bluuebunny/crossref_metadata_2025 huggingface dataset:
Bacterial Ammeline Metabolism via Guanine Deaminase
Authors: Seffernick Jennifer L. , Dodge Anthony G. , Sadowsky Michael J. , Bumpus John A. , Wackett Lawrence P.
Published: December 2009

Melamine toxicity in mammals has been attributed to the blockage of kidney tubules by insoluble complexes of melamine with cyanuric acid or uric acid. Bacteria metabolize melamine via three consecutive deamination reactions to generate cyanuric acid. The second deamination reaction, in which ammeline is the substrate, is common to many bacteria, but the genes and enzymes responsible have not been previously identified. Here, we combined bioinformatics and experimental data to identify guanine deaminase as the enzyme responsible for this biotransformation. The ammeline degradation phenotype was demonstrated in wild-type
Escherichia coli
and
Pseudomonas
strains, including
E. coli
K12 and
Pseudomonas putida
KT2440. Bioinformatics analysis of these and other genomes led to the hypothesis that the ammeline deaminating enzyme was guanine deaminase. An
E. coli
guanine deaminase deletion mutant was deficient in ammeline deaminase activity, supporting the role of guanine deaminase in this reaction. Two guanine deaminases from disparate sources (
Bradyrhizobium japonicum
USDA 110 and
Homo sapiens
) that had available X-ray structures were purified to homogeneity and shown to catalyze ammeline deamination at rates sufficient to support bacterial growth on ammeline as a sole nitrogen source.
In silico
models of guanine deaminase active sites showed that ammeline could bind to guanine deaminase in a similar orientation to guanine, with a favorable docking score. Other members of the amidohydrolase superfamily that are not guanine deaminases were assayed
in vitro
, and none had substantial ammeline deaminase activity. The present study indicated that widespread guanine deaminases have a promiscuous activity allowing them to catalyze a key reaction in the bacterial transformation of melamine to cyanuric acid and potentially contribute to the toxicity of melamine.
A Novel Pheromone Quorum-Sensing System Controls the Development of Natural Competence in
Streptococcus thermophilus
and
Streptococcus salivarius
Authors: Fontaine Laetitia , Boutry Céline , de Frahan Marie Henry , Delplace Brigitte , Fremaux Christophe , Horvath Philippe , Boyaval Patrick , Hols Pascal
Published: December 2009

In streptococcal species, the key step of competence development is the transcriptional induction of
comX
, which encodes the alternative sigma factor σ
X
, which positively regulates genes necessary for DNA transformation. In
Streptococcus
species belonging to the
mitis
and
mutans
groups, induction of
comX
relies on the activation of a three-component system consisting of a secreted pheromone, a histidine kinase, and a response regulator. In
Streptococcus thermophilus
, a species belonging to the
salivarius
group, the oligopeptide transporter Ami is essential for
comX
expression under competence-inducing conditions. This suggests a different regulation pathway of competence based on the production and reimportation of a signal peptide. The objective of our work was to identify the main actors involved in the early steps of
comX
induction in
S. thermophilus
LMD-9. Using a transcriptomic approach, four highly induced early competence operons were identified. Among them, we found a Rgg-like regulator (Ster_0316) associated with a nonannotated gene encoding a 24-amino-acid hydrophobic peptide (Shp0316). Through genetic deletions, we showed that these two genes are essential for
comX
induction. Moreover, addition to the medium of synthetic peptides derived from the C-terminal part of Shp0316 restored
comX
induction and transformation of a Shp0316-deficient strain. These peptides also induced competence in
S. thermophilus
and
Streptococcus salivarius
strains that are poorly transformable or not transformable. Altogether, our results show that Ster_0316 and Shp0316, renamed ComRS, are the two members of a novel quorum-sensing system responsible for
comX
induction in species from the
salivarius
group, which differs from the classical phosphorelay three-component system identified previously in streptococci.
Deletion of the
ftsZ
-Like Gene Results in the Production of Superparamagnetic Magnetite Magnetosomes in
Magnetospirillum gryphiswaldense
Authors: Ding Yao , Li Jinhua , Liu Jiangning , Yang Jing , Jiang Wei , Tian Jiesheng , Li Ying , Pan Yongxin , Li Jilun
Published: December 2009

Magnetotactic bacteria (MTB) synthesize unique organelles termed “magnetosomes,” which are membrane-enclosed structures containing crystals of magnetite or greigite. Magnetosomes form a chain around MamK cytoskeletal filaments and provide the basis for the ability of MTB to navigate along geomagnetic field lines in order to find optimal microaerobic habitats. Genomes of species of the MTB genus
Magnetospirillum
, in addition to a gene encoding the tubulin-like FtsZ protein (involved in cell division), contain a second gene termed “
ftsZ
-like,” whose function is unknown. In the present study, we found that the
ftsZ
-like gene of
Magnetospirillum gryphiswaldense
strain MSR-1 belongs to a 4.9-kb
mamXY
polycistronic transcription unit. We then purified the recombinant FtsZ-like protein to homogeneity. The FtsZ-like protein efficiently hydrolyzed ATP and GTP, with ATPase and GTPase activity levels of 2.17 and 5.56 μmol phosphorus per mol protein per min, respectively. The FtsZ-like protein underwent GTP-dependent polymerization into long filamentous bundles
in vitro
. To determine the role of the
ftsZ
-like gene, we constructed a
ftsZ
-like mutant (Δ
ftsZ
-like mutant) and its complementation strain (Δ
ftsZ
-like_C strain). Growth of
ΔftsZ
-like cells was similar to that of the wild type, indicating that the Δ
ftsZ
-like gene is not involved in cell division. Transmission electron microscopic observations indicated that the Δ
ftsZ
-like cells, in comparison to wild-type cells, produced smaller magnetosomes, with poorly defined morphology and irregular alignment, including large gaps. Magnetic analyses showed that Δ
ftsZ
-like produced mainly superparamagnetic (SP) magnetite particles, whereas wild-type and Δ
ftsZ
-like_C cells produced mainly single-domain (SD) particles. Our findings suggest that the FtsZ-like protein is required for synthesis of SD particles and magnetosomes in
M. gryphiswaldense
.
MreB Drives
De N
ovo
Rod Morphogenesis in
Caulobacter crescentus
via Remodeling of the Cell Wall
Authors: Takacs Constantin N. , Poggio Sebastian , Charbon Godefroid , Pucheault Mathieu , Vollmer Waldemar , Jacobs-Wagner Christine
Published: December 2009

MreB, the bacterial actin-like cytoskeleton, is required for the rod morphology of many bacterial species. Disruption of MreB function results in loss of rod morphology and cell rounding. Here, we show that the widely used MreB inhibitor A22 causes MreB-independent growth inhibition that varies with the drug concentration, culture medium conditions, and bacterial species tested. MP265, an A22 structural analog, is less toxic than A22 for growth yet equally efficient for disrupting the MreB cytoskeleton. The action of A22 and MP265 is enhanced by basic pH of the culture medium. Using this knowledge and the rapid reversibility of drug action, we examined the restoration of rod shape in lemon-shaped
Caulobacter crescentus
cells pretreated with MP265 or A22 under nontoxic conditions. We found that reversible restoration of MreB function after drug removal causes extensive morphological changes including a remarkable cell thinning accompanied with elongation, cell branching, and shedding of outer membrane vesicles. We also thoroughly characterized the composition of
C. crescentus
peptidoglycan by high-performance liquid chromatography and mass spectrometry and showed that MreB disruption and recovery of rod shape following restoration of MreB function are accompanied by considerable changes in composition. Our results provide insight into MreB function in peptidoglycan remodeling and rod shape morphogenesis and suggest that MreB promotes the transglycosylase activity of penicillin-binding proteins.
Functional Analysis of the Stability Determinant AlfB of pBET131, a Miniplasmid Derivative ofBacillus subtilis(natto) Plasmid pLS32
Authors: Tanaka Teruo
Published: December 2009

Bacillus subtilisplasmid pBET131 is a derivative of pLS32, which was isolated from anattostrain ofBacillus subtilis. The DNA region in pBET131 that confers segregational stability contains an operon consisting of three genes, of whichalfA, encoding an actin-like ATPase, andalfBare essential for plasmid stability. In this work, thealfBgene product and its target DNA region were studied in detail. Transcription of thealfoperon initiated from a σA-type promoter was repressed by thealfBgene product. Overproduction of AlfA was inhibitory to cell growth, suggesting that the repression of thealfoperon by AlfB is important for maintaining appropriate levels of AlfA. An electrophoretic mobility shift assay and footprinting analysis with purified His-tagged AlfB showed that it bound to a DNA region containing three tandem repeats of 8-bp AT-rich sequence (here designatedparN), which partially overlaps the −35 sequence of the promoter. A sequence alteration in the first or third repeat did not affect the AlfB binding and plasmid stability, whereas that in the second repeat resulted in inhibition of these phenomena. The repression ofalfA-lacZexpression was observed in the constructs carrying a mutation in either the first or third repeat, but not in the second repeat, indicating a correlation between plasmid stability, AlfB binding, and repression. It was also demonstrated by the yeast two-hybrid system that AlfA and AlfB interact with each other and among themselves. From these results, it was concluded that AlfB participates in partitioning pBET131 by forming a complex with AlfA andparN, the mode of which is typified by the type II partition mechanism.
Production of the Bsa Lantibiotic by Community-Acquired
Staphylococcus aureus
Strains
Authors: Daly Karen M. , Upton Mathew , Sandiford Stephanie K. , Draper Lorraine A. , Wescombe Philip A. , Jack Ralph W. , O'Connor Paula M. , Rossney Angela , Götz Friedrich , Hill Colin , Cotter Paul D. , Ross R. Paul , Tagg John R.
Published: December 2009

Lantibiotics are antimicrobial peptides that have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications. Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, some lantibiotics are produced by pathogens and, rather than contributing to food safety and/or health, add to the virulence potential of the producing strains. Indeed, genome sequencing has revealed the presence of genes apparently encoding a lantibiotic, designated Bsa (bacteriocin of
Staphylococcus aureus
), among clinical isolates of
S
.
aureus
and those associated with community-acquired methicillin-resistant
S. aureus
(MRSA) infections in particular. Here, we establish for the first time, through a combination of reverse genetics, mass spectrometry, and mutagenesis, that these genes encode a functional lantibiotic. We also reveal that Bsa is identical to the previously identified bacteriocin staphylococcin Au-26, produced by an
S. aureus
strain of vaginal origin. Our examination of MRSA isolates that produce the Panton-Valentine leukocidin demonstrates that many community-acquired
S. aureus
strains, and representatives of ST8 and ST80 in particular, are producers of Bsa. While possession of Bsa immunity genes does not significantly enhance resistance to the related lantibiotic gallidermin, the broad antimicrobial spectrum of Bsa strongly indicates that production of this bacteriocin confers a competitive ecological advantage on community-acquired
S. aureus
.
The Genome of the Amoeba Symbiont “CandidatusAmoebophilus asiaticus” Reveals Common Mechanisms for Host Cell Interaction among Amoeba-Associated Bacteria
Authors: Schmitz-Esser Stephan , Tischler Patrick , Arnold Roland , Montanaro Jacqueline , Wagner Michael , Rattei Thomas , Horn Matthias
Published: December 2009

Protozoa play host for many intracellular bacteria and are important for the adaptation of pathogenic bacteria to eukaryotic cells. We analyzed the genome sequence of “CandidatusAmoebophilus asiaticus,” an obligate intracellular amoeba symbiont belonging to theBacteroidetes. The genome has a size of 1.89 Mbp, encodes 1,557 proteins, and shows massive proliferation of IS elements (24% of all genes), although the genome seems to be evolutionarily relatively stable. The genome does not encode pathways forde novobiosynthesis of cofactors, nucleotides, and almost all amino acids. “Ca. Amoebophilus asiaticus” encodes a variety of proteins with predicted importance for host cell interaction; in particular, an arsenal of proteins with eukaryotic domains, including ankyrin-, TPR/SEL1-, and leucine-rich repeats, which is hitherto unmatched among prokaryotes, is remarkable. Unexpectedly, 26 proteins that can interfere with the host ubiquitin system were identified in the genome. These proteins include F- and U-box domain proteins and two ubiquitin-specific proteases of the CA clan C19 family, representing the first prokaryotic members of this protein family. Consequently, interference with the host ubiquitin system is an important host cell interaction mechanism of “Ca. Amoebophilus asiaticus”. More generally, we show that the eukaryotic domains identified in “Ca. Amoebophilus asiaticus” are also significantly enriched in the genomes of other amoeba-associated bacteria (including chlamydiae,Legionella pneumophila,Rickettsia bellii,Francisella tularensis, andMycobacterium avium). This indicates that phylogenetically and ecologically diverse bacteria which thrive inside amoebae exploit common mechanisms for interaction with their hosts, and it provides further evidence for the role of amoebae as training grounds for bacterial pathogens of humans.
CotE Binds to CotC and CotU and Mediates Their Interaction during Spore Coat Formation in
Bacillus subtilis
Authors: Isticato Rachele , Pelosi Assunta , De Felice Maurilio , Ricca Ezio
Published: December 2009

CotE is a morphogenic protein that controls the assembly of the coat, the proteinaceous structure that surrounds and protects the spore of
Bacillus subtilis
. CotE has long been thought to interact with several outer coat components, but such interactions were hypothesized from genetic experiment results and have never been directly demonstrated. To study the interaction of CotE with other coat components, we focused our attention on CotC and CotU, two outer coat proteins known to be under CotE control and to form a heterodimer. We report here the results of pull-down experiments that provide the first direct evidence that CotE contacts other coat components. In addition, coexpression experiments demonstrate that CotE is needed and sufficient to allow formation of the CotC-CotU heterodimer in a heterologous host.
Completed Genome Sequence of the Anaerobic Iron-Oxidizing Bacterium
Acidovorax ebreus
Strain TPSY
Authors: Byrne-Bailey Kathryne G. , Weber Karrie A. , Chair Antinea H. , Bose Saumyaditya , Knox Traci , Spanbauer Trisha L. , Chertkov Olga , Coates John D.
Published: December 2009

Acidovorax ebreus
strain TPSY is the first anaerobic nitrate-dependent Fe(II) oxidizer for which there is a completed genome sequence. Preliminary protein annotation revealed an organism optimized for survival in a complex environmental system. Here, we briefly report the completed and annotated genome sequence of strain TPSY.
Nitrogenase Switch-Off and Regulation of Ammonium Assimilation in Response to Light Deprivation in
Rhodospirillum rubrum
Are Influenced by the Nitrogen Source Used during Growth
Authors: Teixeira Pedro Filipe , Wang He , Nordlund Stefan
Published: December 2009

Nitrogen fixation and ammonium assimilation in
Rhodospirillum rubrum
are regulated in response to changes in light availability, and we show that the response in terms of glutamine synthetase activity and P
II
modification is dependent on the nitrogen source used for growth, N
2
or glutamate, although both lead to nitrogenase derepression.
Is periprocedural sedation during acute stroke therapy associated with poorer functional outcomes?
Authors: Nichols C , Carrozzella J , Yeatts S , Tomsick T , Broderick J , Khatri P
Published: December 2009

Background

To safely perform acute intra-arterial revascularization procedures, use of sedative medications and paralytics is often necessary. During the conduct of the Interventional Management of Stroke trials (I and II), the level of sedation used periprocedurally varied. At some institutions, patients were paralyzed and intubated as part of the procedural standard of care while at other institutions no routine sedation protocol was followed. The aim of this study was to identify patient characteristics that would correlate with the need for deeper sedation and to explore whether levels of sedation relate to patient outcome.

Methods

75 of 81 patients in the Interventional Management of Stroke II Study were studied. Patients had anterior circulation strokes and underwent angiography and/or intervention. Four sedation categories were defined and tested for factors potentially associated with the level of sedation. Clinical outcomes were also analyzed, including successful angiographic reperfusion and the occurrence of clinical complications.

Results

Only baseline National Institutes of Health Stroke Scale varied significantly by sedation category (p=0.01). Patients that were in the lower sedation category fared better, having a higher rate of good outcomes (p<0.01), lower death rates (p=0.02) and higher successful angiographic reperfusion rates (p=0.01). There was a significantly higher infection rate in patients receiving heavy sedation or pharmacologic paralysis (p=0.02) and a trend towards fewer groin related complications.

Conclusion

In this small sample, patients not receiving sedation fared better, had higher rates of successful angiographic reperfusion and had fewer complications. Further examination of the indications for procedural sedation or paralysis and their effect on outcome is warranted.
Prospects for a genetic management strategy to control Gyrodactylus salaris infection in wild Atlantic salmon (Salmo salar) stocks
Authors: Salte Ragnar , Bentsen Hans Bernhard , Moen Thomas , Tripathy Smita , Bakke Tor Andreas , Ødegård Jørgen , Omholt Stig , Hansen Lars Petter
Published: December 2009

We estimated additive genetic variation and heritability of survival after Gyrodactylus salaris infection from survival records in a pedigreed family material of wild Atlantic salmon ( Salmo salar ) in a controlled challenge test. We used a statistical model that distinguishes between survival time for the fish that died and the ability to survive the entire test as two separate traits. Eleven of the 49 full-sib families suffered 100% mortality, 15 families had between 10% and 25% survival, and the four least affected families had survival rates between 36% and 48%. Estimated heritability of survival on the liability scale was 0.32 ± 0.10. Time until death for fish that died during the test and the ability to survive the entire test were not expressions of the same genetic trait. Simply selecting survivors as parents for the next generation is expected to more than double the overall survival rate in only one generation, given similar exposure to the parasite. Improving the genetic capacity to survive the infection will probably not eradicate the parasite, but when used as a disease control measure, such improvement may contain the infection at a level where the parasite ceases to be a major problem.
THE INFLUENCE OF DRUGS AND POTASSIUM ON RESPIRATION AND POTASSIUM ACCUMULATION BY BRAIN TISSUE
Authors: Bilodeau Fernand , Elliott K. A. C.
Published: December 2009

When brain cortex slices in which the potassium content has been reduced by cold-pretreatment are incubated aerobically at 38° in a potassium-free glucose-containing medium, addition to the medium of 3.6 mM potassium causes an increase of the oxygen uptake rate (15) accompanied by a reaccumulation of potassium in the tissue and an approximately equal extrusion of sodium. In the absence of added substrate or in a sodium-free medium, little potassium is reaccumulated in the tissue and no increase of the oxygen uptake rate is observed. Malonate (1 mM), pentobarbital (0.5 mM), and protamine (100 μg per ml), which decrease the potassium uptake, and phenobarbital (0.5 mM), chlorpromazine (0.01 mM), atropine (1 mM), and procaine (0.1 mM), which have no effect on potassium uptake, suppress the stimulatory action of 3.6 mM potassium on respiration and markedly reduce the large stimulatory effect of 60 mM potassium. Protoveratrine (0.001 mM) and 2,4-dinitrophenol (0.05 mM) also exert little or no effect on the non-stimulated respiration, but when they are added with potassium (3.6 mM) the respiration is increased more than by potassium alone. When protoveratrine is added before potassium, it depresses the potassium uptake and suppresses the stimulatory action of potassium on respiration. Interrelations of the actions of drugs and potassium are discussed and the similarities between the biochemical actions of diverse agents are noted.
Biochemistry of Polyglycerophosphatides. Effect of Divalent Cations on the Biosynthesis and Composition of Polyglycerophosphatides in Isolated Mitochondria
Authors: Domazet Z. , Stuhne-Sekalec L. , Davidson J. B. , Stanacev N. Z.
Published: December 2009

The effect of the addition of divalent cations on the formation of phosphatidylglycerophosphate, phosphatidylglycerol, and cardiolipin in isolated mitochondria from rat liver and guinea pig heart was studied. Although it was confirmed that the biosynthesis of phosphatidylglycerol in rat liver mitochondria does not require the addition of divalent cations, the addition of Mn2+ and Mg2+ has a profound effect on the level of synthesis and composition of polyglycerophosphatides. By developing a method for the preparation of membrane-bound labeled phosphatidylglycerol and an assay for the quantitative determination of polyglycerophosphatides, the high conversion of phosphatidylglycerol-2′-3H to cardiolipin-3H was established only in the presence of Mn2+ and/or Mg2+. Zn2+ and Ca2+, in the concentration tested, did not stimulate this conversion, nor were they inhibitory. In addition, the association of formed cardiolipin-3H with the mitochondrial membrane was demonstrated. Some implications of these findings are discussed.
Urea-Induced Inactivation and Dissociation of NAD-Specific Glutamate Dehydrogenase of Neurospora and its Reversal by Substrates
Authors: Grover A. K. , Kapoor M.
Published: December 2009

The NAD-specific glutamate dehydrogenase is partially inactivated by 1.5 M and 2.0 M urea. The same concentrations of urea also catalyze a cleavage of the native enzyme into partially active sub-units, approximately half its size. On removal of urea, by passage of the denaturation mixture through a Sephadex G-25 column, the dissociated subunits undergo reassociation restoring the native structure. The presence of substrates NADH and 2-oxoglutarate during renaturation enhances the rate of reassociation and reactivation considerably.
Biochemical Changes in Progressive Muscular Dystrophy. X. Studies on the Biosynthesis of Protein and RNA in Cellular Fractions of the Skeletal Muscle of Normal and Vitamin E Deficient Rabbits
Authors: Villers André de , Simard Paulette , Srivastava Uma
Published: December 2009

Present investigation was undertaken to elucidate the metabolism of protein and nucleic acids in the skeletal muscle of control and vitamin E deficient rabbits. In order to attain this objective, the content and synthesis of protein and RNA were measured in the homogenate and various subcellular fractions of the skeletal muscle of control and vitamin E deficient rabbits. Our results indicate a possibility of reduction in the muscle volume in the dystrophic muscle since the content of DNA and RNA did not change in such muscles. The protein content, however, decreased considerably in the dystrophic muscle.Incorporation studies indicated a considerable increase in the synthesis of protein and RNA. It was also observed that the excretion of radioactivity was much higher in the urine of dystrophic animals. These findings indicate a rapid turnover of protein in the dystrophic muscle.The sucrose density gradient analysis of polysomal material from the dystrophic muscle indicated a lowering in the concentration of heavier polysomes synthesizing myosin and a considerable increase in the region of lighter polysomes synthesizing tropomyosin. From these findings it is speculated that vitamin E may play an important role in the synthesis of specific muscle proteins. Addition of 250 μg of poly-U restored the polysomal content of dystrophic muscle to that of control, leading to the hypothesis that vitamin E deficient rabbit muscle may be lacking in the messenger RNA content.
Studies on Avian Mucopolysaccharide–Peptide Complexes and on In Vivo Incorporation of D-[1-14C]hexosamine
Authors: Hilborn John C. , Anastassiadis Phoebus A.
Published: December 2009

The mucopolysaccharide–peptide complexes from White Leghorn skin, comb, wattle, heart, liver, and testis were isolated and analyzed for constituent carbohydrate and amino acid units. Individual mucopolysaccharides in each tissue were characterized using several techniques. All tissues examined, except liver, contained at least two mucopolysaccharides, including a low molecular weight component. The low molecular weight component appeared to be the only substance isolated from liver. The incorporation of D-[1-14C]hexosamine into acetone-dried tissues and mucopolysaccharide–peptide complexes was investigated. Intravenously injected D-[1-14C]galactosamine accumulated in the liver to a much greater extent than D-[1-14C]glucosamine. In addition, evidence was provided which indicated that glucosamine was converted to nonhexosamine products. The increase of radioactivity associated with mucopolysaccharide–peptide complexes with increasing time coincided with a decrease in liver radioactivity.
The Conformational State of Methionine Residues in the Temperature-controlled Transition of Chymotrypsinogen and α-Chymotrypsin
Authors: Wasi Safia , Hofmann T.
Published: December 2009

The reaction of the two methionine residues in bovine α-chymotrypsin and chymotrypsinogen A with hydrogen peroxide at pH 3.0 and temperatures varying from 4° to 60° was investigated. Reaction rate constants were determined and compared with those of free methionine. Methionine-192 in α-chymotrypsin has the same reactivity as free methionine over the whole temperature range. This is compatible with the results of X-ray analysis (1) which shows that the sulfur atom is fully exposed. The ratio of the reactivity of methionine-192 in chymotrypsinogen to that of free methionine at temperatures of up to 30° is about 1:40. At higher temperatures this ratio increases markedly. It is concluded that methionine-192 in chymotrypsinogen is partially buried and access to reagents is restricted. No reaction of methionine-180 has been detected up to 25° (α-chymotrypsin) and 30° (chymotrypsinogen), respectively. At higher temperatures there is a steep increase in the reactivity. These results have been interpreted in terms of a two-state transition between a form in which methionine-180 is buried and one in which it is exposed. The calculation of transition temperatures gave a value of 55° for α-chymotrypsin and 60° for chymotrypsinogen. These have been compared with values of 48–51° for α-chymotrypsin and 54–56° for the zymogen obtained from ultraviolet difference spectra and from optical rotatory dispersion studies (2–4).
The Role of Dihydropsoralen in Furanocoumarin Metabolism
Authors: Brown Stewart A.
Published: December 2009

Trapping experiments with [2-14C]umbelliferone and 2′,3′-dihydropsoralen (DHP) administered to Ruta graveolens yielded negative results over metabolic periods of 5–24 h. Specific radioactivities of psoralen were comparable to those previously observed, but those of DHP were about two orders of magnitude lower. These results do not support the theory (Z. Naturforsch. 25b, 700 (1970)) that DHP is the immediate precursor of psoralen in furanocoumarin biosynthesis, and it is proposed instead that DHP is not on the main pathway, but is in an equilibrium with psoralen that greatly favors dehydrogenation.
Electron Paramagnetic Resonance (34 and 55 GHz) Studies of Hemoproteins
Authors: Gray A. L. , Buckmaster H. A.
Published: December 2009

Measurements of the 34 and 55 GHz electron paramagnetic resonance (E.P.R.) spectra of single type A crystals of metmyoglobin, metmyoglobin fluoride and methemoglobin and polycrystalline metmyoglobin and methemoglobin at 77 K are reported. The frequency dependence of the g-values of metmyoglobin, metmyoglobin fluoride, and methemoglobin is used to estimate the zero-field splitting (2D). It is found that (2D) [Formula: see text], (2D) (MbF) = (8.1 ± 1.0) cm−1, and 15 cm−1 <(2D) (HbH2O). The observed angular E.P.R. line width variation of these hemoproteins is compared with the variation predicted by a misorientation hypothesis. The frequency dependence of these line widths is also studied and shown to be consistent with a statistical variation in the zero-field splitting. The g-value and line width measurements are compared with those reported previously and the estimates of the zero-field splitting are compared with the values obtained using other measurement techniques.
Solubilization and Selected Properties of Crambe Seed Thioglucosidase (Thioglucoside Glucohydrolase, EC 3.2.3.1)
Authors: Tookey H. L.
Published: December 2009

Ultrasonic treatment releases thioglucoside glucohydrolase (thioglucosidase) from insoluble particles of Crambe abyssinica seed meal. The crude enzyme is optimally activated by 10−2 M ascorbate, requires a reducing agent for stability, and is inhibited by 10−3 M p-chloromercuribenzenesulfonate.Chromatography of a meal extract on cross-linked dextran separates two enzyme fractions. About 80% of the activity elutes at an apparent molecular weight of 110 000; the remainder elutes at the void volume. Both crude soluble enzyme and the fractions from the dextran column produce goitrin from epi-progoitrin, but in the presence of ferrous ion the chief aglucon product becomes 1-cyano-2-hydroxy-3-butene. The Michaelis constant (of the major peak) at pH 5 is 0.004 M. Ferrous ion produces substrate inhibition at high epi-progoitrin levels. Sequential fractionation of meal extract by ammonium sulfate precipitation and by chromatography on cross-linked dextran effects an 80-fold purification.
Effect of Some Steroidal Compounds on the In Vitro Incorporation of Acetate-1-14C Into Digitonin Precipitable Sterols by Slices of Rat Liver
Authors: Patwardhan Vasant V. , Lanthier André
Published: December 2009

Rat liver slices were incubated in vitro with sodium acetate-1-14C and the effect of some steroids on the incorporation of the substrate into digitonin precipitable sterols (DPS) was studied. Radioactive biosynthetic DPS was isolated from each incubate following digitonin precipitation, hydrolysis of the digitonide, and two silica gel thin-layer chromatographies. The following compounds were found to inhibit the in vitro DPS biosynthesis in this system: medroxyprogesterone acetate, norethynodrel, 17α-ethynyl estradiol, 17-hydroxy-progesterone, progesterone, and dehydroepiandrosterone.
The Transfer of Glucose to Steroids by Sheep Liver Microsomes
Authors: Labow Rosalind S. , Williamson Denis G. , Layne Donald S.
Published: December 2009

Microsomal preparations from the liver of female sheep can effect the transfer of glucose from UDP-glucose to the 17α-hydroxyl group of 17α-estradiol-3-glucuronide, and, in much poorer yield, to some other steroids containing a 17 α-hydroxyl group. No evidence was found for steroid galactosyl or N-acetylglucosaminyl transferase activity in sheep liver. Glucuronyl transferase with an activity of a high order to a variety of substrates was present. Preliminary work indicated that the steroid glucosyl transfer is mediated by an enzyme distinct from glucuronyl transferase.
Effects of Actinomycin D on Purine Ribonucleotide Metabolism in Ehrlich Ascites Tumor Cells In Vitro
Authors: Snyder Floyd F. , Henderson J. Frank
Published: December 2009

Actinomycin D treatment of Ehrlich ascites tumor cells in vitro causes slight to moderate inhibition of purine ribonucleotide synthesis de novo and from purine bases, and strong inhibition of inosinate dehydrogenase activity. These effects have the same dose–response relationship as inhibition of RNA synthesis by this drug. Daunomycin has similar effects on purine metabolism at a concentration that substantially inhibits nucleic acid synthesis. Actinomycin D treatment leads to elevated intracellular concentrations of ATP and GTP, and the effects of this drug on purine metabolism are believed to be mediated by these purine ribonucleoside triphosphates.
Evidence that Ferrihemoglobin May Function as an Intracellular Heme Carrier in Reticulocytes
Authors: Schulman Herbert M.
Published: December 2009

Ferrihemoglobin can (1) stimulate globin synthesis in rabbit reticulocytes and in a reticulocyte cell-free protein synthesizing system, and (2) cause newly synthesized αβ-globin dimers to form tetramers which are indistinguishable from hemoglobin in the ultracentrifuge.These observations are consistent with the idea that a small pool of ferrihemoglobin may act as an intracellular heme carrier in reticulocytes.
A Cytokinin Oxidase in Zea mays
Authors: Whitty C. Dennis , Hall Ross H.
Published: December 2009

An enzyme activity that catalyzes conversion of N6-(Δ2-isopentenyl)adenosine (i6Ado) to adenosine was detected in cultured tobacco tissue by Pačes et al. (1971) (Plant Physiol. 48, 775–778). Purification and characteristics of this enzyme in corn kernels have been studied. Only the naturally occurring cytokinins i6Ado and ribosylzeatin serve as substrates; the nucleoside or free base works equally as well. The reaction requires oxygen. An unstable intermediate appears to be the primary reaction product. This product decomposes to adenosine.The enzyme activity is greatest at pH 5–7. It is independent of added magnesium. The molecular weight of the enzyme is about 88 000. The activity of the enzyme in corn kernels increases from the time of pollination to about 21 days.A nucleoside hydrolase is isolated with i6Ado oxidase. The activities can be partially separated by G-150 gel filtration. The hydrolase is less stable than the i6Ado oxidase and frozen preparations lose their activity after 2 months, whereas i6Ado oxidase is stable under these conditions.
Structural Studies of Staphylococcal Protease. I. Spin Labelling of the Active Site and a Comparison with other Proteases
Authors: Dugas Hermann , Gaudet Fernand
Published: December 2009

Staphylococcus aureus protease has been spin-labelled at the active-site serine residue with the monocyclic-phosphorus spin label (MSL), 1-oxyl-2,2,6,6-tetramethyl-4-piperidinylethylphosphorofluoridate. The electron paramagnetic resonance (E.P.R.) spectra of the protease in different buffers at various pH's have been analyzed and compared with those of trypsin, subtilisin BPN′, and α-chymotrypsin under identical conditions. In a given buffer, the shape of the E.P.R. signals of spin-labelled staphylococcal protease is unaffected by pH changes except below pH 4.0, at which a gradual loss of conformational integrity of the active site occurs. In bicarbonate buffer and particularly in acetate buffer, the mobility of the label is much more restricted than in phosphate buffer or in potassium chloride solution. The implications of this finding are discussed in terms of a model whereby the label is able to orient towards two different but adjacent regions of the active site. The relative population of the label in each of these orientations is believed to be buffer-dependent. An attempt to correlate the shape of the E.P.R. signals with the pH values of maximal proteolytic activity of the enzyme is also presented. These results show that to obtain meaningful information from a comparative spin label study of the geometry of the active site of serine proteases, particular care should be exercised to assure that the different proteases experience identical conditions of pH, buffer, and temperature.
S-Adenosylhomocysteine Metabolism in Various Species
Authors: Walker R. D. , Duerre J. A.
Published: December 2009

Eleven microorganisms, four plants, and major organs from the chicken, dog, rat, and rabbit were assayed for the presence of S-adenosylhomocysteine hydrolase, S-adenosyl-homocysteine nucleosidase, and S-ribosylhomocysteine-cleavage enzyme. All bacteria (procaryotes) were found to possess S-adenosylhomocysteine nucleosidase and S-ribosylhomocysteine-cleavage enzyme but not S-adenosylhomocysteine hydrolase. All eucaryotes tested, including yeasts, plants, birds, and mammals, possessed S-adenosylhomocysteine hydrolase but not S-adenosylhomocysteine nucleosidase or S-ribosylhomocysteine-cleavage enzyme. Of all the organs assayed in the vertebrates, the level of S-adenosylhomocysteine hydrolase was highest in liver, pancreas, and kidney, lower in spleen and testis, and very low in brain and heart. In all systems tested, equilibrium of the hydrolase reaction always favored synthesis over hydrolysis. We studied some of the kinetic properties of the hydrolase from rat liver. In the direction of synthesis, the Km value was 1.5 mM for adenosine and 4.5 mM for L-homocysteine, whereas marked substrate inhibition was observed with L-homocysteine. The condensation reaction is subject to product inhibition, and was inhibited by adenine. Results from in-vivo experiments revealed that the cells of the various organs of the dog are impermeable to the exogenously administered S-adenosylhomocysteine.
Preparation and Properties of Matrix-Supported Horseradish Peroxidase
Authors: Bartling Greg J. , Chattopadhyay Swaraj K. , Barker Charles W. , Brown Harry D.
Published: December 2009

A new method of enzyme immobilization has been described using poly(4-methacryloxybenzoic acid) as the carrier. Activation of the polymer, prior to enzyme attachment, was achieved with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The enzyme coupling step proceeded through nucleophilic attack by the protein on a mixed carbonic anhydride. The degree of polymer activation was determined by analysis for quinoline, a by-product of the reaction. The polymer–enzyme complex was compared to the enzyme in solution in terms of pH optimum, substrate kinetics, and thermal denaturation. Potential uses of the polymer–enzyme system in chemical synthesis of benzoquinone derivatives are discussed.
Inhibition of Cardiolipin Synthesis by End-Products and Other Complex Lipids in Membrane Preparations of Micrococcus lysodeikticus
Authors: Siervo August J. De
Published: December 2009

Using membrane preparations of Micrococcus lysodeikticus, the end-products of cardiolipin synthesis, cardiolipin and glycerol, were shown to inhibit cardiolipin synthetase at several concentrations. Other phospholipids tested for inhibitory effects, phosphatidylethanolamine, phosphatidylinositol, and phosphatide acid were also shown to inhibit cardiolipin synthesis. Phosphatidic acid was considerably more inhibitory than cardiolipin, phosphatidylethanolamine was similar to cardiolipin, and phosphatidylinositol less inhibitory at the same concentrations. A non-phosphate-containing glycolipid was also inhibitory. In contrast, glycerophosphate had no effect on cardiolipin synthesis.
Effect of palmitoyl-CoA and β-oxidation of fatty acids on the kinetics of mitochondrial citrate transporter
Authors: Cheema-Dhadli Surinder , Halperin Mitchell L.
Published: December 2009

The kinetics of the hepatic mitochondrial citrate transporter were studied using 1,2,3-benzene tricarboxylate and the inhibitor-stop technique at 8 °C. The apparent Km for this transporter was 250 μM and the maximum velocity was 2 nmol of citrate transported per minute per milligram of mitochondrial protein. This apparent Km was increased when hepatic mitochondria were preincubated with both L-palmitoylcarnitine and CoA-SH but not with either alone. This rise in apparent Km was accompanied by a rise in the acid insoluble CoA-SH content. Removal of mitochondrial acid insoluble CoA by 'defatted albumin' resulted in a parallel decrease in the apparent Km. The apparent Km for the citrate transporter was increased after coupled β-oxidation of L-palmitoylcarnitine or octanoate without a detectable increase in acid insoluble CoA. Inhibition of β-oxidation of L-palmitoylcarnitine by the D-derivative prevented the rise in the apparent Km. Preincubation with ATP resulted in an increase in this apparent Km. When L-palmitoylcarnitine oxidation occurred without ATP accumulation (hexokinase, glucose, ADP, and inorganic phosphate) the apparent Km for the citrate transporter increased two- to threefold.Therefore, the apparent Km for the citrate transporter varied directly with the acid insoluble CoA content. In addition, this Km was increased as a result of β-oxidation of fatty acids but the mechanism was not solely attributable to a rise in acid insoluble CoA or ATP. The physiological implications of these findings are discussed.
Separation of potato spindle tuber viroid ribonucleic acid from Scopolia sinensis into three infectious forms and the purification and oligonucleotide pattern of fraction II RNA. Part IX
Authors: Singh R. P. , Michniewicz J. J. , Narang S. A.
Published: December 2009

The existence of three infectious forms of potato spindle tuber viroid (PSTV) RNA from Scopolia sinensis was demonstrated by fractionation with high salt, by reverse phase and high pressure liquid chromatography, and by polyacrylamide gel electrophoresis. Purification of fraction II was achieved by the following steps: extraction of nucleic acid with phenol, precipitation of the RNA with cetyltrimethylammonium bromide, fractionation of the RNA with lithium chloride and isopropanol, and finally gel electrophoresis. A procedure using reverse phase chromatography was developed to obtain 70–90% recovery of RNA from polyacrylamide gels. Purified PSTV fraction II RNA was digested with ribonuclease A and T1 and labelled with [γ-32P]ATP using polynucleotide kinase. The labelled digests were separated by the electrophoresis–homochromatography procedures of Sanger. About 20 and 30 spots were obtained with ribonuclease A and T1, respectively.
Circular dichroism and difference spectra of members of the troponin–tropomyosin system in cetyltrimethylammonium bromide
Authors: McCubbin William D. , Kay Cyril M.
Published: December 2009

The detergent cetyltrimethylammonium bromide (CTAB) was used as a perturbant to study protein structure. Low concentrations of CTAB induced difference spectra for Ac-Trp-OEt and Ac-Tyr-OEt. The ΔεM values at their difference maxima were found to be 1300 at 292 nm for Ac-Tyr-OEt and 400 at 287 for Ac-Tyr-OEt. These values were used to determine the number of tyrosine residues exposed in tropomyosin and troponin C, as well as the tyrosine and tryptophan residues exposed in troponin I and troponin T. In tropomyosin and troponin C all of the tyorosine residues were accessible to detergent. For TN-T, three of four tyrosines were free while the tryptophan residues were only partially exposed. In the case of TN-I both tyrosines were fully exposed but again evidence was obtained for a partially buried tryptophan chromophore. The stability of these proteins to CTAB was studied by measuring the far-uv circular dichroism spectra. Tropomyosin was quite sensitive to detergent and suffered a 60% loss in ellipticity at the concentration of CTAB used. The troponins, on the other hand, were affected to a lesser extent.
Pericytes in the macrovascular intima: possible physiological and pathogenetic impact
Authors: Juchem Gerd , Weiss Dominik R. , Gansera Brigitte , Kemkes Bernhard M. , Mueller-Hoecker Josef , Nees Stephan
Published: December 2009

The frequently observed de-endothelialization of venous coronary bypass grafts prepared using standard methods exposes subendothelial prothrombotic cells to blood components, thus endangering patients by inducing acute thromboembolic infarction or long-term proliferative stenosis. Our aim was to gain deeper histological and physiological insight into these relations. An intricate network of subendothelial cells, characterized by histological features specific for true pericytes, was detected even in healthy vessels and forms, coupled to the luminal endothelium, a second leaflet of the macrovascular intima. These cells, and particularly those in the venous intima, express enormous concentrations of tissue factor and can recruit additional amounts of up to the 25-fold concentration within 1 h during preincubation with serum (intimal pericytes of venous origin activate 30.71 ± 4.07 pmol coagulation factor x·min−1·10−6 cells; n = 15). Moreover, decoupled from the endothelium, they proliferate rapidly (generation time, 15 ± 2.1 h, n = 8). Central regions of atherosclerotic plaques, as well as of those of restenosed areas of coronary vein grafts, consist almost completely of these cells. In stark contrast with the prothrombogenicity of the intimal pericytes, intact luminal endothelium recruits high concentrations of thrombomodulin (CD 141) specifically within its intercellular junctions, activates Protein C rapidly (42 ± 5.1 pmol/min·106 venous endothelial cells at thrombin saturation; n = 15), can thus actively prevent coagulatory processes, and never expresses histologically detectable and functionally active tissue factor. Given this strongly prothrombotic potential of the intimal pericytes and their overshooting growth behavior in endothelium-denuded vascular regions, they may play important roles in the development of atherosclerosis, thrombosis, and saphenous vein graft disease.
Cross-bridge cycling gives rise to spatiotemporal heterogeneity of dynamic subcellular mechanics in cardiac myocytes probed with atomic force microscopy
Authors: Azeloglu Evren U. , Costa Kevin D.
Published: December 2009

To study how the dynamic subcellular mechanical properties of the heart relate to the fundamental underlying process of actin-myosin cross-bridge cycling, we developed a novel atomic force microscope elastography technique for mapping spatiotemporal stiffness of isolated, spontaneously beating neonatal rat cardiomyocytes. Cells were indented repeatedly at a rate close but unequal to their contractile frequency. The resultant changes in pointwise apparent elastic modulus cycled at a predictable envelope frequency between a systolic value of 26.2 ± 5.1 kPa and a diastolic value of 7.8 ± 4.1 kPa at a representative depth of 400 nm. In cells probed along their major axis, spatiotemporal changes in systolic stiffness displayed a heterogeneous pattern, reflecting the banded sarcomeric structure of underlying myofibrils. Treatment with blebbistatin eliminated contractile activity and resulted in a uniform apparent modulus of 6.5 ± 4.8 kPa. This study represents the first quantitative dynamic mechanical mapping of beating cardiomyocytes. The technique provides a means of probing the micromechanical effects of disease processes and pharmacological treatments on beating cardiomyocytes, providing new insights and relating subcellular cardiac structure and function.