Biomedical subjects

Vernon E Anderson

Publications and source records attributed to Vernon E Anderson.

35 records · Page 2Linked to original sources

PubMed2004

Raman spectroscopic characterization of secondary structure in natively unfolded proteins: alpha-synuclein.

The application of Raman spectroscopy to characterize natively unfolded proteins has been underdeveloped, even though it has significant technical advantages. We propose that a simple three-component band fitting of the amide I region can assist in the conformational characterization of the ensemble of structures present in natively unfolded proteins. The Raman spectra of alpha-synuclein, a prototypical natively unfolded protein, were obtained in the presence and absence of methanol, sodium dodecyl sulfate (SDS), and hexafluoro-2-propanol (HFIP). Consistent with previous CD studies, the secondary structure becomes largely alpha-helical in HFIP and SDS and predominantly beta-sheet in 25% methanol in water. In SDS, an increase in alpha-helical conformation is indicated by the predominant Raman amide I marker band at 1654 cm(-1) and the typical double minimum in the CD spectrum. In 25% HFIP the amide I Raman marker band appears at 1653 cm(-1) with a peak width at half-height of approximately 33 cm(-1), and in 25% methanol the amide I Raman band shifts to 1667 cm(-1) with a peak width at half-height of approximately 26 cm(-1). These well-characterized structural states provide the unequivocal assignment of amide I marker bands in the Raman spectrum of alpha-synuclein and by extrapolation to other natively unfolded proteins. The Raman spectrum of monomeric alpha-synuclein in aqueous solution suggests that the peptide bonds are distributed in both the alpha-helical and extended beta-regions of Ramachandran space. A higher frequency feature of the alpha-synuclein Raman amide I band resembles the Raman amide I band of ionized polyglutamate and polylysine, peptides which adopt a polyproline II helical conformation. Thus, a three-component band fitting is used to characterize the Raman amide I band of alpha-synuclein, phosvitin, alpha-casein, beta-casein, and the non-A beta component (NAC) of Alzheimer's plaque. These analyses demonstrate the ability of Raman spectroscopy to characterize the ensemble of secondary structures present in natively unfolded proteins.

Amides↗

PubMed2004

Tazobactam inactivation of SHV-1 and the inhibitor-resistant Ser130 -->Gly SHV-1 beta-lactamase: insights into the mechanism of inhibition.

The increasing number of bacteria resistant to combinations of beta-lactam and beta-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections. Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant beta-lactamases provides a rationale for the design of novel compounds. In the present work, SHV-1 and the Ser(130) --> Gly inhibitor-resistant variant of SHV-1 beta-lactamase were inactivated with tazobactam, a potent class A beta-lactamase inhibitor. Apoenzymes and inhibited beta-lactamases were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), digested with trypsin, and the products resolved using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass increases observed for SHV-1 and Ser(130) --> Gly (+ Delta 88 Da and + Delta 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive beta-lactamase. These two mass increments are consistent with the formation of an aldehyde (+ Delta 70 Da) and a hydrated aldehyde (+ Delta 88 Da) as stable products of inhibition. Our results reveal that the Ser --> Gly substitution at amino acid position 130 is not essential for enzyme inactivation. By examining the inhibitor-resistant Ser(130) --> Gly beta-lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser(70) in this enzyme. After acylation of tazobactam by Ser(130) --> Gly, inactivation proceeds independent of any additional covalent interactions.

Binding Sites↗

PubMed2004

Understanding the transition states of phosphodiester bond cleavage: insights from heavy atom isotope effects.

The nucleotides of DNA and RNA are joined by phosphodiester linkages whose synthesis and hydrolysis are catalyzed by numerous essential enzymes. Two prominent mechanisms have been proposed for RNA and protein enzyme catalyzed cleavage of phosphodiester bonds in RNA: (a) intramolecular nucleophilic attack by the 2'-hydroxyl group adjacent to the reactive phosphate; and (b) intermolecular nucleophilic attack by hydroxide, or other oxyanion. The general features of these two mechanisms have been established by physical organic chemical analyses; however, a more detailed understanding of the transition states of these reactions is emerging from recent kinetic isotope effect (KIE) studies. The recent data show interesting differences between the chemical mechanisms and transition state structures of the inter- and intramolecular reactions, as well as provide information on the impact of metal ion, acid, and base catalysis on these mechanisms. Importantly, recent nonenzymatic model studies show that interactions with divalent metal ions, an important feature of many phosphodiesterase active sites, can influence both the mechanism and transition state structure of nonenzymatic phosphodiester cleavage. Such detailed investigations are important because they mimic catalytic strategies employed by both RNA and protein phosphodiesterases, and so set the stage for explorations of enzyme-catalyzed transition states. Application of KIE analyses for this class of enzymes is just beginning, and several important technical challenges remain to be overcome. Nonetheless, such studies hold great promise since they will provide novel insights into the role of metal ions and other active site interactions.

Base Composition↗

PubMed2004

Prevention of artifactual protein oxidation generated during sodium dodecyl sulfate-gel electrophoresis.

Prevention of artifactual protein oxidation occurring during sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis is critical for identifying physiological protein oxidation implicated in human diseases due to the routine use of gel electrophoresis to separate the multiple proteins in proteomic studies. To develop a methodology that completely prevents artifactual protein oxidation in SDS acrylamide gel electrophoresis, cytochrome c was electrophoresed on polyacrylamide gels and subjected to trypsin in-gel digestion followed by tryptic peptide analysis by mass spectrometry. It was found that degassing the acrylamide solution to remove molecular oxygen prior to gel polymerization is a crucial process to protect the electrophoresed protein from reactive oxygen species generated during electrophoresis. However, significant artifactual protein oxidation remains that can only be eliminated entirely, if proteins are electrophoresed on an SDS gel photopolymerized with flavin as the photoinitiator and thioglycolate included in the cathode buffer as a reactive oxygen species scavenger. Using this combination of methodologies, cytochrome c isolated from adult rat heart mitochondria was purified and digested followed by mass spectrometric analysis, demonstrating the requisite high resolution of the polyacrylamide gel and the entire elimination of artifactual oxidation.

Amino Acid Sequence↗

PubMed2004

Tartrate dehydrogenase reductive decarboxylation: stereochemical generation of diastereotopically deuterated hydroxymethylenes.

Tartrate dehydrogenase catalyzes the reductive decarboxylation of meso-tartrate to glycerate. Concomitant with the ketonization of the intermediate enolate the C3 hydroxymethylene of glycerate necessarily acquires a proton from solvent. In D2O, the proton is shown to be added stereospecifically to form (2R,3R)-[3-2H]glycerate. The 1H-NMR assignments of the diastereotopic C3 protons of glycerate were confirmed by the enzymatic conversion of [1R-2H]fructose-6-phosphate to (2R,3R)-[3-2H]glycerate. The decarboxylation-protonation occurs with retention of configuration, implying that the general acid is positioned on the same face of the intermediate as the departing carboxylate. The stereochemically pure (2R,3R)-[3-2H]glycerate is readily synthesized and serves as a chiral hydroxymethylene synthon as demonstrated by the synthesis of (2S,3R)-[3-2H]serine.

Alcohol Oxidoreductases↗

PubMed2003

Understanding resistance to beta-lactams and beta-lactamase inhibitors in the SHV beta-lactamase: lessons from the mutagenesis of SER-130.

Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in kcat/Km for ampicillin. Marked increases in the dissociation constant for the preacylation complex, KI, of clavulanic acid (SHV-1, 0.14 microm; S130G, 46.5 microm) and tazobactam (SHV-1, 0.07 microm; S130G, 4.2 microm) were observed. In contrast, the k(inact)s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for beta-lactams and beta-lactamase inhibitors. These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.

Ampicillin↗

PubMed2003

Metal binding and oxidation of amyloid-beta within isolated senile plaque cores: Raman microscopic evidence.

Alzheimer's disease (AD) is characterized by the deposition of amyloid plaques in the parenchyma and vasculature of the brain. Although previous analytical studies have provided much information about the composition and structure of synthetic amyloid-beta fibrils, there is, surprisingly, a dearth of data on intact amyloid plaques from AD brain. Therefore, to elucidate the structure and detailed composition of isolated amyloid plaque cores, we utilized a high-resolution, nondestructive technique, Raman microscopy. The data are of very high quality and contain detailed information about protein composition and conformation, about post-translational modification, and about the chemistry of metal binding sites. Remarkably, spectra obtained for senile plaque (SP) cores isolated from AD brain are essentially identical both within and among brains. The Raman data show for the first time that the SP cores are composed largely of amyloid-beta and confirm inferences from X-ray studies that the structure is beta-sheet with the additional possibility that this may be present as a parallel beta-helix. Raman bands characteristic of methionine sulfoxide show that extensive methionine oxidation has occurred in the intact plaques. The Raman spectra also demonstrate that Zn(II) and Cu(II) are coordinated to histidine residues in the SP cores, at the side chains' N(tau) and N(pi) atoms, respectively. Treatment of the senile plaques with the chelator ethylenediaminetetraacetate reverses Cu binding to SP histidines and leads to a broadening of amide features, indicating a "loosening" of the beta-structure. Our results indicate that Abeta in vivo is a metalloprotein, and the loosening of the structure following chelation treatment suggests a possible means for the solubilization of amyloid deposits. The results also reveal a direct chemical basis for oxidative damage caused by amyloid-beta protein in AD.

Aged↗

PubMed2003

Measuring gluconeogenesis using a low dose of 2H2O: advantage of isotope fractionation during gas chromatography.

The contribution of gluconeogenesis to glucose production can be measured by enriching body water with (2)H(2)O to approximately 0.5% (2)H and determining the ratio of (2)H that is bound to carbon-5 vs. carbon-2 of blood glucose. This labeling ratio can be measured using gas chromatography-mass spectrometry after the corresponding glucose carbons are converted to formaldehyde and then to hexamethylenetetramine (HMT). We present a technique for integrating ion chromatograms that allows one to use only 0.05% (2)H in body water (i.e., 10 times less than the current dose). This technique takes advantage of the difference in gas chromatographic retention times of naturally labeled HMT and [(2)H]HMT. We discuss the advantage(s) of using a low dose of (2)H(2)O to quantify the contribution of gluconeogenesis.

Animals↗

PubMed2003

Mass spectrometric characterization of mitochondrial electron transport complexes: subunits of the rat heart ubiquinol-cytochrome c reductase.

Complex III of the mitochondrial electron transport chain, ubiquinol-cytochrome c reductase, was isolated by blue native polyacrylamide gel electrophoresis. Ten of the 11 polypeptides present in this complex were detected directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following electroelution of the active complex. Tryptic and chymotryptic digestion of the complex permit the identification of specific peptides from all of the protein subunits with 70% coverage of the 250 kDa complex. The mass of all 11 proteins was confirmed by second dimension Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and elution of the separated polypeptides. Additionally, the identity of the core I, core II, cytochrome c and the Rieske iron-sulfur protein were confirmed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) characterization of the peptides generated by in-gel trypsin digestion of the SDS-PAGE separated proteins. The methodology demonstrated for analyzing this membrane-bound electron transport complex should be applicable to other membrane complexes, particularly the other mitochondrial electron transport complexes. The MS analysis of the peptides obtained by in-gel digestion of the intact complex permits the simultaneous characterization of the native proteins and modifications that contribute to mitochondrial deficits that have been implicated as contributing to pathological conditions.

Amino Acid Sequence↗

PubMed2003

[Inhibition of triclosan to fatty acid synthase from goose uropygial glands and human breast cancer cells in vitro].

BACKGROUND & OBJECTIVE: It has been indicated that fatty acid synthase (FAS) is abnormally overexpressed in human breast cancer compared with normal human tissue. Inhibition of FAS induces apoptosis of human breast cancer cells. The aim of this study was to observe the inhibition of triclosan on FAS from goose uropygial glands for establishing the method and to study the inhibition of triclosan on FAS from human breast cancer SKBr3 cells in vitro. METHODS: The goose uropygial glands FAS was purified by ultra-centrifugation and Superdex PG 200 chromatography; the human breast cancer SKBr3 cell FAS was partially purified by ultra-centrifugation. The FAS was interacted with different concentrations of Triclosan with different times before catalyzing. Then the substrates of FAS were added to the reaction system. The inhibitory activities of triclosan against the FAS were investigated using spectrophotometric assays. RESULTS: In the goose uropygial gland group, FAS was purified as a single band at 250kDa with SDS-PAGE. The inhibitory activities of triclosan(12.5 micromol/L) at 0, 5, and 10 minute on FAS were 26.40%, 28.30%, and 43.93%, respectively. The inhibitory activities of triclosan (25.00 micromol/L) at 0, 5, and 10 minute on FAS were 46.22%, 50.28%, and 97.05%, respectively. The inhibitory activities of triclosan (100.00 micromol/L) at 0, 5, and 10 minute on FAS were 98.11%, 97.75%, and 97.37%, respectively. In human SKBr3 breast cancer cell group, the inhibitory activities of triclosan (25, 50, 100, and 200 micromol/L) at 5 minute on FAS were 20.00%, 26.67%, 60.00%, and 100%, respectively. CONCLUSION: Triclosan inhibits the FAS from goose uropygial glands and human breast cancer SKBr3 cells. The inhibitory activities depended on the concentrations of triclosan and the interaction times between triclosan and FAS before catalyzing.

Animals↗

PubMed2002

Evidence for direct attack by hydroxide in phosphodiester hydrolysis.

Phosphodiester hydrolysis has been the subject of intense study due to its importance in biology. Despite the numerous significant analyses of phosphodiester cleavage mechansim, comparatively little is known about the nucleophiles in these reactions. To determine whether hydroxide acts as a nucleophile or a general base in the hydrolysis of thymidine-5'-p-nitrophenyl phosphate,we determined solvent deuterium isotope effects (D2Ok), ionic strength effects, and 18O isotope effects on the solvent nucleophile (18knuc). The D2Ok for hydroxide-catalyzed phosphodiester hydrolysis is slightly inverse (0.9 +/- 0.1), suggesting that a proton transfer does not occur in the transition state. A significant alpha effect is observed with hydroperoxide, demonstrating that oxyanions can act as nucleophiles in the reaction. Additionally, the ionic strength dependencies of hydroxide and hydroperoxide catalysis are indistinguishable, suggesting that they act by the same mechanism. Finally, the 18knuc for the hydroxide-catalyzed reaction is 1.068 +/- 0.007, well in excess of the equilibrium 18O isotope effect between water and hydroxide (1.040 +/- 0.003). Together, the data are most consistent with direct nucleophilic attack by hydroxide. From the observed 18knuc and the known equilibrium component, the kinetic component of the isotope effect was calculated to be 1.027 +/- 0.010. This large kinetic component suggests that little bond order to the nucleophile occurs in the transition state.

Hydrolysis↗

PubMed2002

Pre-steady-state and stopped-flow fluorescence analysis of Escherichia coli ribonuclease III: insights into mechanism and conformational changes associated with binding and catalysis.

To better understand substrate recognition and catalysis by RNase III, we examined steady-state and pre-steady-state reaction kinetics, and changes in intrinsic enzyme fluorescence. The multiple turnover cleavage of a model RNA substrate shows a pre-steady-state burst of product formation followed by a slower phase, indicating that the steady-state reaction rate is not limited by substrate cleavage. RNase III catalyzed hydrolysis is slower at low pH, permitting the use of pre-steady-state kinetics to measure the dissociation constant for formation of the enzyme-substrate complex (K(d)=5.4(+/-0.6) nM), and the rate constant for phosphodiester bond cleavage (k(c)=1.160(+/-0.001) min(-1), pH 5.4). Isotope incorporation analysis shows that a single solvent oxygen atom is incorporated into the 5' phosphate of the RNA product, which demonstrates that the cleavage step is irreversible. Analysis of the pH dependence of the single turnover rate constant, k(c), fits best to a model for two or more titratable groups with pK(a) of ca 5.6, suggesting a role for conserved acidic residues in catalysis. Additionally, we find that k(c) is dependent on the pK(a) value of the hydrated divalent metal ion included in the reaction, providing evidence for participation of a metal ion hydroxide in catalysis, potentially in developing the nucleophile for the hydrolysis reaction. In order to assess whether conformational changes also contribute to the enzyme mechanism, we monitored intrinsic tryptophan fluorescence. During a single round of binding and cleavage by the enzyme we detect a biphasic change in fluorescence. The rate of the initial increase in fluorescence was dependent on substrate concentration yielding a second-order rate constant of 1.0(+/-0.1)x10(8) M(-1) s(-1), while the rate constant of the second phase was concentration independent (6.4(+/-0.8) s(-1); pH 7.3). These data, together with the unique dependence of each phase on divalent metal ion identity and pH, support the hypothesis that the two fluorescence transitions, which we attribute to conformational changes, correlate with substrate binding and catalysis.

Base Sequence↗

PubMed2002

Structural mechanism of enoyl-CoA hydratase: three atoms from a single water are added in either an E1cb stepwise or concerted fashion.

We have determined the crystal structure of the enzyme enoyl-CoA hydratase (ECH) from rat liver with the bound substrate 4-(N,N-dimethylamino)cinnamoyl-CoA using X-ray diffraction data to a resolution of 2.3 A. In addition to the thiolester substrate, the catalytic water, which is added in the hydration reaction, has been modeled into well-defined electron density in each of the six active sites of the physiological hexamer within the crystallographic asymmetric unit. The catalytic water bridges Glu(144) and Glu(164) of the enzyme and has a lone pair of electrons poised to react with C(3) of the enzyme-bound alpha,beta-unsaturated thiolester. The water molecule, which bridges two glutamate residues, is reminiscent of the enolase active site. However, unlike enolase, which has a lysine available to donate a proton, there are no other sources of protons available from other active site residues in ECH. Furthermore, an analysis of the hydrogen-bonding network of the active site suggests that both Glu(144) and Glu(164) are ionized and carry a negative charge with no reasonable place to have a protonated carboxylate. This lack of hydrogen-bonding acceptors that could accommodate a source of a proton, other than from the water molecule, leads to a hypothesis that the three atoms from a single water molecule are added across the double bond to form the hydrated product. The structural results are discussed in connection with details of the mechanism, which have been elucidated from kinetics, site-directed mutagenesis, and spectroscopy of enzyme-substrate species, in presenting an atomic-resolution mechanism of the reaction. Contrary to the previous interpretation, the structure of the E-S complex together with previously determined kinetic isotope effects is consistent with either a concerted mechanism or an E1cb stepwise mechanism.

Animals↗

PubMed2002

Polarization of cinnamoyl-CoA substrates bound to enoyl-CoA hydratase: correlation of (13)C NMR with quantum mechanical calculations and calculation of electronic strain energy.

When alpha,beta-unsaturated substrates bind to the active site of enoyl-CoA hydratase, large spectral changes can be observed [D'Ordine, R. L., et al. (1994) Biochemistry 33, 12635-12643]. The differences in the isotropic magnetic shieldings of the free and active site-bound forms of the carbonyl, alpha-, and beta-carbons of the substrates, hexadienoyl-CoA, cinnamoyl-CoA, and (N,N-dimethyl-p-amino)cinnamoyl-CoA have been experimentally determined. The carbonyl and beta-carbons are all deshielded, while the alpha-carbons show increased shielding. These chemical shift perturbations are interpreted to suggest that the pi-electrons of the enoyl thiolester are polarized when bound at the active site. Using the crystal structure of (N,N-dimethyl-p-amino)cinnamoyl-CoA bound at the enzyme active site, the shielding tensors were calculated at three different levels of theory, up to a density functional theory model that included all of the contiguous active site residues. These calculations successfully reproduced the observed spectral changes and permitted the electronic polarization of the substrate to be quantified as an electron density difference map. The calculated electron density difference confirms the loss of electrons at the electrophilic beta-carbon and carbonyl carbon, while a slight increase in electron density at the alpha-carbon where proton donation occurs during the hydration reaction and a larger increase in electron density at the carbonyl oxygen are predicted. The energy required to polarize the electrons to the observed extent was calculated to be 3.2 kcal/mol. The force that provides the requisite energy for the polarization is the interaction of the electric field generated by the protein at the enzyme active site with the polarizable electrons of the substrate. Because the induced electronic polarization is along the predicted reaction pathway, the extent of substrate activation by the induced electronic strain is catalytically relevant.

Acyl Coenzyme A↗

PubMed2002

Productive folding to the native state by a group II intron ribozyme.

Group II introns are large catalytic RNA molecules that fold into compact structures essential for the catalysis of splicing and intron mobility reactions. Despite a growing body of information on the folded state of group II introns at equilibrium, there is currently no information on the folding pathway and little information on the ionic requirements for folding. Folding isotherms were determined by hydroxyl radical footprinting for the 32 individual protections that are distributed throughout a group II intron ribozyme derived from intron ai5gamma. The isotherms span a similar range of Mg(2+) concentrations and share a similar index of cooperativity. Time-resolved hydroxyl radical footprinting studies show that all regions of the ribozyme fold slowly and with remarkable synchrony into a single catalytically active structure at a rate comparable to those of other ribozymes studied thus far. The rate constants for the formation of tertiary contacts and recovery of catalytic activity are identical within experimental error. Catalytic activity analyses in the presence of urea provide no evidence that the slow folding of the ai5gamma intron is attributable to the presence of unproductive kinetic traps along the folding pathway. Taken together, the data suggest that the rate-limiting step for folding of group II intron ai5gamma occurs early along the reaction pathway. We propose that this behavior resembles protein folding that is limited in rate by high contact order, or the need to form key tertiary interactions from partners that are located far apart in the primary or secondary structure.

Base Pairing↗

PubMed2002

Triclosan inhibits enoyl-reductase of type I fatty acid synthase in vitro and is cytotoxic to MCF-7 and SKBr-3 breast cancer cells.

BACKGROUND AND PURPOSE: Human type I fatty acid synthase has been proposed as a chemotherapeutic target for the treatment of breast cancer based on the inactivation of human beta-ketoacyl synthase activity by cerulenin. Triclosan, a common antibiotic, functions by inhibiting the enoyl-reductase enzymes of type II fatty acid synthases in susceptible bacteria. If triclosan is an inhibitor of human fatty acid synthase and if inhibition of fatty acid synthase is toxic to breast cancer cell lines, triclosan could prove to be a lead compound for the treatment of breast cancer. Consequently, the inhibitory activity of triclosan against vertebrate type I fatty acid synthases and its effects on breast cancer lines in cell culture were investigated. METHODS: The inhibitory activities of triclosan against human and goose fatty acid synthases and each of the partial reactions were investigated using spectrophotometric assays. The ability of triclosan at various concentrations to inhibit growth and reduce the viability of MCF-7 and SKBr-3 cells in culture was evaluated. RESULTS: Kinetic studies showed triclosan to be a slow binding inhibitor of human and goose type I fatty acid synthase and to inhibit the partial activity of enoyl-reductase with IC(50) values between 10 and 50 microM. Triclosan at similar concentrations was also shown to inhibit both viability and growth of MCF-7 and SKBr-3 cells in culture. CONCLUSIONS: The results corroborate the hypothesis that fatty acid synthase may be a target of breast cancer chemotherapy and suggest that inhibitors of the enoyl-reductase partial activity of fatty acid synthase may have chemotherapeutic potential.

Antineoplastic Agents↗

PubMed2002

Amino acid substitutions at Ambler position Gly238 in the SHV-1 beta-lactamase: exploring sequence requirements for resistance to penicillins and cephalosporins.

Site saturation mutagenesis of the 238 position in the SHV beta-lactamase was performed to identify the complete sequence requirements needed for the extended spectrum beta-lactamase (ESBL) phenotype. MICs (in micrograms per milliliter) in an isogenic background, Escherichia coli DH10B, demonstrated that the Gly238Ala mutation conferred the most resistance to penicillins and cephalosporins. The absolute increase in resistance was greatest against cefotaxime for the Gly238Ala mutant (0.06 to 8 micro g/ml). Except for the strain possessing the Gly238Pro beta-lactamase, ceftazidime MICs were also elevated. None of the mutant SHV beta-lactamases were expressed in as great an amount as the wild-type beta-lactamase. Kinetic analysis of the Gly238Ala mutant revealed that penicillin and cephalosporin substrates have a lower K(m) for the enzyme because of this mutation. Ampicillin and piperacillin MICs were inversely proportional to the side chain volume of the amino acid in cases larger than Ser, suggesting that steric considerations may be a primary requirement for penicillin resistance. Secondary structural effects explain increased resistance to oxyiminocephalosporins. Based upon this study, we anticipate that additional mutations of Gly238 in the SHV beta-lactamase will continue to be discovered with an ESBL (ceftazidime or cefotaxime resistant) phenotype.

Cephalosporin Resistance↗