bluuebunny/crossref_metadata_2025_split huggingface dataset:
Efficient Prenylation by a Plant Geranylgeranyltransferase-I Requires a Functional CaaL Box Motif and a Proximal Polybasic Domain
Authors: Caldelari Daniela , Sternberg Hasana , Rodrı́guez-Concepción Manuel , Gruissem Wilhelm , Yalovsky Shaul 
Published: October 2002

Abstract
Geranylgeranyltransferase-I (GGT-I) is a heterodimeric enzyme that shares a common α-subunit with farnesyltransferase (FTase) and has a distinct β-subunit. GGT-I preferentially modifies proteins, which terminate in a CaaL box sequence motif. Cloning of Arabidopsis GGT-I β-subunit (AtGGT-IB) was achieved by a yeast (Saccharomyces cerevisiae) two-hybrid screen, using the tomato (Lycopersicon esculentum) FTase α-subunit (FTA) as bait. Sequence and structure analysis revealed that the core active site of GGT-I and FTase are very similar. AtGGT-IA/FTA and AtGGT-IB were co-expressed in baculovirus-infected insect cells to obtain recombinant protein that was used for biochemical and molecular analysis. The recombinant AtGGT-I prenylated efficiently CaaL box fusion proteins in which the a2 position was occupied by an aliphatic residue, whereas charged or polar residues at the same position greatly reduced the efficiency of prenylation. A polybasic domain proximal to the CaaL box motif induced a 5-fold increase in the maximal reaction rate, and increased the affinity of the enzyme to the protein substrate by an order of magnitude. GGT-I retained high activity in a temperature range between 24°C and 42°C, and showed increased activity rate at relatively basic pH values of 7.9 and 8.5. Reverse transcriptase-polymerase chain reaction, protein immuno-blots, and transient expression assays of green fluorescent protein fusion proteins show that GGT-IB is ubiquitously expressed in a number of tissues, and that expression levels and protein activity were not changed in mutant plants lacking FTase β-subunit.
Chemical Composition of thePrunus laurocerasusLeaf Surface. Dynamic Changes of the Epicuticular Wax Film during Leaf Development
Authors: Jetter Reinhard , Schäffer Stefanie 
Published: October 2002

AbstractThe seasonal development of adaxial Prunus laurocerasus leaf surfaces was studied using newly developed methods for the mechanical removal of epicuticular waxes. During epidermal cell expansion, more than 50 μg leaf−1 of alkyl acetates accumulated within 10 d, forming an epicuticular wax film approximately 30 nm thick. Then, alcohols dominated for 18 d of leaf development, before alkanes accumulated in an epicuticular wax film with steadily increasing thickness (approximately 60 nm after 60 d), accompanied by small amounts of fatty acids, aldehydes, and alkyl esters. In contrast, the intracuticular waxes stayed fairly constant during development, being dominated by triterpenoids that could not be detected in the epicuticular waxes. The accumulation rates of all cuticular components are indicative for spontaneous segregation of intra- and epicuticular fractions during diffusional transport within the cuticle. This is the first report quantifying the loss of individual compound classes (acetates and alcohols) from the epicuticular wax mixture. Experiments with isolated epicuticular films showed that neither chemical conversion within the epicuticular film nor erosion/evaporation of wax constituents could account for this effect. Instead, transport of epicuticular compounds back into the tissue seems likely. Possible ecological and physiological functions of the coordinate changes in the composition of the plant surface layers are discussed.
The Short-Chain Alcohol Dehydrogenase ABA2 Catalyzes the Conversion of Xanthoxin to Abscisic Aldehyde[W]
Authors: González-Guzmán Miguel , Apostolova Nadezda , Bellés José M. , Barrero José M. , Piqueras Pedro , Ponce María R. , Micol José L. , Serrano Ramón , Rodríguez Pedro L. 
Published: October 2002

AbstractMutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified during the course of two independent screenings and named salt resistant (sre) and salobreño (sañ). The sre and sañ mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay. Complementation analyses revealed that sre1-1, sre1-2, sañ3-1, and sañ3-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene. A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of four new aba2 alleles. The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD- or NADP-dependent oxidoreductases. Recombinant ABA2 protein produced in Escherichia coli exhibits a K m value for xanthoxin of 19 μM and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC–mass spectrometry. The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress. The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin→abscisic aldehyde→ABA transition as the last steps of the major ABA biosynthetic pathway.
Characterization and multiple molecular forms of TorD from Shewanella massilia, the putative chaperone of the molybdoenzyme TorA
Authors: Tranier Samuel , Mortier‐Barrière Isabelle , Ilbert Marianne , Birck Catherine , Iobbi‐Nivol Chantal , Méjean Vincent , Samama Jean‐Pierre 
Published: October 2002

AbstractSeveral bacteria use trimethylamine N‐oxyde (TMAO) as an exogenous electron acceptor for anaerobic respiration. This metabolic pathway involves expression of the tor operon that codes for a periplasmic molybdopterin‐containing reductase of the DMSO/TMAO family, a pentahemic c‐type cytochrome, and the TorD cytoplasmic chaperone, possibly required for acquisition of the molybdenum cofactor and translocation of the reductase by the twin‐arginine translocation system. In this report, we show that the TorD chaperone from Shewanella massilia forms multiple and stable oligomeric species. The monomeric, dimeric, and trimeric forms were purified to homogeneity and characterized by analytical ultracentrifugation. Small‐angle X‐ray scattering (SAXS) and preliminary diffraction data indicated that the TorD dimer is made of identical protein modules of similar size to the monomeric species. Interconversion of the native oligomeric forms occurred at acidic pH value. In this condition, ANS fluorescence indicates a non‐native conformation of the polypeptide chain in which, according to the circular dichroism spectra, the α‐helical content is similar to that of the native species. Surface plasmon resonance showed that both the monomeric and dimeric species bind the mature TorA enzyme, but that the dimer binds its target protein more efficiently. The possible biologic significance of these oligomers is discussed in relation to the chaperone activity of TorD, and to the ability of another member of the TorD family to bind the Twin Arginine leader sequences of the precursor of DMSO/TMAO reductases.
The effect of prime‐site occupancy on the hepatitis C virus NS3 protease structure
Authors: Casbarra Annarita , Piaz Fabrizio Dal , Ingallinella Paolo , Orrù Stefania , Pucci Piero , Pessi Antonello , Bianchi Elisabetta 
Published: October 2002

AbstractWe recently reported a new class of inhibitors of the chymotrypsin‐like serine protease NS3 of the hepatitis C virus. These inhibitors exploit the binding potential of the S′ site of the protease, which is not generally used by the natural substrates. The effect of prime‐site occupancy was analyzed by circular dichroism spectroscopy and limited proteolysis‐mass spectrometry. Generally, nonprime inhibitors cause a structural change in NS3. Binding in the S′ site produces additional conformational changes with different binding modes, even in the case of the NS3/4A cofactor complex. Notably, inhibitor binding either in the S or S′ site also has profound effects on the stabilization of the protease. In addition, the stabilization propagates to regions not in direct contact with the inhibitor. In particular, the N‐terminal region, which according to structural studies is endowed with low structural stability and is not stabilized by nonprime inhibitors, was now fully protected from proteolytic degradation. From the perspective of drug design, P‐P′ inhibitors take advantage of binding pockets, which are not exploited by the natural HCV substrates; hence, they are an entry point for a novel class of NS3/4A inhibitors. Here we show that binding of each inhibitor is associated with a specific structural rearrangement. The development of a range of inhibitors belonging to different classes and an understanding of their interactions with the protease are required to address the issue of the most likely outcome of viral protease inhibitor therapy, that is, viral resistance.
Plasticity in structure and interactions is critical for the action of indolicidin, an antibacterial peptide of innate immune origin
Authors: Nagpal Sushma , Kaur Kanwal J. , Jain Deepti , Salunke Dinakar M. 
Published: October 2002

AbstractThe comparative analysis of two cationic antibacterial peptides of the cathelicidin family—indolicidin and tritrypticin—enabled addressing the structural features critical for the mechanism of indolicidin activity. Functional behavior of retro‐indolicidin was found to be identical to that of native indolicidin. It is apparent that the gross conformational propensities associated with retro‐peptides resemble those of the native sequences, suggesting that native and retro‐peptides can have similar structures. Both the native and the retro‐indolicidin show identical affinities while binding to endotoxin, the initial event associated with the antibacterial activity of cationic peptide antibiotics. The indolicidin–endotoxin binding was modeled by docking the indolicidin molecule in the endotoxin structure. The conformational flexibility associated with the indolicidin residues, as well as that of the fatty acid chains of endotoxin combined with the relatively strong structural interactions, such as ionic and hydrophobic, provide the basis for the endotoxin–peptide recognition. Thus, the key feature of the recognition between the cationic antibacterial peptides and endotoxin is the plasticity of molecular interactions, which may have been designed for the purpose of maintaining activity against a broad range of organisms, a hallmark of primitive host defense.
The role of disulfide bond in the amyloidogenic state of β2‐microglobulin studied by heteronuclear NMR
Authors: Katou Hidenori , Kanno Takashi , Hoshino Masaru , Hagihara Yoshihisa , Tanaka Hiroyuki , Kawai Tomoji , Hasegawa Kazuhiro , Naiki Hironobu , Goto Yuji 
Published: October 2002

Abstractβ2‐Microglobulin (β2‐m) is a major component of dialysis‐related amyloid fibrils. Although recombinant β2‐m forms needle‐like fibrils by in vitro extension reaction at pH 2.5, reduced β2‐m, in which the intrachain disulfide bond is reduced, cannot form typical fibrils. Instead, thinner and flexible filaments are formed, as shown by atomic force microscopy images. To clarify the role of the disulfide bond in amyloid fibril formation, we characterized the conformations of the oxidized (intact) and reduced forms of β2‐m in the acid‐denatured state at pH 2.5, as well as the native state at pH 6.5, by heteronuclear NMR. {1H}‐15N NOE at the regions between the two cysteine residues (Cys25–Cys80) revealed a marked difference in the pico‐ and nanosecond time scale dynamics between that the acid‐denatured oxidized and reduced states, with the former showing reduced mobility. Intriguingly, the secondary chemical shifts, ΔCα, ΔCO, and ΔHα, and 3JHNHα coupling constants indicated that both the oxidized and reduced β2‐m at pH 2.5 have marginal α‐helical propensity at regions close to the C‐terminal cysteine, although it is a β‐sheet protein in the native state. The results suggest that the reduced mobility of the denatured state is an important factor for the amylodogenic potential of β2‐m, and that the marginal helical propensity at the C‐terminal regions might play a role in modifying this potential.
Protein stability indicates divergent evolution of PD‐(D/E)XK type II restriction endonucleases
Authors: Fuxreiter Monika , Simon István 
Published: October 2002

AbstractType II restriction endonucleases recognize 4–8 base‐pair‐long DNA sequences and catalyze their cleavage with remarkable specificity. Crystal structures of the PD‐(DE)XK superfamily revealed a common α/β core motif and similar active site. In contrast, these enzymes show little sequence similarity and use different strategies to interact with their substrate DNA. The intriguing question is whether this enzyme family could have evolved from a common origin. In our present work, protein structure stability elements were analyzed and compared in three parts of PD‐(DE)XK type II restriction endonucleases: (1) core motif, (2) active‐site residues, and (3) residues playing role in DNA recognition. High correlation was found between the active‐site residues and those stabilization factors that contribute to preventing structural decay. DNA recognition sites were also observed to participate in stabilization centers. It indicates that recognition motifs and active sites in PD‐(DE)XK type II restriction endonucleases should have been evolutionary more conserved than other parts of the structure. Based on this observation it is proposed that PD‐(DE)XK type II restriction endonucleases have developed from a common ancestor with divergent evolution.
Evaluation of Antibiotic Susceptibilities of Three Rickettsial Species Including
Rickettsia felis
by a Quantitative PCR DNA Assay
Authors: Rolain Jean-Marc , Stuhl Laetitia , Maurin Max , Raoult Didier 
Published: October 2002

ABSTRACT

Rickettsiae grow only intracellularly, and the antibiotic susceptibilities of these bacteria have been assessed by either plaque, dye uptake, or immunofluorescence assays, which are time-consuming. We used a quantitative PCR (with the LightCycler instrument) to assess the levels of inhibition of
Rickettisa felis
,
R. conorii
, and
R. typhi
DNA synthesis in the presence of various antibiotics. We established the kinetics of rickettsial DNA during growth and showed that
R. conorii
grows more quickly than
R. typhi
in cell culture, with maximum replication occurring after 5 and 7 days, respectively. The MICs of the antibiotics tested for
R. conorii
and
R. typhi
by the quantitative PCR assay were similar to those previously obtained by plaque and dye uptake assays. We found that
R. felis
is susceptible to doxycycline, rifampin, thiamphenicol, and fluoroquinolones but not to gentamicin, erythromycin, amoxicillin, or trimethoprim-sulfamethoxazole. The resistance of this new species to erythromycin is consistent with its current taxonomic position within the spotted fever group. We believe that quantitative PCR could be used in the future to simplify and shorten antibiotic susceptibility assays of other rickettsiae and other strict intracellular pathogens.
Effects of Neutrophils on Cefazolin Activity and Penicillin-Binding Proteins in
Staphylococcus aureus
Abscesses
Authors: Bamberger David M. , Herndon Betty L. , Fitch Jeffrey , Florkowski Aaron , Parkhurst Vera 
Published: October 2002

ABSTRACT

Bacteria survive within abscesses despite antimicrobial therapy, usually necessitating drainage. Our previous work showed that bacterial killing is diminished within the neutrophils of animals with abscesses. To further assess the role of neutrophils in
Staphylococcus aureus
survival and the poor activities of β-lactams in abscesses, tissue cage abscess-bearing rats were given polymorphonuclear leukocyte (PMN)-depleting antibody prior to and several times following inoculation of the tissue cages with
S. aureus.
Cefazolin (300 mg/kg of body weight/day) was administered to all animals in appropriately divided doses. After 7 days of antimicrobial therapy, the 17 animals that received anti-PMN serum had significantly fewer abscess neutrophils than the 18 controls and fewer abscess bacteria (5.55 versus 3.79 log
10
CFU/ml [
P
= 0.04]) than the 18 controls. The data were consistent with the premise that cefazolin is more effective in abscesses depleted of neutrophils. To investigate further,
S. aureus
was incubated with rat peritoneal neutrophils; and bacterial cell membrane proteins were isolated, labeled with biotinylated ampicillin, separated by electrophoresis, blotted onto nitrocellulose, and stained for biotin reactivity. PBP 2 expression was consistently and significantly decreased after a brief, nonkilling PMN exposure. These experiments showed that PMN depletion enhanced the activity of cefazolin in the abscess milieu. Furthermore, altered bacterial cell wall cefazolin targets may be the mechanism by which the PMN diminishes antimicrobial activity, suggesting the importance of the staphylococcus-PMN interaction in the outcome of established infections.
Antistreptococcal Activity of Telithromycin Compared with Seven Other Drugs in Relation to Macrolide Resistance Mechanisms in Russia
Authors: Kozlov Roman S. , Bogdanovitch Tatiana M. , Appelbaum Peter C. , Ednie Lois , Stratchounski Leonid S. , Jacobs Michael R. , Bozdogan Bülent 
Published: October 2002

ABSTRACT

The susceptibilities of 468 recent Russian clinical
Streptococcus pneumoniae
isolates and 600
Streptococcus pyogenes
isolates, from 14 centers in Russia, to telithromycin, erythromycin, azithromycin, clarithromycin, clindamycin, levofloxacin, quinupristin-dalfopristin, and penicillin G were tested. Penicillin-nonsusceptible
S. pneumoniae
strains were rare except in Siberia, where their prevalence rate was 13.5%: most were penicillin intermediate, but for three strains (two from Smolensk and one from Novosibirsk) the MICs of penicillin G were 4 or 8 μg/ml. Overall, 2.5% of
S. pneumoniae
isolates were resistant to erythromycin. Efflux was the prevalent resistance mechanism (five strains; 41.7%), followed by ribosomal methylation encoded by constitutive
erm
(B), which was found in four isolates. Ribosomal mutation was the mechanism of macrolide resistance in three isolates; one erythromycin-resistant
S. pneumoniae
isolate had an A2059G mutation in 23S rRNA, and two isolates had substitution of GTG by TPS at positions 69 to 71 in ribosomal protein L4. All
S. pyogenes
isolates were susceptible to penicillin, and 11% were erythromycin resistant. Ribosomal methylation was the most common resistance mechanism for
S. pyogenes
(89.4%). These methylases were encoded by
erm
(A) [subclass
erm
(TR)] genes, and their expression was inducible in 96.6% of isolates. The rest of the erythromycin-resistant Russian
S. pyogenes
isolates (7.6%) had an efflux resistance mechanism. Telithromycin was active against 100% of pneumococci and 99.2% of
S. pyogenes
, and levofloxacin and quinupristin-dalfopristin were active against all isolates of both species.
Phenylpropenamide Derivatives AT-61 and AT-130 Inhibit Replication of Wild-Type and Lamivudine-Resistant Strains of Hepatitis B Virus In Vitro
Authors: Delaney William E. , Edwards Ros , Colledge Danni , Shaw Tim , Furman Phil , Painter George , Locarnini Stephen 
Published: October 2002

ABSTRACT
The phenylpropenamide derivatives AT-61 and AT-130 are nonnucleoside analogue inhibitors of hepatitis B virus (HBV) replication. They inhibited the replication of wild-type HBV with 50% inhibitory concentrations of 21.2 ± 9.5 and 2.40 ± 0.92 μM, respectively, compared to 0.064 ± 0.020 μM lamivudine. There were no significant differences in sensitivity between wild-type and nucleoside analogue-resistant (rtL180M, rtM204I, and rtL180M + rtM204V) HBV.
Cloning and Expression of the Haloalkane Dehalogenase GenedhmAfromMycobacterium aviumN85 and Preliminary Characterization of DhmA
Authors: Jesenská Andrea , Bartoš Milan , Czerneková Vladimíra , Rychlík Ivan , Pavlík Ivo , Damborský Jiří 
Published: October 2002

ABSTRACTHaloalkane dehalogenases are microbial enzymes that catalyze cleavage of the carbon-halogen bond by a hydrolytic mechanism. Until recently, these enzymes have been isolated only from bacteria living in contaminated environments. In this report we describe cloning of the dehalogenase genedhmAfromMycobacterium aviumsubsp.aviumN85 isolated from swine mesenteric lymph nodes. ThedhmAgene has a G+C content of 68.21% and codes for a polypeptide that is 301 amino acids long and has a calculated molecular mass of 34.7 kDa. The molecular masses of DhmA determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography are 34.0 and 35.4 kDa, respectively. Many residues essential for the dehalogenation reaction are conserved in DhmA; the putative catalytic triad consists of Asp123, His279, and Asp250, and the putative oxyanion hole consists of Glu55 and Trp124. Trp124 should be involved in substrate binding and product (halide) stabilization, while the second halide-stabilizing residue cannot be identified from a comparison of the DhmA sequence with the sequences of three dehalogenases with known tertiary structures. The haloalkane dehalogenase DhmA shows broad substrate specificity and good activity with the priority pollutant 1,2-dichloroethane. DhmA is significantly less stable than other currently known haloalkane dehalogenases. This study confirms that a hydrolytic dehalogenase is present in the facultative pathogenM. avium. The presence of dehalogenase-like genes in the genomes of other mycobacteria, including the obligate pathogensMycobacterium tuberculosisandMycobacterium bovis, as well as in other bacterial species, includingMesorhizobium loti, Xylella fastidiosa, Photobacterium profundum, andCaulobacter crescentus, led us to speculate that haloalkane dehalogenases have some other function besides catalysis of hydrolytic dehalogenation of halogenated substances.
Enhanced Production of
Trichoderma reesei
Endoglucanases and Use of the New Cellulase Preparations in Producing the Stonewashed Effect on Denim Fabric
Authors: Miettinen-Oinonen Arja , Suominen Pirkko 
Published: October 2002

ABSTRACT

Trichoderma reesei
strains were constructed for production of elevated amounts of endoglucanase II (EGII) with or without cellobiohydrolase I (CBHI). The endoglucanase activity produced by the EGII transformants correlated with the copy number of the
egl2
expression cassette. One copy of the
egl2
expression cassette in which the
egl2
was under the
cbh1
promoter increased production of endoglucanase activity 2.3-fold, and two copies increased production about 3-fold above that of the parent strain. When the enzyme with elevated EGII content was used, an improved stonewashing effect on denim fabric was achieved. A
T. reesei
strain producing high amounts of EGI and -II activities without CBHI and -II was constructed by replacing the
cbh2
locus with the coding region of the
egl2
gene in the EGI-overproducing CBHI-negative strain. Production of endoglucanase activity by the EG-transformant strain was increased fourfold above that of the host strain. The filter paper-degrading activity of the endoglucanase-overproducing strain was lowered to below detection, presumably because of the lack of cellobiohydrolases.