bluuebunny/crossref_metadata_2025_split huggingface dataset:
A possible role of neurotensin in NANC relaxation of longitudinal muscle of the jejunum and ileum of Wistar rats
Authors: Yamaji Michiru , Ohta Mayuko , Yamazaki Yasuko , Fujinami Kaori , Fujita Akikazu , Takeuchi Tadayoshi , Hata Fumiaki , Takewaki Tadashi 
Published: October 2002

The mediators of nonadrenergic, noncholinergic (NANC) relaxation in longitudinal muscle of the jejunum and ileum of Wistar rats were examined in vitro.
Treatment of the jejunal and ileal segments with α‐chymotrypsin resulted in decreases in the NANC relaxations induced by electrical field stimulation (EFS) by about one half.
The NANC relaxations were also decreased by about one half after the segments had been desensitized to neurotensin. A neurotensin receptor antagonist, SR48692 (10 μM) inhibited the NANC relaxation by 56 and 34% in the jejunal and ileal segments, respectively.
An inhibitor of small conductance Ca2+‐activated K+ channel (SK channel), apamin (100 nM) also inhibited the NANC relaxation by 83 and 63%, respectively. Exogenous neurotensin‐induced relaxations of the two segments were abolished by apamin.
In the ileal segments, NG‐nitro‐L‐arginine (L‐NOARG, 100 μM), inhibited the NANC relaxation by 43%. L‐NOARG, but not apamin, further inhibited the relaxation which persisted after the desensitization to neurotensin. Apamin with SR48692 inhibited the relaxation only to the same extent as apamin alone.
EFS induced inhibitory junction potentials (i.j.ps) in the longitudinal muscle cells of the ileum. I.j.ps consisted of a rapid and a delayed phase. L‐NOARG significantly inhibited only the delayed phase.
EFS induced only a rapid i.j.ps in the jejunum. SR48692 and apamin inhibited the i.j.ps.
These findings suggest that neurotensin and unknown substance(s) mediate NANC relaxation via SK channels in the jejunum of Wistar rats, and that neurotensin via SK channels and nitric oxide not via SK channels separately mediate the relaxation in the ileum.

British Journal of Pharmacology (2002) 137, 629–636. doi:10.1038/sj.bjp.0704914
Pharmacological approach towards the development of indolequinone bioreductive drugs based on the clinically inactive agent EO9
Authors: Loadman P M , Bibby M C , Phillips R M 
Published: October 2002

The bioreductive drug EO9 (3‐hydroxy‐5‐aziridinyl‐1‐methyl‐2[indole‐4,7‐dione]–prop‐β‐en‐α‐ol) has good pharmacodynamic properties in vitro, modest anti‐tumour activity in experimental tumour models, but failed to show activity in clinical trials. Understanding the reasons for its poor efficacy in vivo is important in terms of progressing second generation analogues into the clinic.
In two human tumour xenografts, direct intra‐tumoural injection resulted in improved anti‐tumour activity compared with intravenous administration suggesting that drug delivery to tumours is suboptimal. Compared with Mitomycin C (MMC) and the experimental agent MeDZQ, EO9 was rapidly cleared from the systemic circulation (t1/2=1.8 min) whereas MMC and MeDZQ had significantly increased plasma t1/2 values (14 and 22 min respectively). These three compounds demonstrated similar pharmacodynamic properties in terms of potency towards the NQO1 (NAD(P)H:Quinone oxidoreductase) rich H460 cell line in vitro but differed significantly in their in vivo activity with growth delays of 17.7, 4.5 and 1.0 days for MMC, MeDZQ and EO9 respectively. EO9 was rapidly metabolized by red blood cells in vitro (t1/2=14.5 min) which must contribute to its rapid pharmacokinetic elimination in vivo whereas MMC and MeDZQ were metabolized at comparatively slower rates (t1/2>120 min and 77.0 min respectively).
In conclusion, the development of second generation EO9 analogues should address the issue of drug delivery and analysis of drug metabolism by murine whole blood in vitro could be utilized as a preliminary screen to identify lead compounds that are likely to have improved pharmacokinetic profiles in vivo.

British Journal of Pharmacology (2002) 137, 701–709. doi:10.1038/sj.bjp.0704916
The impairment of endothelium‐dependent arterial relaxation by7‐ketocholesterol is associated with an early activation of protein kinase C
Authors: Deckert Valérie , Duverneuil Linda , Poupon Sandrine , Monier Serge , Le Guern Naig , Lizard Gérard , Masson David , Lagrost Laurent 
Published: October 2002

Among components of oxidized low density lipoproteins, cholesterol derivatives oxidized in position 7 inhibit endothelium‐dependent arterial relaxation by decreasing the release of the main endothelium‐derived relaxing factor, nitric oxide (NO). The aim of the present study was to bring new insights into the molecular mechanism by which 7‐ketocholesterol can block the endothelium‐dependent arterial relaxation.
Superoxide dismutase did not prevent the inhibitory effect of 7‐ketocholesterol on endothelium‐dependent relaxation, and consistent observations were made whether superoxide dismutase was conjugated or not to polyethylene glycol. In addition, neither glutathione supplementation, nor oxypurinol, i.e. a xanthine oxidase inhibitor could reverse the effect of 7‐ketocholesterol, indicating that NO was not inactivated by superoxide anion.
A direct alteration of the activity of the calcium‐dependent NO synthase could also be ruled out, since identical relaxing effects of the calcium ionophore A23187 were observed whether arterial rings were treated or not with 7‐ketocholesterol.
Whereas the above observations come in support of an early, inhibitory action of 7‐ketocholesterol, the specific blockade of one given subtype of membrane receptors could be discarded, and similar inhibitions were observed when either muscarinic or purinergic receptors were stimulated.
Finally, the blockade of protein kinase C activity by chelerythrine arose as the sole relevant tool in preventing the effect of 7‐ketocholesterol on the endothelium‐dependent relaxation of rabbit aortic rings. In addition, complementary studies on cultured bovine aortic endothelial cells came in direct support of the ability of 7‐ketocholesterol to activate PKC.
In conclusion, 7‐ketocholesterol that is present in human hypercholesterolaemic plasma, in atherosclerotic arteries, and in many processed foods can block the release of NO by vascular endothelial cells through its ability to activate PKC.

British Journal of Pharmacology (2002) 137, 655–662. doi:10.1038/sj.bjp.0704920
The role of helix stabilizing residues in GCN4 basic region folding and DNA binding
Authors: Hollenbeck Jessica J. , McClain Diana L. , Oakley Martha G. 
Published: October 2002

AbstractBasic region leucine zipper (bZip) proteins contain a bipartite DNA‐binding motif consisting of a coiled‐coil leucine zipper dimerization domain and a highly charged basic region that directly contacts DNA. The basic region is largely unfolded in the absence of DNA, but adopts a helical conformation upon DNA binding. Although a coil → helix transition is entropically unfavorable, this conformational change positions the DNA‐binding residues appropriately for sequence‐specific interactions with DNA. The N‐terminal residues of the GCN4 DNA‐binding domain, DPAAL, make no DNA contacts and are not part of the conserved basic region, but are nonetheless important for DNA binding. Asp and Pro are often found at the N‐termini of α‐helices, and such N‐capping motifs can stabilize α‐helical structure. In the present study, we investigate whether these two residues serve to stabilize a helical conformation in the GCN4 basic region, lowering the energetic cost for DNA binding. Our results suggest that the presence of these residues contributes significantly to helical structure and to the DNA‐binding ability of the basic region in the absence of the leucine zipper. Similar helix‐capping motifs are found in approximately half of all bZip domains, and the implications of these findings for in vivo protein function are discussed.
Heterogeneity in the structural dynamics of Sulfolobus solfataricus β‐glycosidase revealed by electron paramagnetic resonance and frequency domain fluorometry
Authors: Lozinsky Evgenia , Febbraio Ferdinando , Shames Alexander I. , Likhtenshtein Gertz I. , Bismuto Ettore , Nucci Roberto 
Published: October 2002

AbstractThe local and global dynamics of the Sulfolobus solfataricus β‐glycosidase were studied by electron spin resonance and time‐resolved fluorescence techniques. For electron paramagnetic resonance (EPR) investigations, the protein was covalently modified by the maleimido nitroxide spin label, which is specific for cysteine ‐SH groups, at position 344 and at position 101, where Ser‐101 was changed into a cysteine by site‐directed mutagenesis. The greater reactivity of exposed Cys‐101 suggested the exclusive modification of this amino acid compared with Cys‐344. The labeled proteins underwent temperature perturbation in the range 290–335 K and the values of the spin‐label rotation correlation frequencies (νc) ranged from 6 × 107 to 2 × 108 sec−1 for the protein labeled at position C344 and from 5.62 × 107 to 1.10 × 108 sec−1 for the protein labeled at C101. These rotation correlation values are related to the local dynamic characteristics of the protein matrix. The temperature dependence of rotation correlation frequencies expressed in terms of Arrhenius coordinates (log (νc) vs. 1/T) for the protein labeled at C344 exhibited a linear dependence but with a change in the slope at 311 K. For the protein labeled at C101, no change in the slope was observed at the same temperature. General dynamic information was deduced from the analysis of the fluorescence emission decay of the tryptophanyl residues that are present in each region of the protein structure. Fluorescence data analysis highlighted a bimodal distribution of fluorescence lifetimes arising from the contribution of two emitting groups: one consisting of closely clustered tryptophans responsible for the long‐lived emission component (7.1 nsec) and the other composed of tryptophans nearer to the protein surface, which can be associated to the short‐lived component (2.5 nsec). The temperature dependence of lifetime distribution parameters linked to the long‐lived and short‐lived components, expressed in Arrhenius coordinates, showed two different points in which the change in the slope occurred (i.e., 328 K and 338 K, respectively). The Arrhenius analysis of data provided the activation energy relative to the conformational changes characterizing the local and global movements running through the protein matrix.
Construction and characterization of protein libraries composed of secondary structure modules
Authors: Matsuura Tomoaki , Ernst Andreas , Plückthun Andreas 
Published: October 2002

AbstractOnly a minute fraction of all possible protein sequences can exist in the genomes of all life forms. To explore whether physicochemical constraints or a lack of need causes the paucity of different protein folds, we set out to construct protein libraries without any restriction of topology. We generated different libraries (all α‐helix, all β‐strand, and α‐helix plus β‐strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (α‐helix, β‐strand, and β‐turn) in various proportions, based primarily on the patterning of polar and nonpolar residues. We wished to explore that part of sequence space that is rich in secondary structure. The analysis of randomly chosen clones from each of the libraries showed that, despite the low sequence homology to known protein sequences, a substantial proportion of the library members containing α‐helix modules were indeed helical, possess a defined oligomerization state, and showed cooperative chemical unfolding behavior. On the other hand, proteins composed of mainly β‐strand modules tended to form amyloid‐like fibrils and were among the least soluble proteins ever reported. We found that a large fraction of members in non‐β‐strand–containing protein libraries that are distant from natural proteins in sequence space possess unexpectedly favorable properties. These results reinforce the efficacy of applying binary patterning to design proteins with native‐like properties despite lack of restriction in topology. Because of the intrinsic tendency of β‐strand modules to aggregate, their presence requires precise topologic arrangement to prevent fibril formation.
Distance‐scaled, finite ideal‐gas reference state improves structure‐derived potentials of mean force for structure selection and stability prediction
Authors: Zhou Hongyi , Zhou Yaoqi 
Published: October 2002

AbstractThe distance‐dependent structure‐derived potentials developed so far all employed a reference state that can be characterized as a residue (atom)‐averaged state. Here, we establish a new reference state called the distance‐scaled, finite ideal‐gas reference (DFIRE) state. The reference state is used to construct a residue‐specific all‐atom potential of mean force from a database of 1011 nonhomologous (less than 30% homology) protein structures with resolution less than 2 Å. The new all‐atom potential recognizes more native proteins from 32 multiple decoy sets, and raises an average Z‐score by 1.4 units more than two previously developed, residue‐specific, all‐atom knowledge‐based potentials. When only backbone and Cβ atoms are used in scoring, the performance of the DFIRE‐based potential, although is worse than that of the all‐atom version, is comparable to those of the previously developed potentials on the all‐atom level. In addition, the DFIRE‐based all‐atom potential provides the most accurate prediction of the stabilities of 895 mutants among three knowledge‐based all‐atom potentials. Comparison with several physical‐based potentials is made.
Construction of a fluorescent biosensor family
Authors: De Lorimier Robert M. , Smith J. Jeff , Dwyer Mary A. , Looger Loren L. , Sali Kevin M. , Paavola Chad D. , Rizk Shahir S. , Sadigov Shamil , Conrad David W. , Loew Leslie , Hellinga Homme W. 
Published: October 2002

AbstractBacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand‐mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share a common physical signal transduction functionality and detect many different analytes. We demonstrate the facility of designing optical biosensors based on fluorophore conjugates using 8 environmentally sensitive fluorophores and 11 bPBPs specific for diverse ligands, including sugars, amino acids, anions, cations, and dipeptides. Construction of reagentless fluorescent biosensors relies on identification of sites that undergo a local conformational change in concert with the global, ligand‐mediated hinge‐bending motion. Construction of cysteine mutations at these locations then permits site‐specific coupling of environmentally sensitive fluorophores that report ligand binding as changes in fluorescence intensity. For 10 of the bPBPs presented in this study, the three‐dimensional receptor structure was used to predict the location of reporter sites. In one case, a bPBP sensor specific for glutamic and aspartic acid was designed starting from genome sequence information and illustrates the potential for discovering novel binding functions in the microbial genosphere using bioinformatics.
Genetic analysis of response regulator activation in bacterial chemotaxis suggests an intermolecular mechanism
Authors: Re Sandra Da , Tolstykh Tatiana , Wolanin Peter M. , Stock Jeffry B. 
Published: October 2002

AbstractResponse regulator proteins of two‐component systems are usually activated by phosphorylation. The phosphorylated response regulator protein CheY∼P mediates the chemotaxis response in Escherichia coli. We performed random mutagenesis and selected CheY mutants that are constitutively active in the absence of phosphorylation. Although a single amino acid substitution can lead to constitutive activation, no single DNA base change can effect such a transition. Numerous different sets of mutations that activate in synergy were selected in several different combinations. These mutations were all located on the side of CheY defined by α4, β5, α5, and α1. Our findings argue against the two‐state hypothesis for response regulator activation. We propose an alternative intermolecular mechanism that involves a dynamic interplay between response regulators and their effector targets.
Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging
Authors: Bogin Oren , Levin Inna , Hacham Yael , Tel‐Or Shoshana , Peretz Moshe , Frolow Felix , Burstein Yigal 
Published: October 2002

AbstractPrevious research in our laboratory comparing the three‐dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224‐Lys254) and a short ion‐pair network (Lys257‐Asp237‐Arg304‐Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site‐directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K‐ and in the double mutant V224E/S254K‐CbADH, but not in the single mutant V224E‐CbADH. Both single amino acid replacements, M304R‐ and Q165E‐CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E‐CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R‐CbADH and solved its three‐dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.
Enhanced Interleukin-12 and CD40 Ligand Activities but ReducedStaphylococcus aureusCowan 1-Induced Responses Suggest a Generalized and Progressively Impaired Type 1 Cytokine Pattern for Human Schistosomiasis
Authors: Montenegro Silvia M. L. , Abath Frederico G. C. , Domingues Ana Lúcia C. , Melo Wlademir G. , Morais Clarice N. L. , Coutinho Eridan M. , Mahanty Siddhartha , Wynn Thomas A. 
Published: October 2002

ABSTRACTWhole-blood-cell cultures from schistosomiasis patients were stimulated with a variety of T-cell-dependent and T-cell-independent stimuli to determine whether the defect in type 1 cytokine expression observed following helminth infection is associated with alterations in interleukin-12 (IL-12) or CD40 ligand (CD40L) responsiveness. Cultures from uninfected individuals produced abundant gamma interferon in response toStaphylococcus aureusCowan 1 (SAC), while patients with intestinal and hepatosplenic disease displayed intermediate and weak responses, respectively. Importantly, the decrease in type 1 cytokine expression was not attributed to defects in IL-12- or CD40L-induced activity. Indeed, schistosomiasis patients displayed heightened responses and even produced more biologically active IL-12 when stimulated with SAC and CD40L than did uninfected controls. Finally, additional studies suggested only a partial role for IL-10, since intestinal patients were the only group that overproduced this downregulatory cytokine. Together, these studies demonstrate that the type 1 deficiency in chronic hepatosplenic schistosomiasis is not related to specific defects in IL-12, IL-10, or CD40L activity, although changes in the functional status of antigen-presenting cells appear to be involved.
The Growth Response of
Escherichia coli
to Neurotransmitters and Related Catecholamine Drugs Requires a Functional Enterobactin Biosynthesis and Uptake System
Authors: Burton Claire L. , Chhabra Siri Ram , Swift Simon , Baldwin Tom J. , Withers Helen , Hill Stephen J. , Williams Paul 
Published: October 2002

ABSTRACT

The neurotransmitter norepinephrine (NE) stimulates the growth of low inocula of
Escherichia coli
in a minimal medium (SAPI) supplemented with serum (SAPI+serum) and induces the production of an “autoinducer” (AI) which, in turn, promotes
E. coli
growth in the absence of NE. Given the importance of NE, epinephrine, and their corresponding adrenergic agonists and antagonists in clinical medicine, we sought to investigate the molecular basis for these observations. Using a variety of NE precursors, metabolites, and therapeutic agents, we demonstrated that their ability to stimulate
E. coli
growth in SAPI+serum is dependent on the presence of a catechol (1,2-dihydroxybenzene) moiety with maximal activity requiring a two-carbon substituent incorporating a terminal primary amine. Serum contains the iron-binding glycoprotein, transferrin, and when SAPI+serum was supplemented with sufficient Fe
3+
to saturate transferrin, growth inhibition was relieved. Other metal cations, including Mg
2+
, Ca
2+
, and Zn
2+
, had no effect. These data suggested that the stimulation of
E. coli
growth by NE in SAPI+serum may involve the catecholate siderophore, enterobactin, a cyclic triester of 2,3-dihydroxybenzoylserine. Consistent with this hypothesis,
E. coli
strains with mutations in ferrienterobactin transport (
fepA
or
tonB
) or enterobactin biosynthesis (
entA
) did not respond to NE. Furthermore, NE induced expression of the ferrienterobactin receptor, FepA, during growth in SAPI+serum. The enterobactin degradation product, 2,3-dihydroxybenzoylserine (DBS) was as effective as NE in stimulating the growth of
E. coli
and mutations in
fepA
or
tonB
abolished the DBS-dependent growth stimulation. In contrast to NE, however, DBS stimulated the growth of the
entA
mutant. Moreover, after growth in an iron-limited M9 medium in the absence of NE, ethyl acetate extracts of the

E. coli entA
+

parent but not of the
entA
mutant contained AI, i.e., stimulated the growth of
E. coli
in SAPI+serum. Taken together, these data show that when low numbers of
E. coli
are inoculated into SAPI+serum, NE, DBS, and related catecholamines induce the enterobactin iron uptake system. This, in turn, facilitates iron sequestration from transferrin and indicates that the AI present in NE-conditioned SAPI+serum medium is enterobactin and its DBS breakdown products.