spot to spot variability of 7.1% across all conditions. Furthermore, when we examined the changes in gene expression that occurred between the two phases of the fermentation, we found that results for the majority of the genes agreed with observations made using other methods. These procedures will be a valuable addition to the metabolic engineering toolbox for the improvement of C. glutamicum amino acid-producing strains.

Corynebacterium↗

PubMed2001

Accumulation of the PhaP phasin of Ralstonia eutropha is dependent on production of polyhydroxybutyrate in cells.

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are granule-associated proteins that accumulate to high levels in strains that are producing PHAs. The accumulation of phasins has been proposed to be dependent on PHA production, a model which is now rigorously tested for the phasin PhaP of Ralstonia eutropha. R. eutropha phaC PHA synthase and phaP phasin gene replacement strains were constructed. The strains were engineered to express heterologous and/or mutant PHA synthase alleles and a phaP-gfp translational fusion in place of the wild-type alleles of phaC and phaP. The strains were analyzed with respect to production of polyhydroxybutyrate (PHB), accumulation of PhaP, and expression of the phaP-gfp fusion. The results suggest that accumulation of PhaP is strictly dependent on the genetic capacity of strains to produce PHB, that PhaP accumulation is regulated at the level of both PhaP synthesis and PhaP degradation, and that, within mixed populations of cells, PhaP accumulation within cells of a given strain is not influenced by PHB production in cells of other strains. Interestingly, either the synthesis of PHB or the presence of relatively large amounts of PHB in cells (>50% of cell dry weight) is sufficient to enable PhaP synthesis. The results suggest that R. eutropha has evolved a regulatory mechanism that can detect the synthesis and presence of PHB in cells and that PhaP expression can be used as a marker for the production of PHB in individual cells.

Acyltransferases↗

PubMed2001

New insight into the role of the PhaP phasin of Ralstonia eutropha in promoting synthesis of polyhydroxybutyrate.

Phasins are proteins that are proposed to play important roles in polyhydroxyalkanoate synthesis and granule formation. Here the phasin PhaP of Ralstonia eutropha has been analyzed with regard to its role in the synthesis of polyhydroxybutyrate (PHB). Purified recombinant PhaP, antibodies against PhaP, and an R. eutropha phaP deletion strain have been generated for this analysis. Studies with the phaP deletion strain show that PhaP must accumulate to high levels in order to play its normal role in PHB synthesis and that the accumulation of PhaP to low levels is functionally equivalent to the absence of PhaP. PhaP positively affects PHB synthesis under growth conditions which promote production of PHB to low, intermediate, or high levels. The levels of PhaP generally parallel levels of PHB in cells. The results are consistent with models whereby PhaP promotes PHB synthesis by regulating the surface/volume ratio of PHB granules or by interacting with polyhydroxyalkanoate synthase and indicate that PhaP plays an important role in PHB synthesis from the early stages in PHB production and across a range of growth conditions.

Bacterial Proteins↗

PubMed2000

Lipases provide a new mechanistic model for polyhydroxybutyrate (PHB) synthases: characterization of the functional residues in Chromatium vinosum PHB synthase.

Polyhydroxybutyrate (PHB) synthases catalyze the conversion of beta-hydroxybutyryl coenzyme A (HBCoA) to PHB. These enzymes require an active site cysteine nucleophile for covalent catalysis. A protein BLASTp search using the Class III Chromatium vinosum synthase sequence reveals high homology to prokaryotic lipases whose crystal structures are known. The homology is very convincing in the alpha-beta-elbow (with the active site nucleophile)-alpha-beta structure, residues 131-175 of the synthase. A conserved histidine of the Class III PHB synthases aligns with the active site histidine of the lipases using the ClustalW algorithm. This is intriguing as this histidine is approximately 200 amino acids removed in sequence space from the catalytic nucleophile. Different threading algorithms suggest that the Class III synthases belong to the alpha/beta hydrolase superfamily which includes prokaryotic lipases. Mutagenesis studies were carried out on C. vinosum synthase C149, H331, H303, D302, and C130 residues. These studies reveal that H331 is the general base catalyst that activates the nucleophile, C149, for covalent catalysis. The model indicates that C130 is not involved in catalysis as previously proposed [Müh, U., Sinskey, A. J., Kirby, D. P., Lane, W. S., and Stubbe, J. (1999) Biochemistry 38, 826-837]. Studies with D302 mutants suggest D302 functions as a general base catalyst in activation of the 3-hydroxyl of HBCoA (or a hydroxybutyrate acyl enzyme) for nucleophilic attack on the covalently linked thiol ester intermediate. The relationship of the lipase model to previous models based on fatty acid synthases is discussed.

Acyltransferases↗

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

Characterization of IS1676 from Rhodococcus erythropolis SQ1.

To develop a transposable element-based system for mutagenesis in Rhodococcus, we used the sacB gene from Bacillus subtilis to isolate a novel transposable element, IS1676, from R. erythropolis SQ1. This 1693 bp insertion sequence is bounded by imperfect (10 out of 13 bp) inverted repeats and it creates 4 bp direct repeats upon insertion. Comparison of multiple insertion sites reveals a preference for the sequence 5'-(C/T)TA(A/G)-3' in the target site. IS1676 contains a single, large (1446 bp) open reading frame with coding potential for a protein of 482 amino acids. IS1676 may be similar to an ancestral transposable element that gave rise to repetitive sequences identified in clinical isolates of Mycobacterium kansasii. Derivatives of IS1676 should be useful for analysis of Rhodococcus strains, for which few other genetic tools are currently available.

Base Sequence↗